Growth hormone and drug metabolism. Acute effects on nuclear ribonucleic acid polymerase activity and chromatin.

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.     

Early effects of oestradiol-17β on the chromatin and activity of the deoxyribonucleic acid-dependent ribonucleic acid polymerases (I and II) of the rat uterus

Oestradiol-17β (1.0μg) was injected intravenously into ovariectomized rats. The earliest detectable hormonal response in isolated uterine nuclei was an increase (10–15min) in RNA polymerase II activity (DNA-like RNA synthesis), which reached a peak at 30min and then decreased to control values (by 1–2h) before displaying a second increase over control activity from 2 to 12h. The next response to oestradiol-17β was an increase (30–60min) in polymerase I activity (rRNA synthesis) and template capacity of the chromatin. The concentrations of acidic chromatin proteins did not begin to increase until 1h after injection of oestradiol-17β and histone concentrations showed no significant changes during the 8h period after administration. The early (15min) increase in RNA synthesis in `high-salt conditions' can be completely eliminated by α-amanitin, an inhibitor of the RNA polymerase II. The exact nature of this early increase in endogenous polymerase II activity remains to be determined, e.g. whether it is caused by the increased availability of transcribable DNA of the chromatin or via direct hormonal activation of the enzyme per se.     

Mouse Brain DNA-Dependent RNA Polymerases After Chronic Morphine Treatment

Abstract: Chronic morphine pellet implantation was found to decrease the specific activity of two forms of mouse brain RNA polymerase I and to alter the requirements of Mg²⁺ and Mn²⁺ for the activities of RNA polymerases II and III. DNA-dependent RNA polymerases were partially purified from small dense nuclei isolated from brains of naive and morphine tolerant-dependent mice, and three RNA polymerases were separated on a DEAE-Sephadex A-25 column. The three fractions, referred to as peak I, peak II, and peak III, were studied, characterized, and identified as being RNA polymerases I, II, and III, respectively. Chronic-morphine pellet implantation resulted in a lower specific activity of RNA polymerase I, but the specific activities of RNA polymerases II and III were not affected. This effect was prevented by preimplantation of a naloxone pellet and thus was narcotic-specific. Chronic morphine treatment lowered the concentration of Mg²⁺ required for optimal activity of RNA polymerase II and elevated the Mn²⁺-Mg²⁺ activity ratios of RNA polymerases II and III. A second DEAE-Sephadex A-25 column chromatography of the peak I RNA polymerase was carried out, revealing five component activity peaks. Two of these contained lower specific activities as a result of chronic morphine pelletimplantation. These specific changes in RNA polymerase function in morphine tolerance-dependence may be associated with the elevated chromatin template activities, altered chromatin phosphorylation, and elevated rates of cell-free translation that have been reported by others.     

The physicochemical properties of the cytoplasmic androgen receptor in the kidneys of normal, carrier female (tfm/+) and androgen-insensitive (tfm/y) mice.

The physicochemical properties of the androgen-receptor complex in mouse kidney were determined. The sedimentation coefficient, 7.9S and Stokes radius of 82A, are compatible with an asymmetric protein [frictional ratio f/fo 1.98; axial ratio, assuming a prolate ellipsoid, 20] having a molecular weight of 270,000 daltons. The kidney receptor is a relatively acidic protein of esoelectric point (pI) 4.8 and readily precipitable with protamine sulfate or ammonium sulfate. Studies with protein-specific reagents suggest that both cysteine and tryptophan residues may be necessary for maintaining the functional configuration associated with androgen binding. The kidney receptor can promote the association of testosterone with purified DNA. These properties of the androgen receptor in mouse kidney are remarkably similar to those of male accessory sexual tissue. The receptor detected in carrier female mice (tfm/+ heterozygous for the gene for testicular feminization (tfm), has the same physical properties as that of normal mice. However, due to a decrease in receptor concentration, binding activity is only 69% that of normal. In cytosol from androgen-insensitive mice (tfm/+), a specific androgen receptor cannot be demonstrated by 5 different techniques.     

