ANTICANCER RESEARCH 31: 3851-3858 (2011)

Mode of Associated with Adenovirus-mediated Suicide in HNSCC Tumor Model

DEEPIKA SRIVASTAVA1, GANESH JOSHI1, KUMARAVEL SOMASUNDARAM2 and RITA MULHERKAR1

1Advanced Centre for Treatment, Research and Education in , Tata Memorial Centre, Navi Mumbai, India; 2Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India

Abstract. Background: Adenoviral mediated suicide gene via chain termination after incorporation of GCV- therapy has been shown to have a tumoricidal effect against triphosphate into replicating DNA as well as inhibition of a variety of tumor models. Although the efficacy of this DNA polymerase alpha (1). Another promising aspect of this treatment regimen has been verified, the molecular mechanism strategy is the “bystander effect” where the cytotoxic of Herpes simplex virus thymidine kinase gene (HSVtk)- and molecules (GCV-triphosphate) are taken up by the ganciclovir (GCV)-induced cell death is still not well neighboring untransduced cells via gap junctions thereby established. Materials and Methods: The mode of cell death enhancing tumour cell kill (2-6). by adenoviral (Adv)-HSVtk/GCV was examined in the head The cytocidal efficacy of the HSVtk/GCV system in the and neck squamous cell carcinoma cell line, NT8e. Results: treatment of various tumors has been demonstrated both in The cell death was independent of apoptotic gene expression. vitro and in vivo (7-9). has been suggested as the Moreover apoptosis was not evident from cell cycle kinetic cytocidal mechanism of Adv-HSVtk/GCV transduced cells analysis. Adv-HSVtk/GCV treated cells showed time dependent (10-13). A study on colorectal cancer (14) showed a accumulation of cells in the S-phase of the cell cycle although combination of apoptosis in the G1 cell cycle phase and late there was no increase in the “apoptotic peak” or sub-G1 apoptotic or necrotic sub-G1 DNA fragmentation, depending population. Swelling of the cytoplasm without apparent on the tumor cell line, upon treatment with Adv- nuclear condensation suggested a possible involvement of HSVtk/GCV. Another study (15) showed that, GCV-induced necrosis. Conclusion: The apoptotic mechanism may not play death of Adv-HSVtk transduced oral SCC cells was mediated a central role in the Adv-HSVtk/GCV induced NT8e cell death through an apoptotic pathway. Contrary to the above, it was and other mechanisms should be considered. suggested that a nonapoptotic mechanism may play a central role in the HSVtk/GCV induced cell death (16, 17). One of the most promising strategies for gene therapy is Although the efficacy of this therapy has been verified, the suicide gene therapy. In this strategy, vectors carrying molecular mechanism of Adv-HSVtk/GCV induced cell metabolic enzyme genes are introduced into the target cells. death is still not well characterized. In the present study the Expression of the enzyme converts an active prodrug to a aim was to determine the mode of cell death induced by toxic product, thus selectively killing the target cell. One Adv-HSVtk/GCV and to verify whether apoptotic or non- such prodrug activation strategy involves the introduction of apoptotic responses are associated with Adv-HSVtk/GCV the herpes simplex virus thymidine kinase gene (HSVtk) into induced cell death in NT8e cells. tumor cells followed by the administration of the prodrug ganciclovir (GCV). HSVtk transduced cells selectively Materials and Methods convert GCV, a guanosine analogue, into a mono- Cell lines and antibodies. The head and neck squamous carcinoma phosphorylated form which is then further phosphorylated cell line, NT8e was used for all the experiments. NT8e is derived by the endogenous kinases. HSVtk/GCV induces cell death from a squamous cell carcinoma of the pyriform fossa from a cancer patient and was developed and characterized in our laboratory (18). Human embryonic kidney 293 (HEK293) cells were used for virus preparation and viral titer estimation. All the cell lines were grown in Correspondence to: Prof. Rita Mulherkar, ACTREC, Tata Memorial Dulbecco’s modified Eagle’s medium (DMEM, GIBCO BRL, Grand Centre, Kharghar, Navi Mumbai, 410210, India. Tel: +91 island, NY, USA) supplemented with 10% FCS (GIBCO BRL) 37˚C 2227405044, Fax: 912227405080, e-mail: [email protected] in a humidified air incubator under 5% CO2 conditions. Caspase-3 antibody was obtained from Cell Signaling Technology, (Beverly, Key Words: Adenovirus, prodrug activation, HNSCC, ganciclovir, MA, USA) and β-tubulin antibody was obtained from Sigma-Aldrich thymidine kinase, apoptosis. (Sigma-Aldrich Corporation, Saint Louis, MO, USA).

