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Gene Therapy and Molecular Biology Vol 11, page 1 Gene Ther Mol Biol Vol 11, 1-14, 2007

Colchicine-mediated focal adhesion formation promotes transient, lipoplex-mediated transfection of A549 cells Research Article

Rajesh R. Nair§, David M. Sherry# and Lindsay A. Schwarz* Department of Pharmacological and Pharmaceutical Sciences (RRN & LAS) and College of Optometry (DMS), University of Houston, Houston, TX 77204-5037

__________________________________________________________________________________ *Correspondence: Lindsay A. Schwarz, Department of Pharmacological and Pharmaceutical Sciences, University of Houston, Houston, TX 77204-5037, USA; Tel: 713-743-1778; Fax: 713-743-1229; E-mail: lschwarz@uh.edu § Current address: 7777 Knight Road, Dept of Cancer Biology, M.D. Anderson Cancer Center, Houston, TX 77054 # Current address: Department of Cell Biology, College of Medicine, University of Oklahoma Health Sciences Center,

P.O. Box 26901, Oklahoma City, OK 73104 Key words: lipoplex, cell signaling, focal adhesion kinase, gene transfection Abbreviations: chlorophenol red-b-D-galactopyranoside, (CPRG); focal adhesion kinase, (FAK); focal adhesion-regulated non-kinase, (FRNK); N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecytoxyl)-1-propanaminium bromide/cholesterol, (DMRIE/C) Received: 5 October 2006; Revised: 5 January 2007 Accepted: 11 January 2007; electronically published: February 2007

Summary Colchicine, a microtubule-disrupting drug, enhances lipoplex-DNA-mediated transfection. Colchicine-mediated increases in transgene expression are dependent on interference with tubulin polymerization, as pretreatment with paclitaxel, a microtubule-stabilizing agent, significantly inhibited the enhancing effects of colchicine. In addition to its interference with tubulin polymerization, colchicine-treatment activates Rho family GTPases, integrin clustering and the non-receptor tyrosine kinase, focal adhesion kinase (FAK), all known to be involved in formation of focal adhesions. We show that colchicine-mediated enhancement of transgene expression required activation of a Rho GTPase, as Clostridium difficle toxin B inhibited enhancement. Activation of a Rho GTPase lead to engagement of integrins, as the RGD-sequence peptide, an inhibitor of integrin clustering, abrogated colchicine-enhanced transgene expression. Genistein, a tyrosine kinase inhibitor, and cytochalasin D, both capable of inhibiting stress fiber formation, abolished colchicine-induced increases in transgene expression and suppressed focal adhesion formation, suggesting enhanced transgene expression involved stress fiber and focal adhesion formation. FRNK is an endogenous regulator of the tyrosine kinase, focal adhesion kinase (FAK). In A549 cells stably overexpressing the negative regulator, FRNK, colchicine pre-treatment did not enhance transgene expression, suggesting a critical role for FAK. Moreover, PP1, a selective, src-family kinase inhibitor also suppressed the ability of colchicine to enhance transgene expression. We propose that Rho-regulated, integrin clustering stimulates FAK and src kinase activation, formation of both focal adhesions and stress fibers, all of which appear critical to colchicine-mediated enhancement of transgene expression, as transfected by lipoplexes.

efficiency. However, exploring the cellular responses that promote uptake, nuclear translocation and expression of transfected genes, should yield strategies for improving transgene expression, as delivered by non-viral systems.

Although interactions between liposome-DNA complexes and the cell remain largely unexplained, it is known that liposome-DNA complexes are taken up by endocytosis. After internalization, the transgene DNA within the endosomal vesicles is transported via the

I. Introduction While newer, “gutless” adenoviral vectors induce less inflammation, safety issues and questions regarding the efficacy of repetitive therapeutic applications of viral vectors persist (Shayakhmetov et al, 2005; van der Linden et al, 2005). Thus, it is still important to refine and optimize gene delivery by non-viral vectors. The utility of cationic liposomes and other non-viral vectors in gene therapy has been limited due to their low transfection 1

Nair et al: Focal adhesion involvement in lipoplex-mediated transfection microtubules to the lysosomes. Much of the DNA transfected by lipoplexes is rapidly degraded in the endolysosomal compartment. Nevertheless, a fraction of this DNA escapes from endosomal vesicles through an acidification process that is aided by cationic liposomes.

Intact transgene DNA is translocated to nucleus where it is transcribed (Zuhorn and Hoekstra, 2002).

The importance of microtubule dynamics in this process was shown by Hasegawa and colleagues using immunocytochemistry to follow lipoplex trafficking.

These authors showed that in cells with intact microtubules, fluorescently-labeled transfected DNA colocalizes with lysosomes. However, when cells are treated with nocodazole, an agent that depolymerizes microtubules, or pacitaxel, an agent that stabilizes microtubules, DNA-lysosomal colocalization was not detected. Interestingly, Hasegawa and colleagues showed that, in cells treated with nocodazole or paclitaxel more transfected DNA accumulated in the nucleus over time and was maximal by 4 h after treatment with either agent (Hasegawa et al, 2001).

Agents that depolymerize microtubules, such as colchicine and nocodazole are known to increase the transfection efficiency of cationic liposomes both in vivo and in vitro. Intraperitoneal injection with colchicine increases transgene expression in liver of Gunn rats (2.5fold) (Chowdhry et al, 1996) as well as in BALB/c mice (2-fold) (Baru et al, 1995). While the mechanisms by which colchicine increases transiently-transfected transgene expression are still unclear, it has been suggested that disruption of microtubules limits the transfer of DNA between endosomes and lysosomes, thus preventing much of the degradation of transfected DNA (Chowdhry et al, 1996). In support of this suggestion, while investigating intracellular transport of transfected DNA, Brisson and colleagues found that inhibiting noncoated pit endocytosis decreased transgene expression but pretreatment of cells with nocodazole, presumed to block late endosome-lysosomal fusion, increased transgene expression by 2- to 3-fold (Brisson et al, 1999). However, Kitson and colleagues found no change in transgene expression in sheep airway epithelia pretreated with nocodazole (Kitson et al, 1999). Furthermore, chloroquine, an agent that prevents acid hydrolysis of lysosomal contents, has been shown to both increase (Baru et al, 1995) or decrease (Brisson et al, 1999) transfection efficiency. Despite the inconsistencies, these findings, nonetheless suggest that interference with microtubule dynamics is critical to improving transient transgene expression when employing non-viral vectors.

In addition to its effects on endo-lysosomal fusion, microtubular disruption also induces cellular signaling events similar to those seen with a variety of growth factors (Bershadsky et al, 1996). These signals ultimately include tyrosine phosphorylation of proteins involved in focal adhesions assembly (Jung et al, 1997; Kirchner et al, 2003). For example, microtubule disruption activates two, focal adhesion-associated tyrosine kinases, c-src and focal adhesion kinase (FAK). Their activation, by colchicine treatment appears to be primarily integrin-dependent and is regulated through the activation of the small GTP-

binding protein, Rho A (Enomoto, 1996). Furthermore, FAK activation, subsequent to microtubule disruption, leads to changes in expression of genes such as COX-2 and urokinase-type plasminogen activator gene (Irigoyen and Nagamine, 1999; Subbaramaiah et al, 2000). In light of these reports, we hypothesized that there may be microtubule-mediated cell signaling events, auxiliary to any role microtubules might play in endo-lysosomal fusion, involved in enhancing transient transgene expression.

The present study was undertaken to investigate the involvement of the Rho family of GTPases, integrins and focal adhesions in the enhancement of transgene expression observed in colchicine-treated A549 cells, transiently transfected with DNA-lipoplexes. We show that colchicine-mediated enhancement of transient, transgene expression requires the formation of focal adhesion complexes and activation of tyrosine kinases, csrc and FAK. These events appear to be dependent on the activation of the Rho family of GTPases and the clustering of integrins.

II. Materials and methods A. Chemicals and reagents Colchicine was purchased from Sigma Chemicals, St. Louis, MO. Genistein, cytochalasin D, PP1, Paclitaxel, RGD and control peptides and Clostridium difficle toxin B were purchased from Calbiochem, San Diego, CA. Dulbecco’s minimal essential medium (DMEM), OPTI-MEM, and cationic lipid N-(2hydroxyethyl) -N,N-dimethyl-2,3-bis(tetradecytoxy)-1propanaminiumbromide /cholesterol (DMRIE/C) and fetal bovine serum (FBS) were purchased from InVitrogen-Invitrogen/ Life Technologies/GIBCO, Carlsbad, CA. pCMV!-galactosidase (pCMV!gal) containing the bacterial LacZ gene was purchased from Clontech, Palo Alto, CA. pCMVFRNK-HA was generously provided by Dr. H. Sheldon Earp (University of North Carolina, Chapel Hill, NC). Rabbit polyclonal anti-FAK IgG and mouse monoclonal anti-phosphotyrosine IgG2b antibodies were purchased from Santa Cruz Biotechnologies, Santa Cruz, CA. The secondary antibodies, goat-anti-rabbit, AlexaFluor-488 was purchased from InVitrogen/Molecular Probes, Carlsbad, CA and the goat-anti-mouse Cy3 was purchased from Jackson ImmunoResearch Laboratories, West Grove, PA.

B. Cells A549 cells (CCL-185), a human lung adenocarcinoma cell line, representative of the distal respiratory epithelium, were purchased from ATCC, Manassas, VA. Cells were maintained in DMEM plus 15% low endotoxin-defined FBS (GIBCO), 200mM L-glutamine and 50Âľg/ml gentamycin and incubated in a humidified 370C incubator containing 5% CO2. Cells were trypsinized (0.25% trypsin, 0.53 mM EDTA) and diluted to a cell concentration of 7.5 " 104 cells/ml and either plated in 12- well culture dishes (1ml/well) for !-galactosidase assays or plated on cover slips (22mm, round) (0.4 ml/cover slip) for immunocytochemistry. For western blotting cells were plated at 2.7 X 105 per T25 cell culture flasks. These cell concentrations provide monolayers approximately 30% confluent 24 hours post seeding.

C. Treatment regimen All treatments with inhibitors were performed before transfection. All inhibitor concentrations were employed at concentrations that did not effect cell viability, as assessed by

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Gene Therapy and Molecular Biology Vol 11, page 3 microscopy and trypan blue exclusion. Cells were treated for 3 hours with indicated concentrations of paclitaxel, for 5 hours with indicated concentrations of genistein, PP1 and cytochalasin D, for 48 hours with indicated concentrations of toxin B and for 24 hours with indicated concentration of RGD-sequence peptide.

The dose response for colchicine has been determined previously and the optimal concentration of colchicine of 5 µM was used for a treatment period of 1 hour. After the various treatments, the cells were washed twice with OPTI-MEM before transfection, unless otherwise indicated.

complexes were adsorbed onto protein A beads (Sigma, St. Louis MO) and washed according to manufacturer’s instructions. The entire eluted fraction was loaded onto 8-16% SDS-PAGE gels and resolved by electrophoresis. All proteins were transferred to polyvinylidene fluoride membrane (PVDF) (Immuno-BLOT, Bio-Rad Lab, Hercules, CA) and blocked with Tris-buffered saline/Tween-20 (0.05%) containing either 5% non-fat dried milk or membrane blocking solution (Zymed Laboratories, San Francisco, CA). The membranes were probed with indicated antibodies and detected by secondary antibodies coupled to horseradish peroxide and ECL Plus detecting reagent (Amersham Biosciences, Piscataway, NJ). Western protein membranes were stripped and probed with mouse anti-glyceraldehyde phosphodehydrogenase (GAPDH) to normalize for loading differences. For all films, densitometry was performed on exposed x-ray film (Alpha-Innotech, San Leandro, CA).

D. Transfection All transfections were performed in OPTI-MEM. Cell monolayer was rinsed with OPTI-MEM prior to transfection with 1 µg pCMV!gal/4 µg DMRIE/cholesterol in 0.5 ml OPTI-MEM. Cells were incubated with DNA-lipid complexes for 2 hrs at 370C in a humidified, 5% CO2 incubator. Transfection complexes were removed and monolayers washed twice with OPTI-MEM and then either immediately subjected to immunocytochemistry and western blotting at 40C or fed with DMEM plus 15% FBS and incubated for 72 hrs for !-galactosidase assay.

H. Preparation of stable transformant A549 cells Cells were plated at 3 " 105 cells/ml in T25 cm2 cell culture flasks. After overnight incubation in growth medium, cells were transfected with 14 µg of pCMV-HA-FRNK plasmid and 2 µg of the selectable vector, pRSVneo, complexed to 64 µg of DMRIE/c in OPTI-MEM. Some cells were transfected with pRSV-neo only and untransfected cells were used as controls.

The cells were incubated in growth medium for the first 48 hrs at 370C. After 48 hrs the growth medium was removed and replaced with growth medium containing 1 mg/ml of geneticin (G418, GIBCO). The selection medium was changed periodically changed and G418 was increased to a final concentration of 1.3 mg/ml. The cells surviving selection were expanded and subsequently, individual colonies derived from individual cells were seeded T25 cm2 cell culture flasks containing 1.3 mg/ml of G418. These cells were employed in experiments investigating the role of FAK in colchicine-mediated enhancement of transgene expression.

E. !-galactosidase assay Cells were lysed in 10 mM Tris, pH 7.6, 0.1% Triton X100. Each clarified cell lysate was first assayed for total protein, using bicinchoninic acid assay per the manufacture’s instruction (BCA assay, Pierce Chemicals, Rockford, IL). After adjusting samples for equivalent protein concentration in 10 mM Tris, pH 7.6, samples were assayed for !-galactosidase activity as described (CPRG, Roche, Nutley, NJ). A !gal standard curve (!galactosidase, InVitrogen) was included with each assay and all experimental !gal values were within the linear slope of the standard curve. The !-galactosidase activity was measured in units/ml (U/ml) and the activity of !-galactosidase in cells treated with growth medium before transfection was set as equal to 1. Fold change of all other drug treatment groups was calculated as: (U/ml !gal in drug treated, transfected)/(U/ml !gal in medium-treated, transfected).

I. Statistical analysis All experiments were independently performed three or more times. !gal expression was measured in U/ml of !galactosidase cell lysates. To obtain fold-change, the !gal U/ml of the treated, transfected lysates were divided by the !gal U/ml in lysates of untreated, transfected controls. The maximal and minimal U/ml for each set of experiments are included in the figure legends as a point of reference. The data were plotted as mean ± SD and analyzed for statistical significance between treatment groups using one-way ANOVA followed by post hoc, Students-Newman-Keuls test (Primer of Biostatistics; Glantz SA, statistical software version 5.0). A P< 0.05 was accepted as statistically significant.

F. Immunocytochemistry Cells were plated on coverslips. Following different drug or inhibitor treatments and transfection, cells were rinsed in PBS. The cells were then fixed with methanol on ice for 5 min, followed by 1 min incubation with NaBH 4 (0.5 mg/ml). The cells were then washed three times with PBS and blocked with PBS containing 2% normal goat serum, and 0.1% Triton X-100 (blocking solution) for 45 min. After blocking, the cells were incubated concomitantly with mouse anti-phosphotyrosine IgG (1:40 dilution) for 48 hours followed by rabbit anti-FAK IgG (1:60 dilution) for the last 24 hours, at 40C. The cells were washed three times with PBS, reblocked and incubated for 45 min with anti-mouse-Cy3 (1:500) and anti-rabbit-AlexaFluor 488 (1:400). The cells were examined, in a blinded manner, for fluoresecence (Olympus IX70 epifluorescence microscope).

III. Results A. Enhancement of transiently transfected reporter gene expression involves cellular mechanisms distinct from endolysosomal fusion

G. Western blotting Cells were seeded into T25 cm 2 cell culture flasks and then treated with drugs and inhibitors prior to transfection. The flasks containing cells were then placed on ice and lysed in 1 ml of iced cold lysis buffer (1% Triton X-100 and 0.01M Tris, pH 7.6) for 30 min. Flasks were scraped and total protein was determined for the unfractionated lysates using BCA assay. Five µg of total protein was applied to an 8-16% SDS-PAGE gel (Gradipore, Forresttown, Australia) and proteins resolved by electrophoresis, For immunoprecipitations, cell lysates were incubated with polyclonal rabbit anti-FAK antibody overnight. Protein antibody

We have previously shown that treatment of A549 cells with 5 µM colchicine for 1 h prior to transfection enhances transgene expression by 15- to 30-fold (Nair et al, 2002). While the literature supports the hypothesis that depolymerizing microtubules favorably alters DNA release from the endo-lysosomal pathway, it is difficult to reconcile the observations that both stabilizing and destabilizing microtubules promote the more rapid release

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Nair et al: Focal adhesion involvement in lipoplex-mediated transfection of DNA from the endo-lysosomes (Brisson et al, 1999; Kitson et al, 1999). Furthermore, in previous studies we observed that paclitaxel, a compound that stabilizes microtubules, also enhances transgene expression in a variety of cell lines, albeit to modest degree when compared to colchicine (Nair et al, 2002). Thus, it was possible that, in addition to enhancing transfection efficiency strictly through an endo-lysosomal pathway, changes in microtubule dynamics influence other aspects of transgene delivery or expression.

To determine the extent of the contribution of additional colchicine-mediated effects that enhance transgene expression, we transfected A549 cells with DMRIE/C-pCMV!gal complexes for two hours, washed away untransfected complexes and then treated the cells at with 5 µM of colchicine at time 0 (immediately after transfection) or 6, 12, 18 and 24 hours after transfection.

Treatment of cells for a period of 1 hour with 5 µM of colchicine immediately after transfection (t=0) and, at each successive treatment time point produced a significant increase in !gal expression, as compared to !gal expression in untreated, transfected cells (33 ± 5.5fold increase; Figure 1). Since endo-lysosomal fusion should be complete within the first few hours after transfection (Hasegawa et al, 2001; Rejman et al, 2005), these studies suggested that enhancement in reporter gene expression mediated by colchicine was not exclusively dependent on inhibition of endo-lysosomal fusion. Rather, colchicine treatment mediated other cellular actions that had significant effects on transgene expression.

Paclitaxel stabilizes microtubules through the direct binding of tubulin but at a site that does not interfere with tubulin binding by colchicine (Kumar, 1981). To determine whether or not microtubule disruption, specifically, was important to enhanced transgene expression, A549 cells were treated with paclitaxel prior to treatment with 5 µM of colchicine. Paclitaxel significantly inhibited the ability of colchicine to increase reporter gene expression (colchicine treatment only, 16-fold; paclitaxel

plus colchicine treatments, 6-fold). At its highest dose, 10 µM, paclitaxel inhibited the colchicine-induced increase by approximately 63% (data not shown). Detection of additional decreases in transgene expression was limited by paclitaxel’s own ability to enhance transgene expression; alone, paclitaxel mediated a 6-fold (+/- 1.4) enhancement of !gal expression (data not shown) (Nair et al, 2002). Thus, the ability of colchicine to enhance transgene expression appears to be dependent, in part, on microtubular disruption.

B. Colchicine-induced enhancement

of reporter gene expression requires activation of Rho family of GTPases, integrin clustering and actin stress fiber formation Colchicine induces integrin clustering and actin stress fiber formation through cellular events that involve Rho A GTPase activation (Enomoto, 1996). Induction of integrin clustering by activated Rho GTPases promotes “outside-in” signaling (Hotchin and Hall, 1995; Clark et al, 1998) known to activate kinase pathways that ultimately alter the cytoskeleton and even gene expression (Schlaepfer et al, 1998; Watanabe et al, 2003). In light of these known activities, specific inhibitors of Rho A activation and events that sequentially follow Rho A activation were employed to determine the involvement of these events in colchicine-mediated enhancement of transgene expression. First, A549 cells were treated with Clostridium difficle toxin B (toxin B) that, through glycosylation of an aspartate residue in the effector region of the Rho GTPases, inactivates GTPase activity in all members of the Rho family (Just et al, 1995). Pretreatment of A549 cells with toxin B alone did not significantly alter transiently transfected !gal transgene expression (Figure 2). A549 cells, pretreated with 5 µM of colchicine only for

Figure 1.

Colchicine-mediated enhancement of reporter gene expression occurs independently of endo-lysosomal fusion. Cells were transfected for two hours as described.

Transfected cells (TFX) were treated with 5 µM of colchicine (TFX, COL) for an interval of 1 h immediately after transfection (T=0 hours) or 6 h, 12 h, 18 h and 24 h after transfection. Cells were harvested 72 hours posttransfection and supernatants analyzed for !gal activity by CPRG assay, as described. *p<0.05, when compared to untreated, transfected cells (n=3).

Average maximal U/ml !gal, 0.023 ± 0.0046; Average control U/ml !gal (fold-increase = 1), 0.00064 ± 0.00008.

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Gene Therapy and Molecular Biology Vol 11, page 5 1 h prior to transfection, caused a 53-fold (± 6.7) increase in !gal reporter gene expression, as compared with untreated, transfected cells (Figure 2). However, toxin B significantly and dose-dependently inhibited the increase in !gal expression induced by colchicine. These results suggested that activation of a Rho GTPase was involved in the colchicine-mediated enhancement transgene expression.

Next, to determine the role of integrin clustering in the colchicine-mediated enhancement of transient transgene expression, we treated A549 cells with the RGD peptide. While the RGD peptide (H-Gly-Arg-Gly-AspSer-OH) is a ligand for integrin proteins, this peptide also prevents integrin-clustering (Pierschbacher and Rouslathi, 1984). An inactive peptide, (H-Gly-Arg-Ala-Asp-Ser-ProOH), and the vehicle, acetic acid, were employed as controls.

For these experiments, A549 cells were treated in suspension to prevent plastic adherence-induced integrin clustering (Ruegg et al, 1992). Cells were treated with either the RGD or the inactive peptides for 30 min at 370C, to allow the binding of the peptides to the integrins. The cell suspension was then plated and incubated in the presence of the RGD peptide for an additional 24 hours.

Figure 3 shows that RGD peptide alone, at 1 mM, did not significantly alter !gal transgene expression, as compared to untreated, transfected cells. Colchicine alone, at 5 µM, resulted in a 14-fold increase in !gal transgene expression.

This colchicine-mediated increase in transgene expression

was similar to that seen in cells treated with acetic acid (the vehicle) or the inactive peptide. In contrast, pretreatment with the active RGD peptide significantly inhibited the colchicine-induced increase in reporter gene expression by 70%, as compared to the controls (Figure 3).

These results suggest that microtubule depolymerization, most likely acting through RhoA GTPase, initiated integrin-clustering.

While it is unclear as to whether existing actin stress fibers participate in integrin clustering or whether actin stress fibers form as a result of integrin clustering, colchicine treatment is known to stimulate actin stress fiber formation (Blystone, 2004; Wozniak et al, 2004). To determine the role of actin polymerization in the colchicine-mediated increase in transgene expression, cells were treated with cytochalasin D, an inhibitor of actin polymerization. Cytochalasin D alone, at the doses employed, did not significantly alter !gal expression; colchicine, at 5 µM, increased !gal expression in A549 cells by 15-fold (± 1.1), as compared to untreated, transfected cells (Figure 4). As seen in Figure 4, at its highest dose of 10 µM, cytochalasin D significantly inhibited the colchicine-induced increase in transgene expression by 71%. These results indicated that actin polymerization was critical for the colchicine-mediated increase in transgene expression, as delivered to A549 cells using cationic liposomes.

Figure 2. Rho GTPases participate in colchicine-mediated enhancement of transgene expression in A549 cells. A549 cells were treated with toxin B (4, 8, 12, 16 and 20 ng/ml) in either in growth medium (Medium) or medium plus 5 µM of colchicine (Colchicine, 1 h) for 48 h. Cells were then transfected with pCMV!gal as described. Cells were lysed and supernatants were harvested and !gal activity determined as described. *p<0.05, when compared to untreated, transfected cells; #p<0.05, when compared to colchicine treated, transfected cells (n=4). Average maximal U/ml !gal, 0.012 ± 0.0013; average control U/ml !gal, 0.00022 ± 0.00011. Inset: Effects of toxin B on transfection (U/ml !gal).

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Figure 3. Colchicine-mediated increases in reporter gene expression involves integrin clustering. Cells were incubated in 0.1% FBS overnight, then treated with 1mM of RGD peptide (HGly-Arg-Gly-Asp-Ser –OH) in growth medium (Medium) or with medium containing 5 µM of colchicine (Colchicine, 1 h) for 24 h. Acetic acid only and an inactive peptide (1mM) (H-Gly-Arg-AlaAsp-Ser-Pro-OH), were employed as controls. Cells were transfected with pCMV!gal as described. Cell supernatants were harvested and !gal levels determined. *p<0.05, when compared with untreated, transfected cells; #p<0.05, when compared with colchicine treated, transfected cells (n=3). Average maximal U/ml !gal, 0.0065 ± 0.006; average control U/ml !gal (fold-change = 1), 0.00043 ± 0.0004.

Figure 4. Stress fiber formation is involved in colchicinemediated enhancement of reporter gene expression in A549 cells. Cells were treated with cytochalasin D (2, 4, 6, 8, and 10 µM) in either growth medium (Medium) or medium containing 5 µM of colchicine (Colchicine, 1 h) for 5 h. Cells were transfected with pCMV!gal as described. Cell supernatants were harvested and !gal levels were determined. *p<0.05, when compared to untreated, transfected cells; #p<0.05, when compared to colchicine-treated, transfected cells (n=3). Average maximal U/ml !gal, 0.00136 ± 0.0013; average control U/ml !gal, 0.00009 ± 0.00004.

adhesions creates a harbor for the docking of various signaling and accessory proteins, including the tyrosine kinases, FAK and c-src, each of which has been implicated in activation of the MAPK pathway (Schlaepfer et al, 1998; Brunton et al, 2004; Graness et al, 2006).

Furthermore, microtubule disruption is known to induce tyrosine phosphorylation of FAK as well as other proteins that participate in the formation of focal adhesions

C. Colchicine-mediated enhancement of reporter gene expression requires src-FAK activation RhoA-regulated integrin clustering and actin stress fiber formation are associated with focal adhesion formation (Schoenwaelder and Burridge, 1999). In addition to their role in anchoring cells to the extracellular matrix, the assembly of integrin-containing, focal 6

Gene Therapy and Molecular Biology Vol 11, page 7 (Bershadsky et al, 1996).

To determine the role of tyrosine phosphorylation in the colchicine-mediated enhancement of reporter gene expression, cells were pretreated with genistein, a broadspectrum inhibitor of tyrosine kinases. Genistein, itself, did not alter !gal transgene expression, as compared to untreated, transfected cells. As seen in previous experiments, colchicine, at 5 µM increased !gal expression (37-fold ± 6.7), as compared to untreated, transfected cells (Figure 5). When used in combination with colchicine, genistein significantly inhibited the ability of colchicine to induce an increase in transgene expression. At its highest dose of 140 µM, genistein blocked the ability of colchicine to induce an increase in transgene expression by 85% (Figure 5). These results suggested that colchicine, either directly or indirectly activated a tyrosine kinase and that tyrosine phosphorylation of some cellular target was involved in enhancing transgene expression.

Colchicine is known to activate FAK and FAK serves as both a tyrosine kinase as well as a scaffold for proteins that direct downstream cell signaling. Thus, our next experiments focused on the role FAK activation in the enhancement of transgene expression. Endogenous FAK activation is regulated, in part, by the cellular molecule, FAK-related non-tyrosine kinase (FRNK) that inhibits FAK tyrosine phosphorylation and interferes with focal adhesion formation (Richardson and Parsons, 1996).

We developed an A549 cell line stably overexpressing FRNK (A549FRNK). A549 cells stably expressing neomycin only, the selectable gene employed in generating A549FRNK, were used as transformation controls (A549NEO). To confirm FRNK overexpression, A549NEO and A549FRNK cells were assessed for FAK immunofluorescence by immunocytochemistry. While FAK-specific puncta resembling focal adhesions were

present in colchicine-treated, untransfected, nontransformed A549 or A549NEO, FAK-specific immunofluorescence was undetectable in the A549FRNK cells (data not shown).

Expression of transiently transfected pCMV!gal in untreated A549NEO cells showed !gal levels similar to those seen in untreated, but transiently transfected, nontransformed A549 cells. Although not statistically significant, !gal expression in A549FRNK cells, in the absence of colchicine, was decreased by 30%, as compared to untreated, transfected non-transformed A549 or A549NEO cells (Figure 6). Treatment of nontransformed A549 cells with 5 µM of colchicine prior to transient pCMV!gal transfection produced a 12.5-fold (± 0.47) increase in !gal expression. A similar increase in !gal expression was observed in colchicine-treated, !gal transfected, A549NEO cells. In contrast, in A549FRNK cells, the colchicine-mediated increase in reporter gene expression was blunted, by 80 % (2.6 ± 0.33- fold increase in !gal transgene expression), as compared to the A549NEO cells (Figure 6).

Western blot analysis was performed to determine whether or not overexpression of FRNK would alter tyrosine phosphorylation of FAK. Lysates obtained from colchicine-treated A549FRNK and A549NEO cells were subjected to SDS-PAGE, probed for tyrosinephosphorylated proteins, then reprobed for FAK, itself and finally probed for GAPDH, as a loading control. In A549NEO cells, colchicine treatment significantly increased the tyrosine phosphorylation of a 125 kD protein (Figure 7, inset), corresponding to FAK (IDVCol 5µM = 4.6 ± 0.2), as compared to untreated, !gal transfected A549NEO cells (T=0, IDVcontrol = 2.1 ± 1.2) (Figure 7).

Figure 5. Colchicine-induced increases in reporter gene expression requires tyrosine phosphorylation. Cells were treated with genistein (60, 80, 100, 120 and 140 µM) either in growth medium (Medium) or with medium containing 5 µM of colchicine (Colchicine, 1 h) for 5 h. Cells were transfected with pCMV!gal as described. Cell supernatants were harvested and !gal levels determined. *p<0.05, when, compared to untreated, transfected cells; #p<0.05, when compared with colchicine treated, transfected cells (n=4). Average maximal U/ml !gal, 0.0048 ± 0.0006; average control U/ml !gal (fold-change = 1), 0.00013 ± 0.00003.

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Figure 6. Activation of FAK is required for colchicine-induced increase in reporter gene expression in A549 cells. Non-transformed A549 cells (A549 cells), A549 cells stably expressing neomycin only (Neo-stables) and A549 cells stably expressing FRNK plus neomycin (FRNK-stables) were incubated in medium containing 0.1% FBS overnight then treated with either growth medium (Medium) or medium containing 5 µM of colchicine (Colchicine) for 1 h. Cells were transfected with pCMV!gal as described. Cell supernatants were harvested and !gal levels were determined. *p<0.05, when compared to respective untreated, transfected cells; #p<0.05, when compared with colchicine-treated, transfected cells (n=3). Average maximal U/ml !gal, 0.0042 ± 0.0033; average !gal, medium-treated A549 cells, 0.00033 ± 0.00025 (foldchange = 1); average !gal, medium-treated A549, stably expressing pRSVneo, (neo-stables), 0.00035 ± 0.00013 U/ml,(fold-change = 1; average !gal in medium-treated, A549 cells stably expressing FRNK, (FRNK-stables) 0.00020 ± 0.00003 U/ml, (fold-change =1).

Figure 7. FAK activation is required for colchicineinduced increase in transgene expression. A549 cells, stably expressing neomycin or FRNK plus neomycin, were incubated overnight in DMEM containing 0.1% FBS, then treated with either medium only, or medium containing either 1 µM or 5 µM of colchicine, for 1 h.

(A) Cell lysates were subjected to SDS-PAGE and proteins blotted as described. NEO, untreated A549 cells stably expressing neomycin only; NEO+Col, 1µM, A549 cells stably expressing neomycin, treated with 1 µM of colchicine; NEO+Col, 5µM, A549 cells stably expressing neomycin treated with 5 µM of colchicine; FRNK, untreated A549 cells stably expressing FRNK+neomycin; FRNK Col, 1 µM, A549 cells stably expressing FRNK+neomycin, treated with 1 µM of colchicine; FRNK Col, 5µM, A549 cells stably expressing FRNK+neomycin, treated with 5 µM of colchicine. Blots were analyzed for phosphotyrosine proteins and FAK. GAPDH was used as loading control.

(B) Immunoblot band intensities were analyzed by densitometry (integrated density values, IDV) and are represented as mean ± SD of the ratio of the IDV of the 125 kD anti-phosphotyrosine (PY) (125 kD)/ IDV of GAPDH, from 3 independent experiments. *p<0.05, when compared with PY-125 kD/ GAPDH IDV ratio of A549 cells stably expressing FRNK.

However, in lysates obtained from A549FRNK cells, colchicine treatment did not increase levels of the tyrosine-phosphorylated, 125 kD protein (IDVCol 5µM = 1.8 ± 0.8); band intensity was similar to that obtained in lysates from untreated, !gal transfected A549NEO cells (IDVControl =1.3 ± 0.4). These combined results suggested that tyrosine phosphorylation, specifically of FAK, was important for the colchicine-mediated enhancement in transient transgene expression in A549 cells.

Phosphorylation of FAK has been shown to be important for the recruitment of src-family kinase.

Specifically, the phosphorylation on FAK tyrosine residue 397 creates a high affinity binding site recognized by the SH2 domain of src family of kinases (Schaller et al, 1994).

In order to elucidate the role of src-kinase in the colchicine-mediated enhancement of reporter gene expression, we utilized with PP1, a more specific inhibitor of src-kinase (Hanke et al, 1996) in subsequent experiments.

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Gene Therapy and Molecular Biology Vol 11, page 9 As expected, 5µM colchicine, significantly increased !gal expression by 21-fold (± 6.4), as compared to untreated, transfected A549 cells (Figure 8). PP1, alone, at the concentrations employed (2-10 µM), did not alter !gal expression, as compared to untreated, transfected cells.

However, when combined with colchicine, PP1 significantly and dose-dependently inhibited the ability of colchicine to mediate an increase in reporter gene expression. The highest dose of PP1 (10 µM,) inhibited the colchicine-mediated increase in !gal expression by 89% (Figure 8). To clarify the relationship between src family tyrosine kinase activity and FAK activation, A549 cells were pretreated with 10 µM of PP1 for 2h prior to treatment with 5 µM colchicine and subsequent pCMV!gal transfection. FAK was immunoprecipitated (IP) with anti-FAK antibody and eluted proteins were resolved by SDS-PAGE and incubated with either antiphosphotyrosine or anti-FAK. A representative FAK-IP, incubated with anti-phosphotyrosine and anti-FAK immunoblot is presented as an inset in Figure 8 shows that PP1, in addition to blocking colchicine-mediated enhancement of transgene expression, significantly inhibited colchicine-mediated FAK tyrosine phosphorylation. These combined results suggest that colchicine-mediated enhancement in reporter gene

expression requires src family kinase activity and that src kinases phosphorylated FAK.

D. Colchicine-induced increase in reporter gene expression involves formation of focal adhesions In order to determine whether or not focal adhesion formation correlated with colchicine-induced enhancement of reporter gene expression, A549 cells were grown on cover slips and treated with colchicine alone or in combination with genistein prior to transfection and then analyzed by immunocytochemistry, using antiphosphotyrosine and anti-FAK antibodies.

Dual labeling of untreated, untransfected A549 cells with anti-phosphotyrosine antibody and anti-FAK antibody revealed little or no co-localized staining at the cell periphery, where focal adhesions typically form. A similar lack of colocalized staining at the periphery was observed in genistein-treated, transfected cells (Figure 9). In contrast, colchicine-treated cells displayed dual-stained puncta localized to the cell membrane (Figure 9. panel L). Addition of genistein to colchicine-treated cells completely abolished the appearance of these focal adhesions, indicating that tyrosine phosphorylation was required to produce colchicine-mediated formation of focal adhesions.

Figure 8. PP1 decreases colchicine-mediated increase in transgene expression through inhibition of FAK activity. Cells were treated with PP1 (2, 4, 6, 8, and 10 µM) for 5 h before transfection. Colchicine (5 µM, final concentration) was added to one set of PP1treated cell cultures and all cultures were incubated for an additional h. PP1 treatment was continued during and for 24 hours after transfection. !gal activity was determined in cell supernatants. *p<0.05, as compared to untreated, transfected cells; #p<0.05, as compared to colchicine-treated, transfected cells (n=3). Inset: A549 cells were treated with medium (Control, Transfection), 5 µ M colchicine (Colchicine, 1 h), 10 µM PP1 for 5 h (PP1) or 10 µM PP1 (5 h) plus colchicine (5 µM, 1h) (Colchicine+PP1). All cultures except “control” were transfected with pCMV!gal. Cell lysates were immunoprecipitated using anti-FAK antibody and then subjected to SDS-PAGE. Blots were incubated first with anti-phosphotyrosine and after film exposure stripped and incubated with anti-FAK, as loading control.

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Nair et al: Focal adhesion involvement in lipoplex-mediated transfection

Figure 9. Colchicine induces focal adhesion formation and FAK tyrosine phosphorylation in A549 cells. A549 cells were plated on coverslips and incubated overnight in 0.1% FBS. The cells were treated with medium, 5 ÂľM of colchicine (1h), alone or in combination with 140 ÂľM of genistein (5 h). Cells were transfected and immunocytochemistry performed as described. (A, B, C), untreated, untransfected cells; (D, E, F), untreated, transfected cells; (G, H, I), genistein-treated, transfected cells; (J, K, L), colchicine-treated, transfected cells; (M, N, O), colchicine plus genistein-treated, transfected cells. (A, D, G, J and M ), were stained with anti-FAK; (B, E, H, K and N), are the same cells additionally stained with anti-phosphotyrosine (anti-PY) and finally (C, F, I, L and O) are respective merged images. The panels shown are representative of three independently- performed experiments.

enhancement of transient, transgene expression by approximately 65%, indicating Rho GTPase activation was involved in enhanced transgene expression. In these experiments, enhancement of transgene expression by colchicine alone appeared greater (53-fold), relative to other experiments presented in this report (average foldchange, ~22-fold). This greater level of enhanced transgene expression is most likely the result of the longer incubation required with toxin B treatment (48 h). During the incubation, both the untreated and treated A549 cells continued to divide in the experimental cell culture dishes.

Thus, at transfection, there were more cells available for transfection than experiments that require a shorter pretreatment incubation.

The mechanism by which microtubular depolymerization causes activation of Rho family of GTPases is not completely understood. However, certain guanine nucleotide exchange factors (GEF), such as GEFH1, an exchange factor for, rac1 and rho A GTPases, are localized and sequestered on the microtubules (Ren et al, 1998). Microtubule disruption liberates these GEFs where they are free to activate Rho GTPases (Ren et al, 1998).

IV. Discussion This is the first report showing FAK/src activation and focal adhesion formation participate in the enhanced expression of lipoplex-mediated, transiently transfected DNA and offers insight into how efficiency of lipoplexmediated transfection can be improved.

Although the ability of colchicine to enhance transgene expression has been well documented (Baru et al, 1995; Chowdhry et al, 1996), the mechanism by which microtubule disruption enhances transgene expression was presumed to be due solely to disruption of endo-lysosomal fusion (Chowdhry et al, 1996; Hasegawa et al, 2001; Wang and MacDonald, 2004). However, we show that in A549 cells enhanced transgene expression occurs even when colchicine was added 24 h after transfection, a time by which endo-lysosomal fusion should be complete (Rejman et al, 2005).

Microtubule-disruption also induces activation of Rho A GTPases, and integrin clustering (Bershadsky et al, 1996; Enomoto, 1996; Kirchner et al, 2003; Graness et al, 2006). Clostridium difficile toxin B, an inhibitor of Rho GTPases (Just et al, 1995) inhibited colchicine-mediated

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Gene Therapy and Molecular Biology Vol 11, page 11 Rho GTPase activation is seen just prior to integrin clustering. When integrins associate with certain extracellular matrix substrates or activated growth factor receptors, conformational changes within the intracellular portion of the integrin promote the binding of a variety of molecules that direct subsequent signaling cascades (Schlaepfer et al, 1998; Martin et al, 2002). RGD peptide, known to block integrin clustering (Pierschbacher and Rouslathi, 1984), inhibited the colchicine-mediated enhancement of transgene expression by as much as 70%, as compared to cells pretreated with a control peptide, suggesting that either integrin clustering itself or some process associated with integrin clustering was important for enhanced transgene expression.

Keller and colleagues previously reported that they could enhance transient, lipoplex-mediated lymphocyte transfection by allowing a non-adherent, hematopoietic cell line to attach to an adherent cell line prior to transfection with pCMV!gal. Transgene expression within the hematopoeitic cell line was significantly enhanced (Keller et al, 1999). While they did not address the role of integrins in transgene expression, it is tempting to consider that integrins may have also played a role in enhancing transgene expression in this notably difficult-to-transfect, non-adherent cell type. In a more recent report, Perlstein and colleagues examined the role of integrin ligands in transfection of smooth muscle cells (Perlstein et al, 2003).

They found that a denatured collagen, a ligand for integrin #$!3 the same integrin expressed on A549 cells (Majda et al, 1994), enhanced liposome-mediated transgene expression 10-20 fold (Perlstein et al, 2003).

Clustered integrins provide a scaffold for actinbinding proteins including talin, filamin and #-actinin (Lee and Juliano, 2004). Whether integrins provide an anchor point for the development of actin filaments (Blystone, 2004) or whether polymerized actin fibers direct integrin clustering (Wozniak et al, 2004) is still debatable. Nonetheless, with cellular adhesion, actin stress fibers are formed (Brakebusch and Fassler, 2003). We show that pretreatment of A549 cells with cytochalasin D prior to the addition of colchicine and transfection, diminished colchicine-mediated enhancement of transgene expression by approximately 70%, indicating that stress fiber formation was also involved in enhancing transgene expression.

In contrast to our results, Perlstein and colleagues found that a 24 h treatment of cells with cytochalasin D modestly enhanced (6-fold), rather than blocked transgene expression, a result they attribute to an increase in the amount of cytoplasmic G-actin, known to act as an inhibitor of DNAse I (Perlstein et al, 2003). These authors also showed that jasplakonide, a drug that stabilizes actin polymers, decreased transfection efficiency (Perlstein et al, 2003). However, Brisson and colleagues using cytochalasin B, which also inhibits stress fiber formation, saw little change in transfection efficiency (Brisson et al, 1999). In addition, in our hands neither cytochalasin D nor jasplakonide enhanced pCMV!gal transfection (data not shown). The contradictions between these studies most likely involve differences in cells employed, cell culture substrates (collagen vs. plastic), drug concentration and

duration of drug exposure. Nevertheless, our experiments employing RGD, toxin B and cytochalasin D, support a model where activation of Rho GTPases, actin polymerization and clustering of integrins accompany microtubule disruption and play an important role in expression of transiently transfected transgenes.

Downstream of the integrins, signaling critical to enhancement of transgene expression appears to involve tyrosine kinase activation. FAK autophosphorylation of tyr 397 creates a high affinity binding site, recognized by the SH2 domain of src family kinases (Schaller et al, 1994).

We show that pretreatment of A549 cells with either genistein, a broad-spectrum tyrosine kinase inhibitor or the more selective PP1, inhibit colchine-mediated enhancement of transgene expression. Notably, colchicine treatment of A549 cells stably expressing FRNK, an endogenous, negative regulator of FAK, failed to enhance transgene expression in these transformants. Lack of enhanced transgene expression correlated with a decrease in colchicine-mediated FAK phosphorylation in A549FRNK cells.

Not only did PP1 dose-dependently decrease the ability of colchicine to augment transgene expression but as shown by FAK immunoprecipitation, PP1 treatment suppressed the degree of FAK tyrosine phosphorylation.

These combined results suggest that both src and FAK tyrosine kinases are involved in enhancing transgene expression and that FAK is a target of a src kinase.

How does activation of this src-FAK pathway lead to increased transgene expression? FAK has been implicated, directly or indirectly, with activating expression of endogenous genes. Watanabe and colleagues showed that FAK overexpression induced expression of endogenous monocyte chemotractant protein-1 (MCP-1). Upregulation of MCP-1 was inhibited by genistein and by overexpression of FRNK. (Watanabe et al, 2003).

