Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 29
Filter
Add more filters










Publication year range
1.
Oncogene ; 32(33): 3829-39, 2013 Aug 15.
Article in English | MEDLINE | ID: mdl-22964639

ABSTRACT

We report on the role of hexamethylene-bis-acetamide-inducible protein 1 (HEXIM1) as an inhibitor of metastasis. HEXIM1 expression is decreased in human metastatic breast cancers when compared with matched primary breast tumors. Similarly we observed decreased expression of HEXIM1 in lung metastasis when compared with primary mammary tumors in a mouse model of metastatic breast cancer, the polyoma middle T antigen (PyMT) transgenic mouse. Re-expression of HEXIM1 (through transgene expression or localized delivery of a small molecule inducer of HEXIM1 expression, hexamethylene-bis-acetamide) in PyMT mice resulted in inhibition of metastasis to the lung. Our present studies indicate that HEXIM1 downregulation of HIF(-)1α protein allows not only for inhibition of vascular endothelial growth factor-regulated angiogenesis, but also for inhibition of compensatory pro-angiogenic pathways and recruitment of bone marrow-derived cells (BMDCs). Another novel finding is that HEXIM1 inhibits cell migration and invasion that can be partly attributed to decreased membrane localization of the 67 kDa laminin receptor, 67LR, and inhibition of the functional interaction of 67LR with laminin. Thus, HEXIM1 re-expression in breast cancer has therapeutic advantages by simultaneously targeting more than one pathway involved in angiogenesis and metastasis. Our results also support the potential for HEXIM1 to indirectly act on multiple cell types to suppress metastatic cancer.


Subject(s)
Mammary Neoplasms, Experimental/metabolism , Neovascularization, Pathologic/metabolism , Transcription Factors/metabolism , Animals , Blotting, Western , Chromatography, High Pressure Liquid , Female , Flow Cytometry , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Immunohistochemistry , Immunoprecipitation , Lung Neoplasms/metabolism , Lung Neoplasms/secondary , MCF-7 Cells , Mammary Neoplasms, Experimental/pathology , Mice , Mice, Transgenic , Neoplasm Invasiveness/pathology , Oligonucleotide Array Sequence Analysis , RNA-Binding Proteins , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Tandem Mass Spectrometry , Tissue Array Analysis , Two-Hybrid System Techniques
2.
Oncogene ; 30(47): 4731-9, 2011 Nov 24.
Article in English | MEDLINE | ID: mdl-21602889

ABSTRACT

We have previously reported that the expression of antioxidative stress enzymes is upregulated by trans-hydroxytamoxifen (TOT) in breast epithelial cell lines providing protection against estrogen-induced DNA damage. This regulation involves Estrogen Receptor ß (ERß) recruitment to the Electrophile Response Element (EpRE) and a novel protein, human homolog of Xenopus gene which Prevents Mitotic Catastrophe (hPMC2). We have also demonstrated that ERß and hPMC2 are required for TOT-dependent recruitment of poly (ADP-ribose) polymerase 1 (PARP-1) and Topoisomerase IIß (Topo IIß) to the EpRE. Sequence analysis reveals that the C-terminus of hPMC2 encodes a putative exonuclease domain. Using in vitro kinetic assays, we found that hPMC2 is a 3'-5' non-processive exonuclease that degrades both single-stranded and double-stranded substrates. Mutation of two conserved carboxylate residues drastically reduced the exonuclease activity of hPMC2, indicating the relative importance of the catalytic residues. Western blot analysis of breast cancer cell lines for Quinone Reductase (QR) levels revealed that the intrinsic exonuclease activity of hPMC2 was required for TOT-induced QR upregulation. Chromatin immunoprecipitation (ChIP) assays also indicated that hPMC2 was involved in the formation of strand breaks observed with TOT treatment and is specific for the EpRE-containing region of the QR gene. We also determined that the transcription factor NF-E2-related factor-2 (Nrf2) is involved in the specificity of hPMC2 for the EpRE. In addition, we determined that the catalytic activity of hPMC2 is required for repair of abasic sites that result from estrogen-induced DNA damage. Thus, our study provides a mechanistic basis for transcriptional regulation by hPMC2 and provides novel insights into its role in cancer prevention.


Subject(s)
Estrogens/pharmacology , Exonucleases/metabolism , Gene Expression Regulation, Enzymologic , NAD(P)H Dehydrogenase (Quinone)/genetics , Amino Acid Sequence , Catalysis , DNA Breaks , Gene Expression Regulation, Enzymologic/drug effects , Humans , Molecular Sequence Data , NF-E2-Related Factor 2/physiology , Response Elements/physiology , Tamoxifen/analogs & derivatives , Tamoxifen/pharmacology , Transcription, Genetic
3.
Oncogene ; 30(33): 3563-9, 2011 Aug 18.
Article in English | MEDLINE | ID: mdl-21423213

ABSTRACT

Tamoxifen resistance is a major problem in the treatment of estrogen receptor (ER)-positive patients. We have previously reported that hexamethylene bis-acetamide-inducible protein 1 (HEXIM1) inhibits ERα activity by competing with ERα for binding to cyclin T1, a subunit of positive transcription elongation b (P-TEFb). This results in the inhibition of the phosphorylation of RNA polymerase II (RNAPII) at serine 2 and the inhibition of transcription elongation of ERα target genes. As HEXIM1 can inhibit ER activity, we examined whether it has a critical role in the inhibitory effects of tamoxifen on ER. We observed that tamoxifen-induced HEXIM1 recruitment to the promoter region of ER target genes and decreased the recruitment of cyclin T1 and serine 2 phosphorylated RNAPII to the coding regions of these genes. Conversely, in cells wherein HEXIM1 expression has been downregulated we observed attenuation of the inhibitory effects of tamoxifen on estrogen-induced cyclin T1 recruitment to coding regions of ER target genes. As a consequence, downregulation of HEXIM1 resulted in the attenuation of the repressive effects of tamoxifen on estrogen-induced gene expression and proliferation. Conferring clinical relevance to our studies is our analysis of human breast cancer tissue samples that indicated association of lower expression of HEXIM1 with tumor recurrence in patients who received tamoxifen. Our studies provide a better understanding of the mechanistic basis for the inhibitory effect of tamoxifen on ER activity and may suggest new therapeutic targets for the treatment of tamoxifen-resistant breast cancer.


