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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