Usability and Suitability of the Omics-Integrating Analysis Platform tranSMART for Translational Research and Education.

BACKGROUND: Platforms like tranSMART assist researchers in analyzing clinical and corresponding omics data. Usability is an important, yet often overlooked, factor affecting the adoption and meaningful use. Analyses on the specific needs of translational researchers and considerations about the application of such platforms for education are rare. OBJECTIVES: The aim of this study was to test whether tranSMART can be used in education and how well medical students and professional researchers can handle it; to identify which kind of translational researchers-in terms of skills, experienced limitations, and available data-can take advantage of tranSMART; and to evaluate the usability and to generate recommendations for improvements. METHODS: An online-based test has been done by medical students (N = 109) and researchers (N = 26). The test comprised 13 tasks in the context of four typical research scenarios based on experimental and clinical data. A web questionnaire was provided to identify both the needs and the conditions of research as well as to evaluate the system's usability based on the "System Usability Scale" (SUS). RESULTS: Students and researchers were able to handle tranSMART well and coped with most scenarios: cohort identification, data exploration, hypothesis generation, and hypothesis validation were answered with a rate of correctness between 82 and 100%. Of the total, 72.2% of the teaching researchers considered tranSMART suitable for their lessons and 84.6% of the researchers considered the platform useful for their daily work; 65.4% of the researchers named the nonavailability of a platform like tranSMART as a restriction on their research. The usability was rated "acceptable" with a SUS of 70.8. CONCLUSION: tranSMART is potentially suitable for education purposes and fits most of the needs of translational researchers. Improvements are needed on the presentation of analysis results and on the guidance of users through the analysis, especially to ensure the compliance of the analysis with the requirements of statistical testing.

Characterization of glucocorticoid-specific binding in the L2C leukemia.

Glucocorticoid-specific binding macromolecules were measured and characterized in L2C cells, a B-lymphocyte guinea pig leukemia that is resistant to administered cortisol or adrenocorticotrophic hormone. L2C cells were harvested by cardiac puncture followed by Ficoll:Hypaque separation techniques. The cells were lysed by sonication, and the cytosol was obtained after centrifugation at 106,000 x g for 60 min at 0 degrees. Cytosol glucocorticoid receptor measurements were obtained by hydroxylapatite assay or column chromatography using Sephadex G-25. Maximal specifically bound [3H]triamcinolone acetonide in the cytosol fraction was 300 fmol/10(8) cells. Scatchard plot of specific L2C cytosol binding gave a Kd of 18 nM and an estimate of 2000 binding sites/cell. The specificity of binding in L2C cytosol was triamcinolone acetonide > dexamethasone > cortisol > progesterone > testosterone = estradiol. Binding of [3H]triamcinolone acetonide to cytosol glucocorticoid receptors was maximal at 22 hr of incubation at 19 degrees, and the receptor complex was stable for 48 hr. The receptor complex was not affected by DNase or RNase, but the receptor complex was lost with pronase or heat denaturation. Whole-cell binding assays were also performed, which resulted in similar quantities of maximally bound glucocorticoid receptor as well as the specificity of binding of various hormone analogs as found in the cytosol receptor assays. Transferring the whole cells from 0 to 22 degrees resulted in the appearance in nuclei of approximately 65% bound receptors. However, these translocated receptor complexes do not appear to affect the viability of the L2C cells as measured by trypan blue exclusion.

Glucocorticoid effects on lipopolysaccharide-stimulated murine B-cell leukemia line (BCL1) cells.

The effects of glucocorticoids were studied in lipopolysaccharide (LPS)-stimulated splenic murine B-cell leukemia line one (BCL1) cells. At 24 hr, LPS caused a 3-fold increase in [3H]-thymidine incorporation compared to similarly cultured unstimulated cells. Triamcinolone acetonide (TA) and dexamethasone at a concentration of 10(-8) M reduced [3H]thymidine incorporation 80 and 53%, respectively, while estradiol at concentrations of 10(-10) to 10(-5) M had no effect. A 500-fold excess of cortexolone or progesterone blocked the response of 10(-8) M TA by 42 and 38%, respectively, indicating that the glucocorticoid response could be inhibited by antiglucocorticoids. The maximum rate of thymidine incorporation in LPS-stimulated cells occurred at 24 hr, a time at which 10(-5) M TA present in parallel cultures from the initiation of LPS stimulation showed a 79% reduction in [3H]thymidine incorporation. If TA was added at any time after the initiation of LPS stimulation, the degree of decrease in nucleotide incorporation was not as marked. Therefore, maximum TA effect in LPS-stimulated BCL1 cells occurred when TA was added to cultures at the onset of mitogen stimulation. We also measured glucocorticoid-specific receptor in whole cells both before LPS stimulation and in BCL1 cells incubated 24 hr in the presence of LPS. The equilibrium dissociation constant, the number of sites/cell, and the hormone specificity of the glucocorticoid receptor were similar prior to and at the peak of mitogen stimulation.