Subject(s)
Antineoplastic Agents, Hormonal/pharmacology , Breast Neoplasms/drug therapy , Estrogen Antagonists/pharmacology , RNA-Binding Proteins/physiology , Tamoxifen/pharmacology , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin T/metabolism , Estrogen Receptor alpha/antagonists & inhibitors , Estrogen Receptor alpha/physiology , Female , Humans , Neoplasm Recurrence, Local/prevention & control , Phosphorylation , RNA Polymerase II/metabolism , Transcription Factors , Transcriptional Elongation Factors/metabolism
4.
Oncogene ; 29(25): 3639-49, 2010 Jun 24.
Article in English | MEDLINE | ID: mdl-20453883

ABSTRACT

Recently, we found that mutation of the C-terminus of transcription factor hexamethylene bisacetamide-inducible protein 1 (HEXIM1) in mice leads to abnormalities in cardiovascular development because of aberrant vascular endothelial growth factor (VEGF) expression. HEXIM1 regulation of some genes has also been shown to be positive transcription elongation factor b (P-TEFb) dependent. However, it is not known whether HEXIM1 regulates VEGF in the mammary gland. We demonstrate that HEXIM1 regulates estrogen-induced VEGF transcription through inhibition of estrogen receptor-alpha recruitment to the VEGF promoter in a P-TEFb-independent manner in MCF-7 cells. Under hypoxic conditions, HEXIM1 inhibits estrogen-induced hypoxia-inducible factor-1 alpha (HIF-1alpha) protein expression and recruitment of HIF-1alpha to the hypoxia-response element in the VEGF promoter. In the mouse mammary gland, increased HEXIM1 expression decreased estrogen-driven VEGF and HIF-1alpha expression. Conversely, a mutation in the C-terminus of HEXIM1 (HEXIM1(1-312)) led to increased VEGF and HIF-1alpha expression and vascularization in mammary glands of heterozygous HEXIM1(1-312) mice when compared with their wild-type littermates. In addition, HEXIM1(1-312) mice have a higher incidence of carcinogen-induced mammary tumors with increased vascularization, suggesting an inhibitory role for HEXIM1 during angiogenesis. Taken together, our data provide evidence to suggest a novel role for HEXIM1 in cancer progression.


Subject(s)
Epithelial Cells/metabolism , Gene Expression Regulation , Mammary Glands, Animal/metabolism , RNA-Binding Proteins/metabolism , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Animals , Carcinogens/toxicity , Cell Hypoxia/drug effects , Cell Line, Tumor , Epithelial Cells/drug effects , Epithelial Cells/pathology , Estradiol/pharmacology , Estrogen Receptor alpha/metabolism , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mammary Glands, Animal/blood supply , Mammary Glands, Animal/drug effects , Mammary Glands, Animal/pathology , Mammary Neoplasms, Experimental/chemically induced , Mammary Neoplasms, Experimental/metabolism , Mammary Neoplasms, Experimental/pathology , Mammary Neoplasms, Experimental/physiopathology , Mice , Mutation , Neovascularization, Pathologic/metabolism , Neovascularization, Physiologic , Positive Transcriptional Elongation Factor B/metabolism , Promoter Regions, Genetic/genetics , RNA-Binding Proteins/chemistry , RNA-Binding Proteins/genetics , Response Elements , Transcription Factors
5.
Oncogene ; 27(49): 6376-84, 2008 Oct 23.
Article in English | MEDLINE | ID: mdl-18663360

ABSTRACT

In the presence of ERbeta, trans-hydroxytamoxifen (TOT) protects cells against 17beta-estradiol (E(2))-induced oxidative DNA damage (ODD) and this correlates with increased expression of the antioxidative enzyme quinone reductase (QR). Here, we investigate the molecular mechanism responsible for ERbeta-mediated protection against ODD. We observe constitutive interaction between ERbeta and the novel protein hPMC2. Using a combination of breast epithelial cell lines that are either positive or negative for ERalpha, we demonstrate TOT-dependent recruitment of both ERbeta and hPMC2 to the EpRE (electrophile response element)-regulated antioxidative enzyme QR. We further demonstrate TOT-dependent corecruitment of the coactivators Nrf2, PARP-1 (poly (ADP-ribose) polymerase 1) and topoisomerase IIbeta, both in the presence and absence of ERalpha. However, absence of either ERbeta or hPMC2 results in nonrecruitment of PARP-1 and topoisomerase IIbeta, loss of antioxidative enzyme induction and attenuated protection against ODD by TOT even in the presence of Nrf2 and ERalpha. These findings indicate minor role for Nrf2 and ERalpha in TOT-dependent antioxidative gene regulation. However, downregulation of PARP-1 attenuates TOT-dependent antioxidative gene induction. We conclude that ERbeta and hPMC2 are required for TOT-dependent recruitment of coactivators such as PARP-1 to the EpRE resulting in the induction of antioxidative enzymes and subsequent protection against ODD.


