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Hormonal requirements for full hepatic expression of alpha(2)-macroglobulin (alpha(2)M), alpha(1)-acid glycoprotein (AGP), haptoglobin (Hp) and gamma-fibrinogen (Fb) were assessed at the level of mRNA. Prior to exposure to turpentine-induced inflammation, rats were either depleted of glucocorticoids by adrenalectomy or supplemented with an excess of dexamethasone. Adrenalectomy alone did not affect the basal level of acute phase protein (APP) expression except for alpha(2)M mRNA, the level of which was enhanced. In contrast, dexamethasone treatment alone promoted full induction of alpha(2)M, significant, but not maximal increase of AGP and Hp mRNAs and suppression of Fb. In adrenalectomized rats, acute phase (AP)-cytokines, released in response to inflammation, promoted full expression of Fb and Hp and increased the level of AGP mRNA whereas alpha(2)M mRNA remained at the basal level. Inflammation in dexamethasone pretreated rats elicited changes which, in comparison to mRNA values for dexamethasone unpretreated inflamed rats, were seen as overexpression of alpha(2)M, full expression of AGP and incomplete expression of Hp, whereas Fb mRNA remained at the basal level. These data suggest that glucocorticoids are the principal inducers of a(2)M and AP-cytokines of Fb. For full induction of AGP, additive actions of glucocorticoids and AP-cytokines are required whereas expression of Hp is predominantly controlled by AP-cytokines. (C) 1998 Elsevier Science Ltd. All rights reserved.

The glucocorticoid receptor (GR) is a phosphoprotein and a member of the steroid/thyroid receptor superfamily of ligand dependent transcription factors. When the glucocorticoid receptor is expressed in yeast (Saccharomyces cerevisiae), it is competent for signal transduction and transcriptional regulation. We have studied the glucocorticoid receptor phosphorylation in yeast and demonstrated that the receptor is phosphorylated in both the absence and presence of hormone, on serine and threonine residues. This phosphorylation occurs within 15 min upon addition of radioactivity in both hormone treated and untreated cells. As reported for mammalian cells, additional phosphorylation occurs upon hormone binding and this phosphorylation is dependent on the type of the ligand. We have followed the hormone dependent receptor phosphorylation by electrophoretic mobility shift assay, and have shown that this mobility change is sensitive to phosphatase treatment. In addition, the appearance of hormone dependent phosphoisoforms of the receptor depends on the potency of the agonist used. Using this method we show that the residues contributing to the hormone dependent mobility shift are localized in one of the transcriptional activation domains, between amino acids 130-247. We altered the phosphorylation sites within this domain that correspond to the amino acids phosphorylated in mouse hormone treated cells. Using phosphopeptide maps we show that hormone changes the peptide pattern of metabolically labelled receptor, and we identify peptides which are phosphorylated in hormone dependent manner. Then we determine that phosphorylation of residues S224 and S232 is increased in the presence of hormone, whereas phosphorylation of residues T171 and S246 is constitutive. Finally, we show that in both yeast and mammalian cells the same residues on the glucocorticoid receptor are phosphorylated. Our results suggest that yeast cells would be a suitable system to study glucocorticoid receptor phosphorylation. The genetic manipulability of yeast cells, together with conservation of the phosphorylation of GR in yeast and mammalian cells and identification of hormone dependent phosphorylation, would facilitate the isolation of molecules involved in the glucocorticoid receptor phosphorylation pathway and further our understanding of this process. (C) 1998 Elsevier Science Ltd. All rights reserved.

The glucocorticoid receptor (GR) signal transduction and transcriptional regulation are efficiently recapitulated when GR is expressed in Saccharomyces cerevisiae. In this report we demonstrate that the in vivo GR phosphorylation pattern, hormone dependency and interdependency of phosphorylation events were similar in yeast and mammalian cells. GR phosphorylation at S246 exhibited inhibitory effect on S224 and S232 phosphorylation, suggesting the conservation of molecular mechanisms that control this interdependence between yeast and mammalian cells. To assess the effects of GR phosphorylation the mutated GR derivatives T171A, S224A, S232A, S246A were overexpressed and their transcriptional activity was analysed. These receptor derivatives displayed significant hormone inducible transcription when overexpressed in S. cerevisiae. We have established an inducible methionine expression system, which allows the close regulation of the receptor protein levels to analyse the dependence of GR function on its phosphorylation and protein abundance. Using this system we observed that GR S246A mutation increased its activity across all of the GR concentrations tested. The activity of the S224A and S246A mutants was mostly independent of GR protein levels, whereas the WT, T171A and S232A mediated transcription diminished with declining GR protein levels. Our results suggest that GR phosphorylation at specific residues affects its transcriptional functions in a site selective manner and these effects were directly linked to GR dosage. Crown Copyright (C) 2010 Published by Elsevier Inc. All rights reserved. Ministry of Science and Technological Development of Serbia [03E24, 143042B, 143044B]; Serbian Academy of Sciences and Arts; Wellcome Trust [069024]

