Differential distribution of glucocorticoid and estrogen receptor isoforms: localization of GRbeta and ERalpha in nucleoli and GRalpha and ERbeta in the mitochondria of human osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cell lines.

The localization of glucocorticoid and estrogen receptors alpha (GRalpha, ERalpha) and beta (GRbeta, ERbeta) in osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cells was studied by immunofluorescence labelling and confocal laser scanning microscopy, as well as by subcellular fractionation and immunoblotting of the proteins of the fractions with respective antibodies. In HepG2 and SaOS-2 cells GRbeta and ERalpha were localized mainly in the nucleus, particularly concentrated in nuclear structures, which on the basis of their staining with antibody against C23-nucleolin, were characterized as nucleoli. A faint, diffuse GRbeta and ERalpha staining was also observed in the cytoplasm. GRalpha and ERbeta were specifically enriched at the site of cell mitochondria, which were visualized by labelling with the vital dye CMX. Immunoblotting experiments corroborated the immunofluorescence labelling distribution of glucocorticoid and estrogen receptor isoforms in the cell lines studied. These findings support the concept of a direct action of steroid/thyroid hormones on mitochondrial functions by way of their cognate receptors and also suggest a direct involvement of GRbeta and ERalpha in nucleolar-related processes in HepG2 and SaOS-2 cells.

Differential subcellular distribution of estrogen receptor isoforms: localization of ERalpha in the nucleoli and ERbeta in the mitochondria of human osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cell lines.

The localization of estrogen receptors alpha (ERalpha) and beta (ERbeta) in osteosarcoma SaOS-2 and hepatocarcinoma HepG2 cells was studied by immunofluorescence labelling and confocal laser scanning microscopy, as well as by subcellular fractionation and immunoblotting of the proteins of the fractions with respective antibodies. In both cell types, ERalpha was localized mainly in the nucleus, particularly concentrated on nuclear structures, which on the basis of their staining with pyronin and with antibodies against the nucleoli-specific Ki67 antigen and C23-nucleolin, were characterized as nucleoli. A faint, diffuse ERalpha staining was also observed in the cytoplasm. ERbeta was specifically enriched at the site of the mitochondria, visualized by labelling with the vital dye CMX and antibody against the mitochondrial-specific cytochrome oxidase subunit I. Immunoblotting experiments corroborated the immunofluorescence labelling distribution of ERalpha and ERbeta. These findings support the concept of a direct action of steroid/thyroid hormones on mitochondrial functions by way of their cognate receptors and also suggest a direct involvement of ERalpha in nucleolar-related processes.

The mitochondrion as a primary site of action of steroid and thyroid hormones: presence and action of steroid and thyroid hormone receptors in mitochondria of animal cells.

Mitochondria are key cellular organelles that regulate events related to energy production and apoptosis. These processes are modulated, in turn, by steroid and thyroid hormones in the course of their actions on metabolism, growth and development. In this context, a direct effect of these hormones on the mitochondrial-linked processes, possibly by way of cognate mitochondrial receptors, has been proposed. In this paper we review data from the literature and present new findings supporting this concept. Receptors for steroid hormones, glucocorticoids and estrogens, and for T(3), have been detected in mitochondria by immunofluorescence labeling and confocal laser microscopy, by Western blotting of mitochondrial proteins and by immunogold electron microscopy. Furthermore, the mitochondrial genome contains nucleotide sequences with high similarity to known hormone-responsive elements, which interact with the appropriate receptors to confer hormone-dependent activation of reporter genes in transfection experiments. Thus, thyroid hormone stimulates mitochondrial transcription mediated by the cognate receptor when added to an in organello mitochondrial system, capable of faithful transcription.

The mitochondrion as a primary site of action of regulatory agents involved in neuroimmunomodulation.

A major system of neuroimmunomodulation is the hypothalamic-pituitary-adrenocortical (HPA) axis, acting through glucocorticoids and their intracellular signaling components, exerting both stimulatory and inhibitory effects on the immune reaction. Glucocorticoids inhibit the production of proinflammatory cytokines by interacting with nuclear transcription factors (nuclear factor [NF]-kappaB, activated protein [AP]-1) and induce the production of several anti-inflammatory cytokines by gene activation. In some cells and/or in extreme stress conditions, apoptosis is evoked. In most processes related to neuroimmunomodulation a prominent role is emerging for mitochondria. These organelles generate more than 90% of the cell's energy requirements through oxidative phosphorylation (OXPHOS), which is regulated by several agents, including steroid and thyroid hormones. These hormones are inducers of nuclear and mitochondrial OXPHOS gene transcription and they exert a primary action not only on nuclear but also on mitochondrial genes by way of cognate receptors. Recently, additional nuclear transcription factors involved in neuroimmunomodulation have been detected in mitochondria (NF-kappaB, AP-1, p53, calcium/cAMP response element binding protein [CREB]), and binding sites of these and putative binding sites of other nuclear transcription factors have been identified in the mitochondrial genome. The interaction of these factors with mitochondrial regulatory proteins, with receptors and with the genome has been shown and, in some cases, modulation of mitochondrial transcription was observed with possible effects on energy yield. The mitochondria store a host of critical apoptotic activators and inhibitors in their intermembrane space and the release of these factors could be another possible mode of action of the mitochondrially translocated regulatory agents and receptors.

