Biomedical subjects

S M Hyder

Publications and source records attributed to S M Hyder.

At least 19 recordsLinked to original sources

PubMed2000

Induction of the angiogenic factor VEGF in the uterus by the antiprogestin onapristone.

Onapristone (also referred to as ZK 98,299) is an antiprogestin that shares a number of structural similarities to mifepristone (RU-486) and other drugs in this class. While investigating the actions of antiprogestins on steroid hormone induced gene expression of angiogenic factors such as vascular endothelial growth factor (VEGF), we noted that onapristone alone induces VEGF transcript levels in the immature, ovariectomized rat uterus. In addition, onapristone induces expression of c-fos mRNA, which is induced by estrogens but not progestins in this target tissue. This induction of VEGF and c-fos by onapristone is inhibited by the antiestrogen ICI 182,780, but not by the antiprogestin RU-486. Both transcripts are very rapidly induced by onapristone, with maximal mRNA levels observed 3-6 h after in vivo administration of the drug. This time course is similar to that for induction of these genes by estrogenic hormones. Dose-response studies show that both these genes are maximally induced by a 2.5 mg/kg dose of onapristone following intra peritoneal injection. These results indicate that onapristone rapidly upregulates several genes normally under estrogenic regulation in the immature rat uterus. Importantly, this is the first report of the induction of a major angiogenic factor by an antiprogestin. Since an increase in vascularity increases tumor expansion and metastasis, the induction of angiogenesis and its regulatory factors such VEGF may be an important end-point to consider in the development and use of antiprogestins for the treatment of neoplastic disease.

Animals↗

PubMed2000

Identification of functional estrogen response elements in the gene coding for the potent angiogenic factor vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) is a potent stimulator of angiogenesis and a prognostic factor for many tumors including those of endocrine-responsive tissues such as the breast and uterus. We and others have previously shown that VEGF is regulated by estradiol and tamoxifen in the uterus and by estradiol in human breast cancer cells, and pharmacological evidence has suggested that this regulation was mediated by transcriptional activation of the estrogen receptor (ER). This prompted us to investigate whether the VEGF gene contains sequences that bind the ER and confer hormonal inducibility to reporter constructs in the presence of the two ER subtypes. These studies identified two sequences homologous to the consensus estrogen response element, GGTCAnnnTGACC, which bind both ER-alpha and ER-beta. One of these elements is located in the 5'-untranslated region of the VEGF gene (GGGCAaagTGACT), and the other is located in the 3'-untranslated region (GAGCAcccTGCCC). Competition with excess unlabeled oligonucleotides indicates that these two elements bind both ERs specifically, mutations in either half-site of the two elements abolish receptor binding, and ER-alpha- and ER-beta-specific antibodies interact with complexes formed with the corresponding receptor subtypes. In cells containing either ER-alpha or ER-beta, the 3'-element behaves as a traditional enhancer that confers hormone inducibility to reporter constructs in an orientation-independent manner, and transcriptional activity is blocked by the pure antiestrogen ICI 182,780. The pattern of transcriptional activity of the element located in the 5'-flanking region is more complex. In the orientation found in the endogenous gene, this element is nonresponsive to ER-beta but confers estrogen-dependent inhibition of transcription with ER-alpha that is blunted by ICI 182,780. In the opposite orientation, the 5'-element confers hormone inducibility with either ER-alpha or -beta, and ICI 182,780 blocks activation by ER-alpha but not by ER-beta. These findings support the hypotheses that estrogens directly regulate VEGF transcription in target tissues and tumors, although such regulation appears likely to involve a complex interplay of cis- and trans-acting elements not previously observed for other hormone-responsive genes.

3' Untranslated Regions↗

PubMed2000

Expression of estrogen receptor coactivators in the rat uterus.