In order to contribute to the understanding of mechanisms by which regulatory proteins recognize genetic information stored in DNA, analyses of their interaction with specific nucleotides are usually performed. In this study, the electrophoretic mobility shift assay (EMSA) was applied to analyze the interaction of nuclear proteins from the liver of rats of different age i.e., young (3-month-old), middle- aged (12-month-old) and aged (24-month-old), with radioactively labelled synthetic oligonucleotide analogues, corresponding to GRE. The levels of GRE binding activity were assessed by quantitative densitometric scanning of the autoradiograms. The results showed statistically significant decreasing values of up to 78% and 49% in middle aged and old animals, respectively, compared to young animals (p < 0.05). The specificity of the nuclear proteins-GRE interaction was demonstrated by competition experiments with unlabelled GRE. In a supershift assay, using the antibody BuGR2, it was shown that the GR proteins present in nuclear extracts have a high affinity for the GRE probe. The stabilities of the protein-DNA complexes were analysed and it was concluded that they changed during ageing. .

Inflammation/Irradiation/Radioprotection/α 2 -Macroglobulin/Dexamethasone. This work was aimed at the radioprotective efficacy of turpentine oil (TO), α 2 -Macroglobulin (α 2 -M), Amifostine (Ami) and/or dexamethasone (Dex). These agents were administrated, alone or in combination, prior to irradiation of rats with 6.7 Gy (LD 50/30 ). The survival was recorded daily for 4 weeks after irradiation and body weight, peripheral leukocytes and thrombocytes were measured. The plasma concentration of α 2 M and other acute phase proteins were determined by crossed immunoelectrophoresis. All rats receiving α 2 M and Ami alone or in combination survived the radiation injury, whereas the rate of survival of TO-treated rats was 90%. Radiation and therapy-induced changes in the expression of acute phase protein genes were atypical for the acute phase reaction. Dex alone was lethal for 45% and 55% of control and irradiated rats, respectively. Pretreatment with 1mg Dex reduced radioprotective efficacy of TO and Ami to 30% and 40%, respectively. Given together TO and Ami provided 70% protection to rats receiving Dex. The TO and α 2 -M enhanced the rate of survival from 50% to 90% and 100%, respectively. In the presence of 1mg Dex the TOinduced radioprotectors and Ami exhibited radiosensitizing rather than radioprotecting activities.

The effect of dexamethasone (DEX) on glucocorticoid receptor (GR)-mediated gene expression was examined in the brain of young and aged rats. Electrophoretic mobility shift assays showed that DEX treatment led to an increase of glucocorticoid response element (GRE) binding activity in aged rats, whereas in young animals GRE binding activity was decreased. Western blot analysis and reverse transcriptase polymerase chain reaction confirmed that, in aged animals, the GR mRNA and the GR protein levels were increased on DEX treatment. The binding activity of GRE activating protein-1 (AP-1) site and cross-competition analysis demonstrated specific pattern of expression during the ageing and DEX treatment, suggesting that GR modulates the activity of transcription factors AP-1 (Fos/Jun proteins) through protein-protein interaction. On the basis of these results, it can be concluded that the composition of transcriptional complexes that bind to GRE and AP-1 regulatory elements changes upon DEX treatment in an age-specific manner.