Irigoyen et al. showed that colchicine-mediated expression of the endogenous urokinase-type plasminogen activator gene involved not only FAK-src activation but subsequently, activation of ERK 1/2 (Irigoyen and Nagamine, 1999). Thus, a potential means whereby FAK increases gene or even transgene expression is via activation of the Ras/MAP kinase cascade. The FAK-srckinase interaction appears to facilitate the assembly of scaffolding proteins that promote MAP kinase binding and subsequent activation of ERK (Schlaepfer et al, 1998), (Irigoyen and Nagamine, 1999). In support of this signaling pathway, an increase in ERK1/2 phosphorylation was seen in A549 cells pretreated with colchicine (data not shown, Ph. D thesis, R R Nair). Moreover, when vinblastine, another agent that disrupts microtubules, was directly incorporated into cationic liposomes, not only was early lysosomal release of transfected DNA seen but vinblastine also increased NF-%B activation (Wang and MacDonald, 2004). Thus, activation of cellular transcription factors, through a microtubule-mediated stimulation of certain kinases might increase the level of transgene transcription. For example, colchicine is known to increase cyclooxygenase-2 gene transcription via activation of the ERK pathway (Subbaramaiah et al, 2000). Notably, colchicine did not increase actin B gene

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Nair et al: Focal adhesion involvement in lipoplex-mediated transfection signaling pathway. Biochem Biophys Res Commun 262, 666-670.

Jung H, Shin I, Park Y, Kang K, Ha KS (1997) Colchicine activates actin polymerization by microtubule depolymerization. Mol Cells 7, 431-437.

Just I, Selzer J, Wilm M, von Eichel-Strieber C, Mann M, Aktories K (1995) Glucosylation of rho proteins by Clostridium difficile toxin B. Nature 375, 500-503.

Keller H, Yunxu C, Marit G, Pla M, Reiffers J, Theze J, Froussard P (1999) Transgene expression, but not gene delivery, is improved by adhesion-assisted lipofection of hematopoietic cells. Gene Ther 6, 931-938.

Kirchner J, Kam Z, Tzur G, Bershadsky A, Geiger B (2003) Live-cell monitoring of tyrosine phosphorylation in focal adhesions following microtubule disruption. J Cell Sci 116, 975-986.

Kitson C, Angel B, Judd D, Rothery S, Severs N, Dewar A, Huang L, Wadsworth S, Cheng S, Geddes D, Alton E (1999) The extra-and intracellular barriers to lipid and adenovirusmediated pulmonary gene transfer in native sheep airway epithelium. Gene Ther 6, 534-546.

Kumar N (1981) Taxol-induced polymerization of purified tubulin. J Biol Chem 256, 10435-10441.

Lee JW, Juliano R (2004) Mitogenic signal transduction by integrin- and growth factor receptor-mediated pathways. Mol Cells 17, 188-202.

Majda J, Gerner W, Vanlingham B, Gehlesen K, Cress AE (1994) Heat shock-induced shedding of cell surface integrins in A549 human lung tumor cells in culture. Exp Cell Res 210, 46-51.

Martin K, Slack J, Boerner S, Martin C, Parsons J (2002) Integrin connections map: to infinity and beyond. Science 296, 1652-1653.

Nair R, Rodgers J, Schwarz L (2002) Enhancement of trangene expression by combining glucocorticoids and anti-mitotic agents during transient transfection using DNA-cationic liposomes. Mol Ther 5, 455-462.

Perlstein I, Connolly J, Cui X, Song C, Li Q, Jones P, Lu Z, Defelice S, Klughery B, Wilensky R, Levy R (2003) DNA delivery from an intravascular stent with a denatured collagen-polylactic-polyglycolic acid-controlled release coating: mechanisms of enhanced transfection. Gene Ther 10, 1420-1428.

Pierschbacher M, Rouslathi E (1984) Cell attachment activity of fibronectin can be duplicated by small synthetic fragments of the molecule. Nature 309, 30.

Rejman J, Bragonzi A, Conese M (2005) Role of clathrin- and caveolae-mediated endocytosis in gene transfer mediated by lipo- and polyplexes. Mol Ther 12, 468-474.

Ren Y, Li R, Zheng Y, Busch H (1998) Cloning and characterization of GEF-H1, a microtubule-associated guanine nucleotide exchange factor for rac and rho GTPases.

J Biol Chem 273, 34954-34960.

Richardson A, Parsons T (1996) A mechanism for regulation of the adhesion-associated protein tyrosine kinase pp125FAK. Nature 380, 538-540.

Ruegg C, Postigo A, Sikorski E, Butcher E, Pytela K, Erle D (1992) Role of integrins #4!7/#4!P in lymphocyte adherence to fibronectin and VCAM-1 and in homotypic cell clustering. J Cell Biol 117, 179-189.

Schaller M, Hildebr J, Shannon J, Fox J, Vines R, Parsons J (1994) Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src. Mol Cell Biol 14, 1680-1688.

Schlaepfer D, Jones K, Hunter T (1998) Multiple GRB2mediated integrin-stimulated signaling pathways to ERK/Mitogen-activated protein kinase: summation of both c-

expression in the A549 cells, indicating that this enhancement does not apply to globally, to every cellular gene (Nair et al, 2002). Further studies will be needed to elucidate these specific mechanisms.

In conclusion, our finding that focal adhesions are involved in the enhancement of transgene expression has important implications for improving the outcome of lipidmediated gene therapy. As the actual targets within this pathway are identified, specific agents that stably mimic these effects may prove useful for improving lipoplexmediated, transient transfection.

Acknowledgments The authors wish to acknowledge the support of this project by the Department of Pharmacological and Pharmaceutical Sciences at the University of Houston.

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Gene Therapy and Molecular Biology Vol 11, page 13 Src and focal adhesion kinase-initiated tyrosine phosphorylation events. Mol Cell Biol 18, 2571-2585.

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Gene Therapy and Molecular Biology Vol 11, page 15 Gene Ther Mol Biol Vol 11, 15-20, 2007

Inhibitory effect of antisense RNA of ornithine decarboxylase gene on human esophageal squamous carcinoma cell line Eca109 Research Article

Hui Tian1,*, Lin Li1, Qing Huang1, Xianxi Liu2, Yan Zhang2 1

Department of Thoracic Surgery, Shandong University Qi Lu Hospital Experimental Center of Medical Molecular Biology, School of Medicine, Shandong University, Jinan 250012, Shandong, China 2

__________________________________________________________________________________ *Correspondence: Tian Hui, Department of Thoracic Surgery, Shandong University Qi Lu Hospital, Jinan 250012, Shandong, China; Tel: 86-531-82169463; Fax: 86-531-86927544; E-mail: tianhuiy@sohu.com Key words: Ornithine decarboxylase; Adenovirus vector; esophageal neoplasms; Eca109 cell line; Gene therapy Abbreviations: 3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide, (MTT); cytomegalovirus, (CMV); Difluoromethylornithine, (DFMO); fetal bovine serum, (FBS); Ornithine decarboxylase, (ODC); triphosphate-biotin nick end-labeling, (TUNEL) Received: 28 November 2006; Revised: 22 December 2006 Accepted: 27 February 2007; electronically published: March 2007

Summary To investigate the in vitro inhibitory effect of rAd-ODC/Ex3as on human esophageal carcinoma cells. The infection rate of rAd-ODC/Ex3as was measured with the aid of GFP expression. Western Blot technique was used to observe the inhibition of ODC expression in infected tumor cells. The malignant phenotype of Eca109 cell line was assessed by growth curve. TUNEL was used to analyze cell apoptosis. Approximate 65% of Eca109 cell line were infected with rAd-ODC/Ex3as when MOI reached 50. The expression of ODC was inhibited in the infected tumor cells. rAdODC/Ex3as could inhibit Eca109 cell line growth and invasive ability at 20 of MOI. TUNEL proved that rAdODC/Ex3as can lead to cell apoptosis. rAd-ODC/Ex3as could inhibit effectively the expression of ODC gene and the growth of of esophageal squamous carcinoma cell line Eca109 in vitro, and induce apoptosis. It may be one of the promising medicines for antisense gene therapy in esophageal cancer.

Complete structure and nucleotide sequence of ODC gene from mammalians is known for human (Moshier et al, 1990), which have 12 exons and 11 introns. Active mammalian ODC is homodimer with 2-fold symmetry.

Subunits have molecular weight of about 51kDa and the polypeptide chain consists of 461 amino acids. ODC becomes activated after treatment with chemical carcinogens and tumor promoters, as well as in cells transformed by various oncogens, such as v-src, neu and ras (Pegg et al, 1988; Sistonen et al, 1989; Auvinen et al, 1992). The level of ODC was reportedly elevated in various cancers (Glikman et al, 1987; Upp JR, Jr. et al, 1988; Love et al, 2003) and related to recurrence (Love et al, 2003). Some chemotherapeutic agents, such as Difluoromethylornithine (DFMO), which aimed to inhibit the activity of ODC have appeared and taken on inhibitory effects on tumor growth in vitro and in vivo (Umemoto, 1989; Zagaja et al, 1998), though showing dose-limiting

I. Introduction The polyamines, spermidine, spermine and the diamine precursor, putrescine, are positively charged aliphatic amines at physiological conditions, have a lowmolecular weight and a simple chemical structure. They interact with various macromolecules, both electrostatically and covalently and, as a consequence, have a variety of cellular effects. They are known to be critically involved in cell growth and have been implicated in the process of cell transformation (Auvinen et al, 1992; Moshier et al, 1993). On the other hand, the level of polyamine is high in cancer cell and tissues, and rapid tumor growth has been associated with remarkable elevation of polyamine biosynthesis and accumulation (Marton and Pegg, 1995; Pegg et al, 1998).

Ornithine decarboxylase (ODC) is the first and the rate-controlling enzyme in polyamine biosynthesis. It decarboxylates L-ornithine to form diamine putrescine.

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Tian et al: Inhibitory effect of antisense RNA of ornithine decarboxylase gene on cell line Eca109 wide range of viral titres, from 1 to 100 pfu/cell (MOI, multiplicity of infection). After 48 hours of incubation, 3-(4,5methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was added (50µg/well) for 4 hours. Formazan products were solubilized with DSMO, and the optical density was measured at 570nm. To observe the effect of adenovirus on cell proliferation, MTT assay was also used to draw cell growth curves. Cells were inoculated at a density of 4000 cells per well, under which control cells remained subconfluent and in exponential phase growth for the duration of the assay. Due to different infective efficiency, A-549 cells were infected by 50 and 25 MOI respectively. All experiments were performed in sextuple. After 24, 48, 72, 96 and 120 hours, cell viability was measured by absorbance at 570nm as described previously.

toxicity. Stable transfection of human lung squamous carcinoma cell line LTEP-78 with antisense ODCexpressing plasmid DNA has been shown too related with the reversion of malignant phenotypes of human lung squamous carcinoma cells (Guan et al, 1996). Taken together, these findings suggest that ODC may provide an important target for the development agents that inhibit carcinogenesis and tumor growth.

Esophageal cancer is one of the most frequently diagnosed cancers in the world. Metastatic esophageal cancer is essentially resistant to systemic cytotoxic chemotherapy, while external beam and radioisotope radiotherapy offers only symptom palliation. Clearly the development of novel therapies, such as gene therapy, is a high priority. Some studies had proved that lung cancer had greater elevated polyamine levels (Carlisle et al, 2002). Because Adenoviral vectors are among the most promising gene transfer vehicles for direct, in vivo gene therapy for the treatment of a diverse array of human disease (Meager, 1999). In this study, we used a replication-deficient recombinant adenovirus to efficiently deliver a 120bp antisense ODC which is complementary to initiation codon and tested the effect of antisense ODC on esophageal cancer. The data presented here show that adenovirus-mediated gene transfer of antisense ODC could significantly inhibit growth of esophageal cancer cells.

D. Western blotting analysis of ODC proteins Eca109 cell line was infected with rAd-ODC/E3as by 50 MOI in 1640 medium containing 5%FCS for 48 hours. The cells were washed three times with ice-cold PBS and collected with a cell scraper. Total cell lysates were prepared in extraction buffer containing 0.05M Tris (pH8.0), 0.15M NaCl, 0.02% Sodium Azide, 0.1% SDS, 100µg/ml PMFS (phenylmethylsulfonyl fluoride), 1µg/ml aprotinin and 1%NP-40. The extracts were subjected to 12% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was blocked for 1h at room temperature in PBS containing 1% powdered milk. Mouse antiODC monoclonal antibody was added at a dilution of 1/500 and incubation was continued overnight at 4$. The secondary antibody was horseradish peroxidase-conjugated antimouse IgG antibody (Zhongshan Beijing China). Antibody reactive bands were revealed using the ECL Western blotting detection system (Santa Cruz CA). The content of each protein sample was controlled by means of "-actin. For quantitation of bands, we used Nikon digital camera and SmartView analysis software.

II. Materials and methods A. Cell culture and reagents Esophageal cancer Eca109 cell line was obtained from Chinese Academy of Science. Cells were cultured in DMEM or RPMI 1640 medium supplemented with 10% heat-inactived fetal bovine serum (FBS), 100U/ml penicillin, and 100!g/ml streptomycin. MTT was purchased from Sigma, MO. "-actin antibody and ECL Western blotting detection system were obtained from Santa Cruz, CA. Monoclonal antibody of ODC was made in our lab. Other reagents were all of reagent grade and obtained from Chinese companies.

E. HPLC analysis of polyamine pools Eca109 cell line was infected with rAd-ODC/Ex3as at the MOI of 50. After 48 hours, cells were trypsined and washed with PBS twice. Intracellular polyamine were extracted from cell pellets with 10% trichloroacetic acid, dansylated, and measured by reverse phase HPLC as described previously (Fu et al, 1998).

F. TUNEL was used to analyze cell apoptosis

B. Adenovirus and infection condition

Terminal deoxynu-cleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) was used to detect apoptotic cells. TUNEL was performed with the kit according to the manufacture’s instruction.

The recombinant adenovirus rAd-ODC/Ex3as, containing the cytomegalovirus (CMV) promoter and GFP gene, was constructed by reversely inserting a 120bp cDNA fragment of ODC into the multiple clone sites (Zhang et al, 2003), rAdODC/Ex3as was purified by ultracentrifugation in cesium chloride step gradients (Prevec et al, 1991). The titer of the viral stock, measured in plaque-forming unit (pfu)/ml, was determined to be 8.5#109pfu/ml by a method published previously (Wei et al, 2000),and the frozen stock was confirmed to have retained their titer. The control virus rAd-GFP was same to rAdODC/Ex3as but no gene inserted in the polylinker. Viral stocks were suitably diluted in serum-free medium to obtain the desired pfu, added to cell monolayers of lung cancer cells and incubated at 37$ for 2 hours. The necessary amount of culture medium with 5% fetal bovine serum was then added and the cells were incubated for the desired times.

G. Statistical analysis Statistical analysis was performed using Statview J 5.0 software (SAS Institute Inc., San Francisco, CA). A significant difference was defined as p<0.05.

III. Results A. Inhibitory effects of rAd-ODC/Ex3as on Eca109 cell line There was dose-dependent growth inhibition in Eca109 cell line, which reflected the transduction efficiency of the adenovirus to esophageal cancer cell line.

We chose 50 MOI of adenovirus to infect Eca109 cell line.

Under these conditions, rAd-ODC/Ex3as was more suppressive of growth than the control rAd-GFP virus, while rAd-GFP had no obviously toxic effect on cells. We

C. MTT assay Firstly MTT assay was employed to assess transduction efficiency of rAd-ODC/Ex3as in Eca109 cell line. Briefly, cells were seeded at density of 5000 cells/well in 96-well plates and grown overnight. On the next day the cells were infected by a

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Gene Therapy and Molecular Biology Vol 11, page 15 examined the in vitro growth inhibition of rAdODC/Ex3as in Eca109 cell line using cell growth curves as described in â&#x20AC;&#x153;Material and Methodsâ&#x20AC;?. Antisense ODC had an impact on the growth of esophageal cancer cells.

RAd-ODC/Ex3as in both of the cells inhibited their proliferation by ~50% when compared with the control virus and no virus-treated groups (Figure 1).

software showed that ODC expression in Eca109 cell line infected with rAd-ODC/Ex3as accounted for 40% of that in cells treated with rAd-GFP (Figure 2). HPLC also exhibited a decrease of concentrations of the three polyamines: putrescine (put), spermidine (spd),spermine (spm), especially of putrescine (Table 1).

C. TUNEL assay for apoptosis

B. Effect of rAd-ODC/Ex3as on expression of the ODC and polyamine pools in the cell lysate

To examine the mechanism by which rAdODC/Ex3as may retard esophageal cancer cell growth in vitro, we used TUNEL to detect the effect of the rAdODC/Ex3as on apoptotic cells at 48 (Figure 3) and 72 hours after infection. As shown in Table 2, the rate of apoptosis in cells infected by rAd-ODC/Ex3as was significantly high in comparison to infected by rAd-GFP or no virus-treated cells (p<0.05).

The ODC proteins produced from Eca109 cell line after infection with rAd-ODC/Ex3as were examined by Western immunoblot analysis. The ODC expression in the cells infected with rAd-ODC/E3as substantially more reduced than in the cells infected with rAd-GFP or no virus-treated cells. The results analyzed by SmartView

Figure 1. The effect of rAd-ODC/Ex3as on growth of Eca109 cell line. Growth curves of cells was drawn after Eca109 cell line were infected with either rAd-ODC/Ex3as or rAd-GFP at MOI of 50, and absorbance was measured everyday in a period of 5 days.RAdODC/Ex3as in both of the cells inhibited their proliferation by ~50% when compared with the control virus and no virus-treated groups.

Figure 2. Western blotting analysis for ODC expression in Eca109 cell line after transduction of rAd-ODC/E3as or rAd-GFP. Eca109 cell lines were infected with adenoviruses at MOI of 50 . After 48 hours, 2#106 cell were collected in 300!l extraction buffer. 40!l protein extraction was added to SDS-PAGE. A. rAd-ODC/Ex3as-infected Eca109 cell lines. B. No virus-treated Eca109 cell lines. C. rAd-GFP-infected Eca109 cell lines

Table 1. Polyamine Pools of Eca109 cell lines Cell line and Treatment Eca109 cell line +rAd-GFP +rAd-ODC/E3as

Put 590 525 254

Polyamine polls (pmol/106cell) Spd Spm 1560 1489 1463 1672 1189 1321

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Tian et al: Inhibitory effect of antisense RNA of ornithine decarboxylase gene on cell line Eca109

Figure 3. The effect of rAd-ODC/Ex3as on the apoptosis of Eca109 cell lines 48 hours after infection. Cells were observed under 100# microscope. Those brown cells were apoptotic cells. A: Eca109 cell lines infected with rAd-ODC/Ex3as, B: Eca109 cell lines infected with rAd-GFP, C: Eca109 cell lines

Table 2. The rate of apoptosis at 48 and 72 hours after infecting rAd-ODC/Ex3as or rAd-GFP (%) (x±s) Cell line and Treatment Eca109 cell line +rAd-GFP +rAd-ODC/E3as

48h

72h

5.8!0.63 9.2!0.87 25.6±1.82

8.2!0.43 12.3!0.54 64.2±2.42

p -

<0.05

substantially more reduced than in the cells infected with rAd-GFP or no virus-treated cells. On the other hand a substantial decrease in ODC expression resulted in the reduction of polyamine biosynthesis. In addition, the reduction of polyamines may contribute to the marked suppression of cancer cell growth and tumor formation.

Resent studies also showed inhibiting mRNA expression of ODC can effectively inhibits the growth of some cancer cells, such as breast, prostate, colorectal, pancreatic cancer and bladder carcinoma cell (Weeks et al, 2000; Love et al, 2003; Subhi et al, 2004; Wolter et al, 2004). These findings suggest that polyamine metabolism and ODC could be potential therapeutic targets in the treatment of some cancer.

To examine the mechanism of antisense ODC inhibiting the growth of esophageal cancer cells, we demonstrated rAd-ODC/Ex3as infection can contribute significantly to cell apoptosis in comparison to rAd-GFP infected or no virus-treated cells by TUNEL. In the last years, some studies had demonstrated the inhibition of ODC could lead to induction of apoptosis of some cancer cells (Feith et al, 2005; Seiler and Raul, 2005; Stanic et al, 2006). So, our previous study indicated the induction of apoptosis was the mechanism of antisense ODC inhibiting the growth of esophageal cancer cells.

In general, Our data suggest that adenoviral vector mediated antisense ODC can lead to induction of apoptosis and inhibition of growth of esophageal cancer cells in vitro. The rAd-ODC/Ex3as could be a potential agent against esophageal cancer, however, further in-depth in vivo studies must be warranted.

IV. Discussion Polyamines are aliphatic cations with multiple functions and are essential for life. In normal cells, ployamine levels are intricately controlled by biosynthetic and catabolic enzymes. Multiple abnormalities in the control of polyamine synthesis, metabolism, uptake and function might be responsible for increased levels of polyamines in cancer cells as compared to that of normal cells, especially in lung cancer cells (Carlisle et al, 2002).

At the same time, targeting specific molecules in cells by antisense inhibition was shown to have potential effectiveness in decreasing the protein expression. ODC is the most important enzyme in polyamine biosynthesis.

More recently, the overexpression of ODC in NIH3T3 cells caused transformation of these cells to a malignant phenotype, in essence qualifying ODC as an oncogene (Auvinen et al, 1997). Inhibition of ODC by DFMO could compromise cell growth and transformation (Metcalf et al, 1978). Schipper’s recent in vitro studies using conformationally restricted polyamine analogues showed that these compounds inhibited cell growth, probably by inducing antizyme-mediated degradation of ODC (Schipper et al, 2000). In addition, Alm and colleageus showed in 2000 that ODC was a well-defined target gene for c-myc and other oncogenes. Therefore, we targeted the ODC by using an antisense gene delivery strategy with a replication-deficient recombinant Ad vector. In the present study, we demonstrated that rAd-ODC/Ex3as could inhibit esophageal cancer growth and lead to the apoptosis of Eca109 cell lines.

MTT assay showed antisense ODC had an impact on the growth of esophageal cancer cells. RAd-ODC/Ex3as in both of the cells inhibited their proliferation by ~60% when compared with the control virus and no virus-treated groups. At the same time, Western blotting showed the ODC expression in the cells infected with rAd-ODC/E3as

References Alm K, Berntsson PS, Kramer DL, Porter CW, Oredsson SM (2000) Treatment of cells with the polyamine analog N,N11-

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Gene Therapy and Molecular Biology Vol 11, page 15 diethylnorspermine retards S phase progression within one cell cycle. Eur J Biochem 267, 4157-4164.

Auvinen M, Laine A, Paasinen-Sohns A, Kangas A, Kangas L, Saksela O, Andersson LC, Holtta E (1997) Human ornithine decarboxylase-overproducing NIH3T3 cells induce rapidly growing, highly vascularized tumors in nude mice. Cancer Res 57, 3016-3025.

Auvinen M, Paasinen A, Andersson LG, Holtta E (1992) Ornithine decarboxylase activity is critical for cell transformation. Nature 360, 355-358.

Carlisle DL, Devereux WL, Hacker A, Woster PM, Casero RA Jr (2002) Growth status significantly affects the response of human lung cancer cells to antitumor polyamine-analogue exposure[J]. Clin Cancer Res ,8, 2684-9.

Devens BH, Weeks RS, Burns MR, Carlson CL, Brawer MK (2003) Polyamine depletion therapy in prostate cancer.

Prostate Cancer Prostatic Dis 3, 275-279.

Feith DJ, Bol DK, Carboni JM, Lynch MJ, Sass-Kuhn S, Shoop PL, Shantz LM (2005) Induction of ornithine decarboxylase activity is a necessary step for mitogen-activated protein kinase kinase-induced skin tumorigenesis. Cancer Res 65, 572-8.

Fu S, Zou X, Wang X, Liu X (1998) Determination of polyamine in human prostate by high-performance liquid chromatography with fluorescence detection. J Chromatogr B Biomed Sci Appi 709, 297-300.

Glikman P, Vegh I, Pollina MA, Mosto AH, Levy CM (1987) Ornithine decarboxylase activity, prolactin blood levels, and estradiol and progesterone receptors in human breast cancer.

Cancer 60, 2237-2243.

Guan J, Fan M, Cao S (1996) Reversion of malignant phenotypes of human lung squamous carcinoma cells by ornithine decarboxylase antisense RNA. Zhonghua Zhong Liu Za Zhi 18, 81-83.

Love RR, Astrow SH, Cheeks AM, Havighurst TC (2003) Orinithine decarboxylase(ODC) as a prognostic factor in operable breast cancer. Breast Cancer Res Treat 79, 329334.

Marton LJ, Pegg AE (1995) Polyamines as targets for therapeutic intervention. Annu Rev Pharmacol Toxicol 35, 55-91.

Meager A (1999) Gene Therapy Technologies, Applications and Regulations: John Wiley & Sons, Ltd; 81 Metcalf B, Bey P, Danzin C, Jung M, Casara P, Vevert J (1978) Catalytic irreversible inhibition of mammalian ornithine decarboxylase by substrate and product analogues. J Am Chem Soc 100, 2551-2553.

Moshier JA, Dosescu J, Skunca M, Luk GD (1993) Transformation of NIH/3T3 cells by ornithine decarboxylase overexpression. Cancer Res 53, 2618-2622.

Moshier JA, Gilbert JD, Skunca M, Dosescu J, Almodovar KM, Luk GD (1990) Isolation and expression of a human ornithine decarboxylase gene. J Biol Chem 265, 4884-4892.

Pegg AE, Madhubala R, Kameji T, Bergeron RJ (1988) Control of ornithine decarboxylase activity in alphadifluoromethylornithine-resistant L1210 cells by polyamines and synthetic analogues. J Biol Chem 263, 11008-11014.

Pegg AE, Xiong H, Feith DJ, Shantz LM (1998) Sadenosylmethionine decarboxylase:structure, function and regulation by polyamines. Biochem Soc Trans 26, 580-586.

Prevec L, Christie BS, Laurie KE, Bailey MM, Graham FL, Rosenthal KL (1991) Immune response to HIV-1 gag antigens induced by recombinant adenovirus vectors in mice and rhesus macaque monkeys. J Acquir Immune Defic Syndr 4, 568-576.

Schipper RG, Deli G, Deloyer P, Lange WP, Schalken JA, Verhofstad AA (2000) Antitumor activity of the polyamine analog N(1),N(11)-diethylnorspermine against human prostate carcinoma cells. Prostate 44, 313-321.

Seiler N, Raul F (2005) Polyamines and apoptosis. J Cell Mol Med 9, 623-42.

Sistonen L, Holtta E, Lehvaslaiho H, Lehtola L, Alitalo K (1989) Activation of the neu tyrosine kinase induced the fos/jun transcription factor complex, the glucose transporter and ornithine decarboxylase. J Cell Biol 109, 1911-1919.

Stanic I, Facchini A, BorzĂŹ RM, Vitellozzi R, Stefanelli C, Goldring MB, Guarnieri C, Facchini A, Flamigni F (2006) Polyamine depletion inhibits apoptosis following blocking of survival pathways in human chondrocytes stimulated by tumor necrosis factor-alpha. J Cell Physiol 206, 138-46.

Subhi AL, Tang B, Balsara BR, Altomare DA, Testa JR, Cooper HS, Hoffman JP, Meropol NJ, Kruger WD (2004) Loss of methylthioadenosine phosphorylase and elevated ornithine decarboxylase is common in pancreatic cancer[J]. Clin Cancer Res 10, 7290-6.

Umemoto S (1989) Antitumor effect of alphadifluoromethylornithine(DFMO) changes in ornithine decarboxylase(ODC) activity and polyamine(PA) levels in human tumor transplanted into nude mice. Nippon Geka Gakkai Zasshi 90, 650-660.

Upp JR, Jr., Saydjari R, Townsend CM, Jr., Singh P, Barranco SC, Thompson JC (1988) Polyamine levels and gastrin receptors in colon cancers. Ann Surg 207, 662-669.

Weeks RS, Vanderwerf SM, Carlson CL, Burns MR, O'Day CL, Cai F, Devens BH, Webb HK (2000) Novel lysine-spermine conjugate inhibits polyamine transport and inhibits cell growth when given with DFMO[J]. Exp Cell Res 261, 293302.

Wei D, Tang Z, Chen S (2000) Construction of recombinant adenovirus vector containing mIL-12 using the method of homogenous recombination in Bacteria and its expression in vitro with high efficient. Chin J Biochem Mol Biol 16, 716721.

Wolter F, Ulrich S, Stein J (2004) Molecular mechanisms of the chemopreventive effects of resveratrol and its analogs in colorectal cancer: key role of polyamines?[J]. J Nutr 134, 3219-22.

Zagaja GP, Shrivastav M, Fleig MJ, Marton LJ, Rinker-Schaeffer CW, Dolan ME (1998) Effects of polyamine analogues on prostatic adenocarcinoma cells in vitro and in vivo. Cancer Chemother Pharmacol 41, 505-512.

Zhang Y, Liu X, Hu H, Geng Z, Wang X, Zhang B (2003) Construction of an antisense RNA recombinant adenovirus vector of the third extron in ODC gene. Journal of Shandong University (Health Sciences) 41, 371-374.

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Gene Therapy and Molecular Biology Vol 11, page 21 Gene Ther Mol Biol Vol 11, 21-26, 2007

Autologous stem cell transplantation for primary refractory or relapsing Hodgkin’s disease: comparison between CD34+ immunoselected and unselected stem cells graft Research Article

Federica Sorà*, Anna Laura Di Febo, Nicola Piccirillo, Luca Laurenti, Patrizia Chiusolo, Silvia De Matteis, Giuseppe Leone, Simona Sica Department of Haematology, CatholicUniversity, Rome

__________________________________________________________________________________ *Correspondence: Federica Sorà, MD, Istituto di Ematologia, Policlinico “A. Gemelli”, Università Cattolica S.Cuore, Largo Gemelli, 8, 00168 Roma; Tel: +39-06-30154278; Fax: +39-06-3017319; e-mail: f.sora@rm.unicatt.it Key words: autologous stem cell transplantation, CD34+, cell immnuoselection, Hodgkin’s disease Abbreviations: Autologous stem cells transplantation, (ASCT); Bloodstream infection, (BSI); Centre for Disease Control, (CDC); Complete response, (CR); disease free survival, (DFS); freedom from progression, (FFP); Hodgkin’s disease, (HD) either; leukapheretic procedure, (LKP); non-prophylactic antibiotics, (NPA); Overall survival, (OS); Partial response, (PR); peripheral blood stem cells, (PBSC); qualitative chain reaction, (PCR) Received: 28 November 2006; Revised: 01 February 2007 Accepted: 05 February 2007; electronically published: March 2007

Summary Autologous stem cells transplantation (ASCT) is widely accepted in the treatment of high risk Hodgkin’s disease (HD) either resistant or relapsing after first-line chemotherapy. Since the presence of Hodgkin/Reed- Sternberg cells in peripheral blood stem cells (PBSC) collection was demonstrated, and CD34+ antigen is not expressed on HD cells, positive selection of CD34+ cells has been demonstrated an efficient purging method to reduce the number of tumour cells in the graft. We conducted a non randomized pilot study using CD34+ selected ASCT in refractory/relapsing HD patients and compared the clinical outcome to HD patients receiving unselected PBSC. Eleven patients received CD34+ selected ASCT (group A) and 11 patients received unmanipulated ASCT (group B).

Patients were matched for age, sex, diagnosis, IPI and response to first-line therapy. Group A received a median number of 4.74 x 106/kg CD34+ selected cell while patients of B group of 8.45 x 106/kg unselected PBSC (p=0.23). No difference was observed between the two groups in any of the endpoints analyzed (haematological engraftment, infections, morbidity and mortality, progression, survival). These results show no advantages for patients receiving CD34+ selected ASCT compared to unselected PBPC transplant at least for refractory/relapsing HD.

high-dose therapy is currently unsettled.

Classic HD cells express CD30 and CD15 antigens on their surface and lack the expression of CD34 antigen.

The positive selection of CD34+ cells from PBSC has been demonstrated an efficient purging method to reduce the number of atypical CD30+ cells (Blystad et al, 2001a,b) in peripheral blood stem cells collection from patients with HD. However the advantage in terms of clinical outcome in patients receiving CD34+ selected PBSC compared to those receiving unselected PBSC has not been established.

In order to elucidate the role of purging in HD we report the results of haematological reconstitution and clinical outcome of patients receiving CD34+ selected PBSC

I. Introduction Autologous stem cells transplantation has been included in the algorithm of treatment of high risk Hodgkin’s disease. In the last years, peripheral blood stem cells (PBSC) have replaced bone marrow as stem cell source because of a faster haematological recovery after reinfusion but some authors suggested a less favourable outcome in patients receiving PBSC compared to bone marrow (Maiolino et al, 1997). An explanation for these results might originate in the presence of neoplastic contamination of peripheral stem cells collection (Wolf et al, 996) although the correlation between the presence of tumour cells in the grafts and the incidence of relapse after 21

Sorà et al: Autologous stem cell transplantation for primary refractory or relapsing Hodgkin’s disease CR in 2 (18%)patients, PR in 5 (45%) patients and 4 (36%) patients showed disease progression; in group B, 2 (18%) patients obtained CR, 6 (55%) patients achieved PR and 3 (27%) patients had disease progression.

transplantation compared to a well-matched group of high risk patients receiving unmanipulated PBSC.

II. Materials and Methods The majority of HD were reconstituted with unmanipulated PBSC after high dose chemotherapy, but from march 1996 a pilot study of CD34+ selection and ASCT was started and positive selection of the PBSC was performed in all patients with poor prognostic features such as refractory or relapsing Hodgkin’s disease with or without bone marrow involvement.

The protocol was approved by the local ethical commitee and patients or their guardians gave their informed written consent.

Eleven patients (group A) were enrolled onto the study which was opened at the beginning of 1996 and closed at the end of 1999. For the purpose of the analysis patients were then matched for demographic and clinical parameters to a group of patients who received unmanipulated PBSC (group B) from March 1992 to October 2001 with Hodgkin’s disease autografted at our Institution and extracted from our database.

Characteristics of patients are showed in Table 1. Median age was 23 years (range 17-42) in group A and 30 years (range 15-35) in group B; sex distribution was: 4 males and 7 females in group A and 6 males and 5 females in group B. The median IPI score at diagnosis calculated according to Bierman et al (Bierman et al, 2002) was 2 in both groups with a range of 1-3 in group A and 1-4 in group B respectively. Disease stage at diagnosis was III-IV in the majority of patients: 7 of 11 patients (64%) had IV stage in the group A and 5 of 11 (45%) in group B and the vast majority (9 out of 11 patients: 81%) had B symptoms in both groups. Nodular sclerosis was the histological subtype in 8 of 11 patients (73%) in both groups. Bone marrow involvement was present in 5 (45%) patients in group A and in 3 patients (27%) in group B. All patients at diagnosis received conventional first-line therapy as reported in table 1. Complementary radiotherapy was administered in 4 patients (36%) in group A and 1 patients (9%) in group B either after first line chemotherapy or for relapse.

Group A included 2 patients (18%) in relapse at 6,5 months (range 3-10), 2 patients (18%) achieving partial remission after first line chemotherapy and 7 patients (63%) with disease progression, Group B included 3 patients (27%) in relapse at 16 (range 3-60) months, 2 patients (18%) achieving partial remission after first line chemotherapy and 6 patients (54%) with disease progression.

C. CD34+ cell harvesting and purging PBSC were mobilized and collected after MiCMA plus GCSF 5mg/kg/day subcutaneously (sc) from day 8 until completion of leukapheretic procedure (LKP). Leukapheres started when the peripheral white blood count was greater than 1.0 x 109/l and CD34+ cells in peripheral blood were >20/ml.

LKP were continued daily until a minimum harvest of 2x106 CD34+/kg was obtained. In patients submitted to immunoselected CD34+ stem cell transplantation the selection was carried out using the Ceprate SC system (Cellpro, Bothell, WA, USA) or the CliniMACS device (Miltenyi Biotech GmbH, Bergish-Gladbach, Germany). All patients had a back up of unfractionated PBSC stored in liquid nitrogen.

D. Stem cell transplantation As conditioning regimen all patients in both groups received BEAM (carmustine 300mg/m2on day –7, etoposide 200mg/m2 on day-6,-5,-4,-3, cytosine arabinoside 200mg/m2 on day-6,-5,-4,-3 and melphalan 140mg/m2 on day –2). In nine out of 11 patients receiving CD34+ selected PBSC, G-CSF was administered at a standard dose of 263 mg daily or on alternate day until neutrophil count was >500/ml for two consecutive days.

All patients received standard antimicrobial, antiviral treatment and Pneumocystiis Carinii prophylaxis. Broad spectrum intravenous antibiotic therapy was started for fever of unknown origin in neutropenic patients.

Empirical antifungal therapy was started for fever not responsive to antibiotics therapy. All patients were CMV seropositive and were regularly examined for CMV infection after transplantation with antigenemia or qualitative chain reaction (PCR) for CMV DNA. Bloodstream infection (BSI) and catheter related infection were defined according to Centre for Disease Control (CDC) (CDC-MMWR, 2002).

Viral infections were defined as the evidence of positive colture from urine, stools or throat in association with symptoms.

CMV infection was defined as either the evidence of any level of pp 65 antigenemia and or a positive culture.

E. Endpoints A. Salvage chemotherapy

During this study we evaluated the following clinical outcomes: time to neutrophil recovery (0.5x109/l and 1.0x109/l), time to platelets recovery >20x109/l and >50x109/l, reticulocyte recovery (>1%), time to untransfused Hgb >10 g/dl, number of pRBCu and SDu infused, length of hospitalization, duration of non-prophylactic antibiotics (NPA), number of days with BT>38°C, incidence of sepsis and viral infections, duration of GCSF administration (in days), overall survival, freedom from progression and disease free survival for patients achieving complete response.

All patients were treated with salvage chemotherapy: mitoxantrone 10 mg/m2 day 1, carboplatinum 100 mg/m2 day 1-4, cytosine arabinoside 2 g/m2 day 5, methylprednisolone 500 mg/m2 day 1-5 (MiCMA) (La Barbera et al, 2000) followed by G-CSF and peripheral blood stem cell collection. After evaluation of response to chemotherapy, autologous stem cell transplantation was carried out in all patients irrespective of their response.

B. Response to salvage chemotherapy and disease status at transplantation

F. Statistical analysis Overall survival (OS) analysis, disease free survival (DFS) and freedom from progression (FFP) analysis were assessed from the first day of transplantation (version 2.01; GraphPad Software Inc., San Diego, CA).

In OS analysis, an event was defined as death from any cause; the DFS was calculated for all patients who had reached CR, FFP was calculated from the date of response until relapse or progression whichever came first. Survival curves were generated using Kaplan-Meier method for survival analysis and log-rank test was performed for survival curve comparison. Chi2

Before transplantation all patients underwent accurate restaging by total body CT scanning and bone marrow biopsy.

Complete response (CR) was defined as complete disappearance by physical exam and radiographic studies of all measurable or evaluable disease with no indication of recurrence. Partial response (PR) was defined as ! 50%, but < 100% improvement in all measurable or evaluable disease, with no indication of tumour regrowth before high dose chemotherapy consolidation.

After a median of 2 cycles (range 1-4) of MiCMA, response and thus, disease status at transplantation, was as follows: in group A,

22

Gene Therapy and Molecular Biology Vol 11, page 23 test was used to analyse the categorical factors. Statistical

significance was defined as p<0.05.

Table 1. Characteristics of patients Characteristics No. of patients(%) Sex (%): Male Female Age, median (range) in year Histology (%): Nodular sclerosis Nodular sclerosis1 Nodular sclerosis2 Mixed cellularity Lymphocyte depletion HD- not otherwise specified Staging (%): I-II III-IV unknown B symptoms (%) Bone marrow involvement (%) unknown Bulky disease (%) unknown IPI (%): I II III IV First-line chemotherapy (%): ABVD ABVD/MOPP VEPEB BEACOPP STANFORD V MOPP Radiotherapy (%): Response to first-line therapy (%): PD CR PR Status at transplantation (%): PD PR CR CD34+ cells reinfused x 10 6 (range) : Engraftment (range) : Days to ANC >0.5x109/l Days to ANC >1.0x109/l Days to haemoglobin level >10g/dl Days to reticulocyte>1% Days to platelet count >20x109/l Days to platelet count >50x109/l Days with fever>38째C Days in hospital No.of platelet units transfused No.of RBC units transfused

PBSC No 11 (100)

CD 34+ No 11 (100)

4 (36) 7 (64) 23 (17-42)

6 (55) 5 (45) 30 (15-35)

5 (45.5) 1 (9) 2 (18.25) 0 (0) 2 (18.25) 1 (9)

6 (55) 0 (0) 2 (18) 3 (27) 0 (0) 0 (0)

3 (27) 7 (64) 1 (9) 9 (81) 5 (45) 1 (9) 7 (64)

1 (9) 10 (91)

5 (45.5) 2 (18.25) 4 (36.25) 0 (0)

5 (45.5) 2 (18.25) 3 (27.25) 1 (9)

5 (45.5) 3 (27.25) 2 (18.25) 1 (9)

4 (36)

5 (45.5) 1 (9) 0 (0) 0 (0) 2 (18.25) 3 (27.25) 1(9)

6 (55) 3 (27) 2 (18)

6 (55) 3 (27) 2 (18)

4 (36.25) 5(45.5) 2 (18.25) 4.74 (1.39-13.8)

3 (27) 6 (55) 2 (18) 8.45 (1.99-19.7)

12(9-20) 13(9-40) 45(14-120) 15(9-34) 13(10-33) 16(13-120) 4(2-20) 27(21-46) 2(0-4) 2(0-6)

13(10-28) 17(10-60) 23(5-90) 14(11-90) 11(7-19) 18(10-60) 4(0-15) 27(21-40) 2(0-4) 1(0-6)

9 (81) 3 (27) 2 (18) 2 (18)

CR= complete remission, PR= partial remission, PD= progressive disease, ANC= absolute neutrophil count, RBC= red blood cell, IPI= international prognostic index.

23

Sorà et al: Autologous stem cell transplantation for primary refractory or relapsing Hodgkin’s disease also liver and lung in all patients. In group B, one relapse was observed (14.28%) in previous sites of the disease at 13 months. Treatment at relapse included further chemotherapy with or without radiotherapy, nonmyeloablative allogeneic stem cell transplantation etc. At the time of this analysis 5 out of 11 patients in group A and 7 out of 11 patients of and group B are alive with a median follow-up of 69 months (range 61-89) and 70 (range 33-121) respectively. Four patients are in CR and 1 patient is in relapse after CD34+ ASCT, whilst 6 patients are in CR and 1 patient is in relapse after unselectedPBSCT. Six patients died in group A and 4 patients in group B at a median of 24 months (range 1-41) and 20 months (range 1-55) respectively after transplantation.

Median survival for patients in PD after transplantation was 16 months (range 1-27) in group A and 7 months (range 1-55) in group B respectively and was significantly shorter only for patients in PD receiving CD34+ PBSCT compared to responding patients (p=0.004). Death was related to Hodgkin’s disease in all patients. Survival analysis resulted in an overall survival (OS) rate of 45.5 % at a median observation time of 70 months from diagnosis in A group and 71.6 % at 70 months in B group (p=ns).

Overall survival rates calculated from transplantation were 45.5% after 41 months in A group and 72.7% after 42 months in B group (p=ns) (Figure 1A). Disease free survival rates (calculated only for patients achieving CR after transplant) were 57.1% after a median observation time of 61 months in patients of A group and 85.7% after 70 months in B group (p=0.24). Time to progression was 5 + months after CD34 ASCT with FFP of 45% and 33 months in group B with a FFP of 53% in the control group (p=ns) (Figure 1B).

III. Results A. Haemopoietic recovery After conditioning chemotherapy the patients in group A received a median number of 4,74 x 106 (range 1.39-13.8) CD34+ cells/kg while the patients in group B received a median number of 8,45 x 106 (range 2.28-13.7) CD34+ cells/kg (p= ns).

