Mechanisms of resistance to anti-thymidylate synthase (TS) chemotherapeutic agents in colon cancer

Abstract

Thymidylate Synthase (TS) catalyzes the final step in the synthesis of thymidylate, a nucleotide required for DNA synthesis. This enzyme is the target enzyme for several important anti-cancer drugs including 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-FUdR). 5-FU and 5-FUdR are metabolized to FdUMP which, in the presence of a folate cofactor, inhibits TS by forming a tight covalent "ternary complex". TS is usually considered to be a cytoplasmic enzyme, but some reports suggest it may also be present in the nucleus. One recent report has indicated that high levels of nuclear TS were associated with poorer clinical response to 5-FU. Using a combination of immunocytochemistry, confocal microscopy, immunohistochemistry, western blotting and cell fractionation techniques, evidence is presented that a fraction of cellular TS is indeed located in the nucleus of some colorectal cancer cell lines and tissues. The specificity for TS of the polyclonal antibody used was first confirmed. The effect of TS overexpression on cellular localization was studied in HeLa-55 cells, a 5-FUdR-resistant line containing an amplified TS gene. Nuclear TS was clearly evident in HeLa-55 but not in parental HeLa cells. Fractionation of HeLa-55 growing in 5-FUdR showed that both the ternary complex and free TS were present in the cytoplasm, while only free TS was present in the nucleus. From a panel of 6 colorectal cancer lines and normal fibroblasts, RKO and HCT-116 clearly showed nuclear TS. Strong evidence of nuclear TS in some colorectal cancer tissues is also presented. A putative leucine-rich nuclear export signal sequence (NES) was identified in TS protein. TS constructs lacking this sequence accumulated more nuclear TS, but the sequence did not function as a nuclear export signal when fused to GFP. A recent report suggests that p53 status can affect the sensitivity of HCT-116 to 5-FUdR. I examined HCT-116 p53 wt and HCT-116 p53 null cells and observed that p53 null cells, in contrast to p53 wt cells, failed to form a ternary complex when exposed to 5-FUdR. p53 wt cells were also more sensitive to killing by 5-FUdR. However, the differences observed were in fact independent of p53 status. 5-FUdR-sensitive cell lines became resistant after several passages in culture. Current evidence suggests that the basis of resistance is related to either drug metabolism or uptake. This thesis has contributed towards a better understanding of TS, a cellular target for an important class of anti-cancer drugs.