The role of the glucocorticoid receptor (GR) in senescence was studied in rats of increasing age. Statistically significant changes in the number of GRs from rat liver were detected, whereas the affinity for the ligand triamcinolone acetonide (TA) did not change with increasing age, and was in the range of 1-2 nM. In all cases the number of receptors was lower in rats treated with hormone in vivo relative to untreated animals. In addition, we have found changes in GR activation, as measured by the binding to DNA cellulose in the mentioned age groups. Furthermore, expression of the glucocorticoid hormone (GH)-inducible gene, tyrosine amino transferase (TAT) also showed age-related alterations. We conclude that receptor function shows oscillatory changes during ageing. In addition, response to GH generally declines towards the older age. This. specific periodicity in functional characteristics of the GR may reconcile conflicting results about the receptor number and properties during the ageing process, and marks particular age at which individual organism shows the highest or the lowest sensitivity to the actions of GH. (C) 1999 Elsevier Science Inc. All rights reserved. null

Activities of superoxide dismutases MnSOD and CuZnSOD were measured in appropriate subcellular fractions prepared from livers of intact and long-term gonadectomized (GX) rats of both sexes, and of GX female and male rats injected sc with a single dose of 5 mu g estradiol benzoate (EB) or 2 mg progesterone (P). In female livers, MnSOD activity did not vary significantly during the estrous cycle, declined after gonadectomy in comparison to proestrus, and was steady in GX females treated with EB or P. The activity of CuZnSOD was lowered at proestrus and elevated after removal of the ovaries in comparison to proestrus value. EB suppressed, and P elevated CuZnSOD activity in GX females. In the liver of male rats, MnSOD was not affected by gonadectomy nor by EB and P treatments. CuZnSOD activity was reduced following orchiectomy and enhanced in GX mates following treatment with P, while EB had no effect. These results suggest that P and EB modulate the activity of CuZnSOD and do not affect MnSOD in the rat liver. The modulatory effects are elicited by P in the males and by P and EB in the females. (C) 1997, Editrice Kurtis.

Continuous secretion of a variety of growth factors, hormones and other regulatory elements is a common characteristic of tumor cells. The inappropriate control of the expression of some tissue-specific proteins and the ectopic synthesis of biologically active molecules such as various autocrine mitogenes, transforming growth factors, and differentiation-inhibiting or differentiation-inducing factors, result from a pleiotropic effect of oncogene activities in malignant cells that derepress and/or keep active a number of genes, which are normally silent in differentiated cells (1). Available literature on growth factors and hormones secreted by both normal and malignant cells is so vast that inevitably a reduced number of contributions will be referenced in this short overview.

When cancer is viewed as a disease of abnormal cell regulation of the growth and differentiation, the importance of signal transduction to cancer is obvious. It has been shown in a number of tissues that steroids play important roles in the mechanism(s) of signal transduction, and are probably through this mechanism(s) involved in the initiation, progression and/or inhibition of carcinogenesis. Steroid receptors belong to a family of regulatory proteins - cellular regulatory transcriptional factors - that interact with specific DNA promoter enhancer elements and regulate gene expression. This ability of steroid receptors depends on the binding of specific hormones and on the activation of steroid-receptor complex. Since the expression of some oncogenes could be subjected to the regulatory action of steroid hormones on one hand and since steroid receptors, and some oncogene products, share striking homology on the other hand, it can be postulated that the mechanism of action of steroid hormones on both receptor and post receptor levels, may be closely related to the cellular mechanism of action of oncogenes and their protein products (for references see 1).

The facts about the role of glucocorticoids in malignant melanoma growth in vivo and in vitro are still controversial. There are a lot of data showing that glucocorticoids cause significant tumor growth inhibition in vivo. Several investigators have demonstrated growth inhibition with glucocorticoids in human melanoma cell lines. On the other hand, in B16 mouse melanoma cells glucocorticoids failed to inhibit the growth in vitro as they did in vivo (1). A dose-related glucocorticoid-induced inhibition has been noted by Benckhuijsen (2) and in serum-free media by Di Sorbo (3). Furthermore, an increase of certain proteins was detected upon glucocorticoid treatment in different B16 cell lines (1). In our previous experiments with different glucocorticoids the inhibition of B16/C3 mouse melanoma cell growth and increased melanin secretion have been observed (4).

