Differential solubilization of nuclear glucocorticoid receptors by DNAase I and DNAase II

This study analyzes the sensitivity of nuclear bound glucocorticoid receptors to solubilization from nuclei by DNAase I and DNAase II. Thymocytes were incubated with 10(-8) M [3H]dexamethasone, [3H]cortisol or [3H]triamcinolone acetonide, without or with 10(-6) M unlabelled dexamethasone, for 30 min at 37 degrees C and nuclei from these cells were digested with either DNAase I and DNAase II. DNAase I for 2 h at 3 degrees C leads to solubilization of 60% of the nuclear DNA and release of 10--20% triamcinolone acetonide-receptor, 30--40% dexamethasone-receptor and 85--90% cortisol-receptor. DNAase II at the same enzymatic concentration solubilizes only 10--20% of the nuclear DNA, but releases 40--50% triamcinolone-receptor, 60--70% dexamethasone-receptor and 100% cortisol-receptor. Release of nuclear bound dexamethasone-receptor by DNAase I parallels the solubilization of DNA, reaching maximum values by 2 h at 3 degrees C, whereas maximal release by DNAase II is obtained within 45 min when DNA solubilization is not complete. When nuclei initially extracted with DNAase I are re-extracted with DNAase II, greater than 65% of the DNAase I residual dexamethasone-receptors are solubilized, whereas DNAase I is ineffective in solubilizing DNAase II residual dexamethasone-receptors. DNAase I solubilizes only 30% of the 0.4 M KCl residual dexamethasone-receptor whereas DNAase II digests over 90% of this fraction. DNAase I extracts of nuclear dexamethasone-receptor chromatograph on G-100 Sephadex as a single radioactive peak just after the void volume, whereas DNAase II extracts of nuclear dexamethasone-receptor chromatograph as two peaks of radioactivity, one which is similar to the DNAase I solubilized receptor and a second broad peak of macromolecular bound radioactivity which is smaller in size.     

Metabolism of dITP in HeLa cell extracts, incorporation into DNA by isolated nuclei and release of hypoxanthine from DNA by a hypoxanthine-DNA glycosylase activity.

dITP may be generated from dATP by a slow, nonenzymatic hydrolysis. While [3H]dITP was degraded rapidly to [3H]deoxyinosine by HeLa cell nuclear extracts, no net degradation of [3H]dITP was observed in the presence of physiological concentrations of ATP, apparently because the extract contained deoxynucleoside diphosphate kinase activity that regenerated [3H]dITP from [3H]dIDP. Isolated HeLa cell nuclei, as well as partially purified DNA polymerase alpha, incorporated [3H]dITP into DNA at 50-60% of the rate of [3H]dGTP incorporation. No rapid release of the incorporated radioactivity was observed. The molecular weight of nascent DNA containing dIMP residues, however, decreased slightly after prolonged incubation in the presence of EDTA, suggesting that a repair process is initiated in dIMP-containing chromatin. Furthermore, release of free [3H]hypoxanthine from [3H]dIMP-containing DNA was detected after incubation with nuclear extracts in the presence of EDTA, suggesting the presence of hypoxanthine-DNA glycosylase activity in HeLa cell nuclei.     

High affinity binders for cyclic adenosine 3', 5'-monophosphate on plasma membranes isolated from rat liver and ascites hepatomas.

Plasma membranes from rat liver were found to contain at least two types of specific binding sites for cyclic [3H] adenosine 3', 5'-monophosphate (c[3H]AMP) with apparent dissociation constants of 0.51 +/- 0.14 and 2.9 +/- 0.6 nM (O degrees), respectively. The levels of these binding sites in liver plasma membranes were about 0.60 +/- 0.20 and 1.3 +/- 0.5 pmole/mg protein. The highest affinity binders for c[3H]AMP were found to be reduced in amount in plasma membranes of ascites hepatomas to 1/3 to 1/4 as compared with liver membranes in the cases of AH-130 and AH-7974 and to an almost undetectable level in the case of AH-130F(N). No difference in the endogenous phosphorylation of plasma membranes by (gamma-32P])ATP was, however, detected among liver and hepatoma plasma membranes. Addition of cAMP or cGMP at various concentrations did not affect the endogenous phosphorylation of plasma membranes of these cells.     

