Biomedical subjects
Publications and source records attributed to C M Klinge.
This paper provides an annotated summary of the current literature detailing what is known about the interaction of antiestrogen-liganded estrogen receptor (ER) vs estradiol-liganded ER with DNA in vitro and in vivo. It describes the current models of the mechanism by which antiestrogen-liganded ER blocks transcription of certain estrogen-regulated genes.
We have recently used DNA containing estrogen responsive element (ERE) sequences for affinity purification to prepare calf uterine estrogen receptor (ER) at near homogeneity. The capacity of this purified ER to alter DNA topology upon binding was examined. Although the ER is not a topoisomerase, the presence of ER changes the distribution of topoisomers generated by incubation of plasmid DNA with excess wheat germ topoisomerase I. This effect is larger in plasmids containing a consensus ERE sequence. Two dimensional gel electrophoretic analysis suggested that interaction of ER and ERE causes negative supercoiling in regions of the plasmid accessible to topoisomerase I, resulting from overwinding of DNA contacting the ER. The extent of topological alteration was dependent on ER concentration. We suggest that the observed conformational changes in the DNA could have a role in regulation of transcription.
The interaction of partially purified calf uterine estradiol-charged estrogen receptor ([3H]ER) with rat nuclei was studied in vitro. We previously observed a significantly greater number of [3H]ER binding sites (at saturation) in nuclei of R3230AC mammary tumors from intact vs ovariectomized (ovex) rats with no difference in the affinity of [3H]ER binding for these nuclei. We now report on the nuclease sensitivity of [3H]ER binding sites in nuclei from these tumors and from normal rat tissues. Digestion of tumor nuclei with deoxyribonuclease I (DNase I) prior to incubation with [3H]ER in vitro resulted in a progressive loss of [3H]ER binding capacity, which was not accompanied by alterations in the affinity of [3H]ER for the nuclei (Kd = 1-3 nM). A significantly lower concentration (P less than 0.005) of DNase I eliminated 50% of the [3H]ER binding sites in nuclei of tumors from intact hosts (8 unit.min/ml) compared to tumors from ovex hosts (22 unit.min/ml). These results indicate that DNA regions capable of binding ER are more susceptible to DNase I digestion in tumors from intact rats than those from ovex hosts, suggesting that the endogenous hormonal milieu is responsible, at least in part, for maintenance of nuclease-sensitive DNA conformations in this hormone-responsive mammary tumor. The amount of DNase I required to eliminate 50% of [3H]ER binding to nuclei from lactating mammary gland, liver, and kidney ranged from 14 to 56 unit.min/ml. Therefore, accessibility of [3H]ER binding sites to nuclease digestion in normal rat tissue is generally less than that of R3230AC tumors.
Reproducible, rapid measurement of estrogen receptor (ER) binding to DNA was accomplished in microtiter wells treated so that ER-DNA complexes or DNA bound in preference to free ER. Mixtures of 35S-labeled DNA and [3H]estrogen-charged ER ([3H]ER), incubated to equilibrium in microfuge tubes, were transferred to microtiter wells previously treated with histone followed by gelatin. After binding of the DNA or ER-DNA complex to the treated wells, free ER was removed by washing. Radioactivity retained in each well was measured by placing individual wells from snap-apart microtiter plates directly in scintillation fluid. Binding of DNA was saturable, and ER-DNA complex binding was complete within 2 h at 4 C. The use of 35S-labeled DNA and [3H]ER allowed stoichiometric determination of ER bound to DNA. The amount of ER specifically bound to a consensus estrogen-responsive element (ERE) containing the inverted repeat GGTCAgagTGACC was determined by comparing ER bound to plasmid containing or lacking the ERE. At saturating concentrations of ER, plasmids bearing one, two, and four EREs in tandem bound approximately one, two, and four dimeric ER molecules, respectively. Scatchard analysis of saturation binding data revealed a Kd of 0.15 nM for specific ER binding to a single ERE site. Thus, the assay detects ER retaining both DNA-binding and estrogen-binding functions. ER complexed with DNA in the well was also detected using a monoclonal antibody specific for the receptor. Simple modifications of this method would allow study of other DNA-protein interactions.
Rapid purification of calf uterine estrogen receptor (ER) to near homogeneity has been accomplished by use of sequence-specific DNA affinity resin. Very high selectivity for the estrogen receptor is achieved through the use of DNA-Sepharose containing eight tandem copies of a consensus estrogen response element (ERE) DNA sequence. The highly purified ER prepared by this new scheme may be labeled economically with ligands of high specific activity. This purification scheme selects for intact receptors retaining function in both estrogen-binding and DNA-binding domains. Purified receptor has an electrophoretic mobility consistent with a molecular weight of 68,000, sediments as a 5S species on sucrose gradients, and reacts with antibody specific to the human estrogen receptor.