Rat liver ribosomes, prepared 1–24 h after intraperitoneal cortisol injection, contain multiple phosphorylated S6 consisting of four distinct derivatives in addition to the original non-phosphorylated S6. 25 h following the hormone injection the extent of S6 phosphorylation, as judged by its electrophoretic pattern in two-dimensional gels, resembles that of untreated rats. Ribosomal subunits with gradually increased degree of S6 phosphorylation, isolated at different time intervals after cortisol injection, exhibit polyphenylalanine polymerization levels inversely proportional to the extent of S6 phosphorylation. In addition, they show an elevated misincorporation of leucine in a poly(U)-programmed in vitro system. The lower amount of polyphenylalanine synthesized by multiple phosphorylated ribosomes in vitro is likely due to an enhanced susceptibility of nascent polypeptide chains synthesized in the in vitro system to proteinases present in the pH 5 and S-100 fractions. Liver polysomes derived from cortisol-treated animals lose their highly phosphorylated derivatives when exposed to S-100 enzymes. The loss can be prevented by concomitant action of proteinase and RNAase inhibitors (phenylmethylsulfonyl fluoride and heparin) but not by an inhibitor of phosphatase (sodium fluoride). In the absence of RNAase and proteinase inhibitors only degradation of old 40 S subunits can be demonstrated. 25 h after the cortisol treatment degradation of liver ribosomes occurs simultaneously with S6 dephosphorylation and is preceded by polysomal breakdown.

In a Yugoslav village, psychosocial, anamnestic medical, and pathophysiological data were recorded for 1,353 persons in a longitudinal study. The role of psychosocial stress in carcinogenesis, as far as we have investigated it, may be described as follows: (1) Psychosocial stress in terms of high hopelessness, high antiemotionality, etc. has a strong relevance for cancer incidence which does not act via one of our physiological variables associated with cancer. This follows from the results of our multivariate analysis. (2) Psychosocial stress is substantially associated with a low lymphocyte percentage, which in turn is a relatively strong risk factor for cancer. (3) Psychosocial stress is relatively weakly associated with the cholesterol minimum; but apart from the fact that the cholesterol characteristics of cancer subjects are more marked descriptively under stress conditions, psychosocial stress significantly enhances the efficacy of the most important physiological risk variables for cancer.

An unlinked regulatory mutation hisT1504, causes an approximate 11-fold derepression of the histidine (his) operon and a linked constitutive mutation hisO1242 causes an approximate 15-fold derepression. In this study we demonstrate that hisT1504 provokes a significant increase in the UV-induced reversion frequency of his ochre and frameshift mutations. Analysis of revertants derived from frameshift mutants show that this increment in derepressed strains compared to the repressed strains is due to better growth of suppressed revertants by weak frameshift suppressors. The frequency of revertants suppressed by strong frameshift suppressors appears to be the same in repressed and derepressed strains. In contrast, intragenic revertants appear at two-fold decreased frequency in derepressed strains carrying either of the histidine constitutive mutations, hisT1504 or hisO1242. A possible competition is indicated between frequently transcribing RNA polymerase and error-promoting recombinational repair within the histidine operon.

Cortisol injected into the rat induces immediate metabolic changes in liver cells, occuring within time intervals shorter than that accounted for by the expression of specific genes. Among these extragenomic acute metabolic responses we describe: the enhanced rate of the synthesis of receptor protein for cortisol, the increase in overall capacity for protein synthesis and increased rate of phosphorylation of histones and nonhistone proteins. All these changes depend on cortisol and occur within the first 30 minutes after hormone administration. All these acute metabolic responses to cortisol might be correlated to the hormone-produced activation of the native macromolecular receptor-system. In an attempt to integrate extragenomic and genetic responses to cortisol a new concept and hypothesis of cortisol action is proposed, which can probably be extended to other steroids and extrapolated to other steroid responsive tissues.

As soon as the first clinico-epidemiology reports appeared describing an association of herpes virus infection with genital cancer, relevant questions were introduced into an ongoing follow-up of a cohort already being studied for psychosocial risk factors in cancer. The study, which was initiated in 1966, comprised 1353 persons; disease incidence was determined in 1976. Incidences of cancer (including extragenital cancer) were found to be directly related to the reported prevalence of genital herpes symptoms, but were related inversely to extragenital herpes prevalence and to previous fever episodes. With regard to the relatively common incidence of "fever blisters", i.e. facial herpes, attributed to HSV-type 1, the latter findings is less surprising than the strong association of HSV type 2 lesions with overall cancer incidence. This association was particularly noticeable in individuals who had not reported episodes of fever earlier. These findings indicate the predictive role of certain host factors which are possibly related to systematic responses of the organism, including a diminished protection against (latent) herpes genitalis infection (recurrences). Further prospective studies are needed to investigate the proper time-relationship between clinically-overt herpes genitalis and the manifestation of cancer and its possible role in provoking the recurrent eruption of latent infections.

