Biomedical subjects
Publications and source records attributed to T C Spelsberg.
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A high-affinity localization of [2H]methylcholanthrene and/or its metabolites to a specific nuclear fraction (Fraction l) between 4 and 72 hr of exposure is described. Other carcinogenic hydrocarbons, such as benzo(a)pyrene and dibenz(a,h)anthracene, demonstrate a similar markded localization in Fraction l after 24 hr of incubation. The weak carcinogen dibenz(a,c)anthracene, as well as steroid hormones, sho little localization in this fraction. Two types of binding are measured:and organic solvent: extractable (non-covalent) binding and a nonextractable (covalent) binding. Maximal levels of the combined extractable and nonextractable binding per mass DNA are found at 24 hr of exposure, while at 48 and 72 hr of exposure the binding is reduced. The highest level of the nonextractable binding per mass DNA is also observed at the 24-hr exposure period. However, as the period of exposure increases, the proportion of the total nuclear-bound radioactivity representing the non-extractable type increases. Analysis by high-pressure liquid chromatography of the extractable radioactivity from the fractions indicates that the longer the period of exposure, the greater the extent of metabolism of 3-methylchol-anthrene. When 7,8-benzoflavone (a flavanoid hydroxylase inhibitor) is included in the incubations, practically all metabolic alterations of the parent compound are prevented. In addition, the time-dependent increase in nonextractable radioactivity from all nuclear subfractions is prevented. A metabolic-dependent "covalent" binding of carcinogenic polycyclic aromatic hydrocarbons to the barious nuclear subfractions of chromatin is suggested. This covalent binding is markedly localized in a specific fraction of the chromatin containing rapidly labeled nascent RNA.
Data are presented to explain discrepancies in the literature involving the in vitro binding of steroid receptor complexes to isolated nuclei and chromatin. The type of binding in vitro of the progesterone-receptor complex to nuclei, chromatin, or DNA of hen organs is largely determined by the ionic strength of the medium. Low ionic conditions (0.01 to 0.05 molar potassium chloride) result in a nonspecific, nonsaturable binding, while high ionic conditions (0.15 to 0.20 molar potassium chloride) create a tissue-specific, saturable binding. Pure DNA binds the steroid receptor complex extensively in low salt but very little in the higher salt conditions.
The effects of estrogen, progesterone and estrogen + progesterone combined on nuclear transcriptional processes in oviducts of immature chicks, previously withdrawn from estrogen, are reported. The responses to the steroids of the endogenous nuclear RNA polymerase activities, both nucleolar (I) and nucleoplasmic (II), the chromatin compositions and template capacities, and the appearance of ovalbumin messenger RNA (mRNA) are compared. When immature chicks (previously treated at 14 days with estrogen) are withdrawn from estrogen treatment, there is a gradual reduction in both polymerase activities. Diurnal variations in polymerase II activties in the oviduct of withdrawn chicks required that subsequent experiments include time-matched controls. The hormones alter RNA polymerase II and II activities in vivo as assayed in isolated nuclei. Progesterone represses the polymerase I and II activities, while estrogen alone and estrogen + progesterone enhance both polymerase activities immediately after injection. Diethylstillbestrol, a synthetic estrogen, causes changes similar to those of estrogen. The effects of these steroids on the polymerases are detected within 15 min of hormone injection. Changes in the capacities of chromatins to serve as template for RNA synthesis in general correlated with changes in polymerase II activities. Interestingly, in the case of estrogen treatment, the acidic chromatin protein (but not histone) levels fluctuate positively with the template capacities of the chromatin. An antagonism between estrogen and progesterone is observed in the responses of both RNA polymerases I and II activities as well as in the chromatin template capacity. Levels of messenger RNA coding for ovalbumin, as detected by hybridization with labeled complementary DNA, increase in oviducts of withdrawn chicks within 2--3 of the injection of estrogen, progesterone or estrogen + progesterone. This rapid accumulation of ovalbumin mRNA is not accompanied in each case by a similar increase in polymerase II activity or chromatin template capacity.