Binding of [3H]triamcinolone acetonide-receptor complexes to chromatin from the B-cell leukemia line, BCL1.

The binding characteristics of partially purified glucocorticoid receptor complexes from hormone sensitive, non-differentiating BCL1 cells to sequentially deproteinized BCL1 chromatin-cellulose was investigated. [3H]Triamcinolone acetonide (TA)-receptor complexes were purified (approx. 30-fold) from DEAE-cellulose columns by salt elution which allowed receptor activation only in the absence of molybdate. Addition of 10 mM molybdate completely blocked salt activation. The binding pattern of the activated [3H]TA-receptor complexes to chromatin-cellulose extracted with 0-8 M guanidine hydrochloride revealed three regions of increased binding activity (acceptor sites), at 2, 5 and 7 M guanidine hydrochloride. Acceptor site binding was markedly reduced for chromatin extracted with 3, 6 and 8 M guanidine hydrochloride. Non-activated receptor complexes demonstrated very low binding to deproteinized chromatin. It was also shown that chromatin binding required glucocorticoid receptors and that free ligand or ligand bound to other proteins did not bind significantly to chromatin. In addition, binding of [3H]TA-receptor complexes to partially deproteinized chromatin was competable by unlabeled TA-receptor complexes. Scatchard analysis demonstrated that chromatin from non-differentiating BCL1 cells possesses multiple, high-affinity binding sites which differ in their affinity for the glucocorticoid receptor. Partially deproteinized chromatin from lipopolysaccharide-stimulated BCL1 cells demonstrated a different pattern of receptor binding, i.e., receptor binding was significantly greater to chromatin previously extracted with 6-8 M guanidine hydrochloride. These results suggest that differentiation alters the state of chromatin and the interaction of non-histone protein/DNA acceptor sites with glucocorticoid receptors. These alterations may play a role in the acquisition of hormone resistance.

Binding of [3H]estradiol- and [3H]H1285-receptor complexes to rabbit uterine chromatin.

In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [3H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [3H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physiochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions.

Androgen induction of a specific uterine protein.

Studies were conducted to determine the ability of androgens in vitro to elicit the induction of a specific uterine protein (IP) normally attributed to estrogens. Both 5alpha-dihydrotestosterone (5alpha-DHT) and testosterone were effective in stimulating IP synthesis in rat uterine tissue in a concentration dependent manner (0.1 muM to 50 muM). 5alpha-DHT was more effective than testosterone and reached approximately 85% of the estradiol stimulated IP response at 10 muM and 50 muM; whereas testosterone was only able to achieve about a 70% IP response at 50 muM. This androgen stimulated IP synthesis was stereospecific since cis-testosterone and 5beta-DHT, inactive androgen isomers, were unable to evoke a detectable IP response at any concentration studied. Antiandrogens were unable to inhibit the 5alpha-DHT stimulated IP synthesis but antiestrogens were able to greatly inhibit the 5alpha-DHT and testosterone stimulated IP responses in a concentration dependent manner. The anti-estrogens, themselves, were very weak inducers of the IP response. The nuclear accumulation of the estrogen receptor by various androgens and inactive androgen isomers was also determined. Approximately 100% nuclear accumulation of receptor was attained with 1 muM 5alpha-DHT, whereas 50 muM testosterone was needed for 100% uptake. 5beta-DHT was unable to translocate the receptor at the lower concentrations tested, but caused a significant nuclear accumulation of 50 muM. Cis-testosterone was unable to cause the nuclear accumulation of the estrogen receptor at all concentrations studied. These studies suggest that some of the estrogen receptors accumulated in the nuclei by androgens, inactive androgen isomers, or antiestrogens may not be capable of eliciting a biological response.