Covalent binding of benzo[a]pyrene metabolites to DNA, RNA and chromatin proteins in the AKR mouse embryo cell-line

A specific fraction from the nuclei of the AKR mouse embryo cell-line (fraction I) displayed a much greater localization of radioactivity compared to fraction II and III when the chemical carcinogen, [3H]benzo[a]pyrene (B[a]P) was incubated with the cells for 24 h. The radioactivity in fraction I consisted of both covalently and non-covalently bound metabolites. Isolation of the DNA, RNA and protein of fraction I revealed that 94% of the covalently bound radioactivity was to protein, 5% to RNA and 1% to DNA. Analysis of the fraction I proteins by SDS gel electrophoresis revealed that there was more radioactivity covalently bound to the larger proteins than to smaller proteins. Isoelectric focusing (IEF) of the purified proteins displayed two peaks of radioactivity, one at a pH of 5 and the other at 11. The former proteins bound more radioactivity per mass of protein than the latter proteins. Analysis of fraction I histones on acid urea polyacrylamide gels showed that the radioactivity coincided with histones H3 and H2B and low levels of radioactivity associated with histones H1, H2A and H4. Two significant peaks of radioactivity closely migrated near but did not co-migrate with histone H1. The distribution of the bound radioactivity is probably a reflection of the availability of the proteins to the reactive carcinogen metabolites. The possible binding of B[a]P metabolites to phosphorylated histones and to the high mobility of group (HMG) proteins 1 and 2 is discussed.     

Dissociation of tumour-promoter-induced effects on prostaglandin release, polyamine synthesis and cell proliferation of 3T3 cells.

The phorbol ester 12-O-tetradecanoylphorbol 13-acetate induces tumour promotion, inflammation, cell proliferation and prostaglandin release. Recent reports suggest that the prostaglandins released by 12-O-tetradecanoylphorbol 13-acetate (TPA) initiate a cascade of events leading to polyamine synthesis and cell proliferation. In experiments designed to test this contention, it was found that addition of TPA (1 microM to 1 nM) to confluent mouse 3T3 fibroblasts successively caused the release of prostaglandins E2 and I2, induction of the enzyme ornithine decarboxylase (EC 4.1.1.17), stimulation of [3H]thymidine incorporation into DNA, and cell proliferation. Pretreatment of the cells with the anti-inflammatory steroid dexamethasone (1 microM) or the non-steroidal anti-inflammatory drug indomethacin (1 microM) inhibited TPA-induced prostaglandin release. However, dexamethasone enhanced the other effects of TPA, whereas indomethacin was ineffective. Addition of prostaglandin E2 to the cultures did not induce ornithine decarboxylase activity and cell proliferation. Pretreatment of the cells with 1,3-diaminopropane (1 mM) or alpha-methylornithine (5 mM), inhibitors of polyamine synthesis, decreased TPA-induced ornithine decarboxylase activity without affecting DNA synthesis. TPA stimulated [3H]thymidine incorporation into DNA, even when the ornithine decarboxylase activity was completely blocked. These data suggest that the proliferative effect of TPA on 3T3 cells is independent of prostaglandin release and polyamine synthesis.     

Interrelationship between nuclear histone binding and cell proliferation

1. Binding of non-enzymatically [methyl-14C]-labeled histone H3 to nuclei isolated from young and old rat livers, regenerating rat liver, and tumor cells has been investigated. 2. Scatchard plot analysis indicated that various cell types had different binding capacity and different dissociation constant (Kd). 3. Nuclei isolated from younger rats had fewer binding sites and lower Kd (or higher Ka) values for [methyl-14C]H3 than those from older rats. 4. Fewer binding sites and lower Kd values were also observed with nuclei isolated from the maximally regenerating liver (24 hr after partial hepatectomy) and the fast-growing ascites tumor and Novikoff hepatomas. 5. These results strongly suggest that the number of binding sites and affinity of histone H3 for nuclei appears to be correlated with the degree of cell proliferation. 6. Fractionation of the [methyl-14C]H3 bound nuclei into nuclear membrane and nucleoplasm demonstrates that approx. 94% of radioactivity is associated with the former in which less than 6% of DNA is found, whereas 94% of total DNA is found in nucleoplasm. 7. This suggests that the binding of [methyl-14C]H3 to nuclei is independent of DNA present in each fraction.     

