Resistance of colorectal cancer cells to 5-FUdR and 5-FU caused by Mycoplasma infection.

BACKGROUND: 5-Fluorouracil (5-FU) is an antineoplastic drug that targets thymidylate synthase (TS). Tumour cells can develop resistance to anti-TS drugs by a variety of mechanisms including up-regulation of TS protein and alterations in drug uptake and degradation. The possible mechanisms of the observed rapid development of resistance to the pyrimidine analogs 5-FUdR and 5-FU in cultured HCT116 colon cancer cells were investigated. MATERIALS AND METHODS: Cell survival was determined in resistant and control HCT116 cells treated with 5-FUdR and 5-FU for 7 days. The ability of the cells to take up and metabolize these drugs was determined by Western blotting and [3H]thymidine incorporation. RESULTS AND CONCLUSION: Resistant HCT116 cells were 5- and 100-fold more resistant to killing by 5-FU and 5-FUdR, respectively, than the parental cells and exhibited impaired uptake. Although the HCT116R cells were initially Mycoplasma free, a low level of Mycoplasma contamination was found in these cells after several weeks in culture. Sensitivity to 5-FUdR was restored by treatment with an anti-Mycoplasma antibiotic. Our observations emphasize the need for frequent testing for Mycoplasma contamination in any cell line under investigation for resistance to anti-TS drugs.

Nuclear expression of thymidylate synthase in colorectal cancer cell lines and clinical samples.

Thymidylate synthase (TS) [TYMS; OMIM reference number (188,350)] is normally considered to be a cytoplasmic enzyme. However, a few reports have suggested it may also be present in the nucleus. To explore this in more detail, we used a highly specific polyclonal antibody to TS and a combination of techniques, including immunocytochemistry, confocal microscopy, cell fractionation, and Western blotting. We developed cell line HeLa-55, a HeLa derivative that grossly overexpresses TS. Although the vast majority of TS was in the cytoplasm, some TS also was seen in the nucleus. TS in parental HeLa cells and in normal human fibroblasts was seen exclusively in the cytoplasm. HeLa-55 cells exposed to 5-fluorodeoxyuridine were fractionated and examined by Western blotting. Interestingly, both free TS and the ternary complex of TS were seen in the cytoplasmic fraction but only free TS was detected in the nuclear fraction. Amongst different cell lines examined, HCT-15 and normal fibroblasts showed no nuclear TS, HCC-2998 and SW-620 showed a small amount of nuclear TS, and HT-29, RKO, and HCT-116 showed a strong nuclear TS signal. Nuclear staining was clearly evident in some clinical colorectal specimens, both normal and malignant. This staining was definitively shown to be TS by competition with recombinant TS protein. A putative leucine-rich nuclear export sequence was identified but its function could not be confirmed. We conclude that small amounts of TS protein is present in the nucleus of some cell types but further work is needed to determine the significance of this observation.