Analysis of two forms of molybdate-stabilized estrogen receptor.

We recently reported that the calf uterine estrogen receptor prepared in 10-mM molybdate elutes as two components (peaks I and II) from DEAE-Sephadex columns with a linear KCl gradient. We have extended these studies and report that unoccupied cytosol receptors also elute as two peaks (0.21 and 0.25 m KCl), as determined by postlabeling experiments with [3H]estradiol. Since molybdate-stabilized cytosol estrogen receptors incubated in the presence of 0.3 M KCl as well as estrogen receptors in peaks I and II did not demonstrate receptor binding to DNA-cellulose, we conclude that both receptor forms are nonactivated. Also, [3H]estradiol-receptor complexes from cytosol prepared in 10 mM molybdate sediment as 6.7S in 5-20% sucrose density gradients containing 0.3 M KCl, whereas receptor complexes from cytosol prepared without molybdate sediment as 4.5S. Both peaks I and II were eluted from DEAE-Sephadex columns prepared with either 10 mM molybdate or 10 mM tungstate, with both phosphatase inhibitors having effectively blocked salt activation of the receptor. However, receptor preparations in the presence of 10 mM arsenate were not eluted from DEAE-Sephadex, and arsenate was unable to inhibit receptor activation by KCl. Sucrose density gradient analysis of peaks I and II indicates that peak I sediments at approximately 4.8S, whereas peak II sediments at approximately 6.3S. If pooled fractions of the leading portion of peak I are rechromatographed on another DEAE-Sephadex column, both peaks I and II are recovered. Likewise, if pooled fractions of the descending portion of peak II are rechromatographed, both peaks I and II were measured. Therefore, we conclude that there may be an equilibrium between two forms of the molybdate-stabilized calf uterine estrogen receptor.

Relationships between mammary estrogen receptor and estrogenic sensitivity. II. Binding of cytoplasmic receptor to chromatin.

Mammary glands from nulliparous mice are responsive to estradiol, whereas mammary glands from lactating mice are unresponsive, despite the presence of high concentrations of estrogen receptors. This study examined the relation between mammary estrogenic sensitivity and ability of mammary estrogen receptors to bind to intact chromatin as well as to partially deproteinized chromatin. Mammary chromatin was prepared from nulliparous and lactating mice, linked covalently to cellulose and deproteinized sequentially by 0-8 M guanidine chloride (Gdn X HCl). The binding of receptors to these various chromatin preparations was determined using partially purified [3H]estradiol-receptor complexes ([3H]ER) obtained by fractionation on diethylaminoethyl cellulose columns. The binding pattern of [3H]ER from nulliparous mice to chromatin fractions from either nulliparous or lactating mice revealed maximal binding activity with chromatin previously extracted with 4-6 M Gdn X HCl. Binding was of high affinity [dissociation constant (Kd) 3.6 X 10(-10) M], saturable and steroid receptor and species specific. However, mammary [3H]ER preparations from lactating mice bound poorly to intact chromatin as well as to the Gdn X HCl extracted chromatin fractions isolated from either mammary gland of nulliparous or lactating mice. In mixing experiments the estrogen receptor preparation from lactating mice decreased substantially the binding activity of [3H]ER from nulliparous mice to chromatin suggesting the presence of an inhibiting factor. Thus, these studies reveal that the unresponsiveness of lactating mammary glands to estradiol coexists with the inability of estrogen receptors from lactating mice to interact with specific high affinity sites on mammary chromatin and also that this impeded interaction of estrogen receptors with chromatin may be due to some inhibitor(s) present in the cytosol of lactating mammary glands.

Estrogen and antiestrogen binding to different forms of the molybdate-stabilized estrogen receptor.

