The Study Of Killing Effect Of HSV1-sr39TK/GCV On Gliomas And The Possible Mechanisms Of Its Bystander Effect

Glioma is the most common type of primary brain neoplasms,and despite advances in neurosurgical techniques,radiation treatment,and chemotherapy,the prognosis of these tumors remains dismal,especially in patients diagnosed as high-grade glioma,with a median survival <1 year.Limitations of surgery, radiotherapy,and chemotherapy make the development of new treatment strategies necessary.Gene therapy may offer a new strategy for treatment of malignant gliomas.The most widely used gene therapy approach involves the transfer of herpes simplex virus-1 thymidine kinase(HSV1-TK)gene,followed by prodrugs administration. HSV1-TK has a very broad substrate specificity,and can catalyze the phosphorylation of nucleoside analogs(e.g.,ganciclovir(GCV),acyclovir(ACV),and so on).Once phosphorylated,these nucleoside analogs are further phosphorylated by cellular kinases to the corresponding nucleoside triphosphates that inhibit host cell DNA replication,resulting in cell death.The combination of HSV1-TK with GCV has demonstrated promise for tumor ablation in a variety of animal models,and has been used in numerous clinical trials.However,the dose of GCV required for tumor regression in clinical settings is immunosuppressive.Although ACV is relatively nontoxic even at high doses,HSV1-TK displays a very high K_m towards ACV that precludes its application in gene therapy clinical settings.As a means to not only improve the overall efficacy of the HSV1-TK/GCV system but also reduce the requirements of high-dose GCV treatment,more and more researchers make attempt to construct novel HSV1-TKs,the HSV1-TK mutants.One potential mutant is HSV1-sr39TK,which contained seven bases substitutions at or near active sites of HSV1-TK gene via semi-random sequence mutagenesis,resulting in five amino acid replacement,and finally improving prodrug sensitivity.With GCV as the substrate,HSV1-sr39TK demonstrated a 14-fold decrease in K_m compared with the wild-type enzyme.Compared with other mutants, HSV1-sr39TK provided a more effective and safer alternative to wild-type TK.During the study of this suicide gene system,it was found that it not only affects cells expressing HSV1-TK gene,but also kills adjacent untransduced cancer cells.This phenomenon,known as the "bystander effect",results in the killing of a larger portion of the cells than that transduced with the suicide gene.Indeed,complete tumor eradication has been demonstrated even when as few as 10%of the tumor cell inoculum is transfected with HSV1-TK gene.However,the mechanism of the bystander effect is not fully understood.The intoxication of untransduced bystander cells is believed to result from a gap junction intercellular transfer of toxic phosphorylated GCV molecules from the cytoplasm of HSV-TK-transduced cells to untransduced cells by a family of connexin proteins.In the present study,we first transfected C6 cells with HSV1-TK or HSV1-sr39TK,named C6-TK and C6-sr39TK respectively,and then assessed the killing effect of HSV1-TK/GCV and HSV1-sr39TK on glioma cells in vitro and in vivo.At last,we examined whether gap junction communication plays an important role in the bystander effect of C6 cells.PARTâ… Construction of C6 cells expressing HSV1-sr39TK geneObjective:To construct C6 cells expressing HSV1-sr39TK gene or HSV1-TK gene.Methods:1.pcDNA3-TK established by subcloning HSV1-TK cDNA into a eukaryotic expressing vector pcDNA3.1,and pcDNA3-sr39TK plasmid,a kind gift from others,were amplified in Escherichia coli DH5α,purified using Hipure Plasmid Midi-prepare Kit,cleaved by restriction endonucleases Xhol and EcoR1 digestion,and identified by electrophoresis.The HSV1-TK and HSV1-sr39TK were further confirmed by sequencing.2.The pcDNA3-TK and pcDNA3-sr39TK plasmids were introduced into C6 cells in vitro using Effectene Transfection Reagent,named C6-TK and C6-sr39TK respectively.RT-PCR,Western blotting and immunocytochemistry were used to detect the expressing level of transfected gene.Results:1.Restriction endonucleases Xhol and EcoR1 showed that HSV1-TK and HSV1-sr39TK gene was correctly inserted into the multicloning site.DNA sequencing data showed that the coding regions of HSV1-TK and HSV1-sr39TK gene were both 1128bp,and were identical to the sequences published in GenBank,apart from a nonsense mutaion of HSV1-TK gene sequence.2.RT-PCR and Western blotting analysis demonstrated expression of TK gene in C6-TK and C6-sr39TK cells 48 hours after transfection,while in untransduced cells no similar bands found.Semiquantitative analysis revealed no significant difference of mRNA or portein expression level existing between the two groups (P=0.058 and P=0.095 respectively).Immunocytochemistry showed(13.25±1.12)%cells of C6-TK cells were TK positive,and(13.76±2.09)%of C6-sr39TK were positive(χ²=0.142,P=0.706),whereas no positive cell was found in untransduced group.Conclusions:We successfully constructed and amplified recombinant eukaryonic expression plasmids pcDNA3-TK and pcDNA3-sr39TK,and effectively transduced them into C6 cells via liposomes,named C6-TK and C6-sr39TK cells respeetively.TK gene expression could be detected in both C6-TK and C6-sr39TK group,and there was no statistical expression difference detected between the two groups.Partâ…¡The treatment effect of HSV1-sr39TK/GCV on gliomasObjective:To explore the killing effect of mutant herpes simplex virus type 1 thymidine kinase(HSV1-sr39TK)and its wild type(HSV1-TK)on rat C6 glioma cells after GCV treatment.Methods:1.C6-TK and C6-sr39TK cells were seeded into 96-well dishes,and the cytotoxicity assay were observed and compared by the standard MTT assay after 72 hours of GCV administration.Absorbance at a wavelength of 570nm was measured to calculate the percentage of live cells against the control group. 