waf1 enhances the cytotoxicity of ganciclovir in HSV-tk transfected ovarian carcinoma cells

Cancer Letters 212 (2004) 43–52 www.elsevier.com/locate/canlet

The cyclin-dependent kinase inhibitor p21cip1/waf1 enhances the cytotoxicity of ganciclovir in HSV-tk transfected ovarian carcinoma cells Christelle Zillera, Hubert Lincetb, Christian D. Mullerc, Cathy Staedelb, Jean-Paul Behra, Laurent Poulaina,b,* a

Laboratoire de Chimie Ge´ne´tique, CNRS UMR 7514, Universite´ Louis Pasteur Strasbourg I, Faculte´ de Pharmacie, 74 route du Rhin, BP 24, 67401 Illkirch, France b GRECAN, Unite´ ‘Biologie et the´rapies innovantes des cancers localement agressifs’, Centre de Lutte Contre le Cancer Franc¸ois Baclesse, Avenue du Ge´ne´ral Harris, 14076 Caen cedex 05, France c Laboratoire de Pharmacologie et Physico-Chimie des Interactions Cellulaires et Mole´culaires, CNRS UMR7034, Universite´ Louis Pasteur Strasbourg I, Faculte´ de Pharmacie, 74 route du Rhin, BP 24, 67401 Illkirch, France Received 16 March 2004; accepted 24 March 2004

Abstract Suicide gene therapy could be an attractive addition to the treatment of ovarian carcinomas, for which acquired chemoresistance frequently results in treatment failure. Here we show that transfection of the HSV-tk gene, followed by incubation with up to 1 mM ganciclovir fails to induce cell death in SKOV3 chemoresistant human ovarian carcinoma cells. However, co-transfection of HSV-tk with Cip1/Waf1 encoding the p21cip1/waf1 inhibitor of cdks, allows 100 mM ganciclovir to eradicate the population of tumor cells. Potentiation of a drug by co-transfer of HSV-tk with Cip1/Waf1could thus represent another therapeutic approach for tumours that are resistant to conventional therapy. q 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Suicide gene therapy; Herpes simplex virus-thymidine kinase; Ovarian carcinoma; Polyethylenimine; p21Cip1/waf1

1. Introduction Suicide gene therapy is based on the transfer of non-mammalian genes encoding enzymes able to selectively convert a prodrug into a highly toxic * Corresponding author. Address: GRECAN, Unite´ ‘Biologie et the´rapies innovantes des cancers localement agressifs’, Centre de Lutte Contre le Cancer Franc¸ois Baclesse, Avenue du Ge´ne´ral Harris, 14076 Caen cedex 05. France. E-mail address: [email protected] (L. Poulain).

metabolite ([18] for a review). Several suicide gene/ prodrug approaches, including Herpes simplex virus thymidine kinase (HSV-tk)/Ganciclovir (GCV), have been developed during the past decade ([31] and [43] for reviews). Adenovirus-based HSV-tk suicide gene therapy was shown to be effective in vitro and in vivo against leukaemia [30], melanoma [48], gliosarcoma [38] colon and colorectal cancers [11,23], mesothelioma [12], and ovarian cancers [4,12,13,23,46]. Phase I clinical trials are being conducted for localized

0304-3835/$ - see front matter q 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2004.03.048

