A new quantitative RT–PCR assay for thymidylate synthase mRNA in blood leukocytes applied to cancer patients and healthy controls

Abstract

Thymidylate synthase (TS) is the target enzyme for 5-fluorouracil (5-FU). TS mRNA and protein levels in colorectal tumours are among the most important determinants for tumour response to 5-FU. TS mRNA levels in blood leukocytes may give information on pharmacokinetic and pharmacodynamic actions of 5-FU on TS as it has previously been shown that inhibition of TS levels by 5-FU in bone marrow leukocytes resembles the degree of TS inhibition in colorectal tumours. The aim of this study was to develop a quantitative high-throughput RT–PCR assay for TS mRNA expression in blood leukocytes (CURT–PCR). Furthermore the TS mRNA levels in blood of patients with colorectal cancer and healthy controls was compared. TS mRNA levels in 17 patients with colorectal cancer did not differ from 20 matched controls whereas a group of 14 younger controls had significantly lower TS mRNA expression than patients and matched controls. In order to investigate the sensitivity of the assay towards cellular reactions such as proliferative stimuli, isolated blood leukocytes were stimulated with phytohemagglutinin both in mitogenic and non-mitogenic concentrations and an induction of TS mRNA expression was measured in both cases. TS activity and cellular proliferation also increased but only at mitogenic concentrations, suggesting that TS mRNA expression is an early leukocyte activation marker. This new CURT–PCR assay may allow improved studies of functional kinetics of drugs with impact upon TS. Further studies are required to establish the possible clinical benefit of TS mRNA measurements in blood leukocytes.