2.Cells were also seeded into 6-well dishes,and stained by Hoechst 33342 and Propidium Iodide(PI)after 72 hours of GCV administration.The percentage of PI positive cells to the total cells of each group were counted under a fluorescence microscope.3.Untransduced cells were inoculated subcutanously in the left frank of 20 BALB/c nude mice,which were randomly assigned to 2 groups and inoculated with C6-TK or C6-sr39TK subcutanously in the right frank,and treated with GCV 7days after inoculation.Measurement of tumor size and ¹⁸F-FDG micro-PET scan were performed before and after GCV treatment.All of animals were sacrificed after the last micro-PET scan,and tumor tissues were extracted to further pathologic examination. The TK gene expression of tumor tissues was evaluated by RT-PCR,Western blotting and immunochemistry.Results:1.As the prodrug GCV concentration increasing from 0μM to 400μM,the cell survival rates in C6-sr39TK group declined from(99.96±3.54)%to(4.75±1.79)%,while in C6-TK group from(100.03±2.95)%to(59.16±3.48)%and in C6 group from.(100.29±1.20)%to(83.62±7.56)%.There was a significant difference in killing effect among C6,C6-TK and C6-sr39TK group after GCV treatment.In addition,significantly morphological changes,characterized with growth inhibition and cell death,were observed among the transfection groups,especially in group of C6-sr39TK after GCV treatment.2.Double labeling with Hoechst 33342 and PI showed that most cells in untransduced group were Hoechst positive,with the nuclei uniformly and hazily emitting blue light,while the red light emitting dead cells were rare.The percentage of PI positive cells increased significantly in both C6-TK and C6-sr39TK,with a percentage of(31.529±0.019)%and(60.960±0.020)%respectively.A significant difference was also observed between C6-TK and C6-sr39TK group(χ²=35.765, P=0.000).Among the Hoechst positive cells,some condensed or fragmented nuclei characteristic of apoptosis were observed.3.In vivo experiment,the rate of tumor formation was 100%.After 10 day treatment of GCV following tumor formation,tumors in animals implantated with C6-sr39TK,C6-TK or C6 got an average volume equaling(574.08±107.72),(928.47±165.61)and(1287.24±364.84)mm³ respectively.The most powerful tumor growth inhibition was observed in C6-sr39TK +GCV group,with an inhibition rate of tumor growth about 55.40%.Mciro-PET scan showed there was no significant difference of T/NT ratios of tumors before GCV treatment among C6,C6-TK and C6-sr39TK group(F=0.150,P=0.861).However,after GCV administation,the T/NT ratios of tumor in C6-sr39TK(3.335±0.700)were significantly lower than C6-TK (3.648±0.743)and untransduced group(4.576±1.100)(F=7.006,P=0.003). Numerous necrotic and hemorrhagic tumor tissues as well as fibroplasia were observed in transfection group,especially in C6-sr39TK group by HE staining. RT-PCR,Western blotting and immunochemistry demonstrated TK gene expression could still be detected in tumor tissues at the end of the experiment.Conclusions:C6 cells transfected with HSV1-sr39TK were more sensitive to GCV than those with HSV1-TK both in vitro and in vivo,and thus HSV1-sr39TK can be considered as a means to improve the overall efficacy of the HSV1-TK/GCV suicide gene system.Partâ…¢The possible mechanism of the bystander effectObjective:To explore the role of gap junctional intercelular communication(GJIC)in the bystander effect.Methods:C6-TK cells were seeded into 96-well dishes,and divided into two groups, AGA⁺ group(dealing with 18α-glycyrrhetinic acid(AGA),an inhibitor of GJIC)and AGA^- group.The cytotoxicity assay were observed and compared by the standard MTT assay after 72 hours of GCV administration.Absorbance at a wavelength of 570nm was measured to calculate the percentage of live cells against the control group.RT-PCR,Western blotting and immunocytochemistry were used to investigate the effect of AGA on the expression level of Connexin 43(Cx43)gene.Results:Cells in AGA⁺ group were not as sensitivity as those in AGA^- group.At the same concentration of GCV,the cell survival rates of the former group were significant higher than the latter one(P＜0.05).However,AGA could not completely inhibit the killing effect of HSV1-TK/GCV.Significantly lower expression level of Cx 43 mRNA was observed after AGA administration(t=37.703,P=0.000).However, there was no significant change of Cx 43 expression level demonstrated either by Western blotting or the immunocytochemistry between the two groups.Conclusions:The fact that AGA could reduce the bystander effect of C6 cells confirmed the important role of GJIC played in the bystander effect,which may be partially mediated by the alteration of Cx 43 expression.However,AGA could not completely block the total effect.Perhaps there were other mechanisms contributing to the bystander effect of HSV1-TK/GCV on C6 cells.