Nuclear receptor coactivators associate in a ligand-dependent manner with estrogen receptors (ER) and other nuclear receptors, and they enhance ligand-dependent transcriptional activation. This study examined basal coactivator expression in rat uterus to investigate if expression of these genes is regulated by estradiol-17 beta or tamoxifen. Ovariectomized mature and immature rats were injected with estradiol-17 beta, tamoxifen, or vehicle (i.e., sesame oil) alone. Uteri were collected and analyzed for changes in coactivator mRNA expression using Northern blot and in situ hybridization analyses. Constitutive uterine mRNA expression of switch protein for antagonist (SPA), SRC-1, GRIP1, RAC3, RIP140, and p300 mRNAs was observed in control uteri, and treatment with ER ligands did not alter coactivator mRNA levels. The data suggest that expression of these coactivator genes is not sensitive to estradiol or tamoxifen in the rat uterus. No cell type-specific pattern of expression was apparent in uterine sections from mature and immature rats; however, silver grains were more abundant in luminal and glandular epithelial cells compared with the stroma and myometrium, indicating that coactivator mRNA levels vary among the uterine compartments. Thus, to our knowledge, we show for the first time that there is constitutive expression of several uterine nuclear receptor coactivators in a physiological setting that remains insensitive to estrogenic regulation. Furthermore, we speculate that higher constitutive levels of coactivator expression in glandular and luminal epithelial cells may be associated with increased hormonal responsiveness by these uterine compartments.

Adaptor Proteins, Signal Transducing↗

PubMed2000

Regulation of VEGF in the reproductive tract by sex-steroid hormones.

Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. In adults, angiogenesis is an infrequent event in the normal tissue except in the female reproductive tract where angiogenesis occurs frequently during the cyclical repair and regeneration of the endometrium as well as in the ovary. Little is known about angiogenesis in the male reproductive tract. The role of VEGF in controlling reproductive tract physiology and the role of hormones in regulating this key regulator of angiogenesis is not well understood. Since reproductive tract physiology is largely under sex-steroid regulation, we have reviewed some recent studies describing the role of sex-steroid hormones in regulating VEGF. We have also included studies on the role of sex-steroids in regulating VEGF and angiogenesis in endometrial, breast and prostate pathologies. We have provided an extensive review of the classical VEGF and VEGF receptors with examples drawn from numerous studies in the literature using diverse biological systems to encourage similar studies in the area of reproductive tract physiology. It is speculated that such studies will provide insights into understanding the role of VEGF in reproductive tract development, causes of infertility, and cancer. Such knowledge would allow us to target VEGF for improving human reproductive tract abnormalities, for enhancing implantation and fertility, and for designing drugs for treatment of endocrine dependent cancers.

Adult↗

PubMed1999

Interaction of human estrogen receptors alpha and beta with the same naturally occurring estrogen response elements.

Estrogen receptors are derived from two different gene products referred to as estrogen receptor-alpha (ER-alpha) and ER-beta. Both receptors bind to the consensus estrogen response element (ERE) present in the vitellogenin gene, but their binding to hormone response elements present in other estrogen responsive genes has not been reported yet. Using in vitro expressed human receptors, we now show that ER-beta binds to a panel of six endogenous hormone response elements (vitellogenin, c-fos, c-jun, pS2, cathepsin D, and choline acetyltransferase) already known to bind ER-alpha and confer estrogen inducibility to reporter constructs. Binding of ER-alpha and ER-beta occurred at similar DNA concentrations for some EREs, but different DNA concentrations were required to form complexes of the two receptors with other elements. These results illustrate for the first time by direct receptor-DNA binding studies that both ER-alpha and ER-beta bind to a number of EREs present in endogenous hormone regulated genes, and further suggest that the two forms of the receptor display different patterns of affinities for naturally occurring hormone response elements.

Animals↗

PubMed1999

The pure antiestrogen ICI 182,780 inhibits progestin-induced transcription.

The pure antiestrogen ICI 182,780 binds to the estrogen receptor with high affinity and is currently in clinical trials for the treatment of human breast cancer. We now show for the first time that ICI 182,780 also exhibits potent antiprogestin activity at doses frequently used in laboratory investigations. The antiprogestin activity of ICI 182,780 was detected in HeLa, HepG2, and CV1 cells transiently transfected with either the A or B forms of the human progesterone receptor and a luciferase construct driven by a progesterone-response element. ICI 182,780 inhibited progesterone-induced gene transcription in a dose-dependent fashion with maximum inhibition obtained at 10(-6) M and an IC50 of approximately 2 x 10(-7) M. The ICI compound produced the same degree of inhibition as that obtained with the antiprogestin RU-486. The antiestrogen tamoxifen did not display antiprogestin activity in this test system, and ICI 182,780 did not inhibit the activity of transfected androgen or glucocorticoid receptors. These results clearly establish that ICI 182,780 has significant antiprogestin activity in addition to its well-documented antiestrogenic activity and raises the possibility that both antihormonal properties of this compound are exhibited in laboratory studies and in the course of treatment of human breast tumors.