Regulation of RNA Synthesis by DNA-Dependent RNA Polymerases and RNases during Cold Acclimation in Winter and Spring Wheat

Chromatin DNA-dependent RNA polymerases and RNases activities were measured in winter and spring varieties to understand the overall regulation of RNA synthesis during cold acclimation. We found that total RNA polymerase activities were significantly higher in chromatin isolated from winter wheat compared to the spring wheat during the acclimation period. This increase was parallel to the increase in protein and RNA contents during hardening. The ratio of RNA polymerase I to RNA polymerase II activity was higher than 2 in winter wheat after 30 days of hardening compared, to a ratio of 0.90 under the nonhardening conditions. The increase in activity and the ratio of polymerase I to polymerase II was maintained after the separation of the enzymes from the template, suggesting that RNA synthesis is regulated in part at the enzyme level. On the other hand, the chromatin associated RNase activity decreased in both varieties during acclimation, indicating a nonspecific inhibition caused by low temperature rather than a selective genetic response associated with cold acclimation.     

Ribonucleic Acid Synthesis by Cucumber Chromatin: Developmental and Hormone-induced Changes

When intact etiolated 2-day cucumber (Cucumis sativus) embryos were treated with indoleacetic acid (IAA), gibberellin A₇ (GA₇), or kinetin, chromatin derived from the embryonic axes exhibited an increased capacity to support RNA synthesis in either the presence or the absence of bacterial RNA polymerase. An IAA effect on cucumber RNA polymerase activity was evident after 4 hours of hormone treatment; the IAA effect on DNA template activity (bacterial RNA polymerase added) occurred after longer treatments (12 hours). GA₇ also promoted template activity, but again only after a prior stimulation of endogenous chromatin activity. After 12 hours of kinetin treatment, both endogenous chromatin and DNA template activities were substantially above control values, but longer kinetin treatments caused these activities to decline in magnitude. When chromatin was prepared from hypocotyl segments that were floated on a GA₇ solution, a GA-induced increase in endogenous chromatin activity occurred, but only if cotyledon tissue was left attached to the segments during the period of hormone treatment.     

In vitro effect of androgen on RNA synthesis in nuclei from androgen-independent subline of Shionogi carcinoma (CS 2)

By serial transplantation of CS 1, a subline of Shionogi carcinoma SC 115, to female mice, another subline was obtained and designated CS2. The subline showed a complete loss of androgen dependency on the growth of the tumor. When male mice bearing the tumor were castrated and treated with testosterone, the activity of RNA polymerase I in isolated nuclei from the tumor hardly varied during the period of the experiments (36 h), while the activity of RNA polymerase II exhibited a transient increase (about 40%) at 6 h after the testosterone injection. The results, together with the previous ones showing 80% and 40% increases in RNA polymerase I activity at 24 h after testosterone administration in the case of SC 115 (androgen-dependent tumor) and CS 1 (less androgen-dependent tumor), respectively, indicate that the stimulation of RNA polymerase I activity by androgen in the tumor tissues is closely related to the androgen dependency on the growth of the tumors.     

Modification of the template capacity of liver chromatin for form-B ribonucleic acid polymerase by food intake in rats under controlled feeding schedules.

Nuclei from liver of rats accustomed to eating during the first 8h of a daily 12h dark period demonstrate an increased capacity to synthesize RNA 6H after the beginning of the feeding period. 2. This increase is accompanied by a higher yield of extractable form-B DNA-dependent RNA polymerase activity. 3. The endogenous RNA polymerase activity associated with nuclear chromatin is also stimulated by food intake. Both purified and chromatin-associated form-B enzyme activities exhibit different ionic strength requirements after food intake. 4. The sensitivity of exogenous (added) form-B-enzyme to changes in ionic strength changes after feeding when chromatin is used as template. 5. Chromatin extracted from the liver of fed rats is a better template for form-B-enzyme than chromatin extracted from starved rats.     

