Thymidylate synthase (TS; EC 2.1.1.45) is an important cellular
enzyme that converts dUMP to
dTMP, which is essential for
DNA biosynthesis. In addition, TS is an important cellular target for the fluoropyrimidine cytotoxic drugs that are widely used in the treatment of solid
tumors. We have generated five
monoclonal antibodies against human TS using a recombinant human TS
enzyme. These
antibodies react specifically with human TS and display negligible cross-reactivity with other cellular
proteins found in human cells. Binding affinity studies demonstrate that all
antibodies form a tight interaction with recombinant human TS
enzyme (Kd range = 0.3-11.0 nM). All
antibodies display reactivity on
enzyme-linked
immunosorbent assay and immunoprecipitation. On Western blot analysis each detects a
protein of approximately 36 kDa molecular mass under denaturing conditions. In addition to their reactivity on immunoprecipitation and Western analysis, two of the
antibodies, TS 106 and TS 109, are reactive on immunohistochemical staining of human colon
carcinoma cell lines and tissue, producing a granular cytoplasmic staining pattern. Specificity for TS is demonstrated by the lack of staining with preimmune
IgG and the disappearance of the signal when the
antibodies are preabsorbed with recombinant human TS
enzyme. Quantitation of TS by Western blot analysis and biochemical
FdUMP binding assay in 5-fluorouracil-resistant colon
carcinoma cell lines (NCI H630R10, NCI H630R1) and a sensitive colon
carcinoma cell line (NCI H630) revealed a 36- and 6-fold increase in TS in the resistant cell line as measured by the biochemical assay compared to a 39- and 10.6-fold increase as measured by densitometric analysis of the Western blot. These comparative studies of immunohistochemical, Western, and biochemical analyses reveal that the immunological detection of TS in human colon cell lines is a sensitive and quantitative assay. Thus the ability of these
antibodies to detect TS in human
cancer cells and tissue may allow measurement of TS in human tissues by quantitative immunohistochemistry in studies of drug resistance and for determination of proliferative rates.