Different mechanisms of acquired resistance to fluorinated pyrimidines in human colorectal cancer cells.
5-Fluorouracil (5-FU), 5-fluoro-2'-deoxyuridine (FdUrd) and 5-trifluorothymidine (F3(d)Thd) are antimetabolites which are metabolized to their corresponding active forms which inhibit DNA synthesis via inhibition of thymidylate synthase (TS). To investigate ways of overcoming 5-FU-resistance, we established acquired-resistant colorectal cancer cell lines against these three drugs by continuous and step-wise escalation of drugs, and analyzed the cytotoxicity and the mechanism of resistance to the drugs. When cells were incubated with the 3 drugs for 72 h, the resistance ratio to parental DLD-1 human colorectal tumor cells was 65.2 for DLD-1/5-FU, 9.7 for DLD-1/FdUrd and 448.6 for DLD-1/F3(d)Thd cells. DLD-1/5-FU cells did not show any cross-resistance against FdUrd and F(3)dThd. However, DLD-1/FdUrd cells showed 3- and 9-fold increased resistance to 5-FU and F3(d)Thd, respectively, and DLD-1/F3(d)Thd cells also showed about 90-fold resistance to FdUrd. Analysis of enzyme activities and gene expression associated with pyrimidine metabolism indicated that a significant decrease in orotate phosphoribosyltransferase activity in DLD-1/5-FU cells, a 7-fold increase of TS mRNA in DLD-1/FdUrd cells, and a 37-fold decrease in thymidine kinase activity of DLD-1/F3(d)Thd cells were the major mechanisms of drug resistance. These findings were closely associated with the cytotoxicity of 5-FU, FdUrd and F3(d)Thd against the established 5-FU-, FdUrd- or F3(d)Thd-resistant cells. When DLD-1/FdUrd cells expressing increased TS mRNA were treated with FdUrd and F3(d)Thd for only 4 h, the resistance ratios of DLD-1/FdUrd cells to parental DLD-1 cells were markedly different for FdUrd and F3(d)Thd, suggesting that the cytotoxicity with short-time exposure to F3(d)Thd is due to a mechanism other than TS inhibition, although the cytotoxicity of F3(d)Thd in the short-time is low compared to that of long-time exposure. In conclusion, F3(d)Thd, an antimetabolite that inhibits TS activity, may be effective against 5-FU and/or FdUrd-resistance in colorectal cancer cells caused by amplification of TS and/or deletion of orotate phosphoribosyltransferase.
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5-Fluorouracil pyrimidine orotate 5-FU FdUrd F3(d)Thd 5-FU FdUrd F3(d)Thd 5-FU FdUrd F3(d)Thd FdUrd FdUrd F3(d)Thd 5-FU FdUrd FdUrd F3(d)Thd F3(d)Thd F3(d)Thd F3(d)Thd 5-FU FdUrd orotate thymidylate 5-fluoro-2'-deoxyuridine 5-FU FdUrd 5-trifluorothymidine FdUrd F3(d)Thd 5-FU FdUrd F(3)dThd FdUrd F3(d)Thd 5-FU F3(d)Thd F3(d)Thd FdUrd fluorinated pyrimidines orotate phosphoribosyltransferase TS thymidine kinase TS TS TS TS orotate phosphoribosyltransferase thymidylate synthase TS