Cellular RNA synthesis in normal and mengovirus-infected L-929 cells.

Cellular RNA synthesis was studied in mouse L-929 cells and in these cells infected with mengovirus. RNA polymerases I, II, and III were partially purified and their chromatographic properties were analyzed by DEAE-Sephadex A-25 chromatography. RNA polymerase II was purified from mouse liver and its subunit structure was compared to that of normal and virus-infected L-929 cells by two-dimensional gel electrophoresis. By these criteria, the enzymes from all three sources were identical. The RNA synthetic activities and capacities of chromatins from normal and virus-infected cells were compared under a variety of conditions. The endogenous activity in chromatin from infected cells was inhibited relative to controls but the residual activity responded normally to stimulation by ammonium sulfate, heparin, and Sarkosyl. The template capacity of the chromatins was compared with added RNA polymerase II and by a rifampicin challenge assay utilizing Escherichia coli RNA polymerase. Identical results were obtained in each case. The number of growing RNA chains and the rates of their elongations were determined. The results showed that nuclei and chromatin from infected cells have a smaller number of RNA polymerase II molecules engaged in RNA synthesis than normal cells do but that the active molecules elongate RNA chains at the same rate.     

The effects of oestradiol-17beta on the ribonucleic acid polymerases of immature rabbit uterus.

Measurements of the endogenous RNA polymerase activities of nuclei isolated from immature rabbit uteri have shown that prior treatment of the animals with oestradiol-17beta has a profound effect on the apparent activities of both RNA polymerases A and B. Within 1 h of hormone treatment, the activity of RNA polymerase A is increased and continues to rise until about 4h when it reaches a plateau and remains steady until at least 8h. The activity of RNA polymerase B increases sharply after oestradiol treatment reaching an early maximum at 30-45 min. Thereafter this activity declines until by 1-2h it approaches control values but a second increase in activity then occurs with a maximum at 3-4h. Treatment of the rabbits with alpha-amanitin before the administration of oestradiol inhibits the hormone-induced stimulation of RNA polymerase A activity in isolated nuclei but when the administration of alpha-amanitin is delayed until after the early rise of RNA polymerase B activity, the oestradiol-induced stimulation of RNA polymerase A is retained. Similar results have been obtained in experiments with cycloheximide suggesting that the stimulation of RNA polymerase A activity by oestradiol is dependent on the hormone-induced stimulation of RNA polymerase B and the subsequent synthesis of protein using the RNA product of the early increase in RNA polymerase B activity. Measurement of the activities of RNA polymerases A and B after isolation of the enzymes from immature rabbit uterine nuclei before and after oestradiol treatment failed to show any differences. Therefore it would appear that the changes in the observed activities of RNA polymerases A and B in isolated nuclei are consequences of changes in the structure and function of chromatin rather than the results of modifications in the RNA polymerases themselves.     

Template activity of chromatin during stimulation of cellular proliferation in human diploid fibroblasts

1. Contact-inhibited confluent monolayers of WI-38 human diploid fibroblasts can be stimulated to divide by replacing the medium with fresh medium containing 30% foetal calf serum. 2. Of the cells 40–75% are stimulated to divide with a peak DNA synthesis between 15 and 21h and a peak mitotic index between 28 and 30h after stimulation. 3. In the first 12h before the initiation of DNA synthesis there is a biphasic increase in the incorporation of [³H]uridine into RNA of whole cells. 4. This is paralleled by a similar biphasic stimulation of chromatin template activity measured in vitro in a system in which purified cell chromatin is incubated with an exogenous RNA polymerase isolated from Escherichia coli. 5. The changes in chromatin template activity are believed to represent activation of the genome, with more sites available for RNA synthesis, and to account almost entirely for the changes in RNA synthesis occurring in the whole cell.