The fluoropyrimidine antimetabolites 5-fluorouracil (FUra) and 5-fluoro-2'-deoxyuridine (FdUrd) have a prominent role in the treatment of human colorectal tumors as a result of their intrinsic cytotoxic activities as well as their ability to modulate the actions of other therapeutic tools (e.g., radiation and radiosensitizers). In many cases the biological activity of these drugs is due to inhibition of thymidylate synthase (T.S.), although the specific consequences of this inhibition, which ultimately lead to loss of viability, are poorly understood. By using pulsed-field gel electrophoresis we have obtained preliminary data showing that FdUrd-induced cytotoxicity in human colorectal tumor (HCT) cells is associated with the formation of large DNA fragments (up to 5 Mbase), which appear to be the result of non-randomly distributed double strand breaks. Furthermore, we have demonstrated major qualitative and quantitative differences in the fragmentation patterns of different HCT cell lines. We postulate that this damage is a key event in turning DNA synthesis inhibition into an irreversible, lethal insult, and that variations in sensitivity to FdUrd among HCT cell lines may be at least partially due to differences in the processes by which these cells convert T.S. inhibition into double strand breaks. Therefore, the major goal of this project is to understand the mechanism(s) responsible for the formation of DNA double-strand breaks induced by FdUrd, in human colorectal tumor cell lines. We will approach this objective by testing a set of specific hypotheses concerning: -the sequence-specificity of double-strand break formation -the relationship of double strand breaks to single strand break formation -the effect of chromatin conformation on fragment formation
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