Nine out of 11 patients receiving CD34+ selected cells received G-CSF after reinfusion for a median number 11 doses (range 4-15). No patients received G-CSF in the B group. All patients engrafted, no patient needed additional back-up of unmanipulated PBSC. The median time to recover a neutrophil count >0.5 x 109 and > 1 x 109 was 12 days (range 9-20) and 13 days (range 9-40) respectively in A group and 13 days (range 10-28) and 17 days (range 10-60) respectively in B group (p=ns).

Median time to achieved a self-sustained platelet count> 20 x 109/l and > 50x 109/l in the patients of A group was 13 (range 10-20) and 16 days (range 13-120) respectively (p=0.04) and 11 (range 8-19) and 18 days (range 10-60) respectively in patients of B group.

Median time to achieve a reticulocyte count > 1% in the patients of A group was 15 days (range 9-34) and 14 days (range 11-90) in patients of B group (p=ns).

Haemoglobin level > 10 g/dl was reached after 45 days (range 14-120) in the patients of A group and after 23 days (range 5-92) in the patients of B group (p=ns).

Median length of hospitalization was 27 days in both groups (p=ns).

No statistically significant difference was observed in terms of transfusion requirements: patients of A group received a median number of 2 SDu of platelets (range 04) and 2 pRBCu (range 0-6); patients of B group received a median number of 2 SDu of platelets (range 0-4) and 1 pRBCu (range 0-6).

Median number of days with BT > 38°C was 4 in both groups.

B. Infectious transplantation

episodes

IV. Discussion High dose chemotherapy with autologous stem cells transplantation has been extensively used in patients with refractory or relapsing Hodgkin’s disease. Data generated from single centre and from international registries clearly show that ASCT in these category of patients may produce 20-30% of long-term disease- free survivors (Hornig et al, 1997; Sweetenham, 1999). Clearly these results are superior to what salvage chemotherapy alone can now offers as shown in a randomised trial from the EBMT group for relapsed HD (Schmitz et al, 2002). Despite these encouraging results, disease relapse is still the major cause of treatment failure in the majority of patients after transplantation. One explanation may originate from the presence of neoplastic cells in the graft and also in the type of stem cell source. The presence of circulating Hodgkin/Reed-Sternberg cells of B-lymphoid origin in a patient with advanced HD has been elegantly demonstrated (Kanzler et al, 1996; Wolf et al, 1996) which were able to establish a HD derived cell line in 1997. In the mean time EBMT data showed poorer results for both progression free and overall survival in HD patients receiving PBSCT instead of bone marrow. Furthermore some studies indicated that atypical CD30+ cells can be detected in the product of aphereses of Hodgkin’s disease patients (Blystad et al, 2001b) and to a lesser extent in bone marrow harvest and their presence correlated with

after

Sepsis was documented in 3 out of 11 patients (27.27%) in both groups. Viral infections, related to adenovirus (2 patients) and CMV (1 patient), were observed in 3 out of 11 patients (27.27% only in A group (p=0.06).

C. Response to transplantation After transplant there were 7 out of 11 patients in CR (63.6%) in both groups; 4 patients (36.4%) in group A and 3 patients in group B were in PD (27.3%), 1 patient in Group B was in PR (9%).

Five patients (45%) were submitted to complementary radiotherapy after transplantation in group A while only one patient (9%) received radiotherapy in group B. In group A, 3 patients relapsed after transplantation (42.85%) at 4, 5 and 19 months respectively. Relapse involved previous sites of the disease but unexpectedly

24

Gene Therapy and Molecular Biology Vol 11, page 25

--!--= patients submitted to CD34+ PBSC; --!--= patients submitted to unmanipulated PBSC. Figure 1. Overall survival analysis (A) and freedom from progression analysis (B).

relapse after transplantation (Sharp et al, 1991; Wolf et al, 1996; Blystad et al, 2001a). A clinical benefit of removing such unwanted cells from the grafts was then suggested in the treatment of high risk patients. Then, considering the lack of CD34 antigen on the surface of Hodgkin/ReedSternberg cells, positive selection of CD34+ cells has been applied (Blystad et al, 2001a; Lakota et al, 2002) although the significance of circulating CD30+ cells is still not clear (Sharp et al, 1999).

In this study we report the results of a pilot trial carried out at our institution for patients with refractory/relapsing HD using CD34+ selected peripheral blood progenitor cells.

The rationale of the study was to reduce the incidence of relapse in advanced stage HD patients undergoing high dose chemotherapy and ASCT. The issue of CD30+ cell contamination in the graft was not addressed in this study.

Patients included in the study were then compared to a group of patients closely matched for demographic and clinical parameters and receiving unmanipulated PBPC. Our results showed no benefit in any of the clinical endpoints in patients receiving CD34+ cells. In particular, with the exception of a faster platelets recovery > 20x 109/l after reinfusion of PBSC no difference was observed in haematological recovery between the two groups. No differences were also found in terms of days with fever, days of i.v. antibiotics, length of hospitalization.

Cautiously, the incidence of viral infections, mostly CMV infections, was felt to be slightly increased in patients receiving CD34+ selected cells (Salutari et al, 1998).

Despite the high efficiency of purging procedure, no evidence of benefit on CR rate, DFS or FFP was detected in patients transplanted with CD34+ cells with nearly a half of the patients relapsing at a median of 5 months. Overall survival was clearly superimposable in both groups and was very prolonged with a median of 20 and 24 months respectively. These data are likely to be influenced by the management of relapse after transplantation with further chemotherapy and or local radiotherapy producing symptomatic responses and they are similar to those

recently reported by other authors (Shamash et al, 2000; Paltiel et al, 2003). Disease progression produced significantly lower survival after CD34+ ASCT. Interestingly relapse after CD34+ transplantation involved lung parenchyma in all patients as a new site and liver in 1 patient. This behaviour also has been described by Shamash et al in up to 53% of patients with lung recurrences after unselected PBSCT (Shamash et al, 2000).

Two clinical reports were recently published using purging procedures in Hodgkinâ&#x20AC;&#x2122;s disease (Blystad et al, 2001a; Lakota et al, 2002). In both studies CD34+ selection was adopted. The first study included 10 patients with advanced HD in 2nd or higher CR at transplantation.

The authors reported a strikingly high response rate with 90% of patients in CR at a median follow-up of 26 months (Lakota et al, 2002). The second study included 21 patients with HD, the majority of whom were in 2nd PR, undergoing CD34+ PBSCT. In this study data were compared to a group of patients treated with unselected PBSCT. Although there was no difference between the 2 groups, EFS was 75% at 4 years in patients receiving CD34+ ASCT (Blystad et al, 2001a). These differences in treatment outcome are probably related to the small number of patients treated in these series, including our own study, the length of follow up and heterogeneity of clinical characteristics and particularly status at transplantation which still remains one of the mayor determinant for the outcome of ASCT (Constans et al, 2003).

Although our data confirm that CD34+ ASCT in high risk HD is feasible and safe, clinical outcome after an appropriate follow up was unaffected by the purging procedure and despite the small number of patients in our series, our results indicated that CD34+ selection may not be efficient in the setting of HD at least in patients with relapsed/refractory disease.

25

Sorà et al: Autologous stem cell transplantation for primary refractory or relapsing Hodgkin’s disease survival in patients with progressive disease following autologous transplant for lymphoma. Bone Marrow Transplant 31, 565-9.

Salutari P, Sica S, Laurenti L, Leone F, Chiusolo P, Piccirillo N, Micciulli G, Leone G (1998) Incidence of sepsis after peripheral blood progenitor cells transplantation: analysis of 86 consecutive hemato oncological patients. Leuk Lymphoma 30, 193-7.

Schmitz N, Pfistner B, Sextro M Schmitz N, Pfistner B, Sextro M, Sieber M, Carella AM, Haenel M, Boissevain F, Zschaber R, Muller P, Kirchner H, Lohri A, Decker S, Koch B, Hasenclever D, Goldstone AH, Diehl V; German Hodgkin's Lymphoma Study Group; Lymphoma Working Party of the European Group for Blood and Marrow Transplantation (2002) Aggressive conventional chemotherapy compared with high-dose chemotherapy with autologous haemopoietic stem-cell transplantation for relapsed chemosensitive Hodgkin's disease: a randomised trial. Lancet 359, 2065-71.

Shamash J, Lee SM, Radford JA, Rohatiner AZ, Chang J, Morgenstern GR, Scarffe JH, Deakin DP, Lister TA (2000) Patterns of relapse and subsequent management following high-dose chemotherapy with autologous haematopoietic support in relapsed or refractory Hodgkin's lymphoma: a two centre study. Ann Oncol 11, 715-9.

Sharp JG, Chan WC (1999) Detection and relevance of minimal disease in lymphomas. Cancer Methastasis Revs 18, 12742.

Sharp JG, Kessinger A, Piruccello SJ (1991) Frequency of detection of suspected lumphoma cell in peripheral blood stem cell collection. In: Dicke KA, Armitage JO, Dicke Evinger MJ (eds) Autologous Bone Marrow Transplantation University of Nebraska Medical Centre: Ohama, 801-810 .

Sweetenham JW, Carella AM, Taghipour G Cunningham D, Marcus R, Della Volpe A, Linch DC, Schmitz N, Goldstone AH (1999) High-dose therapy and autologous stem-cell transplantation for adult patients with Hodgkin's disease who do not enter remission after induction chemotherapy: results in 175 patients reported to the European Group for Blood and Marrow Transplantation. Lymphoma Working Party. J Clin Oncol 17, 3101-9.

Wolf J, Kapp U, Bohlen H Kornacker M, Schoch C, Stahl B, Mucke S, von Kalle C, Fonatsch C, Schaefer HE, Hansmann ML, Diehl V (1996) Peripheral blood mononuclear cells of a patient with advanced Hodgkin's lymphoma give rise to permanently growing Hodgkin-Reed Sternberg cells. Blood 87, 3418-28.

Acknowledgements This work was supported in part by Associazione Italiana per la Ricerca sul Cancro (AIRC) Milan, Italy. We are grateful to the nursing staff of the Divisione di Ematologia, PoliclinicoA. Gemelli.

References Bierman PJ, Lynch JC, Bociek RG, Whalen VL, Kessinger A, Vose JM, Armitage JO (2002) The International Prognostic Factors Project score for advanced Hodgkin's disease is useful for predicting outcome of autologous hematopoietic stem cell transplantation. Ann Oncol 13, 1370-7.

Blystad AK, Holte H, Kvaloy S Smeland E, Delabie J, Kvalheim G (2001a) High dose therapy in patients with Hodgkin's disease: the use of selected CD34(+) cells is as safe as unmanipulated peripheral blood progenitor cells. Bone Marrow Transplant 3, 295-305.

Blystad AK, Torlakovic E, Holte H, Blystad AK, Torlakovic E, Holte H, Kvaloy S, Lenschow E, Kvalheim G (2001b) CD34(+) cell enrichment depletes atypical CD30(+) cells from PBPC grafts in patients with HD. Cytotherapy 28, 849-57.

Centers for Disease Control and Prevention (CDC) (2002) MMWR Morb Mortal Wkly Rep. 50, 27-28.

Constans M, Sureda A, Terol MJ Constans M, Sureda A, Terol MJ, Arranz R, Caballero MD, Iriondo A, Jarque I, Carreras E, Moraleda JM, Carrera D, Leon A, Lopez A, Albo C, DiazMediavilla J, Fernandez-Abellan P, Garcia-Ruiz JC, Hernandez-Navarro F, Mataix R, Petit J, Pascual MJ, Rifon J, Garcia-Conde J, Fernandez-Ranada JM, Mateos MV, Sierra J, Conde E; GEL/TAMO Cooperative Group (2003) Autologous stem cell transplantation for primary refractory Hodgkin's disease: results and clinical variables affecting outcome. Ann Oncol 14, 745-51..

Horning SJ, Chao NJ, Negrin RS Horning SJ, Chao NJ, Negrin RS, Hoppe RT, Long GD, Hu WW, Wong RM, Brown BW, Blume KG (1997) High-dose therapy and autologous hematopoietic progenitor cell transplantation for recurrent or refractory Hodgkin's disease: analysis of the Stanford University results and prognostic indices. Blood 89, 801-13.

Kanzler H, Hansmann ML, Kapp U Wolf J, Diehl V, Rajewsky K, Kuppers R (1996) Molecular single cell analysis demonstrates the derivation of a peripheral blood-derived cell line (L1236) from the Hodgkin/Reed Sternberg cells of a Hodgkin's lymphoma patient. Blood 87, 3429-36.

La Barbera EO, Chiusolo P, Laurenti L Menichella G, Di Febo AL, Piccirillo N, Sora E, Marra R, Teofili L, Leone G, Sica S (2000) MiCMA: an alternative treatment for refractory or recurrent Hodgkin's disease. Ann Oncol 11, 867-71.

Lakota J, Ballova V, Drgona L, Durkovic P, Vranovsky A (2002) Use of selected CD34+ cells in the treatment of relapsed/progressive HD: experiences from a single center.

Cytotherapy 4, 177-80.

Majolino I, Pearce R, Taghipour G, Goldstone AH (1997) Peripheral-blood stem-cell transplantation versus autologous bone marrow transplantation in Hodgkin's and nonHodgkin's lymphomas: a new matched-pair analysis of the European Group for Blood and Marrow Transplantation Registry Data. Lymphoma Working Party of the European Group for Blood and Marrow Transplantation. J Clin Oncol 15, 509-17.

Paltiel O, Rubinstein C, Or R Nagler A, Gordon L, Deutsch L, Polliack A, Naparstek E (2003) Factors associated with

Federica Sorà

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Gene Therapy and Molecular Biology Vol 11, page 27 Gene Ther Mol Biol Vol 11, 27-36, 2007

Gene therapy for arthritis: defining novel gene targets Review Article

Charles J. Malemud* Department of Medicine/Division of Rheumatic Diseases, Case Western Reserve University School of Medicine, Cleveland, Ohio

__________________________________________________________________________________ *Correspondence: Charles J. Malemud, Ph.D., Department of Medicine, Division of Rheumatic Diseases, University Hospitals Case Medical Center, 2061 Cornell Road, Cleveland, Ohio 44106-5076, USA; Telephone: (216) 844-7846; Fax: (216) 844-2288; E-mail: cjm4@cwru.edu Key words: Adenoviral/Adenoviral-associated Vector, Gene Transfer, Inflammation, Osteoarthritis, Rheumatoid Arthritis Abbreviations: adenovirus-FasL (Ad.FasL); angiopoietin (Ang); collagen-induced arthritis (CIA); cytomegalovirus (CMV); diseasemodifying anti-rheumatic drugs (DMARDs); extracellular matrix (ECM); fibroblast-like synoviocytes (FLS); helper Type 2 (Th2); inhibitor of !B (I!B); insulin-like growth factor-1 (IGF-1); Interferon-! (IFN-!); interleukin-1 (IL-1); interleukin-1 receptor antagonist (IL-1Ra); I!B kinase (IKK!); matrix metalloproteinase (MMP); metacarpophalangeal (MCP); non-steroidal anti-inflammatory drugs (NSAIDs); osteoarthritis (OA); receptor activator of nuclear factor-!B ligand (RANKL); rheumatoid arthritis (RA); T-cell receptor (TCR); tissue inhibitor of metalloproteinases (TIMPs); TNF-" receptor-I (TNFR-I); tumor necrosis factor-" (TNF-"); vascular endothelial growth factor (VEGF); VEGF-receptor-I (VEGFR-I) Received: 23 January 2007; Revised: 23 February 2007 Accepted: 19 March 2007; electronically published: March 2007

Summary Rheumatoid arthritis (RA) and osteoarthritis (OA) are debilitating diseases of the musculoskeletal system. RA is characterized by immune dysfunction and the classical cellular and soluble mediators of inflammation, whereas during its early stages, OA is considered a non-inflammatory disorder. However, a common pathway inherent to both RA and OA is articular cartilage and subchondral bone destruction resulting in non-functional synovial joints.

Over the past decade or so, many of the candidate pathophysiologic pathways including those regulated by cytokines, growth factors and transcription factors that promote RA and OA disease progression have been elucidated. Although medical therapies directed at neutralizing cytokine activity have emerged and are now employed in treating RA and OA, there is considerable debate as to how long these biologics can be employed to modify chronic disease progression without emergent serious adverse events. This has led to a significant increase in studies that have employed gene transfer strategies in RA and OA animal models directed at inhibiting inflammatory cytokines, immune-mediated inflammation, growth factors, angiogenesis factors, apoptosis and matrix metalloproteinases that play a prominent role in RA and OA pathology. The significant abrogation of inflammation in arthritis animal models, in particular, by interleukin-1 receptor antagonist (IL-1Ra) and soluble TNF receptor gene constructs makes them potentially useful for treating RA and OA.

synoviocyte proliferation, defects in apoptosis as well as lymphocyte, monocyte and macrophage migration with resultant pannus development (Malemud and Gillespie, 2005). The classical signs and symptoms of inflammation emerge from these events (Firestein, 2005), and, in RA, cartilage destruction and bone resorption result from matrix metalloproteinase (MMP) gene up-regulation and stimulation of other bone resorptive agents (Walsh et al, 2005) which result in bone destruction and ankylosis.

By contrast, the etiopathogenesis of OA is considered to be a functionally non-inflammatory process.

Thus, 20 to 30 years can pass before the clinical symptoms

I. Introduction Rheumatoid arthritis (RA) and osteoarthritis (OA) are among the most debilitating of human musculoskeletal system degenerative disorders. RA is a systemic autoimmune disease of unknown cause with cellular and humoral immune dysfunction as its hallmarks (Feldman et al, 1996; Pope and Perlman; 2000; Malemud, 2007). At the pathophysiologic level, RA is characterized principally by activation of fibroblast-like synoviocytes (FLS) within the synovial tissue which ultimately presents as synovial hyperplasia.

Synovial hyperplasia results from

27

Malemud: Gene therapy for arthritis: defining novel gene targets of OA emerge which include pain, stiffness and reduced range of motion (Veys and Verbruggen, 1999). As such, OA is a common clinical finding in the elderly population.

At the cellular level, OA is characterized by mesenchymal stem cell proliferation within the synovial joint giving rise to osteophytes whose functional significance remains to be fully elucidated. However, it has been postulated that osteophytes could play a role in the reduced range of motion characteristic of OA joints (Malemud et al, 1999).

Further along in the disease process, OA is typically marked by a type of inflammation involving several cellular markers of the classical inflammatory response (Pelletier et al, 2001; Attur et al, 2002a), articular cartilage remodeling, fibrillation, fissuring and dissolution (i.e. eburnation) as well as sclerotic changes in subchondral bone (Malemud et al, 2003).

An eventual pathologic feature common to both OA and RA is activation of the normal synovium (Feldmann et al, 1996). Synovial activation results in pro-inflammatory cytokine gene amplification that is, in part, regulated by stress-activated protein kinases and mitogen-activated protein kinases (Berenbaum, 2004). The resultant cytokine gene up-regulation exemplified by interleukin-1 (IL-1) and/or tumor necrosis factor-" (TNF-") occurs without a concomitant robust stimulation of the endogenous IL-1 antagonist interleukin-1 receptor antagonist (IL-1Ra) or soluble anti-TNF-" neutralizing receptor molecule gene expression, and coupled to pro-IL-1 and pro-TNF-" activation (Attur et al, 2002a; Malemud, 2004) lead to increased levels of IL-1 and TNF-" in arthritis synovial fluid. These events ultimately result in an anabolic/catabolic imbalance with resultant joint destruction (Malemud et al, 2003). Further, stimulation of synovial angiogenic factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor-1, and -2 (Malemud 2007) as well as elevated levels of eicosanoid metabolites such as prostaglandins and leukotrienes (Martel-Pelletier et al, 1999a) accompany arthritis disease progression. In particular, IL-1 and TNF-" both stimulate chondrocyte matrix metalloproteinases (MMPs) as well as a family of proteolytic enzymes with the structural properties of a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS) gene expression while suppressing endogenous MMP inhibitors such as the family of tissue inhibitor of metalloproteinases (TIMPs) (Burrage et al, 2006). The concerted action of several MMPs, most prominently, MMP-2, MMP-3, MMP-9 and MMP-13 (Burrage et al, 2006) in combination with reduced or only slightly elevated TIMP levels appear to be responsible for cartilage extracellular matrix (ECM) protein degradation in arthritis (Burrage et al, 2006).

Adding to cartilage and bone deterioration, a common finding in RA and OA, is ECM protein synthesis suppression. ECM protein suppression occurs as a result of pro-inflammatory cytokine activity as well as the activity of other soluble mediators of inflammation which activate transcription factors causing a decrease in ECM protein gene expression (Martel-Pelletier et al, 1999b; Fernandes et al, 2002). In addition, chondrocyte non-responsiveness to circulating growth factors such as insulin-like growth

factor-1 (IGF-1) also occur which likely compromise articular cartilage and subchondral bone repair pathways.

The possibility that a skewed upward growth hormone to somatostatin ratio plays a critical role in RA inflammation (Denko and Malemud, 2004) and that pituitary/hypothalamic dysfunction in general regulates, in part, arthritis inflammation, pain and disease progression has also recently been reviewed (Denko and Malemud, 2005).

Medical therapies designed to retard arthritis progression have commonly included anti-inflammation strategies such as oral and intra-articular corticosteroids, non-steroidal anti-inflammatory drugs (NSAIDs) (Pelletier et al, 1994) as well as disease-modifying anti-rheumatic drugs (DMARDs) such as methotrexate, hydroxychloroquine, sulfasalazine and leflunomide (Gaffo et al, 2006). More recently, anti-IL-1 [i.e. IL-1 receptor antagonist; anakinra; (Kineret)®], anti-TNF-" neutralizing monoclonal antibodies (i.e. infliximab; (Remicade®); adalimumab; (Humira®), dimeric p75TFNR fusion protein (i.e. etanercept; Enbrel®), anti-T-cell activation (i.e. abatacept; Orencia®) as well as anti-CD20 therapy (i.e. rituximab; Rituxan®) have been employed primarily in RA (Gaffo, 2006). However, a common problem likely to occur in treating OA and RA patients with these biologic agents is the deleterious side-effects and additional comorbid conditions, in particular, infections such as tuberculosis (Weaver, 2003) as well as other emergent medical problems such as malignancies and thrombocytopenia that may associate with employing these biologic agents over the long duration of treating these chronic diseases especially with anti-TNF-" biologics.

Testing of experimental agents in RA and OA animal models that are designed to suppress cartilage ECM degradation and bone resorption has generally involved oral administration of synthetic MMP inhibitors and other anti-resorption compounds with less than ideal efficacy or amelioration of clinicopathologic features (Malemud et al, 2003; Malemud, 2004). Because of the generally unfavorable results obtained by attempting to suppress MMP activity in arthritis animal models, alternative modalities including gene therapy strategies have been developed that stem from elucidating the key intracellular pathways giving rise to inflammation, apoptosis resistance, ECM protein degradation and suppression of ECM protein compensatory biosynthesis in arthritis (Malemud et al, 2003). In addition, successful experimental therapy of arthritis in animal models with recombinant proteins have generally required continuous, multiple injections to achieve any amelioration of clinical inflammation (van de Loo et al, 2006; Moritz et al, 2006; Adriaansen et al, 2006a). Several of these approaches have involved local administration of recombinant proteins to affected joints. However, the identification of novel targets for RA and OA intervention in animal models notwithstanding (Moritz et al, 2006; van de Loo et al, 2006) the problem of achieving sustained biological effects by comparing local with systemic administration of recombinant proteins has generally favored the former rather than the latter mode of recombinant protein

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Gene Therapy and Molecular Biology Vol 11, page 29 administration. Additional overriding issues of potential clinical toxicity and lack of efficacy inherent in the use recombinant proteins have led to a more sustained and systematic effort to employ gene therapy for arthritis intervention.

This review will focus on significant developments in gene therapy strategies for arthritis since 2003 in which gene transfer has been employed to suppress arthritis incidence, progression and pathophysiologic pathways in arthritis animal models. The generally favorable results of gene transfer studies in animals (Campbell et al, 2005; van de Loo et al, 2006) have led to their consideration for use in phase I clinical trials in OA (Evans, 2005; Evans et al, 2006) and RA (Tomita et al, 2003; Muller-Ladner et al, 2003; Van de Loo, 2004, Van de Loo et al, 2004; Adriaansen et al, 2006a; Nakajima, 2006).

From an historical perspective on the use of gene transfer to treat experimental OA, earlier studies where in vivo gene transfer of an IL-Ra gene-construct (Pelletier et al, 1997; Fernandes et al, 1999) or an IL-1!-receptor decoy gene (Attur et al, 2002b) was employed should be appreciated. These gene constructs were successfully implemented in OA animal models and additional in vitro studies also suggested a mechanism for their action.

unwarranted side-effects in some of the study participants characterized as injection site reaction with echymosis. In addition, because leukemia had developed in some children with SCID using cell therapy (Check, 2003) and the same viral backbone employed in the study reported by Evans and colleagues in 2005, a 5-year waiting period ensued before publication of the study results to ensure that unwarranted effects of the IL-1Ra gene transfer did not occur. After more than 5 years, the subjects receiving the IL-1Ra transgene were free of replication-competent virus and no adverse events occurred. These results showed the feasibility of direct gene transfer of FLS containing an IL-1Ra transgene pertinent to suppressing cartilage and bone destruction in human RA.

B. IL-10 gene transfer IL-10 is a cytokine with potent anti-inflammatory properties and the capacity to down-regulate IL-1 and TNF-" in vitro (Fernandes et al, 2002). Of note, IL-10 also suppressed TNF receptor-I expression while increasing the level of TNF receptor-II in human FLS isolated from OA synovium (Alaaeddine et al, 1999). Thus, the capacity to increase IL-10 levels by gene transfer seemed to be a worthy target in RA and OA. In that regard, Woods and colleagues showed in 2005 that when used prophylactically, intranasal delivery of an IL-10 geneconstruct (pG-IL-10) to DBA/1 mice with CIA delayed arthritis onset and reduced arthritis severity. IL-10 expression appeared to target monocytes and macrophages as well as draining lymph nodes. The expression of pG-IL10, however, appeared to be independent of any changes in TNF-". In addition, Khoury and colleagues showed in 2006 that delivery of an IL-10 gene construct using a nonviral in vivo intra-muscular electrotransfer method to mice with CIA was more effective in suppressing inflammation than intra-articular electrotransfer administration.

II. Anti-cytokine approaches A. IL-1Ra gene transfer Ever since cytokines were identified as playing a key role in OA and RA disease progression, these molecules have been targeted for gene therapy intervention in arthritis animal models with a view towards applying the results of these studies to phase I clinical arthritis trials. In that regard, IL-1Ra cDNA was shown to reduce inflammation in an RA animal model characterized by a deficiency in IL-1Ra (Hur et al, 2006). In particular, an adenoviral vector-construct containing human hIL-1Ra and GFP (i.e.Ad.IL-1Ra.GFP) was administered by intraarticular injection into the ankle joints of mice with established chronic inflammatory arthritis induced by Type II collagen (i.e. collagen-induced arthritis; CIA). Not only did the treated mice show abrogation of arthritis inflammation, but these animals also showed reduced levels of helper Type 1 (Th1)-driven IgG2a antibodies to Type II collagen compared to controls and increased levels of helper Type 2 (Th2)-driven IgG1 antibody as well.

These studies established that gene transfer of IL-1Ra could be therapeutically efficacious in suppressing the immunologically-mediated early stages of human RA. In a more recent short-term phase I clinical trial, 9 postmenopausal women with destructive RA underwent unilateral silastic implant surgery. Synovium was recovered from the 2nd-5th metacarpophalangeal (MCP) joints and RA FLS transduced with a retrovirus containing IL-Ra cDNA ex vivo. After intra-articular injection of autologous FLS-containing the IL-1Ra transgene (106-6.5106 cells) into 2nd-5th MCP operated joint of one hand, the IL-1Ra transgene was expressed as IL-1Ra protein at levels significantly higher than joint tissues injected with control vector, but IL-1Ra expression occurred only at the highest cell dose (Evans et al, 2005). Although no adverse events occurred, intra-articular injection did produce

C. IL-13 gene transfer IL-13 is a Th2-produced cytokine with potent antiinflammatory properties that is abundant in the synovial fluid of RA patients (Malemud, 2004). In vitro, IL-13 inhibited IL-1! and TNF-" expression and augmented IL1Ra production in human OA-FLS stimulated with lipopolysaccharide (Jovanovic et al, 1998). In a recent study, Nabbe and colleagues showed in 2005 that an IL-13 gene construct transferred using an adenoviral vector to mice with immune complex-induced arthritis actually increased the number of synovial joint inflammatory cells.

However, IL-13 gene transfer also abrogated chondrocyte apoptosis via down-regulation of FC#RI and MMPmediated proteoglycan degradation induced by immunecomplexes without altering MMP-3, -9, -12 or -13 mRNA levels. These results suggested that IL-13 over-expression in immune-complex-mediated murine arthritis inhibited chondrocyte apoptosis and proteoglycan cleavage despite having little effect on the overall inflammation profile.

These studies concluded that modulation of FC#RI by TH2 cytokines might provide an additional therapeutic approach for modifying cartilage damage in experimental RA (Nabbe et al, 2005).

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Malemud: Gene therapy for arthritis: defining novel gene targets to activate transmembrane TNF-RI in keeping with a model previously proposed by Adam and colleagues, in 1995. In addition, the anti-inflammatory effect of the TNFR-I receptor-construct was long-lasting and compared favorably with repeated intra-muscular treatments with etanercept. These results suggested that targeting TNF-" receptor-I by gene transfer may also prove useful in future arthritis therapy. In addition to studies focusing on the use of soluble TNF-RI gene therapy strategies, there are additional ongoing clinical trials with soluble TNF-RII (see, http://www.wiley.co.uk/genetherapy/clinical).

D. IL-4 gene transfer IL-4 is another anti-inflammatory cytokine that also can spare synoviocytes from nitric oxide-induced apoptosis in vitro (Relic et al, 2001). IL-4 has also been shown to suppress TNF-"-mediated prostaglandin E2 production by OA synovial fibroblasts (Alaaeddine et al, 1999). In a recent study, Haas and colleagues showed in 2006 that an IL-4 gene transferred to rats with adjuvantinduced arthritis significantly reduced synovial vessel density and ankle joint inflammation. Joint homogenates collected from rats containing the IL-4 transgene inhibited both endothelial migration and tube formation in vitro despite high levels of VEGF. The angiostatic effects of the IL-4 transgene correlated in vitro with elevated levels of IL-18, chemokines CXL16 and CXL5 as well as endostatin. Thus, IL-4 transgene strategies that demonstrate suppression of synovial neoangiogenesis might provide some efficacy in RA.

III. Immunomodulatory approaches A. Interferon-" Interferon-! (IFN-!) has potent immunomodulatory properties (Tak, 2004). Its use as a human RA therapy was, however, compromised in two recent clinical trials by apparent attrition problems due to lack of efficacy rather than safety (Genovese et al, 2004) and the failure of subcutaneous administration of IFN-!1 together with methotrexate to alter RA radiographic progression (van Holten et al, 2005). This lack of efficacy may also be attributable to the mode of administration as well as issues of pharmacokinetics (Adriaansen et al, 2006b), even though a prior study had shown that daily recombinant IFN-! injections reduced arthritis severity and inhibited pro-inflammatory cytokine responses, including TNF-" and IL-6, while increasing IL-10 in murine CIA (van Holten et al, 2004). To investigate the possibility that IFN! gene transfer could substitute for administration of recombinant INF-! protein, Adriaansen and colleagues, (2006b) treated rat AIA with an intra-articular injection of adenovirus vector containing an IFN-! construct (i.e.

Ad.IFN-!) at different doses ranging from 1.2 x 109 to 1.2 x 1011 viral particles. The levels of IFN-! synthesis in the arthritic hindpaw peaked 2 days after intra-articular Ad.IFN-Ă&#x; injection and synovial inflammation was significantly reduced. Further, Ad.IFN-! abrogated bone erosions, but only at the highest dose employed (i.e. 1.2 x 1011 viral particles). In addition, Ad.IFN-! reduced the signaling molecules, c-Cbl and CBl-b whose activity is critical for osteoclast differentiation (Miyazaki et al, 2004) as well as reducing the MMP-3/TIMP-1 ratio. A histologic examination of the rat synovium suggested an antiinflammatory effect of Ad.IFN-!.

E. Neutralizing TNF-! by gene transfer Anti-TNF-" monoclonal antibody therapy has proven efficacy as an RA therapy (Hsu et al, 2006). AntiTNF-"-mediated suppression of inflammation is regarded as the major mechanism attributed to these therapies which have also been shown to retard radiologic evidence of RA disease progression, especially when employed in conjunction with methotrexate (Breedveld et al, 2006).

However, there is little evidence that any of the current RA therapies with DMARDs or biologics reverse the damage to joints caused by RA, although aggressive combination therapy (e.g. adalimumab plus methotrexate) appeared to be superior to monotherapy (e.g. adalimumab or methotrexate) in retarding RA progression (Breedveld et al, 2006).

At the cellular level, the mechanisms attributed to anti-TNF-" strategies primarily include, a reduction in circulating TNF-", suppression of nuclear factor-!B activation and down-regulation of MMP gene transcription (Berenbaum, 2004; Roman-Blas and Jimenez, 2006), making use of anti-TNF-" treatment potentially attractive for the therapy of OA as well as RA. Because it is not known how long RA patients can be treated with the current commercially available anti-TNF-" biologics, infliximab, etanercept and adalimumab, that neutralize the downstream effector pathways attributed to TNF-" makes TNF-" gene therapy strategies an attractive target for arthritis intervention. In that regard, Bloquel and colleagues showed in 2004 that a single dose of plasmids encoding 3 soluble TNF-" receptor-I (TNFR-I) variant forms (i.e. monomeric, dimeric and chimeras) delivered intra-muscularly by electrotransfer to mice with CIA reduced the clinical signs of arthritis inflammation.

However, no change in arthritis clinical scores occurred when only the monomeric form of the soluble TNFR-I transgene was employed and only moderate changes occurred with the dimeric form. Thus, it appeared that at least 2 of the 3 variant subunits of TNF-" trimer have to bind to inhibit cell activation by TNF-". Prevention of transmembrane TNF receptor binding was proposed as the putative mechanism of action of these TNFR-I-receptor constructs because the remaining TNF subunit was unable

B. T-cell Receptor (TCR) strategies T-cells containing both TCR" and TCR! accumulate in human RA synovium (Firestein, 2005) whereas the TCR! gene repertoire provided a signature profile for Tcell involvement in the inflamed paws of mice with CIA (Haqqi et al, 1992). To investigate the extent to which TCR recovered from the inflamed joints of mice with CIA could then be employed to suppress inflammation, Fujio and colleagues in 2006 first expanded a clone of cells containing TCR",! genes that were originally isolated from the inflamed paw of a mouse with CIA. The clonotype (i.e. B47) was reconstituted on peripheral CD4+ T-cells and used as the therapeutic cellular vehicle. B47 was autoreactive, but not specific for Type II collagen.

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Gene Therapy and Molecular Biology Vol 11, page 31 Fujio et al, (2006) found that TCR genes from B47transduced T cells accumulated in the inflamed paws of mice with CIA. Of note, injection of cells that had been co-transduced with B47 and a soluble TCRRIg geneexpressing vector by fusing the p75 TNFR (i.e. TNFR-II) with the Fc domain of IgG2a also resulted in significant suppression of paw inflammation. Arthritis suppression correlated with TNFRIg transcripts in the hind paw but not with serum TNFRIg content. In addition, cells cotransduced with B47 and a Foxp3 gene-construct reduced levels of TNF-", IL-17A, IL-1!, suppressed bone destruction and progression of established CIA. These studies suggested that TCR genes might be applied to induce immune suppression in inflammatory arthritis.

C. Kallistatin Kallistatin is a member of the serpin protein family that was identified as a specific inhibitor of kallikrein (Miao et al, 2003). Whereas kallikrein was shown to promote angiogenesis (Emanueli et al, 2002), kallistatin was shown to inhibit angiogenesis and tumor growth (Miao et al, 2002). This evidence suggested that kallistatin might be a potent inhibitor of VEGF-mediated angiogenesis in RA. Thus, Wang and colleagues, showed in 2005 that an adenoviral vector containing the human kallistatin gene (i.e. Ad.H.KBP) suppressed rat ankle arthritis, reduced synovial vessel density and neutrophil levels as well as IL-1! and TNF-", but not VEGF. In addition, Ad.H.KBP markedly inhibited endothelial proliferation in vitro suggesting that kallistatin gene transfer could become an adjunctive angiostatin therapy in RA.

IV. Anti-angiogenesis strategies A. Tie-2 RA progression is dependent on neoangiogenesis (Malemud, 2007). The angiopoietin (Ang)-Tie ligand receptor system plays a key role in vascular integrity by virtue of its ability to regulate permeability, resistance, and, in arthritis to regulate inflammation by its involvement in synovial neoangiogenesis (Fiedler and Augustin, 2006). Tie-2 and Ang-1 were previously shown to regulate angiogenesis in CIA by virtue of the fact that Tie-2 and Ang-1 levels were increased in RA synovium (DeBusk et al, 2003). Further, DeBusk and colleagues found in 2003 that TNF-" promoted Tie-2 deposition in synovium which apparently involved interactions between endothelial cells and FLS, NF-!B activation and Ang-1 expression. The results of that study (DeBusk et al, 2003) provided the impetus for employing an adenovirus construct containing a soluble Tie-2 receptor (i.e.

Ad.ExTek) to treat murine CIA. Thus, Chen and colleagues showed in 2005 that CIA disease severity was considerably reduced when Ad.ExTek was administered after the onset of CIA compared to the control group.

Further, Ad.ExTek protected mice with CIA against bony erosions, suppressed neoangiogenesis in the paws of Ad.ExTek-treated mice and reduced the levels of receptor activator of nuclear factor-!B ligand (RANKL), a key mediator of osteoclastogenesis and bone erosion.

However, no differences in anti-collagen antibodies were noted and suppression of CIA disease activity could not be correlated with suppression of anti-collagen antibodies.

V. Stimulation of apoptosis by gene transfer Defective synovial apoptosis with resultant synovial hypertrophy is one of the hallmarks of RA, and may arise as a result of defects in Fas/Fas ligand-induced apoptosis (Perlman et al, 2001; Baier et al, 2003; Malemud and Gillespie, 2005). Previously, FasL mRNA could be detected in 5 of 6 RA patient synovium but most of the FasL was localized to infiltrating mononuclear cells (Asahara et al, 1997). Thus, correcting a putative defect in the Fas/FasL pathway by FasL gene transfer might be an effective â&#x20AC;&#x153;molecularâ&#x20AC;? substitute for surgical synovectomy in RA. Recently, Kim and colleagues showed in 2006 that synovectomy of hand RA moderately improved hand function although no changes in grip strength or range of motion occurred. Further, synovectomy was not recommended for the RA knee or ankle with radiographically advanced joint space narrowing (Kim et al, 2006). To address the possibility that FasL gene therapy could ameliorate synovial hypertrophy, Zhang and colleagues studied in 2005 whether FasL gene transfer could alter the inflammatory properties of human RA synovia obtained by synovectomy. Zhang and colleagues in 2005 employed repeated administrations of adenovirusFasL (i.e. Ad.FasL) ex vivo and then grafted Ad.FasLtransduced synovia on to C.B-17 SCID mice. After recovering the implants, the number of synoviocytes and mononuclear cells was significantly reduced compared to implants from control animals and an approximately 15fold increase in apoptotic nuclei was seen. This study suggested that intra-articular FasL gene transfer could serve to induce apoptosis in RA synovium and could therefore be conceivably employed to reduce the effects of synovial pannus in RA.

B. VEGF VEGF-B is a significant mediator of neoangiogenesis in RA synovium (Malemud, 2007). In CIA, the arthritis incidence and severity was greatly diminished in VEGF-B -/- mice compared to their wild-type littermates as was synovial vessel density (Roccaro et al, 2005). Further, the finding that an adenoviral VEGF-receptor-I (VEGFR-I)construct delivered to mice with CIA significantly diminished arthritis severity suggested that VEGF blockade via VEGFR-I gene transfer (Afuwape et al, 2003) could be a successful angiostatin mechanism in experimental arthritis.

VI. NF-!B gene therapy Suppression of NF-!B activation is a critical step in modulating the inflammatory response (Malemud et al, 2003). Phosphorylation of the inhibitor of !B (I!B) is a prerequisite event in NF-!B activation during inflammation which is principally regulated by I!B kinase (IKK!) (Chen et al, 2001; Hayden and Ghosh, 2004).

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Malemud: Gene therapy for arthritis: defining novel gene targets Thus, Tas and colleagues (2006) specifically targeted IKK! by employing a recombinant adeno-associated virus type 5 dominant-negative-IKK!-construct (i.e. rAAV5.IKK!.dn) by intra-articular injection into the inflamed right ankle joint of rats with AIA. The results showed that right ankle paw swelling was suppressed and immunohistochemical analysis of the synovium revealed reduced intensity of IL-6 and TNF-", but not IL-10.

However, no significant effect was found on cartilage and bone destruction, MMP-3 or TIMP-1. This study showed that rAAV5.IKK! could be successfully employed to locally reduce experimental arthritis inflammation endpoints in vivo and ex vivo. Although rAAV generally transduces less efficiently than adenoviral vectors expressing the same transgene, stable long-term expression is posited to make rAAV a better candidate than adenovirus for gene transfers in chronic diseases such as RA (Chernajovsky et al, 2004).

collected from synovial cell monolayer cultures was sufficient to stimulate proteoglycan synthesis by normal cartilage explants. This study suggested that IGF-1/IL-Ra co-transduction of synovial membrane may be a feasible gene therapy model for stimulating cartilage ECM proteins during early arthritis.

IX. Conclusions Although several medical therapies appear to be provide symptomatic relief of pain in RA and OA, at present, specific therapies that take advantage of the pathophysiologic events pertinent to RA and OA focus on neutralizing the effects of IL-1 (i.e. IL-1Ra) and TNF-" with soluble TNF receptor monoclonal antibodies. An approach using dominant negative TNF-variants has also been considered (Steed et al, 2003). More recently, targeting the B-cell antigen, CD20 with rituximab has been tested in RA (Dass et al, 2006; Popa et al, 2006).

Since 2003, experimental studies employing a variety of gene therapy strategies have shown that over-expression of transferred genes that inhibit cytokines, growth factors and MMP activity as well as immune-mediated events and inflammation may be useful in the future treatment of RA and OA. However, additional animal studies are warranted so that the long-term safety as well as temporal and spatial consequences of continuous expression of transferred gene-constructs can be monitored. Successful exploitation of IL-1Ra and IGF-1 gene transfers in human tissue in vitro (i.e. the ex vivo ‘approach’) has resulted in the generation of IL-1Ra and IGF-1 gene transfer strategies for eventual use in phase I OA clinical trials (Tomita et al, 2003; Evans, 2005).

The success or failure of specific gene therapy strategies for RA and even OA is likely to depend on the type of vectors employed (Bessis and Boissier, 2006) as well as host immune responses to the transgene including stimulation of innate immunity (Bessis et al, 2004). It also remains to be determined if, for example, rheumatoid factors with the capacity to form immune complexes with the expressed secreted protein directed by the transgene will limit their effectiveness in ameliorating immune cell dysfunction and inflammation in RA and cartilage destruction in OA (Evans et al, 2005).

Gene therapy for arthritis may be the ‘wave of the future’ and clinical trials are warranted to test the efficacy of gene transfer paradigms determined from animal studies. These would include the effectiveness of adenoassociated viral vectors compared to adenoviral vectors and local administration compared to systemic injection.

However, it is still too early to predict the extent to which gene therapy will become an adjunctive arthritis treatment or even replace the current standard of medical care employed in the treatment of clinically active RA or OA (Gaffo et al, 2006).