Effects of dimethylnitrosamine on RNA synthesis and metabolism in mouse liver.

Following i.p. injection of dimethylnitrosamine into male C57BL mice, synthesis of liver nuclear heterogeneous RNA was inhibited significantly, reaching approximately 20% of control within 2 hr of a dose of 40 mg/kg. Synthesis of nucleolar RNA was also inhibited, although to a smaller extent, reaching about 70% of control after the same treatment. These effects were observed both during RNA synthesis in vivo and during in vitro transcription with isolated nuclei and nucleoli. Examination of RNA polymerases I and II, isolated and partially purified by diethylaminoethyl Sephadex column chromatography, did not indicate any change either in their activities in the transcription of exogenous DNA or in their in vivo binding to chromatin. On the other hand, the activity of purified chromatin as a template for transcription by added, partially purified RNA polymerase II was significantly reduced, suggesting that carcinogen-induced damage to chromatin was the cause of the observed inhibition of heterogeneous RNA synthesis. When purified DNA was used in place of chromatin as a template for transcription by partially purified RNA polymerase II, no inhibition was observed. Dimethylnitrosamine treatment had a pronounced effect on the kinetics of appearance of the cytoplasmic RNA species. Four hr after a 40-mg/kg dose of dimethylnitrosamine, the rate of appearance in the cytoplasm of polyadenylate-containing RNA was inhibited by 50%, while that of 4S, 18S, and 28S ribosomal RNA was inhibited by over 80%.

Induction of tryptophan oxygenase and tyrosine aminotransferase by metabolites of hydrocortisone.

Metabolites of hydrocortisone were isolated from rat liver on a preparative scale, fractionated by column chromatography on Sephadex Lh-20 and silica gel and tested for biological activity. Apart from the well known neutral metabolites, steroid glucuronides and sulfates, we obtained metabolite fractions containing non-conjugated steroidal carboxy acids and acid metabolites of unknown structure. One of these fractions induced tyrosine aminotransferase (EC 2.6.1.5) in adrenalectomized female rats but not tryptophan oxygenase (EC 1.13.11.11), whereas another one mainly increased activity of tryptophan oxygenase. The doses necessary to significantly induce both enzymes were much lower in case of these metabolites than in the case of hydrocortisone itself. The active fractions eluting from silica gel column were analyzed by thin-layer chromatography in two different solvent systems. Absence of hydrocortisone in these fractions could be clearly demonstrated. Furthermore, the active fractions eluting from the silica gel column were characterized by treatment with an extract from Helix pomatia and/or diazomethane and subsequent analysis by thin-layer chromatography. We conclude, considering the biological activity of some synthesized derivatives of hydrocortisone, that the biologically active components are acid metabolites of hydrocortisone which are not identical to any of the known metabolites.

Nucleolar RNA synthesis in the liver of partially hepatectomized and cortisol-treated rats.

RNA synthesis by isolated nucleoli from rat liver is significantly enhanced 12--14 h after partial hepatectomy and 4 h after cortisol administration. The increased RNA synthetic capactiy is demonstrable also in the respective high salt nucleolar extracts and in Biogel A-1.5 filtration fractions of the nucleolar extracts. DNA saturation experiments using nucleoli and Biogel fractions from control and treated animals as RNA polymerase source, have demonstrated, that independent of the extent of RNA synthesis, saturation of transcription is reached at the same concentration of exogenous template. We conclude that the activity and not the amount of nucleolar RNA polymerase is increased as a result of partial hepatectomy or cortisol administration. Parallel to the effects on RNA polymerase, the activity, of RNA-degrading enzymes present in nucleoli is also enhanced by the same treatment.

Absence of hydrocortisone from cytoplasmic hormone-protein complexes formed in vivo after administration of biologically active doses of [3H]hydrocortisone.

