Adenoviral-mediated HSV-tk Gene Therapy Controlled By HTERT Promoter For Experimental Bladder Cancer

Malignant tumor is one of the major life threatening diseases, incidence of which is getting higher and higher and difficult to treat. Every year the number of people who were died of malignant tumor exceeded ten million in the world. There are more than one million people suffering form malignant tumours in China. 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.Despite improved surgical techniques and chemotherapy, the long term prognosis for patients with advanced disease has not markedly improved over the past 10 years. Hence,better therapies must be investigated. We must look for more effective treatment for Bladder cancer.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 effective and clinical useful potentiality. Molecular chemotherapy is a gene therapy approach designed to achieve selective eradication of cacinoma cells via a genetically expressed toxin.This method is often named as "suicede gene therapy". 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 high 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 replication halts growth of the developing DNA strands and inhibits DNA polymerase activity. 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 of this approach can be amplified by a "bystander effect",killing the nontransduced neigbouring cells. Thus,suicide gene therapy theoretically is not necessarily targeting 100% of tumor cells for effective treatment. However,gene expression in nontarget cells is a central problem in cancer gene therapy. Efficient gene therapy regiments require transgene expression especially in tumors. There are two strategies to achieve this goal. Transductional targeting can be accomplished by modification of vectors. Another method is transcriptional targeting achieved by regulation of transgene expression. Gene regulatory elements that drive transcription of some special proteins in tumors have the potential capacity to control gene expression in a tumor cell-specific manner. Transcriptional targeting is based on the use of these tissue or tumor-specific elements,mainly promoters. There have been several candidate promoters analyzed in gene therapy studies for specific transcriptional control in bladder cancer cells. Their activity and specificity are still being evaluated. Telomerase is a specialized DNA polymerase responsible for the replication of telomeres. Telomerase is highly active in most immortalized cell lines and more than 85% human cancers but is inactive in most somatic cells. The main components of the human telomerase enzyme are the human template RNA (hTR) component and the human telomerase reverse transcriptase(hTERT). Although hTERT and hTR are both necessary for telomerase activity,expression of hTERT is present specifically in tumor cells whereas hTR is present in both normal and tumor cells. Because the hTERT gene is highly active in tumor cells but repressed in most normal cells and because its expression is regulated at the transcription level,the hTERTpromoter may be used for tumor-specific expression of transgenes. In the present study,hTERT promoter was used to induce exprssion of the HSV-tk gene,and efficiency was tested in cancer gene therapy. Our study includes three parts. The first part: Construction of recombinant adenovirus vector containing hTERT promoter and HSV tk gene Objective: 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 recombinant 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 和3.8×1010pfu/ml respectively. The second part: Killing efficiency of human bladder cancer cells by HSV-tk/GCV system under the control of hTERT promoter in vitro Objective: To observe the selective killing effect of adenoviral mediated herpes simplex virus-thymidine kinase/ganciclovir (HSV-tk/GCV) suicide gene system controlled by human telomerase reverse transcriptase(hTERT) promoter on bladder cancer cells in vitro. Methods: Bladder cancer cell line 253J was cultured in RPMI1640 medium containing 10% FBS, and human fibroblast cell line MRC-5 was cultured in MEM medium containing 10% FBS. They were transfected by therecombinant adenoviru(sAd-CMV-EGFP and Ad-hTERT-EGFP) of different multiplicities of infection(MOI), and the infection rate was measured by observing the expression of enhanced green fluorescent protein(EGFP) under the fluorescent microscopy. Ad-hTERT-HSV/tk and Ad-CMV-HSV/tk were transfected into 253J and MRC-5 followed by GCV treatment. Total RNA was extracted from the transfected cells and HSV-tk mRNA was detected by RT-PCR. The relative survival rate of cells in presence of prodrug GCV was measured with MTT method. The bystander effect was also studied by co-culture of transfected and non transfected 253J cells. Results: MRC-5 cells couldn't be infected by recombinant adenoviru Ad-hTERT-EGFP. 253J cells could be infected selectively by Ad-hTERT-EGFP, with the infection rate associated with the MOI of recombinant adenovirus(p<0.01).When MOI was 1, the infection rate was 8.3%;.When MOI was 100, the infection rate was 85.2%;When MOI was 1000, the infection rate was 100%. However, MRC-5 cells and 253J cells both could be infected by recombinant adenoviru Ad-CMV-EGFP, and there were no obvious difference in the infection rate of two cell lines. By RT-PCR, HSV-tk mRNA could be detected in both cell lines transfected by Ad-CMV-HSV/tk and 253J cells transfected by Ad-hTERT-HSV/tk, but couldn't be detected in MRC-5 cells transfected by Ad-hTERT-HSV/tk. GCV inhibited significantly the growth of both cell lines infected with Ad-hTERT-HSV/tk and the growth of 253J cells infected with Ad-CMV-HSV/tk(p<0.001);The relative survival rate of 253J cells correlated to both the concentration of the predrugs and the MOI of recombinant adenovirus(p<0.01). When MOI was 1, 1μmol/L GCV killed 95.4% 253J; When MOI was 100, 1000μmol/L GCV killed 6.1% 253J. The bystander effect existed. When the transfected cells reached 30%,about 50% cells could be killed. When the transfected cells reached 50% or higher, it could kill about 87% cells. The third part: Killing efficiency of human bladder cancer by HSV-tk/GCV system under the control of hTERT promoter in vivoObjective: To explore the killing effect of adenoviral mediated HSV-tk/GCV suicide gene system controlled by human telomerase reverse transcriptase(hTERT) promoter on bladder cancer cells in vivo. Methods: 2×107 253J cells were inoculated s.c. into the right forelimb armpit of 6-week-old nude mice to establish tumors. After tumors reached 6mm in diameter,mice were divided into 4 groups(Group A,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,Ad-hTERT-HSV/tk,GCV and PBS for 15 days respectively. Tumor sizes were measured once 4 days. 30 days later tumors tissues were observed under light and electron microscope. Expression of PCNA and incidence of apoptosis in bladder cancer cells were detected by immunohistochemistry and in situ 3'-DNA end labaling technique. Results:The heterotransplantative tumourigenecity of the tumour in nude mice was 100% after 253J cells were inoculated.The growth rate of the tumour in nude mice in Group A decreased significantly compared those of tumor in nude mice in Group B,C and D. Average survival time of nude mice in Group A was longer than that in Group B,C and D. Compared with Group B,C and D, PCNA proliferative indexes (PI) were lower(p<0.01), apoptosis indexes(AI) were higher (P<0.05) in Group A after the treatment. There were no obviously difference in Group B,C and D. Conclusions 1 The four recombinant adenovirus Ad-CMV-HSV/tk ,Ad-hTERT-HSV/tk,Ad-CMV-EGFP and Ad-hTERT-EGFP were constructed successfully. After they were propagated in 293 cells and purified by cesium chloride density purification, their titres were very high. 2 Ad-hTERT-EGFP could infected 253J cells selectively, with the infection rate associated with the MOI of recombinant adenovirus. Adenoviral vector containing hTERT promoter for the HSV/TK plus GCV treatment appears to be highly selective in killing bladder cancer cell line in vitro. The relative survival rates of 253J cells were correlated to both the concentration of the predrugs and the MOI of recombinant adenovirus Ad-hTERT-HSV/tk.