Subject(s)
DNA Damage , Estrogen Receptor beta/metabolism , NAD(P)H Dehydrogenase (Quinone)/metabolism , Trans-Activators/metabolism , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cell Line, Tumor , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Estrogen Receptor beta/genetics , Female , Gene Expression Regulation, Enzymologic/drug effects , Humans , NAD(P)H Dehydrogenase (Quinone)/genetics , NF-E2-Related Factor 2/metabolism , Oxidative Stress/drug effects , Oxidative Stress/genetics , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerases/metabolism , Response Elements/drug effects , Retroviridae/genetics , Tamoxifen/analogs & derivatives , Tamoxifen/pharmacology , Trans-Activators/genetics , Transcription, Genetic/drug effects , Transfection , Up-Regulation/drug effects
6.
Oncogene ; 26(24): 3587-90, 2007 May 24.
Article in English | MEDLINE | ID: mdl-17160017

ABSTRACT

We previously reported that antiestrogen-liganded estrogen receptor beta (ERbeta) transcriptionally activates the major detoxifying enzyme quinone reductase (QR) (NAD(P)H:quinone oxidoreductase). Further studies on the functional role of ERbeta-mediated upregulation of antioxidative enzymes indicated protective effects against estrogen-induced oxidative DNA damage (ODD). We now report on in vivo and in vitro studies that show that ERbeta-mediated upregulation of QR are involved in the protection against estrogen-induced mammary tumorigenesis. Using the August Copenhagen Irish (ACI) model of estrogen-induced carcinogenesis, we observed that increased ODD and decreased QR expression occur early in the process of estrogen-induced mammary tumorigenesis. Prevention of ACI mammary gland tumorigenesis by tamoxifen was accompanied by decreased ODD and increased QR levels. These correlative findings were supported by our findings that downregulation of QR levels led to increased levels of estrogen quinone metabolites and enhanced transformation potential of 17beta-estradiol treated MCF10A non-tumorigenic breast epithelial cells. Concurrent expression of ERbeta and treatment with 4-hydroxytamoxifen decreased tumorigenic potential of these MCF10A cells. We conclude that upregulation of QR, through induction by tamoxifen, can inhibit estrogen-induced ODD and mammary cell tumorigenesis, representing a possible novel mechanism of tamoxifen prevention against breast cancer.


Subject(s)
Breast Neoplasms/pathology , Breast Neoplasms/prevention & control , Estrogen Antagonists/pharmacology , Estrogens/pharmacology , NAD(P)H Dehydrogenase (Quinone)/physiology , Tamoxifen/pharmacology , 8-Hydroxy-2'-Deoxyguanosine , Animals , Breast Neoplasms/chemically induced , Cell Transformation, Neoplastic , DNA Damage/drug effects , Deoxyguanosine/analogs & derivatives , Deoxyguanosine/metabolism , Estrogen Receptor beta/drug effects , Estrogen Receptor beta/metabolism , Humans , Mammary Glands, Animal/pathology , Oxidative Stress , Rats , Tamoxifen/analogs & derivatives , Tumor Cells, Cultured
7.
Thorax ; 59(7): 586-90, 2004 Jul.
Article in English | MEDLINE | ID: mdl-15223866

ABSTRACT

BACKGROUND: Tissue inhibitors of metalloproteinases (TIMPs) play a major role in extracellular matrix turnover in the lung. However, in chronic lung disorders such as idiopathic pulmonary fibrosis (IPF) and pigeon breeders' disease (PBD), TIMPs may promote an adverse non-degradative environment. We hypothesised that polymorphisms in TIMP-3 could affect susceptibility to IPF and PBD. METHODS: Two promoter variants, -915A>G and -1296T>C, were genotyped in 323 healthy subjects, 94 subjects with IPF, 115 with PBD, and 90 exposed to avian antigen but without PBD. The severity of fibrosis in lung tissue and the clinical outcome after 1 year was determined in the PBD group. RESULTS: The variants did not influence susceptibility to IPF, but the rare alleles of both variants appeared to be protective against susceptibility to PBD (odds ratio (OR) for carriage of at least one rare allele from either variant 0.48, 95% CI 0.30 to 0.76, p = 0.002). Haplotype analysis of positions -915 and -1296 estimated four haplotypes: *A*T, *G*T, *A*C and *G*C, respectively. Their frequencies differed overall between subjects with PBD and healthy subjects (p = 0.0049) and this was attributable primarily to the *G*C haplotype (OR 0.53, 95% CI 0.36 to 0.77, p = 0.001). The severity of fibrosis correlated with poorer outcome in the PBD group (r = 0.73, p<0.01) but no relationship was seen between the *G*C haplotype and outcome or fibrosis. However, PBD subjects with the *G*C haplotype did have proportionally fewer lymphocytes in their bronchoalveolar fluid than those with the common *A*T haplotype (p = 0.029). CONCLUSIONS: TIMP-3 variants appear to contribute to susceptibility to PBD. This may be through the inflammatory reaction rather than the fibrotic reaction.