Antineoplastic Agents↗

PubMed1999

Estrogen modulates spontaneous alternation and the cholinergic phenotype in the basal forebrain.

We report that a small population of neurons expresses both choline acetyltransferase and classical estrogen receptor immunoreactivity and they are found primarily in the bed nucleus of the stria terminalis. In short-term ovariectomized ageing mice (24 months, n = 5) there were 41.0 +/- 4.1% fewer of these double-labeled cells than in young (five months, n = 5) short-term ovariectomized C57BL/6J mice. To study cholinergic neuron estrogen responsiveness, young mice (n = 8) were ovariectomized at puberty (five weeks). After three months half of the mice (n = 4) were given physiological levels of 17beta estradiol for 10 days. Bed nucleus double-labeled neurons increased by 32.9% (P < or = 0.003) in the young mice given estrogen. In a gel shift assay, double-stranded oligonucleotides with putative estrogen response elements from the choline acetyltransferase gene were used as competitors against estrogen receptor binding to consensus estrogen response elements. A sequence with 60% homology to the vitellogenin estrogen response element was found to compete at 500- and 1000-fold excess. Young mice (five months) with ovaries demonstrated significantly (P < or = 0.04) better performance in the spontaneous alternation T-maze test than did old (19 month) mice with ovaries (young = 66.3 +/- 3.3% correct choices; vs old = 55.0 +/- 4.0% in old mice with ovaries). Young mice (five months old), ovariectomized for one month and treated with estrogen, showed significantly more spontaneous alternation than ovariectomized controls (69.1 +/- 2.8% vs 58.3 +/- 3.9%; P < or = 0.04). Estrogen also increased spontaneous alternation in old, short-term ovariectomized mice (61.5 +/- 2.7% vs 48 +/- 3.3%; P < or = 0.005). In either young or old ovariectomized mice, estrogen increased spontaneous alternation to levels seen in young animals with ovaries. Estrogen increases the number of choline acetyltransferase-immunoreactive and choline acetyltransferase/estrogen receptor-immunoreactive cells in old or young mice lacking estrogen, and enhances working memory in old or young mice lacking estrogen. Our data suggest that estrogen may act at the level of the choline acetyltransferase gene, but in view of the limited distribution of cholinergic cells expressing the classical estrogen receptor, it is unlikely that these cells can account for a memory enhancing effect of estrogen replacement.

Aging↗

PubMed1999

Regulation of angiogenic growth factors in the female reproductive tract by estrogens and progestins.

Proper regulation of angiogenesis and vascular permeability is essential for the physiological functioning of the female reproductive tract, and major health problems in women, such as dysfunctional uterine bleeding, endometriosis, and uterine cancer, involve a vascular component. There is a large body of literature that describes the effects of sex steroids on the vasculature of the reproductive tract, but far less is known about the molecular mechanisms that regulate these important actions. We hope that this minireview will help emphasize the need for mechanistic studies in this area to improve treatment and prevention of these major health problems in women. Specifically, we believe it will be important to 1) define the exact roles of FGF, VEGF, and other factors in physiological and pathological events in the reproductive tract and the cell types and receptors involved; 2) identify estrogen and progesterone receptor subtypes, the DNA elements, nuclear protein factors, and signaling pathways that mediate regulation of these genes by sex steroids; 3) elucidate any mechanisms of cross-talk between sex steroids and other regulatory factors in the overall regulation of FGF, VEGF, and other angiogenic/permeability factors; and 4) eventually understand how genetic polymorphisms of key regulatory elements affect angiogenesis and the regulation of vascular function in the female reproductive tract.