After administration of [3H]hydrocortisone to adrenalectomized rats, hormone-protein complexes were isolated from liver cytosol by DEAE-cellulose chromatography. After application of biologically active and inactive doses of hydrocortisone five binding components were detected eluting at the same salt concentrations as the hormone-protein complexes observed after incubation of cytosol with [3H]hydrocortisone in vitro. The isolated hormone-protein fractions were acidified and extracted with ethylacetate and the steroids were analyzed by thin-layer chromatography. No significant amount of hydrocortisone could be detected in any of the complexes formed in vivo 5-60 min after administration of biologically active doses of hydrocortisone. 3 xi, 11 beta, 17 alpha, 20 xi, 21-Pentahydroxypregnane, steroidal carboxy acids, glucuronides and a very polar conjugate of hydrocortisone were found in the different fractions. After an in vivo dose of hydrocortisone of about 1/5000th of the minimal dose required for enzyme induction, hydrocortisone could be found in all the cytoplasmic hormone-protein complexes formed. In contrast to the cytoplasmic hormone-protein complexes, hydrocortisone could be readily demonstrated in nuclei isolated after the administration of biologically active doses of hormone, although acid metabolites were found to represent the main part of the radioactive compounds present in the nuclei. These acid metabolites were located in the nuclear envelope. These results seem to contradict the generally accepted theory that hydrocortisone induces biosynthesis of proteins via a cytoplasmic hydrocortisone-receptor complex: after administration of biologically active doses of hydrocortisone, no such complex could be detected.

Localization of glucocorticoid hormone receptors in mitochondria of human cells.

Glucocorticoid hormones regulate the transcription of nuclear genes by way of their cognate receptors. In addition, these hormones also modulate mitochondrial gene transcription by mechanisms which are as yet poorly understood. Using immunofluorescence labeling and confocal laser scanning microscopy we show that the glucocorticoid receptor of HeLa and Hep-2 cells is specifically enriched at the sites of the mitochondria which were visualized by labeling with the vital dye CMX and antibodies against cytochrome oxidase subunit I. Immunogold electron microscopy demonstrated that the receptor was located within the inner space of the mitochondria. Immunoblotting experiments also revealed the presence of glucocorticoid receptor in mitochondria isolated from HeLa and Hep-2 cells. Finally, living HeLa cells expressing green fluorescent-glucocorticoid receptor fusion protein revealed a distinct mitochondrial GFP fluorescence. Our results support the concept of a receptor-mediated direct action of steroid hormones on mitochondrial gene transcription.

Interaction of cytosol fractions containing activated glucocorticoid-receptor complexes from rat liver and thymus with heterologous nuclei: effects on transcription.

Two rat liver cytosol fractions containing activated glucocorticoid-receptor complexes are able to stimulate the transcriptional activity of rat liver nuclei; the respective fractions from the cytosol of thymocytes inhibit the capacity of thymus nuclei for RNA synthesis. A similar inhibitory effect on thymus nuclei is exerted by the presence of rat liver cytosol fractions. Spot hybridization using a tyrosine aminotransferase (TAT) probe demonstrates that TAT gene expression is stimulated by the liver cytosol fractions acting on homologous nuclei whereas it is inhibited, in thymus nuclei, by the addition of thymus cytosol fractions. No effect on transcription is observed if the liver or thymus cytosol is heat activated in the presence of the glucocorticoid antagonist, cortexolone. Treatment of liver nuclei, previously subjected to the action of thymus cytosol fractions with the respective liver ones, restores transcriptional activity to control or higher levels. We conclude that rat thymocyte nuclei and cytosol contain transcriptional factors, which in the presence of the glucocorticoid-receptor complex, irrespective of its source, inhibit gene expression, whereas in the absence of such factors, the glucocorticoid-receptor complex positively regulates the respective genes.

Autoantibodies to the core proteins of hnRNPs.

A novel autoantibody reacting the the core polypeptides of hnRNP particles has been detected in the serum of a patient with systemic lupus erythematosus (SLE) and Sjögren's syndrome manifestations. Immunoblot analysis, using either rat liver or HeLa nuclear extracts as the antigen source, demonstrated that the autoantibody interacts with a specific subgroup of the core polypeptides of hnRNP particles, namely A2, B1 and B2, but not with A1, C1 and C2.

Identification of two discrete ribonucleoprotein particles within the monomer population of rat liver nuclear RNPs.