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1. Varied doses of labelled or unlabelled progesterone were injected into immature chicks which had previously been stimulated with oestrogen. The concentrations of nuclear bound [3H]progesterone were correlated with the effects of the hormone on endogenous RNA polymerase I and II activities in isolated oviduct nuclei. 2. The extent of nuclear localization of [3H]progesterone in oviduct (a progesterone target tissue) was shown to be much greater than in lung (non-target tissue). The conccentration of bivalent cations in solvents used in the nuclei isolations has a marked effect on the amount of bound hormone in the nuclei. 3. Evidence for the existence of several classes of binding sites for progesterone in the oviduct nuclei is given. These classes represent about 1000) 10000 and 100000 molecules of the hormone per cell nucleus and are saturated by injecting approx. 10, 100 and 1000 mug of progesterone respectively. 4. When saturation of the first (highest affinity) class of nuclear sites occurs, a marked inhibition in RNA polymerase II (but not RNA polymerase I) activity was observed. When the second class of sites was saturated, alterations in both RNA polymerase I and II activities were observed. Binding to the third class of nuclear binding sites was not accompained by further changes in polymerase activity. It is suggested that the first two classes of nuclear binding sites may represent functional sites for progesterone action in the chick oviduct.
Steroid hormones, including progesterone, are known to bind with high affinity (Kd approximately 1x10(-10)M) to receptor proteins once they enter target cells. This complex (the progesterone-receptor) then undergoes a temperature-and/or salt-dependent activation which allows it to migrate to the cell nucleus and to bind to the deoxyribonucleoproteins. The present studies demonstrate that binding the hormone-receptor complex in vitro to isolated nuclei from the oviducts of laying hens required the same conditions as do other studies of bbinding in vitro reported previously, e.g. the hormone must be complexed to intact and activated receptor. The assay of the nuclear binding by using multiple concentrations of progesterone receptor reveals the presence of more than one class of binding site in the oviduct nuclei. The affinity of each of these classes of binding sites range from Kd approximately 1x10(-9)-1x10(-8)M. Assays using free steroid (not complexed with receptor) show no binding to these sites. The binding to each of the classes of sites, displays a differential stability to increasing ionic concentrations, suggesting primarily an ionic-type interaction for all classes. Only the highest-affinity class of binding site is capable of binding progesterone receptor under physioligical-saline conditions. This class represent 6000-10000 sites per cell nucleus and resembles the sites detected in vivo (Spelsberg, 1976, Biochem. J. 156, 391-398) which cause maximal transcriptional response when saturated with the progesterone receptor. The multiple binding sites for the progesterone receptor either are not present or are found in limited numbers in the nuclei of non-target organs. Differences in extent of binding to the nuclear material between a target tissue (oviduct) and other tissues (spleen or erythrocyte) are markedly dependent on the ionic conditions, and are probably due to binding to different classes of sites in the nuclei.
The multiple classes of binding sites for the progesterone-receptor complex in hen oviduct muclei were found to be of chromatin origin. The highest-affinity, and presumably most physiologically important class, is localized in oviduct chromatin and contains approx. 6000-10000 sites per nucleus. None of these sites is detected in spleen chromatin. Two new techniques were used for assaying rapidly the binding of steroid-receptor complexes to soluble deoxyribonucleoproteins in vito. The extent of high-affinity binding by the nucleo-acidic protein fraction from spleen chromatin is as great as that by the nucleo-acidic protein from oviduct chromatin. Consequently the tissue-specific nuclear binding of the progesterone receptor is found not to be a consequence of the absence of the nuclear binding sites (acceptors) from chromatin of non-target tissue (spleen), but rather a result of complete masking of these sites. In the target-tissue (oviduct) chromatin, approx. 70% of the high-affinity acceptor sites are also masked. Acidic proteins, and not histones, appear to be responsible for the masking of these acceptor sites. In addition, acidic proteins represent (or at least are an essential part of) these high-affinity sites in the oviduct nucleus. Pure DNA displays a few high-and many low-affinity binding sites. In support of previous work with immature chicks, the acidic protein fraction of the nucleo-acidic results thus support the hypotheis that protein complexed with DNA, and not DNA alone, represent the high-affinity binding sites for the steroid-receptor complexes in nuclear chromatin. The lower-affinity classes of binding sites may represent DNA and/or other nuclear components.
