Materials and methods

Chemicals

NS398 was purchased from BioMol, Plymouth Meeting, PA. Nimesulide was a kind gift from Rafa Laboratories, Israel. GCV and AA were purchased from Sigma, St Louis, MO. [8-³H]-GCV (22 Ci/mmol) was from Moravek Biochemicals, Brea, CA. All other reagents and chemicals were of the highest quality available.

Cell culture

The 3-methylcholanthrene-induced murine colon adenocarcinoma cells (MC38) were a generous gift of Dr SA Rosenberg, NCI, USA. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/l D-Glucose (Biological Industries, Beit Haemek, Israel), supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin and 10% fetal calf serum (FCS). Cells were maintained at 37°C in a humidified atmosphere of 95% air and 5% CO₂.

Retroviral vectors and tumor cell transduction

Vector producer cell lines (PA317/STK and PA317/LNL6) were provided by Clinical Gene Therapy Branch, NIH, USA. STK and LNL6 retroviral vectors have been described in detail by Moolten et al.¹ To construct STK vector, HSV-tk sequence was inserted into LNL6 under the control of Simian Virus 40 (SV40) early region promoter and enhancer. LNL6 vector harbors the neomycin resistance (Neo^R) gene, which encodes for neomycin phosphotransferase II and confers resistance to the neomycin analog G418 (Fig 1). MC38 cells transduced with HSV-tk (MC38/HSV-tk) or Neo^R gene (MC38/Neo^R) were generated by stable transduction of MC38 cells as previously described.¹ Briefly, PA317/STK and PA317/LNL6 cells were cultured to 90% confluence for 24 hours. Supernatant medium was then filtered through 0.45 m filter, supplemented with 8 g/ml polybrene (Hexadimethrine bromide, Sigma) and applied to target cells for 48 hours with changing every 12 hours. Thereafter, transduced cells were exposed to 1 mg/ml G418 (Geneticin, Life Technologies, Grand Island, NY) for 2 weeks to select for cells expressing vector-derived genes. Validation of HSV-tk expression in MC38/HSV-tk cells was performed by measuring the antiproliferative effect of GCV and its phosphorylated metabolites in vitro as described below. GCV, when assessed over a 48-hour period, had no cytotoxic effects in MC38 or MC38/Neo^R cells over the dose range studied (0–100 M), therefore its IC₅₀ (the drug concentration resulting in 50% inhibition of cell growth) was >100 M, whereas under the same experimental conditions, GCV inhibited MC38/HSV-tk growth rate with IC₅₀ of 0.94 M.

Figure 1.

Diagrammatic representation of STK and LNL6 retroviral vectors. STK vector contains herpes simplex virus type 1 thymidine kinase (HSV-tk) sequence, derived from pTK retroviral vector, which is inserted into LNL6 vector under control of SV40 promoter. LNL6 vector harbors Neo^R gene only. LTR, long terminal repeat; , packaging signal; Neo^R, neomycin-resistance gene.

Full figure and legend (6K)

GCV-resistant MC38/HSV-tk cell line (MC38/HSV-tk/GCV^R) was generated by continuous (6–8 weeks) exposure of MC38/HSV-tk cells to increasing concentrations of GCV (0.1–10 M) as previously described by Degr'eve et al³⁵. The IC₅₀ of GCV against MC38/HSV-tk/GCV^R cells was 68 M, which is 72 times higher than the IC₅₀ measured in GCV-sensitive MC38/HSV-tk cells.

Effects of GCV on cell proliferation

To assess the antiproliferative effect of GCV, MC38, MC38/Neo^R, MC38/HSV-tk and MC38/HSV-tk/GCV^R cells were incubated in the presence of increasing concentrations of GCV (0–100 M) for 48 hours. Thereafter, cells were harvested by trypsinization, counted using a Coulter counter and cell growth rate was expressed as a percentage of the increase in cell numbers of the untreated control cultures. IC₅₀ values were calculated using the CalcuSyn software.³⁶

