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Background In female patients with systemic lupus erythematosus (SLE), we identified estrogen receptor ERα, ERβ and ERα variant transcripts in peripheral blood mononuclear cells (PBMC). Exon 1 and 2 of ERα gene was subjected to mutation analysis to assess whether possible nucleotide alterations are linked to the disease. Methods The whole coding sequence of ERα was analysed by reverse transcription polymerase chain reaction (RT-PCR) and cDNA sequencing in PBMC prepared from 19 SLE patients and 12 healthy females. ERα exon 1 and exon 2 were subjected to mutation analysis using DNA isolated from whole blood of 21 SLE patients and 29 healthy females. The aminoterminal coding sequence of ERβ was also analysed by RT-PCR. Results Wild type ERα and ERα splicing variants with deletions in exons 2, 5 and 7 were detected both in healthy individuals and in SLE patients, with no qualitative difference in their expression among the two populations. In ERα exon 1, the polymorphisms identified codon 10 and codon 87, both in patients and in healthy individuals who were not associated with the disease. No other mutations were present in ERα exon 1 or ERα exon 2 in all subjects studied. ERβ was expressed in both populations. Conclusion: PBMC of SLE patients express wild type ERα, ERβ and the same ERα variants as do healthy individuals. Genetic alterations in exon 1 and exon 2 of the ERα gene are not linked with SLE disease.

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.

The central role of estrogens and androgens in the male reproductive system has focused attention on the presence and distribution of their cognate receptors [estrogen receptor (ER) alpha, ERbeta and androgen receptor (AR)] in male reproductive tissues and cells. Since the presence of steroid hormone receptors in mitochondria of mammalian cells has been well documented, we investigated the possibility of mitochondrial localization of sex steroid hormone receptors in sperm.Applying immunofluorescence labelling and confocal laser scanning microscopy we show that the estrogen receptor beta and the AR of human sperm are specifically enriched in the midpiece, at the site of the mitochondria, which were visualized by labelling with the vital dye CMX. Nuclear and mitochondrial localization of AR was also detected in LnCap human prostate cancer cells. Differentially, most of the ERalpha immunostaining is in the form of a compact zone at a region corresponding to the equatorial segment of the upper post-acrosomal region of the sperm head. Immunoblotting experiments using sperm extracts revealed the presence of a 66 and a 45 kDa protein reacting with the ERalpha antibody, one 64 kDa protein reacting with the ERbeta antibody and a 110 and a 90 kDa protein reacting with the antibody against AR.Our findings suggest that the differential localization of AR and ER isoforms in human sperm reveals distinct roles of these receptors in the physiology of sperm cells and, perhaps, also in the process of fertilization.

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 (p0.01). 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.

In female patients with systemic lupus erythematosus (SLE), we identified estrogen receptor ERa, ERb and ERa variant transcripts in peripheral blood mononuclear cells (PBMC). Exon 1 and 2 of ERa gene was subjected to mutation analysis to assess whether possible nucleotide alterations are linked to the disease.The whole coding sequence of ERa was analysed by reverse transcription polymerase chain reaction (RT-PCR) and cDNA sequencing in PBMC prepared from 19 SLE patients and 12 healthy females. ERa exon 1 and exon 2 were subjected to mutation analysis using DNA isolated from whole blood of 21 SLE patients and 29 healthy females. The aminoterminal coding sequence of ERb was also analysed by RT-PCR.Wild type ERa and ERa splicing variants with deletions in exons 2, 5 and 7 were detected both in healthy individuals and in SLE patients, with no qualitative difference in their expression among the two populations. In ERa exon 1, the polymorphisms identified codon 10 and codon 87, both in patients and in healthy individuals who were not associated with the disease. No other mutations were present in ERa exon 1 or ERa exon 2 in all subjects studied. ERb was expressed in both populations.PBMC of SLE patients express wild type ERa, ERb and the same ERa variants as do healthy individuals. Genetic alterations in exon 1 and exon 2 of the ERa gene are not linked with SLE disease.

