Estrogen regulation of human breast cancer cell line MCF-7 tissue plasminogen activator.

The human breast cancer cell line MCF-7 produces a number of estrogen-regulated proteins, among which is tissue plasminogen activator (tPA). Increased medium concentrations of PA activity were observed after the addition of 17 beta-estradiol to cultures of MCF-7 cells. However, in the current study these hormone-regulated increases are limited to cultures near or at confluence, but not in the preconfluent period. MCF-7 cell cultures produce either tPA activity alone or in combination with urokinase activators. At confluence, a single exposure to 17 beta-estradiol stimulates a marked transitory rise in tPA activity in the extracellular and cell-associated compartments; the peak increases were at 48 h for medium activity and 24-48 h for cell-associated activity. Sustained exposure to hormone leads to a persistent increase in activity in both compartments. Examination of the structure-function relationships of estrogen agonists, steroidal and nonsteroidal, as well as nonestrogenic steroids indicated that stimulation of PA activity was restricted to estrogen agonists. The increased activity was reflected in enhancement of tissue PA activity when viewed using sodium dodecyl sulfate-polyacrylamide gel zymography. Those cultures expressing both activators revealed no alteration of urokinase activity due to hormone addition. Antiestrogens added to MCF-7 cells not rigorously limited in exogenous estrogens selectively suppressed tissue PA activity, but not that of urokinase. These data indicate that at the point when MCF-7 cell cultures are no longer growing exponentially, addition of estrogen agonists at physiological concentrations elevates tPA activity while not altering expression of urokinase activity. The discussion suggests a possible role that this regulation may subserve in the function of breast epithelial cells.

Suppression of estrogen-regulated extracellular tissue plasminogen activator activity of MCF-7 cells by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) suppresses the estrogen enhancement of tissue plasminogen activator (t-PA) by MCF-7 breast cancer cells. 17 beta-estradiol treatment of MCF-7 cells was previously shown to enhance t-PA secretion in a receptor-mediated process dependent on RNA and protein synthesis. The current studies demonstrate that treatment with TCDD, at a concentration as low as 10(-11) M, reduces the 17 beta-estradiol-induced enhancement of t-PA secretion in these cells. Treatment of MCF-7 cells with TCDD alone does not alter t-PA activity nor was inhibition of t-PA activity observed when TCDD was added directly to the enzyme assay. Kinetic studies and the lack of inhibition following in vitro mixing of conditioned media from TCDD-treated and control 17 beta-estradiol stimulated MCF-7 cells argue against TCDD induction of a plasminogen activator inhibitor. The related polychlorinated dibenzofuran, 2,3,7,8,-tetrachlorodibenzofuran, while also active, is less potent that TCDD. Other polychlorinated dibenzodioxins, polychlorinated dibenzofurans, and polychlorinated biphenyls do not suppress 17 beta-estradiol induction of t-PA over the concentrations tested. These results are in agreement with the structure-activity relationships established using these compounds in other assay systems. Treatment with TCDD does not alter the number or affinity of 17 beta-estradiol receptors of MCF-7 cells. TCDD treatment does not suppress constitutive t-PA activity in the estrogen independent breast cancer line MDA-MB-231 nor the t-PA induced by 12-O-tetradecanoylphorbol-13-acetate in HeLa cells. These effects suggest that TCDD is not acting directly on expression of the t-PA genome. Induction of aryl hydrocarbon hydroxylase by TCDD, a cytochrome P-450 regulated metabolic enzyme for which TCDD is the most potent known inducer, was observed in MCF-7 cells but not in MDA-MB-231 or HeLa cells. A plausible mechanism for the antiestrogenic activity of TCDD is based on the metabolic conversion of 17 beta-estradiol to less active derivatives by TCDD induced cytochrome P-450 metabolic enzymes.

Induction of creatine kinase in immature rat uteri by zearalenone, an estrogenic mycotoxin.

Intraperitoneal administration of alpha-zearalenone (ZEN) to female immature rats induced synthesis of a uterine protein which has been identified as creatine kinase. The induced enzyme was purified to homogeneity by chromatography on DEAE Sephacel and Hydroxyapatite Ultrogel. Guinea pig antiserum against estrogen-induced uterine, rat uterine creatine kinase crossreacted with the ZEN-induced enzyme, indicating that ZEN exhibits an early estrogenic response in a manner analogous to natural estrogens.

Estradiol preferentially enhances extracellular tissue plasminogen activators of MCF-7 breast cancer cells.

MCF-7 human breast cancer cells secrete two immunologic types of plasminogen activator, one related to urokinase, the other unrelated. We have now examined whether estrogen stimulation of secreted plasminogen activator activity reflects an increase in one or both types. Examined semiquantitatively by sodium dodecyl sulfate-polyacrylamide gel electrophoretic zymography, the conditioned media of control cells were seen to contain a major activator band (Mr approximately 54,000) immunologically related to urokinase and a barely discernible doublet (Mr approximately 64,000 and Mr approximately 68,000). Addition of estradiol or, at much higher concentrations, testosterone led to marked enhancement of doublet activity, while the 54-kDa band was invariant. The 64-68-kDa doublet was immunoreactive with antiserum directed against Bowes melanoma tissue plasminogen activator but not with antiurokinase antibodies. Enhancement of doublet activity was correlated with hormone-induced increases in total secreted plasminogen activator activity. Neither progesterone nor dexamethasone increased total activity or the 64-68-kDa zones of lysis. Estradiol and testosterone alterations were blocked by appropriate concentrations of an estrogen antagonist (LY156758), actinomycin D, or cycloheximide. Regulation of MCF-7 cell-secreted tissue plasminogen activators thus appears to be mediated by an estrogen receptor process and to require sustained RNA and protein synthesis.

Purification and characterization of creatine kinase, an estrogen-induced uterine protein (IP) from immature rats.

An estrogen-responsive translational product, the induced protein (IP) first described by Notides and Gorski (8), was obtained solely from the target organ, immature rat uterus, and purified to homogeneity in a procedure using two chromatography steps. The purified IP has a molecular weight of 49,000, and the isoelectric point is 5.2. Creatine kinase activity is associated with the homogeneous IP. There are some differences between the uterine enzyme and the creatine kinase BB isoenzyme, including differences in stability, and sensitivity to mercaptans. Estrogen-induced creatine kinase purified by this simple, reproducible method is a useful antigen for further studies on the translation and transcription processes involved in hormone-modulated synthesis.