High-density lipoprotein-associated 17beta-estradiol fatty acyl ester uptake by Fu5AH hepatoma cells: implications of the roles of scavenger receptor class B, type I and the low-density lipoprotein receptor

17beta-estradiol (E2) fatty acyl esters naturally incorporate into high-density lipoprotein (HDL). The objective was to elucidate mechanisms involved in HDL-associated E2 cellular uptake and to determine the intracellular distribution of E2 and its fatty acyl esters (E2-FAE) after uptake. [3H]E2 or [3H] cholesterol was incubated with human serum for 24 h to allow for fatty acyl esterification. Total-HDL containing [3H]E2-FAE or [3H]cholesterol esters was isolated by sequential density ultracentrifugation and then incubated with Fu5AH rat hepatoma cells for various time points. Cellular uptake was determined by intracellular radioactivity as a percentage of total radioactivity. Chemical inhibition of scavenger receptor class B, type I and low-density lipoprotein (LDL) receptor competition assays were performed to determine cellular uptake mechanisms. Compared to HDL-[3H]cholesterol, cellular uptake of HDL-[3H]E2 occurred at an initially rapid rate. SR-BI inhibition resulted in a decrease in HDL-E2 uptake and LDL impaired this uptake in a concentration-dependent manner. Accordingly, pretreatment of cells with BLT-1 combined with LDL addition significantly attenuated HDL-E2 uptake. HDL-E2-FAE was hydrolyzed into free E2 with the maximum at 24 h. Fu5AH cells facilitate HDL-E2 uptake by at least SR-BI and LDL receptor pathways and intracellular hydrolysis of E2-FAE into free E2 ensues.     

Identification of radioactivity in cell nuclei from brain, pituitary gland and genital tract of male rhesus monkeys after the administration of [3H]testosterone

Enzymes are present in the primate brain that convert testosterone into 17 beta-hydroxy-5 alpha-androstan-3-one (dihydrotestosterone), estradiol-17 beta and 4-androstene-3,17-dione. To identify the metabolites of testosterone that accumulate in cell nuclei obtained from different regions of the brain, 9 adult castrated male rhesus monkeys were injected with 5 mCi [3H]testosterone as an intravenous bolus. After 1 h, brains were rapidly removed and the left halves were used for autoradiography while the right halves were dissected to provide 14 samples. Radioactive metabolites in cell nuclei were identified by high-performance liquid chromatography (HPLC) and by repeated recrystallization. In autoradiograms of brain, most of the labeled neurons were in the hypothalamus, preoptic area and amygdala. These three regions also had the highest levels of radioactivity. The major form of this radioactivity was [3H]estradiol-17 beta (Type I tissues) and the major radioactive androgen present was [3H]testosterone. In all other brain regions and pituitary gland, the major form of radioactivity was unchanged [3H]testosterone (Type II tissues). In genital tract structures, [3H]dihydrotestosterone predominated (Type III tissues). These results suggested that, in contrast to its actions on genital tract structures, testosterone acts on neuronal nuclei mainly in unmetabolized form or after conversion to estradiol-17 beta.     

Evidence for the existence of a stable association between nascent DNA and the nuclear membrane of HeLa cells.

Nascent DNA-nuclear membrane complexes isolated from HeLa cells and solubilized in a sodium dodecyl sulfate-urea solution were examined by gel electrophoresis, column chromatography, isopycnic centrifugation, and by extraction with chloroform/methanol. Radioactivity attributable to [3H]DNA co-migrated with three protein peaks during electrophoresis. This radioactivity was eliminated by prior treatment with DNAase. In addition, all of the radioactivity attributable to nascent DNA eluted with a specific protein on Sepharose 4B columns. This DNA - protein complex banded at a density of 1.58 gm/cm3 in sucrose-CsCl gradients. Treatment with DNAase, phospholipase A and C, and dilute alkali disrupted the complex. Moreover, 93% of the radioactivity attributable to protein and 70% of that attributable to DNA could be extracted from the complex with a chloroform/methanol solution. The results suggest that nascent DNA may be in a stable association with a proteolipid moiety of the nuclear membrane.     