We report that the calf uterine estrogen receptor, prepared in a Tris-molybdate buffer, bound by 10 nM [3H]estradiol and eluted by a KCl gradient from DEAE-cellulose columns, yielded only one very sharp receptor peak. Estrogen receptor prepared in phosphate buffer with molybdate and eluted with KCl also yielded only one sharp peak on DEAE-cellulose. However, if DEAE-Sephadex (with phosphate buffer plus molybdate) was used, the [3H]estradiol-receptor complex eluted with two sharp peaks at approximately 0.21 and 0.25 M KCl (Peaks I and II, respectively). But the high-affinity antiestrogen, [3H]H1285, bound to estrogen receptor, eluted only as Peak I and not as Peak II. Both the [3H]estradiol and [3H]H1285 binding peaks were saturable since they could be eliminated with 200-fold excess estradiol. Therefore, ion exchange chromatography using different resins and/or buffers may be useful for determining physicochemical differences in estrogen versus antiestrogen receptor complexes.

Tissue and species specificity of unmasked nuclear acceptor sites for the estrogen receptor of Squalus testes.

The testicular estrogen receptor of the shark Squalus acanthias is restricted to nuclear subfractions when tissue is homogenized in low salt buffers and adheres tightly to nuclei and DNA-cellulose even when exposed to high salt conditions. Therefore, we examined the binding characteristics of this receptor to chromatin subfractions from homologous and heterologous tissues. Squalus chromatin linked to cellulose and partially deproteinized by 0-8 M guanidine hydrochloride (GuHCl) gave extraction patterns similar to those obtained with mammalian and avian chromatin. Chromatin as prepared in our laboratory contained no bound estrogen receptor. The binding pattern of the [3H]estradiol-labeled nuclear estrogen receptor to chromatin fractions from Squalus testicular zones I/II (containing spermatogonia, spermatocytes, and high receptor levels) revealed maximal binding activity (acceptor sites) on chromatin previously extracted with 2-4 M GuHCl (350% increase over unextracted chromatin), with a 40% decrease from maximal binding at 5-8 M GuHCl. By contrast, binding to chromatin from zone III (containing spermatozoa and little or no detectable receptor) showed no major peak and 4 times less binding at 3 M GuHCl-extracted chromatin. We have previously shown that zones I and II contain the majority of testicular receptors and, presumably, are the primary sites of estrogen action, whereas receptor activity in zone III is minimal (less than 5%), indicating a secondary or nontarget tissue. Squalus testicular [3H]estradiol-receptor complexes bound minimally to rabbit uterine chromatin. Likewise, [3H]estradiol-receptor complexes from rabbit uterus, Squalus oviduct, or mouse testis bound minimally to Squalus testicular chromatin. Thus, maximal binding occurred only with Squalus zones I/II chromatin and Squalus testicular receptor. The binding of [3H]estradiol-receptor complexes to testicular chromatin (zones I/II) was of high affinity (Kd = 1.9 X 10(-10) M) and low capacity and was optimal in the presence of 150 mM KCl, but was unaffected by the addition of urea in an amount similar to that of Squalus body fluids (300 mM). [3H]Estradiol binding to chromatin required undenatured receptor and was competitively inhibited by radioinert estradiol-receptor complexes, confirming saturability of acceptor sites. The affinity of the Squalus estrogen receptor for homologous chromatin was in the same range as that reported for other systems, despite the unusual nuclear extractability characteristics of Squalus receptor. This study provides new evidence for tissue and species specificity of receptor binding to chromatin acceptor sites.

Acceptor sites on chromatin for receptor bound by estrogen versus antiestrogen in antiestrogen-sensitive and -resistant MCF-7 cells.