HPLC separation of GCV phosphorylated metabolites

Preparation of cell extracts

The phosphorylation rate of GCV in the appropriate incubations was measured by HPLC as described by Agbaria et al.³⁷ MC38, MC38/Neo^R, MC38/HSV-tk and MC38/HSV-tk/GCV^R cells were cultured for 6 hours with [8-³H]-GCV, 10 M, 1 Ci/ml. At the end of incubations, cells were washed three times with PBS, and after trypsinization, collected by centrifugation at 1500 g for 10 minutes. The resulting cell pellet was extracted with 0.50 ml of cold (4°C) 60% methanol (HPLC grade) by vigorous vortex mixing, then heated at 95°C for 2.5 minutes. The heated extracts were centrifuged at 12,000 g for 10 minutes, the clear supernatant fraction was evaporated to dryness under nitrogen and redissolved in 250 l water, and aliquots were subjected to gradient anion-exchange chromatography as described below.

Gradient anion-exchange HPLC

Separations of GCV and its phosphorylated metabolites were carried out using a Hewlett-Packard 1100 HPLC system with a diode-array ultraviolet absorption detector and controlled by ChemStation software (Version 6.01). A Whatman Partisil-10 SAX column (250 4.6 mm) was eluted with a flow rate of 2 ml/min employing the following elution program: 0–5 minutes, 100% buffer A (0.01 M ammonium phosphate, native pH); 5–20 minutes, linear gradient to 25% buffer B (0.7 M ammonium phosphate with 10% methanol); 20–30 minutes, linear gradient to 100% buffer B; 30–40 minutes, isocratic buffer B; 40–55 minutes, linear gradient to 100% buffer A and equilibration. One-minute fractions were collected and radioactivity was determined by scintillation spectrometry. The retention times of GCV and its phosphates were as follows: GCV, 3 minutes; GCV-MP, 9 minutes; GCV-DP, 24 minutes; GCV-TP, 33 minutes. Fractions containing radiolabeled GCV nucleotides were quantitated based on the known specific activity of the parent tritiated nucleoside.

Animals and tumor growth in vivo

All animal care and experiments were performed in accordance with the guidelines of the Animal Care and Use Committee of the Ben-Gurion University of the Negev. Male C57BL/6 mice, at 6–8 weeks of age, were inoculated subcutaneously (s.c.) with 0.5 10⁶ of MC38, MC38/Neo^R, MC38/HSV-tk or MC38/HSV-tk/GCV^R cells into the left and right flanks as described by Noy et al.³⁸ Tumor growth rate was monitored by measuring the tumor volume with a sliding caliper once in 3 days. The volume of the cuboid mass was calculated from the major dimension (L) and minor dimension (S) using the following equation: tumor volume (V) = L (S)²/2. To evaluate the effect of COX-2 inhibition on tumor growth rate of MC38 and MC38/HSV-tk tumors, mice were treated daily with intraperitoneal (i.p.) injections of nimesulide (20 mg/kg), starting from the eighth day after inoculation (100 mm³ mean tumor volume). Animals in the control group received dimethyl sulfoxide (DMSO), 50 l/day, i.p.

PGE₂ measurement

PGE₂ that was released into the culture medium was measured by radioimmunoassay (RIA), which allows accurate measurement of PGE₂ in sample (at the range of 0.075–20 ng/ml) as was described in detail by Danon et al.³⁹ The blank values in the absence of PGE₂ are in the range of 2–7% of the total values measured in the presence of PGE₂. Briefly, 100 l medium samples were collected from the appropriate incubations and mixed with 500 l RIA assay buffer (containing K₂HPO₄ 10 mM, KH₂PO₄ 1 mM, 0.9% sodium chloride, 0.1% sodium azide and 0.1% bovine serum albumin, pH 7.4), containing an appropriate amount of reconstituted antiserum. The mixture was incubated at 4°C for 30 minutes and 100 l of radiolabeled PGE₂ was added. Tubes were incubated at 4°C for 1 hour, and then a cold charcoal–dextran suspension was added (0.2 ml). After 10 mintues, the tubes were centrifuged at 3500 rpm for 10 minutes at 4°C. Radioactivity in supernatants was then measured by scintillation spectrometry. Percent binding of radiolabeled PGE₂ was compared to a standard curve and the amount of PGE₂ in the samples was calculated. For the determination of PGE₂ release by MC38, MC38/Neo^R and MC38/HSV-tk tumors ex vivo, C57/BL6 mice were inoculated s.c. with MC38, MC38/Neo^R or MC38/HSV-tk cells and tumors were allowed to grow for seven days, reaching a size of 50–100 mm³. Mice were then killed and tumors were surgically removed. Tumor tissues were incubated with shaking at 37°C in 1 ml of Krebs–Henseleit buffer for 1 hour and PGE₂ that was released to the buffer was then measured by RIA.

