Dissertations / Theses on the topic 'Steroid receptors'

Chapter 1 begins with a brief overview of molecular recognition and then presents a review of recently developed methods for lead generation and the synthesis of self-defining host molecules. Chapter 2 describes a strategy for the synthesis of functionally active molecules which combines advantageous features of some of the methods described in Chapter 1. Relevant literature and precedents are then introduced followed by a discussion of theoretical considerations. In Chapter 3, after an introduction to the use of cholate building blocks in supramolecular chemistry, the synthesis of the new steroidal molecules studied is described, together with a discussion of their structural properties. Chapter 4 outlines the development of an effective method for the reversible macrocyclisation of steroidal building blocks by transesterification. The application of this method to the thermodynamically-controlled, and metal ion-templated, cyclisation of the steroid derivatives is then discussed in Chapter 5. An investigation into the metal ion building properties of the steroid derivatives using electrospray mass spectrometry is described in Chapter 6. The chapter begins with initial experiments to explore the circumstances in which the technique can generate meaningful information and then summarises the metal ion binding characteristics of the steroids investigated. Chapter 7 explores transorthoesterification as a milder reversible reaction for the synthesis of macrocycles and then describes initial studies towards a host capable of binding a porphyrin guest. In Chapter 8, the results of the cyclisation experiments and the binding study are compared with each other and put into the wider context of the long term goals of the project. That is followed by some ideas for future directions of this work.

G protein coupled receptors (GPCRs) are the largest family of transmembrane receptors in the genome and are activated by a multitude of ligands including neuropeptides, hormones and sensory signals. The paraventricular nucleus (PVN) and supraoptic nucleus (SON) of the hypothalamus are important mediators in homeostatic control. Many modulators of PVN/SON activity, including neurotransmitters and hormones act via GPCRs - in fact over 100 non-chemosensory GPCRs have been detected in either the PVN or SON. The introduction to this thesis begins with a comprehensive summary of GPCR expression within the PVN/SON, with a critique of the detection techniques used within the literature. Also discussed are some aspects of the regulation and known roles of GPCRs in the PVN/SON, as well the possible functional significance of orphan GPCRs. Particular interest is paid to the recently 'deorphanised' G protein-coupled oestrogen (E2) receptor, GPER, which is the first receptor to be acknowledged as a steroid binding GPCR (although there are conflicting studies regarding its affinity for E2) and is expressed in the PVN and SON. Steroids are known to have fast non-genomic effects that are thought to be mediated in-part by membrane-associated forms of the traditional steroid receptors (members of a family of transcription factors). However, the possible discovery of a fast E2 GPCR has raised speculation regarding the existence of other steroid binding GPCRs. Thus the experimental Chapters were undertaken to explore the concept of fast steroid receptors, with particular emphasis on their possible roles in neuroendocrine systems. Firstly, the distribution of the putative E2 receptor was investigated to give further insight into its possible in vivo roles. In the rodent, high levels of GPER gene and protein expression were detected in the oxytocin and vasopressin neurones in the PVN and SON, the anterior and intermediate lobe of the pituitary, adrenal medulla and renal medulla and pelvis, suggesting roles for GPER in multiple functions including hormone release. To clarify the controversy surrounding GPER as an E2 receptor, we investigated GPER function in vitro using a series of cell signalling assays. However, E2 did not stimulate GPER-mediated signalling, suggesting that either GPER remains an orphan GPCR, or the cell lines used in this study lacked a vital component for E2 activation of GPER. As the rapid effects of glucocorticoid have been reported in numerous brain regions (including the PVN and SON), endocrine, and other tissues, the second part of this thesis focussed on the search for a possible fast glucocorticoid receptor. We compared the tissue distribution gene expression profiles of approximately 125 orphan GPCRs common to human and rodent with tissues that are known to exhibit fast effects of steroids (e.g., hippocampus, PVN, SON, thymus, kidney, etc.). Of the 125 orphans,3 GPCRs (GPR108, GPR146, and TMEM87B) had distribution profiles that closely matched the regions/tissues of interest. These orphans were tested for glucocorticoid activation using a universal deorphanisation assay. However, the identity of the fast glucocorticoid receptor remains unknown, as none of the candidate orphan GPCRs responded to glucocorticoids.

Thesis (Ph.D.)--Aberdeen University, 2008.
Title from web page (viewed on Feb. 23, 2009). With: Natural disordered sequences in the amino terminal domain of nuclear receptors : lessons from the androgen and glucocorticoid receptors / Iain J. McEwan ... et al. Nuclear Receptor Signalling. 2007: 5. Includes bibliographical references.

A novel bacterial expression system that is capable of producing high levels of soluble, stable, biologically active human vitamin D3 and estrogen receptors has been developed. The method utilizes ubiquitin fusion technology and a low temperature nalidixic acid induction of the lambda PL promoter. This system can produce large quantities of receptor antigen, but only a small fraction displays wild-type DNA and hormone binding properties. Therefore, the use of this system to overproduce receptors for crystallization studies is not practical. To overcome these problems, a 2 um based ubiquitin fusion system which allows regulated expression of the estrogen receptor in yeast (Saccharomyces cerevisiae) was developed. This system produces the estrogen receptor to a level of 0.2% of the total soluble protein. Moreover, this protein is undegradable, soluble, and biologically active. To test the transcriptional activity of the estrogen receptor produced in yeast, a cis-trans transcription assay was developed. This assay revealed that the transcriptional activity of the human estrogen receptor expressed in yeast was similar to that observed in transfected mammalian cells. This reconstituted estrogen transcription unit in Saccharomyces cerevisiae was utilized to examine the regulation of estrogen receptor functions by antiestrogens, to develop a random and rapid approach for identifying novel estrogen response elements, to characterize estrogen receptor variants cloned from human breast tumors, and to examine the effect of estrogen receptor on the regulation of osteocalcin gene.