Subject(s)
Bird Fancier's Lung/genetics , Promoter Regions, Genetic , Tissue Inhibitor of Metalloproteinase-3/genetics , Adult , Bronchoalveolar Lavage Fluid/cytology , Female , Forced Expiratory Volume/physiology , Genetic Predisposition to Disease , Genotype , Humans , Macrophages/pathology , Male , Middle Aged , Prognosis , Pulmonary Fibrosis/genetics , Vital Capacity/physiology
9.
Recent Prog Horm Res ; 55: 163-93; discussion 194-5, 2000.
Article in English | MEDLINE | ID: mdl-11036937

ABSTRACT

The action of nuclear hormone receptors is tripartite, involving the receptor, its ligands, and its co-regulator proteins. The estrogen receptor (ER), a member of this superfamily, is a hormone-regulated transcription factor that mediates the effects of estrogens and anti-estrogens (e.g., tamoxifen) in breast cancer and other estrogen target cells. This chapter presents our recent work on several aspects of estrogen action and the function of the ER: 1) elucidation of ER structure-function relationships and development of ligands that are selective for one of the two ER subtypes, ERalpha or ERbeta; 2) identification of ER-selective co-regulators that potentiate the inhibitory effectiveness of anti-estrogens and dominant-negative ERs and modulate the activity of estrogens; 3) characterization of genes that are regulated by the anti-estrogen-ER versus the estrogen-ER complex; and 4) elucidation of the intriguing pharmacology of these ER complexes at different gene regulatory sites. These findings indicate that different residues of the ER hormone-binding domain are involved in the recognition of structurally distinct estrogens and anti-estrogens and highlight the exquisite precision of the regulation of ER activities by ligands, with small changes in ligand structure resulting in major changes in receptor character. Studies also explore the biology and distinct pharmacology mediated by ERalpha and ERbeta complexed with different ligands through different target genes. The upregulation of the anti-oxidant detoxifying phase II enzyme, quinone reductase, by the anti-estrogen-occupied ER, mediated via the electrophile response element in the QR gene, may contribute to the beneficial antioxidant effects of anti-estrogens in breast cancer and illustrates the activation of some genes by ER via non-estrogen response element sequences. The intriguing biology of estrogen in its diverse target cells is thus determined by the structure of the ligand, the ER subtype involved, the nature of the hormone-responsive gene promoter, and the character and balance of co-activators and co-repressors that modulate the cellular response to the ER-ligand complex. The continuing development of novel ligands and the study of how they function as selective agonists or antagonists through ERalpha or ERbeta should allow optimized tissue selectivity of these agents for hormone replacement therapy and treatment and prevention of breast cancer.


Subject(s)
Receptors, Estrogen/metabolism , Amino Acid Sequence , Animals , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Estrogens/chemistry , Estrogens/metabolism , Estrogens/pharmacology , Female , Humans , Ligands , Models, Biological , Models, Molecular , Molecular Sequence Data , Prohibitins , Receptors, Estrogen/chemistry , Receptors, Estrogen/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism
10.
J Biol Chem ; 275(44): 34306-13, 2000 Nov 03.
Article in English | MEDLINE | ID: mdl-10908561

ABSTRACT

The regulation of the quinone reductase (QR) gene as well as other genes involved in detoxification is known to be mediated by an electrophile/antioxidant response element (EpRE/ARE). We have previously observed that QR is up-regulated by the antiestrogen trans-hydroxytamoxifen in breast cancer cells. QR gene regulation by the antiestrogen-occupied estrogen receptor (ER) is mediated by the EpRE-containing region of the human QR gene, and the ER is one of the complex of proteins that binds to the EpRE. In an effort to further understand the mechanism for ER regulation of QR gene we identified other protein factors that regulate QR gene transcriptional activity in breast cancer cells. One of these protein factors, hPMC2 (human homolog of Xenopus gene which prevents mitotic catastrophe), directly binds to the EpRE and interacts with the ER in yeast genetic screening and in vitro assays. Interestingly hPMC2 interacts more strongly to ER beta when compared with ER alpha. In transient transfection assays using reporter constructs containing the EpRE, hPMC2 alone can slightly activate reporter in ER-negative MDA-MB-231 breast cancer cells. The activation of QR gene activity by hPMC2 is enhanced in the presence of ER beta.


Subject(s)
Cell Cycle Proteins/physiology , Gene Expression Regulation, Enzymologic , NAD(P)H Dehydrogenase (Quinone)/genetics , Transcription, Genetic , Animals , Base Sequence , DNA Primers , Estrogen Receptor Modulators/metabolism , Humans , Receptors, Estrogen/metabolism , Saccharomyces cerevisiae/genetics , Xenopus
11.
Proc Natl Acad Sci U S A ; 96(12): 6947-52, 1999 Jun 08.
Article in English | MEDLINE | ID: mdl-10359819

ABSTRACT

The action of nuclear hormone receptors is tripartite, involving the receptor, its ligands, and its coregulator proteins. The estrogen receptor (ER), a member of this superfamily, is a hormone-activated transcription factor that mediates the stimulatory effects of estrogens and the inhibitory effects of antiestrogens such as tamoxifen in breast cancer and other estrogen target cells. To understand how antiestrogens and dominant negative ERs suppress ER activity, we used a dominant negative ER as bait in two-hybrid screening assays from which we isolated a clone from breast cancer cells that potentiates the inhibitory activities of dominant negative ERs and antiestrogen-liganded ER. At higher concentrations, it also represses the transcriptional activity of the estradiol-liganded ER, while having no effect on other nuclear hormone receptors. This clone, denoted REA for "repressor of estrogen receptor activity," encodes a 37-kDa protein that is an ER-selective coregulator. Its competitive reversal of steroid receptor coactivator 1 enhancement of ER activity and its direct interaction with liganded ER suggest that it may play an important role in determining the sensitivity of estrogen target cells, including breast cancer cells, to antiestrogens and estrogens.