The effects of various hormonal perturbations that alter growth of two different rat mammary tumors in vivo were investigated by study of the interactions of [3H]estradiol-charged estrogen receptors ([3H]ER) with tumor nuclei in vitro. Nuclei from the transplantable R3230AC adenocarcinoma were isolated after ovariectomy, estrogen treatment, or progesterone treatment. Saturable specific binding of [3H]ER to nuclei was assayed in this in vivo-like system. Scatchard analysis of [3H]ER-nuclear binding data indicated that these perturbations did not affect affinity, which ranged from Kd 1.0 to 2.4 nM. However, the number of [3H]ER-binding sites/nucleus was altered according to the treatment: intact rats, 94,500 +/- 4,200; ovariectomy, 70,400 +/- 3,200; ovariectomy plus estradiol, 82,100 +/- 5,800; and ovariectomy plus progesterone, 73,900 +/- 2,500. Nuclei from primary tumors induced by 7,12-dimethylbenz(a)anthracene displayed similar affinities for [3H]ER, although these tumors had fewer binding sites per nucleus. Animals bearing 7,12-dimethylbenz(a)-anthracene-induced tumors were either ovariectomized or made diabetic by administration of streptozotocin, perturbations that cause regression of the majority of tumors. The number of [3H]ER binding sites per nucleus, in tumors classified according to growth characteristics in host animals subsequent to hormonal perturbation, was: intact growing 36,300 +/- 3,400; ovex regressing, 15,400 +/- 3,400; ovex, estrogen-treated growing, 28,100 +/- 2,700; diabetic regressing, 19,500 +/- 2,400; diabetic static, 32,100 and diabetic growing, 42,000 +/- 7,100. These results indicate that (a) the number of nuclear ER-binding sites can be reduced by hormonal interventions that cause tumor regression and (b) endogenous ovarian hormones may play a role in regulating nuclear ER binding.
Interaction of [3H]4-hydroxytamoxifen-charged estrogen receptor [( 3H]AER) with nuclei was compared to that of [3H]17 beta-estradiol-charged estrogen receptor [( 3H]ER) in vitro. Specificity of [3H]AER binding was demonstrated since more than 90% of [3H]AER binding was displaced by ten-fold excess estradiol-charged ER. For R3230AC tumors, the number of [3H]AER binding sites was approximately 40% lower than the number of [3H]ER binding sites. There were no differences in affinity of binding of these receptors complexes (Kd range 0.7-1.6 nM). In contrast 0.7-1.6 nM). In contrast to a reduction of [3H]ER binding after ovariectomy, no difference in the number of [3H]AER binding sites was seen among tumors from intact, ovex, or estrogen-treated ovex rats. These results suggest that [3H]AER bind to 60% of the sites that bind [3H]ER, and that neither tissue type nor host ovarian status affects the number of nuclear [3H]AER binding sites.
To investigate the role of hormones in regulating growth of neoplastic mammary cells, we established a heterologous assay for studying interactions of partially purified calf uterine [3H]estradiol-charged estrogen receptor ([3H]ER) with rat tumor nuclei in vitro. This system displays saturable high affinity binding of [3H]ER which is time and salt dependent. Optimal assay conditions required for the heterologous system were identical to those we reported for the homologous calf nuclear binding system. Specificity of [3H]ER binding was demonstrated; 10-fold excess unlabeled estrogen-charged ER (EcR) competed for greater than 90% of the [3H]ER binding sites and binding of [3H]estradiol (not complexed with ER) was less than 1% of [3H]ER binding. Binding of [3H]ER displayed tissue specificity in decreasing order: R3230AC mammary tumor greater than lactating mammary gland = liver greater than kidney greater than lung. Scatchard analysis of saturation data provided estimates of binding affinity to nuclei from R3230AC mammary tumors [Kd, 2.0 +/- 0.3 (SE) nM); the number of binding sites per nucleus for R3230AC tumors was 95,000 +/- 13,800. [3H]ER binding to nuclei isolated from R3230AC rat mammary tumors grown in intact rats was 40% higher than that observed in tumors from ovariectomized animals. Results of administration of individual pharmacological doses of either progesterone or an estrogen to ovariectomized rats did not restore nuclear ER binding levels in R3230AC tumors to those detected in tumors from intact rats. These results suggest that the physiological levels of endogenous hormones produced by the ovaries are important in regulating the number of ER binding sites in nuclei from these mammary tumors.
Extracts of whole tissue or isolated nuclei from lactating rat mammary gland that has diminished cell replication capacity were more active than the corresponding extracts of pregnant rat mammary gland that contains actively replicating cells in causing a dose-dependent inhibition of DNA polymerase alpha in vitro. Purification of the inhibitor from both tissue and nuclear extracts using a sequence of Sephacryl S200, DEAE-cellulose and CM52 columns confirmed the above assay results. Using the same assay and purification procedures, both tissue and nuclear extracts from the rapidly growing transplanted R3220AC mammary tumors exhibited very little or no inhibitor activity. The partially purified mammary inhibitor (mol. wt of 155kD, high A280 nm/A260 nm ratio, heat labile) was equally inhibitory to the purified DNA polymerase alpha from either R3230AC tumor or calf thymus, and to the nuclear matrix bound DNA polymerase alpha of R3230AC tumor.