30-50 S RNP particles (monoparticles) isolated from rat liver nuclei were submitted to electrophoresis in native 0.5% agarose gels. Two RNP fractions were thus separated, a minor one remaining closer to the top of the gel (MI) and a more abundant one migrating further into the gel (MII). SDS-polyacrylamide gel electrophoresis revealed that MII contains the major monoparticle (Mr 30 000-40 000 or 'core') polypeptides and higher molecular weight proteins, whereas MI contains several minor proteins of Mr greater than 40 000. Some proteins are common to both particle classes. Urea-acrylamide gel electrophoresis revealed that HnRNA is mainly present in MII, whereas snRNA is confined to the MI particle class.

Testosterone induces Ca2+ influx via non-genomic surface receptors in activated T cells.

Using the Fura-2 method we investigated a possible direct action of testosterone on cytosolic free calcium of splenic T cells isolated from female C57BL/10 mice. Testosterone at physiological concentrations of 1-10 nM induces an increase in [Ca2+]i within seconds, which is due to Ca2+ influx and not to Ca2+ release from intracellular stores. In contrast, estradiol induces both Ca2+ influx and Ca2+ release. The testosterone-induced Ca2+ influx is mediated by Ni2+-blockable channels and is not inhibited by cyproterone, a blocker of the classical androgen receptor. Ca2+ influx can also be induced by testosterone conjugated to BSA which is impermeable to the plasma membrane. These data indicate a novel mode of direct action of testosterone on T cells which is not mediated through the classical androgen receptor response, but through unconventional plasma membrane receptors.

Characterization of a monoclonal antibody reacting with histone H3.

A hybridoma cell line, 1GB3, has been obtained from a fusion between SP/O-Ag 14 myeloma cells and lymphocytes from BALB/c mice immunized with rat liver nuclear proteins. This hybridoma secreted a monoclonal antibody of the IgG2b class which reacted specifically with histone H3 in enzyme-linked immunosorbent assay (ELISA) as well as in immunoblotting and immunodot assays. Stringent test conditions were necessary to eliminate the presence of nonspecific or contaminating reactions with other histones than H3. The monoclonal antibody appears to recognize an epitope situated in the N-terminal residues 20-50 of histone H3; it recognizes this epitope in the octamer aggregate of core histones but not in the core particle.

Stabilization and characterization of the dexamethasone-binding proteins in rat liver cytosol.

Conditions were worked out for maximal stabilization of dexamethasone binding activity of rat liver cytosol in the absence of the protective steroid ligand. Important stabilization factors are ionic strength, thiol-protecting agents, glycerol and pH. Maximal stability of the cytosol is observed in a buffer consisting of 20 mM Tris-HCl, pH 7.5, 50 mM KCl, 25 mM beta-mercaptoethanol and 20% glycerol. Chromatography of cytosol on DEAE-cellulose revealed the existence of three dexamethasone receptors, binder DE-1, present in the flow-through fraction and binders DE-2 and DE-3, eluting from the column with salt concentrations of 100 and 190 mM, respectively. Binders DE-2 and DE-3 are not adsorbed on phosphocellulose at pH 7.5, whereas binder DE-1 is. All three receptors are retained to varying degrees on DNA-cellulose columns: binder DE-1 is eluted with salt concentrations of 270 mM, whereas binders DE-2 and DE-3 are eluted between 180 and 200 mM NaCl. The dexamethasone receptors also bind natural glucocorticoids, but to varying degrees, the highest binding being observed to binder DE-2. The receptors obtained after chromatography on DEAE-cellulose, but not on phosphocellulose, cannot be to an appreciable extent charged with dexamethasone.

Hormonal steroids act as tumour promoters by modulating oncogene expression.

Recent advances in the molecular action of steroid hormones and in the role of oncogenes in cell transformation are considered in defining, at the molecular level, the involvement of steroid hormones in tumour formation. In the context of the generally accepted three-stage model of carcinogenesis, it is proposed that the hormonal steroids act as tumour promoters by modulating oncogene expression. It is postulated that the hormonal steroids act on cells in which the initiating carcinogen has either induced mutations in protooncogenes normally hormonally regulated or has induced changes in gene architecture, aligning protooncogenes to hormone-responsive elements, thus placing these genes under non-physiological hormonal control. In contrast to the defined action of solitary carcinogens on the genetic material, tumour promoters appear to act by various molecular pathways, one of which, as hypothesized for hormonal steroids, could be a direct effect on oncogene expression.

Absence of in vitro mutagenicity of the fluid from breast cysts of women with macrocystic disease.

Cystic fluid from 30 Greek women suffering from macrocystic disease was tested for mutagenicity in the Salmonella typhimurium mutagenicity assay using three bacterial strains in the presence or absence of liver homogenate. None of the samples tested showed mutagenic potential in this test supporting the absence of potential carcinogens in the cyst fluids.