Subject(s)
Estrogens/metabolism , Hormone Antagonists/pharmacology , Receptors, Estrogen/antagonists & inhibitors , Receptors, Estrogen/metabolism , Amino Acid Sequence , Animals , Base Sequence , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , CHO Cells , Cricetinae , Estrogens/genetics , Female , Humans , Molecular Sequence Data , Prohibitins , Receptors, Estrogen/agonists , Receptors, Estrogen/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism
12.
J Biol Chem ; 273(39): 25443-9, 1998 Sep 25.
Article in English | MEDLINE | ID: mdl-9738013

ABSTRACT

We have previously reported that antiestrogens stimulate quinone reductase (NAD(P)H:(quinone-acceptor) oxidoreductase (QR or NQO1); EC 1.6.99.2) enzymatic activity, an action that may provide protective effects against the toxicity and mutagenicity caused by quinones. We have now investigated the transcriptional regulation of the QR gene by antiestrogens. In transfection experiments employing the 5'-flanking (863-base pair) region of the human QR gene promoter with its electrophile/antioxidant response element (EpRE/ARE) or deleted or mutated constructs, we observe that antiestrogens induced an increase in QR gene promoter reporter activity in estrogen receptor (ER) negative breast cancer and endometrial cancer cells transfected with ER, and this induction by antiestrogens was repressed by estradiol. The stimulation of QR transcriptional activity required the 31-base pair electrophile-responsive region from the human QR gene promoter and a functional ER. Intriguingly, antiestrogens were stronger activators of the QR EpRE via the ER subtype ERbeta than ERalpha. Oligonucleotide gel mobility and antibody shift assays reveal that the ER binds to the EpRE but is only a minor component of the proteins bound to the EpRE in ER-containing MCF-7 breast cancer cells. While binding of ERbeta to the estrogen response element was weaker when compared with ERalpha, ERbeta and ERalpha showed similar binding to the EpRE. Together these findings provide evidence that QR gene regulation by the antiestrogen-occupied ER is mediated by the EpRE-containing region of the human QR gene and indicate that the ER is one of the complex of proteins that binds to the EpRE. In addition, that ERbeta is a more potent activator at EpRE elements than is ERalpha suggests that the different levels of these two receptors in various estrogen target cells could impact importantly on the antioxidant potency of antiestrogens in different target cells. These findings have broad implications regarding the potential beneficial effects of antiestrogens since EpREs mediate the transcriptional induction of numerous genes, including QR, which encode chemoprotective detoxification enzymes.


Subject(s)
Estrogen Antagonists/pharmacology , Gene Expression Regulation, Enzymologic/drug effects , NAD(P)H Dehydrogenase (Quinone)/genetics , Receptors, Estrogen/drug effects , Transcription, Genetic/drug effects , Base Sequence , Breast Neoplasms/enzymology , Breast Neoplasms/genetics , DNA Primers , Estrogen Receptor alpha , Estrogen Receptor beta , Humans , Nuclear Proteins/metabolism , RNA, Messenger/genetics , Receptors, Estrogen/metabolism , Tumor Cells, Cultured
13.
Breast Cancer Res Treat ; 44(1): 23-38, 1997 May.
Article in English | MEDLINE | ID: mdl-9164675

ABSTRACT

Antiestrogens have proven to be highly effective in the treatment of hormone-responsive breast cancer. However, resistance to antiestrogen therapy often develops. In addition, although tamoxifen-like antiestrogens are largely inhibitory and function as estrogen antagonists in breast cancer cells, they also have some estrogen-like activity in other cells of the body. Thus, recent efforts are being directed toward the development of even more tissue-selective antiestrogens, i.e. compounds that are antiestrogenic on breast and uterus while maintaining the beneficial estrogen-like actions on bone and the cardiovascular system. Efforts are also being directed toward understanding ligand structure-estrogen receptor (ER) activity relationships and characterizing the molecular changes that underlie alterations in parallel signal transduction pathways that impact on the ER. Recent findings show that antiestrogens, which are known to exert most of their effects through the ER of breast cancer cells, contact a different set of amino acids in the hormone binding domain of the ER than those contacted by estrogen, and evoke a different receptor conformation that results in reduced or no transcriptional activity on most genes. Resistance to antiestrogen therapy may develop due to changes at the level of the ER itself, and at pre- and post-receptor points in the estrogen receptor-response pathway. Resistance could arise in at least four ways: (1) ER loss or mutation; (2) Post-receptor alterations including changes in cAMP and phosphorylation pathways, or changes in coregulator and transcription factor interactions that affect the transcriptional activity of the ER; (3) Changes in growth factor production/sensitivity or paracrine cell-cell interactions; or (4) Pharmacological changes in the antiestrogen itself, including altered uptake and retention or metabolism of the antiestrogen. Model cell systems have been developed to study changes that accompany and define the antiestrogen resistant versus sensitive breast cancer phenotype. This information should lead to the development of antiestrogens with optimized tissue selectivity and agents to which resistance may develop more slowly. In addition, antiestrogens which work through somewhat different mechanisms of interaction with the ER should prove useful in treatment of some breast cancers that become resistant to a different category of antiestrogens.


