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Comparison of the activity levels and localization of dipeptidyl peptidase IV in normal and tumor human lung cells Mashenka Dimitrova a,∗ , Ivaylo Ivanov b , Ralitza Todorova a , Nadezhda Stefanova b , Veselina Moskova-Doumanova b , Tanya Topouzova-Hristova b , Veselina Saynova c , Elena Stephanova b a

Institute of Experimental Morphology, Pathology and Anthropology with Museum, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria Faculty of Biology, University of Sofia “St. Kl. Ohridsky”, 1164 Sofia, Bulgaria c Active Therapy Hospital “Doverie”, 1632 Sofia, Bulgaria b

a r t i c l e

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Article history: Received 5 July 2011 Received in revised form 16 November 2011 Accepted 17 November 2011 Available online 18 December 2011 Keywords: Dipeptidyl peptidase IV Fluorogenic substrates Enzyme cytochemistry Lung cancer cells Squamous cell lung cancer

a b s t r a c t Dipeptidyl peptidase IV (DPPIV) was studied in three human lung cells – P (fetal lung-derived cells), A549 (lung adenocarcinoma) and SK-MES-1 (squamous cell carcinoma) using a fluorescent cytochemical procedure developed on the basis of the substrate 4-(glycyl-l-prolyl hydrazido)-N-hexyl-1,8-naphthalimide. The observed differences in the enzyme expression were confirmed by measuring the enzyme hydrolysis of glycyl-l-prolyl-para-nitroanilide. The surface and total dipeptidyl peptidase activities of P cells were correspondingly 7–8 and 3–10 times higher than those of SK-MES-1 and A549 cells. The ratio surface per total activity showed that in P (95%) and A549 (93%) cells the enzyme is associated with the plasmalemma while in SK-MES-1 cells (35%) it is bound to intracellular membranes. In order to compare the results from cell cultures with those in human tumor, the enzyme activity was investigated in cryo-sections of three cases of diagnosed squamous lung carcinoma. DPPIV activity was restricted to the connective tissue stroma surrounding the DPPIV-negative tumor foci. © 2011 Elsevier Ltd. All rights reserved.

1. Introduction Dipeptidyl peptidase IV (DPPIV; EC is a membraneassociated serine-type protease hydrolyzing Xaa-Pro or Xaa-Ala dipeptides from amino-terminals of oligopeptides at pH 7.0–8.5 (Gossrau, 1979; Gutschmidt and Gossrau, 1981). It participates in the hydrolysis of a vast number of biologically active peptides, thus altering their activity or receptor specificity (Mentlein, 1999). Besides its protease activity, DPPIV has other functions as a receptor molecule, co-stimulatory protein and adhesion molecule (Boonacker and Van Noorden, 2003). Alterations of DPPIV activity levels have been reported in malignant, autoimmune, inflammatory and infectious diseases (Antczak et al., 2001a,b; Lambeir et al., 2003). The enzyme has been supposed to be involved in tumor growth and angiogenesis (Bauvois, 2004). Its up-regulation has been suggested as an additional indicator for the differentiation of malignant from benign nodules in thyroid carcinoma (Kholova et al., 2003). Most of the histological sub-types of lung tumors have been found as DPPIV-negative (Asada et al., 1993). The DPPIV mRNA

∗ Corresponding author. Tel.: +359 2 979 23 85; fax: +359 2 719007. E-mail address: (M. Dimitrova). 0040-8166/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tice.2011.11.003

