AN IN-VITRO NUCLEOSIDE ANALOG SCREENING METHOD FOR CANCER GENE-THERAPY

Suicide genes that sensitize cells to drugs that are normally nontoxic at therapeutic levels represent an important approach in human gene t herapy research. We have developed an in vitro screening assay to asse ss the modulation of nucleoside analogs after transfection of a vector expressing the herpes simplex virus thymidine kinase gene (HSV-TK). T he thymidine kinase gene enhances nucleoside phosphorylation to nucleo tides that kill cells by blocking DNA elongation. Cells lines used are 3T3-NIH fibroblasts (parental cells) and 3T3-TKc3 (HSV-TK gene-transf ected 3T3-NIH). Two types of analysis are performed: a cytotoxicity as say, the neutral red uptake assay to assess the IC50 on the two cell l ines, and an HPLC analysis coupled to a radiochemical flow detector to evaluate metabolic proles after incubation of cells with tritiated an alogs. Results show that cells expressing the HSV-TK gene are more sen sitive than the parent cells to the effect of acyclovir or ganciclovir , the reference purine analog drugs, and also to the effect of pyrimid ine analogs, bromodeoxyuridine, bromovinyldeoxyuridine, and ethyldeoxy uridine. Promising nucleoside analogs for gene therapy that can be ach ieved by HSV-TK could be evaluated using this model.