Potential role of autophagy in behavioral changes of the flank organ

The flank organ of the Syrian hamster, which shows a biodynamic response to androgen stimulation, is considered a good model for studying the androgen effect on sebaceous gland and hair. This organ is susceptible to programmed cell death (PCD), a prominent feature associated with sexual organ adjustment. We have recently shown the first direct evidence of a relationship between autophagy and morphological changes in androgen-dependent organs. In this work, animals were stabulated (housed) for two months under control conditions and, after sacrifice, autophagy markers, Beclin 1 and LC3-II, were evaluated. The results revealed autophagic processes in the flank organ of both sexes, especially in females, and no detectable caspase-3 activity. Therefore, these data indicated that macroautophagy, and not apoptosis, was the main mechanism by which the flank organ responds to androgen. Here, we present additional data on the flank organ, showing that the intensity of autophagy between genders depends on the length of stabulation. These data could indicate a role for autophagy in response to behavioral influences. A possible explanation and its implication in Syrian hamster social defeat are discussed in this addendum.     

The effect of ketoconazole related imidazole drugs and antiandrogens on [3H] R 1881 binding to the prostatic androgen receptor and [3H]5 alpha-dihydrotestosterone and [3H]cortisol binding to plasma proteins

Ketoconazole an orally active imidazole drug and bifonazole, clotrimazole, econazole, isoconazole, miconazole and tioconazole are known inhibitors of cytochrome P-450 dependent steroidogenic enzymes. The aim of the present study was to determine whether these imidazole drugs also have an effect on [3H]R1881 binding to the human prostatic androgen receptor, [3H]5 alpha-dihydrotestosterone (5 alpha-DHT) binding to plasma sex hormone binding globulin (SHBG) and [3H]cortisol binding to plasma corticosteroid binding globulin (CBG). In comparison the effect of both steroidal (cyproterone acetate; CPA) and non-steroidal (anandron, flutamide, hydroxyflutamide, ICI 176344) antiandrogens on these steroid binding proteins was also determined. The results of the present study show that the imidazole drugs were without effect on [3H]R1881 binding to the androgen receptor and on [3H]cortisol binding to CBG up to 100 mumol/l. However, they were weak competitors of [3H]5 alpha-DHT binding to SHBG inhibiting 20-53% of binding at 100 mumol/l. In comparison the antiandrogens were strong competitors of [3H]R1881 binding to the androgen receptor, the order of decreasing potency, determined from ID50 (mumol/l) values were CPA (0.073) greater than ICI 176344 (0.4) greater than anandron (0.63) greater than hydroxyflutamide (1) greater than flutamide (greater than 100). The non-steroidal antiandrogens were without effect on [3H]cortisol binding to CBG whereas CPA caused 36% inhibition of binding at 100 mumol/l. Of the antiandrogens studied CPA was the strongest competitor of [3H]5 alpha-DHT binding to SHBG with an ID50 of 23 mumol/l, in contrast the non-steroidal antiandrogens were weak competitors causing less than 40% inhibition at 100 mumol/l. It is concluded that the primary mode of action of the imidazole drugs is through the inhibition of cytochrome P-450 dependent steroidogenic enzymes with little or no effect on steroid binding proteins. In comparison, the antiandrogens were strong competitors of [3H] binding to the androgen receptor but relatively weaker competitors of [3H] steroids binding to plasma binding proteins.     

