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Different lengths of a polymorphic repeat sequence in the thymidylate synthase gene affect translational efficiency
K Kawakami1, D Salonga, J M Park
1Department of Biochemistry and Molecular Biology, School of Medicine, Norris Comprehensive Cancer Center, University of Southern California, Los Angeles, CA 90033, USA. kawakami@med.kanazawa-u.ac.jp
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.