VII. TIMP gene therapy TIMPs are the endogenous MMP inhibitors that maintain normal synovial joint homeostasis and are critical in modulating MMP activity during skeletal development (Malemud, 2006). In early RA, serum MMP to TIMP ratios are generally skewed upward, especially MMP3/TIMP-2 and MMP-9/TIMP-2, compared to an OA control group (Fiedorczyk et al, 2006). This result is likely to be pertinent to the aggressive MMP-mediated degradation of RA cartilage and bone ECM proteins. Of note, methotrexate treatment in RA patients did not alter the MMP-3, -9/TIMP-2 ratios (Fiedorczyk et al, 2006). In OA, synovial fluid MMP levels are also increased (Malemud, 1999) but the mRNA of some TIMP family members (i.e. TIMP-3) are also increased whereas TIMP-1 and TIMP-4 mRNA are decreased (Kevorkian et al, 2004).

To address the possibility that restoration of TIMP levels could ablate MMP effects in inflammatory arthritis, van der Laan and colleagues in 2003 used adenoviral-TIMP-1 and TIMP-3 gene transfer to show that TIMP-1/TIMP-3 over-expression resulted in significant suppression of proliferation and invasiveness in RA-FLS in vitro and also after engrafting on to SCID mice in vivo. Although these studies using SCID were limited with respect to their longterm application to human RA, they may provide the impetus for additional studies in which a TIMP gene transfer to pannus might support a mechanism for suppressing MMP activity and cartilage and bone destruction of RA at that site.

VIII. IGF-1 Chondrocyte unresponsiveness to IGF-1 is considered significant in OA because IGF-1 is crucial for cartilage and bone repair pathways (Denko and Malemud, 2005). To address the possibility that IGF-1 gene therapy could subserve IL-1Ra in OA, Nixon and colleagues in 2005 co-transduced synovial membrane with E1-deleted adenoviral vectors, one containing IGF-1 sequences under cytomegalovirus (CMV) promoter control and the other with IL-1Ra under CMV promoter control. Transduced IGF-1/IL-Ra synovium showed increased IGF-1 and ILRa mRNA. Further, the IGF-1 protein concentration

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Malemud: Gene therapy for arthritis: defining novel gene targets

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Gene Therapy and Molecular Biology Vol 11, page 37 Gene Ther Mol Biol Vol 11, 37-42, 2007

The PPAR-! Pro12Ala allele polymorphism of the Peroxisome Proliferator-Activated Receptor (!)

Gene (PPARG2) is a risk factor with a self-identified obese Dutch population Research Article

Kenneth Blum1,3,4,5,#,*, Thomas JH Chen2, Brian Meshkin3,#, Seth H. Blum4, Julie F. Mengucci4, Alison Notaro5, Vanessa Arcuri5, Roger L. Waite6, Eric R. Braverman5 1

Wake Forest University School of Medicine, NC. USA Chang Jung Christian University, Tainan, Taiwan, Republic of China 3 Salugen, Inc. San Diego, CA, USA 4 Synapatamine, Inc. San Antonio, TX, USA 5 Path Medical Research Foundation, NY, USA 6 GenWellness Inc. San Diego, California 2

__________________________________________________________________________________ *Correspondence: Kenneth Blum, Ph.D. Department Physiology & Pharmacology, Wake Forest University School of Medicine, Medical Center Boulevard, Winstonâ&#x20AC;&#x201C;Salem, North Carolina, 27157-1083, USA; Tel. 210-823-1504; Fax. 210-462-1516; email: drd2gene@aol.com Key words: Obesity, Genotrim, PPAR-! gene, fat regulation Abbreviations: body mass index (BMI); coronary heart disease (CHD); insulin resistance syndrome (IRS); myocardial infarction (MI); peroxisome proliferators-activated receptor (PPAR)

#Conflict of interest disclosure: Both Kenneth Blum and Brian Meshkin are officers and have stock ownership in Salugen, Inc. Received: 12 December 2006; Revised: 22 January 2007 Accepted: 4 April 2007; electronically published: April 2007

Summary Obesity has been identified as a global epidemic and presents a significant increased risk factor for a variety of comorbidities. Positive energy balance and resultant weight gain is largely attributed to a chronic mismatch between energy intake and energy expenditure. The PPAR gene (PPAR-! Polymorphism - Pro12Ala Allele) influences many biological factors including serving as the master regulator of fat-cell formation and influencing insulin resistance.

Based on the literature and proposed associations of the PPAR gene and the Pro12Ala allele polymorphism as a fat regulator gene, we decided to determine its allelic presence in a self-identified (via a cross sectional survey questionnaire) obese Dutch population. In this preliminary observational study, a total of 1,058 subjects were genotyped for the PPARG2 polymorphism. The PPAR gene Ala allele polymorphic frequency was 25.05% of the study subjects (n=1,058) versus 14% of the literature controls (n=2,245). This difference was significant (Z=17.398, p= 0.001). Our results suggest that in a highly motivated (wanting to loose weight) group of self-identified obese subjects (n= 1,058) from the Netherlands, the Pro 12A1a polymorphism of the PPARG2 gene significantly associates with these individuals compared to non-obese controls (n=2,245) and may be considered a risk factor.

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Blum et al: The PPAR-! Pro12Ala allele polymorphism of the (PPARG2) as a risk factor had significantly higher mean BMI compared with those in the lowest quintile (27.3 versus 25.4 kg/m2, respectively; P-trend<0.0001) whereas among PPAR gene variant allele-carriers, there was no significant trend observed between dietary fat intake and BMI (Ptrend=0.99; P-interaction=0.003). In contrast, intake of monounsaturated fat was not associated with BMI among women without the gene, but was inversely associated with BMI among PPAR gene variant allele-carriers (mean in lowest quintile=27.6 versus mean in highest quintile=25.5 kg/m2; P-trend=0.006; P-interaction=0.003).

The relationship between dietary fat intake and plasma lipid concentrations also differed according to the PPAR ! genotype (Pihlajamaki et al, 2004). These data suggest that PPAR-! genotype is an important factor in physiological responses to dietary fat in humans (Memisoglu et al, 2003).

In a case-control study of the Pro12Ala PPAR-! 2 polymorphism, obese subjects with Ala12 were more insulin sensitive than those without. The frequency of Ala12 was significantly lower in the diabetic group, suggesting that this polymorphism protects against Type 2 diabetes. These results revealed that PPAR-! is a thrifty gene mediating Type 2 diabetes. In one study, the authors confirmed a contribution of the PPAR-! 2 Pro12 allele in the genetic risk for type-2 diabetes, especially in obese subjects, where this allele worsens insulin resistance and increases fasting insulin levels (Kadowaki et al, 2002).

Concerning body weight, subjects with a mutation in PPAR- ! (Pro12Ala) had significantly higher body weight than those with a normal genotype (Pro12Pro) (Moon et al, 2005). In another study relating the PPAR gene and weight, the study authors found significantly increased risk for heart disease (nonfatal myocardial infarction (MI) or fatal coronary heart disease (CHD) associated with the A12 allele among individuals with a body mass index > or =25 kg/m2 (women: RR, 1.88; 95% CI, 1.01 to 3.50; men: RR, 1.55; 95% CI, 0.92 to 2.60; pooled: RR, 1.68; 95% CI, 1.13 to 2.50) but not among those <25 kg/m2 (pooled RR, 0.86; 95% CI, 0.37 to 1.97; P heterogeneity overweight versus non overweight 0.16) (Pischon et al, 2005). This has been supported by others (Ghoussaini, 2005; Rhee et al, 2006).

For children and obesity, a study found that the Pro12Ala variant is significantly associated with greater insulin sensitivity in childhood. Because obesity is one of the most important risk factors for cardiovascular diseases and type 2 diabetes, obese children, who are presumably at a higher risk, may be protected from these diseases by the phenotypic effect of the Ala 12 allele on insulin resistance (Buzzetti et al, 2006).

Based on the literature and proposed associations of the PPAR gene and the Pro12Ala allele polymorphism as a fat regulator gene, we decided to determine its allelic presence in a self-identified obese Dutch population.

I. Introduction Obesity has been identified as a global epidemic and presents significant increased risk factors for a variety of co-morbidities. Positive energy balance and resultant weight gain is largely attributed to a chronic mismatch between energy intake and energy expenditure (Cecil et al, 2006).

The Pro12Ala polymorphism peroxisome proliferators-activated receptor (PPAR)-!, caused by a missense mutation in exon B of the adipocyte â&#x20AC;&#x201C;specific ! 2 isoform, was identified in 1997 and is believed to confer transcriptional activity (Yen et al, 1997). Several more genetic variants in PPAR ! are known but are much less frequent (Valve et al, 1999). Since PPAR ! 2 is exclusively expressed in adipose tissue, the prevalent PRO12Ala polymorphism was originally studied for an association with obesity. In recent years evidence pointed to an association of the Ala variant with increased insulin sensitivity in nondiabetic Caucasians.

Most recently, Tonjes et al performed a meta analysis of 57 studies on nondiabetic individuals with pre-diabetic phenotypes and concluded that across all studies, the PRO12Ala polymorphism had no significant effect on diabetic-related traits. Only in selected subgroups, such as Caucasians and obese subjects, did Tonjesâ&#x20AC;&#x2122;s group see an association of the Ala allele with greater body mass index (BMI) and greater insulin sensitivity. Meta analysis of Ala homozygotes more clearly demonstrated the association with greater insulin sensitivity of carriers of the Ala allele (Tonjes et al, 2006).

A. Recent research supporting an antiobesity role for the PPAR gene The PPAR gene (PPAR-! Polymorphism-Pro12Ala Allele) influences many biological factors including serving as the master regulator of fat-cell formation and influence over insulin resistance (Jo-SH et al, 2006).

PPARs exert a measure of transcriptional control regulating glucose transport and insulin sensitivity, lipid metabolism, oxidative stress, and inflammation (Lehrke and Lazar, 2005).

In a study of 311 subjects who participated in a population-based study, weight at birth, 7 years, 20 years, and 41 years, as well as ponderal index at birth, BMI and waist circumference at 41 years were recorded. The PPAR gene was associated with high ponderal index at birth (baby birth weight (p=0.007) and weight at 7 years (p=0.045). The PPAR gene tended to be associated with high birth weight (p = 0.094). Subjects with this gene gained less weight between 7 and 20 years (p = 0.043), more weight between 20 and 41 years (p = 0.001) and ended up having higher waist circumference (p = 0.040) in adulthood than did subjects with the normal genotype. The study authors concluded that the PPAR gene regulates weight and body composition from utero to adulthood (Verreth et al, 2004).

In a published study of 2,141 subjects, the study authors found that PPAR gene impacted responses to dietary fat intake. Among individuals without the mutation, those in the highest quintile of total fat intake

B. The PPAR Gene polymorphisms in screened controls In an attempt to evaluate the potential association of the Pro12Ala polymorphsm in peroxisome prolifeator-

38

Gene Therapy and Molecular Biology Vol 11, page 39 activated receptor ! with our self-identified Dutch population, we decided to utilize literature controls as a comparative baseline frequency of the Ala allele. In our search, we could not find a study that screened for nonobese and related co-morbidities such as diabetes in a Dutch population. Thus we turned to a study involving a large population of Danish people.

The Pro12Ala polymorphism of PPAR-! 2 has been shown to influence insulin sensitivity and the risk of type 2 diabetes in various ethnic populations. Frederiksen and colleagues examined in 2003 whether the polymorphism was related to the insulin resistance syndrome (IRS) among nondiabetic Danish subjects. The Pro12Ala variant was examined using PCR-restriction fragment length polymorphism in a phenotypically well characterized population-based sample of 2.245 nondiabetic subjects.

The study participants were characterized by a number of anthropometric and biochemical measurements and the European Group for the Study of Insulin Resistance criteria enabling a classification of the study population in an IRS group and a non-IRS group. The allelic frequency of the Pro12Ala polymorphism in the total study sample was 14% (95% confidence interval, 13-15%). Two hundred ninety-four subjects fulfilled the European Group for the Study of Insulin Resistance criteria defining the IRS. The frequency of the Ala allele was 12.6% in the IRS group and 14.2% among subjects classified as not having the IRS (P = 0.15). However, the frequency of the variant in the homozygous form was significantly lower in the IRS group [0.7% (0-1.6%)] compared with the frequency in the non-IRS group [2.8% (2.1-3.5%); P = 0.02; odds ratio, 0.24 (0.06-0.99)]. Moreover, in the total study population, homozygous carriers of the variant had lower levels of fasting serum triglyceride [1.1 +/- 0.4 mmol/liter (means +/- SD) vs. 1.4 +/- 0.9 mmol/liter; P = 0.04] and a lower diastolic blood pressure (79 +/- 8 mm Hg vs. 82 +/11 mm Hg; P = 0.01) compared with wild-type carriers.

The same tendency was observed with regard to the homeostasis model assessment estimate of insulin resistance (P = 0.16).

We are cognizant that utilization of this control sample may impact future results and thus, these following results should be interpreted with caution and must await further controlled studies on non-obese and non-IRS Dutch subjects. In this regard, it is noteworthy that the X/Ala frequency varies amongst ethnic groups (Asian lean individuals show only a 5% frequency (Kahara et al, 2003)

and studies on lean Caucasians moderately varied whereby one study (n=280) showed an Ala frequency of 15% (Vaccaro et al, 2000).

II. Materials and Methods A. Subjects In this observational study, a total of 1,058 subjects were genotyped in the nutrigenomics laboratory of Salugen, Inc. (San Diego, CA). Each Caucasian subject self-identified themselves as obese or overweight by selecting GenoTrimâ&#x201E;˘, a DNAcustomized nutraceutical as a potential adjunct to their weight loss efforts. The participants filled out a cross sectional questionnaire related to obesity. The subjects are part of the Dutch Investigation to Evaluate Treatments of DNA-customized nutritional solutions for weight management (D.I.E.T.) Study.

B. Genotyping Each subject was genotyped for the allelic presence of the Peroxisome Proliferator-Activated Receptor-! Gene Pro12Ala polymorphism (PPAR-! Pro12Ala Allele).

DNA was isolated from buccal cells by standard techniques and the Pro-12Ala polymorphism was detected by the PCR utilizing Taq-Man, standard PCR techniques, DNA sequencing and fragmentation that entered the study. The PCR conditions and primers used were those indicated by Hara and colleagues in 2000. The recommendations of Xu and colleagues in 2002 were followed for quality of genotype identification.

C. Statistics The prevalence of these genotypes was measured against literature controls from independent, published clinical studies involving the same genetic mutation in a similar ethnic population. Statistical significance was performed by a biostatistician at Brooklyn College (NY) and determined using the Z-test for two independent proportions (Kanji, 1997).

III. Results In this study, we compared the alleleic frequencies of the self-identified Dutch obese subjects with a well screened non-obese and non insulin resistant Danish control derived from the literature (Frederiksen et al, 2003).

The PPAR gene Ala allele polymorphic frequency was present in 25.05% of the study subjects (n=1.058) versus 14% of the literature controls (n=2,245). This difference was statistically significant (Z=17.398, p= 0.001) (Figure 1).

Figure 1. PPAR-! gene frequency.

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Blum et al: The PPAR-! Pro12Ala allele polymorphism of the (PPARG2) as a risk factor Buzzetti R, Petrone A, Caiazzo AM, Alemanno I, Zavarella S, Capizzi M, Mein CA, Osborn JA, Vania A, di Mario U (2006) PPAR-!2 Pro12Ala variant is associated with greater insulin sensitivity in childhood obesity. Pediatr Res 57, 138140.

Cecil JF, Watt P, Palmer CN, Hetherigngton M (2006) Energy balance and food intake: The role of PPAR-! gene polymorphisms. Physiol Behav 88, 227-233.

Frederiksen L, Brodbaek K, Fenger M, Madsbad S, Urhammer SA, Jorgensen T, Borch-Johnsen K (2003) No interactions between polymorphisms in the #3-adrenergic receptor gene and the PPAR-! gene on the risk of the insulin resistance syndrome in the Danish MONICA cohort. Diabetologia 46,729-31.

Ghoussaini M, Meyre D, Lobbens S, Charpentier G, Clement K, Charles MA, Tauber M, Weill J, Froguel P (2005) Implication of the Pro12Ala polymorphism of the PPAR-! 2 gene in type 2 diabetes and obesity in the French population.

BMC Med Genet 6, 11.

Hansen L, Elkstrom CT, Tabanera y Palacios R, Anant M, Wasasermann K, Reinhardt RR (2006) The Pro12Ala variant of the PPARG gene is a risk factor for Perxisome Proliferator-Activated Receptor–!/a agonist –induced edema in type 2 diabetic patients. J Clin Ednocrinol Metab 91, 3456-3450.

Hara K, Okada T, Tobe K, Yasuda K, Mori Y, Kadowaki H, Hagura R, Akanuma Y, Kimura S, Ito C, Kadowaki T (2000) The Pro12Ala polymorphism in PPAR-! 2 may confer resistance to type 2 diabetes. Biochem Biophys Res Commun 271, 212-216.

Huang C, Zhang Y, Gong Z, Sheng X, Li Z, Zhang W, Qin Y (2006) Berberine inhibits 3T3-LI adpocyte differentiation through the PPAR-! pathway. Biochem Biophys Res Commun 348, 571-578.

Janssens AC, Gwinn M, Valdez R, Narayan KM, Khoury MJ (2006) Predictive genetic testing for type 2 diabetes. BMJ 333, 509-510.

Jo-SH, Yang C, Miao Q, Marzec M, Wasik, MA, Lu P, Wang YL (2006) Peroxisome Proliferator-Activated receptor-!

Promotes Lymphocyte survival through its actions on cellular metabolic activities. J Immunol 177, 3737-3745.

Kadowaki T, Hara K, Kubota N, Tobe K, Terauchi Y, Yamauchi T, Eto K, Kadowaki H, Noda M, Hagura R, Akanuma Y (2002) The role of PPAR! in high-fat diet-induced obesity and insulin resistance. J Diabetes Complications 16, 41-45.

Kahara T, Takamura T, Hayakawa T, Nagai Y, Yamaguchi H, Katsuki T, Katsuki K, Katsuki M, Kobayashi K (2003) PPAR-! gene polymorphism is associated with exercisemediated changes of insulin resistance in healthy men.

Metabolism 52, 209-212.

Kanji GK (1997) 100 statistical tests. Thousand Oaks, California: Sage Publications, Inc. Kepez A, Oto A, Dagdelen S (2006) Peroxisome proliferatorsactivated receptor–!: novel therapeutic target linking adiposity, insulin resistance, and atherosclerosis. Biodrugs 20, 1211-135.

Lehrke M, Lazar MA (2005) The many faces of PPAR-!. Cell 123, 993-999.

Memisoglu A, Hu FB, Hankinson SE, Manson JE, De Vivo I, Willett WC, Hunter DJ (2004) Interaction between a peroxisome proliferator-activated receptor-! gene polymorphism and dietary fat intake in relation to body mass.

Hum Mol Genet 12, 2923-2929.

Meng H, Wang GH, Wang QG, Zhao JG, Gu ZL, Wang YX, Li H (2002) Studies of single nucleotide polymorphism of PPAR gene and its associations with fattiness trait in chicken. Acta Genetica Sinica 29, 119-123.

IV. Discussion Our results suggest that in a highly motivated (wanting to loose weight) group of self-identified obese subjects (n=1.058) from the Netherlands, the Pro 12A1a polymorphism of the PPARG2 gene significantly associates with these individuals compared to non-obese controls (n=2.245) and may be considered as a risk factor.

While this is the first study to genotype highly motivated obese individuals of Dutch descent, many other studies have shown associations with the PPARG2 gene in related conditions. These include type 2 diabetes mellitus (Auboeuf et al, 1997; Rendell, 2004; Wang et al, 2004; Allen et al, 2006; Bergeron et al, 2006; Cecile et al, 2006; Hansen et al, 2006; Soriguer et al, 2006); down regulation of glucose (Roduitt et al, 2000; Xu et al, 2006); childhood obesity (Scaglioni et al, 2006); adiposity (Meng et al, 2002; Kepez et al, 2006); lymphocyte survival (Jo-SH et al, 2006); reduction of obesity-related inflammation in adipose tissue (Tsuchida et al, 2005) and adipocyte differentiation (Huang et al, 2006).

While there have been a number of earlier studies that reveal a significant association of the Ala allele in obesity and related problems, this is the first study to genotype a rather large self-identified and highly motivated Dutch population.

Information derived from this and other studies will further our knowledge related to obesity and the potential development of DNA directed personalized therapeutics to treat obesity as a disease involving neurologic, metabolic, and genetic factors (Blum et al, 2006).

Acknowledgments The authors want to thank the entire staff of Salugen, Inc. and Path Medical and Research Foundation. We especially appreciate all the efforts of Florina Crews of Salugen, Inc. The authors want to thank Rein Norma for financial contribution to this project.

References Allen T, Zhang F, Moodie SA, Clemens LE, Smith A, Gregoire F, Bell A, Muscat GE, Gustafson TA (2006) Halofenate is a selective peroxisome proliferators-activated receptor-! modulator with antidiabetic activity. Diabetes 55, 25232533.

Auboeuf D, Rieusset J, Fajas L, Vallier P, Frering V, Riou JP, Staels B, Auwerx J, Laville M, Vidal H (1997) Tissue distribution and quantification of the expression of mRNAs of peroxisome proliferator-activated receptors and liver X receptor–" in humans: No alteration in adipose tissue of obese and NIDDM patients. Diabetes 46, 1319-1327.

Bergeron R, Yao J, Woods JW, Zycband EI, Liu C, Li Z, Adams A, Berger JP, Zhang BB, Moller DE, Doebber TW (2006) Peroxisome proliferator-activated receptor (PPAR)-" agonism prevents the onset of type 2 diabetes in Zucker diabetic fatty rats: A comparison with PPAR-! agonism.

Endocrinology 147, 4252-4262.

Blum K, Meshkin B, Downs BW (2006) DNA based customized “gene therapy” utilizing a genoscore: A hypothesized paradigm shift of a novel approach to the diagnosis, stratification, prognosis and treatment of inflammatory processes in the human. Med Hypotheses 66, 1008-1018.

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Gene Therapy and Molecular Biology Vol 11, page 41 Moon MK, Cho YM, Jung HS, Park YJ, Yoon KH, Sung YA, Park BL, Lee HK, Park KS, Shin HD. (2005) Genetic polymorphisms in peroxisome proliferator-activated receptor-! are associated with Type 2 diabetes mellitus and obesity in the Korean population. Diabet Med 22, 11611166.

Pihlajamaki J, Vanhala M, Vanhala P, Laakso M (2004) The Pro12Ala polymorphism of the PPAR-! 2 gene regulates weight from birth to adulthood. Obes Res 12, 187-90.

Pischon T, Pai JK, Manson JE, Hu FB, Rexrode KM, Hunter D, Rimm EB (2005) Peroxisome proliferator-activated receptor!2 P12A polymorphism and risk of coronary heart disease in US men and women. Arterioscler Thromb Vasc Biol 25, 1654-1658.

Rendell M (2004) Advances in Diabetes for the millennium: Nutritional Type2 Diabetes. MedGENMed 6(Suppl3), 1017.

Rhee EJ, Oh KW, Lee WY, Kim SY, Oh ES, Baek KH, Kang MI, Kim SW (2006) Effects of two common polymorphisms of peroxisome proliferator-activated receptor-! gene on metabolic syndrome. Arch Med Res, 37, 86-94.

Roduit R, Morin J, Masse F, Segall L, Roche E, Newgard CB, Assimacopoulos-Jeannet F, Prentki M (2000) Glucose down regulates the expression of the peroxisome proliferator – activated receptor–! gene in the pancreatic #–cell. J Biol Chem, 275, 35799-35806.

Scaglioni S, Verduci E, Salvioni M, Biondi ML, Radaelli G, Agostoni C, Giovannini M (2006) PPAR–! 2 Pro12Ala variant, insulin resistance and plasma long chain polyunsaturated fatty acids in childhood obesity. Pediatr Res 60, 485-490.

Soriguer F, Morcillo S, Cardona F, Rojo-Martinez G, de la Cruz Almaraz M, Ruiz de Adana Mde L, Olveira G, Tinahones F, Esteva I (2006) Pro12Ala polymorphismof the PPARG2 gene is associated with type 2 diabetes mellitus and peripheral insulin sensitivity in a population with a high intake of oleic acid. J Nutr 136, 2325-2330.

Tsuchida A, Yamauchi T, Takekawa S, Hada Y, Ito Y, Maki T, Kadowaki T (2005) Peroxisome proliferator-activated receptor (PPAR)-" activation increases adiponectin receptors and reduces obesity-related inflammation in adipose tissue: comparison of activation of PPAR-", PPAR-!, and their combination. Diabetes 54, 3358-3370.

Vaccaro O, Mancini FP, Ruffa G, Sabatino L, Colantuoni V, Riccardi G (2000) Pro12Ala mutation in the peroxisome proloferator-activated receptor !2 (PPAR-!2) and severe obesity: a case control study. Int j Obes Relat Metab Disord 24, 1195-1199.

Valve R., Sivemius K, Miettingen R, Pihlajamaki J, Rissanen A, Deeb SS, Aurwerx J, Uusinupa M, Laakso M (1999) Two polymorphisms in the peroxsomic proliferators-activated receptor –! gene are associated with severe overweight among obese women. J Clin Endocrinol Metab 84, 37083712.

Verreth W, De Keyzer D, Pelat M, Verhamme P, Ganame J, Bielicki JK, Mertens A, Quarck R, Benhabiles N, Marguerie G, Mackness B, Mackness M, Ninio E, Herregods MC, Balligand JL, Holvoet P (2004) Weight-loss-associated induction of peroxisome proliferator-activated receptor-" and peroxisome proliferator-activated receptor-! correlate with reduced atherosclerosis and improved cardiovascular function in obese insulin-resistant mice. Circulation 110, 3259-69.

Wang C, Fengying Z, Chi Y, Wang G (2004) Association of Pro12Ala mutation in peroxisome proliferators-activated receptor-! 2 with obesity and diabetes in Chinese population.

Wei Sheng Yen Chiu 33, 317-320.

Xu J, Turner A, Little J, Bleecker ER, Meyers DA (2002) Positive results in association studies are associated with departure from Hardy-weinberg equilibrium hint for genotyping error? Hum Genet 111, 573-574.

Xu Y, Etgen GJ, Broderick CL, Canada E, Gonzalez I, Lamar J, Montrose-Rafizadeh C, Oldham BA, Osborne JJ, Xie C, Shi Q, Winneroski LL, York J, Yumibe N, Zink R, Mantlo N (2006) Design and synthesis of dual peroxisome proliferators –activated receptors–! and $ agonists as novel euglycemic agents with a reduced weight gain profile. J Med Chem 49, 5649-5652.

Yen CJ, Beamer BA, Negri C, Silver K, Brown KA, Yarmall DP, Burns DK, Roth J, Shuldner AR (1997) Molecular scanning of the human peroxisome proliferators activated receptor-! (hPPAR!) gene in diabetic Caucasians: identification of a Pro12Ala PPAR ! 2 missense mutation. Biochem Biophys Res Commun 241, 270-274.

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Gene Therapy and Molecular Biology Vol 11, page 43 Gene Ther Mol Biol Vol 11, 43-50, 2007

Preliminary study on the recombinant endostatin engineering Lactococcus lactis Research Article

Chongbi Li1,*, Wei Li2, Chunxiang Wang3, Kefei Sun3 1

Biopharmaceutical Research & Development Center of Zhaoqing University, Zhaoqing city 526061, China Department of Obstetrics and Gynecology, First People’s Hospital of Hangzhou, Hangzhou 310006, China 3 GSETS Biopharmaceutical Technology CO.LTD 2

__________________________________________________________________________________ *Correspondence: Chongbi Li, Donggang, Zhaoqing City, Guangdong Province, Biopharmaceutical Research & Development Center of Zhaoqing University, 526061, China; Tel : 0086-0758-2716359; Fax: 0086-0758-2776882; Email: lchongbi@yahoo.com Key words: Endostatin; cloning and expression, Lactococcus lactis, experimental rats, effects, Tumor Abbreviations: 1, 2-dimethylhydrazine, (DMH); Lactococcus lactis, (L lactis), Luria-Bertani, (LB); tumor volume, (TV)

This study was supported by Natural Science Foundation of Guangdong Province 06029354.

Received: 9 January 2007; Revised: 27 February 2007 Accepted: 2 April 2007; electronically published: April 2007

Summary Endostatin is a specific inhibitor of endothelial proliferation and agiogenesis from the COOH-terminal portion of human collagen XVIII. In order to examine the effect on Lactococcus lactis (L. lactis) and endostatin for curing cancer, rat endostatin gene was isolated by RT-PCR from rat kidney and cloned into the plasmid of L lactis and expressed in Lactococcus lactis NZ9000. And the effects were observed by orally L .lactis and recombinant L lactis expressing endostatin for colorectal cancer-induced rats with 1, 2-dimethylhydrazine (DMH) through both the survival and histopathological examination of the rats. The results showed that recombinant endostatin L lactis had a significant effect on the Duke’s stage of the experimental rats (P<0.05). Furthermore, the mean survival of the rats taken orally with recombinant L lactis was longer than that of the rats treated with DMH alone. The study would lay a theoretical foundation for an application of L lactis and endostatin to the anti-tumor.

research to clinical practice. There is therefore a great need to increase the yield and to reduce the cost of the production of recombinant endostatin that is suitable for clinical use. Our strategy centered on the optimization of a probiotic strain, lactococcus lactis expression system because of its probiotic efficiency in expressing foreign proteins as compared with the other systems. At present, few reports have been seen by oral giving medicine using L lactis as a vector. Additionally, lactic acid bacteria (LAB) expression system selected was because of its ability to express heterologous protein in vivo and unnecessary to isolate the protein.

I. Introduction Numerous studies have shown that both primary tumor and metastatic growth are angiogenesis dependant (Folkman et al, 1990,1992; Kim et al, 1993; Millauer et al, 1994). Therefore, the tumor vascular system has become an important target for cancer therapy. An increasing number of antiangiogenic factors have been discovered.

One of the factors responsible for this inhibition was named endostatin, and it was proved that NH2–terminal sequence of endostatin corresponds to the COOH-terminal portion of collagen XVIII (O’Reilly, 1997). It is reported that a recombinant form of this protein expressed in baculovirus-infected insect cells could inhibit the growth of metastases in the Lewis lung tumor model and an insoluble E. coli derived form of this protein was also shown to be efficacious in preventing primary tumor growth in several tumor models (Boehm et al, 1997; O'Reilly et al, 1997). Additionally, it is known that In curing cancer, the application on endostain was very expensive and needed injecting into blood. These barriers have hampered the widespread translation of endostatin

II. Materials and methods A. Bacterial strains and growth conditions E. coli strains Top10 or TG1 were incubated at 37!under aeration, and rendered competent to take up DNA using a CaCl2 method. Lactococcus lactis strains NZ9000 as host bacteria were grown at 30°C in M17 broth (Merck, Darmstadt, Germany) supplemented with 0.5% glucose (GM17). Antibiotics were used at the following concentrations for E. coli: Ampicillin (Am), 100 ug/ml. For L. lactis, the concentrations were as follows:

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Li et al: Preliminary study on the recombinant endostatin engineering Lactococcus lactis erythromycin (Em), 5 µg/ml; chloramphenicol (Cm), 10 µg/ml.

Growth kinetics were determined in GM17(M17 medium in which 1%(wt/vol) glucose) broth as follows. Culture tubes containing 5 ml of prewarmed medium were inoculated with 2% of an overnight culture and incubated at 30°C without shaking in a water bath. Bacterial growth was monitored by spectrophotometric measurements of the optical density at 600 nm (model UV-1205; Japan) every 30 min until the culture reached the stationary phase. The recombinant L lactis production was achieved in either 10% (wt/wt) culture media. All media were heat pasteurized at 90°C for 45 min. Two liters of media was inoculated at 1% (vol/vol) from a fresh GM17 culture.

Then the ferments were enlarged for incubation at 30°C in 30liter BIOTECH-30JS(Shanghai). After finishing fermentation, the strains fermented were deposited through centrifuge and freeze-dried for the experiment.

The endostatin expressed in these cells was monitored by SDS-PAGE. Expressions of endostatin were as follows: Small-scale expression of endostatin in E. coli strain TOP10 harboring the plasmid of interest is grown at 37°C in LB medium with shaking in an air incubator.

When growth is monitored at OD600 until it reaches a value of 1.0 it was induced by a concentration of 0.2 mmol/L Isopropyl-beta-D-thiogalactopyranoside (IPTG) for additional three or four hours. Then 1 mL aliquots of the culture were removed for analysis of protein content by boiling the pelleted cells, treating them with reducing buffer and electrophoresis via SDS-PAGE. The culture was centrifuged (5000 x g) to pellet the cells. Otherwise, The strains carrying endostatin were grown overnight at 30°C in GM17-Cm, diluted 50-fold in the same medium added with 1 ng/ml of nisin (Sigma), and allowed to grow at 30°C to an optical density at 600 nm of 1.0, about 3-4 h of incubation (Steidler et al, 1995, 2000; Sambrook et al, 1989) and then the recombinant L lactis strains were harvested by centrifugation (3000g, 10 min, 4ºC, washed with PBS, resuspended in 1 ml of 10 mM Tris-HCl (pH 7.5), and disrupted with a French press (Bioritech). The cell suspension was centrifuged (10000g, 10 min, 4ºC) to remove cell debris. The samples were mixed in Laemmli buffer and subjected to12% SDS-polyacrylamide gel electrophoresis.

B. Cloning and expression of rat endostatin in L.lactis E.coli Top10 and TG1 cloning efficiency cells were all prepared by ourselves as competent cells and were ready for transformation using the standard protocol.

The total RNA was isolated from a rat kidney tissue using the RNA extracting kit (Promeg). And the sequence encoding the carboxy terminal portion of rat collagen XVIII was got by the method of RT-PCR. AMV reverse transcriptase, Tag DNA polymerase and other reagents were purchased from Boehringer Mannheim or Sigma.

The primers used were: TTT GAA TTC GCC CAC ACC CAC CGC GAC TTC CAG CCG and AAA AGC GCG CGC CTA CTT GGA GGC GGC AGT CAT GAA GCT bases. RT-PCR was carried out using standard conditions.

The amplified fragment was purified using the QIAquick PCR purification kit, and digested with EcoRI and NotI. The plasmids pLa165 and 148 were gifted by Dr Gruss in France. At first, the resulting fragment was ligated into a pre-digested Lactoccus lactis expression and secretion plasmid (pLA165). This plasmid carried a signal peptide based on secretion of the staphyiocuccal nuclease and also contained a nisin-residue promotor from lactic acid bacteria. Additionally, the fragment was also ligated into a pre-digested L.lactis expression plasmid (pla148) without secreting signal peptide of nuclease-residue. Plasmid DNA was purified from E.coli using the alkaline lysis method and was isolated from L.lactis as described previously (Ruyter et al, 1996). Restriction endonucleases, T4 DNA ligase, Tag polymerase and other chemicals used in the test were purchased from Boehringer Mannheim or Sigma and they were operated according to the recommendations of the manufacturer. Transformation of L.lactis NZ9000 was performed by electroporation and selection for recombinants were plated on GM17 agar plates containing the adequate antibiotic (van de Guchte et al, 1989; Wells et al, 1993).

Plasmid DNA can be isolated by a number of different methods and using commercially available kits (Promega Wizard Miniprep kit). The kits follow the manufacturer's suggested protocol for plasmid DNA isolation. DNA samples were sequenced by Shanghai Shenggong Company. The purified plasmid DNA was used for further restriction enzyme digestion with EcoRI and NotI and ligated, additional subcloning.

C. Purification and Western-blotting of Endostatin from L. lactis A purification procedure for recombinant endostatin from L. lactis has been described previously (O’Reilly et al, 1997).

Briefly, bacteria pellet was collected with low-speed centrifugation, followed by lysis with 8 M urea. The lysate was then applied to a heparin column (Qiagen). After washing with 8 M urea containing 10 mM imidazole, endostatin was eluted with 8 M urea containing 250 mM imidazole. Quantification of the endostatin protein before dialysis was performed using the BioRad protein dye method as described by the manufacturer.

Finally, the endostatin product was dialyzed against 1xPBS at 4°C. The polyclonal antiserum was prepared according to the routine protocol from the immunized rabbits with the simply purified endostatin expressed in E.coli. The gel after running SDS-PAGE was transferred onto a nitrocellulose membrane with a Bio-Rad electro-blotter. The blots were developed with BCIP/NBT (Sigma) developing buffer (Sambrook et al, 1989).

D. Fermentation for the Genetic Engineering of Lactococcus lactis Stock cultures of L. lactis strains NZ9000 and the recombinant L. lactis strains were prepared by mixing 10 ml of a fresh culture with 25 ml of 20% skim milk and 25 ml of a 20% glycerol solution and then freezing the mixture at -70°C in 1-ml sterile cryovials. Working cultures were prepared by inoculating 100 ml of LM17 broth with 1 ml of a thawed stock culture, and incubating this mixture at 30°C for 24 h. The growth of L. lactis strains in LM17 broth was evaluated by automated spectrophotometry with a Powerwave unit (Bio-Tek Instrument, Winooski, Vt.) as described previously (Champagne et al, 1999).

Starter production was achieved in either 6% (wt/wt) nonfat dry milk. All media were heat pasteurized at 90°C for 45 min. Two liters of media was inoculated at 1% (vol/vol) from a fresh LM17 culture. The ferments were enlarged for incubation at 30°C in 30liter BIOTECH-30JS (Shanghai). Agitation was kept at 60 rpm, and fermentations were stopped when the pH of the medium

44

Gene Therapy and Molecular Biology Vol 11, page 45 reached 4.7. The time required to complete the various fermentations was registered and will be referred to hereafter as the "fermentation time." Expression and induction of the fermentations were done as above mentioned. The fermentations were precipitated by centrifugation and freeze-dried for tests.

dimensions and distribution of the tumors) were recorded, the colons were fixed flat between pieces of filter paper soaked in 10% phosphate-buffered formalin. And the liver and kidneys were excised and weighed. Other major organs (stomach, small intestine, spleen, lungs and lymph nodes) were also excised and then fixed in 10% phosphate-buffered formalin solution.

Afterward, all tissues were embedded in paraffin and stained with routine hematoxylin and eosin. And then the histopathological analysis was carried out for the correlative colonic tissues.

E. Experimental animals and Experimental protocol 40 male Wistar rats at 5 weeks of age were purchased from the Institute of Animal, Chinese Academy of Medical Sciences, Beijing in China and housed in plastic cages with wood chips in an animal room with a 12 h light/dark cycle at 22±2ºC and 44±5% relative humidity. Rats were fed the basal diet, and water was available. Body weight and food consumption of the rats were measured once a week. DMH was purchased from Tokyo Kasei Co. (Tokyo, Japan). The experimental design is shown in Figure 1. Colorectal cancer inducing was performed as follows.

After the first week acclimatization, forty rats at 6-week-old were randomly divided into 4 groups, 10 rats each group. The rats of 4 groups were given subcutaneous injections of DMH dissolved in normal saline solution, and the dosage is 40mg/kg body weight (wt) once a week for 10 weeks. The fourth group rats were only injected with 0.9% normal saline (vehicle) at the same time.

After the last DMH attacking, the animals in group 1 were fed with 1x108 recombinant L.lactis secreting endostatin protein, the animals in group 2 with the same amount of L.lactis no endostatin gene but containing the plasmids once a day for 22 weeks, and the rats in group 4 were fed with the same amount of solvent without L.lactis (the vehicle control).

Group 3 was taken as a carcinogen control. The length of treatments differed slightly with each other group. The rats were sacrificed under ether anesthesia and checked at week 22nd.

G. Statistical analysis Statistical analysis was carried out using SPSS 9.0 (Statistical Package for the Social Science) software in a computer. The difference between the average values of the groups was analyzed using Cochran’s two-tailed Student’s t-test.

And the difference of lesion incidences between the groups was assessed by chi-square test, and the rat mortality was also counted by the Log Rank method (Peto et al, 1977).

III. Results A. Construction of the expression plasmid The gene endostatin was isolated as about a 0.8kb EcoRI and NotI fragment from rat kidney through RTPCR. And the fragment was introduced into plasmid Teasy vector, and the resulting plasmid, pT-endo, was transferred to E. coli TOP10. The sequence analysis revealed that endostatin was a complete open reading frame. To clone endostatin in L. lactis NZ9000, the fragment cut with EcoRI and NotI was introduced into the vector pLa165. The resulting plasmid, pLa165-endo, was transformed to L. lactis NZ9000 by electroporation.

Transformants were screened by PCR and restriction enzyme analysis. The resulting recombinant strain, containing plasmids pLa165-endo and pLa148-endo, were designated. The plasmid DNA was reisolated and subjected to a restriction analysis. The resulting restriction pattern was identical to the pattern obtained with the plasmid of L. lactis. The result showed that correct coding endostatin sequence has been constructed. Other constructive procedures were done as above mentioned.

As a result, a recombinant L. lactis clone containing endostatin from rat was obtained (Figures 2, 3).

F. Experimental observation The rats treated with DMH-induced colorectal cancer could characteristically develop multiple tumors, and each tumor would be at a different histological stage (Pozharisski, 1975). Therefore, the animals in this experiment were staged (Duke’s stage) with reference to a single index tumor, defined as the largest macroscopically and histologically identifiable colorectal tumor (Dukes et al, 1958).

When the experimental rats were fed till the termination all rats were autopsied. The colons were cut out, flushed with saline and opened along the longitudinal median axis. And then the tumor width (W) and length (L) were measured with calipers. The tumor volume (TV) was determined by the following formula: TV = (L " W 2 ) / 2 . After the gross pathologic changes (number,

!

Figure 1. Experimental design.

45

Li et al: Preliminary study on the recombinant endostatin engineering Lactococcus lactis

Figure 2. Rat endostatin gene by PCR. S: endostatin gene by PCR, Endo:endostatin gene, M: marker.

Figure 3. Expressive plasmid by restriction enzyme. p: expressive plasmid cut by restriction enzyme, e: endostatin gene, M: marker.

food consumption had no significant differences among the groups (Table 1).

Histo-pathological examinations were summarized in Table 2. Adenomas and carcinomas of the rats would be analyzed according to the colonic epithelial lesions. By the end of 22nd week, the examinations indicated that endostatin did not affect the incidence of colon tumors of the rats. However, owing to receiving endostatin, tumor volume of the rats decreased apparently but no significant differences statistically comparing with DMH-treated alone group (p>0.05). But it was found that there was a significant difference in Duke’s stage between the rats treated with DMH alone and with endostatin (p<0.05).

Additionally, liver lesions and lymph nodes metastases of about 30% rats were observed in third group (group 3).

At the termination, the observation and statistics indicated that all the rats treated with endostatin had about 30% survival rates (Figure 6). The macroscopically visible metastases changes were found in their lungs and livers of the rats through the investigation. And all of the rats injected with saline were alive well by the end of the experiment, but none of the rats with DMH-treated alone could survive cancer-induced.

B. Expression of rat endostatin gene in L.lactis The recombinant lactic acid bacteria were incubated and induced by nisin in M17-Glu for 6 hours. The endostatin protein was examined by SDS-PAGE and was identified by Western-blot with the polyclonal endostatin antibody from rabbits. The results showed that rat endostatin protein were expressed obviously in L. lactis as an included body form, and expression of that in superment was much few through examination (Figures 4, 5). The quantity of expression was estimated about 0.1mg/ml .

C. Experiment on animals All rats of groups lived out the termination and maintained a relatively healthy appearance throughout the experiment. No signs of severe toxicity were observed for all the rats after given endostatin, and no tumors were found on the rats treated with normal saline (vehicle). By the end of 22nd week, final average body weights of the rats treated with DMH alone as well as the animals additionally received either endostatin or L. lactis were decreased significantly (p<0.05) comparing with the vehicle control. Relative liver and kidney weights as well

Table 1. Final average body weight, relative liver and kidney weights determination (22wk)a Group- dividing No.

number

n

Final Body Wt, g

Relative Liver Wt, g

Relative Kidney Wt, bg

1 (DMH+ Endostatin)

10

379.0±24.9*

2.94±0.26

0.56±0.12

2 (DMH+ L.Lactis)

10

395.0±36.5 *

3.05±0.25

0.56±0.08

3 (DMH) 4 (Saline+ vehicle)

10 10

383.5±19.2 * 439.5±39.3

3.10±0.40 3.09±0.35

0.55±0.07 0.56±0.12

a: Values are means"SD; b: Kidney weight values are totals for both kidneys. *: P < 0.05 ( t-test) compared with Group 4.