Dexamethasone alters arachidonate release from human epithelial cells by induction of p11 protein synthesis and inhibition of phospholipase A2 activity.

The effect of the glucocorticosteroid, dexamethasone, on arachidonic acid (AA) release and on protein levels of p11 and cytosolic phospholipase A2 (cPLA2) was studied in two epithelial cell lines, HeLa cells and BEAS-2B cells. Dexamethasone treatment of HeLa cells and BEAS-2B cells increased cellular p11 protein and mRNA levels in a time- and dose-dependent manner. It had little effect on levels of cPLA2 protein. In order to determine if increased p11 protein expression resulted in increased interaction between p11 and cPLA2, anti-cPLA2 antibodies were used to immunoprecipitate p11.cPLA2 complexes and Western blots of the immunoprecipitate were used to detect p11. In cells treated with dexamethasone, more p11 was detected in the anti-cPLA2 immunoprecipitate compared with control cells. Dexamethasone treatment of HeLa cells prelabeled with [3H]AA decreased the release of [3H]AA under basal conditions and after stimulation with the calcium ionophore A23187 (10(-6) M). In order to determine if altering the p11 protein levels in HeLa cells independent of glucocorticosteroid treatment could also produce an effect on [3H]AA release, cells were stably transfected with plasmids expressing either p11 antisense mRNA or p11 mRNA. Cloned HeLa cells expressing p11 antisense mRNA exhibited less cellular p11 protein compared with control cells and greater [3H]AA release compared with cells transfected with a control vector. Cloned HeLa cells stably transfected with a p11 expression vector exhibited increased p11 cellular protein and diminished [3H]AA release under basal conditions and in response to A23187. Therefore, dexamethasone alteration of epithelial cell AA release may be due in part to induction of p11 protein expression.     

Author index

Binding of dexamethasone · receptors with isolated nuclei, DNA-cellulose and cellulose has been compared with respect to dependence on salt concentration and resistance to KCl extraction and DNAase I digestion. A solution of cytoplasmic dexamethasone-receptor complexes was prepared by the incubation of rat thymus cells with steroid at 3°C and breaking the cells by hypotonic lysis. Activation of the complexes was accomplished by warming the solution at 25°C for 15 min. Activation significantly increased the ability of dexamethasone · receptors to bind to nuclei and DNA-cellulose but not to cellulose. Dexamethasone-receptor complexes bound to nuclei at 3°C are completely resistant to extraction with 0.1 M KCl, 76% resistant to 0.2 M KCl and 20% resistant to 0.4 M KCl. Dexamethasone · receptors bound to DNA-cellulose are 45% resistant to extraction with 0.1 M and 0.2 M KCl and 29% resistant to 0.4 M KCl extraction. Cellulose-bound dexamethasone · receptors are not resistant to any of these extractions. DNAase I treatment releases 60% of the dexamethasone · receptors bound to DNA-cellulose but only 13% of those bound to nuclei, though at least 60% of the nuclear DNA is solubilized. The presence of 0.15 M KCl decreases binding of activated dexamethasone · receptors to nuclei by 73% but to DNA-cellulose by only 17%. Pretreatment of nuclei with 0.1–0.4 M KCl reduces their capacity to bind activated dexamethasone · receptors by 90% whereas similar treatment reduces the capacity of DNA-cellulose to bind dexamethasone · receptors by only 29%. Nuclei extracted with 0.1 M KCl appear to have a limited capacity to accept dexamethasone · receptors. These studies demonstrate that binding of dexamethasone · receptors to nuclei and DNA-cellulose differs by (a) the higher resistance of nuclear complexes to KCl and DNAase I treatment; (b) the much greater sensitivity of nuclei to KCl treatment.     

