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.