expression has been studied in human bronchial and alveolar cell lines, including A549 cells, by semiquantitative RT-PCR (Baginski et al., 2011). The authors have found obvious differences in the enzyme mRNA levels between different cell lines. DPPIV activity of A549 cells has been evaluated as much lower than that of primary type II rat alveolar cells (Forbes et al., 1999). However, DPPIV activity levels have not been thoroughly studied in human lung cancer cell lines A549 and SK-MES-1 in comparison with a normal human lung cell line. The aim of the present work was to compare DPPIV activity in three types of human lung cell lines – P cells (embryonic diploid cells), A549 (lung adenocarcinoma) and SK-MES-1 (squamous cell carcinoma). For the purpose we used a fluorescent cytochemical procedure developed on the basis of the fluorogenic substrate 4-(Gly-Pro hydrazido)-N-hexyl-1,8-naphthalimide (Gly-Pro-HHNI), recently synthesized by us (Ivanov et al., 2009). The observed differences in the enzyme activity in normal and tumor cells were estimated by measuring surface and total DPPIV activities with Gly-Pro-4-nitroanilide substrate (Gly-Pro-pNA). The decreased levels of the enzyme in tumor cells were readily demonstrated with the fluorogenic substrate, which might be applied as a cyto-diagnostic tool for non-small lung cell carcinoma.

Author's personal copy M. Dimitrova et al. / Tissue and Cell 44 (2012) 74–79

2. Materials and methods 2.1. DPPIV substrates and inhibitor The fluorogenic DPPIV substrate 4-(Gly-Pro hydrazido)-Nhexyl-1,8-naphthalimide (Gly-Pro-HHNI) was synthesized precisely as described before (Ivanov et al., 2009). DPPIV inhibitor N-(H-Phe-Pro-)-O-(4-nitrobenzoyl)hydroxylamine hydrochloride (Phe-Pro-NHONb) was synthesized according to Demuth et al. (1988). Gly-Pro-4-nitroanilide (Gly-Pro-pNA) was purchased from Bachem AG, Switzerland. 2.2. Chemicals Pro-celloidin and the organic solvents were purchased from Fluka (Sigma–Aldrich Chemie GmbH, Germany). All the other chemicals were from Sigma (Sigma–Aldrich Chemie GmbH, Germany) unless otherwise stated.


of 0.5 mmol substrate (Gly-Pro-HHNI) and 0.5 mg/ml piperonal in 0.1 M phosphate buffer, pH 7.8 for 60 min at 37 ◦ C. After the incubation the sections were post-fixed in 4% neutral formalin for 15 min at room temperature, stained with haematoxyline consistent with classical methods of histology and embedded in glycerol/jelly. 2.6. Inhibitor controls Inhibitor controls were pre-incubated in 0.1 M phosphate buffer (pH 7.8) containing 0.5 mM inhibitor Phe-Pro-NHONb for 45 min at room temperature, then transferred to full substrate medium, supplemented with 0.5 mM of the same inhibitor and allowed to stain for an hour at 37 ◦ C. Subsequently, the controls were treated as described above. All the preparations were observed under OPTON IM 35 fluorescent microscope (Carl Zeiss, Germany) or confocal microscope Nikon Eclipse Ti-U. 2.7. Surface DPPIV activity of the lung cells

