The effects of estrogens and antiestrogens on hormone-responsive human breast cancer in long-term tissue culture. (1/13)

We have established or characterized six lines of human breast cancer maintained in long-term tissue culture for at least 1 year and have examined these lines for estrogen responsiveness. One of these cell lines, MCF-7, shows marked stimulation of macromolecular synthesis and cell division with physiological concentrations of estradiol. Antiestrogens are strongly inhibitory, and at concentrations greater than 3 X 10(-7) M they kill cells. Antiestrogen effects are prevented by simultaneous treatment with estradiol or reversed by addition of estradiol to cells incubated in antiestrogen. Responsive cell lines contain high-affinity specific estradiol receptors. Antiestrogens compete with estradiol for these receptors but have a lower apparent affinity for the receptor than estrogens. Stimulation of cells by estrogens is biphasic, with inhibition and cell death at concentrations of 17beta-estradiol or diethylstilbestrol exceeding 10(-7) M. Killing by high concentrations of estrogen is probably a nonspecific effect in that we observe this response with 17alpha-estradiol at equivalent concentrations and in the otherwise unresponsive cells that contain no estrogen receptor sites.  (+info)

Effects of estrogen antagonists and agonists on the ACTH response to restraint stress in female rats. (2/13)

Previous studies have found that female rats are less sensitive than males to hypothalamic-pituitary-adrenal axis feedback inhibition by exogenous glucocorticoid administration. To determine whether estrogen contributes to this sex difference, we examined the effects of the estrogen antagonists tamoxifen and C1628 on the ACTH and corticosterone responses to restraint stress. CI628 increased both the ACTH and corticosterone response to restraint stress, and tamoxifen increased the ACTH response to restraint. Using overiectomized female rats, we also examined the effects of seven days of estradiol and/or progesterone replacement. Low dose estradiol decreased the ACTH but not the corticosterone response to restraint stress while progesterone had no effect on ACTH or corticosterone responses. The combination of estradiol and progesterone also decreased the ACTH response to stress, and the magnitude of the effect did not differ from that found with estradiol treatment alone. These data suggest that in the physiological range estradiol is an important inhibitory factor in the hypothalamic-pituitary-adrenal stress response of females.  (+info)

Uterine peroxidase as a marker for estrogen action. (3/13)

Administration of a single dose of estradiol to immature rats gives rise to the appearance of substantial amounts of peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) enzyme activity in the uterus. This enzyme induction, which is inhibited by administration of actinomycin D and cycloheximide, can be detected at 4 hr after administration of estradiol, reaches a maximum level by 20 hr, and thereafter declines. The amount of uterine peroxidase seen at 20 hr after a single dose increases with dose from 0.1 to 100 microgram of estradiol. Estrone and estriol also show dose-dependent induction of peroxidase, and the quantitative peroxidase responses to these steroids follow their uterotropic capacities. The antiestrogen CI628, capable of low levels of enzyme induction by itself, can inhibit the induction due to estrogen. Solubilization of the uterine enzyme with divalent cations, especially calcium, results in a substantially increased yield of peroxidase. This extraction method provides an enzyme of about 50,000 molecular weight in distinction to the large aggregated form obtained by the usual extraction with sodium chloride.  (+info)

Solubilization of a tamoxifen-binding protein. Assessment of its molecular mass. (4/13)

Recent findings point to a role of Antioestrogen-Binding Site (ABS) in some of the growth-modulatory effects of antioestrogens. In the present study, a method for the solubilization of ABS from rat uterus microsomal fractions by using 3-(3-cholamidopropyl)dimethylammonio-1-propanesulphonate (CHAPS; 20 mM) and KCl (0.4 M) is described. Decreasing the CHAPS concentration below the critical micelle concentration led to long-term stabilization of the protein. All of the membrane-bound ABS was recovered in the extract, and only one class of binding site, with a high affinity for [3H]tamoxifen (KA = 5 x 10(8) M-1) was detectable. This binding was time-dependent and reversible: at 4 degrees C, the association rate constant was ka = 7.2 x 10(4) M-1.s-1, and the reverse rate constant was kd = 1.0 x 10(-4) s-1. Solubilized ABS exhibited an affinity and specificity similar to those of the membrane-bound sites. Under disaggregating conditions, solubilized ABS had an apparent sedimentation coefficient, s20,w, of 5.2 S and a Stokes radius of 6.4 nm. From these two values, molecular masses of 160,000 Da for the detergent-ABS complex, and 110,000 for the protein moiety, were estimated. Assessment of the size of the membrane-bound ABS by a radiation inactivation technique is also described. The 'radiation inactivation size', corresponding to the mass of 1 mol of protein structure(s) whose associated tamoxifen-binding activity is abolished after a single hit by ionizing radiation, was estimated to be 80,000 Da.  (+info)

Role of early and late oestrogenic effects on implantation in the mouse. (5/13)

