Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The antiestrogen tamoxifen is used in the treatment of hormone-responsive breast cancer. However, therapeutic failure has frequently been observed in both patients and animal models after long term treatment. We have studied the effect of a point mutation that leads to the substitution of Val for Gly at codon 400 in the ligand-binding domain of the estrogen receptor (ER) on estrogenic and antiestrogenic activities of 4-hydroxytamoxifen (4-OHT) and its derivatives. Stable ER transfectants derived from MDA-MB-231 CL10A, an ER-negative breast cancer cell line, have been used in these studies. 4-OHT and its fixed ring derivatives showed more estrogen-like activity in ER transfectants than in MCF-7, an ER-positive breast cancer cell line. In this study, 4-OHT was a partial agonist of cell growth in the transfectant S30 cells, which express the wild-type ER. However, it was a full agonist in the mutant ER transfectant ML alpha 2H, which expressed ER with Val at codon 400. The increased estrogenic activity of 4-OHT in ML alpha 2H cells was not due to the preferential isomerization of trans 4-OHT to cis 4-OHT, since the nonisomerizable fixed ring trans 4-OHT was a partial agonist for cell growth in S30 cells and was a full agonist in ML alpha 2H cells. Transient transfection using a reporter plasmid containing an estrogen response element demonstrated that fixed ring trans 4-OHT had estrogenic activity in ML alpha 2H cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1992 Dec
PMID:Point mutation of estrogen receptor (ER) in the ligand-binding domain changes the pharmacology of antiestrogens in ER-negative breast cancer cells stably expressing complementary DNAs for ER. 149 96

In order to better understand the structural requirements for effective high affinity binding of estrogens and antiestrogens by the human estrogen receptor (ER), a comparative study was undertaken in which we examined: 1) native ER from the MCF-7 ER-positive human breast cancer cell line; 2) full length ER expressed in yeast; 3) the ER hormone binding domain (amino acid residues 302-595) expressed in yeast; 4) a bacterially expressed protein A fusion product encoding a truncated ER (amino acid residues 240-595); and 5) a synthetic peptide encompassing amino acids 510-551 of the ER. The binding parameters studied included affinity, kinetics, structural specificity for ligands, and stability. Full length ER expressed in yeast was very similar to the MCF-7 ER in its affinity [dissociation constant (Kd), 0.35 +/- 0.05 nM], dissociation rate (t1/2, 3-4 h at 25 C), and structural specificity for both reversible and covalently attaching affinity ligands. While the truncated ER expressed in yeast was similar to MCF-7 ER in its specificity of ligand binding, it showed a slightly reduced affinity for estradiol (Kd, 1.00 +/- 0.17 nM). The bacterially expressed ER also had a lower affinity for estradiol (Kd, 1.49 +/- 0.16 nM), which may be due in part to an increase in the dissociation rate (t1/2, 0.5 h at 25 C). The attachment of covalent affinity ligands and structural specificity for a variety of reversible ligands was comparable in the bacterially expressed ER to that observed for the receptors expressed in MCF-7 cells and yeast.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1992 Jun
PMID:Structural requirements for high affinity ligand binding by estrogen receptors: a comparative analysis of truncated and full length estrogen receptors expressed in bacteria, yeast, and mammalian cells. 149 91

Previous studies have shown that in the breast there are multiple forms of the enzyme oestradiol dehydrogenase (E2DH), responsible for the interconversion of oestrone (E1) to oestradiol (E2). We have now re-examined oestrogen metabolism in the breast cancer cell lines (T47D and MCF-7) and have shown that steroids previously shown to inhibit the conversion of E1 to E2 in normal breast tissue failed to do so when added to growing monolayers of these malignant cells. In contrast to earlier estimates in normal breast tissues, the apparent Km for this conversion in monolayers of these malignant cells is shown here to be considerably lower, at around 50 nM. Cell free studies on these cell lines have revealed the presence of a high affinity (for E1) form of this enzyme of Mw approximately 80 kDa. The ability to detect this enzyme in soluble cell fractions appears to be critically dependent on buffer composition. Normal breast epithelial cells and adipose tissue appear to be devoid of this form of E2DH. As this form of E2DH has the highest affinity for the substrate E1 of all the forms in the breast, it is probable that this 80 kDa enzyme is responsible for the conversion of E1 to E2 in cell monolayers. If the observation holds that the 80 kDa enzyme is absent in the normal tissues, then the possibility arises that this E2DH may be linked with the neoplastic process in some breast tumours containing malignant epithelial cells of a similar type as studied here.
J Steroid Biochem Mol Biol 1992 Sep
PMID:Oestradiol synthesis from oestrone in malignant breast epithelial cells: studies on a high affinity, 80 kDa form of oestradiol dehydrogenase. 152 49

