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Different Lengths of a Polymorphic Repeat Sequence in the Thymidylate Synthase Gene Affect Translational Efficiency but Not Its Gene Expression

Department of Biochemistry and Molecular Biology, University of Southern California, School of Medicine, Norris Comprehensive Cancer Center, Los Angeles, California 90033 [K. K., D. S., J. M. P., K. D. D., H. U., J. B., P. V. D.], and the Department of Surgery, Kanazawa University School of Medicine, Kanazawa 920-8641, Japan [K. K., K. O., G. W.]

Purpose: Thymidylate synthase (TS) is a target enzyme of 5-fluorouracil. Recently, the TS gene has been shown to contain a polymorphic tandem repeat sequence. The aim of this study was to determine whether differences in the number of tandem repeats could affect gene expression or mRNA translation.

Experimental Design: We quantified TS mRNA isolated from 130 colorectal cancer tissues by real-time reverse transcription-PCR and TS protein in 92 available samples by the fluoro-dUMP binding assay. These values were compared with TS genotypes of the samples determined by a PCR assay.

Results: There was no relation between TS genotype and mRNA expression level. On the other hand, cancer tissues with the 3R/3R genotype had a significantly higher TS protein expression level than did those with the 2R/3R genotype. These results suggest that the efficiency of TS mRNA translation is responsible for the genotype-dependent difference in TS protein expression. Further analysis using TS 5'-untranslated region-luciferase reporter constructs showed that the RNA with the three-repeat sequence was translated three to four times more efficiently than that with two-repeat sequence.

Conclusions: From the results of both in vitro and in vivo study, we conclude that TS mRNA with a three-repeat sequence has greater translation efficiency than that with the two-repeat sequence. The results provide the rationale for comprehensive usage of TS genotyping with quantitation of TS mRNA or TS protein to predict the patient’s response to 5-fluorouracil-based chemotherapy.

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