An inhibitory component that diminishes estrogen receptor (ER) binding to nuclei in vitro is present in cytosol prepared from calf uterus. The inhibitor is heat stable and resistant to enzymatic treatment with trypsin, chymotrypsin, proteinase K, deoxyribonuclease I, or ribonucleases A, T1, and U2. Results of chromatography on DEAE-cellulose and Sephadex G-150 indicate that the factor is a negatively charged macromolecule. Inhibitory activity is sensitive to sequential digestion with chondroitinase ABC, hyaluronidase, and heparinase. Approximately 70% of the inhibitory activity is destroyed by treatment with heparinase alone. Heparitinase destroys only 30% of this activity. Furthermore, the addition of pure hyaluronic acid or chondroitin sulfate to the ER-nuclei binding assay results in little inhibition, whereas addition of heparin inhibits 75% of receptor binding. Overall, these results indicate that glycosaminoglycans, present in bovine uterine cytosol, are capable of inhibiting ER-nuclei interactions. The most potent inhibitory glycosaminoglycan displays heparin-like characteristics.
DNA polymerase alpha activity was markedly higher in all nuclear subfractions, including nuclear matrix, from transplanted R3230AC mammary adenocarcinomas than in the analogous fractions from mammary gland of same tumor-bearing pregnant or lactating rats. Changes in host lactational status had no significant effect on subnuclear distribution of tumor DNA polymerase alpha activity, with the majority (60-75%) localized in soluble nucleoplasm and a significant amount (13-20%) retained in the nuclear matrix. In the host mammary gland, nuclear matrix-bound DNA polymerase alpha was highest, accounting for 48% of total nuclear activity, during late pregnancy when mammary cells undergo rapid raplication. During lactation, when cells in mammary gland cease to divide, only 8% of enzyme activity was in the nuclear matrix, while the majority (60-80%) of DNA polymerase alpha activity was localized in nucleoplasm. In both R3230AC tumor and mammary gland regardless of host's lactational status, the majority (60-80%) of DNA polymerase beta activity was localized in the high salt-soluble chromatin. These present data thus suggest that, regardless of host lactational status, R3230AC tumor has many cycling cells, each with a large pool of DNA polymerase alpha molecules maintaining maximal and constant replicative activity, while normal mammary gland cells have a smaller pool of DNA polymerase alpha which become primarily matrix-bound only during active cell replication during late pregnancy. A constant localization of nuclear DNA polymerase beta in chromatin in both mammary gland and the tumor suggest it is not important in mammary cell proliferation.
We determined that there is a protein in rat liver capable of inhibiting DNA polymerase alpha. To assay for this inhibitor, DNA polymerase alpha was purified from R3230AC rat mammary tumor, a rich source of this enzyme. Protein fractions from Sephacryl S200 gel filtration of total soluble liver extract showing inhibition of DNA polymerase alpha were further chromatographed on DEAE-cellulose. This step revealed two inhibitor protein populations with the major form corresponding to a molecular weight of 143,000 dalton. Soluble extract from isolated rat liver nuclei also showed the presence of at least two inhibitors; the major form was 200,000+ dalton in molecular weight. Both the 143,000 and 200,000+ dalton inhibitor proteins were capable of inhibiting the R3230AC tumor DNA polymerase alpha in a dose-dependent manner. These inhibitors exhibited similar inhibition of nuclear matrix-associated DNA polymerase alpha from either the R3230AC tumor or from regenerating rat liver.
Bovine estrogen receptor (ER) was purified to near homogeneity by estrogen response element (ERE) affinity chromatography, and its ERE binding ability was measured in vitro. Highly purified ER bound EREs with reduced affinity compared to partially purified ER. Partially purified ER contained hsp70, but highly purified ER did not. We examined whether addition of purified recombinant human hsp70 or purified bovine hsp70 would restore the higher ERE binding affinity, stoichiometry, and ligand retention detected with partially purified receptor and how hsp70 affected the rate of ER-ERE association and dissociation. ER-ERE binding was not affected by antibodies to either constitutive or induced forms of hsp70, regardless of ER purity. Addition of purified hsp70, with or without ATP and Mg2+, did not affect the association or dissociation rates of highly purified liganded ER binding to ERE. hsp70 Did not alter the total amount of ER-ERE complex formed. Similarly, hsp70 did not affect the rate of [3H]estradiol (E2) or [3H]4-hydroxytamoxifen (4-OHT) ligand dissociation from ER in the presence or absence of EREs. These data contrast with a report showing that maximal ERE binding by highly purified recombinant human ER required hsp70. We conclude that ER, purified from a physiological source, i.e., calf uterus, does not require hsp70 for maximal ER-ERE binding in vitro. Additionally, once ER is activated and bound by ligand, the receptor assumes its proper tertiary structure, and hsp70 does not impact ER ligand binding domain conformation.