Polymorphic tandem repeat sequences of the thymidylate synthase gene correlates with cellular-based sensitivity to fluoropyrimidine antitumor agents

Abstract

Thymidylate synthase (TS) is one of the target molecules for the antitumor effects of fluoropyrimidine drugs. The cellular thymidylate synthase level is one of the determining factors for the antitumor activity of fluoropyrimidines. TYMS, which encodes TS, has been reported to possess 28-bp tandem repeat sequences in its 5'-untranslated region, the number of which varies. In addition, single nucleotide polymorphisms have also been shown in a triple repeat sequence. In this study, correlation between the polymorphic tandem repeat sequences of the TYMS gene and the antitumor activities of 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (FUdR) were investigated with 30 established human cell lines derived from solid tumors. A reporter assay system was developed in order to compare the ability of the transactivation mediated by the double (2R) and triple (c- or g-type, 3Rc or 3Rg, respectively) repeat sequences using a human colon cancer cell line, DLD-1. The 50% inhibitory concentration (IC(50)) of cell growth by 5-FU and FUdR was measured with 30 different established cell lines of human solid tumors. Genotypes based on the number of the 28-bp TYMS tandem repeat for the above cell lines were determined by electrophoretical analysis of PCR products containing the repeat sequences and nucleotide sequencing. The reporter activity mediated by the 3Rg sequence was significantly higher than that by the 2R and 3Rc sequences. Activities mediated by the 2R and 3Rc sequences were comparable. According to the reporter assay, 2R and 3Rc were judged as low TS expression alleles and 3Rg as a high TS expression allele. On the basis of IC(50) values, cells possessing the 2R/2R and 2R/3R repeat of TYMS were significantly more sensitive to FUdR than those with the 3R/3R repeat. Cells possessing 3Rg/3Rg (a high TS expression genotype) were significantly less sensitive to FUdR than cells with 2R/2R, 2R/3Rc, and 3Rc/3Rc (low TS expression genotypes). Our results of the reporter assays using 2R, 3Rc, and 3Rg repeat sequences prompted us to classify 3Rg as a high TS expression allele, and 2R and 3Rc as low TS expression alleles. The cells with low TS expression alleles were shown to exhibit significantly higher FUdR sensitivity than the cells with high TS expression alleles for the first time. These results were consistent with numerous previous in vitro and in vivo findings that tumors showing high TS expression were less sensitive to fluoropyrimidines. These results support the idea that genotyping the tandem repeat sequences of TYMS in the 5'-untranslated region is useful for individualized therapy involving fluoropyrimidine antitumor drugs.