The oestrogen receptor codon 10 polymorphism detected in breast cancer is also present in non-malignant cells.

The effect of oestrogens on oestrogen-receptive organs and cells is mediated via intracellular receptors (ERalpha and ERbeta). Oestrogen receptor gene polymorphisms in the region encoding the N-terminal portion of the protein are reportedly associated with pathological conditions including breast cancer, hypertension, spontaneous abortion and coronary heart disease. A silent mutation in codon 10 of exon 1, detected in ER-negative and ER-positive human breast cancer cell lines, in breast tumors and blood DNA from breast cancer patients, has been recognized as a polymorphic site. In this study we examined, by denaturing gradient-gel electrophoresis and DNA sequence analysis, the possible presence of a codon 10 polymorphic site in normal oestrogen target organs and cells such as the uterus (myometrium and endometrium), in the placenta and peripheral blood mononuclear cells and in a benign uterus tumour (leiomyoma). We have detected ER codon 10 polymorphism in these samples and have compared them to those observed in breast cancer samples. All tissues and cells studied were homozygous for the wild-type gene, and were heterozygous as well as homozygous for the codon-10-variant type. These results indicate that the presence of the codon-10-variant type is not a characteristic of breast cancer. Out current findings suggest that further investigations are warranted to elucidate the possible linkage of ER codon 10 polymorphism to physiological and pathological conditions.

Detection of oestrogen receptor variants in endometrium, myometrium, leiomyoma and peripheral blood mononuclear cells: comparison to variants present in breast cancer.

Oestradiol has mitogenic and regulatory effects on various organs and cells, mediated mainly by its nuclear receptor (ER). The presence of aberrant ER forms in Oestrogen-dependent tumours has been discussed in correlation with tumour progression. ER variants, generated by alternative splicing, have been detected in human breast cancer, but also in normal mammary glands, therefore their role in tumorigenesis has been questioned. We have investigated, by the use of the reverse transcription polymerase chain reaction amplification technique, the possible existence of ER variants in other normal oestrogen target organs and cells, such as uterus (myometrium and endometrium), in peripheral blood mononuclear cells and in a benign uterus tumour (leiomyoma). We have detected variant ER in these samples and have compared the variant profile to that observed in breast cancer. All tissues and cells studied expressed both wild-type ER and variant species. Variant forms encompassed ER with deletions of exons 2, 5 and 7. Variants with exon 5 deleted were detected only in peripheral blood mononuclear cells and in breast cancer. Variants with exons 2 and 7 deleted were present in all specimens tested. These results corroborate previous findings that the presence of ER variants is not a characteristic of breast cancer. The physiological significance and possible clinical relevance of the variant ER forms remain to be elucidated.

Co-expression of trypsin and tumour-associated trypsin inhibitor (TATI) in colorectal adenocarcinomas.

Trypsin and its specific inhibitor, TATI (tumour-associated trypsin inhibitor), are expressed in normal human pancreas and in a variety of tumours. The aim of the present study was to assess the parallel expression of trypsin and TATI in colorectal cancer, in comparison with their expression in normal epithelial tissue, since proteases and their inhibitors are thought to be co-expressed in malignant neoplasms. We also assessed the possible significance of their expression as a means of differentiation between normal and malignant tissue. We examined qualitatively and semi-quantitatively the immunohistochemical expression of trypsin and TATI on paraffin-embedded serial tissue sections from 91 colorectal adenocarcinomas. The reverse-transcriptase-polymerase-chain reaction (RT-PCR) was also performed on fresh malignant tissue from 55 of the above adenocarcinomas. Normal and non-malignant tissues adjacent to the tumours were also evaluated. Cytoplasmic expression of trypsin (more than 25% of the cancer cells positive) was found in 67 (73.6%) adenocarcinomas, whereas TATI was expressed in the cytoplasm of 59 (64.8%) cases studied. Statistical analysis using Spearman's test has demonstrated a significant correlation between trypsin and TATI immunohistochemical expression (p<0.01). RT-PCR showed co-expression of trypsin and TATI mRNA in all carcinomas studied. Distinct patterns of trypsin and TATI immunohistochemical expression were observed in adjacent, non-malignant tissues, where both trypsin and TATI mRNA were also detected. Normal tissues were negative by immunohistochemistry. Our results indicate co-expression of trypsin and TATI in colorectal tumours both at the mRNA and protein level. We conclude that in colorectal neoplasms, high levels of trypsin and TATI may be important for malignant tumour formation and/or metastatic process.