Wt: weight; g: gram; wk: week.

46

Gene Therapy and Molecular Biology Vol 11, page 47 Table 2. Incidence of colon tumor, classification, multiplicity, tumor volume and stage in rats treated Disposal

DMH+ Endostatin DMH+ L.lactis DMH

Diversitya

Duke‘s stage b

Incidence

Adenoma

Carcinoma

n

n (%)

n (%)

n (%)

No.

mm3

A

B

10

5 (50)

5 (50)1

2.51±1.80

2.37±1.84

1.0

4

-*

10 10

9 (90) 10 (100)

2 (22) 5 (50)

7(78)1 5 (50)

2.66±1.47 4.00±2.96

2.53±2.00 4.30±4.56

4 2

3 -

Tumor volume

C

-* 3

a: Number of tumors/tumor-bearing rat. b: Number of rats with carcinoma for any of three tumor stages. *: P < 0.05 (Chi-Square) compared with DMH–treated group alone.

Figure 4. Rat endostatin protein by SDS-PAGE. S1: from E.coli s2 and s3: purified recombinant. endostatin from L.Lactis; M: marker. Endo: position of endostatin

Figure 5. Rat endostatin protein by western-blot. E: from E.coli S1 and s2 from L lactis. Endo: position of endostatin

Figure 6. Survival rates of the experimental rats in each groups.

Though the survival period of rats administered with DMH could be more long than 22 weeks, the mean survival rates of the group treated with endostatin were higher than one with DMH alone (Figure 6), however, the survival rates had no significant differences in statistics (p>0.05).

IV. Discussion With the aim of studying the functions of the L. lactis and endostatin, well as whether the presence or absence of the recombinant endostatin genes in L lactis would influence the survival or nutrition characteristics of L lactis, we proposed to experimentally construct a probiotic 47

Li et al: Preliminary study on the recombinant endostatin engineering Lactococcus lactis recombinant strain either exerting L lactis or endostatin effect. The design of rational approaches to metabolic engineering and/or natural selection with such an aim requires an in-depth understanding of the pathway, the genes involved, and their regulation. As a result, the industrially recombinant endostatin L. lactis had been obtained and the effects on the experimental animals had been observed.

Endostatin, a 20kDa protein factor responsible for this inhibition has been expressed correctly in E. coli and L lactis. The result was consistent with previous studies in its molecular weight (O’Reilly et al, 1997). Figures 4 and 5 illustrate the presence in which the endostatin gene could be expressed by using a nisin-inducible controlled expression system. In L. lactis, expression of endostatin could contribute to anti-tumor as for previously report (O’Reilly et al, 1997). Furthermore, this expression could not affect the survival of L lactis. Previous work had shown that L. lactis was a probiotic bacterium and could be successfully used in a milk-based medium (O’Reilly et al, 1997), indicating the potential usefulness in fermented dairy foods. It had also been suggested that yogurt bacteria might apply to cure some diseases (Kelkar et al, 1988).

The present study indicated that recombinant engineering strain from L. lactis NZ9000 as a model strain could have either adminstrative heteroprotein into the body or simple application by oral way in spite of its relatively small quatity of expression for endostatin in L lactis comparing with E. coli. It is important to note that the genetic modifications of the endostatin-producing strains (being either chemically induced or genetically engineered) did not appear to affect their acid production during growing period as an important attribute in fermentation of foods.

Furthermore, they have a considerable advantage over the latter since such chemically induced strains are much easier to proliferate from existing industrial strains and are much more likely to be accepted by the public. The present results are thus an important step in the development of recombinant endostatin engineering L lactis application. Though the quantity of endostatin produced from L. lactis entering the stomach and guts have not been known, the preliminary effect of anti-tumor on endostatin in vivo has been identified by the experiment which the rats treated with endostatin sequentially after DMH-treated could prolong the survival effectively. Additionally, animals with less advanced disease (stage A) survived significantly longer than those with more advanced (stage B and C), irrespective of treatment. The Duke’s staging system for human colorectal cancer could provide accurate prognostic information, moreover, in our study, there was not only a significant difference in the levels of differentiation and metastases (Duke’s stage) between the groups treated with DMH alone and with endostatin added but also the rats treated with endostatin showed an elongated survival comparing with that of untreated rats. Additionally, influences of survival for the rats could be also proved by the results of decreasing invasion degree and maintaining highly differentiated malignant tumors.

Therefore, the experiments would, at least in part, explain that the endostatin had a potential antitumor effect.

Furthermore, it is likely to be directly to attribute to induce tumor stabilization and its ability to inhibit specifically endothelial proliferation in endostatin-treated animals through observing the improved survival of the rats.

However, the paper only introduced that oral recombinant L. lactis carrying endostatin could only prolong the survival of tumor-bearing rats but did not introduce whether endogenous endostatin could be produced by nisin induction and no complete cure result. It could be deduced that lack of the significant difference in our study may be small numbers of animals. Instead, the finding more demonstrated that achieving regression of established tumors would be more difficult to be taken on than tumor formation to be inhibited.

One of the most important issues in endostatin therapy was the treatment period. At a typical dose level (20 mg/kg/12 h) previously showed to be active in a large number of studies (Dhanabal et al, 1999). The ultimate goal of antiangiogenic therapy would be to make longterm tumor stabilization (Fortier et al, 1999). The data from the non-primates study indicated that endostatin may be administered for long periods without producing toxicity (Dhanabal et al, 1999), and our experimental result is consistent with the report. Although endostatin prepared from a yeast system is being used in ongoing Phase I clinical trials, the low yield and high cost of the system have made it difficult to produce in quantities that are realistic for comprehensive clinical evaluation and application. Our results presented in this report offer an alternative method that will prove valuable in helping to determine the clinical activity of endostatin. Obviously, it will be of great interest and importance to adopt an effective method given drug in curing the patients with tumors.

Therefore, we propose that this agent might be evaluated in clinical trials as a consolidation drug for the patients who have achieved remission. But it should be not neglected that the production of recombinant endostatin and metabolic activities of intestinal flora of experimental animals were significantly different from those of humans.

The exact change in survival of the animals may be crucial for a sensitive determination of anticancer drugs. And we believe that the effect on endostatin in combination with L. lactis on DMH-induced colon tumor progression was strongly correlated with endostatin other than L. lactis.

The study introduced only the effect of oral L lactis containing endostatin on the experimental rats and not referred to the increase in colonic expression of endostatin.

Therefore, more work needs to be done to investigate the effect of endostatin in recombinant L. lactis on antitumour as well as the precise mechanisms. The results only indicated that the recombinant L lactis would be a potent administrative vehicle because of a probiotics or bacteria that “favor life, and also the way of administration in curing colorectal cancer would have a potential meaning the reason for this is that the survival rates of rats given the probiotics additionally containing endostatin gene rose than the control. And it was also proved that the rats receiving the L lactis containing endostatin had a good effect anti-cancer growth through the preliminary study.

Taken together, these results demonstrate the potential use 48

Gene Therapy and Molecular Biology Vol 11, page 49 administration for 28 consecutive days. Clin Cancer Res 5, 3813s.

Kelkar SM, Shenoy MA, Kaklij GS (1988) Antitumor activity of lactic acid bacteria on a solid fibrosarcoma, sarcoma-180 and Ehrlich ascites carcinoma. Cancer Lett 42, 73-77.

Kim KJ, Li B, Winer J, Armanini M, Gillett N, Phillips HS, Ferrara N (1993) Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumor growth in vivo. Nature 362, 841-844.

Millauer B, Shawver LK, Plate KH, Risau W, Ullrich A (1994) Glioblastoma growth inhibited in vivo by a dominantnegative Flk-1 mutant. Nature 367, 576-579.

Oâ&#x20AC;&#x2122;Reilly MS, Boehm T, Shing Y, Fukai N, Vasios G, Lane WS, Flynn E, Birkhead JR, Olsen BR, Folkman J (1997) An Endogenous Inhibitor of Angiogenesis and Tumor growth.

Cell 88, 277-285.

Peto R, Pike MC, Armitage P (1977) Design and analysis of randomized clinical trials requiring prolonged observation of each patient. Br J Cancer 35, 1-39.

Pgga DR, Kuipers OP, Beerthuyzen MM Boerrigter IA, Devos WM (1996) Functional analysis of promoters in the nisin gene cluster of Lactoccus lactis. J Bacteriol 178,3434-3439.

Pozharisski KM (1975) Morphology and morphogenesis of experimental epithelial tumours of the intestine. J Natl Cancer Inst 54, 1115-1135.

Sambrook J, Fritsch EF, Coulson AR (1989) Molecular Cloning: A Laboratory Manual, 2nd edu. New York: Cold Spring Harbor Laboratory.

Steidler L, Hans W, Schotte L, Neirynck S, Obermeier F, Falk W, Fiers W, Remaut E. (2000) Treatment of murine colitis

by Lactococcus lactis secreting interleukin-10. Science 289, 1352-1325.

Steidler L, Wells JM, Raeymaekers A, Vandekerckhove J, Fiers W, Remaut E (1995) Secretion of biologically active murine interleukin-2 by Lactococcus lactis subsp. lactis. Appl Environ Microbiol 61, 1627-1629 van de Guchte M, van der Vossen JM, Kok J, Venema G. (1989) Construction of a lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis. Appl Environ Microbiol 55, 224-228.

Wells JM, Wilson PW, Lepage RWF (1993) Improved cloning vectors and transformation procedure for Lactococcus lactis.

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of recombinant L lactis containing antiangiogenic endostatin peptide as a novel therapeutic agent in experimental animals with tumor. The results from this study also opened a new avenue for treatment of cancer and provide a hopeful route for promising to overcome drug resistance.

Acknowledgments We thank GSETS Biopharmaceutical Technology CO.LTD for providing the experimental help and thank Prof. Yanfeng Zhong of the Department of Pathology, Beijing University Medical School (China) for histopathological examination.

References Boehm T, Folkman J Browder T, Oâ&#x20AC;&#x2122;Reilly MS (1997) Antiangiogenic therapy of experimental cancer does not induce acquired drug resistance. Nature 390, 404-410.

Champagne, C. P., H. Gaudreau, N. Chartier, J. Conway, and E. Fonchy (1999). Evaluation of yeast extracts as growth media supplements for lactococci and lactobacilli using automated spectrophotometry. J. Gen. Appl. Microbiol. 45:17-21. de Ruyter, P. G., O. P. Kuipers, and W. M. de Vos (1996).

Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin. Appl. Environ.

Microbiol. 62, 3662-3667.

Dhanabal M, Ramchandran R, Volk R, Stillman IE, Lombardo M, Iruela-Arispe ML, Simons M, Sukhatme VP (1999) Endostatin: yeast production, mutants, and antitumor effect in renal cell carcinoma. Cancer Res 59, 189-197.

Dukes CE, Bussey HJ (1958) The spread of rectal cancer and its effect on prognosis. Br J Cancer 12, 309-320.

Folkman J (1990) What is the evidence that tumors are agiogenesis dependent [J]. Natl Cancer Inst 82, 4- 6.

Folkman J.Tumor angiogenesis[A].Holland JF,Frei E,Bast RC,(eds).Cancer Medicine(ed 3)[M].Philadelphia:PA,Lea & Febiger,1992.

Fortier AH, Fogler WE, Tomszewski JE, et al (1999) Recombinant human endostatin protein in cynomolgus monkey produces no toxicological effects following iv

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Gene Therapy and Molecular Biology Vol 11, page 51 Gene Ther Mol Biol Vol 11, 51-60, 2007

Epstein-Barr Virus associated gastric carcinoma Review Article

Hwa Eun Oh1,2, Runjan Chetty1,* 1

Department of Pathology, University Health Network/Toronto Medical Laboratories, University of Toronto, Toronto, Canada 2 Department of Pathology, Myongji Hospital, Kwandong University, College of Medicine, Goyang, Korea

__________________________________________________________________________________ *Correspondence: Dr Runjan Chetty, Department of Pathology, University Health Network, Toronto General Hospital, 200 Elizabeth Street, 11th Floor, Eaton Wing, Room 312, Toronto, ON M5G 2C4, Canada; Fax: 1-416-340-5517; E-mail: runjan.chetty@uhn.on.ca Key words: Epstein-Barr virus, gastric carcinoma, clinicopathologic features, molecular pathology, immunology, environmental factors, EBV-targeted therapy Abbreviations: Burkitt's lymphoma, (BL); CpG island methylator phenotype, (CIMP); cytotoxic T-lymphocyte, (CTL); EBV-associated gastric carcinoma, (EBVGC); EBV-encoded RNA, (EBER); EBV-encoded small RNA 1, (EBNA1); Epstein-Barr Virus, (EBV); gastric carcinomas, (GCs); Hodgkin's disease, (HD); human leukocyte antigen, (HLA); human leukocyte antigen, (HLA); immediate-early, (IE); Immunohistochemical, (IHC); insulin-like growth factor 1, (IGF1); nasopharyngeal carcinoma, (NPC); natural killer, (NK); origin of replication complexes, (ORCs); small interfering RNA, (RNAi);

Received: 22 March 2007; Revised: 11 April 2007 Accepted: 13 April 2007; electronically published: April 2007

Summary Epstein-Barr Virus (EBV) is a ubiquitous human herpesvirus associated with a variety of human malignancies including lymphoma and so-called lymphoepithelial carcinoma seen in a variety of sites, including the stomach.

EBV has been detected in 5-20% of gastric carcinomas worldwide. Evidence is presented which suggests that failure of EBV-specific immunity may play a role in the pathogenesis of EBV-associated malignancy. In this paper, we review the clinicopathologic features, molecular pathology, immunologic aspect, environmental factors in EBVassociated gastric carcinoma and lastly, EBV-targeted therapy.

purpose of this review is to provide an updated comprehensive summary of the clinicopathologic features, molecular pathology, immunologic aspects, environmental factors associated with EBVGC. Another important aspect of this review is to highlight EBV-targeted therapy.

I. Introduction Epstein-Barr Virus (EBV) is a human oncogenic virus, which was identified as herpesvirus-like particles by electron microscopy in a cell line established from a Burkitt's lymphoma biopsy by Epstein, Achong and Barr in 1964 (Epstein et al, 1964). EBV is implicated in the etiology of many human malignancies, including Burkitt's lymphoma (BL), Hodgkin's disease (HD), nasopharyngeal carcinoma (NPC) and EBV-associated gastric carcinoma (EBVGC) (Shibata and Weiss, 1992; International, 1997).

Several studies have revealed that EBV is associated with 5-20% of gastric carcinomas (GCs) worldwide (Shibata and Weiss, 1992; Rowlands et al, 1993; Fukayama et al, 1994). The clinicopathologic features of EBVGC are distinct and include a male preponderance, frequent accompaniment by atrophic gastritis, predominant involvement of the proximal stomach, moderately differentiated tubular or poorly differentiated solid type of histopathology (Kijima et al, 2003; Lee et al, 2004). Although its specific role in gastric carcinogenesis remains unclear, some studies have shown molecular changes that are characteristic of EBVGCs (see later). The

II. Clinicopathologic features EBVGC has been described in different populations from low incidence gastric cancer areas, such as Western Europe and the United States, to high-risk countries, such as Korea and Japan (International, 1997). Now, it is well known that 5-20% of gastric carcinomas throughout the world reveals monoclonal proliferations of EBV-infected carcinoma cells (Shibata and Weiss, 1992; Rowlands et al, 1993; Fukayama et al, 1994; Osato and Imai, 1996).

The EBVGCs have a lymphoid stroma (Kim et al, 2001; Lee et al, 2004) and these tumor-infiltrating lymphocytes are predominantly human leukocyte antigen (HLA) class I restricted CD8 positive cytotoxic T lymphocytes (Tokunaga et al, 1993). EBVGCs are most often poorly differentiated carcinomas, proximal in location, particularly in the gastric cardia and are more

51

Oh and Chetty: Epstein-Barr Virus Associated gastric carcinoma prevalent in male patients (Chang et al, 2001; Kijima et al, 2003; Lee et al, 2004). The mean age was 53.4 years (range: 53.4 Âą 12.7) (Lee et al, 2004). A study showed that gross types were 1 Borrman type I, 2 Borrman type II and 1 early gastric carcinoma type IIc among 4 EBVGCs (Nam et al, 1998). The tumors were composed of syncytial nests of undifferentiated cells having vesicular nuclei with prominent nucleoli, admixed with abundant lymphoplasma cell infiltration in the stroma (Nam et al, 1998). It is also interesting that EBVGC in its intramucosal stage is likely to exhibit a specific histologic pattern with abortive branching-anastomosing tubular structures occupying the middle of the mucosa without destroying the normal mucosal architecture, the so-called lacy pattern (Uemura et al, 1994; Arikawa et al, 1997). Individual carcinoma cells were cuboidal and had oval hyperchromatic nuclei focally with small but distinct nucleoli (Uemura et al, 1994).

The survival rate of the patients with EBVGCs was better than that of the patients with EBV-negative gastric carcinomas, but this was not statistically significant (Kijima et al, 2003; Lee et al, 2004). Even in cases of advanced of EBVGCs, the prognosis was not significantly different from that of patients with EBV-negative carcinomas (Chang et al, 2001).

al, 1998). Some studies showed that the abnormality of Ecadherin expression caused by the aberrant methylation of E-cadherin gene promoter was closely associated with the development of EBVGC. The frequency of this aberrant methylation was significantly higher in EBVGC than in EBV-negative GC (Sudo et al, 2004).

Other studies have shown loss of p73 expression through aberrant methylation of the p73 promoter occurred specifically in EBVGC, together with the global methylation of p14 and p16 (Ushiku et al, 2006). The study also suggested that a specific type of gastritis, prone to a higher grade of atrophy and p73 methylation, might facilitate the development of EBVGC (Ushiku et al, 2006). p73 is mapped to the human chromosome 1p36.2-3, a region which is frequently lost in a wide variety of human tumors including neuroblastoma (Kaghad et al, 1997). The sequence-specific DNA-binding domain, the aminoterminal activation domain and the carboxy-terminal oligomerization domains of p73 are similar to those of p53.

Accompanying these structural similarities, p73 can act as transcription factors and regulate the expression of similar groups of genes by means of direct binding to what were originally identified as p53-binding sites within promoters.

Transcriptional activation of these target genes leads to the induction of cell-cycle arrest and apoptosis (Kaghad et al, 1997; Zhu et al, 1998; Melino et al, 2002). This evidence suggests that p73 may act as a tumor suppressor with an overlapping function with p53. But, interestingly, loss of p73 expression, independent of p53 abnormality, specifically occurred in EBVGC (Ushiku et al, 2006).

Some studies suggested that the accumulation of CpG island methylation simply occurs in the early stage of development CpG island methylator phenotype (CIMP) high gastric carcinoma, without contribution to its further progression (Chang et al, 2006).

There is a negative association between EBV infection and the expression of MUC1, MUC2, MUC5AC, CEA, c-erbB2, smad7 and p53 (Lee et al, 2004).

III. Molecular pathology The exact mechanism by which EBV contributes to the carcinogenesis of the gastric mucosa remains unknown. But, recently, promoter hypermethylation that leads to epigenetic silencing of multiple genes has been recognized as an important mechanism in gastrointestinal carcinogenesis (Kang et al, 2002; Feinberg and Tycko, 2004; Issa, 2004; Kim et al, 2005; Chang et al, 2006; Kusano et al, 2006). In this regard, promoter methylation of the so-called CpG islands, which are CpG dinucleotiderich areas, located within the promoters of approximately 60% of human genes (Feltus et al, 2003), is usually associated with long-term, irreversible epigenetic silencing of X-linked and imprinted genes (Jones and Baylin, 2002).

The p16INK4A gene is a common target of inactivation by epigenetic mechanisms in gastric carcinoma. The product of that gene is an inhibitor of G1/S phase transition, the loss of which promotes uncontrolled cell growth (Toyota et al, 1999; Suzuki et al, 1999). The p16INK4A methylation occurs frequently in EBVGC (Schneider et al, 2000; Kang et al, 2002; Kusano et al, 2006). Thus, it appears that epigenetic silencing of this gene is associated strongly with the development of EBVGC. E-cadherin is also important protein in the carcinogenesis of the stomach. E-cadherin is a Ca2+dependent cell-cell adhesion molecule that plays an essential role in the formation and maintenance of the normal architecture and function of epithelial tissues (Takeichi, 1991; Takeichi, 1995; Bracke et al, 1996).

Abnormalities of the gene and gene expression of Ecadherin have been frequently observed in gastric carcinoma (Oka et al, 1992; Becker et al, 1994; Shino et al, 1995; Tamura et al, 1996; Shun et al, 1998; Machado et al, 1999) and the germline mutation was identified in the hereditary diffuse gastric carcinoma kindred (Guilford et

IV. Immunologic aspects related to EBV EBV is a member of lymphocryptovirus genus of gamma herpes family. The EBV genome is a linear, double stranded, 184-kbp DNA (Kieff and Rickinson, 2001). Like all herpesviruses, EBV can establish either a latent or lytic infection in host cells. In infected cells, the EBV genome enters the nucleus, where it forms a circular episome (Kieff and Rickinson, 2001). Episome formation is mediated by 0.5 kb terminal repetitive sequences located at either end of the linear molecule. Fusion of these sequences results in terminal repetitive regions with variable numbers of repeats (Raab and Flynn, 1986). It is believed that individual infection events lead to episomes which differ in their number of repeat of terminal repetitive region; i.e. episomes within a single cell show the same number of repeats. Thus, analysis of the terminal repetitive region by Southern blot hybridization can provide evidence regarding the clonality of the viral genome (Raab and Flynn, 1986).

In the latent forms of infection, the virus is replicated once per cell cycle as an episome using the viral oriP replication origin, the viral EBNA1 protein, the host cell 52

Gene Therapy and Molecular Biology Vol 11, page 53 DNA polymerase (Kieff and Rickinson, 2001). The latent infection of EBV is characterized by the expression of a limited set of viral genes, the so-called latent genes, including two types of non-translated RNA (EBV-encoded nuclear RNAs; EBER1, EBER2), six EBV-encoded nuclear agents (EBNA1, 2, 3A, 3B, 3C, LP), three latent membrane proteins (LMP1, 2A, 2B), among the nearly 100 viral genes that are expressed during replication infection of EBV (Kieff and Rickinson, 2001). The vast majority of EBV infected tumor cells contain one of the three types of latent EBV infection and expression of the latent EBV gene products is sufficient for immortalization of B cells in vitro (Kieff and Rickinson, 2001). LMP1 is considered to be the major EBV oncogene, although several additional latent viral proteins are also required for EBV immortalization of B cells in vitro (Kieff and Rickinson, 2001). Unfortunately, drugs that specifically inhibit the latent form of EBV infection are not currently available. The role of EBV in causing malignancies is thought to vary according to different types of viral latency and associated histologic type. BL that contains EBV represent type I latency, in which viral gene expression is limited to the EBNA1 protein and untranslated viral transcripts (EBERs). Viral gene expression in tumors with type II latency, as seen in nasopharyngeal carcinoma and EBV-associated HD, is restricted to EBNA1, LMP1, LMP2, BARF0. At the other extreme, type III latency, typified by EBV-transformed lymphoblastoid B cell lines (LCLs) in vitro and EBVpositive diffuse immunoblastic lymphomas, in vivo, is associated with expression of all nine latency associated proteins, including EBNA1 as well as a variety of different EBV proteins (in particular, LMP1 and EBNA2) required for EBV transformation of B cells (Kieff and Rickinson, 2001).

In EBVGC, it has been established that the viral gene expression is restricted to latency I genes quite similar to BL, such as EBV-encoded small RNA 1 (EBNA1), EBV-encoded RNA (EBER), BARF0, BARF1, latent membrane protein 2A (LMP2A) (zur et al, 2000).

The presence of EBER also has been demonstrated in malignant gastric epithelial cells by in situ hybridization (ISH) (Imai et al, 1994).

EBV preferentially infects B-lymphocytes through the binding of the major viral envelope glycoprotein gp350 to the CD21 receptor on the surface of B cells (Nemerow et al, 1987), through the binding of a second glycoprotein, gp42, to human leukocyte antigen (HLA) class II molecules as a co-receptor (Borza and HuttFletcher, 2002). Infection of other cell types, principally epithelial cells, is much less efficient and occurs through separate, as yet poorly defined, pathways (Borza and HuttFletcher, 2002). The presence of EBV in epithelial cells and B-lymphocytes provokes an intense immune response consisting of antibodies to a large variety of viral antigens.

In people with normal immune response, cells expressing EBNAs and LMPs engender EBV-specific, HLA class I restricted, CD8+ cytotoxic T lymphocytes responses (Kieff and Rickinson, 2001). Other defense mechanisms include neutralizing antibodies, cytokines such as interferons, natural killer cells and antibody-dependent-mediated

cytotoxicity (Tang et al, 1993; Kieff and Rickinson, 2001).

The EBNAs in particular, except for EBNA1, have multiple epitopes that are recognized in the context of common class I determinants. The EBNAs and LMP1 also induce the expression of adhesion molecules, rendering the cell susceptible to T lymphocytes adherence and cytocidal effects. As a consequence of immune responses by normal people to primary EBV infection, the number of proliferating virus-infected B-lymphocytes in the peripheral blood rapidly declines to a level of one infected B lymphocyte in 105 or 106. However, cytotoxic T lymphocytes specific for epitopes from five of the EBNAs and the two LMPs persist forever, indicating that cells expressing the EBNAs and LMPs are at least intermittently present in the normal host (Kieff and Rickinson, 2001).

Many reports have indicated that there are defects in the HLA class I-associated antigen processing and presentation pathway in EBV-associated BL, nasal natural killer (NK) cell/T-cell carcinoma (Frisan et al, 1996; Shen et al, 2001) and EBVGC (Dutta et al, 2006). Furthermore, there is evidence of the interference of certain viral antigens in the locus-specific and functional expression of HLA class I antigens in various malignancies (Fruh et al, 1999; Tortorella et al 2000). The triggering of specific cytotoxic T-lymphocyte (CTL) response (largely CD8positive) is dependent on the appropriate presentation of viral or tumor-specific antigens in the context of proper HLA class Ia molecules, giving rise to the first step of immune defence (Hicklin et al, 1999). HLA class Ia molecules that are expressed on the surface of nearly all nucleated cells are composed of a polymorphic transmembrane heavy chain and a monomorphic light chain called !2 microglobulin (!2m). The heavy-chain polypeptides are encoded by 3 closely linked loci, HLA-A, HLA-B, HLA-C. Many alleles are assigned to a particular locus (York and Rock, 1996). Immunohistochemical (IHC) studies in different types of solid tumors have demonstrated defects in HLA class Ia expression (Natali et al, 1989). Moreover, selective down-regulation of the HLA class I A or B locus also has been observed in GC, colon carcinoma, laryngeal carcinoma (Momberg et al, 1989; Lopez et al, 1989). Several other molecules, such as transporter associated with antigen presentation and LMP, have been associated with HLA class Ia antigen presentation (Pamer and Cresswell, 1998). It is noteworthy that malignant cells can escape CTL-mediated immune response by down-regulating HLA class Ia expression; however, then, they may become susceptible to NK cellmediated lysis (Garrido et al, 1997).

A number of reports have suggested that the expression of insulin-like growth factor 1 (IGF1) and IGF2, which function as autocrine/paracrine growth factors, are potent stimuli for tumor cells of varied origin (Macaulay, 1992). The biologic responses of IGF1 and IGF2 are transmitted through the IGF1 receptor (IGF1R), which is a tyrosine kinase transmembrane receptor with expression that has been observed in several types of tumors (Kaleko et al, 1990). It has been reported that the IGF2 and IGF1R genes are overexpressed in GC (Pavelic et al, 2003). Moreover, increased levels of IGF1 in

53

Oh and Chetty: Epstein-Barr Virus Associated gastric carcinoma primary tissue from EBVGC also have been reported using PCR (Iwakiri et al, 2003). EBV-harboring NK cell/T-cell lymphoma, BL, HD, NPC may suppress local immune response to the infiltrating T cell by up-regulating cytokines and cellular growth factors, such as IGF1 (Herbst et al, 1996; Fujieda et al, 1999; Kitagawa et al, 2000; Shen et al, 2001; Iwakiri et al, 2003; Iwakiri et al, 2005). It is noteworthy that IGF1 has been implicated in the modulation of HLA class Ia expression and in the inhibition of apoptosis in glioma cells (Ly et al, 2000).

One possible explanation for the viral-induced, locus-specific down-regulation of HLA class I genes is the interaction of viral LMP2 and cellular nuclear factor "B (NF-"B). In one study, viral LMP2A expression was observed in some EBVGC samples, although the expression level was considerably low compared with LMP2A expression in the positive control Raji cells (Dutta et al, 2006). The "B motif of enhancer A element of the HLA class I gene is the binding site of NF-"!/Rel family transcription factors and is highly conserved and present only in HLA-A and HLA-B gene promoters (Le, 1994).

However, because of the lack of NF-"! binding sites on other HLA class I gene promoters, such as HLA-C, HLAE, HLA-F, they are not regulated by NF-"! (Gobin et al, 1998). Recently, it was demonstrated that EBV-encoded LMP2A expressed in NPC and GC cell lines downregulated cellular NF-"! (Stewart et al, 2004). Analogous to other malignancies, aberrant methylation of HLA class Ia gene promoters may lead to the loss of expression (Nie et al, 2001). Of course, it would be interesting to investigate whether EBV modulates the methylation of HLA genes, as reported for other cellular genes (Kang et al, 2002). Because of the observation that the majority of CTLs recognize peptides presented by HLA-A and HLAB, whereas cells are protected from NK cell cytotoxicity

by HLA-E and HLA-C expression (Littaua et al, 1991; Lee et al, 1998; Yokoyama, 1998), it is necessary to have prior knowledge about locus-specific gene/protein expression to produce a rational immunotherapeutic design.

EBV encodes a unique gene product, BCRF1, that has high amino acid identity with human IL-10 (Moore et al, 1990). Like human IL-10, vIL-10 inhibits the synthesis of IFN-! by lymphocytes and NK cells and suppresses IFN!-mediated cellular events such as the up-regulation of the HLA class I expression and CTL responses. The EBV BARF1 protein functions as a soluble receptor for colonystimulating factor (CSF)-1. Since CSF-1 normally enhances the expression of IFN-" by monocytes, BARF1 protein may function as a decoy receptor to block the activation of the cytokine (Cohen and Lekstrom, 1999).

EBNA1 has been shown to block its own degradation by proteosomes in infected cells (Leviskaya et al, 1997).

Since viral proteins are normally broken down by proteosomes to peptides for presentation to CTL, the ability of EBNA1 to inhibit its degradation may allow the protein to avoid triggering the activation of CTL. Also, EBV can modulate the ubiquitin-proteasome system to manipulate the host immune response, promote viral replication and inhibit apoptosis (Masucci, 2004). The release of virokines and the down-regulation of cell

adhesion molecules are additional strategies for EBVinfected cells to evade the host immune system.

V. Environmental factors Some studies have shown environmental factors may be related to EBVGC. EBVGC is thought to be related to lifestyle, dietary habits and occupational exposure of wood dust, iron filing and tar (Yoshiwara et al, 2005; Koriyama et al, 2005). Although the prevalence of cigarette smoking in EBVGC cases was higher than among non-EBVGC cases, the difference was not statistically significant (Koriyama et al, 2005). Frequent drinking of coffee and high-temperature drinks, as well as frequent intake of salty and spicy foods, were more prevalent among EBVGC cases, but only frequent intake of salty food showed a significant difference between EBVGC and non-EBVGC cases. In addition, patients with EBVGC tended to be exposed to wood dust and/or iron filings and tar (Koriyama et al, 2005). Gastric remnant cancer after a partial gastrectomy for benign gastric disease also shows a statistically higher EBV infection rate than in conventional gastric carcinomas (Yamamoto et al, 1994; Chang et al, 2000). These findings suggest an association between mechanical injuries to the stomach membrane and the high frequency of EBVGC.

VI. EBV-targeted therapy Persistent expression of certain EBV-encoded gene products is likely required for the continued growth of many, if not all, EBV-associated lymphomas. Therefore, EBV-based strategies for treating cancer include prevention of viral oncogene expression, inducing loss of the EBV episome, the purposeful induction of the lytic form of EBV infection, enhancing the host immune response to virally encoded antigens (Israel and Kenney, 2003). Although, currently, EBV-targeted therapies are not made a trial in EBVGCs, some studies tried EBV-targeted therapy in EBV-positive gastric carcinoma cell line in vitro (Feng et al, 2002). EBV-based therapies are currently being developed for the treatment of EBV-positive malignancies. Therefore, the knowledge of this therapy will be useful in the treatment of EBVGC.

A. Loss of the EBV episome Chronic, low-dose hydroxyurea treatment can induce loss of viral episomes in some BLs and LCLs, although the mechanism of loss is not yet clearly defined (Chodosh et al, 1998). Another technique for episomal targeting is suggested by the observation that the cellular genome contains numerous sites for origin of replication complexes (ORCs) to regulate initiation of DNA synthesis, while EBV episomes are thought to contain only one major ORC. Thus, drugs that inhibit various aspects of ORC formation may eventually prove useful for inducing loss of the EBV episome in tumor cells (Dhar et al, 2001).

B. Purposeful induction of the lytic EBV infection The switch from the latent to lytic form of EBV infection is mediated by the two viral immediate-early (IE) 54

Gene Therapy and Molecular Biology Vol 11, page 55 proteins, BZLF1 and BRLF1. The BZLF1 and BRLF1 gene products encode transcription factors that together activate the entire lytic viral cascade of gene expression, ultimately resulting in the production of infectious viral particles (Gutierrez et al, 1996). In vivo, it is thus likely EBV tends to stay in the latent form of infection in quiescent B cells, switches to the lytic form of infection in highly activated B cells. The latent EBV gene product, LMP-2, is thought to play an important role in helping to maintain viral latency in B cells by suppressing the signal transduction cascades which are normally induced by Bcell activation (Miller et al, 1995). Epigenetic factors also play an important role in regulating the state of EBV infection in host cells. In cells containing tightly latent EBV infection, the viral IE promoter DNA is often methylated (Falk and Ernberg, 1999) and the chromatin surrounding the IE promoters is in the unacetylated (inactive) form (Gruffat et al, 2002). Treatment of certain Burkitt’s cell lines in vitro with agents that induce histone acetylation (Westphal, et al, 2000), or reverse DNA methylation (Ben and Klein, 1981) is sufficient to induce the lytic form of EBV infection in a subset of cells, although such agents are generally not very effective in inducing lytic EBV infection in EBV-immortalized lymphoblastoid cell lines (Westphal, et al, 2000).

C. Inhibition properties

of

EBV

CTL have been used successfully for the prophylaxis and treatment of EBV-lymphoproliferative disease post hematopoietic stem cell transplantation (Rooney et al, 1995, 1998). EBNA2, EBNA3a, 3b, and 3c and to a lesser extent LMP2, contain the immunodominant epitopes for latent EBV proteins in normal CTL responses (Murray et al, 1992). Post-transplant-type lymphomas, which typically contain type III latency gene expression, express the full complement of latent virus protein immunodominant epitopes for the host CTL response.

The clinical results with EBV-specific CTL therapy for type II latency tumors such as NPC and EBV-associated HD were less effective than for post-transplant lymphomas (Aisenberg, 1999; Chua et al, 2001). Decreased CTL efficacy most likely reflects immune evasion strategies by tumor cells such as down regulation of immunodominant EBV proteins and secretion of inhibitory cytokines (Poppema et al, 1998). To overcome these immune evasion strategies a number of approaches have been developed including targeting CTL to subdominant EBV antigens and genetically modifying CTL to increase their potency (Gahn et al, 2001; Duraiswamy et al, 2003, 2004; Gottschalk et al, 2003; Bollard et al, 2004; Comoli et al, 2004; Lucas et al, 2004; Straathof et al, 2005).

Burkitt’s lymphoma cells evade the immune system by down regulating the expression of cell adhesion molecules, MHC class I molecules and EBV latency antigens and thus the prospect for the development of an EBV-specific immunotherapy is problematic. The only EBV antigen expressed in Burkitt’s lymphoma, EBNA1, is inefficiently processed for HLA class I presentation due to an internal glycine-alanine repeat region and so far only few endogenously processed HLA class I restricted peptides have been identified that are recognized by CD8 positive T cells (Lee et al, 2004; Voo et al, 2004). Several MHC class II restricted peptides from EBNA1 have been identified, which are recognized by CD4 positive T cells and the potential use of these cells for the adoptive immunotherapy of Burkitt’s lymphoma is being actively explored (Paludan et al, 2002; Munz, 2004).

transforming

Theoretically, inhibiting one or more of the EBV proteins known to be required for transformation of B cells in vitro (including LMP1, EBNA2, EBNA3a and EBNA3c) might reverse the oncogenic phenotype of at least some EBV-associated tumors. For example, antisense RNA directed against LMP1 decreased the expression not only of LMP1 in EBV-positive LCLs, but also cellular proteins induced by LMP1, such as the antiapoptotic proteins Bcl-2 (Kenney et al, 1998). Furthermore, LMP1antisense RNA resulted in decreased LCL proliferation, increased apoptosis, increased sensitivity to the cytotoxic drug etoposide (Kenney et al, 1998). Recently, highly effective selective gene inhibition has been achieved with small interfering RNA (RNAi) technology, in which the expression of short (15-20 bp) double-stranded RNA sequences homologous to the gene of interest results in degradation of the target mRNA. This approach has been used successfully to modulate viral expression in vitro of HIV (Coburn and Cullen, 2002), papillomavirus (Jiang and Milner, 2002), poliovirus (Gitlin et al, 2002) model systems.

Downstream effects of LMP1 are mediated in part through activation of NF-k! (Cahir et al, 2000), inhibition of NF-k! function using an inducible dominant repressor has been shown to result in spontaneous apoptosis of LCLs (Cahir et al, 2000).

VII. Conclusion EBVGC is a unique type of gastric carcinoma that is tagged by clonal EBV. Further studies about EBV pathogenesis, viral gene regulation, immunologic aspect and environmental factors in EBVGC are needed. Finally, additional EBV-targeted therapy of EBVGC and EBVGC prevention program will be developed.

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Gene Therapy and Molecular Biology Vol 11, page 61 Gene Ther Mol Biol Vol 11, 61-74, 2007

Reviewing the role of putative candidate genes in “Neurobesigenics,” a clinical subtype of Reward Deficiency Syndrome (RDS) Research Article

Thomas J.H. Chen1, Kenneth Blum2,4,9,*, Gilbert Kaats3, Eric Braverman4, Dennis Pullin5, Bernard W. Downs6, Manuel Martinez-Pons7, Seth H. Blum8, Julie Mengucci8, Debasis Bagchi10,11, Manashi Bagchi11, Ariel Robarge6, Brian Meshkin9, Vanessa Arcuri4, Michael Varshavskiy4, Allison Notaro4, David E. Comings12, Lisa White13 1

Chang Jung Christian University, Tainan, Taiwan and Changhua Christian Hospital, Changhua, Taiwan Department o Physiology & Pharmacology, University of Wake Forest School of Medicine, Medical Center Boulevard Winston-Salem, North Carolina 3 Health and Medical Research Foundation, San Antonio, Texas 4 Path Medical Foundation, Park Ave., New York, New York 5 Sports Medicine Institute, Baylor College of Medicine, Houston, Texas 6 Allied Nutraceutical Research, Lederach, Pennsylvania 7 Department of Education, Brooklyn College CUNY, New York, New York 8 Synaptamine, Inc., San Antonio, Texas 9 Salugen, Inc. San Diego, California 10 Department of Pharmacy Sciences, Creighton University Medical Center, Omaha, Nebraska 11 InterHealth Research Center, Benicia, California 12 Carlsbad Science Foundation, Monrovia, California and City of Hope National Medical Center, Duarte, California 13 Baylor College of Medicine, Department of Molecular Gentics Houston TX and DNA Services of America, Lafayette, LA. 2

__________________________________________________________________________________ *Correspondence: Kenneth Blum, Ph. D. Department of Physiology and Pharmacology, University of Wake Forest School of Medicine, Medical Center Boulevard, North Carolina, 27157, USA; TEL-1-210-823-1504; FAX- 1-210-694-2680; e-mail: DRD2gene@aol.com Key words: Obesity, percent body fat, dopamine, body mass index, DRD2gene, DEXA, screened controls, Reward Deficiency Syndrome (RDS) Abbreviations: 2’- deoxynucleotide 5’- triphosphates (dNTPs); 2-deoxyglucose (2DG); acetylcholine (ACh); Attention-deficit –disorder with or without hyperactivity (ADHD); body mass index (BMI); dopamine D2 receptor (D2R); dopamineD2 receptor gene (DRD2); dual energy X-ray absorptiometry (DEXA); hydroxycitric acid (HCA-SX); Minnesota Twin and Family Study (MTFS); mitochondrial DNA (mtDNA); neuropeptide Y (NPY); nonpreferring (NP); polymerase chain reaction (PCR); Reward Deficiency Syndrome (RDS); single nucleotide polymorphism (SNP); substance use disorder (SUD)

Received: 16 January 2007; Revised: 7 February 2007 Accepted: 28 March 2007; electronically published: May 2007

Summary While there is a considerable body of literature correlating the role of dopaminergic genes and obesity, body mass index, body type, overeating, carbohydrate binging, energy expenditure and low dopamine D2 receptor (D2R) density, there is a paucity of research concerning the dopamine D2 receptor gene (DRD2) variants and percent body fat. We propose that the degree of obesity involving the interactive relationship of the brain’s reward circuitry and the body’s response to stress and caloric deprivation is represented by a new term Reward Deficiency Syndrome (RDS), which is subject to strong genetic influence. We report here the first potential association of DRD2 genotypes and the percent fat phenotype. We genotyped, at the DRD2 Taq1 A1 polymorphism, 122 obese Caucasian subjects and 135 non-obese controls. The first control group consisted of 30 non-obese Caucasians screened to exclude a wide range of addictive behaviors (Controls A). The second control group consisted of 105 non-obese Caucasians

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Chen et al: Association of The Taq A1 Allele of The Dopamine Receptor Gene (DRD2) and Percent Body Fat in Humans screened to exclude substance abuse and psychiatric disorders (Controls B). Controls A were assessed for weight, body mass index (kg/m2) [BMI] and percent body fat using dual energy X-ray absorptiometry (DEXA). The controls B were assessed for weight and BMI. The sample was separated into two groups, those with the Taq1 A1 allele (A1/A1 or A1/A2) and those without the A1 allele (A2/A2). The controls A had a normal range of body fat (2531% for females and 18-25% for males), a mean % body fat of 28.4±3.4 % and a mean BMI of 22.4 ± 2.9 kg/m2. The controls B had a mean BMI of 21.9 ± 2.9 kg/m2 The obese subjects had a percent body fat value of over 32 % for females and over 25% for males, a mean % body fat of 42.1± 7.5%, and mean BMI of 29.3 ± 6.25 kg/m2. The DRD2 Taq1A1 allele was present in 67% of the obese subjects compared to 3.3 % of the well-screened controls A and 33.3 % for controls B. These differences were significant: Controls A vs. Obese subjects: c2 = 39.6 d.f. =1, p<0.0001, and Controls B vs. Obese subjects c2 = 25.9 d.f. 1, p <.0001. These results are consistent with a role of the DRD2 gene in obesity as measured by percent body fat as well as by BMI. Additionally, it is proposed that since fat distribution is under extensive genetic control (h2 <0.80) one putative gene may be the DRD2. humans, over 250 genes, markers, and chromosomal regions have been associated with obesity and related behaviors (Rose et al, 1998; Perusse et al, 2001). Studies of twins provide the clearest evidence for genes and environment both exerting a significant influence on body composition. In 1997, researches examined data from 25,000 pairs of twins and a total of 50,000 family members. On an average, obesity was 67% genetic and 33% environmental (Evans et al, 2001). A small sampling of important candidate obesity genes presented in Table 1 just touches the surface.

