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S M Hyder

Publications and source records attributed to S M Hyder.

At least 37 records · Page 2Linked to original sources
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↗
PubMed1995

Toxicity of endogenous and environmental estrogens: what is the role of elemental interactions?

Many naturally occurring and man-made chemicals present in the environment possess estrogenic activity. Examples include plant and fungal products, pesticides, plasticizers, and other agricultural and industrial chemicals. These environmental estrogens as well as endogenous ovarian estrogens are thought to initiate their physiological actions in target tissues largely via interactions with a nuclear receptor system. The resultant estrogen-receptor complex in turn affects transcription via its interactions with nucleotide sequences known as estrogen response elements (EREs) present in the regulatory regions of hormone responsive genes. A "consensus" ERE sequence GGTCAnnnTGACC was originally identified in the vitellogenin genes of birds and amphibians, but it is now clear that most naturally occurring EREs differ from this sequence in one or more bases. We and others have obtained both in vivo and in vitro data suggesting a differential interaction of receptor complexes containing different ligands with the multiple EREs present in mammalian systems. This raises the possibility that the toxicity of environmental estrogens may arise in part from a differential pattern of ERE activation by environmental compounds relative to endogenous ovarian estrogens. The experimental basis for such a paradigm and its toxicological implications are discussed in this paper.

Animals↗
PubMed1994

In vitro interaction of uterine estrogen receptor with the estrogen response element present in the 3'-flanking region of the murine c-fos protooncogene.

Estradiol treatment rapidly stimulates transcription of the c-fos protooncogene in the rodent uterus, and transfection analysis previously identified an estrogen response element (ERE) in the 3'-flanking region of the murine gene with the sequence GGTCAnnnCAGCC. We now report that endogenous estrogen receptor (ER) obtained from either mouse or rat uterus binds to this 3'-ERE. Unoccupied receptor, receptor occupied with estradiol and receptor occupied with the antiestrogen tamoxifen all bind to this element, and the binding of receptor exhibits strict sequence specificity. By using a competition binding assay, the affinity of the ER for the c-fos-ERE is estimated to be approximately an order of magnitude less than the affinity for the consensus ERE (GGTCAnnnTGACC) found in the Xenopus and chicken vitellogenin genes. Differences in the electrophoretic mobilities of the c-fos and vitellogenin EREs bound to the ER in band-shift assays also suggest subtle structural differences in the two complexes. Mutations in either half-site of the c-fos-ERE destroy ER binding, suggesting that the receptor binds to this sequence as either a homo- or heterodimer. The 3'-fos-ERE region exhibits some homologies to both AP1 and AP2 consensus sites, but neither AP1-like proteins present in uterine extracts nor recombinant AP2 bind this protooncogene sequence. The finding that the ERE present in the 3'-region of the murine c-fos gene interacts with receptors present in the mouse and rat uterus supports a role for this element in the physiological regulation of c-fos expression in the uterus by estrogens.

Animals↗
PubMed1994

Steroid hormone-induced expression of oncogene encoded nuclear proteins.

In this article we have attempted to review the literature on the regulation of nuclear protooncogene expression by steroid hormones and other small molecules that interact with receptors of the steroid/thyroid superfamily. Until about 5 years ago, there were relatively few reports of steroidal regulation of cellular oncogenes, but hundreds of papers on this topic have appeared since then. This demonstrates the intense interest in this area that has developed recently. It now been demonstrated that all the major classes of steroid hormones control expression of nuclear protooncogenes in one or more systems. Given the actions of these proteins as transcription factors and their central role in cellular communications systems, it seems likely that they play a key role in mediating the biological effects of steroids on processes such as proliferation and differentiation. To date, most of the work in this general area has focused primarily on the regulation of three genes: c-fos, c-jun, and c-myc. However, a quick glance at the table of nuclear protooncogenes in the introduction of this article indicates that over 40 nuclear protooncogenes are now recognized. For the large majority of these, regulatory effects of steroids and related molecules have not yet been reported. Hence, we predict that reports in this general area of research will continue to appear at a very rapid rate over the next few years. In addition, we have tried to provide enough background information for readers to get an overview of the regulation of nuclear protooncogene expression by nonsteroidal factors. We felt this information was important to emphasize that steroid hormones represent only one of the many classes of regulatory molecules that control expression of nuclear protooncogenes. Thus, an important area for future research will be to understand how these multiple regulatory systems interact to control expression of this important class of cellular oncogenes and the biological processes that they mediate.