[3H] Cortisol was found to be bound to two distinct components of receptor protein in hepatic cytosol of both normal and adrenalectomized adult male rats. In liver cytosol of normal rats, 78 % of total bound [3H] Cortisol to receptor was associated with the first receptor component, and about 20% to the second one. However, the first component of cytosol receptor in adrenalectomized rats was found to bind 95% of total [3H] Cortisol bound to both receptor components, while only 4.5% of the bound hormone was found associated with the second receptor component. These alterations in binding capacity are discussed in relation to possible regulation of nuclear RNA synthesis by cortisol-receptor complexes.

In 1965–66, a prospective psychosomatic investigation was started with 1,353 relatively old inhabitants of the village of Crvenka, Yugoslavia. The present article reports on the relevance of smoking for the incidence of lung cancer and cardiac infarct. The main results are: (1) The relevance of smoking is reduced, but not eliminated, by introducing psychosocial control variables, suggesting that the latter have direct influences both on smoking and on the diseases. (2) The relevance of smoking interacts very strongly with psychosocial background conditions: it is nearly reduced to zero when the latter are favorable, and is correspondingly high when they are unfavorable. The results are also interpreted in biochemical terms.

The presence of the histidine operator-constitutive mutationhis01242 increases UV-induced mutability within the histidine operon ofSalmonella typhimurium. The rate of reversion ofhisC andhisF ochre and frameshift mutants is increased 5- to 8-fold when these mutations are coupled withhis01242 which causes 15-fold derepression of the operon. The effect does not extend to the whole chromosome since the rate of UV-induced mutability at the unlinked streptomycin locus is the same in the strains carryinghis0+ orhis01242 alleles. The same phenomenon was observed in Hcr− strains.

A large body of recent data is consistent with the idea that steroids, at least sex steroid hormones, regulate gene expression in eucariotic cells via a common two-step molecular mechanism. This two-step model has acquired the status of a “dogma” and has become a generally accepted theoretical framework for experimental research being currently conducted. However, this model (dogma) does not take into consideration the immediate extragenomic effects caused by steroids in responsive tissues, nor does it offer the tentative link between extragenomic and genomic events occurring in target cells. The results of our recent studies on cortisol specific receptor and its regulatory functions lead us to propose a new speculative model, the postulates of which are aimed at integrating both the extragenomic and genomic events occurring in target cells during the course of steroid hormone action. The key concepts of the proposed model are the following: the native cytoplasmic holoreceptor should be a multimer, consisting of several different subunits and comprising the denned “metabolic code” for various multiple cooperative metabolic functions. The “activation” of the receptor, caused by the binding of appropriate steroid hormone, results in the disaggregation of the receptor into the monomeric subunits, which play the role of regulatory proteins. The released subunits—various regulatory proteins—then exert their control at different levels of the mechanism of genetic expression, including the post-translational level. They modulate the preformed molecules and the activities of the regulatory mechanisms operative in respective target cells like for instance phosphorylation/dephosphorylation mechanism. These events result in modifications of translations on the preformed mRNA's and cell membrane transport. The resulting changes are immediate and underlie the steroid-induced extragenomic effects. The subunit(s), which binds the steroid hormone, so called “steroidophilic”—subunits, regulates the rate of transcription, i.e. selective and specific gene expression in target cells. This subunit is a key stone in the macromolecular organization of the receptor. It may be of an isoproteinic nature and may have different affinities towards other subunits thus determining the assembly and nature of other associated subunits, giving rise to the tissue specific receptor polymorphism and causing variations in the “metabolic code” of receptors. “The activation” of receptor, caused by steroids, and resulting in disaggregation of multimer into the monomeric subunits is the key event in the cell responses to steroids. It triggers, controls and links extragenomic events with gene expression. These extragenomic and genomic events are included in the “metabolic program” encoded in the molecular structure of the receptors. The steroids, as effectors trigger the receptor activation, which controls the sequential and selective metabolic events, underlying the specific physiological “responses” of steroid responsive tissues. In this paper we will present some of our recent data to further substantiate the proposed “model” of the integral steroid hormone action in target tissues.