Hb switching in chickens

We have taken advantage of the preferential digestion of active genes by DNAase I to investigate the chromosomal structure of embryonic and adult β-globin genes during erythropoiesis in chick embryos, and in particular to examine the question of hemoglobin switching during development. DNA in isolated red cell nuclei was mildly digested with DNAase I to about 10–15 kb, purified and restricted with a variety of restriction enzymes. The DNA was then separated on agarose gels, transferred to nitrocellulose filters and hybridized with an adult-specific β-globin cDNA clone or a genomic clone containing the genes coding for both an embryonic and an adult β-globin chain. Preferential sensitivity of the respective globin genes was monitored by the disappearance of specific restriction bands after DNAase I digestion of nuclei. In embryonic red cells, both adult and embryonic β-globin genes are very sensitive to DNAase I; however, in adult erythroid lines, the embryonic β-globin gene becomes relatively more resistant but the adult gene remains highly sensitive. Controls showed that all globin genes were resistant to DNAase I in brain nuclei and nuclei from lymphoid cells. Thus the switch from embryonic to adult globin expression is associated with an apparent change in the chromosome structure of the embryonic globin gene as reflected in the gene becoming less accessible to DNAase I in adult red cell nuclei. Our results also show that the chromosomal structure of both adult and embryonic genes is altered in embryonic red cell nuclei; thus the nonexpressed globin gene (that is, the adult gene in embryonic red cells) has already been “recognized” to some degree and marked by the erythroid compartment. The sensitivity of the adult globin gene in embryonic cells may represent a “pre-activation” state of the chromosome.     

Non-histone proteins in fractionated chromatin before and after DNAse II treatment

Chromatin from spleen cells of normal, non-immunized mice and from mice 3 days after immunization with human immunoglobulin G was fractionated at increasing salt concentrations into three fractions: 0.35 M NaCl-soluble, 2 M NaCl-soluble and a residual fraction, dissociated in 2 M NaCl/5 M urea. The residual fraction of chromatin, homogeneous by ultracentrifugation and containing only 25% of the total chromatin DNA, was associated with proteins strongly labeled with [3H]tryptophan, [3H]methionine and [3H]leucine. This fraction was more sensitive to DNAase II treatment than was native, non-fractionated chromatin and it contained approx. 40% Mg2+-soluble DNA sequences. The template activity of the residual fraction was 6--7-times higher than that of non-fractionated chromatin. Fraction A, characteristic for non-immunized spleen cells, was present in three chromatin fractions and after DNAase II treatment it remained only in the residual fraction, which suggests that this fraction is associated with genes non-transcribed in non-immunized mice. Fractions I and B1 were found mainly in the residual fraction, and only in smaller amounts in the 0.35 M NaCl-soluble fraction. After DNAase II treatment, fractions I and B1 in chromatin from immunized mice disappeared, which suggests that these fractions may be associated with active transcribed sequences during the immune reaction.     

Evidence that [3H]Dopamine Is Taken Up and Released from Nondopaminergic Nerve Terminals in the Rat Substantia Nigra In Vitro