Previous studies in this laboratory identified a series of 7 alpha-alkylamide analogues of 17 beta-oestradiol which are pure antioestrogens. Among this initial lead series of compounds, exemplified by ICI 164,384, none was of sufficient in vivo potency to merit serious consideration as a candidate for clinical evaluation. Further structure-activity studies identified a new compound, ICI 182,780, 7 alpha-[9-(4,4,5,5,5-pentafluoro-pentylsulphinyl)nonyl]oestra-1,3,5(10)- triene-3,17 beta-diol, with significantly increased antioestrogenic potency. The antiuterotrophic potency of ICI 182,780 is more than 10-fold greater than that of ICI 164,384. ICI 182,780 has no oestrogen-like trophic activity and, like ICI 164,384 is peripherally selective in its antioestrogenic effects. The increased in vivo potency of ICI 182,780 was also reflected, in part, by intrinsic activity at the oestrogen receptor and in the growth inhibitory potency of ICI 182,780 in MCF-7 human breast cancer cells. ICI 182,780 was a more effective inhibitor of MCF-7 growth than 4'-hydroxytamoxifen, producing an 80% reduction of cell number under conditions where 4'-hydroxytamoxifen achieved a maximum of 50% inhibition. Sustained antioestrogenic effects of ICI 182,780, following a single parenteral dose of ICI 182,780 in oil suspension, were apparent in both rats and pigtail monkeys. In vivo, the antitumour activity of ICI 182,780 was demonstrated with xenografts of MCF-7 and Br10 human breast cancers in athymic mice where, over a 1 month period, a single injection of ICI 182,780 in oil suspension achieved effects comparable with those of daily tamoxifen treatment. Thus, ICI 182,780 provides the opportunity to evaluate clinically the potential therapeutic benefits of complete blockade of oestrogen effects in endocrine-responsive human breast cancer.
J Steroid Biochem Mol Biol 1992 Sep
PMID:ICI 182,780, a new antioestrogen with clinical potential. 152 58

Liarozole reduced tumor growth in the androgen-dependent Dunning-G and the androgen-independent Dunning MatLu rat prostate carcinoma models as well as in patients with metastatic prostate cancer who had relapsed after orchiectomy. In vitro, liarozole did not have cytostatic properties, as measured by cell proliferation in breast MCF-7 and prostate DU145 and LNCaP carcinoma cell lines. It did not alter the metabolism of labeled testosterone i.e. the 5 alpha-reductase in cultured rat prostatic cells. In mouse F9 teratocarcinoma cells liarozole did not show any retinoid-like properties but enhanced the plasminogen activator production induced by retinoic acid. Furthermore, liarozole and retinoic acid similarly reduced the growth of the androgen-dependent Dunning-G tumor in nude mice and inhibited tumor promotion elicited by phorbol ester in mouse skin. These data have raised the hypothesis that the antitumoral properties of liarozole may be related to inhibition of retinoic acid degradation, catalyzed by a P-450-dependent enzyme that is blocked by the drug.
J Steroid Biochem Mol Biol 1992 Sep
PMID:Experimental studies with liarozole (R 75,251): an antitumoral agent which inhibits retinoic acid breakdown. 152 60

