Experiments will be carried out to determine the mechanism of action of and modes of resistance to nucleoside analogs using mammalian cells in culture. The experiments will utilize the techniques of biochemistry, somatic cell genetics, and recombinant DNA to identify and possibly isolate the cellular genes that mediate the effects of these nucleoside analogs. In this proposal, two different analogs will be investigated: 1) the anti-herpes virus guanine analog, 9-(2-hydroxyethoxymethyl) guanine (acycloguanosine, acGuo); and 2) the thymidine analog 5-hydroxymethyldeoxyuridine (hmdUrd). We have already obtained preliminary data on the metabolism of these analogs by wild-type cells as well as by mutants which have been selected for resistance to the cytotoxic effects of the analogs. In both systems, we have found human cell lines which are intrinsically resistant to the cytotoxic effects of the analogs. Through the use of biological and biochemical analyses of cells exposed to the analogs we propose to determine the metabolic fate of the analogs. These experiments will include determining survival under various conditions and analysing the composition of nucleotide pools and DNA by HPLC techniques. The use of genetic analyses of analog resistant cells will allow us to determine the genetic characteristics of the cellular response to the analogs. These experiments will utilize the technique of somatic cell hybridization and DNA-mediated gene transfer. The identification and cloning of the human genes mediating the intrinsic human resistance to the analogs will allow us to determine the modes of resistance and to isolate and characterize the gene products. The anti-herpes virus analog is currently in limited clinical use. Its therapeutic effect is largely based upon the intrinsic resistance of human cells to its effects. These studies will be useful in understanding the mechanism of this resistance and the genes involved. The response of cells to hmdUrd may involve a mechanism for detoxification of the nucleoside by cleavage to the free pyrimidine which is not toxic. Such a mechanism and the genes involved may act to detoxify potentially toxic or mutagenic modified nucleosides generated in cells by radiation or the action of chemicals.