Western blot analysis

Western blot analysis of COX-1 and COX-2 protein expression was performed as previously described,⁴⁰ with slight modifications. Briefly, tumors were excised from mice and homogenized, and cells (1.5 10⁷) were lysed in 200 l of ice-cold lysis buffer (50 mM Tris-HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 50 mM sodium fluoride, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 1 M leupeptin, 20 u/ml aprotinin and 1 mM sodium vanadate). Cytoplasmic fraction was obtained by centrifugation at 12,000 g for 10 minutes at 4°C. Protein concentration in samples was determined by the Lowry method.⁴¹ Thereafter, aliquots of cell lysates/tumor homogenates, reconstituted with an appropriate amount of 3 Laemmli's loading buffer, were resolved on 10% SDS-polyacrylamide minigels along with 5 l of MW standard (Fermentas, MD) and transferred onto nitrocellulose membranes (Bio-Rad, CA). Membranes were incubated overnight at 4°C with either anti-COX-1 or anti-COX-2 monoclonal IgG (BD Transduction Laboratories, NJ) followed by incubation with anti-mouse IgG, peroxidase-linked whole antibody (Jackson Immunoresearch Laboratories Inc., PA) for 90 minutes. The immunocomplexes were visualized by ECL chemiluminescence (Pierce, IL) using exposure to Super RX film (Fuji Photo Film, Japan) for 5 minutes. To confirm equal loading in all lanes membranes were blotted with anti--tubulin monoclonal antibody (Oncogene, CA). Quantitation of the immunoblots was performed by densitometric scanning using 202D Bio Imaging System (Pharmacia) with a Fujifilm Thermal Imaging System FTI-500 (Fuji, Japan) and TINA software (Raytest, Germany).

Statistical analysis

Results are expressed as means SE. Statistical evaluation was carried out using functional analysis (ANOVA) and Student's t-test (two-tailed), to test for differences between the control and experimental results. Values of P <.05 were considered statistically significant.

Results

Effect of HSV-tk gene transduction on growth rate of MC38 tumors in vivo

As shown in Figure 2, tumors derived from HSV-tk-transduced MC38 cells exhibited enhanced growth rate in mice compared to tumors derived from nontransduced MC38 cells (63% increase in mean tumor volume) at 27 days. In order to evaluate the specificity of HSV-tk gene transduction, we also tested the growth rate of tumors arising from MC38/Neo^R cells. MC38/Neo^R cell line was generated by stable transduction of MC38 cells with LNL6 retroviral vector. LNL6 vector that harbors Neo^R gene differs from the STK vector, which was used for HSV-tk gene delivery, only by lacking the HSV-tk sequence insert (Fig 1). Contrary to HSV-tk gene transduction, Neo^R gene transduction resulted in a decline (53%) at 27 days in the tumor growth rate of MC38 tumors (Fig 2). These findings suggest that the enhanced growth rate of MC38/HSV-tk tumors is probably related to the presence of HSV-tk gene in the transduced tumor cells.

Figure 2.

Effect of HSV-tk gene transduction on growth rate of MC38 tumors. C57BL/6 mice were inoculated s.c. in the right and left flanks with MC38, MC38/HSV-tk or MC38/Neo^R cells (0.5 10⁶). Tumor growth rate was determined as described in Materials and methods. Data shown are means SE (n = 8). ^*P <.05, MC38/HSV-tk vs. MC38; ^**P <.05, MC38/Neo^R vs. MC38.