We investigated binding characteristics of basic fibroblast growth factor (bFGF) on membranes prepared from 4 human breast cancer cell lines and 38 primary BC biopsies. Competitive binding experiments were performed and analyzed using the "Ligand" program. Furthermore bFGF mitogenic activity was measured by [3H]thymidine incorporation into DNA from breast cancer cell lines. The presence of high-affinity binding sites was demonstrated in each cell type (MCF-7: Kd = 0.60 nM; T-47D: Kd = 0.55 nM; BT-20: Kd = 0.77 nM; MDA-MB-231: Kd = 0.34 nM). The presence of these high-affinity binding sites was confirmed with saturation experiments. A second class of low-affinity binding sites was detected in the 2 hormone-independent cells (BT-20: Kd = 2.9 nM; MDA-MB-231: Kd = 2.7 nM). bFGF stimulated the proliferation of MCF-7, T-47D, BT-20 but not MDA-MB-231 cell lines. With competition experiments, binding sites were detectable in 36/38 breast cancers; high-affinity binding sites (Kd less than 1 nM) were present in 19/36 cases and low-affinity binding sites (Kd greater than 2 nM) were present in 29/36 cases (the two classes of binding sites were present in 12 breast cancers). No relation between bFGF binding sites and node involvement, histologic type or grading of the tumor was evidenced. There were negative correlations (Spearman test) between total bFGF binding sites and estradiol receptor (P = 0.05) or progesterone receptor (P = 0.009). The demonstration of (1) bFGF specific binding sites in breast cancer membranes, and (2) bFGF growth stimulation of some breast cancer cell lines indicates that this factor may be involved directly in the growth of some breast cancers.
J Steroid Biochem Mol Biol 1992 Sep
PMID:Basic fibroblast growth factor (bFGF): mitogenic activity and binding sites in human breast cancer. 152 70

Increased expression of certain glutathione S-transferase (GST) isoenzymes has frequently been associated with the development of resistance to alkylating agents and other classes of antineoplastic drugs in drug-selected cell lines. The question arises whether this phenomenon is causal or is a stress-induced response associated with drug resistance in these cell lines. We have constructed mammalian expression vectors containing the human GST mu and GST alpha 2 (Ha2) cDNAs and stably transfected them into the human breast cancer cell line MCF-7. Whereas the parental and pSV2neo-transfected cell lines display low GST activity, three individual transfected clones were identified in each group that expressed either GST mu or GST alpha 2. The range of GST activities was similar to those observed in cells selected for anticancer drug resistance. The GST mu specific activities were 56, 150, and 340 mlU/mg, compared with 10 mlU/mg of endogenous GST mu in control lines. Specific activities in GST alpha 2-transfected clones were 17, 28, and 52 mlU/mg, compared with no detectable alpha class GST in control lines. These clonal lines and the parental and pSV2neo-transfected control lines were tested for sensitivity to antineoplastic agents and other cytotoxic compounds. The clones with the highest activity in each group were 1.7-fold (GST alpha 2) to 2.1-fold (GST mu) resistant to the toxic effects of ethacrynic acid, a known substrate for GSTs. However, the GST-transfected cell lines were not resistant to doxorubicin, L-phenylalanine mustard, bis(2-chloroethyl)-1-nitrosourea, cisplatin, chlorambucil, or the GST substrates 1-chloro-2,4-dinitrobenzene or tert-butyl hydroperoxide. Thus, although L-phenylalanine mustard, bis(2-chloroethyl)-1-nitrosourea, chlorambucil, tert-butyl hydroperoxide, and 1-chloro-2,4-dinitrobenzene are known to be metabolized by glutathione-dependent GST-catalyzed reactions, there was no protection against any of these agents in MCF-7 cell lines overexpressing GST mu or GST alpha 2. We conclude that, at the levels of GST obtained in this transfection model system, overexpression of GST mu or GST alpha 2 is not by itself sufficient to confer resistance to these anticancer agents. These studies do not exclude the possibility that GST may be a marker of drug resistance or that other gene products not expressed in MCF-7 cells might cooperate with GST to confer drug resistance.
Mol Pharmacol 1992 Feb
PMID:Expression of human mu or alpha class glutathione S-transferases in stably transfected human MCF-7 breast cancer cells: effect on cellular sensitivity to cytotoxic agents. 153 4