Breast cancer is a great source of morbidity and mortality in the developed world, being the most common cause of cancer death in Australian women and affecting roughly 1 in 10 women. The development and progression of cancers is a multi-stage process, involving numerous perturbations to normal cellular functions, especially to those genes which control cellular growth, cellular differentiation and DNA repair. Over time, these alterations combine to change normal cells into cancerous ones that typically no longer respond to normal control stimuli and grow with great rapidity. The nuclear receptors are a family of proteins which accept incoming signals from various molecules, and then alter gene expression or affect cell behavior directly. The steroid nuclear receptors receive transducer signals from hormones such as estrogen and testosterone and are intimately involved in affecting cellular growth and differentiation. Stimulation of the steroid receptors have been used as successful treatment avenues, so how these genes behave in cancer is of great interest. Accordingly, this study has examined the expression of various nuclear receptors and some related genes in a number of tissue samples derived from breast tumours and from surrounding areas, and examined how various pathological parameters affect their expression. Tissue samples were first microdissected to separate tumour tissue from the surrounding stroma and then subjected to RNA extraction procedures to allow gene expression to be quantitated. RNA was then converted into cDNA by reverse transcription and the genes of interest amplified and quantitated using semiquantitative polymerase chain reaction. Expression data was then normalized by expressing it as a ratio of the gene of interest to the 18S ribosomal gene and differences in expression analysed by analysis of variance (ANOVA) testing. The first portion of the study dealt with the progesterone and glucocorticoid receptors in tumour tissue. Progesterone is an anti-mitogenic signal for breast tissue and the stimulation of the receptor is a useful part of combined hormonal therapy for breast cancer. The glucocorticoid receptor plays a similar role to the progesterone receptor, slowing breast tissue growth and promoting apoptosis. The complete tissue population of 25 samples from control, grade 1, grade 2 and grade 3 tumours underwent PCR for the glucocorticoid and progesterone receptor genes and was normalized using 18S. Analysis of the expression data by ANOVA showed that the progesterone and glucocorticoid receptors were more highly expressed in grade 3 tumour tissue (p=0.023 and p=O.00033, for the progesterone and glucocorticoid receptors, respectively). Most advanced tumours cease being responsive to growth repressing hormones, so these results indicated that the control of the expression of these receptors in tumour tissue was more complicated than might have been expected. The increase in expression observed may be the result of intact growth preventing feedback mechanisms which have malfunctioned in some manner. There are several mechanisms the tumours may be using to escape an increase in progesterone or glucocorticoid sensitivity. The mRNA detected may simply not be being translated into completed proteins or be being spliced into isoforms of the receptors that favour tissue growth. The second portion of the study dealt with the estrogen receptors alpha and beta in tumour tissue. The estrogen receptors control estrogen signaling and are highly important in breast cancer, since estrogen is the primary growth inducing hormone for breast tissue. For the estrogen receptors alpha and beta, the complete tissue population of 25 samples from control, grade 1, grade 2 and grade 3 tumours underwent PCR and were normalized using 18S. ANOVA analysis found that the expression of the estrogen receptors did not change significantly in any grade of cancer (p= 0.057 and p=O.7'38, for ESRα and ESRβ, respectively) nor in tissues that are negative for the estrogen receptor alpha protein, a poor prognostic factor in breast cancer (p= 0.794 and p=O.7l6, for ESRα and ESRβ, respectively). These results indicated that the loss of estrogen receptor in advanced cancers is not controlled at the level of mRNA. The mRNA observed in this study may be being spliced into alternate and possibly inactive isoforms, or may be being degraded post transcriptionally, preventing estrogen stimulation in these tumours. This could prove an excellent area for further study, since if the mechanism that prevents or distorts ESR mRNA translation can be discovered, it would allow manipulation of one of the most important treatment avenues for breast cancer. The third portion of the study dealt with the expression of the androgen receptor in tumour tissue. Androgens are a strong anti-growth signal in breast tissue and despite the side effects that androgens have on women, they have been used to treat breast cancer with some success. The complete tissue population of 25 samples from control, grade 1, grade 2 and grade 3 tumours underwent PCR for the androgen receptor gene and normalized using 18S. The results obtained for androgen receptor expression in tumour tissue showed that androgen receptor expression was significantly elevated in grade 2 and grade 3 tumour tissue, as well as in ESRα negative tumours (p= 0.014 and p=O.O25, respectively). An increase in expression in late stage tumours would seem to be unusual for an anti-mitogenic receptor, however many advanced breast tumours have been found to be receptive to androgen stimulation, even when they no longer respond to other hormones. The increased expression of AR may be a normal response to cellular over-growth, or it may be a mechanism by which the tumour prevents stimulation by other growth retarding hormones, by sequestering all available receptor co-enzymes with a receptor that is unlikely to be stimulated. The fourth portion of the study examined the expression of the estrogen alpha, estrogen beta, progesterone, glucocorticoid and androgen nuclear receptors in stromal tissue derived from the tumours studied in previous chapters. Tumours have been observed in other studies to manipulate the activities of the cells that surround them through the release of cofactors and vice versa. These cofactors include the steroid hormones, among others, and hence the study of how the tumour and stroma interacts is a valuable extension to the results obtained in the previous sections. PCR was performed for all nuclear receptors, except for the estrogen receptor alpha, in the complete tissue population of 25 samples of tissue derived from the stroma of the grade 1, grade 2 and grade 3 tumours used in the previous studies as well as the control tissues. Due to difficulties in PCR optimization for estrogen receptor alpha, only three stromal samples from each grade and four controls were able to produce results, for a total population of 13 samples. Of all the receptors tested, only the progesterone and glucocorticoid receptors displayed significant changes in expression in stromal tissue, with PgR having significantly lower expression in all stromal samples compared to control, while GR was more highly expressed in stroma derived from high grade tumours (p= 5.908x107 and p=2.761x105, for PgR and GR, respectively). UR expression was also increased in stroma derived from ESRα negative tumours (p=5.85x105). These alterations reflect the kind of stimulation a tumour is likely to apply to the surrounding stroma, using progesterone to stimulate the cells into differentiating to provide a more suitable environment, hence the loss in PgR expression. The increase in GR expression may be the result of the high level of growth stimulating factors that tumours produce, priming the local cells to be more sensitive to growth suppressors, a situation that is also mirrored in results previously obtained for the tumour tissue. The fifth part of the study concerned the expression of the nuclear receptor coactivator 1 and nuclear receptor co-activator 3 genes. These proteins are required for the activation and function of the nuclear receptors and both have been implicated in cancer development, being found to be over-expressed in several tissues and cell lines. As integral parts of the nuclear receptor pathway, their level of expression is important for determining how effective any nuclear receptors present will be when stimulated. The complete tissue population of 25 samples from control, grade 1, grade 2 and grade 3 tumours underwent PCR for both nuclear receptor co-activator genes and normalized using 18S. The result of ANOVA analysis on the NCoA data showed that NCoA3 expression remained unaltered in all grades of cancer and stroma and in both ESRα positive and negative tissue. NCoA1 however, was significantly upregulated in grade 3 tumours as compared to grade 1 tumours and also in ESRα negative tumours. This increase in expression would seem to indicate that these tissues would be more capable of acting on any received hormonal stimulation. That this increase in expression occurs in more advanced cancers could be evidence that the nuclear receptor expression observed in prior sections is resulting in NR splice variants that favour, rather than repress, growth, as advanced cancers usually do not respond normally to hormonal stimulation. The final part of the study investigated the possibility of correlations between the expression of the nuclear receptors, between the nuclear receptor co-activators and between all of the tested genes and other pathological parameters, including tumour size, metastasis, site of tumour, carcinoma in situ invasiveness, age of patient and the presence of calcification. The data generated in the prior studies was analyzed using ANOVA for categorical data and correlation analysis for numerical data. The ANOVA and correlation analysis revealed a number of interactions between these factors, which provide additional information on the relationships between the tested genes. Expression of the progesterone receptor was found to be correlated with the expression of GR, AR and NCoA1 (p= 0.022, p=O.OO3, and p=O.0i9, respectively). Likewise the expression of GR and AR were found to be correlated (p=O.O29). Additionally, AR was found to be associated with tumour size (p=O.O36) while GR was found to be associated with both tumour size and metastasis (p= 0.006 and p=7.6x106, respectively). ESRα and ESRβ expression were found to be negatively correlated (p=O.O44M), as were patient age and the amount of ductal carcinoma in situ invasion. Given the results of previous analyses, it is not surprising that PgR, GR, AR and NCoA1 expression are related, and the negative correlations between ESRα and ESRβ expression, as well as between age and ductal carcinoma in situ invasion have been documented in other studies. Hence, these results provide reinforcement for previous observations, as well as providing new information, particularly on AR and GR.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Full Text

For my dissertation research I explored the diversity and functional evolution of steroid hormone receptors (SRs) in animals and the physiological implications of the evolution of antifreeze proteins in Antarctic notothenioid fishes. For the former, I discovered multiple new SRs from the vast and under-sampled swath of animal diversity known as invertebrates. I used the sequences of these and other newly discovered related receptors in combination with genomic data and molecular phylogenetic techniques to revise the understanding of the evolutionary history of this important gene family. While previous studies have suggested that vertebrate SR diversity arose from a gene duplication in an ancestor of all bilaterian animals, my work presents strong evidence that this duplication occurred much later, at the base of the chordates. Furthermore, to determine the implications of added diversity and a revised phylogeny on inferences of the functional evolution of SRs, I functionally characterized heretofore-unknown SRs from hemichordates, an acoelomate flatworm, and a chaetognath and statistically reconstructed and functionally characterized ancestral SRs. My results expand the known sequence and functional repertoire of SRs in animals while reinforcing the previous inference that all SRs evolved from an estrogen-sensitive ancestral receptor. I also explored the consequences of the evolution of antifreeze proteins in Antarctic notothenioid fishes, a crucial adaptation to their icy, polar environment. These special proteins adsorb to ice crystals that enter a fish's body and prevent further growth, thereby averting death. I discovered that, in addition to their lifesaving growth-inhibiting ability, AFPs also prevent the melting of internal ice crystals at temperatures above the expected equilibrium melting point. Together with a decade-long temperature record of one of the coldest fish habitats on earth, my experimental results show that the evolution and expression of antifreeze proteins is accompanied by a potentially detrimental consequence: the lifelong accumulation of ice inside these fishes' bodies. This dissertation includes previously published co-authored material as well as unpublished co-authored material.
2017-01-14

The significance of the hormonal milieu for endometrial changes is as well-known as its link with endometrial carcinoma. Unopposed oestradiol treatment is shown to increase the incidence for this cancer. Obesity leads to elevated levels of oestrogens and is a risk factor for endometrial carcinoma. An association between high tumour proliferation and prognosis is a general feature of human cancer. Tumour growth can be expressed as proliferation rate and flow cytometry (FCM) is a sensitive and reproducable method to estimate S-phase fraction (SPF) and ploidy level. Both parameters have been shown to correlate with prognosis. Sex steroid hormone levels were analysed together with clinical parameters, SPF, and receptors in established endometrial carcinoma. The study consisted of postmenopausal women with endometrial adeno­carcinoma. H ormones were analysed in 127 patients, 99 were analysed for FCM and 60 for oestrogen and progesterone receptors. RIA technique was used for hormone assay of oestrone, oestradiol, progesterone, androstenedione and testosterone plasma levels. The receptors were analysed with an immunohistochemical method, and SPF and ploidy level by flow cytometry. A wide range of oestrogen concentrations was found. Some patients had levels comparable to fertile women. Strong correlations were found between body mass index, weight and depth of uterine cavity. No relations were found between receptors and SPF, apart from oestrogen- receptor positive tumours having a lower SPF when compared with receptor negative tumours. The influence of oestradiol on tumour proliferation expressed as SPF was ambiguous. SPF was increased with higher oestradiol levels in the group of peri-diploid, well-differentiated tumours, while a negative correlation was found for the peri-diploid, moderately differentiated tumours. The aneuploid and poorly differenti­ated tumours had a high SPF regardless of oestradiol concentration. The association between progesterone concentration and SPF was of a more general nature. Progesterone above a threshold level was related to a lower SPF in well-differentiated and moderately differentiated tumours. Thus endogenous progesterone seems to play a role in controlling the tumour’s proliferation activity, in contrast to oestradiol, that had a role which did not appear to relate to proliferation activity in any specific direction. The only stimulative association was seen in well-differentiated tumours, but SPF was still below the mean value for all diploid tumours.

Diss. (sammanfattning) Umeå : Umeå universitet, 1993, härtill 5 uppsatser.

