Quantification of thymidylate synthase gene expression in human gastrointestinal carcinoma tissues using competitive PCR

BACKGROUND/AIMS: Thymidylate synthase (TS) is a target enzyme for 5-fluorou racil (5-FU). It is important to know the TS expression level in tumor tiss ue for chemotherapy. We evaluated the TS expression level using competitive polymerase chain reaction (cPCR), and estimated the reliability and reprod ucibility of this method. METHODOLOGY: TS expression was assessed by both Fluorodeoxyuridine-monophos phate (FdUMP) binding assay and cPCR in 9 human adenocarcinoma cell lines a nd 50 human adenocarcinoma tissues obtained by surgical resection. TS expre ssion was also assessed by cPCR in nine biopsy specimens obtained before su rgery. We synthesized TS and beta-actin competitors. Following cPCR, PCR pr oducts were quantified on ethidium bromide-stained gels using a digital ima ge analyzer. TS mRNA/beta-actin mRNA ratio was used to determine relative T S expression level. RESULTS: The number of FdUMP binding sites on TS and TS mRNA/beta-actin mRN A ratio were significantly correlated in both cell lines and surgically res ected specimens (p<0.01). A linear regression line formed from the data poi nts was obtained for TS mRNA/beta-actin mRNA ratios in surgical specimens v ersus TS mRNA/beta-actin mRNA ratios in biopsy specimens (p<0.01). CONCLUSIONS: Assessment of TS expression by cPCR using our competitors was accurate and reproducible.