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Adenovirus preparation and titre determination. The adenovirus Western blotting for capase-3. The NT8e cells were treated with was constructed previously in our laboratory by a method published 1×105 gfu Adv-HSVtk/GCV for different intervals including 2 h, earlier (5). The recombinant human adenovirus type 5 carries 6h, 12 h, 18 h, 24 h, 36 h, 48 h and 72 h. NIH3T3 (mouse HSVtk and green fluorescence protein (GFP) under two separate embryonic fibroblast) cells and NT8e cells treated with 100 nM cytomegalovirus virus (CMV) promoters. To prepare bulk staurosporine (Sigma-Aldrich Corporation) for 24 h were used as quantities of purified virus, 20×100 mm plates of 3×106 HEK293 positive control. Untreated NT8e cells served as negative control. cells were transduced with sufficient viruses to demonstrate Each time-point lysate was resolved on 12% SDS-PAGE. The cytopathic effect within 36 h. Harvested infected cells and media proteins were transferred onto polyvinylidene difluoride membrane were pooled and centrifuged at 1400 rpm for 10 min. The cell (PVDF, GE Healthcare, Piscataway, NJ, USA) membrane and pellet was resuspended in 100 mM Tris (pH 8) and lysed by rapid probed with 1:1000 dilution of anti-caspase-3 antibody (Cell freeze-thaw. The lysate was centrifuged and the supernatant was Signaling Technology). The blots were developed using the ECL overlaid on a caesium chloride density gradient, centrifuged at Plus system (GE Healthcare). The blot was stripped using stripping 32,000 g for 1 h and the lower virus band was collected and mixed buffer and stained for β-tubulin (anti β-tubulin antibodies at 1:1000, with 2× glycerol buffer and stored at –20˚C. To determine the titre Sigma-Aldrich Corporation) for loading equalization. of adenovirus stock the method of Gueret et al. (19) was followed. Briefly, 5×105 HEK293 cells were plated in a 6-well dish. Cells DNA ladder assay. The NT8e cells were seeded in a 60 mm plate were infected with Adv-HSVtk at various dilutions, harvested 17- and were infected with 1×105, 1×106 or 1×107 gfu Adv-HSVtk 20 h post infection and analyzed for GFP+ cells by flow cytometry. followed by 2 μg/ml GCV treatment for 72 h. The DNA Ladder The titre was reported as green fluorescent units per ml (gfu/ml) assay was performed as reported previously (21). Briefly, on day 3 and calculated by: cells were harvested and treated with incubation buffer (10 mM Tris pH 7.4, 10 mM EDTA, 0.5% Triton-X100) to remove cell debris. % infection × total cell no. × dilution The supernatant was treated with RNase (1 mg/ml, Sigma-Aldrich Titre (gfu/ml) = Corporation) and proteinase-K (20 mg/ml, Sigma-Aldrich 100 × volume Corporation) for 2 h at RT. DNA was extracted using isopropanol precipitation. The pellet was dissolved in TE (10 mM Tris pH 7.4, 9 A titre of ~1×10 gfu/ml was achieved. 1 mM EDTA) and run on a 2% agarose gel at 100V for 1 h 30 min. NIH3T3 cells and NT8e cells treated with 100 nM staurosporine Cell viability assay. The NT8e cells were seeded in a 96-well plate. (Sigma-Aldrich Corporation) for 24 h were used as positive control. The next day Adv-HSVtk at various dilutions was added to each well. Six hours post adenoviral transduction, ganciclovir (GCV, Quantitative real time PCR for apoptotic genes. RNA was extracted Roche pharmaceuticals, Switzerland) at a concentration of 0, 0.5, 1 from untreated NT8e cells and from NT8e cells treated with Adv- or 2 μg/ml was added to the corresponding wells. Cell viability was HSVtk alone or Adv-HSVtk/GCV for 72 h using TRIzol Reagent assessed 72 h after GCV treatment using the Sulpho-rhodamine B (Invitrogen, Carlsbad, CA, USA). The cDNA was made using a (SRB) assay (20). Briefly, at the end of the treatment, cells were Superscript First strand synthesis kit (Invitrogen). Ten nanogram of overlaid with 100 μl of 50% Trichloroacetic acid and were cDNA was used for real time PCR. A real time PCR array was incubated at 4˚C for 1.5 h. The cells were washed with water and carried out using the method patented by Dr. K. Somasundaram, allowed to air dry and 50 μl SRB dye was added to each well. After IISc, Bangalore. The expression analysis for 96 apoptotic genes was incubation in the dark for half an hour the plate was washed with studied and the fold change with respect to untreated control NT8e 1% acetic acid and air dried. The bound dye was solubilized using cells was reported. 10 mM Tris pH 10.5 and absorbance was measured at 540 nm. Cell viability was expressed as relative percent to control. The results represent the mean±SE of triplicates. Results