digitalisering@umu

Les androgens(ADs) et les glucocorticoïdes (GCs) sont des hormones stéroïdiennes qui exercent des effets pléiotropes chez les mammifères. Leurs effets sont relayés par deux récepteurs nucléaires, le récepteur des androgènes (AR) et le récepteur des glucocorticoïdes (GR), respectivement. Même si les GCs sont fréquemment utilisés pour traiter les maladies inflammatoires et les antiandrogènes pour le cancer de la prostate, les traitements à long terme induisent des effets secondaires majeurs, notamment l'atrophie musculaire.Afin de préciser les mécanismes d’action de ces hormones, nous avons réalisé des analyses phénotypiques, transcriptomiques et cistromiques. La première partie de ce travail démontre que GR des myofibres contrôle négativement la masse et la force musculaire aux niveaux physiologiques de GCs. La perte de GR dans les muscles squelettiques n'affecte pas les voies cataboliques, mais augmente l’expression de facteurs anaboliques et réduit celle de facteurs anti-anaboliques. Nous avons également montré que GR se lie à des éléments de réponse du GR (GREs) situés aux enhancers, en association avec Myod1 et Foxf2, et interagit avec des facteurs liés aux promoteurs, tels que Nrf1, pour favoriser la transcription des gènes.Dans la deuxième partie de ce travail, nous avons comparé le cistrome et le transcriptome du GR dans la prostate et le muscle squelettique, et identifié des sites de liaison pour d'autres facteurs de transcription proche des GREs, indiquant que ces facteurs contribuent à la spécificité tissulaire. De plus, en comparant les cistromes et transcriptomes d’AR et de GR dans la prostate, nous montrons que les éléments de réponse liés par les deux récepteurs sont distincts de ceux liés uniquement par AR ou GR, et que la sélectivité du récepteur dépend de la liaison d’autres facteurs de transcription.Enfin, nous avons comparé les données transcriptomiques et épigénétiques du tissu musculaire squelettique et de myoblastes et myotubes C2C12, et nous fournissons une description détaillée de gènes, voies de signalisation et facteurs de transcription exprimés de façon différentielle pendant la différenciation myogénique.En conclusion, nos travaux ont permis de clarifier les mécanismes moléculaires régulant l'homéostasie musculaire et ont établi la base d'une compréhension moléculaire des effets spécifiques des ADs et des GCs dans divers types cellulaires
Androgens (ADs) and glucocorticoids (GCs) are steroid hormones exerting pleiotropic effects in mammals. Their effects are mediated by two nuclear receptors, the androgen (AR) and the glucocorticoid (GR) receptor, respectively. Although GCs are extensively used to treat inflammatory diseases and antiandrogens for prostate cancer, long-term treatments induce major side effects such as muscle atrophy.To determine the mechanisms underlying their effects in muscle, we performed phenotypic, transcriptomic and cistromic analyses. The first part of this work demonstrates that myofiber GR negatively controls muscle mass and strength under physiological GCs levels. GR loss in skeletal muscle did not affect catabolic pathways, but enhanced the expression of anabolic factors and reduced that of anti-anabolic ones. We also showed that myofiber GR binds DNA to GR response elements (GREs) located at enhancers, in association with Myod1 and Foxf2, and interact with promoter-bound factors such as Nrf1 to promote gene transcription.In the second part of this work, we compared GR cistromes and transcriptomes in prostate and skeletal muscle, and identified binding sites for additional transcription factors in the vicinity of GREs, indicating that they contribute to the tissue specificity. In addition, by comparing the AR and GR cistromes and transcriptomes in prostate, we show that the response elements bound by both receptors are distinct from those bound by either AR or GR, and that the receptor-selectivity depends mostly on the surrounding factors.Finally, we compared transcriptomic and epigenetic data of skeletal muscle tissue and C2C12 myoblasts and myotubes and provide a detailed description of genes, signaling pathways and transcription factors that are differentially expressed during myogenic differentiation.In conclusion, our work allowed to clarify the molecular mechanisms regulating muscle homeostasis and provides the basis of a molecular understanding of tissue- and/or promoter-specific activity of ADs and GCs

Steroid action is vital for the development and function of male gonads. Androgen and the androgen receptor (AR) are critical for the initiation and maintenance of complete spermatogenesis and male fertility. Despite this fundamental role, the precise AR-regulated pathways controlling spermatogenic development have yet to be resolved. A major focus of this research was to use a unique gain-of-function transgenic (Tg) mouse model to determine the temporal role of Sertoli cell-specific AR expression in testicular development. The Sertoli cell-specific rat Abpa promoter directed human Tg AR [Tg Sertoli cell (SC)-specific AR (TgSCAR)] expression, providing strong premature postnatal AR immunolocalized to Sertoli cell nuclei. This novel transgenic model provides unique opportunity to directly differentiate vital AR-regulated genes and regulatory pathways involved in optimal Sertoli cell maturation, and meiotic-postmeiotic germ cell development. The current research has also been investigated the functional role of the Sertoli cell glucocorticoid receptor (GR) in vivo using a Tg Cre-loxP approach to conditionally disrupt GR expression. Sertoli cell GR knock-out (SCGRKO) was shown by absent Sertoli cell-specific GR immunolocalization. This novel SCGRKO model has revealed that Sertoli cell-mediated GR actions support normal testicular function. Sertoli cell GR is required to maintain normal testicular Sertoli/germ cell numbers and circulating gonadotrophin levels, as well as optimal Leydig cell maturation and steroidogenesis, providing new insight into gluocorticoid-mediated impact on male reproduction.

[Truncated abstract] Steroid receptors belong to the superfamily of nuclear receptors, and include the androgen receptor (AR), estrogen receptors (ER[alpha] and ER[beta], glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and the progesterone receptors (PRA and PRB). Before binding ligand, the receptor undergoes biochemical and structural modifications through a series of interactions with molecular chaperones and cochaperones all within a receptor heterocomplex. The mature receptor complexes with the major chaperone Hsp90, the stabilising cochaperone p23, and one member of a group of cochaperones termed immunophilins. Steroid receptor-associated immunophilins include the cyclophilin, CyP40, two FK506-binding proteins, FKBP51 and FKBP52, and the protein phosphatase, PP5. Immunophilins are characterised by the presence of TPR domains which compete directly for the TPR-acceptor site within Hsp90. This leads to mutually exclusive, immunophilin-containing receptor complexes. While PP5 contains a C-terminal phosphatase domain, CyP40, FKBP51 and FKBP52 each contain an N-terminal peptidyl prolyl isomerase (PPIase) domain, which catalyses the cis/trans isomerisation of prolyl peptide bonds. FKBP52 has been demonstrated to potentiate the ligand-dependent activity of AR, GR and PR, but not ER[alpha]. Knowing that CyP40 is the preferred immunophilin associated with the ER[alpha] heterocomplex, it was hypothesised that this immunophilin plays a role in ER[alpha] function. ... As all mutants maintained this potentiating activity it was concluded that the five altered residues found within gpGR do not contribute to the altered interaction of FKBP52 and receptor. However, it cannot be discounted that FKBP51 is more competitive for gpGR. Immunophilins are hormonally regulated, with FKBP52 found to be essential for female fertility in mice. It was hypothesised that levels of immunophilins, associated with steroid receptors important in the menstrual cycle, would be regulated to reflect hormonal activity within cycling endometrium. Human pre-menopausal endometrial sections taken from different phases of the menstrual cycle were examined immunohistochemically for expression of CyP40, FKBP51, FKBP51 and PP5. Immunophilin levels peaked at the mid-secretory phase correlating with stromal decidualization, a process essential for eventual blastocyst implantation. The importance of immunophilins to steroid receptor action was therefore reinforced by the observation that immunophilins appear to be hormonally regulated in cycling pre-menopausal human endometrium. Further studies into the effects of immunophilin loss and knockdown on steroid receptor-mediated responses in specific mouse tissues, knockout-derived mouse embryo fibroblasts and cancer cell lines may contribute to our understanding of the receptor-selective and tissue-specific actions of the immunophilins. Elucidation of the mechanisms through which they modulate receptor function may provide opportunities for therapeutic intervention in steroid-related disease.

Nuclear receptors including estrogen receptors (ERs) and progesterone receptors (PRs) are activated by their ligands as well as by signaling pathways in response to peptide hormones and growth factors. In gonadotrophs, gonadotropin releasing hormones (GnRHs) act via the GnRH receptor (GnRHR). Both GnRH-I and GnRH-II activate an estrogen response element (ERE)-driven luciferase reporter gene in LβT2 mouse pituitary cells, and GnRH-I is more potent in this regard. The ERα is phosphorylated at Ser¹¹⁸ in the nucleus and at Ser¹⁶⁷ in both nucleus and cytoplasm after GnRI-I treatments, and this coincides with increased ERct binding to its co-activator, the P300/CBP-associated factor (PCAF). Most importantly, both GnRH subtypes robustly up-regulate expression of the immediate early response gene, Fosb, while co-treatment with ERα siRNA or PCAF siRNA attenuates this effect. This appears to occur at the transcriptional level because co-recruitment of ERα and PCAF to an ERE within the endogenous Fosb promoter is increased by GnRH treatments, as shown by chromatin immunoprecipitation assays. Furthermore, cross-talk between GnRH-I and PR accentuates gonadotropin production. GnRH-I activates a progesterone response element (PRE)-driven luciferase reporter gene and gonadotropin a subunit (Gsua) gene expression in two mouse gonadotroph cell lines, αT3-1 and LβT2. Up-regulation of the PRE-luciferase reporter gene by GnRH-I is attenuated by pre-treatment with protein kinase A (H89) and protein kinase C (GF109203X) inhibitors, while only GF109203X inhibits GnRH-1-induced Gsua mRNA levels. In both cell lines within the same time-frame, knockdown of PR levels by siRNA reduces GnRH-I activation of Gsua mRNA levels by approximately 40%. Both GnRH-I and GnRH-II also increase mouse Gnrhr-luciferase promoter activity and this is significantly reduced by knockdown of PR in LβT2 cells. We conclude that the effects of GnRH-I on Fosb and Gsua expression, as well as mouse Gnrhr promoter activity in mouse gonadotrophs are mediated by ligand-independent activation of ERα and PR. These ligand-independent effects of GnRHs on steroid hormone receptor function may influence the magnitude of changes in the expression of specific genes in the pituitary during the mouse estrous cycle, which in this context may serve as a model in the human menstrual cycle.

Avian intestinal cytosoluble receptors for 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃) were subjected to limited trypsin digestion, endogenous proteolytic action, as well as carboxypeptidase treatment, and the physical and functional properties of the resulting discrete polypeptide fragments were identified and contrasted with the native 1,25(OH)₂D₃ receptor. Resultant fragments were followed by tracing either radioactive 1,25(OH)₂D₃ or by probing with anti-receptor monoclonal antibodies. Two differentially trypsin-sensitive effects on the 1,25(OH)₂D₃ receptor were noted when fragments were detected by their ability to bind 1,25(OH)₂[³H]D₃. Two hormone-bound fragments of 40 and 30 kDa were formed; neither bound to DNA-cellulose nor anti-receptor monoclonal antibodies. Immunoblot technology was used to show the disappearance of the 60 kDa receptor with increasing trypsin concentrations, paralleling the appearance of an immunoreactive 20 kDa fragment. The 20 kDa fragment did not bind hormone but was capable of interacting with DNA-cellulose in a fashion identical to that of the 60 kDa receptor. This fragment is likely the complementary fragment to the hormone-bound fragment of 40 kDa that is described above. In contrast to the exogeneous effect of trypsin, incubation of chick intestinal cytosol resulted in the time-dependent formation of an endogenous protease-derived fragment of 45 kDa. This species retained the hormone-binding site and the antibody determinant, but was devoid of DNA-binding activity. Moreover, it did not generate the trypsin-dependent 20 kDa fragment and therefore was derived from the opposite end of the receptor molecule. Carboxypeptidase treatment of the 1,25(OH)₂D₃ receptor produces a 56 kDa fragment which does not retain hormone, but which does bind to DNA-cellulose and monoclonal antibody. These combined data from various limited enzymatic cleavage studies of the receptor have facilitated the construction of a schematic model of the chick receptor in which the immunoreactive epitope is located between the N-terminal DNA-binding domain and the C-terminal hormone-binding domain. This map for the 1,25(OH)₂D₃ receptor protein is consistent with the general structure of steroid and thyroid hormone receptors and places the vitamin D hormone receptor in a class of macromolecules that are postulated to bind enhancer regions of responsive DNA and thereby control target gene transcription.