Given the complex array of metabolic systems that contribute to overeating, it is not surprising that a number of neurochemical defects have been implicated.

Carbohydrates cause the release of the pleasure-inducing brain chemical dopamine. There is general agreement that other pleasure-inducing substances such as alcohol and nicotine, like glucose, exert an effect on the dopaminergic pathways of the brain. This shows the common genetic thread of multiple addictions (Blum et al, 1996). There are a number of investigators that have observed a significant association between a variant of the dopamine D2 receptor gene and time of the onset of obesity, carbohydrate preference or craving, high body mass index, co-morbid drug abuse, energy expenditure, hyperphagia (Blum et al, 2000) and low dopamine D2 receptors (Wang et al, 2001).

In 1990, Blum and colleagues, reported a strong association between a virulent form of alcoholism and the Taq1 A1 allele of the DRD2 gene (Blum et al, 1990).

Other more recent studies further support an association of this allele with substance abuse vulnerability and other compulsive behaviors (Xu et al, 2004). In this regard, the National Institute on Alcohol Abuse and Alcoholism recently reported data that strongly suggests that DRD2 is a susceptibility gene for substance abuses across multiple populations. Specifically, a haplotype block of 25.8 kb region of the DRD2 gene was highly associated with alcohol dependence and heroin addiction. A low number of dopamine D2 receptors in individuals carrying the Taq1 A1 variant suggest a hypodopaminergic function, as described by Eliot Gardner in a series of published works (Gardner, 1997). When there is a paucity of dopamine receptors the person will be more prone to seek any substance (including glucose) or behavior that stimulates the dopaminergic system as a form of self-healing. In this regard, we know that substances such as alcohol, cocaine, heroin, nicotine and glucose, as well as a number of

I. Introduction Overeating is a biogenetic condition that comes in many forms (Wang et al, 2001). Large family studies in different populations have consistently demonstrated a familial correlation in adult body mass index (BMI) at about 0.2 between parents and offspring and about 0.3 between siblings. Moreover, according to many twin and adoption studies, these correlations are attributable mainly to genetic influences rather than to effects of the shared environment (Males et al, 1997). Fewer studies have addressed the genetic and environmental influences on body shape, assessed by body circumferences, skinfold measurements, and on body composition of fat and lean mass (Carey et al, 1996; Rice et al, 1997; Rose et al, 1998). Most recently, Schousboe and colleagues in 2004 using a more stringent design, found in both Caucasian women and men, heritability for BMI was 0.58 and 0.63, for body fat %, 0.59, and 0.63, and for lean body mass, 0.61 and 0.56 respectively. The same authors found no strong evidence of common environmental effects but did find a significant decrease in heritability with advancing age (Schousboe et al. 2004). Similar findings were found among African- American twins (Nelson et al, 2002), however, there are differences between Blacks and Caucasian probands where certain gene polymorphisms are concerned such as leptin expression and genes associated with BMI (Chagnon et al, 2000). There are certain environmental factors which may reduce body fat, body mass and fat-free mass in both adults and children.

These include but are not limited to low-intensity, longduration exercise, aerobic exercise combined with highrepetition resistance training and exercise programs combined with a behavioral-modification component.

Moreover, genetics may even play a more significant role (LeMura and Maziekas, 2002).

Because of the complexity of compulsive eating disorders, it is likely that more than one defective gene is involved. Indeed at least twelve genes [see below] involved in the neurochemistry of brain reward have been associated with morbidly obese people. Moreover, three metabolic type genes have been identified-one associated with cholesterol production, one with fat transport, and one related to insulin production (Perusse et al, 2001).

Twenty-four different Mendelian disorders have been reported exhibiting obesity as one clinical manifestation.

From animal research we know of 115 genetic sites associated with obesity and related problems. Moreover, in

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Gene Therapy and Molecular Biology Vol 11, page 63 behaviors like gambling and sex, preferentially release dopamine at the nucleus accumbens. In one study (Wang et al, 2001), striatal dopamine D2 receptor availability was significantly lower in ten obese subjects compared to normal non-obese controls. In these obese subjects, BMI

correlated negatively with the measures of D2 receptors.

The individuals with the lowest D2 values had the largest BMI. Table 2 illustrates the role of dopaminergic genes in obesity and related behaviors.

Table 1. Example of a number of Candidate obesity genes.

PATHWAY CNS-Neurotransmitter Mesolimbic “reward” system and appetite regulatory pathway.

GENE Leptin (OB)

POLYMORPISM(S) OB1875 < 208-bp allele

REFERENCE (S) Comings et al, 1996

Serotonergic pathway involving “sweet tooth” and appetite regulation. Implicated in Bulimia nervosa and anorexia nervosa Serotonin concentrating substance, Percent body fat reduction, cholesterol reduction, glucose regulation, reduction of glucose craving in atypical depression.

Association with total cholesterol and triglyceride levels.

Serotonin (5HT2A)

-1438G/A and 102/C

Fuentes et al, 2004; Roy et al, 2004; Tochigu et al, 2005

Acid phosphatase (ACPI)

A*/A*, A/B and A/C and non-allele genotypes B/B, B/C, C/C. These genotypes have been associated with total cholesterol and triglycerides. The ACP1 * A allele may be partially protected against developing Reward Deficiency Syndrome (RDS) G-148A of PNMT

Bottini et al, 2002

Neurotransmitter gene interaction especially serotonin precursors

Phenylethanolamine NMethyltransferase (PNMT)

G/G, A/A and G/A. Compared with the heterozygous PNMT variant, G/A, the presence of the homozygous PNMT variant, either G/G or A/A, was associated with a statistically significant weight loss when challenged with Sibutramine (adrenergic/serotonergic) after 6 months.

Peters et al, 2003

Receptor is involved in both pleasure and antianxiety. It is involved in craving for sugar as well as other addictive substances. Dopamine is a major neurotransmitter released at the n. accumbens and acts as a reward substance in the brain.

Dopamine receptor (DRD2)

The Taq1A1 allele as well as the B1 allele has been associated with a number of Reward Deficiency behaviors including eating and craving for glucose. It has also been associated with obesity, elevated BMI and increased fat storage. The Ser311Cys of the D2gene has been associated with low energy expenditure.

Noble et al, 1994; Tataranni et al, 1996; Blum et al, 2000

Neurotransmitter synaptic clearance

Monoamine Oxidase A (MOA-A)

MOA-A repeat polymorphisms include 2 allele; 3 allele; 3 allele; 4 allele, 5 allele; Short (2,3) and Long (4,5).

Tsugeno and Ito, 1997; Manor et al, 2002; Ito et al, 2003; Manoli et al, 2005

DHEA a natural substance effects female belly fat and is associated with the

Steroid sulfatase (STS)

Absence of site assoc with low MAO activity. STS "G" allele (n = 36) had greater acute changes in DHEA [+4.4 (0.7) vs. +2.0 ng/ml (0.5), S1; +3.2 (0.6) vs. +1.0 ng/ml (0.4), S30; P < 0.01] and

Riechman et al, 2004; Villareal and Holloszy, 2004

receptor

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Chen et al: Association of The Taq A1 Allele of The Dopamine Receptor Gene (DRD2) and Percent Body Fat in Humans STS gene.

DHEAS:DHEA [-37 (11) vs. 5 (7), S30, P < 0.05] than those subjects with only an "A" allele (n = 84).

Interaction of DHEA and adipose tissue as correlated with the PPAR! gene.

Peroxisome ProliferatorActivated Receptor ! (PPAR!)

A corresponding increase in PPAR! mRNA expression suggests that PPAR! may be involved in the up-regulation of adiponectin gene expression after DHEA treatment. Pro12Ala polymorphism of PPAR! gene is a major variant.

Dillon et al, 2000; Karbowska and Kochan, 2005; Blum et al, 2007

Glucose conversion to fat is controlled by the ChREBP gene.

The ChREBP is a very important gene which controls glucose metabolism and may be a key in the etiology of obesity. A high fat diet inhibits glucose metabolism, and is sometimes referred to as the “fatty acid sparing effect of glucose’. It has been showed that the activity of ChREBP was inhibited by a high fat diet.

Therefore glucose stimulates ChREBP activity while fat inhibits ChREBP activity which in turn reduces glucose conversion to fat. In terms of health risk DNA analysis of the ChREBP gene will provide very important information which may result in the proper adjustment of known glucose reducing substances.

Carbohydrate responsive elementbinding protein (ChREBP).

Carbohydrate responsive protein(ChREBP) gene.

Uyeda et al, 2002

element-binding

This is an important gene which controls the end step in the conversion of glucose to fat. The mechanism of glucose activation appears to involve the import of ChREBP protein from the cytosol to the cell nucleus.

This involves the dephosphorylation of the phosoSER196 of ChREBP. A Ser568ASP mutant shows weak DNA-binding. There appears to be two levels of regulation of ChREBP by cAMP-dependent protein kinase (PKA)–mediated phosphorylation as a result of a rise in cAMP. One is the phosphorylation of Ser196, which inhibits nuclear import, and the other is Thr666 which inhibits DNA-binding activity. A Ser 626 ASP (with Ser196Ala) mutant loses transcriptional activity, as a result of DNA-binding activity. because of the presence of the similar double mutant of Thr666Ala (Ser196Ala) which inhibits DNA-binding.

Table 2. Mean (SD) baseline demographic data for subjects for non obese and obese subjects.

Control Subject A (N = 30) Control Subject B (N = 105) Obese Subjects (N = 122)

AGE (Y)

WEIGHT (kg)

BODY FAT (%)

BMI (kg/m2)

44.8 ± 7.1

61.3 ± 6.7

28.4 ± 3.4

22.4 ± 2.9

36.5 ± 6.9

60.5 ± 6.1

Not tested

21.9 ± 1.7

42.3 ± 8.8

82.7 ± 21.7

42.1 ±7.5

29.3 ± 6.2

We are making progress in our understanding of the cerebral mechanisms underlying the behaviors that lead to pathological overeating and obesity. Dopamine, a neurotransmitter that is influenced by and in turn influences other neurotransmitter pathways, modulates

rewarding properties of food and is likely to be involved.

To test the hypothesis that obese individuals who have a high percent body fat compared to non-obese controls that may also have a high presence of the dopamine DRD2 A1 allele, we measured percent body fat and genotyped

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Gene Therapy and Molecular Biology Vol 11, page 65 the normal range (BMI normal range below 25 kg/m2, and had a normal range of body fat between 25-31% for females and 18-25% for males). The percent body fat as assessed by Dual Energy X-Ray Absorptiometry (DEXA) was below 31%. Over a five-year period we studied a total of 1506 subjects (91% Caucasian, 1% black, 7% Hispanic, less than 1% Asian), attending the Path Medical Clinic utilizing DEXA and found the average percent body fat to be 28.7.

individuals for the presence or absence of the dopamine D2 receptor A1 allele.

II. Research Methods and Procedures This study was conducted with IRB approval of both the Path Research Foundation (registration # IRB00002334) and the City of Hope National Medical Center. All patients filled out and signed an approved consent form prior to entering this study. The genotyping was performed at the Department of Medical Genetics at the City of Hope National Medical Center, Duarte, California. Lean controls were provided by Dr. Mathew McGue of the University Of Minnesota ( see control B). The recruitment and characterization of the obese subjects were accomplished at the Health and Medical Research Foundation, San Antonio, Texas and the Sports Medicine Institute, Baylor College of Medicine, Houston, Texas. The recruitment and characterization of the well-screened controls were accomplished at the PATH Medical Research foundation, New York City, New York. The statistical analysis was performed in part at the University Of Texas School Of Public Health, San Antonio, Texas and Brooklyn College, CUNY, New York.

B. Controls B This sample consisted of 105 non-Hispanic Caucasian female adults from the Minnesota Twin and Family Study (MTFS) (Iacono et al, 1999). The MTFS is a large, multidiscipline, multi-year study to examine the interaction between genetic and environmental risk factors in the development of adolescent and adult alcoholism and drug abuse. The advantage of the study is that it uses a population based twin ascertainment in which all same-sex twins born in the state of Minnesota are identified by public birth records, thus providing a measure of a random ascertainment. Since most of the other individuals in this study were females (13.6% males and 86.4% females), genotyping of control B subjects was restricted to the mothers of the twins. In terms of inclusion/exclusion criteria for the control subjects they were administered the parent version of the DICAR (Diagnostic Interview for Children and Adolescents (Welner et al, 1987)) and the Structured Clinical Interview for DSM-III-R (SCID-R) (Spitzer et al, 1987). Subjects with any substance abuse or other DSM III-R diagnosis and subjects with a BMI of greater than 24 were excluded. The range of BMI was 19 to 24.

A. Controls A In order to perform scientifically sound genetic association studies in a complex disease such as obesity, certain exclusion/inclusion criteria must be satisfied. It is now known that polymorphisms of the DRD2 gene (A1, B1, C1 and the haplotype In6-Ex7 and other variants) are associated with a number of impulsive-addictivecompulsive disorders. These include severe alcoholism, polysubstance dependence, crack/cocaine dependence, smoking, obesity (BMI over 25), carbohydrate binging, conduct disorder, defense style personality, schizoid/avoidant personality, violent crimes, pathological gambling, autism, Tourette Syndrome, Attention-deficit – disorder with or without hyperactivity (ADHD), severe withdrawal depression, posttraumatic stress disorder, parental history of alcoholism, drug abuse, obesity in Caucasians, and inability to cope with stress. Therefore, we decided to develop stringent non-Hispanic Caucasian “super normal” controls (Blum et al, 1996, 1990, 2000; Gardner, 1997; Noble, 2003; Xu et al, 2004). Specifically, the exclusion criteria included careful assessment of alcoholism, substance use disorder, family history of chemical dependence, obesity, nicotine dependence (smoking behavior), BMI over 25, carbohydrate binging, Autism, Tourettes, ADHD, mood disorders, personality disorder (novelty seeking), schizophrenia, movement disorders, migraine, pathological gambling and posttraumatic stress. Thus, we carefully stratified 184 individuals attending the Path Medical Clinic of New York City. These patients were stringently assessed to eliminate any impulsive, addictive (including carbohydrate bingeing behavior and obesity as defined by BMI, and scale weight), or compulsive behaviors including absence of both Axis 1 and Axis 2 diagnosis.

Computer analysis revealed a sub population that fit the above exclusion/inclusion criteria. This sub-population consisted of a total of 30 non-Hispanic Caucasians subjects (4 males and 26 females) with an average age of 44.8 (46.3 females and 43.4 males) recruited from the total patient population of the Path Medical Clinic in New York City. Both BMI and body fat of the 30 controls was within

C. Obese subjects A total of 130 unrelated non-Hispanic Caucasian obese subjects (BMI above 25 Kg/m2 and percent body fat above 32%.) were enrolled in the study; 122 subjects were genotyped for the DRD2 gene polymorphisms (17 men and 105 women [mean age, 42.3 years ± 8.8 S.D.]). Subjects were recruited from a variety of fitness and athletic clubs in San Antonio and Houston, Texas, by fitness instructors and sales personnel who provided information about the study to potential participants. In order to ensure compliance, the fitness instructors were paid to monitor the subjects as they progressed through the study to ensure that the subjects reported their physical activity levels and caloric intake and completed the testing. All subjects were asked to consult with their personal physician before giving written informed consent. In terms of inclusion/exclusion criteria the experimental subjects were identified as being obese and had multiple failures in their attempts at dieting. The minimum BMI was 25kg/m.2

D. Dual energy x-ray absorptiometry (DEXA) A number of studies have shown that DEXA can accurately measure fat and lean content of skeletal mass with a typical precision error for total body bone mineral content <1% (Tataranni and Ravussin, 1995). DEXA has also been shown to be a precise method for assessing body composition on obese and non-obese subjects. DEXA correlates highly with underwater weighing, deuterium dilution, and total potassium. The reliability of DEXA makes it possible to monitor the effects of relatively short-term dietary restrictions and exercise on both regional and total body composition.

DEXA provides a three-compartment model of body composition: fat, lean tissue mass and bone mineral content.

Measurements are made using a constant potential energy source at 78kVp and a K-edge filter (cerium) to achieve a congruent, stable, dual-energy beam with effective energies of 40 to 70 keV. The unit performs a series of transverse scans moving form head

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Chen et al: Association of The Taq A1 Allele of The Dopamine Receptor Gene (DRD2) and Percent Body Fat in Humans to toe at 1-cm intervals. The area being scanned is approximately 60 x 200cm. Data is collected for 120 pixel elements per transverse, with each pixel approximately 5 x 10 mm. Total body measurements are completed in 10 to 20 minutes with a scan speed of 16 cm/s, or in 20 minutes with a scan speed of 8 cm/s.

The rate value (ratio of low – to high – energy attenuation in soft tissue) ranges from 1.2 to 1.4.

digested with 5 U of Taq 1 for 22 hours at 65°C for the Taq1A polymorphism. Digestion products were then resolved on a 3% agarose gel (5V/cm) containing 0.65 µg/ml ethidium bromide.

There were three DRD2 Taq1A genotypes: 1) the predominant homozygote A2/A2, which exhibits two restriction fragments of 180 and 130 bp; 2) the heterozygote A1/A2, which exhibits three restriction fragments of 310, 180, and 130bp; and 3) the rare homozygote A1/A1, which produces only the uncleaved 310-bp fragment.

E. Genotyping All subjects were genotyped based on a neutral identification number and read without knowledge of the individual being typed. Total genomic DNA was extracted from each coded blood sample, and aliquots were used for polymerase chain reaction (PCR) analysis. The oligo- nucleotide primers 5’CCGTCGACCCTTCCTGAGTGTCATCA-3’ and 5’CCGTCGACGGCTGGCCAAGTTGTCTA-3’were used to amplify a 310-base pair (bp) fragment spanning the polymorphic Taq1A1 site of the DRD2 gene. The D2A1 and D2A2 genotyping were performed by a PCR technique (Blum et al, 1990; Comings et al, 1996, 2000). PCR was performed in 30- µL reaction mixtures containing 1.5mM MgCl2, 2mM 2’- deoxynucleotide 5’triphosphates (dNTPs), 05 µM primers, 1 ug of template DNA 1, 5U of Taq polymerase (Boehringer Mannheim Corp., Indianapolis, IN), and PCR buffer (20 mM Tris-HCL [pH 8.4] and 50mM KCL. After an initial denaturation at 94°C for 4 minutes, the DNA was amplified with 35 cycles of 30 seconds at 94°C, 30 seconds at 58°C, and 30 seconds at 72°C, followed by a final extension step of 5 minutes at 72°C. The PCR product was

F. Statistical analysis Demographic, clinical, laboratory, interview, and questionnaire data were coded and entered into a computer database. DRD2 allelic prevalence, obtained by personnel blinded to the aforementioned information, was also coded. The chi-square statistic with Yates’ correction for continuity (Siegel, 1956), as appropriate, was used for group comparisons using SPSS statistical software (SPSS, Inc, Chicago, IL).

III. Results Table 2 and Figure 1 summarize the subject demographics and results. For controls A (N =30) the mean (± S.D.) age was 44.8 ± 7.1 years, weight 61.3 ± 6.7 Kg, percent body fat 28.4 ± 3.4, and BMI 22.4 ± 2.9 kg/M2. For controls B (N = 105) the mean age was 36.5 ± 6.9, weight 60.5 ± 6.1 Kg, and BMI 21.9 ± 2.9 kg/m2.

Table 3. The role of dopaminenergic pathways in obesity and related behaviors.

DRDS Receptors and Psychoactive Drugs Certain ant-psychotropic drugs increase feeding behavior and cause weight gain because they block dopamine D2 receptors and dopamine response.

Drugs that stimulate either dopamine D1 and/or D2 receptors reduce overeating Dopamine D2 Receptors and Gene Dopamine stimulators normalize overeating, body fat and blood glucose levels by increasing serum dehydroepiandrosterone (DHEA) There is an evidence that BMI and even personality disorders (temperament) are directly correlated with the ability of dopamine to bind to its respective brain receprots Certain dopamine gene mutations associate with BMI The A1 form of the dopamine D2 receptor gene is associated with an increase of fat storage The A1 form of the dopamine D2 receptor gene is associated with an increase a high BMI In humans using PET scan low dopamine d2 receptors have been found with obese individuals not lean controls Glucose causes a release of dopamine at brain sites Dopamine itself increases the release of glucose from blood cells (hepatocytes) Other Dopamine Gene Other genes such as the dopamine 4 receptor was also associated with obesity and a high BMI Both dopamine D1 and d2 receptors are linked to glucose metabolism and subsequent feeding bahevior Certain dopamine gene forms associate with low energy expenditure.

Source: Welner et al, 1987; Spitzer et al, 1987; Noble, 2003

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Gene Therapy and Molecular Biology Vol 11, page 67

Figure 1. Percent prevalence of the DRD2 gene Taq 1 allele in super controls ( N= 30), regular controls ( N = 105 ) and Obese subjects (N= 122l ). For Controls A vs. Obese subjects X2 = 39.6 d.f. = 1, p = < .0001. For Controls B vs. Obese subjects X2 = 25.9 d.f. = 1, p = < .0001.

plausible mechanisms that may contribute. They include: (1) role of the DRD2 gene polymorphisms in obesity and related behaviors; (2) role of the DRD2 mutations and energy expenditure; (3) the interaction of glucose and dopamine release and hyperphagia; (4) role of dopamine and hyperglycemia; and (5) the importance of using â&#x20AC;&#x153;superâ&#x20AC;? normal controls in association studies.

IV. Discussion This is the first study to associate the prevalence of the DRD2 A1 allele and percent body fat. Our findings of a 67% A1 allele prevalence in obese subjects compared to 33% A1 allele prevalence in non-obese controls appears to be in agreement with the work of Evans et al, 2001.

While there is increasing evidence that genetic factors can influence differences in vulnerability to obesity, there still is controversy as to the exact genetic mechanism. Dopamine, acting through many receptors, can modulate the activity of neuronal reward pathways and thus affect a number of impulsive, addictive and compulsive behaviors, including overeating and carbohydrate bingeing. While obesity is a heterogeneous and prevalent disorder with both genetic and environmental components, the causes of this disease are still unknown. Over the last decade, a number of genetic variants have been associated with obesity and related substrates. Included in this list is CNS regulatory genes such as the OB (LEP) gene and the dopamine D2 receptor gene. In this regard a number of studies (Comings et al, 1993, 1996; Noble et al, 1994; Blum et al, 1996; Levitan et al, 2000; Wang et al, 2001) (Table 1) have reported a positive association of the Taq1 dopamine D2 receptor A1 allele and low density of D2 receptors in obesity, body mass index, carbohydrate binging, parental history of obesity, co-morbid substance use disorder (SUD) and reduced energy expenditure.

In this study we found a significant association of the DRD2 A1 allele and percent body fat. In terms of the phenomena observed herein, there are a number of

A. Role of the DRD2 gene polymorphisms in obesity and related behaviors Specifically, the reinforcing properties of food have also led to an examination of the involvement of DRD2 polymorphisms in obesity. Haplotype 4 (GT) of intron 6 and exon 7 of the DRD2 gene were found to be associated with increasing risk for obesity (Comings et al, 1993). In another study, the DRD2 A1 allele was present in 45.2% of obese subjects (Noble et al, 1994), a prevalence similar to that found in alcoholics, nicotine and other drug dependent subjects. In addition, the A1 allele was significantly associated with carbohydrate cravings.

Variants of the human leptin (LEPT, OB) and the DRD2 genes have been examined in relationship to obesity.

Polymorphisms of the OB gene and the DRD2 A1 allele each associated significantly with obesity (Comings et al, 1996). These two polymorphisms together accounted for about 20% of the variance in BMI, particularly in younger women. Another study has ascertained the relationship of the DRD2 A1 allele in obese subjects with and without comorbid substance use disorders (Blum et al, 1996). In obese subjects, A1 allelic prevalence was significantly higher than controls (P< 10-4). Moreover, the progressive

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Chen et al: Association of The Taq A1 Allele of The Dopamine Receptor Gene (DRD2) and Percent Body Fat in Humans increase in co-morbid substance use disorders in these obese subjects was positively related to increased A1 allelic prevalence (P < 10-6). Furthermore, another case control study (Blum et al. 1996) compared variants of the DRD2 gene in obese (BMI > 30) and non-obese control subjects. The prevalence of the DRD2 A1 allele was significantly higher in obese subjects compared to controls (P = 2 x 10-3) as was the DRD2 B1 allele (P = 3 x 10-3).

The odds ratio for obesity associated with the DRD2 A1 genotype was 3.48 compared to 4.55 for the DRD2 B1 genotype. Moreover, Thomas et al, (2000) and Rosmond et al, (2001), assessed Taq1 A DRD2 alleles in 484 obese and 506 non-obese Chinese subjects. Obese subjects, using either BMI or waist-to-hip ratio criteria, had a significantly higher prevalence of the A1 allele (P=0.02) and A1 allelic frequency (P= 0.03) than non-obese subjects.

glucose utilization. These results suggest that feeding behavior is tied into the stimulation of both D1 and D2 receptors and provides metabolic evidence for the importance of D1 and D2 functional linkage in the brain, which relates to hyperphagia or overeating.

D. Role of dopamine and hyperglycemia The direct effect of dopamine on glucose release from primary cultured rat hepatocytes was studied in Japan by Shiroyama and colleagues in 1998. In this regard, dopamine is known to induce hyperglycemia in both animals and man. Their study investigated whether dopamine has any direct effect on glucose release from hepatocytes through the glycogenolytic and/or gluconeogenic pathways, and at the same time determined the main type of adrenergic receptor involved in glucose release. Glycogen-rich and gluconeogenic-depleted hepatocytes were prepared in order to study glycogenolytic and gluconeogenic-depleted glucose release, respectively. Dopamine caused release of glucose which was inhibited by the beta blocker propranolol. Their study demonstrates that dopamine has a direct effect on hepatocytes, increasing glucose release via both glycogenolytic and gluconeogenic pathways and mediated by beta adrenergic receptors.

B. Role of the DRD2 mutations and energy expenditure Two studies (Jenkinson et al, 2000; Tataranni et al, 2001), assessed the role of other DRD2 mutations on weight and energy expenditure in Pima Indians.

Individuals with a Cys-encoding allele had a higher BMI than those homozygous for the Ser-311 allele (Jenkinson et al, 2000). Further, total energy expenditure and 24 hour resting energy expenditure were lower in homozygotes for the Cys311 allele when compared to heterozygotes and homozygotes for the Ser-311-encoding allele (Tataranni et al, 2001).

C. The interaction of glucose dopamine release and hyperphagia

E. “Super” versus normal controls in association studies.

This is the first report that provides direct genetic evidence that the dopamine D2 receptor A1 allele, which has been associated with lower D2 receptors in humans (Noble et al, 1991; Wang et al, 2001), is positively associated with increased percent body fat. The process of fat storage and the role of genes are poorly understood.

However, with confirming data, our findings point to a significant involvement of human percent body fat and dopamine functionality. In regards to using highly screened, “super” controls, using similarly well screened controls, Neiswanger and colleagues found a strong association of the D2 A1 allele and alcoholism (Neiswanger et al, 1995). Hill suggested failures reported in the literature were due to poor assessment of controls. Their suggestion significantly bolsters the appropriate use of "super" controls to more accurately assess a true phenotype. This is especially important when studying complex behavioral diseases (Hill, 1998). The same researchers found evidence for linkage between the dopamine D2 receptor gene and severe alcoholism, early onset, physical dependence symptoms, and Antisocial Personality Disorder (Hill et al, 1999). However, the use of “super” controls may not be appropriate because these individuals do not mirror the general population, and similar comorbid disorders were not eliminated from the obese group. Thus these results should be regarded as preliminary. Moreover, we also examined a non-super control group, screened only to exclude obesity and DSMIIIR axis I disorders. There the prevalence of the DRD2 A1 allele was still significantly lower than for the obese group.

and

Moreover, it is well known that pharmacologic doses of the glucose analogue, 2-deoxyglucose (2DG), cause acute glucoprivation and are associated with enhanced dopamine turnover in pre-clinical studies. In fact, lines of evidence indicate that a variety of metabolic stressors, including acute glucose deprivation are associated with dopamine release. Using PET, Adler and colleagues (2000) found that 2DG administration enhanced synaptic dopamine concentrations. The administration of 2DG is associated with significant striatal dopamine release even in healthy volunteers. These studies are important because they further closely tie glucose levels to dopaminergic activity and strengthen our understanding of the interactive symbiotic relationship between insulin, serotonin and dopamine. As such, there is a relationship between insulin levels and dopamine release in the tuberonfundibular neurons. The insulin effect is dependent on CA++ ions, protein kinase C, and the Na+-H+ exchange system.

Additionally, when there is lower glucose in the brain leading to cerebral global transient ischemia, monoamine release, especially dopamine, is inhibited. In this regard, Trugman and James (1993) showed D1 antagonists lowered glucose utilization by 24 %-28 % in the globus pallidus, entopeduncular nucleus, subthalmic nucleus, substantia nigra, and even the motor cortex, suggesting that stimulation of the D1 receptor by endogenous dopamine contributes to basal metabolism in these regions. In contrast both D1 and D2 agonists increase

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Gene Therapy and Molecular Biology Vol 11, page 69 average daily food intake was cut in half. This was to be carried out for six months so the researchers could study an extended physiological and biochemical response to semi-starvation. The men were provided with 1600 calories a day including the vitamins, minerals and protein calculated to meet needs. Over the course of the six months all the subjects lost on average a quarter of their initial body weight with over half of the weight being from lean tissue. They also experienced a 40% decrease in basal metabolic rate and a significant increase in food and weight obsession. All of the men began to sneak food, binge eat, obsessively collect recipes and food became the sole topic of conversation. They also experienced a loss of interest in sex and daily activities. By the end of the study all of the men were extremely apathetic. Interestingly, this “old” research still provides insight into the repercussions of low calorie dieting as a method of weight loss.

More recently, Dulloo and Jacquet reexamined in 1998 data from this study and confirmed that semistarvation lowers BMR and thermogenesis. However, they noted that upon refeeding it was observed that fat deposition was accelerated in a preferential fashion over lean tissue synthesis. They further noted that the rate and amount of fat deposition, termed catch-up fat gain, was proportional to the degree of initial fat storage. In other words, the greater the pre-starvation BMI, the greater the suppression of thermogenesis, and the greater the rate and quantity of fat deposition upon refeeding. In addition, they found that the lower protein ratio during refeeding was not due to a shift in the individual’s energy partitioning characteristic, but was attributed to other mechanisms operating via the suppression of thermogenesis in response to severe fat depletion, with the energy thus conserved being directed specifically towards accelerating fat (and not protein) recovery. Under conditions whereby the refed animals were pair fed with weight-matched controls, the rate of protein deposition was the same as in the controls but that of fat deposition was increased by 3-fold as a result of energy spared from a 10–15% lower energy expenditure during the early phase of weight recovery (Dulloo and Girardier, 1990; Dulloo and Jacquet, 1998). A number of animal studies and human research by some of our own authors (already cited elsewhere) demonstrates that calorie reductions result in an attempt to increase dopamine, and/or dopamine stimuli, i.e. hyperphagia, sugar, alcohol, nicotine, gambling, etc (Hao et al, 2000; Johansen et al, 2001). We have already shown that calorie deprivation or restriction promotes abnormal dopamine activity that, especially in individuals carrying the DRD2 Taq 1 A1 allele, leads to a number of excessive RDS behaviors like hyperphagia, sugar cravings, binge eating, etc.

F. The ANNKI gene: A potential candidate dopaminergic linkage site It is noteworthy, that regarding the observed polymorphic association in this paper, a major difficulty with an association of the DRD2 Taq1 A1 allele with other complex behaviors such as alcoholism, is that the Taq1 A polymorphism is located more than 10kb downstream from the coding region of the DRD2 gene (Johnson, 1996) and a mutation at this site would not be expected to lead to any structural change in the dopamine receptor. The most likely explanation for an association is that Taq1 A polymorphism is in linkage disequilibrium with an upstream regulatory element, or a 3’ flanking element, or another gene which confers susceptibility to RDS behaviors. Several linkage disequilibrium studies have found strong linkage disequilibrium between Taq1 A1 allele and the Taq1 B allele and the SSCP 1 allele (Blum et al, 1991; Hauge et al, 1991; Goldman et al, 1993; O'Hara et al, 1993; Johnson, 1996). As we have pointed out, the dopamine D2 receptor has been implicated extensively in relation to alcoholism, substance use disorder, nicotine dependence, anxiety, memory, glucose control, pathological aggression, pathological gambling, and certain sexual behaviors; all RDS behaviors. The most frequently examined polymorphism linked to this gene is the Taq1 A restriction fragment length polymorphism which has been associated with a reduction in D2 receptor density. In a recent study, within the 10kb downstream region of the Taq1 A1 RFLP, Neville and associates identified a novel kinase gene, named ankyrin repeat and kinase domain containing 1 (ANKK1), which contains a single serine/threonine kinase domain and is expressed at low levels in placenta and whole spinal cord RNA but its presence has not localized to brain tissue as yet. This gene is a member of an extensive family of proteins involved in signal transduction pathways. The DRD2 Taq1A allele is a single nucleotide polymorphism (SNP) that causes an amino acid substitution within the 11th ankyrin repeat of ANKK1 (p. Glu713lYs), which, while unlikely to affect structural integrity, may affect substrate-binding specificity. If this is the case, then changes in ANKK1 activity may provide an alternative explanation for previously described associations between the DRD2 gene and RDS behaviors (Neville et al, 2004). Most recently additional studies of this gene and nicotine dependence suggests that the ANNK1 flanking gene is closely tied to the DRD2 gene and it is strongly associated with smoking behavior (Gelenter et al, 2006).

G. Reexamining the consequences of calorie deprivation on obesity and related RDS characteristics

H. Metabolic and neurogenomic integration: future application and perspective

Some of the earliest research providing data still relevant today assessed the effects of starvation and subsequent refeeding in healthy volunteers (Keys et al, 1950). Keys and colleagues reported in 1950 that semistarvation dieting resulted in a down regulation of metabolic rate, reduced thermogenesis and subsequent phase 2 accelerated fat deposition. Thirty two men volunteered to participate in an experiment in which their

The body's number one "Prime Directive" is survival.

There are many aspects of gene induced behavior, from normal metabolism to emergency failsafe protective metabolism that can be and are induced to ensure survival.

As previously shown, there is a clear connection between 69

Chen et al: Association of The Taq A1 Allele of The Dopamine Receptor Gene (DRD2) and Percent Body Fat in Humans dopamine D2 Receptor gene polymorphisms and excessive craving-induced aberrant behaviors. In addition, as stated earlier, chronic stress causes a phase 2 reduction in dopamine concentration and the number of active dopamine cells (Gambarana, 2001; Moore et al, 2001; Miyasaka et al, 2005). As such, in a population carrying a particular polymorphism (i.e. DRD2 Taq1 A1 allele), our notion is that chronic stress (or stressors) causes reduced dopamine secretion/receptivity, thereby increasing the craving or need for reward compensation.

Evidence has been demonstrated that persistent stress can lead to a self-sustaining pattern of abnormal craving behavior in both animals and humans, such as carbohydrate bingeing or long-term abuse of sugar.

Animal model support for this premise can be derived from a series of experiments carried out by Li and associates (Mcbride et al 1990, 1997; Hwang et al, 1990; Volkow and Li 2005, 1990, Russell et al, 2004) upon substance-preferring (P) [seek carbohydrates, alcohol, opiates, etc.] and nonpreferring (NP) rat lines. Among a number of other factors, they found that P rats had a reduced dopamine supply at the nucleus accumbens and reduced densities of dopamine D2 receptors in the mesolimbic areas, accompanied by lower serotonin neurons in the hypothalamus, higher levels of enkephalin in the hypothalamus (due to a lower release) and more GABA neurons in the nucleus accumbens.

Crossing a certain stress threshold not only affects dopamine, but can induce survival gene expressions that affect neurogenobolic signaling, which can retard phase 2 energy, fat and endocrine metabolism (the aftermath of initial stress response).

Work by Kogan et al. confirms that the drug DR4004, a putative 5-HT7 receptor antagonist, also has functional activity at the dopamine D2 receptor (Kogan, et al, 2002). It is of interest that neuroanatomical data suggest a potentially interactive role between accumbens acetylcholine (ACh) and dopamine. There is evidence that Nacc ACh is apparently related to neural processes underlying not only psychostimulant reward but also natural consumption behavior (i.e. feeding). In this regard, Hajnal and colleagues found in 2000 that accumbens cholinergic interneurons play a role in the regulation of body weight and metabolism. In this context both stress and the role of dopamine play an important part in the Ach response (Hajnal et al, 2000).

It has also been known for some time that stress induces the preferential release of the circulatory hormone cortisol in humans (Reddy, 2006). Additionally, lipolysis is the major activity involved in the burning of fat in adipose tissue. Ottosson and colleagues clearly showed in 2000 that cortisol significantly reduced the basal rate of lipolysis (p<0.01) and the catecholamine lipolysis stimulators isoprenaline and noradrenalin in vitro (Ottosson et al, 2000). Furthermore, stress induction studies in animals showed a significantly higher food intake than controls within a few days following the stressinduced events (Miyasaka et al, 2005). These findings are consistent with those in humans demonstrating that stress alters dopamine metabolism, contributing to overeating and increases in other aberrant craving behaviors. In

contrast, a reduction in stress, via massage therapy, was shown to reduce cortisol and increase levels of serotonin and dopamine (Field et al, 2005). In addition, dopamine has been associated with pleasure, and has been called the “anti-stress molecule” and/or the “pleasure molecule” (Blum et al, 2000). When dopamine is released into the synapse, it stimulates a number of receptors (D1-D5) which results in increased feelings of well-being and stress reduction.

Sufficient stress also increases cell turnover, induces apoptosis and causes a cascade of survival events. Genemediated apoptosis (programmed cell suicide) is an attempt to "get rid of the bad" so as not to encumber or interfere with and/or essentially "make room" for the good. Recent research in mice corroborates that stressinduced mutations in mitochondrial DNA (mtDNA) accumulate in tissues of mammalian species and are believed to be a significant contributor to aging.

Accumulation of mtDNA mutations was not associated with increased markers of oxidative stress defects in cellular proliferation, but was correlated with the induction of apoptosis, particularly in tissues characterized by rapid cellular turnover. The levels of apoptotic markers were also found to increase during aging in normal mice (Dulloo and Jacquet 1998). The more severe and stressful the adverse environment in the body/tissues, the more aggressive is apoptosis, as has been shown by research in which apoptosis is curtailed (with appropriate genes being "silenced") as a result of exposure to certain nutraceutical substances (i.e. botanicals/nutrients) (Chanvitayapongs et al, 1997; Jang et al, 1997; Joshi et al, 1999; Ray et al, 1999; Criswell et al, 2005; Kujoth et al, 2005).

While in the past it was thought that DNA was a static non-changeable constituent of the organism, today it well established that environmental, pharmaceutical and nutraceutical elements can indeed have profound effects on altering polymorphic gene expressions. One example of these interesting phenomena is the influence of folic acid upon the methylation of certain DNA. Other examples include predisposition to aberrant sugar craving behavior, such as with excessive carbohydrate/alcohol binging and development of the polymorphism for the Dopamine Receptor D2 A1 Taq1 allele, which increases pleasure cravings in the reward circuitry of the brain (the dopamine system). The actual gene expression depends on an individual’s life style and nutritional status rather than the DNA per se. As way of an example, this known and established polymorphism requires an almost excessive need for certain precursor amino acids to assist in the synthesis of certain brain neurotransmitters and minerals to "over nourish" and answer the excessive needs of this pathway and reduce, eliminate and/or normalize the excessive behavior that results from those demands (Blum et al, 2000).

We propose that a complete understanding of neurometabolic systems, as related to obesity and all of its behavioral subsets when coupled with genomic principles will provide novel targets to combat genetic, physiological, neurological, nutritional and peripheral metabolic dependent deficiencies.

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Gene Therapy and Molecular Biology Vol 11, page 71 HCA-SX significantly inhibits serotonin re-uptake and increases serum serotonin levels in the body. Another benefit of increasing serotonin levels may be in addressing many of the emotional issues overweight people face, including binge-eating and depression. HCA-SX has also been shown to reduce levels of neuropeptide Y (NPY) in the hypothalamic tissue of animals. Earlier research has shown that NPY significantly increases appetite in animals that were pre-satiated with a meal. These new studies provide powerful new evidence on HCA-SX’s ability to influence brain chemicals and neuropeptides involved in appetite control and eating behavior. Another recent finding is HCA-SX’s ability to increase fat oxidation and modulate blood lipid levels. Human clinical trials have shown that HCA-SX significantly increases output of urinary fat metabolites. Following exercise or other fat oxidation (burning) processes, fat tissue breaks down into small molecular components, including malondialdehyde, formaldehyde, acetaldehyde and acetone. Increased urinary levels of these fat metabolites indicate increased fat degradation. Thus, HCA-SX is a potent modulator of various metabolomic and genomic systems involved in behavior, healthy body composition and obesity management (Chanvitayapongs et al, 1997; Jang et al, 1997; Joshi et al, 1999; Ray et al, 1999; Preuss et al, 2004a,b, 2005; Criswell et al, 2005; Downs et al, 2005; Kujoth et al, 2005).

Moreover, obesity is the result of a breakdown in the harmonious (interdependent) symbiosis of multiple systems of genetically regulated neurometabolic signaling due for the most part to deficiencies in nourishment, excesses in burdensome environmental factors and/or inadequate system responsiveness necessary for that symbiotic competence between the energy management system (with regard to energy intake, expenditure and storage), the stress and inflammation management system, the pleasure management system, the immune management system, and the neuro-endocrine system. As such, tactics that address less than all of these multiple systems simultaneously can achieve only limited or no success in sustainable healthy body recomposition. The phenomenon known as “Yo-yo rebound weight gain” causes increases in fat storage that occur as a result of “successful” early onset Phase 1 weight loss efforts (from a variety of conventional “deprivation/stimulation” tactics) followed by cessation of the tactic(s) and/or the body’s genetic survival response to such tactics by lowering the neurometabolic rate, increasing fat storage and increasing appetite (Keys et al, 1950; Dulloo and Jacquet, 1998; Dulloo and Girardier, 1990). Over the last few decades this phenomenon has resulted in an obesity epidemic that is spreading worldwide. Even with the overwhelming arsenal of modalities and their endless combinations, obesity is the second leading cause of preventable death in the United States after tobacco with nearly 200 million Americans (roughly 60%) categorized as overweight or obese, according to the Centers for Disease Control and Prevention.

I. Reward deficiency syndrome (RDS): A paradigm shift Taken together, results of research indicate a profoundly integrated, interdependent and compensatory relationship between the brain’s reward/pleasure circuitry, stress management and the neuroendocrine system (i.e Cortisol), and the energy management system, all of which are affected by lifestyle factors and regulated by genetic “oversight” influenced by particular gene polymorphisms.

The dopamine system appears to be a pivotal factor in the sequela of response initiators that, based on specific polymorphisms, results in particular types of behavior described above. We further propose that Reward deficiency Syndrome (RDS) (Blum et al, 1996) is a subset of disorders contributing to a condition we propose be termed Neurobesigenics. The condition RDS accounts for the deficiencies in metabolic competence brought on by 1) the “excessive compensatory” expression of genetic survival mechanisms provoked by chronic and significant dietary stasis and concomitant nutrient deficiencies, 2) exacerbated by yo-yo BMI induced unhealthy deprivation/stimulation tactics, 3) processed through genetic predispositions involving the brain’s reward management system, energy management system, stress and inflammation management system, immune system, and 4) the interplay of each system as they’re manifested through the endocrine and metabolomic system. While the net release of dopamine is important for normal neurological and metabolic functioning, other neurotransmitters are involved in dopaminergic activation through a brain reward cascade. One of the most important is serotonin, especially as it relates to sugar cravings and the so called sweet tooth phenomena. The interplay of serotonergic and dopaminergic systems are well known and established throughout the neuropharmacological literature. While this strongly suggests that dopamine metabolism should be a primary target for therapeutic intervention, and potentially the ANNKI protein, it is also evident that therapeutic address needs to include pathways like serotonin, leptin, Neuropeptide Y, and potentially other systems that exert an influence on and/or are influenced by dopamine.