DAPI and acridine orange/ethidium bromide (AO/EB) staining. The NT8e cell death upon Adv-HSVtk/GCV treatment. The SRB NT8e cells were seeded in a 60 mm - plate and were infected with data demonstrated that GCV inhibited the growth of Adv- 1×105 gfu Adv-HSVtk followed by 2 μg/ml GCV treatment for 72h. HSVtk transduced NT8e cells in a dose dependent manner For DAPI staining, the cells were washed with 1× PBS and fixed in (Figure 1). A concentration of 2 μg/ml GCV reduced survival 4% Paraformaldehyde for 10 min at RT. The cells were stained with to almost 50% when 0.25 μl or 0.125 μl of the stock virus DAPI (10 μg/ml) for 1 min followed by three washes with 1× PBS 5 5 and examined on a LSM510Meta Confocal microscope. For AO/EB (which corresponded to 2.5×10 and 1.25×10 gfu staining, the cells were washed with 1× PBS and AO/EB (1 mg/ml) respectively) was used for transduction. There was minimal was added to each plate and the cells were examined on an Axiovert nonspecific toxicity by GCV alone as shown by the 200 inverted microscope. untransduced NT8e cells. Therefore in subsequent experiments, the NT8e cells were treated with 1×105 gfu Cell cycle kinetics. To quantify the percent apoptosis and population Adv-HSVtk and 2 μg/ml GCV for three days. of cells in S-phase of the cell cycle by flow cytometry, the NT8e DAPI-staining. Fluorescent microscopic studies using DAPI cells were treated with 1×105 gfu Adv-HSVtk and 2 μg/ml GCV. The cells were harvested at 0 h and on day 1, 2 and 3. staining revealed swelling of the cytoplasm and that the size Corresponding NT8e untreated controls were also harvested. The of the nuclei of the cells treated with GCV for 3 days was cells were fixed with 70% ethanol, stained with 4 mg/ml PI and larger than that of the control untreated cells, but nuclear assessed on FACS Calibur (Becton Dickinson, San Jose, CA). fragmentation and chromatin condensation which are

3852 Srivastava et al: Cell Death by Adv-HSVtk/GCV

including BIK, BNIP3, Bcl10 and TNFRSF9 showed approximately 2 to 3 fold up-regulation. The anti-apoptotic genes such as NOL3, Bcl2 and BIRC1 showed 3 to 4 fold down-regulation. However, the majority of the important genes involved in apoptosis such as caspase-9, caspase-7, FADD, BAK1, BID etc. did not show any differential expression with respect to the untreated control (Figure 7).