The active form of vitamin D is the steroid hormone 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. Central to the mechanism of action of 1,25(OH)₂D₃ is its specific, high affinity intracellular receptor. This research focused on the participation of this receptor in the biology, biochemistry, and molecular biology of the vitamin D regulatory system. The effects of 1,25(OH)₂D₃ on the differentiation of hematopoietic cells were investigated using the cultured human promyelocytic leukemia cell line, HL-60, as a model. It was observed that 1,25(OH)₂D₃ induced macrophage differentiation in HL-60 cells and that a direct biochemical correlation existed between 1,25(OH)₂D₃ receptor saturation and a 1,25(OH)₂D₃-stimulated bioresponse. These data implicate 1,25(OH)₂D₃ as a natural cell differentiating agent and the 1,25(OH)₂D₃ receptor as the mediator of this hormone's action. Since the most fundamental level of control occurs by the regulation of gene expression, studies were undertaken to define the transcriptional control by 1,25(OH)₂D₃ over a known vitamin D-regulated endpoint protein. This work resulted in the molecular cloning of cDNAs to two avian intestinal calcium binding proteins, vitamin D-dependent calcium binding protein and a novel calmodulin-like protein. To gain further insight into the role of the 1,25(OH)₂D₃ receptor as a transcriptional regulator, avian and mammalian 1,25(OH)₂D₃ receptor mRNAs were characterized extensively by the techniques of in vitro translation and immunoprecipitation. These mRNAs were then utilized to construct cDNA libraries from which avian and human intestinal 1,25(OH)₂D₃ receptor cDNAs were isolated and their identity verified by hybrid-selected translation, sequencing, and Northern analysis. It was concluded that demonstrated 1,25(OH)₂D₃ receptors are polypeptides of 52-60 kDa whose activity is regulated by 1,25(OH)₂D₃ at both an mRNA and posttranslational level. Furthermore, the deduced amino acid sequence of receptor mRNA included a highly conserved cysteine, lysine, and arginine rich region that is homologous to other steroid receptors and the oncogene product v- erbA. Thus, the vitamin D receptor to be a specific trans -acting factor, modulating the pleiotropic effects of vitamin D including calcium homeostasis, and cellular differentiation.

Our lab has previously reported the identification of a novel endogenous 19-nor steroid, estradienolone (ED), in pregnant women that strongly bound to sex hormone binding globulin. Estrogen-receptor related receptors (ERRs), which have no known natural ligands, are a family of orphan receptors consisting of 3 isoforms: ERRalpha, ERRbeta and ERRgamma. The ERRs have been shown to actively modulate estrogenic responses, to play an essential role in pregnancy, and are implicated in breast cancer prognosis. My results show that ED acts as an antagonist of the ERRalpha confirming preliminary results obtained by our group. Studies of cellular responses demonstrate that ED has strong anti-mitogenic properties. ED inhibited the growth of both estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) breast cancer cells in a dose-dependent manner but did not have any effects on the proliferation of the non-cancerous immortalized epithelial breast MCF-10A cells. The finding that ED inhibits proliferation of both ER negative and ER positive breast cancer cells, and regulate ERR transcriptional activity may have important ramifications in breast cancer therapy.

Estrogens, such as the biologically active 17-b estradiol (E2) have many actions in the male and female brain. Not only does E2 regulate reproductive behavior in adults, it organizes and activates the brains of younger animals in a sex-specific manner. In addition, many human studies have shown E2 to provide protection against a variety of neurological disorders, including stoke. These studies have been controversial and depend largely on the type and timing of hormone replacement. Animal studies are much less controversial and clearly demonstrate a neuroprotective role for E2 following ischemic brain injury. Because much of E2 neuroprotection requires sex steroid hormone receptors, it is essential to understand expression patterns of these receptors. For the current studies, I evaluated estrogen receptor alpha (ER α), estrogen receptor beta (ER β) and androgen receptor (AR) expression in the cortex. It is known that these receptors change in expression at several times in an animal’s life span including during early postnatal development and following ischemic brain injury. Here I used an in vitro cortical explant model to further examine how these receptors change both during development and following injury. This in vitro model is important because it provides a way to investigate changes in receptor expression pattern in the cortex without input from other brain regions. In addition to characterizing this model, I also evaluated the contribution of E2 to changes in receptor expression and on cell death following injury in the explants. To begin to decipher mechanisms for E2 mediated neuroprotection, I added antagonist for each of the receptors before and after injury. In each these experiments, I also examined potential sex differences by separating the female and male brains before I cultured the explants. Overall, these experiments showed that cortical explants are a good in vitro model. Here we found that E2 was protective in female, but not male cortical explants following injury. However, the exact mechanisms of E2-mediated neuroprotection are still to be deciphered.

Among heat shock proteins, Hsp90 is unusual because it is not required for the proper folding of most cellular proteins but rather is disproportionally linked to the activation of signal transduction proteins including over forty kinases and many steroid hormone receptors. Mutated forms of many Hsp90 clients are causative agents in cancer, making Hsp90 a promising pharmacological target. Many small molecular inhibitors have been identified that competitively bind to the ATP binding site of Hsp90, some of which are in clinical trials as anticancer agents. Although the activation of kinase and hormone receptor clients by Hsp90 and its co-chaperones has been extensively studied, the molecular mechanism of client protein activation is poorly understood. Hsp90 is a dimeric chaperone containing three domains: the N-terminal (N) and middle (M) domains contribute directly to ATP binding and hydrolysis and the C-terminal (C) domain mediates dimerization. At physiological concentration, Hsp90 predominantly forms dimers, but the possibility that full-length monomers might also function in cells has not been tested. In Chapter 3, we used a single-chain strategy to design a full-length Hsp90 monomer (NMCC). The resulting construct was predominantly monomeric at physiological concentration and did not function to support yeast viability as the sole Hsp90. NMCC Hsp90 was also defective at ATP hydrolysis and the activation of kinase and steroid hormone receptor clients in yeast cells. The ability to support yeast growth was rescued by the addition of a coiled-coil dimerization domain, indicating that the parental single-chain construct is functionally defective because it is monomeric. After finding that a full-length Hsp90 monomer containing only one ATPase site was unable to support yeast viability or activate Hsp90 clients, we set out to further explore the role of ATPase activity in client protein activation. Approximately 10 % of the yeast proteome binds to Hsp90 making it important to study Hsp90 function in the cellular environment where all binding partners are present. In Chapter 4, we observed that co-expression of different Hsp90 subunits in Saccharomyces cerevisiae caused unpredictable synthetic growth defects due to cross-dimerization. We engineered super-stabilized Hsp90 dimers that resisted cross-dimerization with endogenous Hsp90 and alleviated the synthetic growth defect. We utilized these super-stabilized dimers to analyze the ability of ATPase mutant homodimers to activate known Hsp90 client proteins in yeast cells. We found that ATP binding and hydrolysis by Hsp90 are both required for the efficient maturation of the glucocorticoid hormone receptor (GR) and v-src confirming the critical role of ATP hydrolysis in the maturation of steroid hormone receptors and kinases in vivo. In addition to its role in the activation of signal transduction client proteins, Hsp90 has been shown to suppress the in vitro aggregation of numerous hard-to-fold proteins. In Chapter 5, we examine the role of charge in Hsp90 anti-aggregation activity. The charge on Hsp90 is largely concentrated in two highly acidic regions. We found that deletion of both charge-rich regions dramatically impaired Hsp90 anti-aggregation activity. Addition of an acid-rich region with a distinct amino acid sequence to our double-deleted Hsp90 construct rescued the anti-aggregation activity of Hsp90 indicating that the net charge contributes to its anti-aggregation activity. The in vitro anti-aggregation activity of Hsp90 studied in Chapter 5 occurs in the absence of ATP. However, all of the biologically important functions of Hsp90 in cells identified to date, including the maturation of kinases and nuclear steroid hormone receptors, clearly require ATP hydrolysis. Why does Hsp90 robustly hinder the aggregation of hard-to-fold proteins without ATP in vitro, but in vivo uses ATP hydrolysis for all of its essential functions? By utilizing separation of function Hsp90 variants (that specifically lack in vitro anti-aggregation activity) we have begun to address this question. We find that anti-aggregation deficient Hsp90 is unable to support yeast growth under stressful conditions, potentially due to reduced cellular expression. Interestingly, the ATP-independent anti-aggregation activity of Hsp90 has no measureable impact on cellular function. Thus, hindering the aggregation of most hard-to- fold proteins by Hsp90 (independent of ATP hydrolysis) does not appear to be important for cell function. These results suggest a cellular model where the Hsp40/60/70 machinery is responsible for hindering the aggregation of most hard-to-fold proteins while Hsp90 assists in the maturation of a select set of clients in an ATP-dependent fashion, potentially aided by its inherent anti-aggregation properties.