2.3. Cells P cells (human fetal lung-derived diploid cells), A549 (human lung adenocarcinoma, ATCC® number: CCL-185TM ) and SK-MES-1 (human lung squamous cell carcinoma, ATCC® number: HTB58TM ) were kindly provided by the National bank for industrial microorganisms and cell cultures (Sofia, Bulgaria). The tumor cells were routinely grown in Dulbecco’s Modified Eagle’s Medium (DMEM), supplemented with 10% Fetal Bovine Serum (FBS) and antibiotic–antimycotic solution (BioWittaker, Cambrex BioScience, Belgium) at 37 ◦ C in humidified atmosphere with 5% CO2 . The A549 cells were additionally grown in the same medium but containing 5% FBS or 10% Newborn Calf Serum (NCS). The P cells were cultured at the same conditions, but the medium was supplemented with 10% NCS. All the cells were grown until 90–95% confluence. For the cytochemical visualization of DPPIV activity, the cells were grown on cover slips until 95–100% confluence. 2.4. Tumor tissue Cryostat sections of tumor tissue extracted at surgery from three patients with diagnosed squamous cell carcinoma of the lung were obtained from Pulmonary Clinic “St. Sofia”, Sofia. The protocol was approved by an independent Ethics Committee. The study was conducted in compliance with the principles of the Declaration of Helsinki 1964 and its amendments. 2.5. Cytochemistry and histochemistry Cells grown on cover slips were washed with PBS and fixed in paraformaldehyde vapors for 5 min at room temperature. Then, they were air-dried and covered by celloidin (0.5% celloidin in absolute ethanol/diethyl ether/acetone 3:3:4) for 30 s at room temperature. The preparations were incubated in a substrate solution containing 0.3 mM substrate (Gly-Pro-HHNI) and 0.3 mg/ml piperonal (the substrate and the aldehyde were pre-dissolved in a minimum amount of dimethylformamide; the incubation solution was filtered before use) in 0.1 M phosphate buffer, pH 7.8 for an hour at 37 ◦ C. After the incubation, they were post-fixed in 4% neutral formalin for 15 min at room temperature, stained in 1 !M Hoechst 33342 aqueous solution for 20 min at room temperature and embedded in glycerol/jelly (glycerol/15% gelatin 1:1 (Lojda et al., 1979)). Cryostat sections of tumor tissue on glass slides were covered by celloidin (1% celloidin in absolute ethanol/diethyl ether/acetone 3:3:4) for a minute at room temperature. DPPIV activity was localized in the sections incubated in a substrate solution, consisting

The cells were grown in 24-well plates, until they reached 90–95% confluence corresponding to a density of 1 × 105 cells per well (defined by counting parallel control wells by the Burger’s camera). Then, the medium was removed, the plates were washed with 0.1 M phosphate buffer (pH 7.8) and 3 ml solution containing 0.25 mM substrate (Gly-Pro-pNA) in the same buffer was added to each well. The reaction was carried out at 37 ◦ C and samples were collected every 30 min. Enzyme reaction was stopped by adding equal volume of 1.0 M acetate buffer, pH 4.0. The enzyme-catalyzed release of 4-nitroaniline (pNA) from the substrate was monitored on Ultrospec® 3000 spectrophotometer (Pharmacia Biotech, Sweden) at 405 nm against a control of substrate solution added to the cells, collected immediately and blocked with equal volume of 1.0 M acetate buffer, pH 4.0. The results were statistically estimated by regression analysis and curves showing the time-dependence of the adsorption at 405 nm were built by means of Sigma Plot 9.0. In the cases of non-linear correlation, the enzyme activity was determined from the initial rate of the reaction. One unit of enzyme activity was defined as the amount of enzyme liberating 1 nmol product (pNA) per minute, per 1 × 105 cells at 37 ◦ C. 2.8. Total DPPIV activity of the lung cells Cells were grown in 10 cm Petri dishes until they reached 90–95% confluence. Then, the cells were harvested by means of a rubber policeman and homogenized mechanically in an ice-bath in 0.1 M phosphate buffer (pH 7.8). After a spectrophotometric measurement of protein amount (Layne, 1957), the lysates were diluted by the same buffer to four varying protein concentrations to final volume of 1.5 ml. To every dilution, 1.5 ml 0.5 mM Gly-Pro-pNA in 0.1 M phosphate buffer (pH 7.8) was added to obtain a final substrate concentration of 0.25 mM. The reaction was carried out at 37 ◦ C. Aliquots were collected every 60 min and the reaction was stopped as described above. Absorption of the samples at 405 nm was measured spectrophotometrically against a control of substrate solution, diluted with equal volume 1.0 M acetate buffer (pH 4.0). One unit of enzyme activity was defined as the amount of enzyme liberating 1 nmol product (pNA) per minute per 1 mg protein at 37 ◦ C. The results were statistically estimated as described for the surface DPPIV activity. 3. Results Recently, we developed a specific fluorogenic substrate (GlyPro-HHNI) for the histochemical localization of DPPIV (Ivanov et al., 2009). For the purposes of the present experiment we extended