In this regard, obesity is a result of a constellation of metabolic dysfunctions influenced by variation of and/or amplified genetic expressions (polymorphisms), as well as poor lifestyle decisions. Thus, successful body recomposition requires the simultaneous address of multiple systems. A novel Ca2+/K+ salt of (–)-hydroxycitric acid (HCA-SX) from Garcinia cambogia, has shown to be effective in obesity management. Appetite suppression of HCA is thought to result from increased glycogen production and concomitant stimulation of glucoreceptors in the liver, which sends signals of satiety to the brain.

However, recent clinical and pre-clinical studies show that HCA-SX increases levels of serotonin (5-HT), a vital neurotransmitter involved in a wide range of behavioral functions in the body, including mood, sleep and appetite control. Studies show that serotonin affects eating behavior and body weight. Increased plasma levels of serotonin are associated with decreased food intake, reduced weight gain and increased energy expenditure.

V. Conclusion The old adage “as the head goes, so goes the body, is 71

Chen et al: Association of The Taq A1 Allele of The Dopamine Receptor Gene (DRD2) and Percent Body Fat in Humans Blum K, Chen Tjh, Meshkin B, Blum Sh, Mengucci Jf, Notaro A, Arcuri V, Waite Rl, Braverman Er (2007) The PPAR-!

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Downs BW, Bagchi M, Subbaraju GV, Shara MA, Preuss HG, Bagchi D (2005) Bioefficacy of a novel calcium-potassium salt of (-)-hydroxycitric acid. Mutat Res 579, 149-62.

Dulloo A and Jacquet J (1998) Adaptive reduction in basal metabolic rate in response to food deprivation in humans: a role for feedback signals from fat stores. Am J Clin Nutr 68, 599-606.

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a simple but appropriate characterization of the symbiosis that exists between the brain, the genome and the resulting metabolomic symphony. We propose that while obesity is a polygenic disorder, our findings indicate a putative important role of the dopamine D2 receptor gene in morbid obesity, especially in high risk populations (Jenkinson et al, 2000; Noble, 2003), and that high to low quality lifestyle factors can exert a positive to negative influence respectively on gene expressions accordingly. Most recently it has been determined by Malis and colleagues (2005) that total and regional fat distribution is strongly influenced by genetic factors in young and elderly twins.

Specifically, the hereditability factor h2=0.83 (young) and 0.86 (elderly). In this regard, our results further suggest that certain polymorphisms of the DRD2 gene observed to associate with percent body fat in this study and possibly fat distribution, may provide the first evidence for involvement of this gene in neurobeisigenics (a proposed new name for Obesity and related disorders) and warrants further independent systematic investigation.

Acknowledgements The concept and design of the study was developed

by Kenneth Blum. THJ Chen, K Blum, G Kaats, D Pullin, ER Braverman, DE Comings, contributed to the execution of the study. K Blum, DE Comings, G Kaats, D Pullin and ER Braverman were involved in patient recruitment. The concept of “Neurobesigenics” was initiated by K Blum, BW Downs and B Meshkin. Editorial input was contributed by Debasis Bagchi, Manashi Bagchi, Ariel Robarge, J. Mengucci, BW Downs, B Meshkin, and DE Comings. Reference styling to conform with journal guidelines was contributed by SH Blum, Vanessa Arcuri, Michael Varshavskiy. The manuscript was drafted by K Blum, TJH Chen, DE Comings, ER Braverman, BW Downs, B Meshkin. All authors commented on the text and the team revised accordingly. K Blum had full access to all the data in the study and had final responsibility for the decision to submit for publication.

The authors would like thank Salugen, Inc., San Diego, California, Path Medical Research Foundation, New York, NY, for their financial support and acknowledge the editorial assistance of Gina S. Bender, Edward N. Bender, Randy Smart and Kristi Brandon. The authors want to thank Dr. Mathew Mcgue for allowing us to utilize the lean controls.

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Gene Therapy and Molecular Biology Vol 11, page 75 Gene Ther Mol Biol Vol 11, 75-78, 2007

Genetically modified stem cells for cellular therapy Review Article

Philippe Taupin National Neuroscience Institute, Singapore and National University of Singapore and Nanyang Technological University, Singapore

__________________________________________________________________________________ *Correspondence: Philippe Taupin, Ph.D., National Neuroscience Institute, Singapore, 11 Jalan Tan Tock Seng, Singapore 308433; Tel: (65) 6357 7533; Fax: (65) 6256 9178; e-mail: obgpjt@nus.edu.sg Key words: embryonic stem cells, neural stem cells, somatic cell nuclear transfer, cellular therapy, gene therapy Abbreviations: altered nuclear transfer (ANT); central nervous system (CNS); green fluorescent protein (GFP); inner cell mass (ICM); Neural stem cells (NSCs); somatic cell nuclear transfer (SCNT) Received: 3 December 2006; Revised: 1 April 2007 Accepted: 11 April 2007; electronically published: May 2007

Summary Stem cells carry the promise to cure a broad range of diseases and injuries, from diabetes, to neurological diseases and injuries. Over the past decade, significant progresses have been made in stem cell research; the derivation of embryonic stem cells (ESCs) from human tissues, the development of somatic cell nuclear transfer (SCNT) technology, and the confirmation that neurogenesis occurs in the adult mammalian brain, including in human.

Despite these advances, there may be decades before stem cell research translates into therapy. Beside the scientific and technical challenges, there are ethical and political constraints and debates over stem cell research, particularly on ESCs and SCNT. In this manuscript, I will discuss how gene therapy is applied to stem cell research, in an attempt to unlock some of the technical, ethical and political hurdles associated with stem cell research.

brain and NSCs reside in the adult central nervous system (CNS) in mammals, including in human (Gage, 2000; Taupin and Gage, 2002; Ming and Song, 2005). Hence, the CNS may be amenable to repair. Neural progenitor and stem cells have been isolated from adult tissues (Reynolds and Weiss, 1992; Gage et al, 1995), including human postmortem (Palmer et al 2001), providing a source of tissues for the treatment of diseases and injuries of the nervous system. The origin, identity and potential of adult-derived neural progenitor and stem cells remain to be fully and unequivocally characterized before adult NSCs could be brought to therapy (Taupin, 2006a).

Genetically modifying cells has been determinant for the study of gene function, and as a therapeutic tool to restore gene function and produce biologically active substances, like neurotransmitter synthesizing enzymes and trophic factors (Verma and Weitzman, 2005). In this manuscript, I will review and discuss recent studies involving genetically modifying stem cells in aim to circumvent some of the technical, political and ethical hurdles of ESC research, and to bring NSC research to therapy.

I. Introduction ESCs are self-renewing pluripotent cells that generate cells from the three germ layers of embryos; neurectoderm, mesoderm and endoderm. ESCs carry the hope to cure a broad range of diseases and injuries, like diabetes, heart diseases, Alzheimerâ&#x20AC;&#x2122;s disease, Parkinsonâ&#x20AC;&#x2122;s disease and spinal cord injuries (Wobus and Boheler, 2005). ESCs are derived from the inner cell mass (ICM) of blastocysts, and have been derived from human donated embryos produced by in vitro fertilization (Thomson et al, 1998). The generation of hESCs provides an unlimited source of tissues for cellular therapy. Because their derivation involves the destruction of blastocysts, there are technical, political and ethical debates and constraints over the use of human ESCs (hESCs) for clinical research and therapy (Wobus and Boheler, 2005). To overcome these issues, investigators are devising strategies and protocols to derive ESCs that genetically matched the patients and without the destruction of embryos.

Neural stem cells (NSCs) are self-renewing multipotent cells that generate the main cell types of the nervous system; neurons astrocytes and oligodendrocytes.

Contrary to a long-held belief, neurogenesis occurs in the

75

Taupin: Genetically modifying stem cells for RNAi cdx2 and the green fluorescent protein (GFP), flanked by two LoxP sequences. The nuclei of genetically engineered fibroblasts, selected by means of GFP fluorescence, were transferred into enucleated oocytes, to produce eggs by ANT. The eggs divided, produced cloned blastocysts that were morphologically abnormal and lacked functional trophoblasts. The cloned blastocysts did not implant into the uterus, but ESCs could be derived from their ICMs. To maintain the developmental potential of the generated ESCs, the expression of Cdx2 was reestablished by deleting the cassette RNAi cdx2, using a lenti virus (Meissner and Jaenisch, 2005).

ANT is a source of controversies and debates; it is argued that ANT is “a flawed proposal”, as there is no basis for concluding that the action of CDX2 or any other gene, represents a transition point at which a human embryo acquires moral status (Melton et al, 2004). So, ANT does not resolve the ethical and political issue over the derivation of ESCs without the destruction of embryos.

In addition, though the expression of Cdx2 is reestablished in the cloned cells, it remains to further evaluate whether cloned ESCs with a temporarily inactivated gene CDX2 have the same developmental potential as ESCs derived from donated eggs. Studies have also reported that SCNT may alter the developmental potential of ESCs (Wakayama et al, 2006). All of which may affect the developmental and therapeutic potential of ESCs generated by ANT. Nonetheless, this study highlights the potential of genetically modifying cells for the advancement of research in stem cell biology.

In all, the therapeutic potential of SCNT combined with gene therapy is enormous. It has not only the potential to treat genetic and gene deficient diseases, but also to circumvent the ethical and political issues currently limiting ESC research. However, developmental issues and acceptance of these techniques remain the main concerns over their applications for the treatment of human diseases. Resolving the issues over the potential of ESC generated by SCNT will involve a deep understanding of the cells’ developmental mechanisms.

The acceptance of SCNT and ESCs for therapy will require further proofs of their potential to treat human diseases and strong legislation supporting and defining the research practice.

II. Genetic engineering to derive ESCs without the destruction of blastocysts Transplantations of ESCs derived from human embryos would require to genetically matching the grafts with the patients and/or use immune-suppressive drug, to avoid the rejection of the grafts by the patients. With the recent advance in SCNT, there is the potential to generate stem cell lines, tissues and organs that would have the patient own genetic make up, and thus not be rejected.

SCNT is a cloning strategy in which nuclei are isolated from a donor’s somatic cells, like fibroblasts, and are transferred into enucleated oocytes from female donors (Campbell et al, 1996). By mechanisms yet to be unraveled, the cytoplasm of the oocytes reprograms the chromosomes of the somatic cell nuclei and the cloned cells develop into blastocysts, from which ESCs can be derived (Wakayama, 2006). Thereby, by isolating nuclei from the patients’ somatic cells, there is the potential to generate isogenic ESCs, carrying a set of chromosomes identical to that of the patients. The potential of SCNT for therapy is further highlighted by the study of Rideout et al.

(2002). In this study the authors combined SCNT and gene therapy to develop strategies for the treatment of genetic diseases. The authors derived ESCs by SCNT from immunedeficient Rag2(-/-) mice, as a model of genetic disease. After correction of the ESCs’ gene defect by homologous recombination, transgenic mice were generated by tetraploid embryo complementation and hematopoietic precursor cells differentiated in vitro were grafted in mutant mice, from the ESCs. An immuno-competent phenotype was restored after tetraploid embryo complementation, whereas grafting of genetically engineered ESCs leaded to immuno-competent leaded immunoglobulins detetectable in the host (Rideout et al, 2002). This show that SCNT combined with gene therapy has the potential to treat genetic and gene deficient diseases. There are however, ethical and political debates over the use SCNT and

ESCs for therapy (Trounson and Pera, 1998; Jaenisch and Wilmut, 2001). Particularly, the generation of ESCs by SCNT, is subject to the same limitations as for their derivation from donated eggs, as it also involves the destruction of embryos.

Altered nuclear transfer (ANT) is a variation of SCNT proposed by Hurlbut in 2005. In ANT, the gene CDX2, a gene crucial for trophectoderm development, is inactivated in vitro in the donor cells. CDX2 encodes the earliest-known trophectoderm-specific transcription factor and is essential for establishment and function of the trophectoderm. Inactivating the gene CDX2 eliminates formation of the fetal-maternal interface, but spares the ICM from which ESCs could be derived. The nuclei deficient for CDX2 are then transferred into enucleated oocytes from female donors, and submitted to the same protocols as for SCNT. Because the eggs created from nuclei deficient for CDX2 produce embryos that are unable to implant into the uterus and do not pursue their developments, ANT has been proposed as a variation of nuclear transfer to derive ESCs, without the destruction of embryos (Hurlbut, 2005).

In 2005, Meissner and Jaenisch reported the use of ANT, to derive ESCs in mice. Meissner and Jaenisch, 2005 genetically modified the donor cells, mouse fibroblasts, by inserting in their genome a cassette coding

III. Genetically derived NSCs

modifying

adult-

Contrary to a long-held belief, neurogenesis occurs in the adult mammalian brain, including in human (Gage, 2000; Ming and Song, 2005). Neurogenesis occurs primarily in two areas of the adult brain, the dentate gyrus of the hippocampus and the subventricular zone. It is hypothesized that newly generated neuronal cells originate from stem cells in the adult brain (Gage, 2000). Neural stem and progenitor cells have been isolated and characterized in vitro from various regions of the adult CNS, including the spinal cord, supporting the existence of NSCs in the CNS (Taupin and Gage, 2002). The generation of new neuronal cells in the adult brain and the isolation and characterization of neural stem and progenitor cells from the adult CNS suggest that the adult 76

Gene Therapy and Molecular Biology Vol 11, page 77 brain may be amenable to repair. Cell therapy in the adult CNS could involve the stimulation of endogenous neural progenitor or stem cells, or the transplantation of adultderived neural progenitor and stem cells (Taupin, 2006b).

Adult-derived neural progenitor and stem cells have been transplanted in animal models, and shown functional engraftment, supporting their potential use for therapy (Shihabuddin et al, 2000).

Adult neural progenitor and stem cells can be genetically modified by retroviral-mediated infection, rendering them a vehicle for gene therapy (Gage et al, 1995). Adult-derived stem cells can be genetically engineered to boost or force their differentiation into a specific pathway. To this aim neural progenitor and stem cells can be genetically engineered to express gene synthesizing enzyme or key transcription factors involved in stem cell differentiation. Adult-derived neural progenitor and stem cells genetically engineered to express the transcription factor Nurr1, a nuclear receptor involved in the differentiation of dopaminergic neurons, have been successfully grafted in animal model of Parkinson’s disease and shown to improve functional deficits (Shim et al, 2007). Adult-derived neural progenitor and stem cells genetically modified to express acid sphingomyelinase reverse lysosomal storage pathology when transplanted into animal models of Niemann-Pick's disease (Shihabuddin et al, 2004). This highlights the potential of genetically modified NSCs for the treatment of neurodegenerative diseases, lysosomal storage diseases and other genetic diseases of the CNS. Fetal-derived neural progenitor and stem cells have been grafted in various models of neurological diseases and injuries, like Parkinson’s disease and spinal cord injury, and shown to improve their neurological deficits (Ourednik et al, 2002; Yan et al, 2004). In these studies, the most likely mechanism of functional recovery is through the synthesis and release of neuroprotective substances by the grafted cells. Genetically modifying neural progenitor and stem cells could therefore also be applied for delivering trophic factors for the treatment for neurodegenerative diseases.

These data highlight the potential therapeutic of genetically modifying neural progenitor and stem cells for the treatment of CNS diseases and disorders. The potential of genetically modified NSCs is further highlighted by their potential for the treatment of brain tumors. Neural progenitor and stem cells migrate to tumors, injured, diseased sites when transplanted in the CNS, either by systemic injection, or through the cerebrospinal fluid (Brown et al 2003; Fujiwara et al 2004). The injected cells migrate to the diseased or degenerated areas where they integrate the host tissue. The properties of NSCs to be genetically modified and to migrate to tumor sites have been proposed for the treatment of brain tumors. It is proposed to genetically modified NSCs with “suicide genes”, like genes coding for cytolytic activities or antitumor cytokines, to attack and destroy brain tumor cells (Yip et al, 2003). This further extends the use of cell engineering of NSCs for cancer therapy in the CNS.

In all, adult neural stem cells have the potential to treat a vast array of neurological diseases, without the ethical and political and ethical issues surrounding ESC research. However, NSC remains an elusive cell. Further studies will aim at identifying and characterizing neural progenitor versus stem cells, at generating homogenous populations of neural progenitor or stem cells, and devising protocols to further enhance the differentiation potential of neural progenitor and stem cells.

IV. Conclusion Stem cell therapy holds the promise to treat a broad range of diseases and injuries. The promise of stem cell research and therapy is to regenerate and reconstruct the original pathway to promote functional recovery, but it may be years away before it emerges as a viable therapy.

Genetically modifying cells has proven valuable to understand gene function, and to deliver trophic factors or neurotransmitter synthesizing enzymes in the CNS. The studies reported show that genetically modifying stem cells may therefore offer an opportunity to bolster stem cell research and therapy. Further studies involving stem cell research and gene therapy will aim particularly at devising strategies to derive pluripotent stem cells without the destruction of embryos that are suitable for therapy, at understanding the role of trophic factors in the in mediating recovery in stem cell transplant and developing vectors allowing sustained expression of the transgene of interest.

Acknowledgments P.T. is supported by grants from the NMRC, BMRC, and the Juvenile Diabetes Research Foundation.

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Campbell KH, McWhir J, Ritchie WA and Wilmut I (1996) Sheep cloned by nuclear transfer from a cultured cell line Nature 380 64-66.

Fujiwara Y, Tanaka N, Ishida O, Fujimoto Y, Murakami T, Kajihara H, Yasunaga Y and Ochi M (2004) Intravenously injected neural progenitor cells of transgenic rats can migrate to the injured spinal cord and differentiate into neurons, astrocytes and oligodendrocytes Neurosci Lett 366 287-291 Gage FH (2000) Mammalian neural stem cells Science 287 1433-1438.

Gage FH, Coates PW, Palmer TD, Kuhn HG, Fisher LJ, Suhonen JO, Peterson DA, Suhr ST and Ray J (1995) Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain Proc Natl Acad Sci USA 92, 1187911883.

Hurlbut WB (2005) Altered nuclear transfer N Engl J Med 352, 1153-1154.

Jaenisch R and Wilmut I (2001) Developmental biology. Don't clone humans! Science 291, 2552.

Meissner A and Jaenisch R (2006) Generation of nuclear transfer-derived pluripotent ES cells from cloned Cdx2deficient blastocysts Nature 439, 212-215.

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Taupin: Genetically modifying stem cells Melton DA, Daley GQ and Jennings CG (2004) Altered nuclear transfer in stem-cell research - a flawed proposal N Engl J Med 351, 2791-2792.

Ming GL and Song H (2005) Adult neurogenesis in the mammalian central nervous system. Annu Rev Neurosci 28, 223-250.

Ourednik J, Ourednik V, Lynch WP, Schachner M and Snyder EY (2002) Neural stem cells display an inherent mechanism for rescuing dysfunctional neurons Nat Biotechnol 20, 11031110.

Palmer TD, Schwartz PH, Taupin P, Kaspar B, Stein SA and Gage FH (2001) Cell culture. Progenitor cells from human brain after death Nature 411, 42-43.

Reynolds BA and Weiss S (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system Science 255, 1707-1710.

Rideout WM 3rd, Hochedlinger K, Kyba M, Daley GQ and Jaenisch R (2002) Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy Cell 109, 17-27.

Shihabuddin LS, Horner PJ, Ray J and Gage FH (2000) Adult spinal cord stem cells generate neurons after transplantation in the adult dentate gyrus. J Neurosci 20, 8727-8735.

Shihabuddin LS, Numan S, Huff MR, Dodge JC, Clarke J, Macauley SL, Yang W, Taksir TV, Parsons G, Passini MA, Gage FH and Stewart GR (2004) Intracerebral transplantation of adult mouse neural progenitor cells into the Niemann-Pick-A mouse leads to a marked decrease in lysosomal storage pathology J Neurosci 24, 10642-10651.

Shim JW, Park CH, Bae YC, Bae JY, Chung S, Chang MY, Koh HC, Lee HS, Hwang SJ, Lee KH, Lee YS, Choi CY and Lee SH (2007) Generation of Functional Dopamine Neurons from Neural Precursor Cells Isolated from the Subventricular Zone and White Matter of the Adult Rat Brain using Nurr1 Overexpression. Stem Cells Epub ahead of print.

Taupin P (2006a) The therapeutic potential of adult neural stem cells. Curr Opin Mol Ther 8, 225-231.

Taupin P (2006b) Neurogenesis in the adult central nervous system C R Biol 329, 465-475.

Taupin P and Gage FH (2002) Adult neurogenesis and neural stem cells of the central nervous system in mammals J Neurosci Res 69, 745-749.

Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS and Jones JM (1998) Embryonic stem cell lines derived from human blastocysts Science 282, 1145-1147. Erratum in: (1998) Science 282, 1827.

Trounson A and Pera M (1998) Potential benefits of cell cloning for human medicine Reprod Fertil Dev 10, 121-125.

Verma IM and Weitzman MD (2005) Gene therapy: twenty-first century medicine. Annu Rev Biochem 74, 711-738.

Wakayama S, Jakt ML, Suzuki M, Araki R, Hikichi T, Kishigami S, Ohta H, Van Thuan N, Mizutani E, Sakaide Y, Senda S, Tanaka S, Okada M, Miyake M, Abe M, Nishikawa S, Shiota K and Wakayama T (2006) Equivalency of nuclear transfer-derived embryonic stem cells to those derived from fertilized mouse blastocysts Stem Cells 24, 2023-2033.

Wakayama T (2006) Establishment of nuclear transfer embryonic stem cell lines from adult somatic cells by nuclear transfer and its application Ernst Schering Res Found Workshop 60, 111-123.

Wobus AM and Boheler KR (2005) Embryonic stem cells: prospects for developmental biology and cell therapy.

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Yan J, Welsh AM, Bora SH, Snyder EY and Koliatsos VE (2004) Differentiation and tropic/trophic effects of exogenous neural precursors in the adult spinal cord J Comp Neurol 480, 101-114.

Yip S, Aboody KS, Burns M, Imitola J, Boockvar JA, Allport J, Park KI, Teng YD, Lachyankar M, McIntosh T, O'Rourke DM, Khoury S, Weissleder R, Black PM, Weiss W and Snyder EY (2003) Neural stem cell biology may be well suited for improving brain tumor therapies Cancer J 9, 189204.

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Gene Therapy and Molecular Biology Vol 11, page 79 Gene Ther Mol Biol Vol 11, 79-92, 2007

Gene therapy trials for the treatment of high-grade gliomas Review Article

Adam M. Sonabend, Ilya V. Ulasov, Maciej S. Lesniak* Division of Neurosurgery, The University of Chicago, Chicago, Illinois, USA

__________________________________________________________________________________ *Correspondence: Maciej S Lesniak MD, The University of Chicago, Division of Neurosurgery 5841 S. Maryland Avenue, MC 3026, Chicago, IL 60637, USA; Tel: (773) 834-4757; Fax: (773) 834-2608; E-mail: mlesniak@surgery.bsd.uchicago.edu Key words: Glioblastoma multiforme (GBM), brain tumor, glioma, clinical trial, gene therapy, virus Abbreviations: adenoviral vectors with HSV-tk, (Ad-HSV-tk); billion infectious units, (BIU); central nervous system, (CNS); Conditionally-replicative adenoviral vectors, (CRAd); coxsackie and adenovirus receptor, (CAR); ganciclovir, (GCV); glioblastoma multiforme, (GBM); Herpes simplex virus type 1 thymidine kinase, (HSV-tk); herpes simplex virus, (HSV); neural stem cells, (NSCs); Newcastle disease virus, (NDV); plaque forming units, (p.f.u.); retrovirus, (RV); retrovirus-mediated herpes simplex virus type 1 thymidine kinase gene therapy, (RV HSV-tk); viral producing cells, (VPC) Received: 26 April 2007; Accepted: 7 May 2007; electronically published: June 2007

Summary High-grade gliomas remain relatively resistant to current therapy. Local recurrence is a common feature and the majority of patients progress despite conventional therapy. One modality-gene therapy-has shown a lot of promise in early preclinical and clinical studies aimed at advancing the treatment of this disease. In this review, we provide a comprehensive overview of clinical trials involving gene therapy in the field of neuro-oncology. The use of different delivery vehicles, including liposomes, cells, and viruses, as well genes, especially cytokines and suicide genes, are explored in detail. The unique features and advantages/disadvantages of the different vectors employed are compared based on results of human studies. We discuss both the limitations and successes encountered in these clinical trials, with an emphasis on the lessons learned and potential ways of improving current gene therapy protocols.

A significant increase in survival of patients with malignant brain tumors is a major goal of therapy and thus, a wide variety of strategies are being explored. Some of the experimental treatments are based on immunotherapy, stem cell therapy, local chemotherapy and radiotherapy (Liau et al, 1999; Lesniak et al, 2001; Yu et al, 2001, 2004; Ehtesham et al, 2002; Ehtesham et al, 2005). In addition, gene therapy is becoming a promising therapeutic alternative. Indeed, the fact that the majority of brain tumors do not metastasize outside of the CNS may allow for local delivery of vectors carrying therapeutic genes (Immonen et al, 2004; Pulkkanen and Yla-Herttuala 2005).

In the last few decades, a considerable amount of research dealing with gene therapy for glioma has been conducted in vitro and in animal models. In the case of human studies, the first clinical trials involving gene therapy for gliomas were published in the 1990â&#x20AC;&#x2122;s. These pioneer studies consisted of retrovirus-mediated herpes simplex virus type 1 thymidine kinase gene therapy (RV HSV-tk) delivered by intra-tumoral injections of viral producing cells (VPC) followed by systemic

I. Introduction Gliomas are the most common form of primary intracranial malignancy. Unfortunately, high-grade gliomas like glioblastoma multiforme (GBM, WHO grade IV) are the most frequently encountered and carry the worst prognosis (Annegers et al, 1981; Louis et al, 2001).

The characteristic resistance to treatment shown by highgrade gliomas resides in their biological behavior and their location within the central nervous system (CNS). As most cancers, gliomas are subject to constant genotypic and phenotypic alterations that can lead to treatment resistance. Resistant cell populations get selected once a therapy is administered.

In addition, most chemotherapeutic agents cannot effectively reach all tumor cells as the blood-brain-barrier limits the penetration of these drugs to brain tumors (Lesniak and Brem 2004). With respect to surgical treatment, the complete resection of high-grade gliomas remains a virtually impossible task since the nature of these tumors is to infiltrate diffusely within surrounding brain parenchyma (Ehtesham et al, 2005).

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Sonabend et al: Gene therapy for glioma administration of ganciclovir (GCV) (Oldfield et al, 1993; Raffel et al, 1994; Kun et al, 1995). Since then, a growing number of trials have gathered information regarding the efficacy and safety of this emerging therapeutic approach (Table 1, Figure 1). In this text, we will focus on the

clinical trials of gene therapy for the treatment of brain tumors. The main vehicles and transgenes employed for the purpose of gene therapy for gliomas will be explored under this scope.

Table 1. Clinical adenovirus-mediated gene therapy studies for the treatment of malignant glioma. Abbreviations: Ad, adenovirus; pfu, plaque-forming unit; Rv, retrovirus; Ab, antibody; vp, viral particle. Reproduced from Pulkkanen and YlaHerttuala 2005 with kind permission from Macmillan Publishers Ltd: [Molecular Therapy].

Figure 1. Median survival in clinical gene therapy trials for malignant glioma. Trials involving less than nine patients and/or trials where full survival data is not available were excluded. Filled bars represent VPCs-mediated RV-HSV-tk gene therapy; open bars randomized controlled group. (â&#x20AC;˘) Adenovirus-mediated HSV-tk gene therapy; (o) ONYX-015 therapy; (!) adenovirus-mediated p53 gene therapy; (") Herpes simplex type 1 mutant therapy Reproduced from Pulkkanen and Yla-Herttuala 2005 with kind permission from Macmillan Publishers Ltd: [Molecular Therapy].

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Gene Therapy and Molecular Biology Vol 11, page 81 entrapped components into cells, compared to other types of liposomes (Felgner et al, 1987; Felgner et al, 1989; Shi and Pardridge 2000; Yoshida et al, 2004b). Moreover, delivery of genes via DNA/liposome complexes to the brain could be achieved by incorporating antibodies to the transferrin receptor in order to facilitate passage across the BBB (Shi and Pardridge 2000, Yoshida et al, 2004b).

Liposomes - as gene therapy vehicles for brain tumors - have been tested in humans. A pilot clinical trial to evaluate the safety and effectiveness of interferon # gene therapy with a liposomal vector was performed in five patients with recurrent malignant glioma (glioblastoma multiforme or anaplastic astrocytoma) (Yoshida et al, 2004a). Transgene expression and antitumor activity were detected in four patients. The general and neurological condition of all patients was the same or had improved 3 months after starting therapy, except for one patient. Two patients showed a partial response (50% tumor reduction) and two others had stable disease 10 weeks after beginning therapy. No direct toxicity attributable to the liposomes was seen in the study.

A phase I/II clinical trial involving liposomal delivery of HSV-tk followed by treatment with i.v. GCV for 14 days has also been performed in patients with recurrent glioblastoma (Jacobs et al, 2001). Transgene expression was assessed by positron emission tomography using a radiolabeled substrate for HSV-tk. In this study, there was evidence of HSV-tk expression in one of the five patients evaluated. Similarly, in a prospective phase I/II clinical study, eight patients bearing recurrent glioblastoma multiforme were treated with stereotactic intra-tumoral convection-enhanced delivery of an HSV-tk gene-bearing liposomal vector followed by systemic GCV (Voges et al, 2003). Treatment was well tolerated without major side effects. In two out of eight patients, a reduction of tumor volume greater than 50% was noted.

Some authors argue that liposome-based systems are less efficient for gene transfer than viral vectors. On the other hand, the former have the hypothetical advantage of being relatively safe (Schatzlein, 2001). Regardless of the view, liposomes are not likely to surpass viral vectors as tools for most of cancer gene therapy strategies (Pulkkanen and Yla-Herttuala 2005). Due to the relative safety that viral vectors have achieved in recent human studies, the main reason for using liposomes seems to be fading away in the context of the much greater transgene expression achieved by viral vectors. Nevertheless, as long as the lack of an effective therapy for gliomas remains, the suitability of the best vector remains a matter of open debate.

II. Strategies and approaches for gene therapy of gliomas Gene therapy consists of the delivery of a gene of interest to tumor cells in order to control and when possible, kill the growing tumor. The strategies chosen to this end vary. In some cases, the idea is to reestablish a tumor suppressor gene like p53 that is frequently disrupted in a malignancy (Kim et al, 2001; Lang et al, 2003; Geoerger et al, 2004). In other cases, the transgene is an enzyme that elicits a toxic effect in the presence of a drug.

Herpes simplex virus type 1 thymidine kinase (HSV-tk) in combination with ganciclovir is an illustrative example of the latter principle; this system is one of the first and most extensively studied gene therapy approaches in human trials for gliomas (Klatzmann et al, 1998, Kun et al, 1995, Oldfield et al, 1993, Prados et al, 2003, Raffel et al, 1994).

Other transgenes tested in the preclinical and phase 1 clinical settings are used to evoke an effective anti-tumor immune response; these include interleukins and interferons (Eck et al, 2001, Kunwar 2003, Okada et al, 2001, Okada et al, 2000, Yoshida et al, 2004a, Yoshida et al, 2004b). Finally, antisense oligonucleotides have also been explored as transgenes since these sequences can shut down oncogenes that play a significant role in the neoplastic phenotype (Zhang et al, 1998; Andrews et al, 2001; Ly et al, 2001; Matsuno and Nagashima 2004).

Although many of these modalities appear interesting and highly sophisticated, only few turn out useful at the bedside.

In order treat a brain tumor with gene therapy, a transgene needs to be delivered into the tumor cells with the aid of a vehicle. Vehicles vary; they include liposomes, cells and viruses. In some cases, distinct vehicles are combined and complemented by each other. For instance, cells have been transfected to generate viral particles that can target tumors. In this text, even though many transgenes are explored, gene therapy systems for glioma are discussed under the scope of the employed vectors.

III. Liposomal vectors DNA injection has been shown to induce transgene expression in target cells when introduced with the aid of a carrier molecule. Liposomes are an illustrative example of such principle. These vectors consist of artificially produced lipid vesicles that can entrap drugs in either their aqueous compartment or their lipid bi-layer (Yoshida et al, 2004a). In order to achieve transgene expression, liposomes need to attach to the target cell surface, be internalized, escape from endosomes, find a way to the nucleus and finally, keep their DNA available for transcription (Thomas and Klibanov, 2003).

Liposomes possess several characteristics that render them suitable for clinical gene transfer trials: they are simple and easy to prepare in large quantities; the prepared liposomes can be sterilized; and they show no intrinsic toxicity or tissue specificity (Yoshida et al, 2004a).

Molecular modifications can add special features to liposomes in order to enhance their transgene delivery capacity. For instance, liposomes bearing positively charged molecules provide more efficient delivery of their

IV. Cells as vectors Most recently, cells have been used as vehicles to carry and produce viral vectors. Indeed, as discussed elsewhere in this text, RV-based gene therapy trials (Oldfield et al, 1993; Raffel et al, 1994; Kun et al, 1995; Palu et al, 1999; Rainov, 2000; Colombo et al, 2005) injected PA317 cells to produce viral particles. These VPC derive from an embryonic mouse fibroblast cell line (Lyons et al, 1995) and have a limited reach since they are unable to migrate. In contrast, stem cells offer the capacity 81

Sonabend et al: Gene therapy for glioma to migrate towards tumor cells. In fact, neural stem cells (NSCs) are a promising tool to target disseminated tumor cells. NSCs have a tropism for infiltrating cancer cells, so they can be used to deliver therapeutic agents directly to tumor pockets that reside beyond the healthy appearing surgical margin (Yip et al, 2003; Ehtesham et al, 2005).

Additionally, mesenchymal stem cells can also be used for this purpose since these cells can migrate to encounter malignant cells in the CNS (Nakamizo et al, 2005, Nakamura et al, 2004).

In addition to fibroblasts and stem cells, genetically modified glioma cells have been evaluated as gene therapy vectors for brain tumors. In fact, a protocol for a phase I study involving genetically modified autologous tumor cells has been reported. Specifically, IL-4 and HSV-tk were carried as transgenes (Okada et al, 2000). The same group also announced a pilot study of vaccination with irradiated autologous glioma and dendritic cells admixed with IL-4 transduced fibroblasts to elicit an immune response (Okada et al, 2001). The therapy was well tolerated and there was no incidence of autoimmune encephalitis in enrolled subjects (Okada personal communication). From that trial, a case report has been published (Okada et al, 2003). The therapy consisted in the combination of fibroblasts transfected with IL-4 and irradiated autologous glioma cells injected intradermally.

With respect to the immune response, the patient showed infiltration of dendritic cells (CD1a+), CD4+ and CD8+ T cells. In fact, the cells increased proportionally to the amount of IL-4 produced at the each site. This patient demonstrated partial clinical response. Treatment was well tolerated and the patient survived for 10 months since the initiation of the treatment.

A. Non-replicating viral vectors Non-replicating vectors were initially considered safer since the risk of uncontrolled infection leading to neural and systemic toxicities is theoretically lower when compared to their oncolytic counterparts. On the other hand, transgene expression of non-replicating adenoviral and RV vectors has been shown to be too deficient to translate into significant clinical outcome.

Retroviruses have been proven safe for intracranial injection and for a long time have been a popular vehicle for gene therapy trials for glioblastoma multiforme.

Replication deficient RV in combination with VPC were initially used for gene therapy trials. These cells were injected into the tumor cavity and subsequently produced and secreted the vectors (Oldfield et al, 1993; Raffel et al, 1994; Kun et al, 1995).

Interestingly, some studies have tested a bicistronic RV-based system that carries interleukin-2 and HSV-tk in patients with recurrent glioblastoma multiforme. A pilot study with four patients showed varying transgene activity in the tumors of the treated patients. (Palu et al, 1999). The study was then extended to a larger population of patients and evaluated safety, feasibility and biological activity of treatment. This time a total of 12 patients received intratumoral injection of VPC followed by intravenous GCV. Treatment was well tolerated with only minor, grade 1 and 2, adverse events. These included transient increase of liver transaminases and transient leukocytosis.

Transduction of tumor cells was demonstrated in tumor biopsies. A marked and persistent increase of intratumoral and plasma Th1 cytokine levels was demonstrated after treatment. Results of this trial suggest that the combined delivery of a suicide and a cytokine gene is safe, it is capable of inducing transgene transduction, it can lead to the activation of systemic cytokine cascade and there are tumor responses in up to 50% of cases (Colombo et al, 2005).

In 2000, Rainov published one of the most representative RV-based studies; the vector consisted of a recombinant replication-deficient RV with HSV-tk transgene as insert. The study consisted of a multicenter, randomized, controlled phase III clinical trial (Rainov, 2000). RV vector particles were injected during surgery, followed by systemic treatment with GCV. Unfortunately, clinical outcomes including time to tumor progression, progression-free median survival, median survival and 12month survival rates showed no significant differences between treatment and control groups (Figure 2). This study was significant because it was one of the few phase III gene therapy trials performed in the setting of malignant glioma which failed to reveal any efficacy.

Retroviruses have a series of disadvantges that make them inefficient gene therapy vectors. For instance, RVs exhibit a low transduction efficacy (Vile and Russell 1995; Rainov and Ren 2003; Pulkkanen and Yla-Herttuala 2005). Moreover, RV can only integrate into the genomes of replicating cells since they require the dissolution of the nuclear membrane. This is important since gliomas also contain non-cycling cells (Fueyo et al, 2000), implying ineffective transgene expression when these vehicles are used. In addition, the low transgene carrying capacity (8

V. Viral vehicles Viral vehicles are naturally capable of transferring genes into target cells. There are many kinds of viruses that have been explored for the purpose of gene therapy for brain tumors. These include herpes simplex virus (HSV) (Markert et al, 2000; Rampling et al, 2000; Papanastassiou et al, 2002; Kambara et al, 2005), retrovirus (RV) (Oldfield et al, 1993; Raffel et al, 1994; Rainov, 2000), measles virus (Phuong et al, 2003), reovirus (Coffey et al, 1998; Yang et al, 2004), adenoassociated virus (Mizuno et al, 1998), Newcastle disease virus (NDV) (Csatary and Bakacs 1999; Russell 2002), Semliki Forest virus (Ren et al, 2003), vaccinia virus (Gridley et al, 1998; Timiryasova et al, 1999; Chen et al, 2001), poliovirus (Gromeier et al, 2000; Jackson et al, 2001) and adenovirus (Miller et al, 1998; Dmitriev et al, 2000; Fueyo et al, 2000; Suzuki et al, 2001; Lamfers et al, 2002; van Beusechem et al, 2002; Chiocca et al, 2004).

Viral vectors can be divided into those that can replicate and lyse tumor cells, also known as oncolytic vectors, and those that do not replicate but can carry transgenes into their targets, known as non-replicating vectors. In the case of the latter kind, the therapeutic effect is solely achieved due to the activity exerted by the transgene expressed in target cells.

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Gene Therapy and Molecular Biology Vol 11, page 83 kb approximately) and the risk of insertional mutagenesis add to the list of disadvantages of these vectors.

Besides RV, adenoviral vectors constitute a popular option for gene therapy of brain tumors. In fact, these vehicles are currently being used in approximately one quarter of all gene therapy trials (McConnell and Imperiale, 2004). These vectors are rendered replication deficient by deletion of early regions of viral genomes. In contrast to RV, non-replicating adenoviruses offer a considerable deal of advantages. Adenoviral vectors transduce both dividing and quiescent cells (both kind found in gliomas) (Pulkkanen and Yla-Herttuala 2005; Sonabend et al, 2006), are efficient with regard to transgene expression and exhibit a safety record as shown in human trials (Sandmair et al, 2000; Chiocca et al, 2004; Lichtenstein and Wold 2004). In addition, adenoviruses can be engineered to restrict transduction (Fueyo et al, 2003, van Beusechem et al, 2002) and transgene

expression to the desired target cell population (Parr et al, 1997; Shinoura et al, 2000; Vandier et al, 2000; Kambara et al, 2005). These features are important in order to decrease toxicity.

In the year 2000, Sandmair et al., published the first clinical trial involving adenoviral vectors (Sandmair et al, 2000). The study compared retroviral vs. adenoviral delivery of HSV-tk. It included 21 patients with primary (n=8) and recurrent (n=13) gliomas that were treated with intra-operative viral injection followed by i.v. GCV as adjuvant therapy. As control, one group of patients received Ad-LacZ (no GCV in these patients). Follow-up included MRI scans to assess disease status (Figure 3). No serious secondary effects were reported. Surprisingly, median survival time of the group treated with Ad-HSV-tk (15 months) was significantly longer than that of RVHSV-tk (7.4 months) and Ad-LacZ (8.3 months) (p< 0.012) (Figure 4) (Sandmair et al, 2000).

Figure 2. Kaplan-Mayer survival graphs of patients with RV HSV-tk and GCV treatment vs. standard treatment from a phase III clinical trial with patients with GBM (Rainov, 2000). (A) Graph showing time to death (overall survival time). (B) Graph showing time to death (overall survival time) for all patients in whom GBM was confirmed by central pathology review. Differences between groups are not significant. Reproduced from Rainov, 2000 with kind permission from Human Gene Therapy.

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Figure 3. VPC-mediated RV-HSV-tk gene therapy was compared to Ad-HSV-tk gene therapy (Sandmair et al, 2000). MRI follow-up 3 months after treatment. (A-C) MRI of patient 10 shows a right temporal GBM, (A) before, (B) day 1 and (C) 3 months after VPC mediated RV-HSV-tk treatment. Shown is a subtotal surgical resection and fast regrowth of the tumor despite the gene therapy. (D-F) MRI of patient 19 shows a left recurring frontal anaplastic astrocytoma before (D), 1 day (E) and 3 months (F) after the operation, radiation and adenovirus-mediated gene therapy. Shown is a total tumor resection and no signs of tumor regrowth 3 months after the treatment. (J-L) MRI of patient 21 shows a left recurring frontoparietal GBM before (J), 1 day (K) and 3 months (L) after reoperation and adenovirus mediated gene therapy. Shown is a subtotal tumor resection and no signs of tumor regrowth 3 months after treatment.

Reproduced from Sandmair et al, 2000 with kind permission from Human Gene Therapy.

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Figure 4. Kaplan-Mayer survival graph of patients after Ad or VPC-RV-mediated or HSV-tk gene therapy for GBM. The difference in outcome between the patients with adenovirus-mediated gene therapy (n=7) and VPC-RV-mediated gene therapy (n=7) was statistically significant (p < 0.012, Fisher exact test). Patients who received #-galactosidase marker gene (n=7) served as controls. X, Means survival time for each group. Reproduced from Sandmair et al, 2000 with kind permission from Human Gene Therapy.

These results encouraged Immonen and colleagues to continue the evaluation of Ad-HSV-tk in a phase IIb randomised, controlled trial (Immonen et al, 2004). Thirtysix patients with operable primary or recurrent malignant glioma were randomized to receive AdvHSV-tk injection (n=17) followed by i.v. GCV or standard care consisting of radical excision (additional radiotherapy in patients with primary tumors) (n=19). The primary end-point was survival as defined by death or surgery for recurrence.

Secondary end-points were all-cause mortality and tumor progression as determined by MRI. Findings were also compared with historical controls (n = 36) from the same unit over 2 years preceding the study. Ad-HSV-tk treatment produced a clinically and statistically significant increase in mean survival from 39.0 +/- 19.7 to 70.6 +/52.9 weeks (P = 0.0095). The median survival time increased from 37.7 to 62.4 weeks (Figure 5). The treatment was well tolerated (Immonen et al, 2004).