Animals↗
PubMed1994

Improved accuracy in direct automated DNA sequencing of small PCR products by optimizing the template concentration.

Data are presented illustrating the optimum concentration range of reverse transcription PCR-generated products under 500 bp for accurate base calling with direct automated DNA sequencing. A 357-bp fragment of the human estrogen receptor, which includes the DNA binding domain of the protein, was used as a representative example of a gene fragment that can be rapidly amplified and sequenced. Using the Taq DNA polymerase dye terminator sequencing protocol and automated sequencing apparatus from Applied BioSystems, 0.1 to 1.0 pmol of PCR product in a 20-microL reaction volume provided > 97% accurate base detection. Concentrations greater or lower than this range increased the number of ambiguous bases due to alterations in the signal-to-noise ratios. This procedure has been successfully utilized with 140-440-bp PCR products within the optimum concentration range. These results show that low amounts of PCR products are necessary and sufficient for direct sequence analysis.

Base Sequence↗
PubMed1993

Creation of an active estrogen-responsive element by a single base change in the flanking sequence of a cellular oncogene: a possible mechanism for hormonal carcinogenesis?

Estrogens are considered to act as promoters in a multistep process of hormonal carcinogenesis, although the molecular mechanisms by which these hormones act in tumorigenesis are unclear at present. Estradiol is known to induce expression of certain proto-oncogenes, and this led us to examine potential regulatory regions of the cellular c-fos oncogene. The 5'-flanking region of the murine c-fos contains a 13-bp palindromic sequence (GGTCTnnnAGACC) with striking homology to the consensus estrogen-responsive element (ERE) GGTCAnnnTGACC. However, the c-fos sequence did not bind the human estrogen receptor or confer hormonal responsiveness in a yeast-based transcriptional test system. Importantly, a single base change in the fifth position of the c-fos sequence (GGTCTnnnAGACC to GGTCA/GnnnAGACC) produced an element that bound the estrogen receptor and conferred estrogen-dependent transcriptional activation of a reporter gene. This suggests a specific hypothesis by which estrogens could act as tumor promoters. In this paradigm, the regulatory region of the cellular oncogenes, tumor suppressor genes, and growth-factor genes contain inactive sequences with close homologies to hormone-responsive elements. Initiation occurs when some agent (e.g., a chemical carcinogen) causes a mutation in such a sequence to create a functional hormone-responsive element. Estrogens, acting through their receptors and the mutated element, can then activate the target gene to stimulate cell proliferation and increase the population of initiated cells.

Animals↗
PubMed1992

Identification of an estrogen response element in the 3'-flanking region of the murine c-fos protooncogene.

We have used transient transfection assays with reporter plasmids expressing chloramphenicol acetyltransferase, linked to regions of mouse c-fos, to identify a specific estrogen response element (ERE) in this protooncogene. This element is located in the untranslated 3'-flanking region of the c-fos gene, 5 kilobases (kb) downstream from the c-fos promoter and 1.5 kb downstream of the poly(A) signal. This element confers estrogen responsiveness to chloramphenicol acetyltransferase reporters linked to both the herpes simplex virus thymidine kinase promoter and the homologous c-fos promoter. Deletion analysis localized the response element to a 200-base pair fragment which contains the element GGTCACCACAGCC that resembles the consensus ERE sequence GGTCACAGTGACC originally identified in Xenopus vitellogenin A2 gene. A synthetic 36-base pair oligodeoxynucleotide containing this c-fos sequence conferred estrogen inducibility to the thymidine kinase promoter. The corresponding sequence also induced reporter activity when present in the c-fos gene fragment 3 kb from the thymidine kinase promoter. Gel-shift experiments demonstrated that synthetic oligonucleotides containing either the consensus ERE or the c-fos element bind human estrogen receptor obtained from a yeast expression system. However, the mobility of the shifted band is faster for the fos-ERE-complex than the consensus ERE complex suggesting that the three-dimensional structure of the protein-DNA complexes is different or that other factors are differentially involved in the two reactions. When the 5'-GGTCA sequence present in the c-fos ERE is mutated to 5'-TTTCA, transcriptional activation and receptor binding activities are both lost. Mutation of the CAGCC-3' element corresponding to the second half-site of the c-fos sequence also led to the loss of receptor binding activity, suggesting that both half-sites of this element are involved in this function. The estrogen induction mediated by either the c-fos or the consensus ERE was blunted by the antiestrogen tamoxifen. Based on these studies, we believe the 3'-fos ERE sequence we have identified may be a major cis-acting element involved in the physiological regulation of the gene by estrogens in vivo.