Cultured human skin fibroblasts: a model for the study of androgen action

Human skin may be considered as a target organ for androgens, as are male sex accessory organs, since all events involved in testosterone action have been observed in this tissue. As a corollary, the mechanism of androgen action can be studiedin vitro in cultured skin fibroblasts. The advantages of this system are that studies can be performed with intact human cells under carefully controlled conditions, differentiated genetic and biochemical characteristics of the cells are faithfully preserved and the biological material is renewable from a single biopsy specimen. The metabolism of androgens, in particular the 5α-reduction of testosterone to the active metabolite, dihydrotestosterone, the intracellular binding of androgen to its specific receptor protein and its subsequent translocation to the nucleus have been studied in skin fibroblasts. The intracellular androgen receptor content of genital skin fibroblasts is higher than that from nongenital skin sites. In addition, the androgen receptor has been characterized as a specific macromolecule with properties of high affinity and low capacity similar to that of other steroid hormone receptors. The pathophysiology of three genetic mutations which alter normal male sexual development and differentiation has been identified in the human skin fibroblast system. In 5α-reductase deficiency, an autosomal recessive disorder in which dihydrotestosterone formation is impaired, virilization of the Wolffian ducts is normal but the external genitalia and urogenital sinus derivatives are female in character. At least two types of X-linked disorders of the androgen receptor exist such that the actions of both testosterone and dihydrotestosterone are impaired and developmental abnormalities may involve both Wolffian derivatives and the external genitalia as well. These two forms of androgen insensitivity result from either the absence of androgen receptor binding activity (receptor(−)form) or apparently normal androgen receptor binding with absence of an appropriate biological response (receptor (+) form). In addition, studies with human skin fibroblasts may also be of value in defining the cellular mechanisms underlying the broad spectrum of partial defects in virilization. In summary, we have correlated our studies of the molecular mechanism of androgen action in human genital skin fibroblasts with those of other investigators as these studies contribute to our understanding of male sexual development and differentiation.     

Inhibition of androgen binding in human foreskin fibroblasts by antiandrogens

Antiandrogen effects on androgen receptor binding and androgen metabolism were studied in cultured human newborn foreskin fibroblasts. Three different antiandrogens were tested in this system: (a) cyproterone acetate (CA); (b) RU23908; and (c) R2956. CA and R2956 were equipotent inhibitors of androgen binding to its intracellular receptor. The magnitude of this action was nearly twice as great against the endogenous androgen ligands, dihydrotestosterone (DHT) or testosterone (T), than with the synthetic ligand, methyltrienolone (R1881). Whereas the relative binding affinities of CA and R2956 were approximately 5-10 times less than T or DHT, RU23908 was another order of magnitude less effective as an inhibitor of androgen binding. The lower relative binding affinity determined for RU23908 could not be explained on the basis of a requirement for metabolic activation. Subcellular fractionation studies and sucrose density gradient analysis further confirmed the rank order of antiandrogenic potency. None of the antiandrogens influenced the rate or profile of metabolites from cellular metabolism of T or DHT. We propose that cultured human genital skin fibroblasts may serve as a valuable system for the future evaluation of antiandrogens in intact ells under physiologic conditions.     

In vitro metabolism and biological activity of all-trans-retinoic acid and its metabolites in hamster trachea

The in vitro metabolism of all-trans-[11,12-3h]retinoic acid to several more polar compounds has been demonstrated in a hamster tracheal organ culture system. The production of these metabolites is dependent on the presence of tissue. The physiological significance of these compounds is shown by the cochromatography of several of the in vitro formed metabolites synthesized from [carboxy-14C]retinoic acid with metabolites isolated from the intestine and urine of hamsters previously injected with 0.1 to 1.5 microgram of [3H]retinoic acid. One of the metabolites shows about one-tenth the biological activity of all-trans-retinoic acid when tested in a hamster tracheal organ culture assay. This biologically active metabolite is converted by the hamster trachea in vitro to a biologically inactive metabolite.     

Effect of total profound hypothermia on the relation between cells in the skin of the white rat at different ages

In experiments performed on 190 white inbred rats of various age, effects of total deep hypothermia on mitotic activity of basal epidermocytes, quantity of non-pigmented granular dendrocytes and lymphocytes of epidermis, tissue dermal basophils has been studied. Age difference in changes of the parameters examined at cold effect have been stated. A suggestion is made on existence in the skin of a local regularity system "basal epidermocyte--non-pigmented granular dendrocyte--intraepidermal lymphocyte--tissue dermal basophils", participating in regulation of protective properties of the organ.     