Evidence suggesting the presence in rat liver nuclear extracts of a new RNP complex of 70-110S has been provided [Hatzoglou, M., Adamtziki, E., Margaritis, L. and Sekeris, C.E (1985) Exp. Cell. Res. 157, 227-241]. Biochemical features unique to this RNP were its stability to salt and RNase digestion and the presence of a pair of polypeptides of 72/74 kDa. By producing antibodies against the 72/74 kDa polypeptides these proteins have been defined as integral components of the 70-110S RNP complex. They comprise two immunologically related polypeptides with an exclusively nucleoplasmic localization, giving a speckled pattern in a diffuse background, similar, but not identical, to the Sm antigen. The 70-110S RNP complex, referred to as large heterogeneous nuclear RNP (LH-nRNP), has a simple protein pattern that includes, in addition to the 72/74 kDa proteins, three stably associated polypeptides of apparent molecular size 110, 61 and 59 kDa. The bulk of its RNA component represents a discrete RNA population of 10-20S, belonging to a subset of the RNA detected within immunopurified HeLa hnRNP complexes. These RNA species are RNA polymerase II transcripts of greater stability relative to the bulk of hnRNA, containing oligo(A) or poly(A) sequences. Immunodepletion and/or antibody addition studies in HeLa splicing extracts using antibodies with specificity for the 72/74 kDa proteins revealed a rather strong inhibition of splicing activity, suggesting participation of the LH-nRNP complex in in vitro splicing.

Immunostaining of estrogen receptors (ERs) was carried out on imprints of 62 breast carcinomas using monoclonal antibodies and a sensitive immunoperoxidase technique (the Abbott ER-ICA kit). The results were compared to those obtained by the conventional biochemical analysis of cytosol proteins and to the degree of tumor differentiation. The cytologic specimens were insufficient for analysis in 6 cases; of the remaining 56 cases, 37 (66%) showed a positive ER reaction. In 51 cases with both types of ER analysis, the immunocytochemical staining of the imprints correlated strongly with the biochemical analysis in 44 cases and weakly in 3. Four cases were negative immunocytochemically and positive biochemically. Among the ductal carcinomas, well-differentiated tumors had higher percentages of ER-positive cells than did poorly differentiated tumors. These results show that the immunoperoxidase method is a highly specific and sensitive technique for the evaluation of ER content; it may be applicable to small samples of tumor tissue and may provide additional information for identifying hormonally responsive breast tumors.

Sequences showing partial homology to glucocorticoid and estrogen responsive elements have been detected in the human and mouse mitochondrial genome. Considering the well established role of hormone responsive elements in steroid hormone action, it is postulated that the mitochondrial genome could be a primary site of action of steroid hormones.

Hormone and growth factor receptors elicit changes in gene expression which regulate cellular homeostasis, proliferation and differentiation. Many oncogenes code for aberrant hormone and growth factor receptors (e.g. v-erbA and v-erbB). The deregulated action of these products interferes with normal signal transduction for cellular proliferation and differentiation, thus leading to cellular transformation. In this context, receptor-mediated transcriptional effects are directly related to the molecular mechanisms underlying oncogenic transformation.

A heterogeneous RNP structure has been isolated from rat liver nuclei by a method previously used for the isolation of 30S RNP complexes carrying heterogeneous RNA (hnRNA) [1]. The RNP sediments in sucrose gradients with s-values of 70–110S. Formaldehyde-fixed preparations band at ρ = 1.40 in isopycnic CsCl gradients. The RNP structure is composed of a heterogeneous population of polypeptides, prominent among which are two proteins with Mr 74 000 and 72 000. It contains both rapidly labelled RNA as well as several species of snRNA, as demonstrated by double-labelling experiments and gel electrophonesis. Treatment of rats with α-amanitin leads to a significant decrease in the amount of recovered RNP. In the presence of 0.7 M NaCl the s-value of the complex changes from 70–110S to 40–80S. The RNP structure is stable to mild RNase A or micrococcal nuclease digestion. Transmission electron microscopy reveals the presence of a heterogeneous population of particles with a mean diameter of 300–360 A. The isolated RNP structure differs completely from the well-known monoparticle or polyparticle hnRNP complexes and from the 30S or smaller snRNP particles but could be similar to or identical with the heterogeneous complex described by Jacob et al. [29].