We examined the chromatin binding characteristics of estrogen receptor from MCF-7 cells when bound by [3H] estradiol vs. the high affinity antiestrogen [3H]H1285 [4-(N,N-diethylaminoethoxy) 4'-methoxy-alpha-(p-hydroxyphenyl)alpha-ethylstilbene]. Two sublines of MCF-7 cells were used: E-3, which is sensitive to antiestrogens, and RR, which is antiestrogen resistant and was selected for its ability to grow in the presence of tamoxifen. Chromatin was prepared from both E-3 and RR cells, linked covalently to cellulose and deproteinized sequentially by 0-8 M guanidine hydrochloride (Gdn X HCl). The chromatin acceptor activity unmasked by Gdn X HCl was determined using partially purified (30-fold) activated [3H]estradiol- or [3H] H1285-receptor complexes obtained by KCl step elution from DEAE-cellulose columns. With chromatin from E-3 cells, maximal binding (acceptor activity) for [3H]estradiol-receptor complexes prepared from either type of MCF-7 cells (E-3 or RR) was unmasked by 1 and 6 M Gdn X HCl, whereas [3H]H1285-receptor complexes exhibited maximal binding to 1 and 4 M Gdn X HCl-extracted chromatin subfractions. Chromatin prepared from RR cells was similar to that from E-3 cells in its binding activity for [3H]estradiol-receptor complexes. It differed, however, in that [3H]H1285-receptor complexes showed less chromatin acceptor site binding in general to 1-8 M Gdn X HCl-deproteinized RR chromatin, and the binding peak unmasked by 4 M Gdn X HCl was absent in chromatin from these cells. Receptor binding to chromatin was stable and was competitively inhibited by radioinert estradiol- or H1285-receptor complexes (but not by denatured receptors), demonstrating the saturability and specificity of these acceptor sites. Thus, estrogen receptor binds differently to chromatin depending on whether estradiol or an antiestrogen is bound to it. In addition, the acquisition of antiestrogen resistance by the RR subline of MCF-7 cells appears to result from alterations in the state of its chromatin rather than changes in the receptor itself. Finally, the observation that the chromatin from the resistant cells differs from that of the sensitive cells suggests that antiestrogens may be able to inhibit the growth of MCF-7 and other antiestrogen-sensitive cells not only by antagonizing the stimulatory effect of estrogens, but also by exerting some separate effect of their own.

Interrelationships between nuclear structure and ligand-activated intracellular receptors.

The role of ligand-activated intracellular receptors in activation of gene expression most probably involves a multistep process that requires alteration in chromatin structure. Results of studies with members of the steroid hormone receptor superfamily as well as the ligand-activated member of the basic region/helix-loop-helix family of transcriptional activators suggests that two different nuclear structures and their associated nuclear proteins are important in the initial stages of gene activation: the nuclear matrix and nucleosomes. Cell- and tissue-specific nuclear matrix proteins and the variant and modified histones appear to be important for tissue and species specificity of ligand-induced responses. Because the function of a receptor may be limited in vivo by promoter and transcription factor accessibility, the various roles of nuclear ligand-receptor complexes may involve interaction with nuclear matrix proteins and/or nucleosomes. Tissue-specific structural nuclear proteins could control the conformation (looping through matrix attachment regions) of the DNA and unwinding or rearrangement of nucleosomes, thus providing specificity to the expression of certain genes. Modulation of cooperative elements required for gene activation may involve association of the gene promoter with the nuclear matrix together with the presence of nucleosomes. Thus, the series of events involved in ligand-receptor activation of genes requires alterations in chromatin structure, which allow access of the receptor complex to elements within the gene.

Effect of the histone deacetylase inhibitor trichostatin A on the responsiveness of rat hepatocytes to dioxin.

Since histone acetylation has been implicated in the facilitation of specific gene transcription, we investigated the effect of increasing histone acetylation through inhibition of histone deacetylase on 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induction of P4501A activity in cultured rat hepatocytes. Inhibition of histone deacetylation was accomplished with addition of trichostatin A (TSA) to the incubation medium, and P4501A activity was measured spectrofluorometrically by determination of the rate of resorufin formation by ethoxyresorufin-O-deethylase (EROD). While TSA alone (5-200 ng/mL) had no effect on EROD activity, TSA potentiated the effect of various concentrations (10(-12) to 10(-10) M) of TCDD. Addition of 200 ng TSA/mL with TCDD resulted in an increased EROD activity of approximately 200% compared with TCDD alone. When TSA was removed from the cells after various incubation times (2, 6, 24 hr) by successive washings with TSA-free medium, it was determined that TSA was required for 24 hr in order to potentiate the effects of a 48-hr incubation with TCDD. In addition to measurement of EROD activity, P4501A1 and 1A2 microsomal protein were determined by western immunoblotting analysis. While neither P4501A1 nor 1A2 was detectable in the presence of TSA alone, P4501A1 was present after incubation of cells with TCDD in the presence or absence of TSA. TCDD plus TSA also resulted in the formation of P4501A2. The results of this study suggest an important role for histone acetylation in the action of TCDD on induction of P4501A enzymes.

Binding of the Ah receptor to receptor binding factors in chromatin.