Adult male rats were subjected either to sham operation or to hypophysectomy and adrenalectomy and maintained for a total of 10 days before treatment with growth hormone. Results of the early effects of growth hormone on the activities of the mixed-function oxidases in rat liver over a 96h period after growth-hormone treatment are presented. 2. Hypophysectomy and adrenalectomy result in decreased body and liver weight and decreased drug metabolism (mixed-function oxidases). Concentrations of electron-transport-system components are also decreased. 3. In the hypophysectomized/adrenalectomized rats, growth hormone decreases the activities of the liver mixed-function oxidases and the cytochrome P-450 and cytochrome c reductases, as well as decreasing the concentration of cytochrome P-450 compared with that of control rats. Similar but less dramatic results are obtained with sham-operated rats. 4. It is concluded that whereas growth hormone enhances liver growth, including induction of many enzyme activities, it results in a decrease in mixed-function oxidase activity. Apparently, mixed-function oxidase activity decreases in liver when growth (mitogenesis) increases.
Adult male rats, subjected either to sham operation or to hypophysectomy and adrenalectomy were maintained for 10 days before treatment with growth hormone. Results of the acute effects of growth hormone on the rat liver nuclear RNA polymerase I (nucleolar) and II (nucleoplasmic) activities as well as the chromatin template capacity were then studied and compared with the growth-hormone effects on the drug metabolism described in the preceding paper (Wilson & Spelsberg, 1976). 2. Conditions for isolation and storage of nuclei for maintenance of optimal polymerase activities are described. It is verified that the assays for polymerase activities require a DNA template, all four nucleoside triphosphates, and a bivalent cation, and that the acid-insoluble radioactive product represents RNA. Proof is presented that under high-salt conditions DNA-like RNA (polymerase II) is synthesized, and that under low-salt conditions in the presence of alpha-amanitin, rRNA (polymerase I) is synthesized. 3. In the livers of hypophysectomized/adrenalectomized rats, growth hormone increases the activity of both RNA polymerase enzymes and the chromatin template capacity within 1h after treatment. The effects last for 12h in the case of polymerase II but for only 6h in the case of polymerase I. Sham-operated rats respond to growth hormone in a manner somewhat similar to that shown by hypophysectomized/adrenalectomized rats. These results, which demonstrate an enhancement of RNA polymerase I activity in response to growth hormone, support those from other laboratories. 4. Growth-hormone enhancement of the chromatin template capacity in the liver of hypophysectomized/adrenalectomized rats contrasts with previous reports. The growth-hormone-induced de-repression of the chromatin DNA could represent the basis of the growth-hormone-induced enhancement of RNA polymerase II activity in the hypophysectomized/adrenalectomized rats, although some effect of growth-hormone on the polymerase enzymes is still suggested.
