Stable expression of human aromatase complementary DNA in mammalian cells: a useful system for aromatase inhibitor screening.
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A mammalian cell expression plasmid, pH beta-Aro, containing the human placenta aromatase complementary DNA was constructed. The prepared plasmid was used to transfect breast cancer cells (MCF-7), noncancerous breast cells (HBL-100), and Chinese hamster ovary cells by a stable expression method. While the maximum velocities for aromatase expressed in three types of cells were different (10-201 pmol of [3H2O] formed/h/mg) using [1 beta, 2 beta-3H]androst-4-ene-3,17-dione as the substrate, the apparent Michaelis-Menten constants were found to be similar (39.9-57.8 nM) and were within the range determined for the enzyme existing in human placenta. The expressed activities were inhibited by the known aromatase inhibitors, 4-hydroxyandrostenedione and aminoglutethimide, at concentrations that normally inhibit the human placental aromatase. However, it was found that the inhibition profiles were different for aromatase expressed in different types of cells, suggesting that other factors, such as the uptake of the inhibitor, may also play a role in determining the inhibition efficiency. These constructed aromatase expressing mammalian cell lines will be very useful tools for aromatase inhibitor screening. (+info)
Medical management of Cushing's disease: what is the future?
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Corticotropin regulation of the synthesis of a specific rat adrenal cytosolic protein. Effects of hypophysectomy and actinomycin D.
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The mechanism of corticotropin stimulation of the synthesis of a specific rat adrenal cytosolic protein was investigated. This protein (protein E) has a mol.wt. of approx. 30000. It is detected by polyacrylamide-gel electrophoresis of cytosol prepared from adrenal slices from rats treated with corticotropin in vivo and control rats, the slices being incubated with [(3)H]- and [(14)C]-leucine respectively. In rats 1-15 days after hypophysectomy, corticotropin, like dibutyryl cyclic AMP, induces an increase in protein E similar to that induced in control rats, even though both compounds no longer stimulate total protein synthesis. Corticotropin stimulation of protein E synthesis is mediated by cyclic AMP but not by corticosterone, since aminoglutethimide, a steroidogenic inhibitor, does not affect corticotropin stimulation, and dexamethasone alone has no effect. Actinomycin D, when injected in vivo 1h before or after corticotropin injection, prevents the effect of corticotropin on protein E synthesis, which is interpreted as evidence that mRNA synthesis is necessary for the stimulation of protein E synthesis. When injected more than 2h after corticotropin, actinomycin D does not prevent corticotropin stimulation of protein E synthesis, but completely blocks corticotropin stimulation of total protein synthesis. This is interpreted as meaning that, after stimulation of mRNA coding for protein E, corticotropin has no effect on the synthesis of protein E. On the other hand, corticotropin stimulation of protein E synthesis persists after hypophysectomy even though it no longer stimulates total protein synthesis. These data suggest that the factor(s) involved in the synthesis of protein E are more stable than those involved in total protein synthesis. (+info)
Efficacy of tamoxifen +/- aminoglutethimide in normal weight and overweight postmenopausal patients with hormone receptor-positive breast cancer: an analysis of 1509 patients of the ABCSG-06 trial.
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Decreased serum concentrations of tamoxifen and its metabolites induced by aminoglutethimide.
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The antiestrogen tamoxifen and the aromatase inhibitor aminoglutethimide show similar response rates when used in the endocrine management of advanced breast cancer. However, numerous clinical trials have demonstrated no increase in response rate from treatment with the drug combination of tamoxifen plus aminoglutethimide. We investigated the possibility of a pharmacokinetic interaction between these two drugs in six menopausal woman with breast cancer. All patients were investigated under three different conditions (termed phases A, B, and C). The steady state kinetics of tamoxifen were determined when administered alone (phase A) and after coadministration of aminoglutethimide for 6 weeks (phase B). In phase B, the pharmacokinetics for aminoglutethimide were determined and compared with these parameters after a tamoxifen washout of 6 weeks (phase C). The serum concentration of tamoxifen and most of its metabolites ([trans-1(4-beta-hydroxy-ethoxyphenyl)-1,2-diphenylbut-1-ene], 4-hydroxytamoxifen, 4-hydroxy-N-desmethyltamoxifen, N-desmethyltamoxifen, and N-desdimethyltamoxifen) were markedly reduced following aminoglutethimide administration, corresponding to an increase in tamoxifen clearance from 189-608 ml/min. The amount of most metabolites in serum increased relative to the amount of parent tamoxifen. These data are consistent with induction of tamoxifen metabolism during aminoglutethimide exposure. We found no effect of tamoxifen on aminoglutethimide pharmacokinetics or acetylation. We conclude that this aminoglutethimide-tamoxifen interaction should be taken into account when evaluating the clinical effect of this drug combination relative to monotherapy. (+info)
Immunocytochemical assay for estrogen receptor in patients with breast cancer: relationship to a biochemical assay and to outcome of therapy.
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We developed an immunoperoxidase technique using a monoclonal antibody to the estradiol receptor (ER) to identify immunoreactive ER (iER) in breast carcinomas and compared this with the conventional dextran-coated charcoal (DCC) steroid binding assay. We also examined the relationship between the iER and response to therapy in patients with advanced breast cancer. We found iER-positive cells in 60 of 90 carcinomas (66.7%); this correlated with the DCC assay (r = 0.76; P less than .001). Of these, 56 patients were found to be assessable for response to endocrine therapy. Twenty-two showed an objective response to some form of endocrine manipulation, and all these had positively stained carcinomas. By deriving a staining intensity index (SII) we observed that 21 of 22 responders (95%) had an SII of greater than or equal to 0.5, whereas only 8 of 34 nonresponders (24%) had an SII of greater than or equal to 0.5. This difference is highly significant (P less than .001). None of the 17 patients with negatively stained carcinomas responded to endocrine therapy. We conclude that the monoclonal antibody to ER can help identify breast cancer patients who may respond to endocrine therapy. (+info)
Epidermal growth factor activates steroid biosynthesis in cultured Leydig tumor cells without affecting the levels of cAMP and potentiates the activation of steroid biosynthesis by choriogonadotropin and cAMP.
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The studies presented herein were designed to investigate the effects of mouse epidermal growth factor (mEGF) on steroid biosynthesis in a clonal strain of cultured murine Leydig tumor cells (designated MA-10). We show that in short-term incubations (up to 8 h), mEGF activates steroid biosynthesis without affecting cAMP levels. The maximal activation of steroid biosynthesis by mEGF (about 10-fold) is, however, much lower than the maximal activation detected with human choriogonadotropin (hCG) or cAMP analogues (about 1000-fold). We also show that mEGF has two (opposing) effects on the activation of steroidogenesis by hCG. Initially, it transiently attenuates the increase in intracellular cAMP and steroid biosynthesis provoked by submaximal concentrations of hCG. At later times, however, it potentiates the stimulatory effects of submaximal concentrations of hCG on steroid biosynthesis in a synergistic fashion. Last, we show that mEGF and submaximal concentrations of cAMP analogues also activate steroidogenesis in a synergistic fashion and that the degree of synergism attained with cAMP analogues plus mEGF is much higher than that attained with hCG plus mEGF. Taken together, our results show that mEGF (i) activates steroidogenesis without affecting cAMP levels and (ii) modulates the activation of steroidogenesis by the cAMP second messenger system. (+info)