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

Thymidylate synthase (TS) is the target enzyme for 5-fluorduracil (5-FU). T S mRNA and protein levels in colorectal rumours are among the most importan t determinants for tumour response to 5-FU. TS mRNA levels in blood leukocy tes 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 col orectal tumours. The aim of this study was to develop a quantitative high-t hroughput RT-PCR assay for TS mRNA expression in blood leukocytes (CURT-PCR ). Furthermore the TS mRNA levels in blood of patients with colorectal canc er and healthy controls was compared. TS mRNA levels in 17 patients with co lorectal cancer did not differ from 20 matched controls whereas a group of 14 younger controls had significantly lower TS mRNA expression than patient s and matched controls. In order to investigate the sensitivity of the assa y towards cellular reactions such as proliferative stimuli, isolated blood leukocytes were stimulated with phytohemagglutinin both in mitogenic and nb n-mitogenic concentrations and an induction of TS mRNA expression was measu red in both cases. TS activity and cellular proliferation also increased bu t only at mitogenic concentrations, suggesting that TS mRNA expression is a n early leukocyte activation marker. This new CURT-PCR assay may allow impr oved studies of functional kinetics of drugs with impact upon TS. Further s tudies are required to establish the possible clinical benefit of TS mRNA m easurements in blood leukocytes. (C) 2000 Elsevier Science B.V. All rights reserved.