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Fig. 1. Localization of DPPIV activity with the substrate Gly-Pro-HHNI in cultured human lung cells. The nuclei are stained with Hoechst 33342. At UV-excitation (!exc = 340–390 nm) the cells nuclei are visible (A, D, G, and J) surrounded by low-fluorescent final reaction product (A, D, and G). At green light excitation (!exc = 540–580 nm) the highly fluorescent enzyme reaction product is observed (B, C, E, F, H, and I). High enzyme activity in P cells (A–C). Amorphous pattern of the reaction product in P cells (C). Moderate (arrows) or low (arrowheads) enzyme activity in A549 cells (D–F). Fluorescent precipitates in A549 cells are amorphous (F) or slightly granular (F insertion). Cell clusters of moderate (arrows) or very low (arrowhead) DPPIV activity in SK-MES-1 cell line (G–I). Highly granular fluorescent product in this cell line (I). Lack of enzyme activity in the inhibitor experiment in P cells (J and K). Bars = 25 !m.

the application of the same substrate for the visualization of the enzyme activity in cultured cells. Best results were obtained using cells fixation in paraformaldehyde vapors and embedding the samples in 0.5% celloidin. The cells nuclei stained by Hoechst 33342 were surrounded by a low fluorescent yellow-orange precipitates, which represented the enzyme activity product (Fig. 1A, D, and G). Upon excitation by green light (!ex = 540–580 nm) the brilliant red fluorescence marking DPPIV activity locations could be viewed in

the cells (Fig. 1B, C, E, F, H, and I). The fluorescence intensity of the final enzyme reaction product was visibly higher in normal than in tumor lung cells pointing out to a higher DPPIV activity in the P cell line in comparison to tumor cell lines (Fig. 1A–I). In the diploid embryonic P cells the reaction product was evenly deposited in the cells (Fig. 1A, B, and C). This reaction pattern most probably indicated that the enzyme was uniformly expressed in the cell periphery. Both tumor cell lines showed a variability between

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Fig. 2. DPPIV activity in SK-MES-1 cells visualized by the substrate Gly-Pro-HHNI. The nuclei are stained with Hoechst 33342. Confocal microscopy. Optical sectioning shows the existence of intracellular enzyme depots. Bar = 15 !m.

individual cells. A549 cells were either moderately DPPIV-positive or DPPIV-negative (Fig. 1D, E, and F). The fluorescent product was of a homogenous (Fig. 1F) or slightly granular appearance (Fig. 1F insertion). SK-MES-1 cells displayed a tendency to gather into clusters of moderate or low to none enzyme activity (Fig. 1G, H, and I). The accumulated fluorescent product was granular probably indicating intracellular enzyme localization (Fig. 1I). The optical sectioning upon confocal microscopy confirmed the presence of cytoplasmic depot of DPPIV in this cell type (Fig. 2). The precise enzyme locations within SK-MES-1 cells remain to be identified in the future. The use of inhibitor in different cell lines lead to an absence of fluorescent reaction product pointing to a total suppression of the enzyme activity (Fig. 1J and K). Quantitative estimation of DPPIV activity levels in normal and cancer cell lines were performed with the substrate Gly-Pro-pNA, containing the same amino acid sequence as the fluorogenic substrate. The activity of the plasma membrane-associated DPPIV and the total enzyme activity, i.e. the plasmalemma-associated plus intracellular DPPIV were determined. Both tumor cell lines showed a linear time-dependence of the surface enzyme reaction product accumulation during the whole 6 h incubation period (Fig. 3). The reaction rate was estimated as nearly similar in the two cancer cell lines (Table 1). In the case of embryonic cells, a non-linear character of the enzyme reaction product accumulation was detected – it reached a saturation level showing a high enzyme activity (Fig. 3). The activity of the enzyme (from the initial rate of the reaction) in the P cells was estimated to be 8 times higher than this in A549 and 7 times than that of SK-MES-1 cells (Table 1). Measurement of the total DPPIV revealed that the relative enzyme activity was linearly dependant on the protein concentration. In these experiments the time-dependent product accumulation in A549 cells had a linear character in the range of used protein concentrations (Fig. 4). On the other hand, SK-MES-1 and P cells displayed a non-linear reaction progress at higher protein concentrations (Fig. 4). The P cell line exhibited the highest total activity (from the initial rate of the reaction) – the amount of the released product was ten times higher in comparison to A549 cells and three times as compared to that, released by SK-MES-1 cells (Table 1). The obtained experimental data about the protein amount per 1 × 105 cells and surface and total enzyme activities were used to calculate the relative surface activity of DPPIV for the three cell lines (Table 1).