Subject(s)
Breast Neoplasms/drug therapy , Estrogen Antagonists/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Drug Resistance , Estrogen Antagonists/therapeutic use , Female , Humans
14.
Proc Natl Acad Sci U S A ; 94(5): 2056-61, 1997 Mar 04.
Article in English | MEDLINE | ID: mdl-9050904

ABSTRACT

On the basis of results of studies using high doses of estrogens, exposure to estrogen during fetal life is known to inhibit prostate development. However, it is recognized in endocrinology that low concentrations of a hormone can stimulate a tissue, while high concentrations can have the opposite effect. We report here that a 50% increase in free-serum estradiol in male mouse fetuses (released by a maternal Silastic estradiol implant) induced a 40% increase in the number of developing prostatic glands during fetal life; subsequently, in adulthood, the number of prostatic androgen receptors per cell was permanently increased by 2-fold, and the prostate was enlarged by 30% (due to hyperplasia) relative to untreated males. However, as the free serum estradiol concentration in male fetuses was increased from 2- to 8-fold, adult prostate weight decreased relative to males exposed to the 50% increase in estradiol. As a model for fetal exposure to man-made estrogens, pregnant mice were fed diethylstilbestrol (DES) from gestation days 11 to 17. Relative to controls, DES doses of 0.02, 0.2, and 2.0 ng per g of body weight per day increased adult prostate weight, whereas a 200-ng-per-g dose decreased adult prostate weight in male offspring. Our findings suggest that a small increase in estrogen may modulate the action of androgen in regulating prostate differentiation, resulting in a permanent increase in prostatic androgen receptors and prostate size. For both estradiol and DES, prostate weight first increased then decreased with dose, resulting in an inverted-U dose-response relationship.


Subject(s)
Diethylstilbestrol/pharmacology , Estradiol/pharmacology , Fetus/metabolism , Prenatal Exposure Delayed Effects , Prostate/drug effects , Animals , Cell Differentiation/drug effects , Diethylstilbestrol/administration & dosage , Dose-Response Relationship, Drug , Drug Implants , Estradiol/administration & dosage , Estradiol/blood , Female , Image Processing, Computer-Assisted , Male , Mice , Mice, Inbred Strains , Organ Size/drug effects , Pregnancy , Prostate/embryology , Prostate/metabolism , Prostatic Hyperplasia/chemically induced , Receptors, Androgen/metabolism
15.
Mol Endocrinol ; 11(3): 330-41, 1997 Mar.
Article in English | MEDLINE | ID: mdl-9058379

ABSTRACT

The estrogen receptor (ER) is a ligand-regulated transcription factor that acts at the promoters of estrogen-regulated genes to modulate their expression. In the present study, we examined three estrogen-regulated promoters, namely the rat progesterone receptor gene distal (PRD) and proximal (PRP) promoters and the human pS2 gene promoter, and observed marked differences in their sensitivity to stimulation by estrogen and repression of estrogen-stimulated transcription by antiestrogen (AE)-occupied ER. ER-containing MCF-7 human breast cancer cells were transfected with reporter gene constructs containing estrogen response elements upstream of the three gene promoters. In this system, PRP and PRD showed similar dose-response curves for stimulation by estradiol whereas pS2 was activated by even lower concentrations of estradiol. By contrast, PRD was much less sensitive to repression of estrogen-stimulated activity by all AEs studied, relative to the PRP and the pS2 promoters. Using deletion and mutational analysis, we have identified a transferable cis element at -131 to -94 bp in PRD that is involved in modulating the sensitivity of this promoter to both estrogens and AEs. The element reduced the magnitude of estrogen-stimulated activity, enhanced the ability of AEs to repress estrogen-stimulated activity, and elicited similiar effects when transferred to the promoter of another estrogen-responsive gene. Thus, removal of this region from PRD further accentuated the insensitivity of this promoter to AE while enhancing its sensitivity (both EC50 and fold induction) to estrogen. Gel mobility shift assays showed that proteins from nuclear extracts of MCF-7 cells interact with this element and that the binding of these proteins is inversely correlated with the transcriptional effectiveness of the ER. The findings demonstrate that a specific cis element from the promoter of an estrogen-responsive gene can alter the transcriptional activity of hormone and antihormone-occupied receptor bound at its response element near the promoter. Such ligand response modulatory elements, and changes in the levels and activity of factors that bind to such elements, may underlie the different sensitivities of steroid hormone-regulated genes to both hormones and antihormones.


Subject(s)
Carrier Proteins/antagonists & inhibitors , Estrogen Antagonists/pharmacology , Estrogens/pharmacology , Neoplasm Proteins/antagonists & inhibitors , Promoter Regions, Genetic , Proteins , Receptors, Estrogen , Animals , Carrier Proteins/genetics , Chloramphenicol O-Acetyltransferase/genetics , Chloramphenicol O-Acetyltransferase/metabolism , DNA Mutational Analysis , Dose-Response Relationship, Drug , Gene Expression Regulation/drug effects , Genes, Reporter/genetics , Humans , Neoplasm Proteins/genetics , Rats , Transfection , Trefoil Factor-1 , Tumor Suppressor Proteins , beta-Galactosidase/metabolism
16.
Proc Natl Acad Sci U S A ; 94(6): 2581-6, 1997 Mar 18.
Article in English | MEDLINE | ID: mdl-9122238

ABSTRACT

Antiestrogens are thought to exert most of their beneficial effects in breast cancer by antagonizing the actions of estrogen. We report here that antiestrogens also stimulate the expression of quinone reductase (QR) [NAD(P)H:quinone oxidoreductase, EC 1.6.99.2], which may provide protective effects against the toxicity and mutagenicity caused by quinones. QR is up-regulated by low concentrations of antiestrogens (trans-hydroxytamoxifen, tamoxifen, and ICI182,780) in estrogen receptor (ER)-containing breast cancer cells, and this increase is suppressed by estrogen via an ER-dependent mechanism. Since regulation of the QR gene, as well as other genes involved in detoxification such as the glutathione S-transferase Ya subunit (GST Ya) gene, is known to be mediated by an electrophile/antioxidant response element (EpRE/ARE), we examined the effects of antiestrogens on a 41-bp electrophile responsive region derived from the GST Ya gene. Transfection of this EpRE-containing region into ER-negative breast cancer cells in the presence or absence of an expression vector for the human ER, as well as mutagenesis studies, revealed that the EpRE-containing construct was activated by antiestrogen to the same extent as by tert-butylhydroquinone (TBHQ), a known activator of EpREs; however, only the stimulation by antiestrogen, and not TBHQ, required ER and was repressed by estradiol, although activation by both inducers mapped to the same 10-bp EpRE consensus sequence. Thus, there appear to be two pathways for QR induction, one that is activated by electrophile inducers such as TBHQ and is ER independent, and a second that is antiestrogen regulated and ER dependent; both pathways act through the EpRE. The anticancer action of antiestrogens may thus derive not only from the already well-known repression of estrogen-stimulated activities but also from the activation of detoxifying enzymes, such as QR, that may contribute to the beneficial antioxidant activity of antiestrogens.