Discussion

Figure 1. NT8e cell kill following Adv-HSVtk/GCV treatment. Cell kill The NT8e cells transduced with Adv-HSVtk showed was assessed by SRB assay after 72 h of Adv-HSVtk/GCV treatment. A susceptibility to GCV in a dose dependent manner but no dose dependent increase in cell kill was observed upon Adv-HSVtk/GCV toxicity was shown in the untransduced NT8e cells. A treatment of NT8e cells. V: virus stock; Error bars represent standard error of means across three experiments. survey of the literature revealed that while some studies have suggested induction of apoptosis on HSVtk/GCV treatment (10, 11, 22, 23), other reports have indicated the involvement of non-apoptotic cell death upon the same characteristic of apoptosis were not seen (Figure 2). Likewise treatment (17, 24). While trying to determine the cause for AO/EB staining also revealed areas of red stained cells, variation in HSVtk/GCV mediated cell death, Katabi et al. which are indicative of necrosis, upon treatment with Adv- (17) reported that interaction with adenoviral early region HSVtk/GCV for three days (Figure 3). E4 encoded viral proteins significantly reduced toxicity mediated by HSVtk/GCV and proposed that apoptosis is not Effect of treatment with Adv-HSVtk/GCV on cell cycle the only method of cell death. Although the role of kinetics. Flow cytometry data revealed that the cells treated adenoviral early region E4 gene product was not currently with Adv-HSVtk/GCV for a period of 3 days, showed a studied, from our earlier studies HSVtk/GCV mediated gradual increase in the proportion of cells in the S-phase of apoptosis was higher when retroviral vectors were used as the cell cycle (Figure 4A). At 0 h 13% of cells were in S- against adenoviral vectors carrying the same HSVtk gene phase but the proportion of S-phase cells on day 1, day 2 and (data not shown). day 3 increased to 48%, 58% and 69% respectively (Figure Surprisingly, no nuclear condensation in Adv-HSVtk/GCV 4B). Only a small proportion of cells (~9%) were seen in the transduced NT8e cells was observed when the cells were sub-G1-phase (Figure 4C) indicating that apoptosis may not stained with DAPI, while, AO/EB staining indicated the be the major form of cell death upon treatment of NT8e cells presence of necrotic cells (red labeled cells). However in a with Adv-HSVtk/GCV. previous report by Li et al., AO/EB staining disclosed morphological features typical of apoptosis in Adv- Caspase-3 cleavage and DNA fragmentation. Western blot HSVtk/GCV treated MDA-MB-468 and MCF-7 cells (25) analysis did not show activation of caspase-3 (cleaved although a very high concentration of GCV (9 μg/ml) was caspase-3 band at ~17KD) on Adv-HSVtk/GCV treatment at used in the study. different time points (Figure 5). Agarose gel electrophoresis The present cytometric data indicated an increase in the demonstrated that the DNA ladder characteristic of apoptosis proportion of cells in the S-phase of the cell cycle upon Adv- was observed in the NT8e cells treated with 1×105 and 1×106 HSVtk/GCV treatment. Further, in accordance with our gfu Adv-HSVtk and 2 μg/ml GCV for three days indicating previous data no sub-G1 fraction “apoptotic peak” was the presence of a small proportion of apoptotic population in observed. There is a possibility that the S-phase arrested cells the treated cells (Figure 6). Cellular DNA preparations from might gradually die through an apoptotic or a non-apoptotic untreated NT8e cells, which were included as the negative pathway, on increasing the duration of the treatment. Earlier control, did not display DNA ladder pattern. The positive studies of flow cytometry by Wei et al. (26) demonstrated that control i.e. 100 nM staurosporine treated NIH3T3 cells and HSVtk/GCV treatment caused either S- and/or G2/M-phase 100 nM staurosporine treated NT8e cells, showed a cell cycle arrest before cell death. They showed an increase distinctive DNA ladder pattern. and accumulation of S-phase B16F10 melanoma cells upon HSVtk/GCV transduction along with a prominent increase in Quantitative real time PCR array analysis. A fold increase the apoptotic population at 72 h. However, Kuratate et al. of 2 or more with respect to the level of the gene expression reported cell death without cell cycle arrest (16). in untreated NT8e control cells was taken as indicative of Caspase-3 is one of the key effector caspases in the cell. On differential gene expression. The pro-apoptotic genes activation, procaspase is cleaved into a lower molecular weight

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Figure 2. Effect of HSVtk/GCV on cell morphology. DAPI staining of Figure 3. Cell death determined by AO/EB staining following Adv- untreated NT8e cells and cells treated with either Adv-HSVtk or Adv- HSVtk/GCV treatment of NT8e cells for 72 h. Images were acquired at HSVtk along with GCV for 72 h. Images acquired at ×63 magnification. ×10 magnification. Necrotic cells, yellow arrows; apoptotic cells, red arrow. Green staining indicates live cells.