Fig. 3. The time-course of reaction product accumulation after the substrate GlyPro-pNA (0.25 mM) hydrolysis by plasma membrane-bound DPPIV of different cell lines. The process was monitored for 6 h at 30 min interval (each result was obtained on the basis of triplicate experiment).

The use of different types of sera in the culturing medium of A549 cells did not change the above results for this cell line. Thus, neither the serum concentration, nor the type of serum used changed the enzyme activity levels. The histochemical demonstration of DPPIV activity in tumor tissues showed that the tumor parenchyma was DPPIV negative (Fig. 5A and B). Tumor stroma contained lots of DPPIV-positive cells enclosing the dark zones of carcinoma foci. In the resection line without tumor infiltrates DPPIV-positive cells were abundant in all the tissue structures (Fig. 5C and D). In the resection line with tumor cells infiltrates DPPIV-negative carcinoma foci were seen surrounded by diffusely distributed enzyme expressing cells (Fig. 5E and F).

Fig. 4. The time-course of reaction product accumulation after the substrate GlyPro-pNA (0.25 mM) hydrolysis by DPPIV in cell lysates. The process was monitored for 6 h at 60 min intervals (each result was obtained on the basis of triplicate experiment). The protein quantity in samples was 0.94 mg for cancer cells and 0.66 mg for P cells.

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Table 1 Determination of DPPIV activity in human lung cells. Cell line A549 SK-MES-1 P a b

Surface activity (nmol/min/1 × 105 cells) a

0.280 ± 0.006 (1) 0.345 ± 0.015 (1.2)a 2.31 ± 0.21 (8.2)a

Total activity (nmol/min/mg protein) b

0.87 ± 0.09 (1) 3.30 ± 0.03 (3.7)b 9.0 ± 0.6 (10.3)b

Protein amount per 1 × 105 cells (mg)

Surface/total activity (%)

0.34 ± 0.02 0.30 ± 0.01 0.27 ± 0.01

93 35 95

Relative surface activity of different cell lines. Relative total activity of different cell lines.

Inhibitor treatment led to a total abolishment of the enzyme activity, represented by the lack of red fluorescence in the samples (cultured cells or tissue sections). The inhibitor used is known as irreversible and specific for DPPIV (De Meester et al., 1992). Thus, that result proved the specificity of the observed enzyme reaction. 4. Discussion DPPIV is known to have a soluble and membrane-bound form. The soluble DPPIV is present in the serum and body fluids (Vanhoof et al., 1992). The membrane-associated enzyme has a ubiquitous distribution in the mammalian organs and tissues and is usually

expressed in the apical periphery of epithelial cells (Gossrau, 1985; Lambeir et al., 2003). The membrane-anchored DPPIV has been found also in the endosomes of BHK cells (Horstkorte et al., 1996) and rat hepatocytes (Kreisel et al., 1993) obviously due to the process of internalization-reexpression, as well as in the lysosomes and trans-Golgi of human hepatocytes (Kyouden et al., 1992; Fukui et al., 1990). In the human lung, DPPIV is restricted to the endothelial cells of blood vessels, sub-mucosal serous glands and alveolar epithelial cells, whereas the bronchial epithelium, fibroblasts and smooth muscles are DPPIV-negative (Van der Velden et al., 1998). The enzyme activity has been detected also in primary pulmonary type II cells in culture (Forbes et al., 1999). On the other hand,