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cancer diseases ([43] for a review). Indeed, when tested on animal models of peritoneal and pleural carcinomatosis, this system frequently led to tumour regression, although poor transfection was observed in most cases. Success could be due to the bystander effect, which leads to cell death in the neighborhood of a transfected cell undergoing apoptosis after exposure to ganciclovir. The bystander effect which might involve intercellular transport of metabolites ([2,6,17,29] for reviews), yet remains to be fully understood. Ovarian carcinomas represent the leading cause of death by gynaecological cancer in women. Acquired resistance after chemotherapy is frequent, resulting in treatment failure. Suicide gene therapy could therefore represent an attractive addition to classical treatments, as demonstrated in previous studies [4,28,39,45,46]. In contrast to virus-based suicide gene delivery to ovarian cancer, non-viral approaches are poorly documented. Considering that non-viral vectors present an excellent safety profile, are easy to produce and are straightforward to use in a clinical environment, we investigated the efficiency of non-viral HSV-tk gene therapy approach on an ovarian carcinoma cell line, SKOV3. We used linear polyethylenimine (L-PEI Mw ¼ 22 kDa, ExGen 500) as transfection reagent. Indeed, PEI is among the most efficient DNA delivery reagents, in vitro [7,8] and especially in vivo [1,14,19,54], mainly as a consequence of its endosome swelling properties [5]. Moreover, we previously demonstrated efficient in vitro and ex vivo [35] transfection of ovarian tumour cells using L-PEI. In the present report, we show that exposure to GCV induces cell cycle arrest in HSV-tk transfected cells. Replication was inhibited, as expected, but was unable to lead to apoptosis even at concentrations of GCV up to 1 mM. We previously reported that apoptosis was induced in SKOV3 cells treated by sub-lethal doses of cisplatin following cip1/waf1 gene transfer [25]. Based on these observations, the cdk inhibitor p21cip1/waf1 could lead HSV-tk-expressing cells to undergo apoptosis as well. We thus co-transfected SKOV3 cells with HSV-tk and cip1/waf1 genes, and studied the fate of the transfected cells following exposure to GCV. Here we show that cip1/waf1 gene transfer potentiates HSV-tk-based suicide gene therapy in SKOV3 cells.

2. Materials and methods 2.1. Chemicals L-PEI (linear polymer, mean MW 22 kDa, ExGen 500) was obtained from Euromedex (Souffelweyersheim, France); GCV was obtained from Roche Diagnostics (France). 2.2. Cell lines SKOV3 cell line was established from human ovarian adenocarcinoma [15] and was obtained from ECACC (Cerdic, Nice, France). Cells were grown in RPMI 1640 medium (Gibco BRL, Lyon, France) supplemented with 10% fetal calf serum (Gibco BRL), 2 mM glutamine (Gibco BRL), 33 mM sodium bicarbonate, 20 mM HEPES, 100 units/ml penicillin (Gibco BRL) and 100 mg/ml streptomycin (Gibco BRL). Cells were maintained at 37 8C in a 5% CO2 humidified atmosphere, and splited twice a week by trypsinization. 2.3. Plasmids pCMV-Luc was provided by Prof. B.Demeneix (Paris, France). pEGFP-C3 were obtained from Clontech (Palo Alto, USA), pcDNA-p21cip1/waf1 has been constructed as previously described [25]. pUT 649 (CMV-HSVtk) were obtained from CAYLA (Toulouse, France). 2.4. PEI – DNA complex formation and transfection Transfections were carried on SKOV3 cells 24 h after plating, at a cell density of 5.104. PEI – DNA complexes were formed with a N/P ratio ¼ 5 (3 ml of 10 mM L-PEI 22 kDa for 2 mg DNA). The plasmids and the corresponding amount of L-PEI were diluted separately in a 5% glucose solution. After 10 min, PEI was added to the DNA, the solution was homogenized and let for 10 min at room temperature. The PEI/DNA complexes were added to the cells in the absence of serum and the plates were incubated at 37 8C in an humidified atmosphere containing 5% CO2 for 2 h, before addition of 10% FCS. The culture medium was changed the next day.