Animals↗
PubMed1991

Presence of an estradiol response region in the mouse c-fos oncogene.

We have previously shown that the intracellular content of c-fos mRNA is rapidly induced (within 1 to 3 hours) in ovariectomized rat or mouse uteri following administration of estradiol. This induction is sensitive to actinomycin D but not to protein synthesis inhibitor puromycin, indicating an effect of estradiol at the transcriptional level, possibly mediated by the estrogen receptor. We have used transient transfection assays with defined regions of the mouse c-fos gene ligated to a reporter plasmid expressing chloramphenicol acetyl transferase to study regulation of this gene by estrogens. These recombinants were transfected in two different estrogen-responsive cell lines, GH4 and MCF-7, and stimulated with estradiol. A two- to five-fold induction of chloramphenicol acetyl transferase activity was observed with a construct containing the intact c-fos promoter and 351 bases of 5'-flanking sequence (-351/+44). A similar induction by estrogen is observed with the endogenous c-fos gene in the two cell lines as determined by RNA blot analysis. Estrogen induction is lost when a construct containing -135/+44 region of the c-fos gene is transfected. Plasmid containing the consensus estrogen response element GGTCAnnnTGACC derived from vitellogenin gene is induced 10- to 50-fold in both estrogen-responsive cell lines. Under identical conditions, the oligonucleotide containing the perfect palindrome GGTCTnnnAGACC, present around the -209 region of the c-fos gene, is completely silent when transfected under the control of thymidine kinase promoter. Additional transfection analysis with a number of c-fos promoter constructs has narrowed the estrogen response region to within the -278 to -135 region upstream of the c-fos promoter.

Animals↗
PubMed1989

Separation of two molecular forms of human estrogen receptor by hydrophobic interaction chromatography. Gradient optimization and tissue comparison.

High-performance hydrophobic interaction chromatography (HPHIC) was used to separate and characterize two molecular forms of estrogen receptor with a SynChropak propyl hydrophobic column (300 A pore size). The linear gradient utilized earlier with a polyether-bonded column (2 to 0 M) ammonium sulfate in 40 min, gave poor resolution with the propyl column. However, resolution was maximized with either an initial ammonium sulfate concentration of 1 M (40-min gradient) or with a two-phase gradient (2 to 0.5 M in 10 min, 0.5 to 0 M in 30 min). This indicated that the propyl column was more hydrophobic than the polyether column. Estrogen receptor separated into two isoforms, either in the presence [MI, retention time (tR) = 13-14 min; MII, tR = 20-21 min] or absence (I, tR = 21-23 min; II, tR = 31-33 min) of the estrogen receptor stabilizing reagent, sodium molybdate. Similar isoforms were observed in cytosols from human breast tumors, uterus, and MCF-7 breast cancer cells. Unlike others, MCF-7 estrogen receptor did not show MI. Since MCF-7 cells contain 90,000 dalton heat shock proteins (HSP-90), HSP-90 is probably not directly involved in MI formation. Sodium molybdate selectively interacted with isoform II and converted it to MI. All isoforms appeared to be high-molecular-weight proteins (greater than 60 A) when subsequently analyzed by high-performance size-exclusion chromatography. Interestingly, when estrogen receptor was immobilized on the stationary phase, no change was detected in either hydrophobicity or steroid-binding capacity. After 16-18 h, immobilized receptor was eluted with a slightly longer tR. During incubation on the column, component MI was converted into I and/or II. HPHIC appears to be a rapid, yet gentle procedure for isolating large receptor complexes in significant quantities with high recoveries. This allows one to discern the complicated structure-function relationships of estrogen receptor and associated non-receptor proteins and provides information about the on-column behavior of complex proteins.

