The combination of fluoropyrimidines and radiation has been demonstrated in numerous randomized clinical trials to increase the survival of patients with gastrointestinal cancers. Our long-term goal is to better protect normal tissue and overcome tumor cell resistance to fluoropyrimidine-mediated radiosensitization through understanding the mechanism. Our data suggest that the classic hypotheses which have been used to explain fluoropyrimidine-mediated radiosensitization have important flaws. We propose a novel hypothesis that radiosensitization results from defective repair of radiation-induced DNA damage which occurs in cells that enter S phase in the presence of fluoropyrimidines and continue to progress in S phase after irradiation. Furthermore, we hypothesize that S-phase entry in the presence of FdUrd depends on both p53 dependent and independent mechanisms. The goal of this proposal is to test this model using human colorectal cancer cells. This goal will be carried out through 2 specific aims using the fluoropyrimidine fluorodeoxyuridine (FdUrd). Specific Aim 1 is to determine the mechanisms (prior to irradiation) which control the entry of cells into S phase in the presence of FdUrd. We hypothesize that two distinct forms of cell cycle arrest can protect cells from FdUrd-mediated radiosensitization. The first depends on wild type p53 and may explain the relative protection seen in the clinic from fluoropyrimidine-mediated radiosensitization of normal (p53 wild type) cells compared to most tumor cells (p53 mutant). The second is p53 independent and may explain resistance demonstrated by some (p53 mutant) tumors to fluoropyrimidine-mediated radiosensitization. We will focus on the roles of cyclin E and p53 on entry of cells into S phase in the presence of fluoropyrimidines. Specific Aim 2 is to determine the key events after irradiation in the presence of FdUrd which lead to sensitization. We hypothesize that cells which progress in S after radiation in the presence of FdUrd demonstrate decreased ability to repair DNA damage. This aim will assess a) the role of S phase progression after irradiation, b) the effect on DNA repair processes and c) the role of DNA single strand breaks in FdUrd-mediated sensitization.