A messenger RNA fraction has been isolated from the liver of cortisol treated rats which when added to an in vitro protein synthesizing sytem gives rise to tyrosine-α-ketoglutarate transaminase activity. Development of enzyme activity is dependent on active protein synthesis and is inhibited by puromycin and erythromycin. It is concluded that the messenger RNA fraction contains the information for the formation of tyrosine transaminase and that the enzyme is formed in vitro by de novo synthesis.

The enzyme RNA polymerase is difficult to extract from most mammalian cells due to its firm association with chromatin. By a combination of intensive ultrasonic treatment and extraction of the nuclei with 0.75 M NaCl at 0° and pH 8.4 for 1.5 hours approximately 80% of the enzyme can be extracted in a soluble, DNA-dependent form. The extracted enzyme has been purified by DEAE-cellulose chromatography and centrifugation on sucrose gradients (5–20%, w/v) containing 10% glycerol. A purification of approximately 250-fold has been achieved in the peak fraction of the gradient. The enzyme requires all four nucleoside triphosphates, mercaptoethanol, a DNA template and either Mg++ or Mn++, the latter being the preferred ion. Enzyme activity can be stimulated by (NH4)2SO4. No direct stimulation of enzyme activity by cortisol phosphate could be observed.

In vitro transcription was used to show that RNA polymerase III is responsible for the initiation of transcription at a position 200 bp upstream from the epsilon-globin major cap site. High levels of -200 transcription interferes with the RNA polymerase II major cap site transcription. Using DNA mediated transient expression, the ratio of -200 to +1 transcription can be modulated by either the direction of replication or the presence of an enhancing element in the vector. We suggest that this heterogeneous usage of cap sites is not related to epsilon-globin gene transcription in vivo, but is instead the result of a combination of factors inherent to transient expression experiments.

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%.

The modulation of tryptophan-oxygenase (TO) and tyrosine amino-transferase (TAT) in the rat liver after a single dose of hydrocortisone has been studied under various physiological conditions. Differences in the induction behavior of the two enzymes have been observed dependent on sex, age, amount of administered hormone, and presence or absence of the adrenals. Some of the results observed are the following: 1) TO reached its maximum induction level 3 h prior to TAT in normal male rats. This difference disappeared when adrenalectomized male rats or when female animals were tested. 2) The maximal induction values of both enzymes were 50--80% higher in adrenalectomized rats than in normal rats. This effect was independent of sex. 3) Higher doses of hydrocortisone were necessary for optimal induction of TO and TAT in normal than in adrenalectomized rats. 4) The minimum dose of hydrocortisone necessary for enzyme induction was significantly lower for TO than for TAT. 5) Actinomycin D caused a complete inhibition of the induction of TO and TAT when given simultaneously with the glucocorticoid. The inhibition was less complete the longer the interval between hormone and actinomycin D administration. The activity of TAT was suppressed to a larger extent than TO.

Glucocorticoids rapidly affect nuclear RNA synthesis by binding, as hormone-receptor complexes, to Glucocorticoid Responsive Elements (GRE), differently positioned in glucocorticoid inducible genes. Glucocorticoids also affect, within minutes, mitochondrial RNA synthesis. We therefore searched for GREs in the mitochondrial genome of human (H), rat (R) and mouse (M) and found a number of such potential elements as follows: one within the 12s - rRNA gene (H1, R1 and M1) one within (H2), or at the start (R2, M2), of the presumptive protein 1 gene and three within the mouse cytochrome oxidase subunit 1 gene (COX1, COX2 and COX3). The nucleotide sequence of H1, R1 and M1 reveals the possibility of the formation of hairpin structures, stabilized by hydrogen bond formation, between three or four consecutive bases. The presence of potential GREs in the mitochondrial genome suggests an adjustment of mitochondrial metabolic control to the general control mechanisms of the cell.

