The long-term goal of this application is to determine the physiologic function of the rTS gene and its relevance to cancer therapeutics. rTS is a gene which overlaps the human thymidylate synthase (TS) gene and is encoded by the opposite strand of DNA. rTS is transcribed so as to produce one RNA (rTS-alpha) which is complementary to TS and to another, rTS-beta, which is not. The rTS gene codes for at least two proteins, rTS-alpha and rTS-beta. rTSB has been found to be overexpressed in three cell lines resistant to: 5-fluorouracil (H630-1, colon cancer), methotrexate (K562 B1A, chronic myelogenous leukemia and fluorodeoxyuridine plus ZD 1694 (HCT-8DF2, ileocecal carcinoma). Elevation of rTSB protein levels in the methotrexate-resistant, and fluorodeoxyuridine plus ZD 1694 cell lines are accompanied, respectively, by a complete and partial loss of growth regulation of TS activity. rTSB protein levels also increase as cells progress from log to stationary phase growth. Immunoprecipitation data indicate that rTSB, and a newly discovered protein, rTS, are complexed with TS and dihydrofolate reductase (DHFR). Taken together, the applicant has presented substantial preliminary data that suggest that rTS proteins appear to play a role in growth regulation of TS and in the development of cellular drug resistance.
Four specific aims are proposed to investigate how rTS may impact on TS gene expression, cellular physiology, and sensitivity to TS inhibitors. They are as follows: 1) Extended characterization of rTS protein expression and identification of and subcellular localization of complex formation of rTS proteins with TS and DHFR, respectively, as a function of growth; 2) Effect of altered expression of rTS-alpha and rTS-beta in transfected human cancer cells on properties of cell growth, TS enzyme activity and TS protein expression, formation of rTS:TS complex and subsequent sensitivity of cancer cells to TS inhibitors; 3) Expression and purification of rTS-beta and rTS-alpha from Pichia pastoris and to determine the effects of purified rTS protein on TS and DHFR enzyme activity and on sensitivity to TS inhibitors in vitro; and 4) Mapping and cloning of the rTS gene in order to characterize the rTS gene structure and organization.