Subject(s)
Estrogen Antagonists/pharmacology , Gene Expression Regulation, Enzymologic/physiology , NAD(P)H Dehydrogenase (Quinone)/biosynthesis , Receptors, Estrogen/metabolism , Animals , Base Sequence , Binding Sites , Breast Neoplasms , Cell Line , DNA-Binding Proteins/metabolism , Estradiol/analogs & derivatives , Estradiol/pharmacology , Female , Fulvestrant , Gene Expression Regulation, Enzymologic/drug effects , Genes, Reporter , Genetic Vectors , Glutathione Transferase/biosynthesis , Growth Hormone/biosynthesis , Growth Hormone/genetics , Humans , Kinetics , Mice , Molecular Sequence Data , Oligodeoxyribonucleotides , Promoter Regions, Genetic , Receptors, Estrogen/biosynthesis , Recombinant Proteins/biosynthesis , Recombinant Proteins/metabolism , Tamoxifen/analogs & derivatives , Tamoxifen/pharmacology , Transfection
17.
Endocr Res ; 23(1-2): 37-57, 1997.
Article in English | MEDLINE | ID: mdl-9187537

ABSTRACT

We examined the effect of glucocorticoids on the proliferation and differentiation of skeletal muscle cells using the C2C12 cell line. We found that treatment with glucocorticoids enhanced muscle cell differentiation but had only minor effects on the clonal growth rate of C2C12 cells. The stimulatory effect of glucocorticoids on myogenic differentiation was reflected in the increased expression of muscle-specific genes, creatine kinase (CK) and acetylcholine receptor gamma subunit (AChR). Dexamethasone had no effect on CK and AChR mRNA stability and enhanced transcription from a CAT reporter genes containing the 3.3kb 5' flanking region of the murine CK gene (-3300MCK-CAT). Since dexamethasone did not affect the expression levels of the myogenic regulatory genes such as myoD and myogenin, the enhancement of muscle-specific transcription might reflect an increase in the functional activity of the regulatory proteins. Other possible mechanisms involved in the differentiation-enhancing effect of glucocorticoids are discussed.


Subject(s)
Dexamethasone/pharmacology , Genes, Regulator , Glucocorticoids/pharmacology , Muscle, Skeletal/drug effects , Transcription, Genetic/drug effects , Cell Differentiation/drug effects , Cell Division/drug effects , Cell Line , Clone Cells/drug effects , Creatine Kinase/genetics , Muscle, Skeletal/cytology , MyoD Protein/genetics , Myogenin/genetics , Receptors, Cholinergic/genetics
18.
Mol Endocrinol ; 10(3): 230-42, 1996 Mar.
Article in English | MEDLINE | ID: mdl-8833652

ABSTRACT

The estrogen receptor (ER) is a transcription factor whose activity is normally activated by the hormone estradiol and inhibited by antiestrogen. It has been found that certain mutational changes in the activation function-2 region in the hormone-binding domain of the human ER result in ligand activity inversion mutants, i.e. receptors that are now activated by antiestrogen and inhibited by estrogen. The ER point mutant L540Q is activated by several antiestrogens (the more pure antiestrogens ICI 164,384 and RU 54,876 or the partial antiestrogen trans-hydroxytamoxifen) but not by estradiol. The presence of the F domain and an intact activation function-i in the A/B domain are required for this activity, as is the DNA-binding ability of the receptor. This inverted ligand activity is observed with several estrogen-responsive promoters, both simple and complex; however, the activating ability of antiestrogens is observed only in some cells, highlighting the important role of cell-specific factors in ligand interpretation. The introduction of two additional amino acid changes close to 540 results in receptors that are still not activated by estradiol but are now able to distinguish between partial antiestrogens (which remain agonistic) and pure antiestrogens (which show a greatly reduced stimulatory activity). These ligand activity inversion mutants remain stable in cells in the presence of the antiestrogen ICI 164,384, as does a related ER mutant receptor that shows the normal, wild type ER ligand activity profile in which ICI 164,384 is transcriptionally inactive. Thus, the presence of adequate levels of mutant ER may be necessary but not sufficient for ICI 164,384 to elicit transcriptional activity. These findings highlight the means by which the carboxyl-terminal region in domain E functions to interpret the activity of a ligand, and they demonstrate that rather minimal changes in the ER can result in receptors with inverted response to antiestrogen and estrogen. Such point mutations, if present in estrogen target cells, would result in antiestrogens being seen as growth stimulators, rather than suppressors, with potentially detrimental consequences in terms of breast cancer treatment with antiestrogens.