Neuropeptide Y (NPY)-like immunoreactivity in peripheral and central nerve fibres of the golden hamster (Mesocricetus auratus) with special respect to pineal gland innervation

Information on the ambient lighting conditions is conveyed from the retina to the pineal organ by a neuronal pathway involving the suprachiasmatic nucleus (SCN) which acts as a circadian pacemaker. In the hamster, circadian rhythms have been shown to be influenced by injection of neuropeptide Y (NPY) into the SCN. Since NPY-immunoreactive nerve fibres are present in the rat and guinea-pig pineal glands it appeared of interest to investigate the hamster pineal as part of the circadian rhythm generating/regulating system. For comparison kidney, small intestine and cerebral cortex were studied. Like in the other rodent species so far investigated only a few of the abundant sympathetic nerve fibres in the hamster pineal gland are NPY-immunoreactive, in contrast to the relatively rich innervation of the other organs. This speaks in favour of a possible central origin of pineal NPY-immunoreactive fibres. These may either exert vasoregulatory effects on pineal vasculature or be involved in the modulation of alpha-adrenergic receptor mediated regulation of pineal metabolism.     

Towards the mapping of the progesterone and androgen receptors

At a time when the secondary structures of receptor proteins are being predicted from sequence data by modeling techniques, knowledge of the ligand characteristics compatible with high-affinity binding to the receptor and with efficient receptor function is indispensable. We have already compared progesterone receptor (PR) ligands in attempts to map the PR hormone-binding site. In the present study, the relative binding affinities (RBAs) of 33 steroid ligands for the cytosol androgen receptor (AR) of rat prostate, measured in a routine screening system, have been compared. Special emphasis has been given to the effects of modifications (unsaturation, methylation, substitution by halogens) that might influence AR recognition by the ring A carbonyl and also to the consequences of these changes on binding specificity. Nonsteroid antiandrogens are reputed to compete with labelled testosterone (or methyltrienolone) binding to AR. Their RBAs, however, are very low compared to those of steroid antiandrogens. It is feasible that such molecules might occupy and interact with the AR site that binds hormone. The solvent accessible surface of one Anandron conformer is highly similar to that of testosterone and this conformer can be adequately superimposed upon the structure of testosterone and of antiandrogenic Des-A steroid derivatives. The nitro group might assume the role of the ring A carbonyl of steroids; reduction of this group to an amine or a hydroxylamine completely suppresses binding. These observations, however, do not eliminate the hypothesis of interference with AR function, and consequent antiandrogenic activity, by interaction with other (adjacent) sites on AR.     

Platelet Dynamics during Natural and Pharmacologically Induced Torpor and Forced Hypothermia

Hibernation is an energy-conserving behavior in winter characterized by two phases: torpor and arousal. During torpor, markedly reduced metabolic activity results in inactivity and decreased body temperature. Arousal periods intersperse the torpor bouts and feature increased metabolism and euthermic body temperature. Alterations in physiological parameters, such as suppression of hemostasis, are thought to allow hibernators to survive periods of torpor and arousal without organ injury. While the state of torpor is potentially procoagulant, due to low blood flow, increased viscosity, immobility, hypoxia, and low body temperature, organ injury due to thromboembolism is absent. To investigate platelet dynamics during hibernation, we measured platelet count and function during and after natural torpor, pharmacologically induced torpor and forced hypothermia. Splenectomies were performed to unravel potential storage sites of platelets during torpor. Here we show that decreasing body temperature drives thrombocytopenia during torpor in hamster with maintained functionality of circulating platelets. Interestingly, hamster platelets during torpor do not express P-selectin, but expression is induced by treatment with ADP. Platelet count rapidly restores during arousal and rewarming. Platelet dynamics in hibernation are not affected by splenectomy before or during torpor. Reversible thrombocytopenia was also induced by forced hypothermia in both hibernating (hamster) and non-hibernating (rat and mouse) species without changing platelet function. Pharmacological torpor induced by injection of 5′-AMP in mice did not induce thrombocytopenia, possibly because 5′-AMP inhibits platelet function. The rapidness of changes in the numbers of circulating platelets, as well as marginal changes in immature platelet fractions upon arousal, strongly suggest that storage-and-release underlies the reversible thrombocytopenia during natural torpor. Possibly, margination of platelets, dependent on intrinsic platelet functionality, governs clearance of circulating platelets during torpor.     

