Killing Efficiency Of Human Ovarian Cancer Cells By HSV-tk/ GCV System Under The Control Of HTERT Promoter Combined With Topotecan

Ovarian cancer remains the leading cause of death from gynecological malignancies. Due to the lack of effective prevention and screening modilities, the majority of patients with epithelial ovarian cancer are at advanced-stage of the disease when they are diagnosed. Despite improved surgical techniques and chemotherapy, the long term prognosis for patients with advanced disease has not markedly improved over the past 10 years. The 5-year survival rate for patients with advanced-stage ovarian cancer is approximately 30% and most patients ultimately succumb to the disease. Hence, better therapies must be investigated. The confinement of ovarian cancer within the peritoneal cavity in the majority of cases suggests the possibility of effective locol gene therapy.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 utilizedsystem 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 ovarian 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 thehuman 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 hTERT promoter 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 combined with topotecan.The first part: Cloning and identification of human telomerasereverse transcriptase gene promoter Methods1. Preparation of standard protein curve: Standard protein curve was prepared with different bovine concentration in Coomassie Brilliant Blue G-250. Afetr abstracting protein from human ovarian cancer...