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Effect of HSV-tk gene transduction on PGE₂ release in vitro and ex vivo

PGE₂ content in samples of medium taken from cultured MC38, MC38/Neo^R or MC38/HSV-tk cells was measured. As shown in Figure 3a, transduction of MC38 cells with HSV-tk gene led to an almost five-fold (162 23 vs. 37 3 ng/10⁶ cells for MC38/HSV-tk and MC38 respectively) increase in the 24 hour release of PGE₂ as compared to MC38 cells, while Neo^R gene transduction actually decreased PGE₂ release (17 1 ng/10⁶ cells). The time course of PGE₂ release during 24-hour also showed that PGE₂ release into the culture medium of MC38/HSV-tk cells was approximately 3–5-fold higher than that of MC38 cells over the entire follow-up period, while transduction with Neo^R gene alone decreased PGE₂ levels (Fig 4). HSV-tk gene transduction of additional two cell lines: namely rat gliosarcoma (9L) and human urinary bladder carcinoma (T-24) also resulted in a significant increase in PGE₂ release (18-fold for 9L and 8-fold for T24, data not shown). Evaluation of PGE₂ release by subcutaneously implanted MC38, MC38/Neo^R and MC38/HSV-tk tumors ex vivo yielded similar results. PGE₂ release by tissues taken from MC38/HSV-tk tumors was significantly higher than that from MC38 tumors (556 16 vs. 299 42 pg/mg tissue for MC38/HSV-tk and MC38 tumors, respectively), whereas PGE₂ release by tissues taken from MC38/Neo^R tumors was nearly half of that released by MC38 tumors (Fig 3b).

Figure 3.

Effect of HSV-tk gene transduction on PGE₂ release. (a) MC38, MC38/Neo^R and MC38/HSV-tk cells were seeded in 24 -well culture plates (10⁵ cells/well) and allowed to attach. Fresh medium was then added and 24 hours later, medium samples were collected and analyzed for PGE₂ concentration by RIA. Data shown are means SE (n = 3). (b) C57BL/6 mice were inoculated in their flank regions with MC38, MC38/Neo^R or MC38/HSV-tk cells. Tumors were grown for 7 days to reach the size of 50–100 mm³. Thereafter, mice were killed; tumors were surgically removed and incubated in 1 ml Krebs–Henseleit buffer for 1 hour. PGE₂ concentration in the buffer was then measured by RIA. Data shown are means SE (n = 5). ^*P <.05, MC38/HSV-tk vs. MC38; ^**P <.05, MC38/Neo^R vs. MC38.

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Figure 4.

Time-course of PGE₂ release by MC38, HSV-tk and Neo^R gene-transduced MC38 cells. MC38, MC38/HSV-tk and MC38/Neo^R cells were seeded in 24 -well culture plates (10⁵ cells/well), allowed to attach and fresh medium was then added. Medium samples were collected at the indicated time points and analyzed for PGE₂ concentration by RIA. Data shown are means SE (n = 3). ^*P <.05, MC38/HSV-tk vs. MC38; ^**P <.05, MC38/Neo^R vs. MC38.

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Overall these findings show that HSV-tk gene transduction resulted in enhanced PGE₂ release in murine colon cancer cells in vitro and in ex vivo tumor models. The enhanced PGE₂ release was concomitant with the augmented growth rate of HSV-tk-transduced tumors.

Effect of exogenous arachidonic acid on PGE₂ release by MC38, MC38/Neo^R and MC38/HSV-tk cells

Prostaglandin synthesis depends on the availability of AA that is released from the phospholipids of cell membrane and rapidly metabolized to oxygenated products by cyclooxygenase. In order to find out whether HSV-tk gene transduction affects the synthesis of PGE₂ upstream or downstream from COX, MC38 and MC38/HSV-tk cells were incubated for 30 minutes with increasing concentrations (0–50 M) of AA. As expected, the addition of AA to the culture medium enhanced the secretion of PGE₂ in both MC38 and MC38/HSV-tk cells, in a dose-dependent manner (Fig 5). However, over the entire AA concentration range, PGE₂ release by MC38/HSV-tk cells was several-fold higher than that of the MC38 cells, indicating that the increased PGE₂ synthesis observed in MC38/HSV-tk cells is downstream of AA release from the membrane phospholipids, therefore reflecting increased COX activity.