Breast Neoplasms↗
PubMed1989

Detection of two high molecular weight hydrophobic forms of the human estrogen receptor.

The human estrogen receptor gene encodes a single protein of molecular weight 65,000 daltons. However, using a sensitive and rapid technique of high-performance hydrophobic interaction chromatography we have detected two distinct estrogen receptor species both of which are high molecular weight proteins (ca. 60A) as determined by high-performance size-exclusion chromatography. These are detected either in the presence or absence of sodium molybdate; rechromatography of individual isoform indicates that the two protein complexes have independent hydrophobic contact points. Consistent elution patterns of the two receptor species indicates they are formed selectively. We conclude that different post-translational modifications of the estrogen receptor protein could allow their specific interaction with non-receptor components resulting in the formation of two distinct high molecular weight complexes which would be rapidly resolved by high-performance hydrophobic interaction chromatography.

Animals↗
PubMed1988

High-performance hydrophobic interaction chromatography as a means of identifying estrogen receptors expressing different binding domains.

Methodology for high-performance hydrophobic interaction chromatography (HPHIC) of estrogen receptors (ER) was developed, utilizing a polyether-bonded stationary phase, which was non-ionic in nature. Using a descending salt gradient (2 M to 0 M ammonium sulphate in 40 min), ERs from human breast cancer separated into two isoforms, which retained ligand-binding domains. The same isoforms were observed with ER preparations from rat uterus. When sodium molybdate, a stabilizer of receptor structure, was incorporated into the mobile phase, it altered the ER characteristics, producing an earlier elution of one component, while the other one remained unchanged. Treatment of breast cancer cytosol with RNase A did not alter ER elution from either the hydrophobic or size-exclusion (TSK 3000 SW) columns. Modification of cysteine residues with N-ethylmaleimide led to a broad elution pattern of receptor from the hydrophobic column, implying the existence of multiple conformations of ER. Limited trypsin treatment of ER, which removes the DNA binding domain, led to the elution of only one receptor peak from the hydrophobic column. The receptor eluted at 24 min both in the presence and in the absence of sodium molybdate. Thus, at least one mechanism of the sodium molybdate effect must involve its direct interaction with ER to influence the sequence between the DNA-binding domain and the N-terminus. This also indicates that the most hydrophobic species of ER (sodium molybdate sensitive) may arise due to the interaction of the DNA-binding site with the stationary phase. Other possibilities, such as differential post-translational modifications of the receptor protein could also account for the two isoforms of ER, observed in HPHIC analysis.

Animals↗
PubMed1988

High-performance hydrophobic interaction chromatography of estrogen receptors and magnesium dependent protein kinase(s): detection of two molecular forms of estrogen receptors in the presence and absence of sodium molybdate.

The separation characteristics of estrogen receptors (ER) from human breast cancer were evaluated based on their hydrophobic properties. Results show that (1) two distinct hydrophobic isoforms of ER exist either in the presence of sodium molybdate (peaks MI and MII with retention times of 15-17 min and 24-26 min) or in its absence (peaks I and II with retention times of 25-27 min and 34-36 min respectively); (2) this is observed whether molybdate (MoO2-4) is added to prepared cytosol or to the buffer prior to homogenization; (3) isoform MII and I separated with similar retention times suggesting they are the same ER species; and (4) isoform MI (Rt = 15-17 min) is a distinct ER species from either MII/I (Rt = 25-28 min) or II (Rt = 34-36 min). The latter isoform represents a highly hydrophobic species seen only in the absence of MoO2-4. Finally, (5) MoO2-4 ions appear to interconvert the most hydrophobic species (II) into the least hydrophobic isoform (MI) with virtually no change in the quantity of isoform(s) MII/I. However, it cannot be ascertained if the II----MI interconversion proceeds via isoform MII/I. Isoform II may result from the interaction with the stationary phase via its DNA binding site since MoO2-4, which is suggested to directly interact with this site, selectively interacts with peak II. These results imply the usefulness of inclusion of receptor stabilizing reagents in the mobile phase for preserving receptor integrity and in elucidating the interrelationships of ER isoforms and associated macromolecules.

Breast Neoplasms↗