We investigated the relation of psychosocial risk factors to mortality in a prospective study of 1353 inhabitants of Crvenka, 619 of whom died between 1966 and 1976. All 38 lung cancer deaths occurred in those with high scores for rationality and antiemotionality (R/A), a factor related to suppression of aggression. Compared with lower R/A, high R/A was also associated with a relative risk of mortality of 29 for other cancer, 4.3 for ischaemic heart disease and 6.5 for stroke. Standardising for R/A reduced the smoking/lung cancer association, virtually eliminated the smoking/other cancer and smoking/heart disease relationships and reduced the association of heart disease with blood cholesterol, blood sugar and hypertension. Long lasting hopelessness was also independently associated with cancer as was anger with heart disease, though not so strongly as for R/A. Psychosocial variables are important predictors of mortality and decisively modify the effect of physical risk factors such as smoking.

tsAEV-LSCC HD3 chicken erythroid cells transformed by the avian erythroblastosis virus (AEV) secrete an autocrine differentiation-inhibiting factor, ADIF, which blocks differentiation without affecting proliferation of the chicken erythroid cells that synthesize and secrete it into the culture medium. The chicken erythroleukemia cell ADIF activity is not restricted to avians. It prevents dimethylsulfoxide (DMSO) from stimulating murine Friend erythroleukemia cells to synthesize hemoglobin. ADIF also blocks erythroid differentiation in normal human and murine bone marrow where it selectively targets the early BFU-E (burst-forming) erythroid precursor cells without affecting the more advanced CFU-E erythroid precursor cells or cells of the different granulocyte-macrophage lineage.

The core of the authors' research program consists in the assumption that complex statistical interactions of sociological and psychological factors with pathophysiological and standard risk factors are more important for the early detection, prediction and prevention of cancer than one or just a few medical risk factors, e.g. the relationship between smoking and lung cancer. As the analysis of these relationships can only be done in interdisciplinary research, interpretation and discussion of social scientific, molecular biological and epidemiological aspects was attempted. In the first part we explicate a model of relevant propositions from the psychosomatic literature to explain the relationship between psychosocial factors of carcinogenesis. A discussion of the authors' research program contains additional variables and propositions. It appears to the authors that among the psychosocial and sociological factors of carcinogenesis, the molecular biological variables are especially relevant, as they form the intervening link between the social science variables and cancer. Discussion of the methodology and the sampling of the authors' empirical studies follows. Two prospective studies, carried out in Yugoslavia and West Germany, are described in detail. The data of these studies are used in the empirical analysis. In the next part the empirical results of different multivariate analyses are presented: The effects of the sociological and psychological variables on carcinogenesis are described; standard risk factors and biochemical-molecular biological factors are brought together, special attention being given to the interaction between these two groups of variables.

The cortisol binding compound, from the cytoplasm of rat liver, was purified by gel filtration and precipitation with ammonium sulphate. The binding compound from rat liver cytoplasm, has been found to consist of two distinct protein fractions. The proteins eluted from DEAE-cellulose column chromatography at 0.04 M and 0.18 M concentrations of KCl, have two different isoelectric points, one at pH 4.85–5.00, and another at pH 5.85–6,10, but only the fraction eluted at 0.04 M KCl was found to be able to stimulate the incorporation of 32 P into RNA of incubated rat liver nuclei. The highest values of 32 P incorporation in the nucleic acids of incubated nuclei were obtained with partially purified hormone protein (s) complexes wich were precipitated with ammonium sulphate saturation between 20–25% and 25–30%.

Survival curves were established for Escherichia coli b/r (CSH). Cells were then inoculated into media containing 50, 100, 200, and 300 mu g/ml of 5- bromodeoxyuridine (5-BUDR). Treatment with increasing amounts of 5-BUDR was followed by a decrease in radioresistance. A second series of cultures was supplemented with 100 mu g/ml of 5BUDR during log phase. A decrease in radioresistance proportional to the number of cell divisions in the presence of 5- BUDR was observed. (H.M.G.)