Detection and quantitation of thymidylate synthase mRNA in human colon adenocarcinoma cell line resistant to 5-fluorouracil by competitive PCR.

BACKGROUND: Thymldylate synthase (TS) is an important target of cancer chemotherapeutic agents, such as 5-fluorouracil (FU). To investigate mechanisms of resistance to FU, we tried to detect TS mRNA in the human colon adenocarcinoma cell lines. MATERIALS AND METHODS: SNU-C1 (C1) and its FU-resistant cell line, SNU-C1/FU (C1/FU) were used for this study. Total RNA was isolated by the AGPC method, then competitive PCR and northern blot were done to detect TS mRNA. RESULTS: Using sets of primers covering the 3'-untranslated region of TS mRNA, PCR products were amplified from cDNA prepared from both C1 and C1/FU in their logarithmic growth phases. However, only cDNA prepared from C1/FU was amplified in the stationary phase. The amount of mRNA was quantified by competitive PCR technique in both cell lines, using another set of primer to amplify the product in the stationary phase. The amount of TS mRNA in C1/FU was found to be four times more than that found in C1. In addition, TS catalytic activity of C1/FU was approximately 2-times higher than that of C1. Southern blot analysis revealed that no TS gene amplification or rearrangement in genomic DNA was detected in these cell lines. CONCLUSIONS: This PCR technique is applicable for detecting TS mRNA, and the TS mRNA level was found to be increased 1.5-fold (as detected by northern blot analysis) and 4-fold (measured by competitive PCR), leading to enhanced TS catalytic activity in C1/FU in contrast to its parent cell line, C1; thus accounting for one possible resistant mechanism to FU.