Quantitative Measurement of Thymidylate Synthase and Dihydropyrimidine Dehydrogenase mRNA Level in Gastric Cancer by Real‐time RT‐PCR

Abstract

1, 2) Thus, it is important to select patients in whom treatment is likely to be effective in order to improve the results of chemotherapy. In vitro anticancer drug sensitivity tests, which we developed with this goal in mind, have already yielded favorable results.3) However, tests that involve cell culturing procedures are complicated by the need for special facilities, and there can be problems with failures attributable to low cell growth.Standard regimens for gastric cancer involve some form of concomitant treatment that includes biomodulated 5-fluorouracil (5-FU) chemotherapy. 4,5) The mechanism of action of 5-FU has been explained in terms of incorporation of fluorouridine 5′-triphosphate (FUTP) into RNA, resulting in altered gene expression, or in terms of inhibition of thymidylate synthase (TS) by the active metabolite 5-fluorouridine monophosphate (FdUMP).6-8) Several reports have indicated that TS expression in the tumors was related to the sensitivity to 5-FU-based chemotherapy both in the laboratory and clinically. [9][10][11][12] On the other hand, dihydropyrimidine dehydrogenase (DPD) which is both an initial and a rate-limiting catabolic enzyme of 5-FU has been reported to play an important role in the pharmacokinetics of 5-FU and was correlated with the antitumor effectiveness of 5-FU in cancer cell lines and tumors. 9,[13][14][15][16][17][18] Recently, both basic and clinical researches have shown a correlation between the antitumor effectiveness of 5-FU and TS or DPD mRNA levels in tumors by using semiquantitative reverse-transcription polymerase chain reaction (RT-PCR). 11,12,16,[19][20][21] One of the major advantages of these methods is that they can be performed with ultralow-volume samples as compared with measurement of the enzyme activities and in vitro chemosensitivity tests.A real-time RT-PCR method based on TaqMan fluorescence methodology requires fewer steps after PCR than conventional PCR methods, simplifying the procedures 5 To whom correspondence should be addressed.

Prediction of sensitivity to fluoropyrimidines by metabolic and target enzyme activities in gastric cancer

“…Recently, a more detailed procedure, in which the precise amount of mRNA expression is determined by using a real-time reverse transcription-polymerase chain reaction assay, has been developed for evaluating the mRNA expression of these enzymes [25]. With this technique, we can predict tumor sensitivity to 5-FU using a minute amount of a biopsy specimen, and we can establish a tailor-made treatment for gastric cancer patients.…”

“…TS activities in MKN-74, GCIY, and GT3TKB were 0.094, 0.311, and 2.621 pmol/mg protein, respectively. Similarly, DPD activities in MKN-74, GCIY, and GT3TKB were 6.0, 277.8, and 287.8pmol/ mg per min, respectively [25].…”

Resistance May Not Be Futile: microRNA Biomarkers for Chemoresistance and Potential Therapeutics

“…These include (a) high expression of the MDR1 and ERCC1 genes in association with reduced response to cisplatin therapy in advanced bladder cancer (9); (b) high expression of ERCC1, LRP, and MRP1 genes in non-small cell lung carcinoma (NSCLC) patients predicting response to cisplatin-based therapies (10); (c) EGFR, ERCC1, MDR1, XRCC1, XRCC3, XPA, and XPD gene polymorphisms as predictive biomarkers for treatment response to epidermal growth factor receptor (EGFR) kinase inhibitors and platinum-based therapies in NSCLC patients (11,12); (d) low levels of dehydropyrimidine dehydrogenase and thymidylate synthase mRNA expression in colorectal and gastric cancers predicting sensitivity to 5-fluorouracil (13,14); (e) genotyping of CYP2D6 in breast cancer patients to identify those most likely to respond well to adjuvant tamoxifen therapy (15). Thus, there are a number of mechanisms for drug resistance.…”

CDK inhibitor enhances the sensitivity to 5-fluorouracil in colorectal cancer cells

“…An equivalent volume (1 μl) of cDNA solution was used for the quantification of specific cDNAs by quantitative real-time RT-PCR. The primer sequences used for the TS and GAPDH genes were described previously (19). Quantitative real-time RT-PCR was carried out using an RT-PCR system GeneAmp5700 (Applied Biosystems, Foster, CA).…”