Resistance of a human ovarian cancer line to 5-fluorouracil associated with decreased levels of 5-fluorouracil in DNA

Two human ovarian cancer cell lines were established from a patient before (PEO1) and after (PEO4) the onset of resistance to 5-fluorouracil (5-FU)/cisplatin-based chemotherapy. Using growth inhibition assays, we determined that the PEO4 line was almost 5-fold more resistant to 5-FU than the PEO1 line. The addition of either 1 or 20 μM leucovorin did not enhance the growth-inhibitory effects of 5-FU against the resistant PEO4 line. In characterizing the potential mechanisms of 5-FU resistance, we found no differences in thymidylate synthase activity between the two lines using both the 5-fluoro-2'-deoxyuridine-5'-monophosphate-binding and catalytic assays. A 4-hr exposure to 1 μM 5-FU resulted in greater ternary complex formation in the resistant line, and we observed no differences between the two lines in 5-FU incorporation into RNA. However, a 4-hr exposure to 1 μM [³H]5-FU resulted in a 3-fold decrease in 5-FU accumulation in the DNA of the resistant PEO4 line. Cesium sulfate gradient centrifugation was used to more accurately separate and analyze for DNA-incorporated 5-FU metabolites and confirmed that the absolute level of 5-FU in the DNA of the PEO4 cells was markedly decreased (6.5-fold) compared with that of the sensitive PEO1 cell line. Moreover, time course studies demonstrated that the accumulated 5-FU in the DNA of the PEO4 cells was more rapidly removed compared with that in the PEO1 cells. Our findings suggest that decreased 5-FU levels in DNA, in part due to an enhanced removal from DNA, represent a mechanism by which the human ovarian cancer PEO4 line expresses decreased sensitivity to 5-FU.