Figure 5.

Effect of exogenous arachidonic acid on PGE₂ release by MC38 and MC38/HSV-tk. MC38 and MC38/HSV-tk cells were cultured for 30 minutes with increasing concentrations of AA. At the end of incubations, PGE₂ concentration in the medium was determined by RIA. Data shown are means SE (n = 3). ^*P <.05.

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Effect of selective COX-2 inhibitors on PGE₂ release by MC38 and MC38/HSV-tk cells

In order to find out whether the constitutive (COX-1) or the inducible (COX-2) isoform of cyclooxygenase are involved in the elevated PGE₂ secretion in HSV-tk transduced cells, the effect of the selective COX-2 inhibitors, nimesulide and NS398, on PGE₂ release by MC38 and MC38/HSV-tk cells was examined. Cells were cultured for 24 hours with medium containing 1 M of either nimesulide or NS398. Both COX-2 inhibitors completely abolished PGE₂ release from both HSV-tk transduced and non-transduced cells (Fig 6). These results indicate that PGE₂ release in both cell lines is COX-2-dependent. Moreover, trace levels of PGE₂ observed in the COX-2 inhibitors treated, MC38 cells, suggest a major role for COX-2 rather than COX-1 in the secretion of PGE₂ in these cells.

Figure 6.

Effect of COX-2 inhibitors on PGE₂ release by MC38 and MC38/HSV-tk cells. MC38 and MC38/HSV-tk cells were cultured for 24 hours in the presence of 1 M of nimesulide (NIM) or NS398. At the end of the incubations, PGE₂ concentration in the medium was determined by RIA. Data shown are means SE (n = 6). ^*P <.05, MC38 treated vs. control; ^**P <.05, MC38/HSV-tk treated vs. control.

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Effect of HSV-tk gene transduction on COX-1 and COX-2 protein expression

In order to confirm that the upregulation of PGE₂ release seen in HSV-tk transduced cells is indeed related to COX-2 overexpression, we carried out a Western blot analysis, which showed that HSV-tk gene transduction significantly (two-fold) enhanced the expression of COX-2 protein in MC38 cells (Fig 7a). Similar findings were obtained with 9L and T-24 cells (data not shown). Conversely, Neo^R gene transduction actually reduced COX-2 protein expression in MC38 cells (Fig 7a). Western blot analysis of COX-2 expression in tumor tissues obtained from tumor bearing mice, showed increased (1.5-fold) expression of COX-2 protein in MC38/HSV-tk tumors and diminished expression of COX-2 protein in MC38/Neo^R tumors as compared to tumors of MC38 cells (Fig 7b). These results further support the notion that the enhancement of PGE₂ release following HSV-tk gene transduction is caused by COX-2 overexpression. Under our experimental conditions, COX-1 protein in both HSV-tk transduced and non-transduced cells was undetectable (data not shown).

Figure 7.

Effect of HSV-tk gene transduction on COX-2 protein expression. (a) Western blot analysis of COX-2 expression in MC38, MC38/Neo^R and MC38/HSV-tk cells. Cells were lysed as described in Materials and methods and aliquots of total cell protein (75 g per lane) were separated on 10% SDS/polyacrylamide gel. COX-2 protein (72 kDa) was identified with specific anti-COX-2 antibody. (b) Western blot analysis of COX-2 protein expression in tissues derived from MC38, MC38/Neo^R and MC38/HSV-tk tumors grown in C57BL/6 mice. Homogenized tumor tissues were lysed as described in Materials and methods and aliquots of total homogenate protein (50 g per lane) were resolved on 10% SDS/polyacrylamide gel. COX-2 protein was identified as described. Bars represent average of relative COX-2 expression, quantitated densitometrically from three independent experiments for each immunoblot. ^*P <.05, MC38/HSV-tk vs. MC38; ^**P <.05, MC38/Neo^R vs. MC38.