Neuropeptide Y (NPY)-like immunoreactivity in peripheral and central nerve fibres of the golden hamster (Mesocricetus auratus) with special respect to pineal gland innervation

Summary Inforimation on the ambient lighting conditions is conveyed from the retina to the pineal organ by a neuronal pathway involving the suprachiasmatic nucleus (SCN) which acts as a circadian pacemaker. In the hamster, circadian rhythms have been shown to be influenced by injection of neuropeptide Y (NPY) into the SCN. Since NPY-immunoreactive nerve fibres are present in the rat and guinea-pig pineal glands it appeared of interest to investigate the hamster pineal as part of the circadian rhythm generating/regulating system. For comparison kidney, small intestine and cerebral cortex were studied. Like in the other rodent species so far investigated only a few of the abundant sympathetic nerve fibres in the hamster pineal gland are NPY-immunoreactive, in contrast to the relatively rich innervation of the other organs. This speaks in favour of a possible central origin of pineal NPY-immunoreactive fibres. These may either exert vasoregulatory effects on pineal vasculature or be involved in the modulation of alpha-adrenergic receptor mediated regulation of pineal metabolism.Supported by the Deutsche Forschungsgemeinschaft, grant Schr 283/1-1     

Glutamic acid 709 substitutions highlight the importance of the interaction between androgen receptor helices H3 and H12 for androgen and antiandrogen actions

The mutation of a single amino acid in the ligand binding domain of the human androgen receptor (AR) can induce functional abnormalities; for example, in androgen binding or interactions with coregulators. We report here on the structure/function analysis of the ARE709K substitution that is associated with partial androgen insensitivity syndrome. We introduced several mutations at position 709 and tested the consequences of these changes on AR structure and activity in the presence of androgen and antiandrogens. Our results demonstrate that a strong interaction between helix H12 and residue 709 in H3 is required to obtain a fully functional AR. We show that glutamic acid 709 can be replaced by a bulky tyrosine residue without significant effect on the activation by agonists. In contrast, smaller or linear residues that are unable to maintain a tight interaction with H12 induce a substantial loss of androgen-induced AR activity. We also show that the agonist activity of partial antiandrogens is dependent on the side-chain residue at position 709. Strikingly, the ARE709Y substitution causes the conversion of cyproterone acetate into a pure antiandrogen and bicalutamide into a partial agonist. Together, our structural and functional data reveal the key role of glutamic acid 709 in androgenic and antiandrogenic activities.     

Molecular determinants of resistance to antiandrogen therapy

Using microarray-based profiling of isogenic prostate cancer xenograft models, we found that a modest increase in androgen receptor mRNA was the only change consistently associated with the development of resistance to antiandrogen therapy. This increase in androgen receptor mRNA and protein was both necessary and sufficient to convert prostate cancer growth from a hormone-sensitive to a hormone-refractory stage, and was dependent on a functional ligand-binding domain. Androgen receptor antagonists showed agonistic activity in cells with increased androgen receptor levels; this antagonist-agonist conversion was associated with alterations in the recruitment of coactivators and corepressors to the promoters of androgen receptor target genes. Increased levels of androgen receptor confer resistance to antiandrogens by amplifying signal output from low levels of residual ligand, and by altering the normal response to antagonists. These findings provide insight toward the design of new antiandrogens.     

Long-term treatment with antiandrogens

Since the first report in 1966 by Scott and Schirmer on the clinical use of antiandrogens in patients with prostatic cancer, several studies have been published. Most of these deal with short-term treatment and include only a limited number of patients.

Steroid antiandrogens have demonstrable progestational and antigonadotrophic effects, whereas the non-steroid “pure” antiandrogens mainly act peripherally on androgen-dependent accessory genital organs and thus preserve libido and sexual potency in most patients.