Oestrogen action in the uterus is expressed in an early phase (Phase I) and a late phase (Phase II). The role of this biphasic oestrogen action in implantation is not clear. To determine the relative importance of Phase I and II responses, triphenylethylene compounds (CI-628, LY-117018, nafoxidine, clomiphene citrate and tamoxifen) and oestrogens (oestriol and oestradiol-17 beta) were used in a physiologically relevant experimental system for studying implantation. All compounds elicited uterine water imbibition to various degrees in ovariectomized-progesterone-treated mice at 6 h (Phase I response) and their effectiveness in inducing implantation in delayed implanting mice correlated with their respective potency to increase uterine wet weight. This suggests that Phase I might be an essential component of oestrogen action in implantation and that the efficiency of a compound to elicit a Phase I response might serve as a predictive indicator of its potential action on implantation.  (+info)

Metabolism of nitromiphene (CI 628) in the immature female rat: role of gastrointestinal microflora in the biotransformation of a triarylethylene antiestrogen. (6/13)

Nitromiphene (NIT; CI 628) is a triarylethylene antiestrogen shown to be effective in treatment of experimental breast cancer. We have studied the fate of NIT in the immature female rat, the animal model in which most of the biochemical studies of NIT have been carried out. NIT was eliminated mainly via the feces after i.p. administration, primarily as metabolites. One of these, a diphenylmethane derivative, p-[2-(N-pyrrolidinyl)ethoxy]-p'-methoxybenzophenone (PMB), was also eliminated in urine as such and as its O-demethyl and keto-reduced metabolites. In uterine and liver tissue, unchanged NIT was accompanied by demethyl NIT (CI 628M), PMB, and a diarylacetophenone derivative, p-[2-(N-pyrrolidinyl)ethoxy-p'-hydroxybenzhydryl phenyl ketone (demethyl KET). In vitro studies showed that O-demethyl NIT was produced in the presence of liver enzymes and that PMB and demethyl KET were produced in the presence of intestinal bacteria. These results suggested that PMB and demethyl KET accumulate in uterine and liver tissue due to reabsorption from the intestine after having been produced there from NIT and demethyl NIT, respectively. The effects of antiestrogens and their metabolites may be due in part to interaction with antiestrogen binding sites. Both demethyl KET and PMB had good affinity for such sites. Thus, these enteric bacterial metabolites not only have the ability to accumulate in vivo, but could, together with demethyl NIT, contribute to the antiestrogenic effects observed with NIT.  (+info)

Dose-dependent inhibition of the preovulatory surges of gonadotropins and prolactin by the antiestrogen CI-628: possible sites of action. (7/13)

The present series of experiments was conducted in an attempt to correlate previously reported dose-dependent and site-selective inhibitory effects of an antiestrogen, CI-628, on 17 beta-estradiol (E2)-receptor interactions in the anterior pituitary gland (AP) and hypothalamus with its effects on the preovulatory surges of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and prolactin. The effects of CI-628 on the response of the AP to luteinizing hormone-releasing hormone (LHRH) and thyrotropin-releasing hormone (TRH) also were examined. In the first study, rats exhibiting 4-day estrous cycles were injected with various doses (0.02, 0.20, 2.0, and 20 mg/kg) of CI-628 or vehicle at 0900 h on diestrus-2 and proestrus. The preovulatory LH surge and both preovulatory and secondary FSH surges were marginally affected by 0.02 mg/kg CI-628, but were completely abolished by higher doses. In contrast, a dose of 0.20 mg/kg only delayed the prolactin surge; however, higher doses were effective in extinguishing cyclic prolactin release. In a second experiment, CI-628 in rats treated on diestrus-2 and proestrus exerted a dose-dependent suppression of the AP LH response to an initial injection of LHRH on proestrous afternoon in rats whose endogenous LH surges were blocked by phenobarbital. However, AP LH responses to a second LHRH injection to assess the self-priming capacity of LHRH were attenuated only in rats given 0.20, 2.0, and 20 mg/kg CI-628. Contrastingly, the AP prolactin response to TRH was suppressed only in rats given 0.20 mg/kg CI-628.(ABSTRACT TRUNCATED AT 250 WORDS)  (+info)

Role of embryonic oestrogen in rabbit blastocyst development and metabolism. (8/13)

Rabbit morulae were grown for 24 h in Ham's F12 medium supplemented with BSA. CI-628 citrate (1.5 micrograms/ml), a specific oestrogen antagonist, significantly inhibited the transformation of morulae to blastocysts. This inhibition was reversed with oestradiol-17 beta (1 micrograms/ml) but not oestradiol-17 alpha (1 micrograms/ml) added to the culture medium. The specific activities of phosphofructokinase, lactic dehydrogenase, malate dehydrogenase and alkaline phosphatase in blastocysts grown in vitro for 24 h in medium TC 199 + BSA showed significant elevation with blastocyst growth and expansion, while that of acid phosphatase revealed no change, and leucine aminopeptidase activity declined significantly. These changes were markedly inhibited by CI-628 citrate (2 micrograms/ml) and were reversed by oestradiol-17 beta (0.4 micrograms/ml) but not by oestradiol-17 alpha (0.4 micrograms/ml). Our findings suggest a role of oestrogen present in the rabbit morula and blastocyst in the triggering of embryonic differentiation and metabolic functions.  (+info)

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