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Effect of nimesulide on growth rate of MC38 and MC38/HSV-tk tumors

To clarify that COX-2 overexpression is accountable for the enhanced growth rate of MC38/HSV-tk tumors, we evaluated the effect of nimesulide, a selective COX-2 inhibitor, on growth rate of both MC38 and MC38/HSV-tk tumors. C57BL/6 mice were inoculated s.c. into the flank regions with either MC38 or MC38/HSV-tk cells and tumors were allowed to grow for one week to reach a volume of 50–100 mm³. Upon tumor establishment, starting from eighth day postimplantation, nimesulide (20 mg/kg, i.p.), or an identical volume of vehicle, were injected daily. Treatment with nimesulide led to a significant decline in the growth rate of both MC38 and MC38/HSV-tk tumors (53 and 45% decrease in mean tumor volume for MC38 and MC38/HSV-tk respectively), suggesting that COX-2 is indeed involved in the growth of both types of tumors (Fig 8). However, it must be noted that treatment with nimesulide did not reduce the growth rate of MC38/HSV-tk tumors to an extent similar to that of MC38 tumors, suggesting that additional factors, but not solely COX-2, are probably involved in the expanded growth rate of MC38/HSV-tk tumors.

Figure 8.

Effect of nimesulide on growth rate of MC38 and MC38/HSV-tk tumors. C57BL/6 mice were inoculated with MC38 or MC38/HSV-tk cells as described in Materials and methods. Treatment was started upon the establishment of tumors, with daily i.p. injections of nimesulide (NIM, 20 mg/kg body weight); while the control group received an identical volume of vehicle (50 l of DMSO). Tumor volume was calculated as described in Materials and methods. Data shown are means SE (MC38/HSVtk, n = 18 in each group; MC38, n = 14 in each group). ^*P <.05, MC38/HSV-tk + NIM vs. MC38/HSV-tk + DMSO; ^**P <.05, MC38 + NIM vs. MC38 + DMSO.

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Tumor growth rate and PGE₂ release by MC38/HSV-tk cells resistant to GCV

HSV-tk-transduced tumor cells that develop resistance to the cytotoxic action of GCV are not accessible to HSV-tk/GCV gene therapy. Therefore, we examined whether the changes in tumor growth rate and PGE₂ release persist upon the development of GCV resistance. The IC₅₀ of GCV on cell growth rate of MC38/HSV-tk/GCV^R cells was 72-fold higher than that of MC38/HSV-tk cells (68 and 0.94 M, respectively), confirming resistance. GCV resistance in our experiments is probably related to loss or decrease of GCV phosphorylation by HSV-tk, as confirmed by the diminished ability of MC38/HSV-tk/GCV^R cells to generate high levels of phosphorylated intracellular GCV metabolites compared with that of GCV-sensitive MC38/HSV-tk cells (Table 1). As shown in Figure 9a, the growth rate of MC38/HSV-tk/GCV^R tumors implanted s.c. in C57BL/6 mice remained elevated (compared with MC38 tumors) to a similar extent as that of MC38/HSV-tk tumors. Likewise, the enhancing effect of HSV-tk gene transduction on PGE₂ release persisted in MC38/HSV-tk/GCV^R cells in culture (Fig 9b). In addition, we examined the effect of GCV on PGE₂ secretion in MC38/HSV-tk cells. GCV did not significantly alter the release of PGE₂ by the HSV-tk-transduced cells (data not shown). Taken together, these results suggest that HSV-tk-transduced tumor cells resistant to GCV possess an enhanced tumor growth rate and increased PGE₂ release similar to those of GCV-sensitive HSV-tk transduced cells.

Figure 9.

Growth rate of MC38/HSV-tk/GCV^R tumors and PGE₂ release by MC38/HSV-tk/GCV^R cells. (a) The growth rate of MC38/HSV-tk/GCV^R tumors in C57BL/6 mice was evaluated as described in Materials and methods. Tumor growth rate was monitored once a week, starting from eighth day postimplantation and expressed as percent increase in mean tumor volume of each group. Data shown are means SE (n = 8). (b) MC38, MC38/HSV-tk and MC38/HSV-tk/GCV^R cells were seeded in 24 -well culture plates (10⁵ cells/well) and allowed to attach. Fresh medium was then added and 24 hours after medium samples were collected analyzed for PGE₂ concentration by RIA. Data shown are means SE (n = 4). ^*P <.05.

