The long-term objectives of the proposal are to develop highly effective therapy for the treatment of colon carcinoma. This will be achieved 1) by understanding how specific genetic mutations that deregulate growth control mechanisms influence cell death processes and sensitivity to cytotoxic agents, and 2) by developing new therapeutic strategies based upon specific molecular characteristics. The proposal will elucidate the role of the p53 tumor suppressor gene in influencing specifically the process of thymineless death, an important mechanism of FUra-LV cytotoxicity. Our hypothesis is that deregulation of this G1 checkpoint by mutations that may cooperate with activated K-ras, may determine whether therapy is cytostatic or cytotoxic. Studies will be initiated in thymidylate synthase-deficient (TS) GC3/cl human colon carcinoma cells axotrophic for dThd, and Thy4, a subpopulation that demonstrates significant delay in commitment to thymineless death due to arrest in late G1 upon dThd withdrawal. Overexpression of wtp53 in an inducible vector system may induce G1 arrest in TS affording protection from thymineless death, whereas expression of mp53 + activated k-ras may allow progression of Thy4 into S during 2dTTP depletion, thereby initiating a cytotoxic response. Studies will also extend to FUra-LV cytotoxicity in parental GC3/cl cells, and in normal cells expressing two wtp53 alleles. Additionally, the role of p53 expression, cyclin E-cdk-2 and cyclin A-cdk-2 interactions in the mechanism of late G1 arrest of Thy4 cells will be elucidated. The mechanism buy which changes in deoxyribonucleoside triphosphate pools (dNPT) signal and regulate G1 arrest or G1 to S progression will be determined. The second focus is to develop biochemical rationales for effective combination of FUra-LV with inhibitors of topoisomerase I, which have significant activity in colon carcinoma xenografts. Synergistic or antagonistic interactions between inhibitors and thymineles stress or FUra- lV-induced dTTP depletion will be defined in cultured cells, and mechanisms that define these interactions will be determined. Potential interactions at the level of cell cycle arrest, DNA replication, or gene transcription afford possibilities for differential responses in neoplastic and normal tissues. Synergistic combination will be evaluated for enhanced curative potential of colon carcinoma xenografts, which are highly predictive of clinical efficacy. It is anticipated that the studies proposed will identify new therapeutic targets related to the deregulation of growth control processes in colon carcinoma, and be of significance in the identification and development of highly effective regimens for the successful treatment of colon carcinoma in man.