Subject(s)
Estrogen Antagonists/pharmacology , Estrogens/pharmacology , Receptors, Estrogen/genetics , 3T3 Cells/drug effects , Animals , Breast Neoplasms/pathology , CHO Cells/drug effects , Cricetinae , Cricetulus , Estradiol/analogs & derivatives , Estradiol/metabolism , Estradiol/pharmacology , Estrogen Antagonists/classification , Estrogen Antagonists/metabolism , Estrogens/metabolism , Female , Humans , Ligands , Mice , Neoplasms, Hormone-Dependent/pathology , Point Mutation , Polyunsaturated Alkamides , Promoter Regions, Genetic , Protein Binding , Receptors, Estrogen/drug effects , Receptors, Estrogen/metabolism , Recombinant Fusion Proteins/biosynthesis , Structure-Activity Relationship , Substrate Specificity , Tamoxifen/analogs & derivatives , Tamoxifen/metabolism , Tamoxifen/pharmacology , Transcription, Genetic , Tumor Cells, Cultured/drug effects
19.
Biol Reprod ; 53(5): 1198-207, 1995 Nov.
Article in English | MEDLINE | ID: mdl-8527526

ABSTRACT

Circulating estradiol is assumed not to contribute to sexual differentiation of the brain or other estrogen target tissues. The only estradiol available for binding to estrogen receptors is thought to be produced within brain cells by the aromatization of testosterone to estradiol as part of the action of androgen in the brain. However, we report that the concentration of free, biologically active serum estradiol (the concentration not bound to plasma proteins) was 0.54-2.17 pg/ml during the fetal and early neonatal period of sexual differentiation. These values were within the same concentration range for free estradiol observed in adult female rats throughout the estrous cycle (diestrus = 0.53 pg/ml; proestrus = 2.26 pg/ml), and estradiol clearly has physiological effects during diestrus as well as proestrus in adult females. When a stable, physiological blood concentration of [3H]estradiol of 49 pg/ml total (0.61 pg/ml free) was achieved with Silastic capsules in 2-day-old female pups, [3H]estradiol was recovered specifically bound to brain cell nuclei at approximately 2.7 fmol per pup brain or 12.4 fmol/mg DNA. The finding of brain uptake of circulating estradiol is contrary to current hypotheses. These findings suggest that estradiol in the fetal and neonatal circulation may be able to interact with testosterone and its metabolites to regulate sexual differentiation of the brain and other estrogen target tissues.


Subject(s)
Animals, Newborn/physiology , Brain/metabolism , Estradiol/blood , Estradiol/metabolism , Fetus/physiology , Sex Differentiation , Animals , Brain/embryology , Brain/growth & development , Diethylstilbestrol/administration & dosage , Diethylstilbestrol/pharmacology , Drug Implants , Estradiol/administration & dosage , Estriol/metabolism , Estrone/metabolism , Female , Kinetics , Rats , Rats, Sprague-Dawley , Receptors, Estrogen/metabolism
20.
Mol Endocrinol ; 9(7): 814-25, 1995 Jul.
Article in English | MEDLINE | ID: mdl-7476965

ABSTRACT

Of the steroid hormone receptor family members, the estrogen receptor (ER) is notable in containing a sizable (42-amino acid) C-terminal region, denoted domain F. This F region differs from its adjacent hormone-binding domain, domain E, in that it is not well conserved among different vertebrate ER species, and its role in the biological activity of the ER is not well defined. We report an important role for the F domain of the ER in modulating the magnitude of gene transcription by estrogen and antiestrogen, and in determining the effectiveness of antiestrogens in suppressing estrogen-stimulated gene transcription. Using transient transfections, we have examined, in several cell types, the transcriptional activity of the full-length wild type human ER and ER lacking the carboxy-terminal F domain (delta F ER, containing amino acids 1-554) or ER altered in the F domain by point mutations. In some cells, namely Chinese hamster ovary (CHO) cells and MDA-MB-231 human breast cancer cells expressing wild type ER or delta F ER, estradiol (E2) stimulates equally transcription of several estrogen-responsive promoter-reporter gene constructs [estrogen ca-18119 element, (ERE)2-TATA-CAT, (ERE)2-pS2-CAT, (ERE)2-progesterone receptor(distal)-CAT]; however, the antiestrogens trans-hydroxytamoxifen and ICI 164,384, which stimulate transcription of some of these reporter constructs with the wild type ER, were unable to stimulate transcription with delta F ER. In addition, these antiestrogens were more effective antagonists of E2-stimulated transcription by delta F ER than by wild type ER. By contrast, in HeLa human cervical cancer cells and 3T3 mouse fibroblast cells, the delta F ER exposed to E2 is much less effective than wild type ER in stimulating transcription, and antiestrogens were less potent in suppressing E2-stimulated transcription by the delta F ER. These differences in response of the delta F and wild type ER to estrogen or antiestrogen do not appear to be due to a change in receptor expression level, binding affinity for ligands, or binding to estrogen response element DNA. Our data support the supposition that the conformation of the receptor-ligand complex is different with estrogen vs. antiestrogen and with wild type vs. delta F ER, such that its potential for interaction with protein cofactors or transcription factors is different and is markedly influenced by cell context. Thus, the F domain of the ER has a specific modulatory function that affects the agonist/antagonist effectiveness of antiestrogens and the transcriptional activity of the liganded ER in cells.


Subject(s)
Estrogen Antagonists/pharmacology , Estrogens/pharmacology , Receptors, Estrogen/metabolism , Transcriptional Activation , 3T3 Cells , Animals , Binding, Competitive , CHO Cells , Cricetinae , Gene Deletion , Gene Transfer Techniques , HeLa Cells , Humans , Mice , Mutagenesis, Site-Directed , Receptors, Estrogen/chemistry , Receptors, Estrogen/genetics , Tumor Cells, Cultured
SELECTION OF CITATIONS
SEARCH DETAIL
...