Figure 4. Flow cytometric analysis of Adv-HSVtk/GCV treated NT8e cells. (A) Flow cytometric analysis of Adv-HSVtk/GCV treated or untreated NT8e cells; (B) Proportion of cells in S-phase; (C) Percent of sub-G1-phase, apoptotic cells. protein which further cleaves other protein substrates to trigger suggests that NT8e cells are either resistant to apoptosis or the apoptotic process. A ~17KD band corresponding to there could be an alternate mechanism, other than apoptosis, cleaved caspase could not be detected in the Adv-HSVtk/GCV which might be playing a role in the cytocidal effect of transduced NT8e cells. Likewise, Kuratate et al. have also HSVtk/GCV treatment on NT8e cells. reported that upon HSVtk/GCV treatment no cleaved or The DNA ladder assay is considered to be one of the activated caspase-3 band was observed (16). All this data reliable methods for detecting apoptosis. Wei et al. and

3854 Srivastava et al: Cell Death by Adv-HSVtk/GCV

Figure 5. Caspase-3 detection in Adv-HSVtk/GCV treated NT8e cells Western blot of caspase–3 (35KD) and cleaved caspase-3 (17KD) after Figure 6. Apoptotic DNA ladder assay following Adv-HSVtk/GCV Adv-HSVtk/GCV treatment up to 72 h. Untreated NT8e cells, negative treatment of NT8e cells. Apoptotic DNA ladder pattern 72 h after Adv- control and NIH3T3 cells treated with 100 nM STS for 24 h, positive HSVtk/GCV treatment of NT8e cells. Untreated NT8e cells, negative control. control; NT8e and NIH3T3 cells treated with 100 nM STS for 24 h, positive control.

Figure 7. Real time PCR array of apoptotic gene expression in Adv-HSVtk/GCV treated NT8e cells. (A) Down-regulated anti-apoptotic genes. (B) Up-regulated pro-apoptotic genes. (C) Unregulated genes.

Samejima et al. and Sung-Jen (26, 27), have independently HSVtk/GCV transduction. It is known that the same stimuli reported a typical apoptotic DNA ladder pattern upon can induce both apoptosis and necrosis under different treatment with HSVtk/GCV as was also observed when the conditions (31). Tumour Necrosis Factor is one such NT8e cells were treated with 1×105 or 1×106 gfu of Adv- molecule which is known to induce apoptosis or necrosis HSVtk and GCV in the present study. Thus there could be a depending on the type of cell line used (32). Other non- small apoptotic population in the present treatment group apoptotic modes of cell death such as caspase independent which was not identified by the other techniques. programmed cell death and autophagy have also been The real time PCR data did not reveal differential expression reported (33-35) and although these modes of cell death were of any significant genes involved in apoptosis. Key players of not investigated in this study, there is a possibility that these apoptosis such as caspase 9, caspase-1, FADD and BID did not could also play a role in Adv-HSVtk/GCV induced NT8e show any differential regulation upon Adv-HSVtk/GCV cell death. A recent report by Jiang et al. demonstrated that transduction in the NT8e cells. However, down regulation in human oncolytic adenovirus type 5 induces cell lysis through some anti apoptotic genes such as NOL3 and BIRC1 was autophagy and autophagy-triggered caspase activity (36). observed. Also a few pro-apoptotic genes such as NOXA and Using a simple and basic approach to understand the mode BNIP3, which are reported to be involved in p53 mediated of cell death in NT8e cells upon Adv-HSVtk/GCV apoptosis (28, 29) and hypoxia (30) respectively, were up- transduction, the data clearly suggest that Adv-HSVtk/GCV regulated. Despite the differential expression of these genes treatment induces cell death in the NT8e cells and although apoptosis, was not observed as a major mode of cell death. the presence of a small proportion of apoptotic cells cannot The present report contradicts earlier published reports be ruled out, apoptosis does not seem to be the central which establish apoptosis as the mode of cell death upon pathway through which cell death occurs. The triggering

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