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2.5. Luciferase assay Twenty four hours after the transfection, cells were lysed and luciferase gene expression was quantified using a commercial kit (Promega, Cergy Pontoise, France) and a luminometer (Mediators PhL, Wien, Austria). Results were expressed as light units integrated over 10 s/mg of protein measured in whole cell lysate using the BCA assay (Pierce, Paris, France). 2.6. Detection of the green fluorescent protein EGFP expression was evaluated in isolated cells by flow cytometry with a FACStar Plus cytometer (Becton Dickinson, San Jose, CA, USA) using 400 mW of 488 nm light from an argon ion laser. Sort windows were used on forward and side scatter to eliminate debris. Granulation, size and fluorescence emission at 515 nm were recorded at a rate of , 800 cells/s. 2.7. Exposure to ganciclovir (GCV) 105 cells/well were seeded in 6 well tissue culture plates and grown for 24 h at 37 8C prior to transfection with 8 mg DNA per well 24 h after transfection, culture media were replaced by GCV solution prepared at 10 to 1000 mM in culture medium. Cells were continuously exposed 4 days to GCV, and the culture media containing were replaced every two days. 2.8. Cell cycle analysis by flow cytometry Cell layers were dissociated by trypsin/EDTA. Cell viability was assessed on the trypan blue exclusion assay. Cell cycle analysis was performed by flow cytometry and morphological characterization of apoptotic cells by nuclear staining with DAPI, as previously described [34].

3. Results 3.1. Transfection efficiency using L-PEI We first determined the percentage of cells expressing the transgene using the Green Fluorescent

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Protein (EGFP) reporter gene. As shown in Fig. 1A, 45% of the SKOV3 cell population expressed the transgene 24 h after transfection. Moreover, using a luciferase reporter system, we found that transgene expression remained constant for at least 6 days after transfection (Fig. 1B). 3.2. GCV is unable to induce cell death in SKOV3 cells transfected with HSV-tk We next tested the efficiency of the HSV-tk/GCV approach, using a pCMV-HSV-tk expression vector. The cytotoxicity of GCV was also evaluated on luciferase-transfected cells as a control. Luciferase gene transfer did not potentiate GCV cytotoxicity for drug concentrations up to 1000 mM (Fig. 2A). In contrast, HSV-tk gene transfer induced morphological changes in the cell nucleus after GCV exposure. The size of nuclei started to increase significantly for concentrations as low as 10 mM, suggesting that cells were arrested in the S phase as expected. However, despite morphological changes, the number of condensed or fragmented nuclei (associated with apoptotic cell death) was very low for drug concentrations below 100 mM (Fig. 2B). Finally, even for the highest concentration of GCV tested (1000 mM), numerous cells presenting nuclei with a normal appearance remained. The HSV-Tk/ GCV system thus appears to exert the expected effect on the cell cycle, but seems unable to lead to apoptotic cell death in SKOV3 cells. 3.3. Cytotoxicity of GCV after co-transfection of HSV-tk with cip1/waf1 Since the apoptotic signal induced by incorporation of GCV into chromatin does not lead to apoptosis in SKOV3 cells, we investigate whether transfection of cip1/waf1 could potentiate the cytotoxicity of HSV-tk/GCV. Results obtained after exposure of SKOV3 cells to 10 mM GCV are presented in Fig. 3 Gene transfer of cip1/waf1 led to sustained growth inhibition, probably due to cell cycle arrest irrespective of the phase, as suggested by flow cytometry analysis (Fig. 3C). Carefull examination of nuclear morphology showed few condensed or fragmented nuclei in these conditions (Fig. 3D).

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Fig. 1. (A) Quantitative evaluation of the percentage of GFP-expressing SKOV3 cells by flow cytometry. Cells were transfected with pCMV-EGFP-C3 complexed to the desired amount of L-PEI (ratio N/P ¼ 5), and GFP expression was evaluated 24 h later by measuring specific fluorescence, as described in material and methods. (B) kinetic of transfection efficiencies of L-PEI in SKOV3 cells. Cells were transfected with 4 or 8 mg of pCMV-Luc complexed to the L-PEI (N/P ¼ 5), and luciferase activity was measured after 24, 48 and 144 h as described in Section 2.