Recently reported data demonstrate that Vav3, a Rho Guanine Nucleotide Exchange Factor (Rho GEF) is overexpressed in breast tumors, coexpressed with ER, necessary for proliferation in breast cancer cells, and predictive of response to neoadjuvant endocrine therapies in patients with ER+ tumors. Such data beg the question as to what roles Vav3 plays in modulation of steroid receptor activity in breast cancer and in resistance to current hormonal therapies. Using reporter assays, I provide novel evidence that Vav3 potentiates Estrogen Receptor activity and represses Androgen Receptor activity in breast cancer cells. Vav3 potentiates ligand-dependent estrogen receptor activity in the MCF-7. A truncated, constitutively active form of Vav3, caVav3 potentiates ligand dependent ER activity in both MCF-7 and T47D. Vav3 activates Rho GTPases through its GEF domain. ER potentiation by caVav3 is dependent upon GEF activity. A caVav3 mutant with defective GEF function represses basal and ligand-mediated ER activity in T47D. Although other studies have shown that Vav3 could activate various Rho GTPases, only constitutively active Rac1 mutants potentiated ER activity in both cell lines. Contrastingly, reporter assays were used to show that caVav3 inhibits ligand-mediated AR activity in the AR+ T47D cell line by both R1881 and DHT stimulation. caVav3-mediated repression of AR activity is GEF-dependent, as caVav3 GEF mutants potentiate AR activity. Constitutively active forms of Rho GTPases were found to repress AR activity to different extents, but R1881-mediated AR activity was only significantly repressed by caCdc42. My studies of the effect of androgens on AR protein by western blot show that androgens downregulate AR protein in the highly Vav3 positive T47D cell line. Previous studies have demonstrated that androgens stabilize AR protein in MCF-7, and I now provide evidence that overexpression of Vav3 or caVav3 reverses hormone-mediated AR protein stabilization in MCF-7. These data are especially relevant given recently published data that decreased AR protein levels contributed to failure of response to MPA in patients with metastatic breast cancer. Further breast cancer studies may prove Vav3 to be a potential drug target in hormone dependent, hormone independent, and metastatic disease.

The ring dove (Streptopeliafi sotia) has proved an excellent animal model for studies in reproductive endocrine-behaviour interactions for many years. It shows bi-parental care and has a stereotyped breeding cycle. Androgen receptor and progesterone receptor immunoreactivity (AR-ir and PR-ir) were localised in the brain of male and female ring doves. Nuclear AR-ir in courting birds was widespread throughout the telencephalong diencephalon, and mesencephalon. Nuclear PR-ir was only localised in discrete regions of the preoptic-hypothalamus of both sexes. In the anterior and posterior hypothalamus, high density of AR-ir and PR-ir neurones was concentrated and co-localised in the nucleus preopticus anterior (POA), nucleus preopticus mediahs (POM), nucleus medialis, pars medianis (POMm), nucleus preopticus paraventricularis magnocellularis (PPM), nucleus hypothalami laterahs posterioris (PLH), and tuberal. hypothalamus (Tu). Investigation of the effects of the breeding cycle on anterior pituitary gland and hypothalamic AR-ir and PR-ir showed a significant (p<0.001) increase in AR-ir cells in courting birds when compared with birds at other stages of the breeding cycle. AR-ir cells in birds brooding young was significantly reduced or absent altogether. In the anterior pituitary gland of both sexes, PR-ir cells increased during courtship and significantly decreased in brooding birds. In the anterior hypothalamus of both sexes, no significant change in PR-ir density was observed. However, a significant increase (p<0.05) of PR-ir neurones was seen 'in the ventral and lateral Tu of incubating male and female doves, respectively, whilst a significant decrease in PR-ir neurones was seen in the ventral region of the Tu in birds brooding young (p<0.001). Although no sex difference was obtained in the number of AR-ir neurones, significantly higher staining intensity for AR-ir was measured in male than female doves, in all hypothalamic regions investigated. Similarly, increased PR-ir staining intensity was measured in courting birds than in birds at other breeding stages. In the anterior pituitary gland and all regions of the hypothalamus the percentage co locahsation between AR-ir and PR-ir changes With breeding cycle. The highest percentage of PR-ir and AR-ir co-localisation (70-909/6w) as obtained in courting birds and the lowest (0.27-24%) in birds brooding young. The association between steroid receptor expression and aromatase P450 enzyme(P450AROM)activity in the brain of the ring dove was also investigated. Sexually experienced ring doves were injected intramuscularly with the aromatase inhibitor fadrozole (0.2 ml of 1 mg n-A-') and paired with saline injected controls in a cage containing a nest bowl and nesting material. The fadrozole/ saline vehicle was administered for 3 days at 12-hour intervals. Saline injected control males displayed aggressive and nest-orientated behaviours whereas fadrozole treated males showed none of these behaviours. Similarly, saline injected control females displayed nest-onented behaviours and were sitting on the nest by day 2, whereas fadrozole injected females showed none of these behaviours. On day 4 of courtship, birds were perfuse-fixed and the brains and anterior pituitary glands subjected to immunocytochernistry for androgen receptor (AR), progesterone receptor (PR) and aromatase (P450ARONýIn* saline treated birds of both sexes, nuclear AR-ir and PR-ir were seen in the PON, PPM, POM, POMm, PLH and Tu. In contrast, fadrozole treatment in both sexes resulted in the complete absence of detectable nuclear AR-ir expression, together with a significant decline in the expression of PR-ir in the POM and POMm, and all regions of the Tu (p< 0.001). A sex difference in PR-ir expression was seen in the POA and PPM (p<0.001) of fadrozole treated birds. Fadrozole administration caused decrease in the percentage co-localisation of AR-ir and PR-ir in both sexes. A high density of AROMir neurones was observed in the POM and POMm of saline injected males, which was found to co-express PR-ir (99%). The viability of maintaining in vitro brain slice preparations was studied with the aim to identify and confirm neural circuits involving the anterior and posterior hypothalamus in the initiation of sexual and parental behaviours in this species. Sections were pressure injected with fluorogold (FG) or Dil (8 -15 nl) in the POA, POM, and the ventral, lateral and dorsal regions of the Tu. Results obtained confirm previous reports of neural connections between the Tu and POA. Neural connections were also seen between the Tu and the nucleus medialis hypothalan-ii posterioris (PMH), and several other regions of the mesencephalon and telencephalon, including the nucleus intercollicularis JCo), nucleus ovoidalis (Ov), dorsolateralis posterior thalami (DLP), archistriatum, pars ventrale (Av) and archistriatum, pars dorsalis (Ad). It is concluded that in the dove, central AR-ir and PR-ir expression and co-localisation are closely associated with the sexual stages of the reproductive cycle. Increases 'in AR-ir neurones in the preoptic-hypothalamus in courting birds are consistent with the behavioural role of androgen acting in this brain area during this time. Similarly, the high PR-ir neurones in the preoptic-hypothalamus are consistent with the progesterone-induced initiation of incubation in this species. The high percentage of AR-ir and PR-ir colocalisation in the preoptic-hypothalamus of courting doves supports previous reports involving progesterone acting in these brain regions to terminate the androgen-dependent aggressive courtship behaviour in male doves. The decrease in the number of co-localised AR-ir and PR-ir neurones M the preoptic-hypothalamus following oestrogen synthesis inhibition suggests a role for oestrogen in the progesterone-dependent termination of aggressive courtship display in males, and models proposing a mechanism involving oestrogen in the orchestration of central systems associated with courtship behaviour in the ring dove are presented. Neural connections between the POA and Tu, and Tu with the PMH, ICo, Ov, DLP, Av and Ad may represent the neuroanatomical pathways involved in the integration of courtship display, vocalisation (nest-coos and/or bow-coos), auditory, propriosceptive signals and gonadotropM secretion in the reproductive cycle of the ring dove. These results are consistent with the hypothesis that the POA, Tu and the midhypothalamus are involved in the orchestration of sexual and parental behaviour of the ring dove.