Animals↗

PubMed1999

Synthetic estrogen 17alpha-ethinyl estradiol induces pattern of uterine gene expression similar to endogenous estrogen 17beta-estradiol.

17alpha-Ethinyl estradiol is one of most widely prescribed estrogens. We compared the effects of this synthetic estrogen to those of the endogenous ovarian hormone 17beta-estradiol on the expression of four estrogen-inducible genes in the rat uterus. The genes examined include c-fos, c-jun, vascular endothelial growth factor, and creatine kinase B, which are all known to be primary responses to estrogen administration. Both estrogens induced the four target genes with similar time courses and produced the same pattern of cell-specific expression of c-fos and vascular endothelial growth factor in the uterine epithelium and stroma, respectively. Dose-response studies established that the potency and efficacy of both estrogens in the uterus were the same for all four hormone-regulated genes. These studies suggest that 17alpha-ethinyl and 17beta-estradiol produce similar if not identical patterns of gene expression in the uterus.

Animals↗

PubMed1998

Progestin regulation of vascular endothelial growth factor in human breast cancer cells.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor associated with the degree of vascularity, progression, and metastasis of breast cancer, and cases of this disease with increased vascular density have a poor prognosis. We show that in T47-D human breast cancer cells, progesterone induces a dose-dependent increase of 3-4-fold in media VEGF levels, with a maximum response occurring at a concentration of 10 nM. This effect is blocked by the antiprogestin RU 486. In addition to progesterone, a number of synthetic progestins used in oral contraceptives (e.g., norethindrone, norgestrel, and norethynodrel), hormone replacement therapy (medroxyprogesterone acetate), and high-dose progestin treatment of breast cancer (megestrol acetate) also increase VEGF in the media of cultured T47-D cells. This effect is hormone specific and is not produced by estrogens, androgens, or glucocorticoids. Collectively, these observations suggest that the increase in VEGF caused by progestins is mediated by progesterone receptors present in T47-D cells. The induction of VEGF by progestins is also cell type specific and does not occur in human breast cancer cell lines MCF-7, ZR-75, or MDA-MB-231, nor in Ishikawa cells derived from a human endometrial carcinoma. This is the first report that progestins regulate VEGF expression in human breast cancer cells and raises the possibility that increased angiogenesis in response to endogenous progesterone or its therapeutically used analogues may play a role in cell growth or metastasis in a subset of human breast tumors.

Breast Neoplasms↗

PubMed1998

The 3-flanking region of the mouse c-fos gene contains a cluster of GGTCA hormone response-like elements.

We previously identified an estrogen response element in the 3'-flanking region of the c-fos protooncogene [1, 2]. This element, GGTCAnnnCAGCC, has one half-site identical to that of the consensus ERE (GGTCAnnnTGACC) but only limited homology to the second half-site. Because of this non-canonical sequence and atypical location in the 3'-untranslated region of an estrogen target gene, we decided to analyze sequences adjacent to this element for the possible presence of other regulatory elements. We now report that the 635 base pairs downstream of the c-fos ERE contain: (i) an unusual cluster of 7 GGTCA hormone response-like elements; (ii) potential binding sites for other known DNA binding proteins; and (iii) a sequence specific binding site for a non-estrogen receptor protein present in hormone target tissues.

Adaptor Protein Complex alpha Subunits↗

PubMed1998

Phorbol ester inhibition of estrogen-induced uterine deoxyribonucleic acid synthesis.

Protein kinase C (PKC) is a key regulatory enzyme in the control of growth and differentiated function in many cell types. Recently it has become clear that cross talk occurs between PKC and steroid hormone-activated signaling pathways. In this work we have thus used the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to investigate the relationship between PKC activation and estrogen-induced proliferation in an in vivo model of hormone action, the immature rat uterus. A single injection of estradiol (E2) to immature female animals increases DNA synthesis in all major uterine cell types. Administration of TPA alone, simultaneous administration of TPA and E2, or administration of TPA 12 h after E2 did not alter uterine DNA synthesis. However, administration of TPA 6 h after E2 markedly decreased [3H]thymidine incorporation and the labeling indices in uterine epithelial, stromal, and myometrial cells. This inhibition represents a decrease in DNA synthesis per se rather than a change in the time course of the tissue response to the hormone. The inhibitory effect of TPA was reversible within 72 h and did not appear to be due to a decrease in the level or degree of occupancy of uterine estrogen receptors. These results suggest that a discrete regulatory event(s) in the pathway of estradiol-induced proliferation is inhibited by PKC activation approximately 6 h after hormonal stimulation.

