The Selective Killing Effect Of Adenoviral Mediated HSV-TK/GCV Suicide Gene System Controlled By HTERT Promoter On Bladder Carcinoma Cells

Objective: Bladder carcinoma is the most common urological malignancy in china. Many methods have been used to the therapy for bladder carcinoma. Gene therapy is one of the most important progress in the past years. 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 "suicide 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,mainly 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. In recent years the specific promoters are often used to control the therapeutic gene expression by regulation of transcription in order to achieve tumor specific target in cancer gene therapy. It has been already confirmed that the activity of human telomerase reverse transcriptase (hTERT) rise in approximately 90% human tumors whereas most normal cells do not express the activity and it becomes a hotspot in transcriptional target of cancer gene therapy. Virus-directed enzyme prodrug therapy (VDERT) has been popular in cancer gene therapy due to its potential foe tumor-specific cytotoxicity. Several biologic features of adenovirus have made such viruses the vectors of choice for certain of these applications. For example, adenoviruses transfer genes to a broad spectrum of cell types, and gene transfer is not dependent on active cell division. Additionally, high titers of viruses and high levels of transgene expression generally can be obtained. To investigate the efficacy of adenovirus -mediated gene therapy using herpes simplex thymidine kinase (HSV-tk) gene and ganciclovir (GCV) treatment for human bladder carcinoma cell line (253J). Methods: we use a recombinant adenovirus vectorcontaining the enhanced green fluorescent protein (EGFP) gene. One construct with a cytomegalovirus (pCMV)-driven enhanced green fluorescent protein (EGFP) reporter gene (Ad-CMV-EGFP), the other construct with a human telomerase reverse transcriptase gene promoter (hTERTp )-driven enhanced green fluorescent protein (EGFP) reporter gene (Ad-hTERT-EGFP) transfected 253J and MRC-5 cells. The percent of transfection was determined by the detection of flurorescence microscopy. Human bladder carcinoma cell line 253J and MRC-5 was infected with Ad-hTERT-HSV/TK and Ad-CMV-HSV/TK, then various amount of GCV was added. Using the hTERT as a promoter for the herpes simplex virus thymidine kinase (HSV-TK) gene in an adenoviral vector(Ad-hTERT-HSV/TK) , the therapeutic efficacy of adenovirus mediated HSV-TK gene transduction,followed by ganciclovir(GCV)administration,was studied. As a control,a recombinant adenoviral vector(Ad-CMV-HSV/TK) which was driven by the CMV promoter,was constructed. The percentage survival of cells is presented as a percentage of the survival cell in the GCV-treated cells divided by that in the cells without GCV treatment(mean 士SD).The relative survival rate of cells in presence of prodrug GCV was tested using MTT method. Different mix culture cells , which contained different per...