Orientadores: Luis Otávio Zanatta Sarian, Sophie Françoise Mauricette Derchain
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Introdução: o câncer de ovário é comumente diagnosticado em estágios avançados, sendo atualmente a neoplasia ginecológica de maior letalidade. As pesquisas têm direcionado esforços na tentativa de descobrir novos fatores prognósticos e métodos terapêuticos. Muitos trabalhos estudam a expressão dos receptores de esteróides (dentre eles, estrógeno (RE), progesterona (RP) e andrógeno (RA)) e HER2 (receptor estimulador de crescimento epitelial - subtipo 2) como fatores prognósticos e associados à resposta terapêutica, apresentando; entretanto, resultados conflitantes. Até onde conhecemos, não há estudos desta natureza no Brasil. Objetivo: Correlacionar à expressão dos RE, RP, RA e HER2 aos fatores clínico patológicos, ao intervalo livre de doença e à sobrevida nos cânceres epiteliais de ovário. Material e métodos: Este é um estudo observacional de coorte retrospectiva. Foram incluídas 152 mulheres com tumores epiteliais malignos, selecionados através dos prontuários no período de 1993 a 2008, no Centro de Atenção Integral à Saúde da Mulher (CAISM) da Universidade Estadual de Campinas - UNICAMP, São Paulo, Brasil. A avaliação da expressão dos RE [subtipos alfa (RE?) e beta (RE?)], RP, RA e HER2 foram realizadas por imunoistoquímica através da construção de microarranjos de tecidos (TMA). Inicialmente, foi realizada análise uni variada da expressão dos receptores acima quanto à idade, estado menopausal, índice de massa corpórea (IMC), tipo histológico, grau histológico, estadiamento inicial de acordo com a classificação proposta pela FIGO e presença de doença residual pós-tratamento cirúrgico. Em seguida, as pacientes foram divididas em dois grupos: ausência da expressão de RE?, RP e HER2 (triplo negativo) e positividade para pelo menos um desses receptores (não triplo negativo); e, avaliadas em relação aos critérios clínicos e epidemiológicos acima. Foram, então, realizadas análises multivariadas dos padrões de expressão de RE ? e ?, RP, RA, HER2 e triplo negativo em relação a estes mesmos critérios. Por fim, análise de sobrevida multivariada foi realizada utilizando-se todos os padrões de expressão e os fatores clínicos epidemiológicos estudados. Resultados: Nas análises multivariadas, mostraram-se significativas as seguintes associações: do receptor de estrógeno alfa (ER?) com tumores de grau histológico menos diferenciado (p=0.01); do receptor de progesterona (RP) à obesidade (p= 0.01); do receptor de andrógeno (RA) com tumores do subtipo seroso (p= 0.01); do receptor de HER2 com tumores dos graus histológicos II-III (p=0.02); do subgrupo triplo negativo com tumores de grau histológico menos diferenciado (II-III) (p<0.01). Não houve associação do RE? com nenhum dos fatores estudados. Quanto à análise multivariada de sobrevida livre de doença e sobrevida global; dentre os padrões de expressão de receptores, apenas o RE? esteve associado com melhor sobrevida livre de doença (RR=0.39; 95%CI 0.17 -0.90). Conclusões: A expressão do RE? esteve mais significativamente associada a fatores clínicos de pior prognóstico. O RP esteve associado à obesidade. O RA esteve significativamente mais presente nos tumores serosos. A expressão do HER2 e a presença de tumores triplo negativo foram maiores em tumores menos diferenciados. Paradoxalmente; o RE? foi o único receptor a apresentar associação com maior sobrevida livre de doença apesar de sua relação significativa com fatores reconhecidos de pior evolução clínica. Não houve diferença estatística significativa na análise multivariada de sobrevida total e sobrevida livre de doença quanto ao grupo de tumores triplo negativo
Abstract: Introduction: Ovarian cancer is commonly diagnosed in advanced stages and currently is the most lethal gynecological malignancy. Surveys have focused efforts in an attempt to discover new prognostic and therapeutic methods. A plenty of studies investigates the expression of steroid receptors (among them, estrogen (ER), progesterone (PR) and androgen AR)) and HER2 (epidermal growth receptor stimulator - subtype 2) as prognostic factors and associated to therapeutic response, presenting, however, conflicting results. As far as we know, there are no studies of this nature in Brazil. Objective: The aim of this study was to correlate the expression of ER (subtypes ? (ER ?) and ? (ER ?), PR, AR and HER2 to clinical pathological factors, the disease-free survival and overall survival of epithelial ovarian cancers. Methods: This is a retrospective observational cohort study. The study included 152 women with malignant epithelial tumors, selected through the records from 1993 to 2008, in the Centro de Atenção Integral à Saúde da Mulher (CAISM) at the State University of Campinas - UNICAMP, São Paulo, Brazil. The expression of ER (? and ?), PR, AR and HER2 was evaluated by immuno histochemistry through tissue microarray (TMA) technique. Initially, univariate analyses were performed, evaluating the expression of each receptor mentioned above to age, menopausal status, body mass index (BMI), histological type, histological grade, initial staging as preconized by FIGO staging of ovarian tumors and presence of residual disease after surgical treatment. Then, the patients were divided into two groups: absence of the expression of ER?, PR and HER2 (triple negative) and positive for at least one of these receptors (not triple negative), and evaluated in relation to the clinical and epidemiological criteria mentioned above. Multivariate analyzes were performed with ER ?, ER?, PR, AR, HER2 and triple negative towards these same criteria. At last, multivariate survival analyses were conducted using all the patterns of receptors' expression, epidemiological and clinical factors studied. Results: In multivariate analyzes, there were the following significant associations: of the estrogen receptor alpha (ER?) with less differentiated histological grade tumors (p = 0.01); of the progesterone receptor (PR) to obesity (p = 0.01); of the androgen receptor (AR) with the serous tumors (p = 0.01); of the HER2 receptor with tumors of histological grades II-III ( p = 0.02); of the triple negative subgroup with less differentiated histological grade tumors (II-III) (p <0.01). There was no association of the ER? with any of the factors studied. In the multivariate analysis of disease-free survival and overall survival; considering the patterns of receptors' expression, only the ER? expression was associated with better disease-free survival (RR= 0.39, 95% CI 0.17 to 0.90). Conclusions: The expression of ER? was more significantly associated with clinical factors of poor prognosis. The PR was associated with obesity. The AR was significantly more prevalent in serous tumors. The HER2 expression and the presence of triple negative epithelial ovarian cancer tumors were higher in less differentiated tumors. Paradoxically, the ER? was the only receptor which showed association with better disease-free survival despite its significant relationship with recognized factors of worse clinical outcome. There was no statistically significant difference in multivariate analysis of overall survival and disease-free survival regarding to the triple negative group
Mestrado
Oncologia Ginecológica e Mamária
Mestra em Ciências da Saúde

Steroid hormone receptor analysis has emerged as an important aid to prognosis in human breast cancer. The availability of ¹²⁵I-labeled estradiol led to the development of a dual-label assay for the simultaneous determination of estrogen and progesterone receptor content. There are indications that plasminogen activator activity may also serve as a prognostic indicator in breast cancer. Two methods, one fluorometric and the other spectrophotometric were examined for their suitability in assaying for plasminogen activator activity. Both assays were found to be equally suitable for the determination of this enzyme in breast tumour cytosols. Cytosol from tumour samples stored at -70°C for 18 - 52 months was assayed for steroid receptor content and plasminogen . activator activity. Estrogen receptor (ER) values determined at the time of the original assay were compared with the re-assayed estrogen receptor content. The results showed that this steroid receptor is remarkably stable to storage at -70°C. Comparisons between the two steroid receptors and plasminogen activator (PA) activity showed significant correlations between estrogen receptor and progesterone receptor (PgR) content and between progesterone receptor and plasminogen activator activity.There was no significant correlation between ER and PA activity. Survival curves were constructed to examine the effect of steroid receptor content and plasminogen activator activity on the survival of breast carcinoma patients. Due to the limited number of patients available, statistically significant differences in survival were not evident. However, there was an indication that estrogen receptor positive patients survived longer than their estrogen receptor negative counterparts. PA activity and progesterone receptor content did not appear to influence survival.
Medicine, Faculty of
Pathology and Laboratory Medicine, Department of
Graduate

In Part 1 of the study methods for estimating the concentrations of the total and free specific steroid receptors for oestradiol and progesterone in the cytoplasm from endometrial tissue are assessed and novel features integrated. The cytosol is treated with Dextran coated charcoal (DCC), and after removing the DCC the steroid receptor proteins are precipitated from the cytosol with a protamine sulphate solution prior to incubation of the precipitate with 3H-labelled steroids in a phosphate buffer. Conditions for the optimum binding of the 3H-labelled steroids are investigated. These include the constituents of the DCC and protamine sulphate solution, and the pH and molarity of the incubation buffer and the concentrations of the 3H-labelled and non-labelled steroids for maximum specific binding. Temperature and duration of incubation are established for the maximum binding of the 3H-labelled steroids to their respective receptors for the estimation of the total and free receptor concentrations. The differences between the two is a measure of the endogenously bound or filled receptor. A novel feature of the methods is that the precipitation of the cytosol, incubation of the precipitate with 3H-labelled steroid and the subsequent counting of the bound radio-activity are performed in the same vial. In Part 2 methods are used to measure the concentrations of the total and free cytoplasmic receptors for oestradiol and progesterone in normal and pathological human endometrial tissue. Also the relationship between the receptor fractions for the two steroids is examined. The binding of 3H-oestrone to the receptor is studied and it is established that this binding is separate from the oestradiol binding. Even though statistically significant differences are found between the receptor concentrations for different types of endometrial tissue, the level of the bound oestradiol receptor, particularly in cases of high oestrogenic stimulation do not agree with those expected from the accepted idea of translocation of oestradiol and receptor from the cytoplasm to the nucleus. To explain the values for the two oestrogen receptors it is suggested that oestradiol and its receptor enter the nucleus separately before combining and binding to the nuclear acceptor. The action of the binding alters the oestradiol receptor to a receptor which does not recognise oestradiol but binds oestrone. The free oestrone receptor is reconverted into an oestradiol receptor in the cytoplasm. Therefore the total available receptor for binding oestradiol is the sum of the concentrations of the free receptor of oestradiol and oestrone. Therefore, if no oestrone receptor is found then the attachment of oestradiol to the nuclear acceptor is not functioning properly.

The effectiveness of estrogen replacement therapy in the prevention of postmenopausal osteoporosis has led to its current widespread use throughout the United States and much of Western Europe, and recently, clinical correlations between circulating androgen levels and structural bone integrity have been presented. Nevertheless, the biochemical mechanism through which estrogens and androgens act to protect and maintain bone has remained unclear. One possibility is that these hormones directly modulate the activity of cells responsible for bone formation. Therefore, studies were conducted to examine the effects of sex steroids on human osteoblast-like cells. In the first set of experiments, a finite human cell line was established from trabecular bone explants obtained from a 48 year-old woman. These cells, designated BG688, were characterized as osteoblast-like in phenotype using several independent criteria. In addition to classical osteoblast markers, BG688 cells also possess approximately 2400 high affinity (K(d) = 0.45nM) 17-β estradiol (E₂) binding sites per cell. The binding of E₂ to a subset of these sites was specific. BG688 cells were also shown to respond to a physiological concentration (10nM) of E₂, which elicits pleiotropic changes in several mRNA levels including a 2-fold increase in the steady state concentration of α₁(I)-procollagen mRNA. These results indicate that human osteoblast-like cells respond to E₂ via a receptor mediated mechanism, but that, unlike the reproductive tissues, osteoblasts are a less sensitive target. In the second series of experiments, the effects of androgenic hormones on the osteoblast-like, human osteosarcoma cell line, HOS TE85 were evaluated. Employing radiolabelled dihydrotestosterone (DHT), 2800 saturable, high-affinity (K(d) = 0.66nM) androgen binding sites were detected per HOS TE85 cell. Androgen binding was specific. The expression of androgen receptors in HOS TE85 cells was further substantiated by Northern analysis. Physiological concentrations of DHT and testosterone decreased HOS TE85 cell proliferation. This finding suggests that androgens may also play a role in osteoblast differentiation. In support of this hypothesis, treatment with testosterone enhanced the abundance of both α₁ (I)-procollagen mRNA and transforming growth factor- β mRNA. The non-aromatizable androgen DHT also elicited an increase in the steady state concentration of α₁(I)-procollagen mRNA. The findings presented herein are significant within the field of bone cell biology in that they demonstrate that osteoblasts are a target cell for the action of sex steroids, via their cognate, high-affinity receptors. These results also have important implications within the broader context of bone pathophysiology in that they suggest a direct modulation of bone forming and bone remodeling activity by sex steroids.