Dioxin induces biological responses through interaction with a specific intracellular receptor, the Ah receptor, and the subsequent interaction of the Ah receptor with chromatin. We report the binding of the Ah receptor, partially purified from rabbit liver, to receptor binding factors in chromatin. Rabbit liver chromatin proteins (CP) were isolated by adsorption of chromatin to hydroxylapatite followed by sequential extraction with 1-8 M GdnHCl. To assay for receptor binding a portion of each CP fraction was reconstituted to rabbit double-stranded DNA using a reverse gradient dialysis of 7.5 to 0 M GdnHCl. These reconstituted nucleoacidic proteins were then examined for binding to [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD)-receptor complexes by the streptomycin filter assay. Prior to the binding assay, [3H]TCDD-receptor complexes were partially purified by step elution from DEAE-cellulose columns. CP fractions 2, 5, and 7 were found to bind to the Ah receptor with high affinity. Scatchard analysis yielded Kd values in the nanomolar range. Competition with 2-fold excess unlabeled TCDD-receptor complexes was demonstrated, and binding was reduced markedly when the receptor was prepared in the presence of 10 mM molybdate. Such chromatin receptor binding factors (RBFs) may participate in the interaction of receptor with specific DNA sequences resulting in modulation of specific gene expression.

Estrogen receptor interaction with specific histones. Binding to genomic DNA and an estrogen response element.

Chromosomal proteins that impart high affinity and specificity to the binding of the estrogen receptor (ER) to DNA are termed estrogen receptor binding factors (ERBFs). Certain partially purified chromosomal protein fractions obtained from rabbit uterine chromatin by extraction with various molarities of GdnHCl when reconstituted to double-stranded DNA demonstrated high affinity binding for the ER. We report the purification and characterization of ERBFs in the chromosomal protein fraction extracted with 4 M GdnHCl (CP4) after large scale purification. These protein fractions were further purified by CL-Sepharose 6B column chromatography which resolved fractions from CP4 that recognized the ER bound by estrogen only or antiestrogen only. Thus, these hydrophobic chromosomal proteins enhanced the binding of the ER to reconstituted chromatin. To further investigate the interaction of ERBFs with ER, gel mobility shift assays were performed. The highly purified CP4 fraction with ERBF activity in the binding assay with reconstituted chromatin caused an increase in the formation of the retarded ER-estrogen responsive element (ERE) band. Thus, chromatin contains specific ERBFs for ER bound by estrogen which enhance the binding of ER to genomic DNA and a target ERE sequence. Further purification of the CL-Sepharose fraction with ERBF activity was achieved by preparative SDS-PAGE. ERBF activity was attributed to proteins with approximate molecular weights of 16,000, 13,000, and 12,000 and a pl of > 9.0. Peptides were partially sequenced by Edman degradation and were found to have identity with histones H2B and H4. A 17 kDa protein without ERBF activity was identified as H3. Since these histones were not readily extracted from chromatin with 3 M NaCl or 1-3 M GdnHCl, we postulate that some ERBFs may be histone variants or modified histones that display a very high affinity for DNA and ER.

Naringenin: a weakly estrogenic bioflavonoid that exhibits antiestrogenic activity.

Treatment of immature 21-day-old female Sprague-Dawley rats with 17 beta-estradiol (E2) (0.5 microgram/rat) caused a significant increase in uterine wet weight, DNA synthesis, progesterone receptor (PR) binding, and peroxidase activity. At doses as high as 40 mg/rat, the bioflavonoid naringenin did not cause a significant increase in any of these E2-induced responses. However, in rats cotreated with E2 (0.5 microgram/rat) plus naringenin (30 mg/rat); there was a significant decrease in E2-induced uterine wet weight, DNA synthesis, PR binding, and peroxidase activity, indicating that naringenin exhibits antiestrogenic activity in the immature rodent uterus. The binding of uterine nuclear extracts to a 32P-labeled estrogen responsive element (ERE) or progesterone responsive element (PRE) was determined using gel electrophoretic band shift assays. Incubation of [32P]ERE with uterine nuclear extracts from rats treated with naringenin or E2 resulted in the formation of estrogen receptor (ER):ERE complexes; a higher mobility complex was prominent in the extracts from E2-treated rats, whereas a lower mobility complex was observed using nuclear extracts from naringenin-treated animals. There was a significant decrease in the intensity of the E2-induced complex using nuclear extracts from rats treated with E2 plus naringenin. In contrast, transformed cytosol from control rats gave an intense ER:ERE complex, whereas the intensity of the band was decreased markedly using transformed uterine cytosol from treated rats. Formation of a PR:PRE complex was also determined using transformed uterine cytosol. Cytosol from E2-treated rats gave an intense retarded band, whereas only weak bands were observed using cytosols from DMSO- (solvent), naringenin-, or naringenin plus E2-treated cells. The results of in vitro studies showed that 1 nM E2 increased (3- to 4-fold) the growth of MCF-7 human breast cancer cells, whereas 1-1000 nM naringenin had no effect on cell proliferation. In cells cotreated with 1 nM E2 plus 1000 nM naringenin, there was a significant decrease in E2-induced cell growth. In MCF-7 cells transiently transfected with a pS2 promoter-regulated luciferase reporter gene, naringenin exhibited weak estrogenic activity. In cells cotreated with 0.1 or 1.0 microM naringenin plus 1 nM E2, naringenin inhibited E2-induced luciferase activity. The results of these studies confirmed that naringenin is a weak estrogen that also exhibits partial antiestrogenic activity in the female rat uterus and MCF-7 human breast cancer cells.