Animals↗

PubMed1997

Triphenylethylene antiestrogens induce uterine vascular endothelial growth factor expression via their partial estrogen agonist activity.

Estradiol induces vascular endothelial growth factor (VEGF) expression in the rat uterus and this may contribute to the hyperemia and increased vascularity produced by estrogens in this target tissue. Triphenylethylene antiestrogens such as tamoxifen have mixed agonist/antagonist activity and their specific effects are tissue and gene specific. These drugs exhibit primarily antiestrogenic actions in mammary tissue and are thus used for the treatment of breast cancer. These drugs are also suggested to be inhibitors of angiogenesis. However, uterine side effects of tamoxifen are thought to stem largely from the agonist activity of the drug in this tissue. Since side effects of tamoxifen such as uterine bleeding and endometrial cancer seem likely to have an angiogenic component, we have examined the effects of this drug, its metabolite, 4-hydroxy-tamoxifen and two additional triphenylethylene antiestrogens, nafoxidine and clomiphene, on the expression of VEGF and another estrogen regulated gene, c-fos, using the rat uterus as an experimental system. All four compounds increase uterine VEGF and c-fos mRNA levels indicating that the triphenylethylene class of antiestrogens are predominantly agonists for the induction of these genes in the uterus.

Animals↗

PubMed1997

Selective inhibition of estrogen-regulated gene expression in vivo by the pure antiestrogen ICI 182,780.

Estrogen-stimulated cell proliferation is thought to be mediated by the induction of growth-regulatory factors. Abnormal regulation of such factors may be associated with tumorigenesis. Two such factors, vascular endothelial growth factor and c-fos, are rapidly induced in the uterus by estrogens. We show that the induction of these transcripts by 17beta-estradiol or tamoxifen is selectively blocked by the pure antiestrogen ICI 182,780 in a dose-dependent manner. This indicates that induction of the two genes requires different levels of estrogen receptor or that their induction occurs by different mechanisms. This suggests that selective dose-dependent antagonism of estrogen-dependent transcription may be possible in target tissues and tumors.

Animals↗

PubMed1996

Uterine expression of vascular endothelial growth factor is increased by estradiol and tamoxifen.

Vascular endothelial growth factor (VEGF) is an endothelial-specific mitogen with potent angiogenic activity. Because vascular growth accompanies normal endometrial regeneration and may also be involved in uterine tumor growth, we studied VEGF regulation by 17 beta-estradiol (E2) and tamoxifen, two agents that can increase uterine cell proliferation and tumor incidence. In immature, ovariectomized rats, E2 elevates uterine VEGF mRNA transiently, with a peak induction of 15-20-fold within 1 h. A maximum response is produced at a dose of 4 micrograms/kg E2, and induction is specific for estrogenic steroids. E2-dependent VEGF induction is inhibited by actinomycin D but not puromycin, suggesting that the effect is due at least in part to direct estrogen receptor regulation of VEGF transcription. PCR amplification and DNA sequencing indicated that VEGF188, VEGF164, and VEGF120 are all induced by E2, but the latter two are the predominant forms in the uterus. In situ hybridization shows a predominantly stromal expression of VEGF mRNA. The antiestrogens tamoxifen, 4-OH tamoxifen, and nafoxidine produce similar increases in uterine VEGF mRNA levels within 6 h, with 1 mg/kg tamoxifen producing a maximum response of 15-20-fold. The tamoxifen response was also inhibited by actinomycin D but not by puromycin, again suggesting direct transcriptional regulation of VEGF expression by antiestrogens. These findings raise the possibility that estrogen and antiestrogen effects on uterine edema, proliferation, and tumor incidence may involve local increases in tissue VEGF production.

Animals↗

PubMed1996

Expression of estrogen receptor variants in normal and neoplastic human uterus.