Orientador: Francisco Eduardo Martinez
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O alcoolismo crônico está associado a distúrbios no sistema reprodutor feminino como disfunção hormonal, alteração na expressão dos receptores esteróides, produção de espécies reativas de oxigênio (ERO), entre outros. A melatonina, hormônio secretado pela glândula pineal, possui função moduladora no ciclo reprodutivo e têm papel importante no combate as ERO. Os estudos envolvendo o alcoolismo crônico e sua interação com a melatonina, em fêmeas, são ainda inconclusivos. O presente trabalho tem como objetivo investigar os efeitos da administração exógena da melatonina sobre os hormônios sexuais, os receptores esteróides sexuais (AR, ER-?, ER-?, PRA e PRB) no ovário, oviduto e útero, além do perfil nutricional e o estresse oxidativo nos ovários de ratas adultas UChB (consumidoras voluntárias de etanol a 10%). Foram utilizadas 60 ratas UChB, distribuídas nos seguintes grupos: UChB Co: sem acesso ao etanol; UChB EtOH: consumo diário de 4 - 5 g etanol/100g de peso corpóreo (PC), ambos recebendo solução veículo. Concomitantemente, os grupos UChB Co+M e UChB EtOH+M receberam injeções diárias de melatonina (100?g/100g PC) via i.p, a partir dos 90 dias de idade, durante 60 dias consecutivos. Aos 150 dias de idade, os animais foram eutanasiados em estro (4a.m) e os materiais coletados e processados. A melatonina aumentou os níveis de progesterona, 6-sulfatoximelatonina e reduziu 17?-estradiol, enquanto a combinação entre etanol + melatonina causou uma queda significativa nesses hormônios. Apesar do receptor androgênico (AR) ovariano não ter sido influenciado pela melatonina, os grupos UChB EtOH e UChB EtOH+M mostraram uma diminuição no AR do oviduto. Ambos os receptores de estrogênio (ER-? e ER-?) no oviduto foram pouco expressos em animais recebendo etanol ou melatonina enquanto somente o ER-? uterino foi reduzido. Por outro lado, receptores de progesterona (PRA e PRB) foram positivamente regulados no ovário por etanol ou etanol + melatonina, enquanto PRA foi negativamente regulado no útero e oviduto, exceto quando o etanol e melatonina foram combinados. Os níveis do receptor de melatonina (MT1R) foram maiores no ovário e útero de ratas tratadas com melatonina, independentemente do consumo de etanol. O peso corpóreo dos animais foi reduzido após interação do etanol e melatonina após 40 dias de tratamento. Em ambos os grupos tratados com melatonina, observou-se redução no consumo energético e líquido. Houve diminuição da quantidade de etanol consumida durante o tratamento e o ciclo estral foi maior em ratas que receberam etanol e melatonina, evidenciado por diestro prolongado. Os níveis de hidroperóxido de lipídio foram maiores nos ovários de ratas UChB EtOH e diminuiu após o tratamento com melatonina. Atividades antioxidantes da superóxido dismutase, glutationa peroxidase e glutationa redutase foram aumentadas nos grupos tratados com melatonina. Conclui-se que a melatonina tem efeito oposto ao etanol sobre os hormônios sexuais. Melatonina e etanol regulam diferencialmente os receptores de esteróides sexuais nos tecidos reprodutivos, atuando principalmente através de seu receptor MT1R. Além disso, a melatonina é capaz de alterar a eficiência alimentar, o ciclo estral, e, contudo, protege os ovários contra o estresse oxidativo resultante do consumo de etanol
Abstract: Chronic ethanol intake is associated with female reproductive disturbances including hormonal dysfunction, changes in the steroid receptors expression, production of reactive oxygen species (ROS), among others. Melatonin, an indolamine secreted by pineal gland, plays key roles in the reproductive cycle, besides having an important function in scavenging ROS. Studies focusing chronic alcoholism and its interaction with melatonin, in females, are still inconclusive. This study aims to investigate the effects of exogenous melatonin administration on sex hormones, sex steroid receptors (AR, ER-?, ER-?, PRA and PRB) in the ovary, oviduct and uterus, as well as the nutritional profile and oxidative stress in the ovaries of adult UChB rats (10% (v/v) ethanol voluntary intake). 60 UChB female rats were divided into the following groups: UChB Co: without access to ethanol (used as control); UChB EtOH: drinking daily ethanol at 4 - 5 g ethanol/100g body weight (BW), both receiving vehicle solution. Concomitantly, UChB Co + M and UChB EtOH + M groups received daily injections of melatonin (100?g/100g BW) via i.p, starting from 90 days old and during the next 60 consecutive days. At 150 days of age, all animals were euthanized in estrus (4a.m). Melatonin increased progesterone, 6- sulfatoximelatonin and decreased 17?-estradiol, while the ethanol+melatonin combination caused a significant fall in these hormones. Despite androgen receptor (AR) in ovary has not been influenced by melatonin, ethanol and ethanol+melatonin led to a decrease in oviduct AR. Both estrogen receptors (ER-? and ER-?) were underexpressed by either ethanol or melatonin in oviduct and only uterine ER-? was downregulated. Conversely, progesterone receptors (PRA and PRB) were positively regulated in the ovary by ethanol or ethanol+melatonin, whereas PRA was downregulated in uterus and oviduct, except when ethanol+melatonin were combined. Additionally, melatonin receptor (MT1R) was increased in ovary and uterus of melatonin-treated rats, regardless of ethanol consumption. Body weight gain was reduced with ethanol plus melatonin after 40 days of treatment. In both melatonin-treated groups, it was observed a reduction in food-derived calories and liquid intake toward the end of treatment. The amount of consumed ethanol dropped during the treatment. Estrous cycle was longer in rats that received both ethanol and melatonin, with prolonged diestrus. Following to oxidative status, lipid hydroperoxide levels were higher in the ovaries of ethanol-preferring rats and decreased after melatonin treatment. Additionally, antioxidant activities of superoxide dismutase, glutathione peroxidase and glutathione reductase were increased in melatonin-treated groups . We conclude that melatonin has opposite effect on sex hormones to those of ethanol consumption. Together, melatonin and ethanol differentially regulates the sex steroid receptors in the reproductive tissues, mostly acting "in situ" through its MT1R receptor. Finally, melatonin is able to affect feed efficiency and, conversely, it protects the ovaries against the oxidative stress arising from ethanol consumption.
Doutorado
Biologia Celular
Doutor em Biologia Celular e Estrutural

Orientador: Sebastião Roberto Taboga
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: A próstata, glândula do sistema genital que tem origem embrionária a partir do seio urogenital, não é exclusiva do sistema reprodutor masculino, sendo também encontrada em fêmeas de vários mamíferos, incluindo humanos e roedores. No macho ela pode apresentar-se altamente desenvolvida em razão da maior quantidade do hormônio testosterona, e, apesar de pouco desenvolvida em fêmeas, devido à baixa quantidade desse mesmo tipo hormonal, é uma glândula funcional. Em fêmeas adultas de gerbilos, a próstata possui uma localização parauretral, exibindo íntimo contato com a parede proximal e medial da uretra, a qual é homóloga a próstata ventral de roedores machos. Embora se conheça a influência da progesterona na fisiologia do sistema reprodutor feminino e masculino, poucos estudos exploram a sua influência, especificamente, sobre a glândula prostática. Desta forma, este trabalho avaliou os aspectos morfofuncionais da glândula prostática masculina e feminina, resultantes da influência da progesterona, e de suas interações com o estradiol e a testosterona. Para isso, gerbilos machos e fêmeas foram castrados cirurgicamente no início do período puberal, aos 45 dias de idade. Ao completarem 90 dias de idade, os gerbilos receberam doses subcutâneas de progesterona ou desse somado a estradiol e testosterona, durante 14 dias. Nos animais castrados de ambos os sexos a próstata mostrou uma morfologia regredida, com uma redução significativa na sua capacidade de secreção, da quantidade de células receptoras de androgênio (AR) e receptoras de estrógeno ? (ER?), porém sem alterar a marcação para receptor de estrógeno ? (ER?). Dessa forma, a castração cirúrgica foi bastante importante, uma vez que permitiu mimetizar de forma satisfatória um ambiente prostático com baixos níveis hormonais. Nos dois sexos, a administração de progesterona isoladamente conseguiu reverter alguns desses efeitos, com uma melhora considerável no padrão secretório da glândula, porém estruturalmente essas mudanças ocorreram de forma moderada. Nesses animais, foi observado um aumento expressivo dos ERs ? e ?, além da presença de células receptoras de progesterona (PR). Em relação aos ARs foi evidenciado que a progesterona pode apresentar características indutoras ou inibitórias dependendo da sua concentração. O tratamento, simultâneo com a progesterona, de estradiol e testosterona desencadeou uma reestruturação glandular mais intensa nos machos e fêmeas, resultando em hipertrofia e hiperplasia do epitélio e do estroma glandular, recuperação do padrão de secreção e amplitude alveolar. Essas características, no entanto, foram acompanhadas pelo surgimento de lesões prostáticas como neoplasias intraepiteliais e o surgimento de debris celulares. A interação da progesterona com o estradiol também regulou positivamente os AR, ER? e ER?, porém não apresentou qualquer efeito sobre os PRs quando comparado aos animais tratados somente com progesterona. Em adição, nesses animais houve um aumento acentuado da proliferação celular, o qual foi contrabalanceado pelo aumento também do índice de morte celular. Nos animais tratados com progesterona e testosterona, a próstata também se desenvolveu e mostrou um aumento das células AR-positivas e do índice apoptótico, havendo, entretanto uma redução dos ER?, ER? e PR. Dessa forma, é razoável concluir que a próstata feminina e masculina comporta-se de forma bastante semelhante frente à ação dos hormônios progesterona, estradiol e testosterona. Ademais, embora a progesterona apresente efeitos estruturais razoáveis na glândula prostática, a sua interação com o estrógeno e a testosterona é capaz de promover uma intensificação desses efeitos, sem recriar, porém um ambiente homeostático semelhante aos dos animais intactos. A progesterona também mostrou ser um fator regulador em potencial da atividade proliferativa e apoptótica prostática, opondo-se aos efeitos da testosterona e do estradiol. Outro fator importante é a descoberta de que a progesterona pode induzir ou inibir a presença de células AR-positivas na glândula, e que esse dualismo funcional é resultado do efeito dose-dependente desse hormônio sobre a próstata
Abstract: The prostate is a gland of reproductive system that arises from the urogenital sinus, being located around the urethra below the bladder. The existence of this gland is not exclusive of the male reproductive system, being found in females of various species, including rodents and humans. In the male, it can be highly developed due to the increased amount of the testosterone, and although poorly developed in females, due to the low quantity of this hormone, it is a functional gland. The prostate of female gerbils has a paraurethral location, showing a closer contact with the proximal and medial urethra wall, being homologous to the ventral prostate of male rats. This study evaluates the morphofunctional aspects of the prostate gland in males and females, regarding the influence of progesterone, and their interactions with estradiol and testosterone. For this, male and female gerbils were surgically castrated in early pubertal period, at 45 days of age. At 90 days of age, the gerbils received subcutaneous doses of progesterone alone or associated to testosterone or estradiol during 14 days. In castrated animals of both sexes, prostate showed a regressed morphology, with a significant reduction in its secretion capacity, the amount of androgen receptor cells (AR) and estrogen receptor ? (ER?), but without changing the labeling for estrogen receptor ? (ER?). Thereby, surgical castration was very important, since it allowed mimetize a prostatic environment with low hormone levels. In both sexes, the administration of progesterone alone could reverse some of these effects with a considerable secretion improvement, but structurally these changes occurred in a moderate way. In these animals, we observed a significant increase of ER ? and ?, besides the presence of progesterone receptor (PR) cells. Regarding ARs, it was shown that progesterone can have inductor or inhibitory characteristics depending on its concentration. The treatment with progesterone plus estradiol and progesterone plus testosterone triggered a more intense prostate restructuration in male and female, resulting however in a hypertrophy and hyperplasia of the glandular epithelium and stroma, besides recovery of the alveoli amplitude and pattern of cellular secretion. These characteristics, however, were followed by the development of prostatic lesions like intraepithelial neoplasia and cellular desquamation. The progesterone and estradiol interaction also upregulated the AR, ER? and ER?, however had no effect on the PRs when compared to the animals treated with progesterone alone. In addition, in these animals there was a marked increase in cellular proliferation, which was counterbalanced by increased cell death. In animals of either sex treated with progesterone and testosterone, the prostate also became developed and showed an increase of AR-positive cells and apoptotic index, although there was a reduction of ER?, ER? and PR. Thus, it is reasonable to conclude that the female and male prostate behaves similarly after the progesterone, estradiol and testosterone administration. Moreover, although the progesterone has reasonable structural effects on the prostate gland, its interaction with estrogen and testosterone can intensificate these effects, but do not recover a homeostatic environment similar to that of intact animals. The progesterone also proved to be a potential regulatory factor of the proliferative and apoptotic activity, opposing the effects of testosterone and estradiol. Another important finding is that progesterone can induce or inhibit the presence of ARpositive cells in the gland, and this functional dualism is the result of dose-dependent effect of this hormone on the prostate
Doutorado
Biologia Celular
Doutor em Biologia Celular e Estrutural

