ANTICANCER RESEARCH 31: 3851-3858 (2011) Mode of Cell Death Associated with Adenovirus-mediated Suicide Gene Therapy in HNSCC Tumor Model DEEPIKA SRIVASTAVA1, GANESH JOSHI1, KUMARAVEL SOMASUNDARAM2 and RITA MULHERKAR1 1Advanced Centre for Treatment, Research and Education in Cancer, 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). Apoptosis 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). 0250-7005/2011 $2.00+.40 3851 ANTICANCER RESEARCH 31: 3851-3858 (2011) 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.