amanitins and Breast-Neoplasms

The mechanism by which estrogen and antiestrogens modulate cytoplasmic thymidine kinase (TK) activity has been studied in MCF-7 cells. Using a cloned cDNA probe for human TK, we have identified a single 1500-nucleotide transcript as the cytoplasmic TK-mRNA in MCF-7 cells. In normally cycling or synchronously growing cells, the level of this mRNA maximally increased 2-3-fold after 24 h of estradiol-17 beta (E2) stimulation and decreased below control level in the presence of antiestrogens. Neither E2 nor antiestrogens altered the size of TK-mRNA. Hormonal regulation of TK-mRNA paralleled changes in TK enzyme activity and [3H]thymidine incorporation. Using endogenous nuclear run-off transcription to investigate the expression of the human TK gene, we demonstrate that TK-mRNA levels were regulated by transcriptional control. Modulation of TK gene activity during the process of estrogen stimulation or antiestrogen inhibition was not accompanied by changes in the methylation pattern at internal sites of the TK gene. These results suggest a transcriptional control of human TK gene by E2 and antiestrogens in MCF-7 cells.

Topics: Amanitins; Breast Neoplasms; Cell Cycle; Cell Line; DNA; Estradiol; Estrogen Antagonists; Estrogens; Gene Expression Regulation; Humans; Methylation; Pyrrolidines; RNA; RNA, Messenger; Tamoxifen; Thiophenes; Thymidine; Thymidine Kinase; Time Factors

The growth of MCF-7 cells was arrested by 24 h of isoleucine deprivation. Following replenishment of the medium, the incorporation of uridine and thymidine into trichloroacetic acid-precipitable material began to increase slowly and gradually rose to the level of cycling cells. The addition of 5 X 10(-9) M estradiol to growth-arrested cells dramatically shortened the time of onset of macromolecular synthesis and increased the overall amount of precursor incorporation 2- to 4-fold over the level obtained by arrested control cells. The increase in uridine incorporation preceded the increase in thymidine incorporation by 6 h. Inhibition of protein synthesis with cycloheximide blocked the recovery of macromolecular synthesis in both control and estrogen-treated cells. Actinomycin D was ineffective in blocking the estrogen-stimulated recovery of macromolecular synthesis at concentrations known to inhibit pre-rRNA synthesis (10(-8) M). At higher concentrations, uridine and thymidine incorporation were inhibited in a dose-dependent manner. Inhibition of RNA polymerase II activity with alpha-amanitin similarly blocked both the recovery of the cells from isoleucine starvation and the potentiation of this by estradiol. Dihydrofolate reductase and thymidine kinase activities are both stimulated by estradiol in MCF-7 cells. In cycling cells, estrogen stimulates a 2-fold increase in their messenger RNAs (mRNAs) within 24 h. The level of dihydrofolate reductase mRNA is unaffected by isoleucine starvation, and estrogen caused no change in dihydrofolate reductase mRNA levels over a 24-h period following reversal of growth arrest. Similar results were observed for the 600-nucleotide pS2 mRNA that has been identified as an estrogen-induced RNA in MCF-7 cells. In contrast, thymidine kinase mRNA was found to be increased by estrogen at 24 h, but not at 12 h, following reversal of growth arrest. This increase correlates with increases in thymidine, but not uridine incorporation. These data indicate that the estrogen-stimulated increase in thymidine incorporation following release from growth arrest is dependent on new RNA synthesis. However, the hormone did not increase the levels of three estrogen-regulated mRNAs coordinately with the increases observed in uridine incorporation.

Topics: Amanitins; Breast Neoplasms; Cell Division; Cells, Cultured; Cycloheximide; Dactinomycin; DNA, Neoplasm; Estradiol; Estrogens; Female; Genes; Humans; Kinetics; Neoplasm Proteins; RNA, Messenger; RNA, Neoplasm; Tetrahydrofolate Dehydrogenase; Thymidine Kinase