Estrogen receptor variants lacking internal exons and representing dominant positive and negative activity may be involved in the initiation and/or progression of endocrine dependent tumors. To assess the role of estrogen receptor in uterine disease, we have analyzed both normal and neoplastic uterine samples for the presence of variant estrogen receptors using the sensitive technique of RT-PCR and direct automated DNA sequencing of the amplified products. Our analysis was conducted to determine the presence of spliced variants lacking exons 3 through exon 8. We demonstrate that both the normal and neoplastic human uterus contains a number of spliced variants of the estrogen receptor that co-exist with the wild type receptor. Variants lacking exons 4, 5 and 7 but not exons 3 and 6 were detected. Also, a novel partial deletion in exon 8 was detected in both the normal and neoplastic tissues, although a total deletion of this exon was not observed. In addition another region of exon 8 deletion was found to be present in one tumor tissue which also contained an insertion within this region, however, other tumors did not contain this variant. In addition, double exon deletion variants were observed lacking exons 3 and 4, exons 4 and 5, and exon 7 with part of exon 8. Although our data represents a limited number of samples it suggests that splicing of the estrogen receptor message occurs in the normal physiological setting. There does not appear to be any association between the presence or absence of spliced variants of estrogen receptor and uterine tumor formation at the mRNA level.

Adenocarcinoma↗

PubMed1995

The protooncogene c-jun contains an unusual estrogen-inducible enhancer within the coding sequence.

Estrogens have previously been shown to induce c-jun mRNA levels in target cells during hormone induced proliferation, and this appears to be a primary hormonal response involving transcriptional activation. In this report we have now identified an estrogen dependent enhancer within the coding sequence of c-jun. This element has the sequence GCAGAnnnTGACC which is identical to the consensus estrogen response element GGTCAnnnTGACC in the second half site, but varies considerably in the first half site. Synthetic oligodeoxynucleotides containing this jun sequence bind the estrogen receptor in cell-free studies using a competitive band shift assay with the consensus element. The jun element also confers hormone inducibility to reporter plasmids in yeast and mammalian based transcriptional systems. Structure-function studies illustrate that the TGACC half-site and its immediate flanking dinucleotides, but not the GCAGA half-site, are required for estrogen receptor binding. In contrast, both the GCAGA and TGACC half-sites are obligatory for hormone-inducible transcriptional activation. These results suggest a model in which the estrogen receptor functions as a heterodimer to regulate transcription of the c-jun protooncogene. Coupled with reports of estrogen response elements in c-fos and estrogenic induction of c-fos and c-jun in vivo, these findings also support a role for AP-1 components as early response genes in estrogen-induced proliferation.

Animals↗

PubMed1995

Estrogen action in target cells: selective requirements for activation of different hormone response elements.

RENE1 cells, an estrogen receptor positive rat uterine endometrial cell line immortalized with the E1A oncogene, were analyzed for the presence of estrogen-dependent signal transduction pathways using the induction of transfected as well as endogenous genes. RENE1 cells express the estrogen receptor as analyzed by Northern blots and ligand binding assays (40 fmoles/mg protein). The receptor system appears functional, based on the induction of reporter constructs containing the consensus estrogen response element (ERE) in transient transfection assays and alterations in endogenous transcripts visualized by utilizing differential display methodology. However, neither transfected repoter constructs containing the c-fos ERE, nor the endogenous c-fos, c-jun, or c-myc genes are induced by estrogens in these cells despite being induced by estrogens in the uterus in vivo. In addition, estradiol did not induce endogenous c-fos expression or the activity of CAT reporters containing the c-fos ERE in a stable transfectant of RENE1 cells with a 3-fold elevation in estrogen receptor content. Under identical conditions, TPA and serum rapidly induce c-fos transcription in RENE1 cells, indicating that the lack of inducibility by estradiol is not due to a general inhibitor of transcription of these genes. These results suggest that RENE1 cells lack factors present in normal uterine cells which are required for the estrogenic induction of a specific subset(s) of EREs. These observations support the generally evolving hypothesis that steroid hormones may act through composite response elements via interactions with other transcription factors, in addition to functioning as homodimers at classical palindromic response elements.

Adenovirus E1A Proteins↗