Potassium chloride (25 mM) and (+)-amphetamine (100 microM) both stimulated the release of radioactivity from slices of substantia nigra preincubated with [3H]3,4-dihydroxyphenylethylamine [( 3H]dopamine). Potassium chloride (25 mM) released radioactivity from slices of both zona compacta and zona reticulata. Prior 6-hydroxydopamine (6-OHDA) lesions of one nigrostriatal pathway did not reduce the spontaneous release of radioactivity, or the potassium chloride- or amphetamine-induced release of radioactivity from slices of nigra ipsilateral to the lesion after preincubation with [3H]dopamine. The accumulation of radioactivity following incubation of nigral slices from 6-OHDA-lesioned animals with [3H]dopamine was increased when compared to uptake into slices from intact tissue. In synaptosomal preparations of striatum, nomifensine but not desipramine or fluoxetine inhibited [3H]dopamine uptake. In contrast, nomifensine, desipramine, and fluoxetine all inhibited [3H]dopamine uptake in nigral synaptosomal preparations. Following 6-OHDA lesions of one nigrostriatal pathway the uptake of [3H]dopamine into nigral synaptosomal preparations was unchanged but uptake into striatal preparations was substantially decreased. In contrast, bilateral electrolesions of the dorsal and medial raphe nuclei reduced [3H]dopamine uptake into nigral preparations but not into striatal synaptosomes. The uptake of [3H]5-hydroxytryptamine ([3H]5-HT) into synaptosomal preparations of substantia nigra was abolished by fluoxetine and reduced by desipramine, but was unaffected by nomifensine. In contrast, fluoxetine, desipramine, and nomifensine all inhibited [3H]5-HT uptake into striatal synaptosomal preparations. Following 6-OHDA lesions of one nigro-striatal pathway the uptake of [3H]5-HT into nigral synaptosomal preparations was unchanged but uptake into striatal preparations was reduced.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of phosphatidyl-dCMP in sarcoma 180 cells. Effect of chlorpromazine, phosphatidic acid and inositol

The synthesis and degradation of phosphatidyl-dCMP was studied in intact and permeabilized Sarcoma 180 cells as well as in isolated nuclei. It was verified that chlorpromazine greatly enhanced phosphatidyl-dCMP synthesis and completely abolished its hydrolysis in intact cells. The former effect was reversible and was partially lost upon permeabilization or isolation of nuclei. Phosphatidic acid also increased liponucleotide synthesis and the combination of phosphatidic acid with chlorpromazine was not additive. When inositol was added to cells which had accumulated phosphatidyl-[3H]dCMP, the recovery of radioactivity in the organic phase decreased; this effect was dose-dependent and specific for inositol, and was accompanied by an increased release of [3H]dCMP to the cell medium. In isolated microsomes, addition of Ptd-dCMP determined incorporation of [3H]inositol into phosphatidyl-inositol. These results strongly suggest that phosphatidyl-dCMP is utilized for the synthesis of phosphatidyl-inositol.     

Nonactivated and activated glucocorticoid receptor complexes from human salivary gland adenocarcinoma cell line

[3H]Triamcinolone acetonide glucocorticoid receptor complexes from human salivary gland adenocarcinoma cells (HSG cells) were shown to be activated with an accompanying decrease in molecular weight in intact cells, as analyzed by gel filtration, DEAE chromatography, the mini-column method and glycerol gradient centrifugation. Glucocorticoid receptor complexes consist of steroid-binding protein (or glucocorticoid receptor) and non-steroid-binding factors such as the heat-shock protein of molecular weight 90,000. To determine whether the steroid-binding protein decreases in molecular weight upon activation, affinity labeling of glucocorticoid receptor in intact cells by incubation with [3H]dexamethasone 21-mesylate, which forms a covalent complex with glucocorticoid receptor, was performed. Analysis by gel filtration and a mini-column method indicated that [3H]dexamethasone 21-mesylate-labeled receptor complexes can be activated under culture conditions at 37 degrees C. SDS-polyacrylamide gel electrophoresis of [3H]dexamethasone 21-mesylate-labeled steroid-binding protein resolved only one specific 92 kDa form. Furthermore, only one specific band at 92 kDa was detected in the nuclear fraction which was extracted from the cells incubated at 37 degrees C. These results suggest that there is no change in the molecular weight of steroid-binding protein of HSG cell glucocorticoid receptor complexes upon activation and that the molecular weight of nuclear-binding receptor does not change, although the molecular weight of activated glucocorticoid receptor complexes does decrease. Triamcinolone acetonide induced an inhibitory effect on DNA synthesis in HSG cells. Dexamethasone 21-mesylate exerted no such effect and blocked the action of triamcinolone acetonide on DNA synthesis. These results suggests that dexamethasone 21-mesylate acts as antagonist of glucocorticoid in HSG cells. The fact that dexamethasone 21-mesylate-labeled receptor complexes could be activated and could bind to DNA or nuclei as well as triamcinolone acetonide-labeled complexes suggests that dexamethasone 21-mesylate-labeled complexes can not induce specific gene expression after their binding to DNA.