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Table 1 - Levels of intracellular GCV-metabolites in MC38, MC38/Neo^R, MC38/HSV-tk and MC38/HSV-tk/GCV^R cells treated with GCV (in pmole/10⁶ cells).

Full table

Discussion

In this study, we report that tumors derived from murine colon cancer cells transduced with HSV-tk gene, exhibit enhanced growth rate upon subcutaneous implantation in mice as compared to tumors derived from wild-type MC38 cells. To elucidate whether the observed alteration in tumor growth rate is specific to the HSV-tk gene, or is the result of the gene transduction procedure itself, we utilized the LNL6 vector. The later, originally used by Moolten et al¹ to construct STK retroviral vector for HSV-tk gene transduction in tumor cells, is frequently applied for the construction of numerous retroviral vectors in gene delivery techniques. The LNL6 vector construct basically encodes for neomycin phosphotransferase II, which confers resistance to the neomycin analog G418 and thereby allows selection for cells that express vector-derived genes. Moolten inserted the HSV-tk sequence, driven by SV40 promoter, into LNL6 vector, resulting in the STK vector construct which harbors both Neo^R and HSV-tk genes. Thus, LNL6 differs from the STK vector only by lacking the HSV-tk sequence. We demonstrated that while stable transduction of murine colon cancer cells with the STK vector construct yielded enhancement of growth rate of tumors derived from these cells, the growth rate of tumors originating from cells transduced with LNL6 vector was actually slower than that of wild-type MC38 tumors. This observation suggests that the enhancement of growth rate observed in tumors transduced with STK vector is related to the specific presence of HSV-tk sequence insert in the vector used for HSV-tk gene-delivery (Fig 2). Obviously, one would expect that Neo^R gene-transduced cells would behave as MC38 cells in these circumstances. The question of why Neo^R gene transduction inhibits PGE₂ release and tumor growth rate remained unsettled.

Numerous investigations have established that upregulation of the COX-2 pathway affects development and growth of solid tumors.^{27, 42} Therefore, we explored in detail both prostaglandin synthesis and cyclooxygenase expression in cultured cells and in tumors transduced with STK and LNL6 retroviral vectors. Evaluation of PGE₂ release by cultured MC38 cells and by tissues derived from MC38 tumors revealed that transduction with the HSV-tk gene effected a significant enhancement of PGE₂ release (Fig 3). On the other hand, transduction of MC38 cells with the LNL6 vector, which lacks the HSV-tk gene but contains the Neo^R gene, resulted in the decline of PGE₂ release as compared to MC38 cells (Fig 3). The complete inhibition of PGE₂ synthesis by specific COX-2 inhibitors (Fig 6), together with the failure to detect COX-1 expression in MC38 cells by Western blot analysis, indicates that COX-2 is probably responsible for PGE₂ synthesis in these cells. Analysis of COX-2 protein expression in cultured MC38 cells and in MC38 tumor models revealed a similar pattern: the presence of the HSV-tk gene resulted in a significant increase in the expression of COX-2, while transduction with LNL6 vector resulted in a significant reduction in the expression of COX-2 (Fig 7). These results further support the notion that upregulation of the PG biosynthetic pathway is related to the presence of the HSV-tk gene. Moreover, COX-2 expression and PGE₂ production in HSV-tk transduced tumors is directly correlated with their growth rate.

Numerous studies have demonstrated that treatment with COX-2 inhibitors leads to a marked reduction in the growth of a variety of neoplasms including colon,^{43, 44} breast,⁴⁵ and others cancers.^{46, 47, 48} Moreover, selective COX-2 inhibitors have been reported to protect from tumorigenesis in vitro and in in vivo cancer models.^{49, 50, 51} These pieces of evidence, as well as the established association of enhanced COX-2 expression with increased tumor growth, suggested that inhibition of PG synthesis would attenuate the enhancement of tumor growth rate that accompanies the presence of the HSV-tk gene. In order to test this possibility, we monitored the growth rate of MC38 and MC38/HSV-tk tumors in mice treated with the selective COX-2 inhibitor, nimesulide, which has been previously demonstrated to exert antitumor activity.^{50, 51} In the current study, nimesulide treatment caused a significant inhibition in growth rate of both MC38 and MC38/HSV-tk tumors. However, nimesulide did not decrease the growth rate of MC38/HSV-tk tumors to a level similar to that of nimesulide-treated MC38 tumors (Fig 8). This finding may suggest that additional procarcinogenic factors besides COX-2 are involved in the overexpanded growth rate of HSV-tk-transduced tumors. Since recent publications^{52, 53} suggest several cyclooxygenase-independent actions of cyclooxygenase inhibitors, one cannot exclude the possibility that attenuation of tumor growth rate by nimesulide is not necessarily related to direct inhibition of COX-2 activity.

