Studies Of The Efficacy And Targeting Of Drug Sensitive Genes("Suicide" Genes) In The Experimental Gene Therapy Of Pancreatic Carcinoma

Drug sensitive gene can convert nontoxic prodrugs into toxic drugs thus kills the cells,therefore it is also called "suicide" gene. With its unique bystander effect(local and distant),drug sensitive gene thereby will probably become the most sensitive and most promising gene therapy for those intractable cancers such as pancreatic carcinoma. It is very important to achieve the specificity of suicide gene expression in targeted cells because its expression in normal tissues will also result in the injury of normal tissues by prodrug. The objectives of our present work are : (1)To study the therapeutic effect on pancreatic carcinoma of three prodrug sensitivity genes,which are herpes simplex virus thymidine kinase (HSV-TK), E.coli. cytosine deaminase (CD) and human thymidine phosphorylase (TP) genes and the mechanism of bystander effect; (2) In order to achieve selective killing of transduced cells, a mosiac promoter containing Myc-Max response element CACGTG and part domain of HSV-TK promoter was constructed , and its tumor-specific transcriptional property in myc-overexpressing cells was studied; (3) To study targeted gene therapy of pancreatic carcinoma mediated by mosiac promoter-driven HSV-TK gene. The main works and results include:1.Experimental therapeutic effect of HSV-TK/GCV,CD/5-FC and TP/5'-DFUR systems on pancreatic carcinoma.1). Three recombinant retroviral vectors expressing HSV-TK, CD and TP gene separately were constructed and used to transfect pancreatic carcinoma cell line PC-2. Following G418 selection, G418 resistant colonies were isolated and identified as PC-2/LXTK, PC-2/LXCD and PC-2/LXTP respectively. The integrations of retrovirus vectors were verified by PCR and Southern blot analysis, and the expressions of exogenous genes in the transformant PC-2 cells were confirmed by in situ hybridization, Northern blot hybridization and assay of enzyme activity. There was no difference in either doubling time or ~3H-TdR incorporation rate between the transformant cells and parental cells, which indicate that there was no influence on cell biology by the expression of foreign genes.2). In vitro sensitivity assay of PC-2/LXTK to GCV showed that expression of HSV-TK in PC-2 cells increased the sensitivity of these cells to prodrug GCV. Clonogenic ability was reduced, and the percentage of colony forming inhibition was 80% in the presence of...