The Role Of A Adenoviral-mediated HSV-tk Gene And GCV System, Scopadulciol, In Improving The Efficacy Of Bladder Cancer Gene Therapy

Bladder cancer is the common malignant tumor in urinary system.It ranks as the eighth highest incidence in all of the malignant tumours.The main characteristic of bladder cancer is easy to recur and transfer.Standard therapies include deforming radical surgical procedures,radiotherapy and chemotherapy.Although conventional cancer therapies produce a high rate of cure for patients with early-stage disease,many cancers recur and the majority of patients with advanced cancer eventually succumb to the disease.With the development of molecular biological technology,gene therapy had become the fourth model in the treatment of cancer.To date the most promising strategies of gene therapy is the use of suicide gene therapy,which is the most effecteve and clinical useful potentiality.One of the strategies utilizes a enzyme/prodrug system which can convert a prodrug into a toxic metabolite leading to cell death.The most frequently utilized system is the thimidine kinase (tk) gene from the herpes simplex virus (HSV),given in combination with ganciclovir (GCV).The HSV-tk product has ligh affinity for acyclovir and its analogues,including ganciclovir,maimly producing the phosphorylated product.Subsequent modification by the host cell to a triphosphorylated form and incorporation during riplication halts growth of the deceloping DNA strands and inhibits DNA polymerase activety.Mammalian tk has a much lower affinity for the prodrug such that tumor cells once transduced are selectively killed in the presence of ganciclovir.The efficiency ofthis approach can be amplified by a by"stander effect", killing the nontransduced neigbouring cells.Telomerase is a specialized DNA polymerase responsible for the replication of telomeres.The selective reactivation of telomerase in tumor cells offers an attractive therapeutic target for developing new broad-spectrum antitumor agents.Although hTERT and hTR are both necessary for telomerase activity,expression of hTERT is present specifically in tumor cills whereas hTR is present in both normal and tumor cills.Because the hTERT gene is highly active in tumor cells but repressed in most normal cills and because its expression insregulated at the transcription level,the hTERT promoter may be used for tumor-specific expression of transgenes.In the present study,hTERT promoter was used to induce expression of the HSV-tk gene,and efficiency wsa tested in cancer gene therapy.Our study includes three parts.The first part:Construction of recombinant adenovirus vector containing hTERT promoter and HSV-tk geneObjective:Construction of recombinant adenovirus vector containing hTERT promoter and HSV- tk gene.Methods:At first the plasmid containing hTERT promoter and HSV-tk gene was constructed by DNA recombinant technology and transfected into 293 cells by using lipofectamine.The crcombinant vector was named Ad-hTERT-HSV/tk.Meanwhile,the recombinant adenovirus Ad-hTERT-HSV/tk,Ad-CMV-EGFP and Ad-hTERT-EGFP were constructed respectively.Then,DNA of 4 recombinant adenovirus were extracted and PCR identification was conducted.In the end,these four recombinant adenovirus were propagated in 293 cells and purified by cwsium chloride density purification,titrated by TCID50 method.Results: The four recombinant adenovirus were constructed successfully,which were propagated in 293 cells and purified by cesium chloride density purification,titrated by TCID50 method.The titre of Ad-CMV-HSV/tk,Ad-hTERT-HSV/tk,Ad-CMV-EGFP and Ad-hTERT-EGFP were 4×1010pfu/ml,1×1011pfu/ml,1.4×1011pfu/ml and 3.8×1010pfu/ml respectively.The seccond part: The role of a Adenoviral-mediated HSV-tk Gene,scopadulciol,in improving the efficacy of Bladder Cancer gene therapy in vitro Objective: The most extensively investigated strategy of suicide gene therapy for treatment of bladder cancer is the transfer of the herpes simplex virus thymidine kinase (HSV-TK) gene followed by administration of antiviral prodrugs such as ganciclovir (GCV). The choice of the agent that can stimulate HSV-TK enzymatic activity is one of the determinants of the usefulness of this strategy. Previously, we found that a diterpenoid,scopadulciol (SDC), produced a significant increase in the active metabolite of ACV. This suggests that SDC may play a role in the HSV-TK/prodrug administration system.Methods:The anticancer effect of SDC was evaluated in HSV-TK-expressing (TK+)253J bladder cancer cells and nude mice bearing TK+ tumors. In vitro and in vivo enzyme assays were performed using TK+ cells and tumors. The phosphorylation of ACV monophosphate (ACV-MP) was measured in TK?cell lysates. The pharmacokinetics of prodrugs was evaluated by calculating area-under-the-concentration-time-curve values. Results:SDC stimulated HSV-TK activity in TK+ cells and tumors, and increased GCV-TP levels.The SDC/GCV combination altered the pharmacokinetics of the prodrugs. In accord with these findings, SDC enhanced significantly the cell-killing activity of prodrugs. The bystander effect was also significantly augmented by the combined treatment of GCV and SDC.The third part: The role of a Adenoviral-mediated HSV-tk Gene,scopadulciol,in improving the efficacy of Bladder Cancer gene therapy in vivo.Objective: In order to extend the in vitro results mentioned above to in vivo situations.Methods:We used nude mice bearing bladder tumors of 253J cells. After the cells were injected subcutaneously, when tumors reached 6mm in diameter,mice were divided into 4 group(GroupA,B,C and D).There were 4 nude mice in each group. Nude mice in Group A,B,C and D were given Ad-hTERT-HSV/tk+GCV+SDC,Ad-hTERT-HSV/tk+GCV, Ad-hTERT-HSV/tk+SDC and PBS for 15 days respectively.Tumor sizes were measured once 4 days.Results: SDC in combination with Ad-hTERT-HSV/tk /GCV resulted in more significant suppression of bladder tumor growth as compared with the other control groups throughout the period of 6 weeks (p < 0.001).Conclusions:SDC was shown to be effective in the HSV-TK/prodrug administration system and improved the efficiency of the bystander effect of GCV. The findings will be considerably valuable with respect to the use of GCV in lower doses and less toxic. This novel strategy of drug combination could provide benefit to HSV-TK/prodrug gene therapy.