Purification and properties of estrogen-responsive cytoplasmic thymidine kinase from human breast cancer

Abstract

The effect of 17 beta-estradiol on cytoplasmic thymidine kinase activity was studied in MCF-7, a human breast cancer cell line in culture which responds to estrogens with an increase in the rate of growth. Levels of 17 beta-estradiol which maximally stimulate [3H]thymidine incorporation into DNA also maximally stimulate thymidine kinase activity. The Vmax for thymidine increased while the Km was not affected by estrogen stimulation when performed on nonpurified enzyme. Tamoxifen, an antiestrogen, decreased the specific activity of the enzyme. To further study its hormonal regulation, cytoplasmic thymidine kinase was purified greater than 2000-fold by affinity column chromatography. The purified preparation migrated in one band to a pI of 8.5 on an isoelectric focusing gel. The purified thymidine kinase was further characterized by examining its molecular weight, pH optimum, heat stability, utilization of phosphate donors, inhibition by nucleotides, and the effect of pyrimidine nucleoside analogs.