Purification and characterization of an Ah receptor binding factor in chromatin.

Dioxin induces biological responses through interaction with a specific intracellular receptor, the Ah receptor, and the subsequent interaction of the Ah receptor with chromatin. We previously reported the binding of the Ah receptor, partially purified form rabbit liver, to receptor binding factors (termed AhRBFs) in chromatin. Rabbit liver chromatin proteins (CP) were isolated by absorption of chromatin to hydroxylapatite followed by sequential extraction with 3 M NaCl and 1-8 M guanidine hydrochloride (GdnHCl). In the present study, we continued the purification of the CP5 fraction, which exhibited AhRBF activity. The proteins in CP5 were separated by CL-Sepharose 6B column chromatography resolving lower molecular weight fractions. To assay for receptor binding, a portion of each Cl-Sepharose 6B fraction was reconstituted to rabbit double-stranded DNA (dsDNA) using a reverse gradient dialysis of 7.5 to 0.0 M GdnHCl. These reconstituted chromatins were then examined for binding to [3H]-2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD)-receptor complexes by the streptomycin filter binding assay. Two protein fractions with a molecular weight in the range of 10,000-14,000 demonstrated high affinity binding to the Ah receptor. The binding of AhRBFs reconstituted to dsDNA was shown, by competition experiments with Ah receptor bound by unlabeled TCDD (TCDD-R), to be > 90% specific for [3H]TCDD-R. Further purification was achieved by preparative ADS-PAGE, and AhRBF activity was attributed to two fractions with molecular weights between 12,000 and 10,000. A kDa protein with AhRBF activity was found to have an isoelectric point (pI) of > or = 10. The 12 kDa AhRBF was sequenced by Edman degradation after cyanogen bromide cleavage and identified as histone H4. Although histone H4 has been postulated to interact with transcription factors in a variety of systems, this is the first report of a specific interaction of AhR with histone H4.

Failure of cannabinoid compounds to stimulate estrogen receptors.

delta 9-Tetrahydrocannabinol (THC), the primary active compound in Cannabis sativa (marihuana), and other cannabinoid receptor agonists exert potent effects on luteinizing hormone and prolactin release in animal models and humans. Compounds possessing the tricyclic cannabinoid structure, including delta 9-THC and cannabidiol, have been reported to interact with rodent uterine estrogen receptors in ligand binding assays. The present study tested the hypothesis that cannabinoid compounds produce a direct activation of estrogen receptors. We investigated whether cannabinoid compounds exhibit estrogen-induced mitogenesis in MCF-7 breast cancer cells. Under conditions in which 10 pM estradiol promoted MCF-7 cell proliferation, no response was observed with biologically relevant concentrations (< = 10 microM) of delta 9-THC or its tricyclic analog desacetyllevonantradol. No response was observed with cannabidiol, a bicyclic cannabinoid compound that exhibits no cannabimimetic behavioral effects but has been reported to bind to the estrogen receptor in vitro. delta 9-THC also failed to antagonize the response to estradiol under conditions in which the antiestrogen LY156758 (keoxifene; raloxifene) was effective. The phytoestrogen formononetin behaved as an estrogen at high concentrations, and this response was antagonized by LY156758. We also investigated the ability of cannabinoid compounds to stimulate transcription of an EREtkCAT reporter gene transiently transfected into MCF-7 cells. Neither delta 9-THC, desacetyllevonantradol, nor cannabidiol stimulated transcriptional activity. We conclude that psychoactive or inactive compounds of the cannabinoid structural class fail to behave as agonists in appropriate assays of estrogen receptor responses in vitro.