Although a similar growth inhibition was observed in HSV-tk-transfected cells, the underlying mechanism may be different. HSV-tk-transfected cells progressed slower through the cell cycle, mainly as a consequence of an elongated S phase. This was shown both by flow cytometry (Fig. 3C) and by microscopic observation of large nuclei (Fig. 3D). Remarkably, no sign of apoptosis was observed in this cell population. When p21cip1/waf1 and HSV-tk were co-transfected into SKOV3 cells, the cytostatic effect was observed

for only 24 h, as a drastic cytotoxic effect took place thereafter (Fig. 3A). Co-transfection indeed led to an increased sub-G1 peak as compared to control cells (Fig. 3C), suggesting apoptotic cell death. Again this was in agreement with the observation of nuclear condensation and fragmentation (Fig. 3D). As a consequence, less than 20% of cells remained viable after 4 days (Fig. 3B). Potentiation of the cytotoxic effect of GCV by co-transfection of p21cip1/waf1 with HSV-tk was also

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Fig. 2. Nuclear morphology of SKOV3 cells (after DAPI staining) transfected either by pCMV-Luc or by pUT 649 (HSV-tk gene) and exposed 24 h later to various concentrations of ganciclovir for 96 h as detailed in Section 2.

observed with higher doses of GCV. However, even 1000 mM GCV did not lead to elimination of SKOV3 cells transfected with HSV-tk or cip1/waf1 alone; after 4 days of exposure to GCV, cells with giant nuclei and cells presenting a normal morphology remained simultaneously visible. On the contrary, cotransfer of p21cip1/waf1 with HSV-tk allowed to induce massive cell death with 100 mM GCV (Fig. 4). Finally, incubation with 1000 mM GCV led to total cell disappearance.

4. Discussion Late discovery of the disease and chemoresistance are the main causes of failure during therapy of epithelial ovarian cancer. Indeed, despite recent advances in chemotherapeutic treatment, the overall

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survival of patients with advanced stage ovarian carcinoma does not exceed 25%. Among the various new strategies that are being developed for the treatment of ovarian cancer, suicide gene therapy appears to be one of the most promising. Adenovirus-based HSV-tk gene therapy was shown to lead to tumour regression in nude mice in colon and colorectal cancers [11,23], mesothelioma [12], and ovarian cancers [4,12,13,23,46]. Efficient liposomemediated HSV-tk gene transfer was reported in peritoneal dissemination of pancreatic origin [3], but no feasibility data were available concerning non-viral HSV-tk gene therapy in ovarian carcinoma. Using linear polyethylenimine (L-PEI) as DNA vector, we tested the efficiency of HSV-tk/GCV to kill SKOV3 ovarian tumour cells, for which efficient transfection using polyethylenimines was reported [35]. We showed that GCV leads to growth inhibition and S-phase elongation in HSV-tk transfected cells. However, apoptosis was not observed in these cells even at high GCV concentrations. In agreement with previous results obtained for SKOV3 cells transfected with Ad(HSV-tk) [4], the IC50 of GCV was found to be ca. 10 mM for SKOV3 cells after non-viral HSV-tk gene transfer. However, ovarian carcinoma cells are ‘resistant’ to GCV after HSV-tk transfection when compared to other tumour cell types [3,11,23,44,50], in agreement with other reports [4,44]. The low cytotoxicity of GCV may result from a defective apoptotic pathway or an altered cell cycle control. We previously showed that ovarian carcinoma cells present alterations in their cell cycle control, thus allowing chemoresistance to occur [34]. We described similar alterations associated with a lack of apoptosis in SKOV3 cells [25]. Ovarian carcinoma cells thus appear to progress through the cell cycle under conditions that normally induce cell cycle arrest. HSV-tk/GCV treatment could therefore be partially ineffective in these cells since it is based on cell death following cell cycle arrest. We previously showed that cip1/waf1 overexpression led to apoptosis in response to cisplatin exposure in cells which do not undergo apoptosis in the presence of cisplatin alone. Moreover, PlisieckaHalasa et al. [32] reported the predictive importance of high p21cip1/waf1 expression in a clinical study including 204 ovarian carcinomas treated with platinum (complete remission with a longer