Short-term treatment with antiandrogens have exerted responses similar to those achieved with conventional endocrine therapy. Because of relatively fewer side effects of these drugs, there is an increasing interest in assessment of the long-term effect, and some studies have been initiated.     

Effects of steroidal and non-steroidal antiandrogens on the androgen binding properties of the rat ventral prostate androgen receptor

Steroidal (cyproterone acetate) and non-steroidal (RU23908 and hydroxyflutamide) antiandrogens are able to block testosterone-induced increases in nuclear androgen receptor (AR) in the prostate of 1-day orchidectomized rats, but when given alone, RU23908 and hydroxyflutamide increase nuclear AR (RU23908 greater than hydroxyflutamide) in the same animal model. The increases in nuclear AR induced by antiandrogen alone or with testosterone alone are blocked by cycloheximide 1 h after administration, suggesting that androgen or antiandrogens induce de novo AR synthesis. Concomitant to nuclear AR accumulation, testosterone is able to induce depletion of cytosol and microsomal AR. Blockade of testosterone-induced depletion of microsomal AR, but not of cytosol AR, occurs in the presence of antiandrogens. Cyproterone acetate has a higher relative binding affinity (RBA) for microsomal AR and cytosol AR than RU23908 or hydroxyflutamide. This phenomenon is in good agreement with the degree of inhibition by these compounds of the association rate of androgen for the microsomal AR. This correlation between RBA and inhibition of the initial rate of hormone binding to the receptor is not found for cytosol AR. The results show that antiandrogens are not 'pure' antagonists of androgen action and they are potent agonists in the absence of testosterone. Furthermore, testosterone alone or antiandrogens per se regulate AR levels acutely by protein-synthesis dependent mechanisms of action, in rat ventral prostate.     

Structure–activity relationship analysis of carbobicyclo and oxabicyclo succinimide analogs as potential androgen receptor antagonists

Prostate cancer (PCa) is a frequently diagnosed male cancer and the second leading cause of cancer-related death in many countries. Due to various amino acid mutations that occurred in the ligand binding domain of androgen receptor (AR), the patients were observed insensitive, even resistant to the marketed antiandrogens such as bicalutamide and enzalutamide, which emphasizes the urgent need for novel antiandrogens to solve drug resistance problem. Recently a series of carbobicyclo and oxabicyclo succinimide analogs were reported to effectively antagonize AR. In this study, to explore the structural requirements for these AR antagonists, we performed quantitative structure–activity relationship analysis on carbobicyclo and oxabicyclo succinimide analogs by using two-dimensional multiple linear regressions (MLR) method and three-dimension comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods. The obtained models show satisfactory results with proper reliabilities and powerful external predictability. Moreover, the CoMFA and CoMSIA contour maps can intuitively represent key features associated with bioactivities. These models may offer guidance for the rational design and modification of new lead compounds for antiandrogens.     

Pyrimidine nucleoside, pseudouridine, and modified nucleoside excretion by growing and resting fibroblasts.

We are examining the relationship of RNA metabolism and de novo pyrimidine synthesis as parameters of malignant transformation. These initial experiments on normal hamster embryo fibroblasts have shown that excreted nucleosides are markers for intracellular RNA metabolism. We employed affinity chromatography to concentrate the nucleosides in the medium and sensitive column chromatographic procedures to quantitatively measure them. The excretion of pyrimidine nucleoside from hamster embryo fibroblasts in sulture was found to be dependent on the growth state of the cells, with the greatest accumulation occurring cell quiescence. The major nucleoside excretion products, uridine and cytidine, were both normal end products of RNA metabolism and the major nucleoside excretion products from cultured cells. The modified nucleosides N-1-methylguanosine, N-2-methylguanosine, N-2-dimethylguanosine, N-4-acetylcytidine, N-1-methylinosine, pseudouridine, N-1-methyladenosine, N-3-methylcytidine, and 5-methyleycytidine were found, as were several unidentified nucleosides.