Nevertheless, as in the case of RV, replication deficient adenoviruses are not very efficient in their capacity for transgene expression. Indeed, a phase I clinical trial of p53 gene therapy was performed using a replication-defective adenoviral vector with wild type p53 (Ad-p53, INGN 201) against malignant brain tumors (Lang et al, 2003). The vector was stereotactically injected intratumorally via an implanted catheter. Treated tumor specimens were obtained and analyzed afterwards. In all patients, exogenous p53 protein was detected within the nuclei of astrocytic tumor cells and transgene expression induced apoptosis of targeted cells. However, with the use of this replication-defective vector, transgene expression was limited to within 5 mm of the injection site.

B. Replicating viral vectors Many scientists favor replication competent vectors over replication defective counterparts. It is thought that since oncolytic viruses exhibit higher replication, infectivity and transgene expression, these vectors could offer a significant advantage. The Ad-p53 cited INGN 201 trial, among other comparative studies suggest this observation (Ichikawa and Chiocca 2001; Lang et al, 2003). Nevertheless, these theoretical advantages remain to be proven by efficacy endpoints such as patient survival.

In the oncolytic virus category, the vectors that have made it to the bedside are based on HSV, the adenovirus and New Castle virus. In the case of HSV oncolytic vectors, a few have been tested in phase I clinical trials where their safety has been proven. Even though only modest results have thus far been obtained with these viruses, their effectiveness remains to be tested in future trials (Markert et al, 2000; Rampling et al, 2000; Papanastassiou et al, 2002).

G207 is one of the HSV-based oncolytic vectors.

This virus has deletions of both $(1)34.5 loci and a lacZ insertion disabling the UL39 gene. With these deletions, HSV-1 no longer produces hemorrhagic, necrotizing encephalitis characteristic of wild-type HSV-1 infection in human CNS (Shah et al, 2003). This vector was tested in a dose-escalation phase I study for patients with malignant glioma. The study included 21 patients with recurrent malignant glioma. Dosage varied from 1x106 plaque forming units (p.f.u.) inoculated at a single site to 3x109 p.f.u. at five sites. No serious adverse effects were attributed to this treatment and no patient developed HSV encephalitis. Radiographic and neuropathologic evidence

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Sonabend et al: Gene therapy for glioma suggestive of anti-tumor activity and long-term presence of viral DNA was documented in 8/20 cases (Markert et al, 2000). There are current plant to continue the evaluation of this vector in Phase Ib and Phase II clinical trials and recruitment for these studies has begun (Shah et al, 2003).

HSV 1716, an oncolytic vector based on wild type HSV-1 17 strain, contains a single deletion in ICP34.5.

This vector was also tested in a phase I clinical trial (Rampling et al, 2000). A total of nine patients with relapsed malignant glioma received intra-tumoral injections of doses up to 1x105 p.f.u. There were no cases of encephalitis and no adverse clinical symptoms. Of nine patients treated, four remained alive and neurologically stable at 14 to 24 months after the vectorâ&#x20AC;&#x2122;s administration (Rampling et al, 2000). In addition, the same group conducted another phase I study with HSV 1716 in twelve patients with recurrent malignant glioma (same dose of 1x105 p.f.u.) (Papanastassiou et al, 2002). In the latter study, the authors documented viral replication in tumors resected four to nine days posterior to injection date. HSV

DNA was assessed by PCR at the sites of inoculation in ten patients and at distal tumor sites in four. Interestingly, viral replication took place in both HSV-seropositive and seronegative patients (Papanastassiou et al, 2002).

Conditionally-replicative adenoviral vectors (CRAd) have been tested in clinical trials as well. CRAd vector dl1520, so called ONYX 015 is a pioneer in this category.

Its replication selectivity is explained as follows: E1B 55K is an early expression adenoviral protein that binds and inhibits p53, consequently prolonging host cell life and favoring viral progeny (Bischoff et al, 1996; Heise et al, 1997; Ramachandra et al, 2001). ONYX 015 has a deletion in the viral genomic region coding E1B 55K. Such deletion restricts the replication of this CRAd to neoplastic cells with defective p53 pathway (Bischoff et al, 1996; Heise et al, 1997; Hall et al, 1998; Habib et al, 2002). Nevertheless, more recently it has been suggested that E1B 55kd deleted ONYX 015 replicates in neoplastic cells due to aberrations in cancer cell nuclear mRNA export rather than by p53 alteration (O'Shea et al, 2004; O'Shea et al, 2005; Parato et al, 2005).

Figure 5. Kaplan-Mayer survival graphs for patients treated with Ad-HSV-tk gene therapy and randomized controls bearing malignant gliomas. (A) All patients. (B) Patients with GBM. Log-rank regression analysis was performed. Reproduced from Immonen et al, 20045 with kind permission from Macmillan Publishers Ltd: [Molecular Therapy].

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Gene Therapy and Molecular Biology Vol 11, page 87 A phase I clinical trial of ONYX 015 injection into peri-tumoral regions of 24 patients with recurrent malignant gliomas was published (Chiocca et al, 2004).

Treatment consisted of intra-cerebral injections of ONYX015. Patients were assigned into cohort groups to test different doses (n=6 per cohort). Doses varied from 1x107 p.f.u. to 1x1010 p.f.u. into a total of 10 sites within the resection cavity. None of the 24 patients experienced serious adverse events related to ONYX-015. The median time to progression after treatment with ONYX-015 was 46 days (range 13 to 452 + days). The median survival time was 6.2 months (range 1.3 to 28.0 + months). This trial proved that injection of up to 1x1010 p.f.u. of ONYX 015 into brain surrounding a resected malignant glioma is safe in humans.

In addition to those discussed in this text, several human trials involving adenoviral vectors for glioma have been published to date (Trask et al, 2000; Eck et al, 2001; Germano et al, 2003; Smitt et al, 2003) (Table 1).

Although conclusions regarding therapeutic effectiveness vary, in the general, these adenoviral vectors appear safe for intracranial injection.

In the case of NDV, the virus was used for a pediatric patient bearing glioblastoma multiforme. The vector was administrated intravenously on a daily basis; neurologic improvement and tumor regression were reported (Csatary and Bakacs 1999). In 2006, a phase I/II trial of intravenous NDV oncolytic virus in recurrent glioblastoma multiforme was released (Freeman et al, 2006). The study included patients diagnosed with recurrent GBM based on imaging studies. NDV-HUJ, the oncolytic HUJ strain of Newcastle disease virus was administrated. The first part of the study utilized an accelerated intrapatient dose-escalation protocol with one-cycle dosage steps of 0.1, 0.32, 0.93, 5.9 and 11 billion infectious units (BIU) of NDV-HUJ (1 BIU = 1X109 EID (50) 50% egg infectious dose) followed by three cycles of 55 BIU. The virus was administered by intravenous infusion over 15 min. In the second part, patients received three cycles of 11 BIU. All patients without progressive disease were maintained with two doses of 11 BIU i.v. weekly. Eleven of the 14 enrolled patients (11-58 years, Karnofsky performance scale 5090%) received treatment. Toxicity was minimal (fever was seen in 5 patients). Maximum tolerated dose was not achieved. Anti-NDV hemagglutinin antibodies appeared within 5-29 days. NDV-HUJ was recovered from blood, saliva and urine samples and one tumor biopsy. One patient achieved a complete response. Intravenous NDVHUJ was found to be well tolerated. The authors concluded that the high tolerability of NDV warrants future studies to asses the clinical anti-tumoral effect of this therapy.

modifications in their genomes and their structural proteins.

Among the CRAd that can potentially be tested in humans, Ad5-!24 is an interesting example. This adenoviral vector carries a 24-bp deletion in the E1 viral genoma. This deletion impairs the vectorâ&#x20AC;&#x2122;s capacity to interfere with Rb protein. Nevertheless, Ad5-!24 replicates in and destroys cancer cells with deficient Rb (Fueyo et al, 2000), a pathway that is commonly altered in gliomas (Hamel et al, 1993; Ueki et al, 1996). Ad5-!24 has been tested in vivo in human glioma xenografts in nude mice. A single dose of this vector induced 66.3 % inhibition and multiple injections an 83.8 % inhibition of tumor growth in this model. On the other hand, normal fibroblast or cancer cells with restored Rb activity were resistant to this virus (Fueyo et al, 2000).

In spite of these advances, a major limitation of currently available adenoviral vectors is the poor infection of tumor cells. The initial transduction process of adenoviruses requires the knob domain of fiber protein to bind the coxsackie and adenovirus receptor (CAR). Even though CAR is widely present in most tissues, it is poorly expressed in gliomas (Bergelson et al, 1997; Tomko et al, 1997; Miller et al, 1998; Asaoka et al, 2000; Grill et al, 2001; Lamfers et al, 2002). Our group has previously examined whether changes in adenoviral tropism can enhance gene transfer in the context of malignant glioma (Zheng et al, 2007). For this purpose, we assessed several receptors that are over-expressed on tumor cells and tested a series of adenoviral vectors that recognize these receptors and carry luciferase trangene: Ad5-RGD which binds %v#3/%v#5; Ad5/3 which contains adenovirus serotype 3 knob and binds to CD46; Ad5-Sigma which incorporates the reovirus sigma knob and binds to junctional adhesion molecule-1; and Ad5-pk7 which contains the polylysine motif and binds heparan sulfate proteoglycans. We also investigated the Ad5-CAV1 vector, which contains the knob of canine adenovirus type 1, a virus previously shown to infect glioma via an unknown mechanism. To evaluate the efficiency of viral transduction associated with all these structural modifications, expression of luciferase transgene both in vitro and in vivo was studied on glioma cell lines. Our results indicate that all five modified vectors attained higher mean luciferase activity vs. control. Among them, Ad5-CAV1 and Ad5-pk7 attained the highest transduction efficiency independent of different tumor lines or infection time. Most importantly, Ad5-pk7 achieved 1000-fold increased transgene expression in human glioma xenografts in vivo (Zheng et al, 2007).

Ad5-!24RGD, a new adenoviral vector with a viral fiber modification, was obtained by insertion of an ArgGly-Asp (RGD) motif into the fiber knob of vector !24 (Suzuki et al, 2001). Such fiber modification enhances viral infection of the target cells by interaction of the inserted motif with %v integrins abundantly expressed in glioma (Lamfers et al, 2002). Ad5-&24RGD exhibited higher oncolytic activity than Ad5-!24. This vector was tested in vivo in nude mice with s.c. human malignant glioma IGRG121 xenografts. Intratumoral injection resulted in complete tumor regression in 9 of 10 mice and

C. Future perspectives Since the use of adenoviruses in human trials for gliomas has led to promising results in clinical outcomes, a considerable variety of CRAd are being developed and tested to further investigate their use. These novel vectors are constructed to preferentially target glioma cells and remain relatively harmless to the surrounding tissue. To this end, adenoviral vectors have been subject to 87

Sonabend et al: Gene therapy for glioma long-term survival in all treated mice compared to controls. The oncolytic activity of this vector was shown to be enhanced by irradiation such that the same therapeutic effect was achieved when a 10-fold lower viral dose was applied (Lamfers et al, 2002).

In addition to the modification of viral structural proteins, transcriptional targeting has been explored as a strategy to achieve specificity of gene therapy in gliomas.

To this end, we have explored various promoters based on their activity in these tumors relative to normal tissues (Ulasov et al, 2007a,b). Some of these promoters could be incorporated into the genome of viral vectors or elsewhere in other gene therapy systems to restrict the expression of transgenes into cells that present high activity of the promoter. For instance, we have evaluated midkine, survivin and CXCR4 promoters in the context of their tumor specificity for gliomas (Ulasov et al, 2007b).

Among these, the survivin promoter demonstrated the highest level of mRNA expression in primary tumor samples and cell lines. Transcriptional targeting was confirmed by infection of glioma cells with an adenovirus expression vector containing a survivin-driven luciferase reporter gene. Of the tested promoters, minimal level of survivin activity was detected in normal human liver and brain (Ulasov et al, 2007b).

with retrovirus VPCs HSV-tk system, showed that no prolongation of survival was achieved.

In the proximate future, questions related to clinical efficacy will be answered since some of the vectors recently tested in phase I clinical trials will be subject to clinical outcome assessment in phase II and phase III studies that are on the way. As the first generations of gene therapy constructs are been evaluated, new and more sophisticated systems are approaching the patient bedside.

For all these reasons, clinicians that treat patients with malignant brain tumors should follow the course of gene therapy for this devastating disease.

Acknowledgments This work was supported by a grant from the National Institute of Neurological Disorders and Stroke (NINDS), K08 NS046430 (ML).

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VI. Conclusions There are a wide variety of strategies for gene therapy of gliomas. In addition to the large number of vectors and their features, different transgenes offer distinct ways of eliciting anti-tumoral response. Although no ideal vector has been yet developed, it seems that in the context of transgene expression as well as tumor cell lysis, oncolytic viruses offer advantages over other vehicles. In order to improve gene therapy efficiency, new vectors should overcome the limited transgene expression that takes place after intra-tumoral injection.

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Gene Therapy and Molecular Biology Vol 11, page 93 Gene Ther Mol Biol Vol 11, 93-112, 2007

Preliminary association of both the Dopamine D2 Receptor (DRD2) [Taq1 A1 Allele] and the Dopamine Transporter (DAT1) [480 bp Allele] genes with pathological aggressive behavior, a clinical subtype of Reward Deficiency Syndrome (RDS) in adolescents Research Article

Thomas JH Chen1, Kenneth Blum2,*, Daniel Mathews3, Larry Fisher3, Nancy Schnautz4, Eric R. Braverman5, John Schoolfield6, Bernard W. Downs7, Seth H. Blum8, Julie Mengucci8, Brian Meshkin9, Vanessa Arcuri5, Anish Bajaj5, Roger L. Waite10, David E. Comings10 1

Chang Jung Christian University, Tainan, Taiwan Republic Of China and Changhua Christian Hospital, Changhua, Taiwan, Republic of China, 2 Department Physiology & Pharmacology Wake Forest University School of Medicine, Medical Center Boulevard, Winstonâ&#x20AC;&#x201C;Salem, North Carolina, 3 Department of Research, UHS Neurobehavioral Systems. Austin, Texas, 4 The Brown School, San Marcos, Texas, 5 Path Medical Foundation, New York, NY, 6 Department of Academic Information Services, University Of Texas, Health Science Center, San Antonio, Texas, 7Allied Nutraceutical Research, Lederach, PA, 8 Synapatamine, Inc. San Antonio, Texas, 9 Salugen, Inc, San Diego, CA, 10 Genwellness, Inc. San Diego, CA, 11 Carlsbad Science Foundation, Emeritus Director, Department of Medical Genetics, City Of Hope National Medical Center, Duarte, CA

__________________________________________________________________________________ *Correspondence: Kenneth Blum, Ph.D. Department Physiology & Pharmacology, Wake Forest University School of Medicine, Medical Center Boulevard, Winstonâ&#x20AC;&#x201C;Salem, North Carolina, 27157-1083, USA; email: drd2gene@aol.com Key words: Dopaminergic genes, Polymorphisms, Pathological Aggression, Super controls, Dopamine D2 receptor, Dopamine Transporter, Aggression Abbreviations: 3-4- dihydroxyphenylacetic acid, (DOPAC); 5-hydroxyindoleacetic acid, (5-HIAA); analyses of covariance, (ANCOVs); ankyrin repeat and kinase domain containing 1, (ANKK1); attention-deficit hyperactivity disorder, (ADHD); brain-derived neurotropic factor, (BDNF); dopamine D2 receptor gene, (DRD2); dopamine transporter gene, (DAT1); dopamine, (DA); Epidemiological Catchment Area, (ECA); monoamine oxidase enzyme, (MAOA); Norepinephrine, (NE); nucleus accumbens, (NAc); PathologicalViolent, (PV); Reward Deficiency Syndrome, (RDS); substance use disorder, (SUD)

There is no author conflict of interest.

Received: 17 March 2007; Revised: 10 May 2007 Accepted: 21 May 2007; electronically published: June 2007

Summary Advances in our knowledge of the neurobiology of aggression have given rise to rational pharmacological treatments for these behaviors. The main biological systems which are known to be involved are certain reward neurotransmitters that include serotonin (5HT), opioid peptides (END), gamma-aminobutyric acid (!ABA), and the

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Chen et al: DRD2 and DAT1genes as a clinical subtype of RDS in adolescents catecholamines (dopamine [DA] and Norepinephrine [NE]). We hypothesize that pathological aggression in adolescents may in part involve polymorphisms of genes linked to the dopaminergic system. It is our notion that pathological aggression is in part similar mechanistically to other forms of impulsive behaviors such as pathological gambling. By analogy to drug dependence, it has been speculated that the underlying pathology in pathological gambling is a reduction in the sensitivity of the reward system. The potential correlation of both the dopamine D2 receptor gene (DRD2) and the dopamine transporter gene (DAT1) polymorphisms with pathological aggression in adolescents was investigated in a total of 291 subjects. Only eleven Caucasian adolescent subjects were diagnosed to have impulsive-aggressive behavior or pathological aggression. For this study 30 super normal controls were screened to exclude a number of reward deficit behaviors including pathological aggression and were genotyped for the DRD2 gene only (the DAT gene genotyping was not similarly genotyped in the super control sample). In the “super normal control group” only one person out of the 30 individuals genotyped carried the A1/ A2 genotype (3.

3%). Additionally, 91 controls were screened to exclude only Attention Deficit Hyperactivity Disorder (ADHD), pathological aggression, alcohol, drug dependence and tobacco abuse. In the present study, 6 out of 11 of the pathological aggressive subjects had the DRD2 A1 allele (55%), 11 out of 11 of the subjects (100%) carried the DAT1 10 allele, whereas 7 out of 11 or 64% carried the 9 allele. When the DRD2 A1 allele (A1/A1 or A1/A2) genotype in these subjects was compared to the super controls (1/30 or 3. 3%) a significant association was observed (Fisher’s exact test p= 0.0006); a similar trend was found with DAT1 480 bp 10/10 genotype when compared to controls (Fisher’s exact test p=0.089). However, when the DAT1 9/10 and 10/10 geneotypes were compared with controls a significant association was observed (Fisher’s exact test p= 0.00006). Albeit the small number of subjects, this is the first report on DNA polymorphisms to suggest a role for both the DRD2 and DAT genes in pathological aggressive behavior and warrants further investigation.

reduction in the sensitivity of the reward system. Studying pathological gamblers and controls during a guessing game using functional magnetic resonance imaging, Reuter and colleagues observed in 2005 a reduction of ventral striatal and ventromedial prefrontal activation in the pathological gamblers that was negatively correlated with gambling severity, linking hypoactivation of these areas to disease severity.

Advances in our knowledge of the neurobiology of pathological (impulsive) aggression have given rise to rational pharmacological treatments for these behaviors.

The main biological systems known to be involved are certain reward neurotransmitters which include serotonin (5HT), opioid peptides (END), gamma-aminobutyric acid (!ABA), and the catecholamines (dopamine [DA] and Norepinephrine [NE]). Normal aggression can be premeditated (offensive) or impulsive (defensive). We refer to impulsive aggression (or explosive aggression) as pathological when there is little or no provocation and the aggressive behavior is repetitive. For the purpose of this paper, the terms pathological aggression and impulsive aggression will be used interchangeably. Typically, the animal literature refers to normal animal aggression as offensive or defensive. In contrast, the human literature refers to either pathological aggression, which is impulsive, or violent offending, which is often premeditated (Broderick et al, 1973; Linnoila et al, 1983; Brunner et al, 1993; Muhlenkamp et al, 1995; YaryuraTobia et al, 1995; Jacobs et al, 2007; Fischer et al, 2007).

I. Introduction In terms of pathological aggressive behavior a number of neurotransmitter systems (Broderick et al, 1973; Linnoila et al, 1983; Brunner et al, 1993; Muhlenkamp et al, 1995; Yaryura-Tobia et al, 1995) are involved. We and others have hypothesized polygenic inheritance for complex behaviors such as pathological (impulsive) aggression. One major pathway that may play a pivotal role in aggressive behavior should include the dopaminergic system. In this regard, Berton and colleagues recently established in 2006 an essential role for the neurotropic factor BDNF (brain-derived neurotropic factor) in the mesolimbic dopamine pathway in social defeat stress in mice. BDNF is a key regulator of the mesolimbic dopamine pathway and potentiates dopamine release in the nucleus accumbens (NAc) through activation of the TrkB receptors on dopaminergic nerve terminals (Goggi et al, 2003).Aversive stimuli such as aggression and subordination also activate the mesolimbic dopamine pathway and have been linked to chronic alterations in dopaminergic function (Insel and Fernald, 2004). Dopaminergic abnormalities have been linked to a number of disorders including schizoid-avoidance behavior (Blum et al, 1997), human affective disorders, such as depression, social phobia, post-traumatic stress disorder (Tiihonen et al, 1997; Schneier et al, 2002; Keedwell et al,2005) as well psychosis, including paranoid schizophrenia (Aragues et al, 2005). Activation of this neural circuit has been characterize extensively in relation to drugs of abuse and other addictive behaviors, but the genetics of this system have been less characterized in pathological aggressive behavior especially in adolescents.

It is our notion that pathological aggressive behavior is in part similar mechanistically to other forms of impulsive behaviors such as pathological gambling. By analogy to drug dependence, it has been speculated that the underlying pathology in pathological gambling is a

A. Serotonin A large body of data has emerged linking impulsive aggression in humans with low serotonergic function.

Yaryura-Tobias and colleagues reported in 1995 higher levels of aggression in adults with low blood levels of serotonin. Linnoila and colleagues reported in 1983 that impulsive aggression was associated with low levels of the

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Gene Therapy and Molecular Biology Vol 11, page 95 serotonin metabolite 5-hydroxyindoleacetic acid in the cerebrospinal fluid. Moreover, others reported on a Dutch family in which a gene mutation in the monoamine oxidase enzyme (MAOA), resulting in a defect in the breakdown of dopamine, serotonin and noreponephrine, was associated with markedly increased aggressive behaviors in teenagers (Brunner et al, 1993). Moreover, Muenlenkamp and colleagues have reported in 1995 that stimulation of the 5-HT1A, 5-HT1B and 5-HT2 receptors reduces offensive aggression, while defensive aggression is reduced only by stimulation of the 5-HT2 receptor. In muricidal rats, 5-HT was higher in the hypothalamus compared to non-muricidal animals as well as higher levels of 5-HT in the amygdala. The serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) was also found to be higher in the hippocampus of the muricidal rats (Broderick et al, 1973).

mechanisms in aggressive behavior (Tazi et al, 1983).

Moreover, Comings and colleagues also associated in 1999 the enkephalinase gene with low amplitude P300 waves, which has been associated with violent offenders (Figure 1 P300 wave map in a violent subject).

D. GABA GABA is found ubiquitously in the central nervous system; its function is reducing neuronal activity.

Eichleman concluded in 1988 that GABA stimulation centrally reduces aggression, but some studies showed a significant percent of patients treated with benzodiazepines becoming more aggressive. Moreover, GABA receptor agonists seem to attenuate heightened alcohol aggressive behavior. It has been suggested that the benzodiazepine chloride ion channel may be a potential anti-aggressive target site (Miczek et al, 1995).Animal studies support a role glutamate in offensive aggressive behavior. Results of a recent study suggest that altered glutamate synthesis and GluR1 receptor expression in specific aggression areas may be involved in adolescent anabolic steroid induced offensive aggression (Fischer et al, 2007).

B. Catecholamines In animal studies Haller and colleagues found in 1996 that enhancing catecholamine function by treatment with alpha-2 adrenergic receptor antagonists’ increased aggressive responses to intruders. Further experiments (Eichelman et al, 1975) in rodents revealed that tricylics and MAO inhibitors, which increased both DA and NE activity, also enhanced aggressive behavior in these animals. In humans, the NE metabolite 3-methoxy-4hydroxyphenylglycol correlated with a positive history of aggressive behavior (Brown et al, 1979) and a positive correlation between aggression and blood levels of phenylethylamine was also found in humans (Sandler et al, 1978). Acute isolation–induced fighting in mice produced a striking “dose–dependent” increase in K M and Vmax for dopamine uptake in mesocortical nerve endings (synaptosomes) but no significant changes for these uptake constraints in nigrostriatal terminals (Hadfield, 1983).

Moreover, the dopamine metabolite 3-4dihydroxyphenylacetic acid (DOPAC) was significantly lower in muricidal rats compared to nonmuricidal animals.

The hippocampus of muricidal rats showed significantly higher DA levels, and higher levels of the NE metabolite homovanillic acid (HVA) were found in the hippocampus of muricidal rats (Broderick et al, 1973). Breese and associates provide evidence to suggest that lack of brain dopamine during development increases the susceptibility for aggression and injurious behavior by influencing D1 dopamine receptor function (Breese et al, 1990).

Furthermore, work from Comings and colleagues showed in 1997 a strong association between aberrant drug seeking behavior and polymorphisms of the D1-dopamine receptor gene.

E. Natural Vs unnatural rewards Grasping the mechanism of motivated behavior requires an understanding of the neural circuitry of rewards (Robbins and Everitt, 1996), otherwise called positive reinforcers. A positive reinforcer is operationally defined as an event that increases the probability of a subsequent response, and drugs of abuse are considered to be stronger positive reinforcers than natural reinforcers (e.g. food and sex) (Cooper et al, 1995; Epping-Jordan et al, 1998; Wightman and Robinson, 2002). The distinction between “natural rewards” and “unnatural rewards” is an important one. Natural rewards include satisfaction of physiological drives (e.g. hunger and reproduction), and unnatural rewards are learned and involve satisfaction of acquired pleasures such as hedonic sensations (Suhara et al, 2001) derived from alcohol and other drugs, as well as from gambling and other risk-taking behaviors (Hodge et al, 1996, 1998; Robbins and Everitt, 1996).

In discussing RDS, we refer specifically to an insensitivity and inefficiency in the acquired (or unnatural) reward system (Blum et al 1996; Blum and Braverman, 2000; Comings and Blum, 2000). RDS also encompasses the acquired need to escape or avoid negative affects created by repeated cycles of alcohol abuse (Koehnke et al, 2002) and dependence.

In view of this evidence of the role of dopamine and aggressive behavior, we decided to carry out association studies of polymorphisms of the DRD2 and DAT1 genes with pathological aggressive behavior in a small adolescent cohort.

C. Opioid peptides The role of opioid peptides has also been studied with regard to aggressive behavior and fighting in animals.

Beta-endorphin blocked the development of shock– induced fighting, while Naloxone facilitated it but only when shock induced fighting occurred at a low rate. The beta-endorphin induced reduction of fighting behavior was blocked by naloxone, suggesting opiate induced receptor

!!. Methods ". Subjects A total of 291 individuals were carefully accessed in this study at three different institutions, The Brown School, San Marcos, Texas, (N =11), The Path Medical Clinic, New York,

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Chen et al: DRD2 and DAT1genes as a clinical subtype of RDS in adolescents New York (N =189), and the City Of Hope National Medical Center, Duarte, California (91). For this study the experimental group consisted of eleven male Caucasian adolescents between the ages of 12 and 19 with an average mean age (+ SD) of 13.87 (+0.30), who were in residential treatment program at the Brown School in San Marcos, Texas. These subjects were selected on the basis of a one-hour structured interview and were all diagnosed to have repetitive, unprovoked, impulsive aggressive behavior (pathological aggression), and their DNA was collected for genotyping. Moreover, each subject selected had a 2.5 SD abnormal brain electrical activity during long latency (500 msec) auditory evoked responses compared to an age, sex and handedness matched normal control group measured by the Nicolett™ BEAM instrument. Only eleven subjects out of a cohort of over 100 patients attending the Brown School of San Marcos, Texas, met the study criteria. Six of the subjects had, in addition, abnormal (absent) P300 responses to auditory oddball paradigm cognitive evoked response testing. A typical electrical brain map of P300 response in an aggressive subject is presented in Figure 2. These subjects are very difficult to find even in a cohort of compromised mentally disturbed adolescents (Only 11% met the criteria).

The control group consisted of thirty super normal controls. These individuals were selected from a total of 189 individuals attending the PATH Medical Clinic in New York City, for both neurological and non-neurological problems.

These individuals were carefully screened to exclude a number of reward behaviors including but not limited to alcoholism, substance use disorder (SUD), smoking behavior, carbohydrate binging, obesity, attention-deficit hyperactivity disorder (ADHD), posttraumatic stress disorder, conduct disorder, antisocial behavior, pathological gambling, aggressive offenses, pathological aggression, deviant sexual behavior, schizoid/avoidant behavioral cluster and other axis 1 and axis 11 mental disorders. These subjects were only genotyped for the DRD2gene polymorphisms. Those who showed a family history of substance abuse/dependence and other defined reward behaviors including major psychiatric disorders (i.e. schizophrenia or mood disorders) were excluded. At a different time for another study, we also genotyped 91 controls that were

screened to exclude only ADHD, alcohol and other drugs of abuse, tobacco abuse and dependence and pathological aggression for the DAT1 9 and 10 alleles. The study protocol was approved by the PATH Foundation IRB (registration number IRB00002334) and Ethics Committee and each participant signed an informed consent.

#. Genotyping Buccal epithelial cells were collected by cotton swabs for DNA isolation. In some subjects a blood sample was obtained for DNA isolation. The construction of primers and methods for both the DRD2 and DAT genes were previously described by Grandy et al, 1989 and Comings et al, 1996.

1) DRD2 polymorphism: The D2A1 and D2A2 genotyping was performed by hybridization of Southern blots as described previously (Comings et al, 1996). A number of samples were also genotyped by a PCR technique (Comings et al, 1996).

2) DAT1 polymorphism: The DAT1 alleles (VNTR situated at the 3’ end of the gene) were determined according to the described procedure of Comings et al, 1996.

C. Statistics DRD2 gene polymorphisms were classified and assessed as follows: Taq1 A (A1 + {A1/A1 and A1/A2 genotypes} vs. A1{A2/A2 genotype}). DAT1 VNTR alleles were classified and assessed on the basis of the presence or the absence of the 10repeat (10/10 or 9/10) vs. the 9 repeat (9/9).

Due to the small sample size of 11 pathological aggression cases, one-tailed Fisher’s exact tests were used to test the association between genotype distribution and diagnosis (pathological aggression vs. control), with p<0.05 considered statistically significant. Since the three tests that were performed were orthogonal, no correction for multiple testing was necessary. 95% confidence intervals based on the binomial distribution were also calculated for each prevalence percentage estimate when possible. Statistical analyses were carried out using Stata 9.0 for Windows.

Figure 1. (A) BEAM of violent and aggressive person, (B) BEAM of non-violent person.

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Figure 2. Illustrates the genotypic prevalence of the eleven Caucasian males with pathological aggressive behavior of both the DRD2A1 polymorphism of the dopamine D2 receptor gene and the 10/10 and 9/10 alleles of the dopamine transporter gene (DAT1) in control subjects. The experimental subjects possessing the A1 allele of the DRD2 gene was compared against the 30 super controls and the prevalence of the 10/10 and 9/10 DAT1 alleles of the experimental subjects were compared against the 91 screened controls.

exact test p= 0.0006); a similar trend was found with DAT1 480 bp 10/10 genotype when compared to controls (Fisher’s exact test p=0.089). However, when the DAT1 9/10 and 10/10 genotypes were compared with controls a significant association was observed (Fisher’s exact test p= 0.00006). (Figure 2).

The below topographical brain map (Figure 1) of a pathological aggressive and violent substance abusing teenager, is similar for aggressive and violent substance abuse offenders in treatment at the PATH Medical Clinic over a ten year period (Blum and Braverman, 2000).

III. Results We found the super normal controls to carry the A1 allele in only one person out of 30 (1/30) for an A1+ rate of 3.3% with 95% c,i. of (0.1, 17.2). The A1+ is a minor form and genotype data from over 3,329 results occurs in approximately 30 percent of the unscreened global population (Noble, 2003). It is to be noted that the A1- is a major allele found in approximately 66% of the unscreened American population. Additionally, we found the less rigorous 91 controls to carry the DAT1 10/10 at 37.4% with 95% c.i. of (27.4, 48.1); the 9/10 at 43.9% with 95% c.i. of (33.6, 54.8) and the 9/9 at 13.1% with 95% c.i. of (7.0, 21.9). Based on genotype data for the DAT1 gene on 3,080 subjects, the 480bp 10/10 allele occurs in approximately 55% of the unscreened American population; the 9/10 occurs in approximately 38% of unscreened Americans; and the rare 9/9 allele occurs in 7% of unscreened Americans (Comings et al, 1996).

Eleven Caucasian adolescent subjects were diagnosed to have impulsive-aggressive behavior or pathological aggression. For this study 30 super normal controls were screened to exclude a number of reward deficit behaviors including pathological aggression and genotyped for the DRD2 gene only. Additionally, 91 controls were screened to exclude only ADHD, pathological aggression, alcohol, drug dependence and tobacco abuse. While six out of 11 of these subjects, all of whom had an absence of P300 responses, had the DRD2 A1 allele for a prevalence of 54.5% with 95% c.i. of (23.4, 83.3), 11 out of 11 of the subjects carried the DAT1 10 allele for a prevalence of 100% (95% c.i. cannot be calculated), whereas seven out of 11 carried the 9 allele for a prevalence of 63.6% with 95% c.i. of (30.8, 89.1). When the DRD2 A1 (A1/A1 or A1/A2) genotype in these subjects were compared to the super controls (1/30 or 3.3%) a significant association was observed (Fisher’s

IV. Discussion This paper presents the first study to demonstrate a putative positive association of dopaminergic polymorphisms and pathological aggressive behavior in adolescents. While these results are preliminary due to the low number of experimental subjects, the careful selection and exclusion criteria, the comprehensiveness of the research methodology and the specificity of the genetic biomarkers, provides strong insights into the etiology of pathological aggression and merits further investigations.

It is well documented that aggressive behavior represents a very complex phenotype. The first question that we must raise is – What constitutes an adequate sample of persons with a given psychiatric diagnosis such as aggression and pathological aggression, and what should we demand of our control groups if we are interested in testing population–based associations? For population–based studies in which the investigator requires a representative control sample, the obvious limitation of this strategy is that this sample may contain individuals with the same disease as that being studied in the experimental group, potentially reducing the power to detect differences between the groups. Thus, removing these confounding cases from the control group may 97

Chen et al: DRD2 and DAT1genes as a clinical subtype of RDS in adolescents improve chances of finding significant differences between experimental and control groups, but risks the lack of representativeness in the control group. Even the use of stratified samples (weighting samples) may not be good enough (Hill and Neiswanger, 1997).

In the case of finding a “pure” phenotype, especially in the psychiatric arena, we really do not know if nature carved out the psychiatric disorders in the same fashion as is seen in DSM-1V, -genes for behavioral tendencies (anxiety, impulsivity, compulsivity, harm avoidance, aggressiveness) may be the ultimate source for our understanding of psychiatric disorders.

One major problem is to recognize that when we consider the pathological aggression phenotype it may be represented by some combination of a number of behavioral tendencies. In this regard, when we consider reward dependence behaviors an emerging concept called Reward Deficiency Syndrome (RDS) (mentioned earlier) may help define this complex array of behaviors (Blum et al, 1996; Comings and Blum, 2000). RDS broadly defines a common genetic tendency whereby the individual may be predisposed to a number of addictive, impulsive and compulsive behavioral tendencies. The need for homogeneity in the affected phenotype is important not only for population-based association studies but also for linkage analysis. Under the RDS concept we are dealing with a list of behavioral tendencies including dependence on alcohol, psychostimulants (cocaine), opiates, marijuana, nicotine (smoking), carbohydrates (sugar binging), pathological gambling, sex addiction, premeditated aggression and pathological aggression, all sharing some genes in common (Hill, 1998). While there are polygenes involved, there is close similarity in terms of all of these substances and behaviors that induce presynaptic dopamine release at the n. accumbens. A screened control group is essential for uncovering population-based associations where the disease in question may be very common. Why is this so? We know that approximately one-third of the population meet lifetime criteria for common psychiatric disorders according to the results of the Epidemiological Catchment Area (ECA) survey. Since these are polygenic disorders requiring a threshold number of polygenes, unaffected individuals in the population also carry some of these genes. The dopamine D2 receptor gene (A1 allele) is present in about one-third of unscreened Americans.

The use of a super control has been criticized by some on the grounds that their relatives will have rates of comorbid disorders lower than that in the general population and may produce spurious co-aggregation of disorders within families. This argument is valid only if the same psychopathology which is removed from the control group is not excluded from among the probands and their relatives. This provides the rational to encourage others to begin to carefully select true controls especially when dealing with complex traits (Hill, 1998).

We believe that our preliminary finding of an association of both the DRD2 and DAT1 polymorphisms provide the first DNA evidence that these two dopaminergic genes play a significant role in pathological aggression. In support of these findings the DRD2 A1

alleles have been found to be associated with not only with personality disorders, (Noble et al, 1998; Mulde, 2002) but with alcoholism (Blum et al, 1990) and dopamine density (Noble et al, 1991; Hietala et al, 1994). Furthermore, dopamine transporter receptor sites are significantly increased in violent compared to non violent alcoholics (Tiihonen et al 1995). In other studies, a significant association has been found between the DRD2 A1 allele and pathological schizoid/avoidant cluster (Blum et al, 1997). While schizoid/avoidant individuals initially tend to be languid, remote, passionless, depersonalized, conflicted, hypersensitive, phobic, and self-deserting, the literature indicates these people tend to alleviate these dysphonic symptoms and seek out pleasure through outrageous acts of aggression with a pervasive pattern of social discomfort followed by substance use disorder. It is noteworthy that Pontius, reported in 1996 on a condition called “limbic psychotic trigger reaction’, supporting the link between limbic dysfunction, unexplained murders, and schizoid/avoidant personality traits.

As we see from the data presented herein, genes play a role in aggressive and violent behavior (Clark and Grunstein, 2000). Studies at the University Of Wisconsin (Bouchard and Loehlin, 2002), using identical twins raised in different families, who had parallel lives, showed that about half of human behavior (including aggression, sexuality, mental function, eating disorder, alcoholism and drug abuse or generalized RDS) can be accounted for by genes (Bouchard and Loehlin, 2002). Very few behaviors depend upon a single gene. Complexes of genes (polygenic) drive most of our heredity-based actions.

However, it is also important to distinguish between pathological aggression and violent offenses, since the former represents impulsive aggression and the latter is generally premeditated. These two potentially distinct behaviors are not understood but may warrant additional research.

Certainly abnormal functioning of these brain systems can be due to specific genetic factors interacting with environmental factors such as abuse of various psychoactive substances, particularly alcohol and stimulants. In this regard, it has been shown that these individuals may have a reduced number of dopamine D2 receptors (Noble et al, 1991; Hietala et al, 1994) and a high number of dopamine transporter sites (Tiihonen et al, 1995). Certainly the finding of hypodopaminergic function as discovered in pathological gambling (Reuter et al, 2005), an example of a RDS behavior, helps us understand the potential driving force of some to induce activation of the dopamine system. Pathological aggression and violent offending behaviors may induce such dopaminergic activation thereby reducing a pathological reward deficiency. Understanding the interaction of these components is likely to lead to better treatment.

Regarding the polymorphic association, a major difficulty with an association of the DRD2 TaqA1 allele with any reward dependent behavior including alcoholism and in this case pathological aggression, is that the Taq1 A polymorphism is located more than 10kb downstream from the coding region of the DRD2 gene (Johnson, 1996) and a mutation at this site would not be expected to lead to

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Gene Therapy and Molecular Biology Vol 11, page 99 and treatment of impulsive, addictive and compulsive behaviors. J Psychoactive Drugs 32, 1-112.

Blum K, Braverman ER, Wu S, Cull JG, Chen TJ, Gill J, Wood R, Eisenberg A, Sherman M, Davis KR, Matthews D, Fischer L, Schnautz N, Walsh W, Pontius AA, Zedar M, Kaats G, Comings DE (1997) Association of polymorphisms of dopamine D2 receptor (DRD2 ) and dopamine transporter (DAT1) genes with schizoid/ avoidant behaviors (SAB) Mol Psychiatry 2, 239-246.

Blum K, Cull JG, Braverman ER, Comings DE (1996) Reward Deficiency Syndrome. The American Scientist 84, 132-45.

Blum K, Noble EP, Sheridan PJ, Montgomery A, Ritchie T, Ozkaragoz T, Fitch RJ, Wood R, Finley O, Sadlack F (1993) Genetic predisposition in alcoholism: Association of the D2 dopamine receptor TaqI B1 RFLP with severe alcoholics.

Alcohol 10, 59-67.

Blum K. Noble EP, Sheridan PJ, Montgomery A, Ritchie T, Jagadeeswaran P, Nogami H, Briggs AT, Cohn JB (1990) Allelic association of human dopamine D2 receptor gene in alcoholism. JAMA 263, 2055-2060.

Bouchard TJ Jr, Loehlin JC (2001) Genes, evolution, and personality. Behav Genet 31, 243-273.

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Chen TJ, Blum K, Mathews D, Fisher L, Schnautz N, Braverman ER, Schoolfield J, Downs BW, Comings DE. (2006) Are dopaminergic genes involved in a predisposition to pathological aggression? Hypothesizing the importance of "super normal controls" in psychiatricgenetic research of complex behavioral disorders. Med Hypotheses, 65, 703-707.

Clark WR, Grunstein RR (2000) Are We Hardwired? The Role Of Genes In Human Behavior. Oxford University Press, New York.

Comings DE, Wu S, Chiu C, Ring RH, Gade R, Ahn C, MacMurray JP, Dietz G, Muhlman D (1996) Polygenic inheritance of Tourette syndrome, stuttering, attention deficit hyperactivity, conduct, and oppositional defiant disorder: The additive and subtractive effect of the three dopaminergic genes DRD2, DBH, and DAT1.Am J Med Genet 67, 264288.

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Comings DE, Dietz G, Johnson JP, MacMurray JP (1999) Association of the enkephalinase gene with low amplitude P300 waves. Neuroreport 10, 2283-2285.

Comings DE, Gade R, Wu S, Dietz G, Muhlman D, Saucier G, Ferry L, Rosenthal TJ, Lesieur HR, Rugle LJ, MacMurray P (1997) Studies of the potential role of the dopamine D1 receptor gene in addictive behaviors. Mol Psychiatry 2, 4456.

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any structural change in the dopamine receptor. The most likely explanation for an association is that the Taq1 A polymorphism is in linkage disequilibrium with an upstream regulatory element, or a 3’ flanking element, or another gene which confer susceptibility to RDS behaviors. Several linkage disequilibrium studies have found strong linkage disequilibrium between the Taq1 A1 allele and the Taq1B allele and the SSCP 1 allele (Blum et al, 1993; Goldman et al, 1993; O’Hara et al, 1993; Johnson 1996; Hill et al. 1999). As we have pointed out, the dopamine D2 receptor has been implicated extensively in relation to alcoholism, SUD, nicotine dependence, anxiety, memory, glucose control, pathological aggression, pathological gambling, and certain sexual behavior all of which are RDS behaviors. The most frequently examined polymorphism linked to this gene is the Taq1 A restriction fragment length polymorphism, which has been associated with a reduction in D2 receptor density. In a recent study, within the 10kb downstream region of the Taq1 A1 RFLP, Neville and associates identified a novel kinase gene, named ankyrin repeat and kinase domain containing 1 (ANKK1), which contains a single serine/threonine kinase domain and is expressed at low levels in placenta and whole spinal cord RNA. There have been no studies to date that linked the presence of this gene to brain tissue.

This gene is a member of an extensive family of proteins involved in signal transduction pathways. The DRD2 Taq1A allele is a single nucleotide polymorphism (SNP) that causes an amino acid substitution within the 11th ankyrin repeat of ANKK1 (p. Glu713lYs), which while unlikely to affect structural integrity, may affect substratebinding specificity. If this is the case, then changes in ANKK1 activity may provide an alternative explanation for previously described associations between the DRD2 gene and RDS behaviors (Neville et al, 2004).

V. Conclusion Thus, these results preliminarily support the concept that dopaminergic genes, in particular the DRD2 and DAT1 polymorphisms, are significantly associated with the reward–dependent traits (Blum et al 1996; Blum and Braverman, 2000; Comings and Blum, 2000) such as pathological aggression and warrants further research (Chen et al, 2005). Moreover, these results may have direct implications for both the diagnosis and targeted treatment of pathologically aggressive and violent offending behaviors.

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