This study was initiated with the objective of separating and characterizing two or more nuclear subfractions, which could then be compared with respect to their relative propensity for binding carcinogenic polycyclic aromatic hydrocarbons. Nuclei were isolated from cloned AKR-2B mouse embryo cells, which are susceptible to transformation by chemical carcinogens. The nuclei were mechanically sheared and subfractions were separated by sedimentation through a 0.17 to 1.7 M sucrose gradient. When the cells were treated with [3H]uridine for 30 min, most of the label incorporated into RNA was recovered in the top region of the gradients, which represented Nuclear Subfraction I. The majority of the chromatin DNA, however was localized in the bottom region (Subfraction II) and the pellet (Subfraction III). Precipitation (with CaCl2) of the rapidly labeled RNA of Subfraction I along with the chromatin DNA suggested that the label was present in nascent RNA chains still attached to the chromatin. Thus, the transcripitionally active chromatin seemed to be localized in Nuclear Subfraction I. The chromatin of Subfraction I was also the best template for RNA synthesis in vitro with exogenous bacterial polymerase. The protein and RNA content of subfraction I was greater than that of the other two subfractions and whole chromatin. Electron microscopy revealed the presence of membrane material in Subfraction I and II, with little such material in Subfraction III. Subfraction I differed from Subfractions II and II and whole chromation with respect to thermal denaturation of the DNA and histone composition (as determined by gel electrophoresis). The acidic protein composition (as determined by gel electrophoresis) differed for the chromatin of all three nuclear subfractions.
The objective of this study was to examine the binding of carcinogenic polycyclic aromatic hydrocarbons in well-characterized nuclear subfractions from transformable cells in culture. A cloned line of AKR mouse embryo cells was exposed to culture medium containing [3H]-3-methyl-cholanthrene (MC) (0.4 mug/ml) 670 Ci/mole). Cellular uptake and nuclear binding were determined after 4 hr of exposure. The addition of unlabeled MC up to 10 mug/ml did not cause reduction of [3H]MC cellular uptake or nuclear binding. From 2 to 5% of the total cellular MC was localized in the nuclei. All nuclear subfractions obtained from mechanically sheared nuclei and separated on sucrose gradients showed some MC binding; however, a high-affinity, high-specific-activity binding of MC was associated only with the slower-sedimenting component shown to represent that fraction of nuclear chromatin that is transcriptionally active. Conditions that caused the precipitation of this chromatin also resulted in the precipitation of the radioactive compound, thus suggesting that the MC was physically bound to the chromatin. Unlabeled MC (10 mug/ml) saturated this high-affinity MC binding to the transcripitionally active chromatin but did not saturate the binding to the other nuclear fractions. The binding of another potent carcinogen, [3H]-1,2,5,6-dibenzanthracene, and the "weak" carcinogen, [3H]-1,2,3,4-dibenzanthracene (3,4-DBA), to whole nuclei and nuclear subfractions was also determined. The concentration, specific activity, and time of treatment were identical with those used for MC. The level of binding of [3H]-1,2,5,6-dibenzanthracene was approximately 3-fold greater in whole nuclei on a per mass DNA basis than in those of either the MC or the 3,4-DBA. The binding of MC and 3,4-DBA to whole nuclei was approximately equal. As with MC, the [3H]-1,2,5,6-dibenzanthracene demonstrated a peak of high specific activity binding to the slower-sedimenting fraction of chromatin while the 3,4-DBA displayed considerably less binding to this fraction.
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Evidence from several model systems suggests that nonhistone chromosomal proteins may regulate gene expression in eukaryotic cells. The data indicate that the synthesis of new species of nonhistone chromosomal proteins as well as modifications of preexisting nonhistone chromosomal proteins are involved in the control of transcription. However, from the vast number of proteins included in this class, it is apparent that, in addition to regulating the transcription of defined genome loci, the nonhistone chromosomal proteins include enzymes that have a general function, proteins that are involved in determining the structure of chromatin, as well as proteins that serve as recognition sites for binding of regulatory macromolecules. The presence of a nucleoplasmic pool of nonhistone chromosomal proteins which may exchange with the chromatin has also been reported (89). While it is clear that the nonhistone chromosomal proteins play a key role in the regulation of gene expression, the exact manner in which they interact with the genome to initiate, modify, or augment the transcription of specific RNA molecules remains to be resolved.
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