MC38 cells expressing the HSV-tk gene that develop resistance to GCV (MC38/HSV-tk/GCV^R) exhibited an enhanced growth rate and augmented prostaglandin synthesis similarly to GCV-sensitive MC38/HSV-tk cells (Fig 9). These observations are clinically significant, because the prognosis of HSV-tk-transduced tumors may seriously deteriorate when they become resistant to GCV with enhanced growth rate without a benefit of a prodrug to accomplish the suicide mission of this treatment.

GCV-resistant MC38/HSV-tk cell lines can be obtained by two different methods. According to the method described by Degr'eve et al³⁵ and employed by us resistance was obtained by prolonged incubation of the HSV-tk-expressing cells in dose-escalating concentrations of GCV. The HSV-tk gene in the resistant cells generated by this method was silenced but not deleted from the cell genome.³⁵ On the other hand, exposure of HSV-tk expressing cells to high concentration of GCV, results in deletion of fragments in the HSV-tk gene sequence as demonstrated by Garin et al.¹⁰

The observation that MC38/HSV-tk/GCV^R exhibit enhanced PGE₂ production and growth rate, in spite of the lack of detectable phosphorylated GCV metabolites suggest that the HSV-tk kinase activity is not required for inducing these effects. Further experiments employing siRNA and expressing HSV-tk mutations with complete lack of HSV-tk-dependent kinase activity could elucidate the link between the presence of HSV-tk gene and the overexpression of COX-2 in the MC38/HSV-tk/GCV^R.

At present, we cannot say how HSV-tk transduction induces COX-2 and PGE₂ production in tumor cells. COX-2 behaves as an "immediate early" gene and is subject to rapid regulation at the transcriptional level.⁵⁴ Sequence analysis of the human COX-2 promoter 5'-flanking region has shown several potential transcription regulatory sequences, including a TATA box, C/EBP and CRE motifs, AP-2, SP1, NF-kappa B and Ets-1 sites,⁵⁵ which have been found to be differentially responsive to various stimuli. Transcriptional regulation of COX-2 protein expression is a complex event and appears to involve diverse mechanisms in different cell types under various conditions. Preliminary results of ongoing research in our laboratory show that HSV-tk gene transduction is associated with elevated NF-kappa B transcriptional activity, which may lead to transcriptional upregulation of COX-2 expression, and probably of other procarcinogenic factors. Currently, we are exploring the exact molecular mechanism by which HSV-tk gene transduction augments COX-2 protein expression.

In this study, we tested the effect of HSV-tk transduction only in three different cell types and we employed only a retrovirus as a gene carrier. Future experiments on a larger variety of cell types and additional gene carriers, such as adenovirus, are required before we can generalize our observations as a general phenomenon, which is not dependent on cell type or the gene carrier.

In summary, the present report shows that tumor growth rate, COX-2 protein expression and PG synthesis are upregulated by HSV-tk gene transduction. These data may partially explain some problems facing HSV-tk/GCV "suicide" gene therapy, such as resistance to chemotherapy and high immunogenicity of the transduced cells. Overexpression of COX-2 may decrease the efficacy of the HSV-tk/GCV system since it can enhance tumor promotion, differentiation, metastatic potential and resistance to chemotherapy. It is therefore tempting to suggest that combining HSV-tk/GCV therapy with COX-2 inhibitors may improve the therapeutic efficacy of this system.

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Acknowledgements

This research was supported by the ISRAEL SCIENCE FOUNDATION (Grant No 500/02).