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Fig. 3. Effect of a 4 days exposure to GCV 10 mM on SKOV3 cells transfected either with pcDNA-p21 alone, pUT 649 alone or both plasmids in combination. (A) Percentage of viable cells as compared to initial number in the flask after various time of GCV exposure, (B) Percentage of viable cells as compared to untransfected-untreated cells after 4 days of GCV exposure, (C) Histograms of DNA contents obtained by flow cytometry and (D) Nuclear morphology observed after DAPI staining.

disease-free survival). We thus wondered whether we could restore a normal apoptotic pathway and cell cycle control by transfection of p21cip1/waf1. In SKOV3 cells (in which p53 gene is deleted [52]) neither endogenous p21cip1/waf1 mRNA nor the corresponding protein were detected prior to transfection [25]. We hoped to convert the inhibition of replication observed in HSV-tk-transfected cells

exposed to GCV to apoptosis by means of p21 overexpression. Exogenous p21cip1/waf1 expression was found to lead to growth arrest in vitro and in vivo in melanoma [53], breast carcinoma [22,42], lung cancer [21] and colorectal cancer [9]. However, the effect of p21cip1/waf1 upregulation on induction of apoptosis remains unclear. P21cip1/waf1 failed to induce apoptosis in normal and tumour cells of

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Fig. 4. Effect of a 4 days exposure to GCV 100 mM on SKOV3 cells transfected either with pcDNA-p21 alone, pUT 649 alone or both plasmids in combination. (A) Histograms of DNA contents obtained by flow cytometry (B) Nuclear morphology observed after DAPI staining.

epithelial origin [22], whereas it induced the formation of giant cells which eventually died from apoptosis in breast and colorectal carcinoma [41,42]. Moreover, It has been demonstrated recently that

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whereas p21cip1/waf1 was dispensable for cell cycle arrest, it was indispensable for apoptosis induction following sodium butyrate treatment in breast cancer cells [10]. This reinforces the idea that the role of p21cip1/waf1 in apoptosis could be essential and should be dissociated from its role in cell cycle control ([26] for a review). In our study, overexpression of p21cip1/waf1 did not induce apoptosis per seduring 5 days following transfection. Only growth inhibition was observed, as previously described [25]. Remarkably, co-transfection of p21cip1/waf1 and HSV-tk led to apoptosis in a GCV dose-dependent manner. In the presence of both p21cip1/waf1 and HSV-tk, an exposure to 100 mM GCV induced the apoptotic cell death of most SKOV3 cells, whereas HSV-tk alone did not kill these cells even in the presence of 1000 mM GCV. It is surprising to observe that apoptotic cell death concerns more than the apparently transfected population. However, we previously reported that PEI/DNA complexes reach every cell present in the flask at the beginning of transfection, and that some of the cells that did not express the transgene 24 h later were still able to express it for several days thereafter [27]. It is difficult to evaluate the percentage of the cells that express the transgene in a period included in the 96 h following the beginning of transfection. Considering the transient aspect of transfection and the plasmid dilution phenomenon occurring during cell proliferation, the observed results could mean that at least 70% of the cells express the transgene during the days following the transfection. In addition to these findings, the bystander effect must also be taken into account. Indeed, as previously described ([33] and [47] for reviews), the bystander effect can lead to death of neighbouring untransfected cells and eradicate the total cell population even if transfection does not exceed 10% of the cells. Taken together, these phenomena could be responsible for the death of a proportion of cells largely exceeding that of transgene-expressing cells observed at 24 h in our protocol. However, in SKOV3 untransfected cells, the expected effect of GCV metabolites transfer should be limited to inhibition of proliferation. Thus, the observed bystander effect could concern the transfer of GCV metabolites through gap junctions, as well as other unidentified mechanisms involving apoptosis-inducing components, such as