Fig. 5. Localization of DPPIV in cryostat sections of squamous lung carcinoma with the substrate Gly-Pro-HHNI. The nuclei are stained with haematoxyline. (A and B) High enzyme activity in tumor stroma (TS); lack of activity in tumor cells (TC) foci. (C and D) Diffuse DPPIV distribution in all the tissue structures in the resection line without tumor infiltrates. (E and F) DPPIV-negative tumor cells (TC) foci in the resection line with tumor infiltrates and diffuse enzyme reaction in the surrounding tissue. (A, C, and E) Light microscopy; (B, D, and F) fluorescent microscopy. Bars = 50 !m.

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analyses of DPPIV expression in different histological sub-types of lung cancer have revealed that some adenocarcinomas express the enzyme, but large cell carcinomas, small cell cancers, squamous cell carcinomas and carcinoid tumors lack DPPIV expression (Asada et al., 1993). Wesley et al. (2004) have reported that non-small cell lung carcinoma cells express much lower DPPIV than the normal human lung cells both compared at mRNA and protein levels. In our experiments, normal fetal lung-derived P cells show a constantly high DPPIV activity, the enzyme reaction product being evenly distributed in the cells. The relative surface activity is 95% and this result is in agreement with the common view that in normal tissues DPPIV is usually most abundant in the plasmalemma. A549 adenocarcinoma cells show 8–10 times lower surface and total DPPIV activity than the normal embryonic cells. The observed varying enzyme activity levels in individual A549 cells could be due to the high heterogeneity of this cell line. For example, Watanabe et al. (2002) have shown that A549 cell line consists at least of two sub-clones exhibiting different susceptibility towards some drugs. A549 cells are derived from alveolar pneumocytes type II and maintain many of their morphological and biochemical characteristics. They have specific lamellar bodies and well developed intracellular membrane system engaged in synthesis, secretion, endocytosis and recycling of pulmonary surfactant as well as membrane proteins (Balis et al., 1984). Our results demonstrate that the relative surface activity in A549 is 93%, which can be explained with the quick externalization of synthesized DPPIV. SK-MES-1 cells also have a low DPPIV in the cytochemical studies and their surface DPPIV activity is 7 times lower than that of P lung cells. Their relative surface activity is 35%, indicating that the main quantity of the active enzyme is restricted within the cells. SK-MES-1 cells have a very low secretion activity (Finkbeiner et al., 1995) and we suggest that the enzyme distribution could possibly correlate with disturbances in intracellular membrane transport system. The low secretory activity of the cells possibly interferes with the enzyme expression on the cell surface. Our preliminary experiments on three diagnosed cases of squamous cell lung carcinomas show a high DPPIV activity in the connective tissue stroma of the carcinomas and lack of enzyme activity in tumor foci. In our study fluorescent observations are more demonstrative than light microscopy due to the possibility of unspecific staining of the histochemical samples. These results indicate that the possible diagnostic value of DPPIV deserves to be studied in more details. In conclusion, our results presented here considered together with previous studies on other types of lung tumor cells and tissues lead to the reasonable assumption that most lung tumor cells are deficient in DPPIV activity. The possible therapeutic value of DPPIV activators remains to be studied in the future. On the other hand, DPPIV expression levels and localization pattern need to be analyzed in more details to estimate their application for diagnostic purposes in human lung cancer. Acknowledgments This work is supported by the Bulgarian Ministry of Education and Science, National Science Fund, Grand nr 1527/05. References Antczak, C., deMeester, I., Bauvois, B., 2001a. Ectopeptidases in pathophysiology. Bioassays 23, 251–260.


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Comparison of the activity levels and localization of dipeptidyl peptidase IV