The distribution and properties of cytoplasmic binding sites for the synthetic glucocorticoid dexamethasone and the natural glucocorticoid corticosterone in the brain and the pituitary were studied in detail. Cortisol-17 beta acid, a derivative which does not bind to the glucocorticoid receptor but is a competitor of corticosterone binding to plasma, was used to overcome plasma interference. In vitro competition assays in the presence of excess cortisol acid reveal that dexamethasone is as effective a competitor for [3H]corticosterone binding as corticosterone itself. Scatchard analysis of equilibrium experiments with both steroids, using cytosol from various brain areas and from the pituitary yielded linear plots, suggesting one class of binding sites. The quantitative distribution of the sites follows the pattern: cortex greater than hippocampus greater than or equal to pituitary greater than hypothalamus greater than brain stem white matter. Furthermore, kinetic analysis of corticosterone dissociation showed a first order reaction, thus indicating the presence of one type of receptor in all brain areas examined. Rat brain cytosolic receptors for corticosterone and dexamethasone elute from DEAE-Sephadex A-50 anion exchange columns at 0.3 M NaCl in the presence of stabilizing sodium molybdate and at 0.15 M NaCl and/or in the buffer wash when heat-activated, thus exhibiting the characteristic activation pattern of rat liver cytosolic glucocorticoid receptor. The ratio of the buffer wash to the 0.15 M NaCl form is low for dexamethasone and very high for corticosterone. Receptor complexes from various brain parts showed the same activation pattern. In our experiments, brain corticosterone and dexamethasone receptors stabilized by sodium molybdate are indistinguishable by a number of techniques, thus indicating that it is unnecessary to evoke specific binding sites for each glucocorticoid.

An important aspect of the use of inhibitors for the elucidation of mechanisms of hormone action is the sequence of administration of the inhibitor and of the hormone.

Some recent hypotheses on the mode of action of hormones are reviewed. One concept, arising from experiments with artificially cleaved ribonuclease, namely that hormones may »complete« some proteins to form an active enzyme, is regarded as unlikely. Moreover, the idea that hormones act as »allosteric effectors« of enzymes is not well substantiated. A third hypothesis, i. e. that hormones may act as gene activators, is discussed at some length. Gene activation would lead to production of messenger-RNA and induced enzyme synthesis. For ecdysone, the moulting hormone of insects, every step of this reaction sequence has been demonstrated experimentally.

The rapidly labelled RNA extracted from rat liver homogenate after a 4 min pulse sediments heterogeneously from 10 S to 80 S. After 30 min labelling time an accumulation of radioactivity between 30 S and 50 S is observed. A comparison of these sedimentation patterns of RNA derived from different labelling times indicates a greater metabolic instability of the rapidly labelled heterogeneously sedimenting RNA in relation to that of the 45 S r-precursor. The RNA sedimenting heavier then 45 S has a base composition which resembles that of DNA. The rapidly labelled RNA is resistant to DNase, pronase and EDTA treatment and thus aggregate formation with protein, DNA and bivalent cations is unlikely. Experiments with actinomycin D and cold orotic acid show a shift of radioactivity to lower sedimentation values which also reflects the metabolic instability of the heavy RNA. Labelling of the ribosomal peaks after actinomycin D treatment could be achieved only after the 30 min pulse, and not after the 4 min pulse, suggesting the absence of an appreciable amount of r-precursor in the shorter pulse. The RNA extracted from nuclei prepared with Triton X-100 remained in the upper fractions when analyzed on a sucrose gradient, reflecting the high susceptibility to degrading enzymes. The major part of the rapidly labelled RNA remains associated with the nuclear fraction.