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release of apoptotic vesicles by dying cells and their subsequent phagocytosis by tumour cells in the neighborhood ([33,47] for reviews). Cell death could occur after co-transfection of p21cip1/waf1 and HSV-tk in any phase of the cell cycle. Indeed, p21cip1/waf1 was shown to arrest cells in G1 and G2 [31], as well as in S phase through interaction with PCNA [49]. However, the relationship between p21cip1/waf1 and cell death occurring after ganciclovir exposure remains unclear. In this study, p21cip1/waf1 overexpression could lead to apoptosis in cells arrested in S-phase after ganciclovir exposure of HSV-tk expressing cells. Alternatively, apoptosis might occur in G2M and G1 phases in cells that escaped from this S-phase arrest. In both cases, p21cip1/waf1 could allow the signal constituted by ganciclovir-induced DNA-modifications to lead to apoptosis, which does not occur in HSV-tk-only transfected cells. Our previous work concerning the combination of cip1/waf1gene transfer with cisplatin in ovarian carcinoma cells [25], as well as a related study by Qin et al. [36] in hepatoma cells are in favour of this explanation. Moreover, p21cip1/waf1 up-regulation has been shown to be involved in apoptosis induced by various therapeutic agents ([26] for a review). Induction of apoptosis could also involve p21-related modifications of gene expression, such as Fas and FasL. The Fas/Fas-L system has indeed been shown overexpressed concomitantly with p53/p21cip1/waf1 in response to cisplatin in hepatoma cells by Qin et al. [37]. Overexpressions were only observed in cisplatin-sensitive cells, and appeared to be involved in cisplatin-induced cell death. Similar observations were reported in ovarian carcinoma by Schneiderman et al. [40] which described defective Fas-L expression in response to cisplatin in resistant cells, despite the presence of functional Fas receptors. This was recently confirmed by Mansouri et al. [27], and reviewed by Li et al. [24] and Fraser et al. [16]. The Fas/Fas-L system could also allow HSV-tk induced cell death, as suggested by Hall et al. [20]. Such a possibilities remain to be investigated further. It has been recently demonstrated that p53-independent p21cip1/waf1 upregulation can directly modulate transcriptional activity of various genes (repression as well as activation), particularly in ovarian carcinoma in conditions in which p21cip1/waf1

overexpression induced apoptosis [51]. These results as well as screening of gene expression patterns in response to p21cip1/waf1 overexpression could subsequently enrich our observations and provide new elements about the role of p21cip1/waf1 in apoptotic decision in the presence of various apoptotic stimuli. Most of the current work in this field aims at optimizing transfection with viral vectors or developing new substrates for HSV-tk with improved cytotoxicity. Potentiation of HSV-tk/GCV gene therapy by co-transfer with apoptosis or cell cyclecontrolling genes is not well documented. HSV-tk suicide gene delivery could allow treatment of cancer diseases that are resistant to conventional chemotherapy (as is the case for peritoneal carcinomatosis of various origins). Considering this latter element and the possibility to increase the efficiency of this treatment by co-transfer of genes involved in the control of cell cycle and apoptosis, such therapeutic combinations have now to be investigated further. Non viral techniques for gene delivery are especially well suited for simultaneous delivery of several genes. Combined with our recent results and with previous reports indicating that HSV-tk may enhance sensitivity of ovarian tumour cells to chemotherapeutic agents [45], this suggests that co-transfer of HSV-tk with apoptosis or cell cycle-modulating genes could be efficiently combined with conventional chemotherapy.

Acknowledgements We wish to thank Prof. Pascal Gauduchon for helpful discussions and critical reading of the manuscript, and Liliane Italiano for technical assistance. This work was supported by the Ligue Nationale contre le Cancer (LNC) and the Association pour la Recherche contre le Cancer (ARC). LP and PE were recipients of post-doctoral fellowships from LNC and ARC, respectively. HL was recipient of doctoral fellowship from LNC (Comite´ du Calvados).

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