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Establishment of enzyme-linked immunosorbent assays for thymidylate synthase and dihydropyriminide dehydrogenase in cancer tissues.

Thymidilate synthase (TS) is a major target of 5-fluorouracil (5-FU) and dihydropyrimidine dehydrogenase (DPD) is a rate-limiting enzyme in the degradation of 5-FU. Intratumoral activity and expression levels of both enzymes are suggested to be predictive markers for response to 5-FU-based chemotherapy in cancer patients. Several different methods are used to measure intratumoral levels of TS and DPD. We developed new enzyme-linked immunosorbent assays (ELISAs) for these enzymes. New ELISAs were produced using anti-TS and anti-DPD polyclonal antibodies obtained using recombinant human TS and purified DPD from pig liver, respectively. Intra-assay coefficients of variations (CVs) were less than 5% in both ELISAs. Inter-assay CVs tested using the extract from 20 breast cancer specimens were 1.5-24.2% for TS-ELISA and 0.4-7.2% for DPD. Importantly, TS and DPD levels measured by the respective ELISAs closely related to TS activity (r(2)=0.8492) and DPD activity (r(2)=0.7666), respectively, measured by the respective substrate assays. To investigate the correlations between clinicopathological characteristics and intratumoral TS and DPD levels, data on 52 breast cancer patients were analyzed. Estrogen receptor (ER)-negative tumors had significantly higher TS and DPD levels than ER-positive tumors. Progesterone receptor (PgR)-negative tumors showed a significantly higher DPD level than PgR-positive tumors. There was no significant correlation of the TS or DPD levels with other clinicopathological factors in this preliminary study. Further studies are warranted to investigate predictive and prognostic values of intratumoral TS and DPD levels in various malignancies using these ELISAs.