Estradiol 17 beta-hydroxysteroid dehydrogenase (E2DH) is the enzyme responsible for the interconversion of estrone (E1), and the more biologically potent steroid, estradiol (E2), and has a crucial role in regulating breast tissue concentrations of E2. It has previously been shown that breast tumor cytosol is able to preferentially stimulate the reductive conversion of E1 to E2 in cultured MCF-7 breast cancer cells. In this study the stimulatory factor(s) from breast tumor cytosol have been partially purified by gel filtration and affinity chromatography. Human serum albumin (HSA) has been identified as a component of this bioactive fraction. Subsequent testing of commercially purified HSA preparations has revealed the ability of some preparations to be highly stimulatory. The albumin present in breast tumor cytosol may therefore be a contributing factor to the observed stimulation of reductive E2DH activity in cultured MCF-7 cells. Such a mechanism may account in part for the higher concentrations of E2 which are observed in breast tumors in vivo.
Mol Cell Endocrinol 1992 Jan
PMID:Identification of albumin in breast tumor cytosol as a factor involved in the stimulation of estradiol 17 beta-hydroxysteroid dehydrogenase (reductive) activity. 155 73

The mechanisms underlying loss of oestrogen responsiveness in breast cancer are not well-defined. Potential mechanisms include loss of receptor expression, alterations in the oestrogen receptor (ER) gene producing proteins with abnormal function, or changes to receptor-dependent or -independent pathways controlling cell proliferation. Examination by Southern analysis of the ER gene in a series of ER-negative and -positive breast tumour biopsies failed to provide evidence of gross rearrangements and in only one of thirty seven tumour DNA samples was significant gene amplification observed. No restriction fragment length polymorphisms were detected for the restriction enzymes EcoR I, Pst I or Hind III. Methylation of the ER gene as assessed by Hpa II and Msp I restriction enzyme digests varied between tumours but the degree of methylation was not correlated with levels of expression of the receptor protein. Similar findings applied in a series of ER-negative and -positive breast cancer cell lines and clonal lines of MCF-7 cells, which were developed as an in vitro model for the acquisition of oestrogen and antioestrogen resistance. In this model there was no evidence that changes to ER receptor function and/or structure at the level of the ER gene, mRNA, ligand binding, and ability to induce progesterone receptor might account for the development of hormone resistance. However, the ability of ER to interact with a DNA sequence containing the vitellogenin promoter oestrogen response element, as assessed by gel retardation assay, was impaired in the clone showing the greatest degree of oestrogen and antioestrogen resistance.
J Steroid Biochem Mol Biol 1992 Mar
PMID:Oestrogen receptor gene structure and function in breast cancer. 156 23

MCF-7 cells were grown in serum free medium (Dulbecco MEM without phenol red, supplemented with Costar SF-1 without insulin). Insulin was added as required and gave dose dependent growth stimulation at concentrations between 5 and 10,000 nM. Identical growth response curves were obtained for thymidine uptake and cell number. Oestradiol and insulin-like growth factor I (IGF-I) added individually both gave a dose dependent stimulation of cell growth in serum free medium containing 50 nM insulin. The growth stimulatory effect of oestradiol was to a large extent inhibited with suramine, a general inhibitor of growth factors, indicating that the effect of oestradiol was mediated through stimulating autocrine secretion of a growth factor. To investigate a possible link between the effects of oestradiol and IGF-I, a specific IGF-I receptor antibody (alpha IR-3), 10 micrograms/ml was used. These experiments were carried out with 2.5 nM insulin in the medium, a concentration at which insulin had no growth stimulatory effect. Stimulation was carried out for 18 h before assay of thymidine uptake. The effect of oestradiol was not significantly reduced by alpha IR-3, indicating that IGF-I was not an autocrine mediator of oestradiol stimulation of cell growth under these conditions, whereas alpha IR-3 extensively reduced growth stimulation by IGF-I. On long term stimulation (5 days) oestradiol had a marked stimulatory effect on cell number and alpha IR-3 almost totally abrogated this effect. When oestradiol (1 nM) and IGF-I (2.5 nM) were added together, the combined effect on thymidine incorporation and cell number was significantly greater than additive. This synergistic effect on the IGF-I growth response was totally abolished by the IGF-I receptor antibody. The results suggest a cooperative interaction of oestradiol and IGF-I. It is concluded that growth stimulation of MCF-7 cells by long term treatment with oestradiol may be mediated through autocrine secretion of IGF-I. the effect of short term stimulation of thymidine incorporation suggest that the growth response of oestradiol is more complex, and indicate that a cooperative interaction with IGF-I is involved, which is unrelated to stimulated autocrine secretion.
J Steroid Biochem Mol Biol 1992 Mar
PMID:Oestradiol treatment increases the sensitivity of MCF-7 cells for the growth stimulatory effect of IGF-I. 156 24


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