Effect of estrogens on IL-1beta promoter activity.

It is well documented that steroid hormones modulate cytokine gene expression. In some tissues estrogens are known to suppress cytokine production while in other tissue types, cytokine expression is enhanced by the hormone. This study was conducted to investigate the regulatory mechanisms which underlie the modulation of the interleukin-1beta (IL-1beta) gene at the transcription level. To accomplish this, the macrophage cell line RAW264.7, which appeared insensitive to 17beta-estradiol (E2) treatment, was stably transfected with the human estrogen receptor (ER) and an IL-1beta promoter-CAT reporter construct. E2 markedly enhanced LPS-induced IL-1beta promoter-driven CAT activity in an E2 dose dependent manner. This responsiveness was estrogen specific since no synergism was observed between LPS and the sex steroids testosterone or progesterone while the estrogen analogue 17alpha-estradiol stimulated only at 10 to 100 times the amount required for 17beta-E2. Several antiestrogens, H1285, ICI 182 780, and tamoxifen inhibited the estrogen stimulated enhancement of IL-1beta promoter activity in a dose-dependent manner, indicating that this effect was indeed mediated through the ER in a ligand dependent manner. The estrogenic effect appeared to be indirect and time dependent since the addition of E2 was required hours prior to LPS stimulation; addition of E2 and LPS at the same time resulted in a greatly reduced estrogenic effect. The estrogen metabolites 17-epiestriol and 16-keto-17beta-E2 displayed an estrogenic response virtually indistinguishable from E2. 4-Hydroxyestradiol displayed activity only at 100-fold the concentration of E2 while 2-hydroxyestrone showed no activity at any of the concentrations tested. Overall the results demonstrate that E2 and some metabolites of E2 synergize with LPS to markedly enhance IL-1beta promoter activity through ER mediated processes.

Effects of antiestrogen versus antiprogestin on transformed and nontransformed steroid receptors.

In order to determine if different physicochemical properties exist among antihormone-receptor complexes, we have compared the interaction of the antiprogestin RU486 with progesterone receptor (PR) versus the triphenylethylene antiestrogen H1285 (4-(N,N-diethyl-aminoethoxy)-4'-methoxy-alpha-(p-hydroxyphenyl-alp ha'- ethylstilbene] with estrogen receptor (ER) from rabbit uterine tissue. Contrary to other reports, we observed no difference in the sedimentation properties of transformed PR (4S) when bound by the antagonist RU486 versus the progesterone agonist R5020 in either cytosol or DEAE partially-purified receptor preparations analyzed on sucrose gradients containing 0.3 M KCl. In addition, we found no difference in the sedimentation properties of these receptor preparations in the presence of 10 mM sodium molybdate: the nontransformed RU486-PR and nontransformed R5020-PR both sedimented as a 6S species. These same results were obtained when the receptor preparation and gradient analysis were performed in the absence of monothioglycerol. Likewise, there was no change in the sedimentation properties of the transformed PR when the receptor, partially purified in the absence of molybdate, was analyzed on sucrose gradients containing 10 mM sodium molybdate to prevent receptor alteration during centrifugation. From DNA-cellulose assays performed with partially purified PR in the absence of molybdate we determined that the 4S form of R5020-PR and RU486-PR is transformed receptor; whereas in the presence of molybdate, the 6S species is nontransformed. In contrast, we found a different pattern of sedimentation when comparing transformed antiestrogen-receptor complexes with transformed estrogen-receptor complexes. In this case, transformed H1285-ER sedimented as 6S and estradiol-ER sedimented as 4S. We conclude from these experiments that these two antihormones, RU486 and H1285, may have different mechanisms of action in their antagonism of steroid hormone action. Antiestrogen stabilizes the salt-transformed ER as a dimer while antiprogestin appears to permit dissociation of the oligomeric form of the receptor to the monomeric form.