O tumor venéreo transmissível (TVT) é uma das neoplasias de maior incidência na espécie canina acometendo principalmente o trato genital masculino e feminino. Embora o tratamento de escolha seja a quimioterapia, a resistência às drogas tem sido um fenômeno freqüentemente observado na prática clínica. A determinação dos receptores esteróides (estrógeno e progesterona) por imunoistoquímica em tecidos normais e tumorais de humanos e várias espécies animais, tem mostrado grande importância tanto na avaliação prognóstica da doença tumoral, como também na possibilidade de tratamentos hormonais como adjuvantes à quimioterapia. Os objetivos desta pesquisa foram avaliar a expressão e comparar a concentração dos receptores de estrógeno α (RE-α) e progesterona (RP4) no tecido vaginal íntegro de fêmeas hígidas e no tecido vaginal e tumoral de fêmeas portadoras de TVT, nas fases do ciclo estral. Foram utilizadas 58 cadelas divididas em grupo experimental (portadoras de TVT) e grupo controle (fêmeas hígidas), agrupadas segundo a fase do ciclo estral determinada por citologia vaginal, dosagem hormonal e aspecto macroscópico dos ovários. Sob anestesia geral, foram colhidos fragmentos da vagina e do tecido tumoral e estes fixados, blocados em parafina e posteriormente submetidos a imunoistoquímica. Nas cadelas do grupo controle houve maior expressão dos RE-α no anestro, proestro e estro (p<0,05) em relação ao diestro. Nas fêmeas do grupo experimental houve maior expressão dos RE-α no diestro (p<0,05) em relação ao estro, porém não mostrou diferença significante quando comparada com o anestro. Não houve diferença estatística para os RP4, nas fases do ciclo estral para os animais do grupo controle e no epitélio vaginal das fêmeas do grupo experimental não houve positividade em nenhum dos fragmentos avaliados. Não se observou positividade para RE-α e RP4 no TVT, porém o endotélio de vasos sanguíneos do tumor apresentou expressão de RE-α em algumas amostras. Concluiu-se que a expressão dos RE-α nos tecido vaginal das cadelas hígidas e de cadelas portadoras de TVT, tem expressão diferente em função do hormônio esteróide circulante, porém não se expressa no tecido tumoral
One of the most frequent canine neoplasias is the Transmissible Venereal Tumour (TVT) which affects male and female genital tract. In clinical routine the standard treatment is chemotherapy, to which nevertheless drug resistance is a common feature. Immunohistochemical determination of steroid hormone receptors (estrogen and progesterone) in normal and tumoral tissues of humans and several other species is of great relevance for prognostic evaluation of oncological patients, as well as for the decision of whether to make use of hormonal therapy in association to chemotherapy. The aim of this research was to determine immunohistochemically estrogen (ER-α) and progesterone (PR4) receptors expression in vaginal tissue of non-affected bitches and in vaginal and tumoral tissues of TVT affected bitches. Fifty-eight bitches were equally divided in 2 groups: experimental group (TVT) and control group (non-affected). Canine estrous cycle phases were determined by means of vaginal cytology, hormonal assay and macroscopic appearance of ovaries. Samples from vaginal and tumoral tissues were obtained with bitches submitted to general anesthesia. Samples were fixed in 10% buffered formaldehyde and then mounted in paraffin-embedded sections. Anestrous, proestrous and estrous control females presented higher ER-α expression than diestrous bitches. Within the experimental group, there was statistical difference between estrous and diestrous phases, being ER-α expression higher for diestrous bitches. However, no significant difference in relation to anestrous females was established. In relation to RP4 expression, no difference among estrous cycle phases of the control group was verified. Furthermore, no RP4 expression was noted in vaginal tissue of TVT affected females. In tumoral tissues ER-α and RP4 were not expressed, although some samples presented ER-α expression in blood vessels’ endothelium. In conclusion, different ER-α expression was verified in vaginal tissue of experimental and control bitches under distinct steroid influence, whereas no ER-α expression in tumoral tissues was evidenced

Steroid hormone receptors (SHRs) are well validated therapeutic targets in a number of diseases. Current therapies competitively antagonise the ligand binding domain (LBD), blocking activation of the receptor and downstream signalling pathways. However cross-reactivity can be seen amongst the antagonists of different SHRs eliciting unwanted side effects. Additionally the acquisition of resistance to current therapies in diseases such as prostate cancer limits their use. The amino-terminal domain (NTD) of SHRs provides an alternative target for antagonism by allowing potential therapies to block receptor transactivation and inhibit interactions with co-activator proteins. Reduced homology between different SHR NTDs also increases the specificity of drug interactions. However development of targeted therapies using rational drug design has been hindered by its intrinsically disordered structure. Establishing cell lines which stably express a SHR responsive reporter gene alongside variants of SHRs lacking the LBD provides a method by which small molecules specifically targeting the NTD of each receptor can be identified. This assay has been designed to overcome the barriers to drug discovery that are presented by an intrinsically disordered protein. The project follows the design, development, optimisation and implementation of a high throughput screening assay with the potential to identify novel small molecule inhibitors of SHRs. The applications of these inhibitors are highlighted throughout, with specific reference to their potential to inhibit the androgen receptor in prostate cancer.

Cytochrome P-450, the terminal oxidase of the metabolic mono-oxygenase system, is thought to exist in multiple forms, which have differing substrate specificities, and are variably inducible by different enzyme inducers. Many mutagens, themselves unreactive, require metabolic activation by one or more of these cytochrome P-450-dependent microsomal enzymes for mutagenic activity. Such mutagens may be detected in the Salmonella mutagenicity test only by the incorporation of an hepatic microsomal (59) fraction into the assay (as a first approximation to in vivo metabolism). Induction of the microsomal enzymes by different agents enhances the metabolic activation of mutagens; in fact, many mutagens are only detected when the 59 fraction has been induced by appropriate agents. Inducers of the phenobarbital-type are known to enhance microsomal steroid hydroxylation when administered at supraphysiological levels, inducers of several mono-oxygenase activities. In turn, the steroids, have been reported to be The inductive effects of the female sex-steroids and the combined effects of steroid and phenobarbital (PB) pretreatment on the metabolic activation of four mutagens have been investigated using the Salmonella assay. Female Sprague-Dawley rats were pret reated with 17a-oestradiol (E2) or progesterone (PRG) , at a level of either 1 mg/kg or 20 mg / kg daily for 14 days. A duplicate set of similarly pretreated groups were also induced with PB. Hepatic microsomal fractions were prepared from each group and incubated with each of the te st mutagens in the presence of a tester strain known to detect each particular type of mutagen. Induction of the hepatic metabolizing system by PB increased the activation of the mutagens significantly (as reflected by an increased number of revertant prototrophic S .typhimurium colonies). The administration of PRG also caused significant, and dose-dependent, induction of the activation of af l atoxin B1, benzo(a)pyrene, and dimethylnitrosamine. In general, E2 exhibited no inductive effect, but it did produce an increase in the activation of aflatoxin B1 (a reaction which is known to be catalysed by a mono-oxygenase prefe rentially inducible by PB). When use was made of a microsomal fraction that was prepared from animals which were both steroidpretreated and induced by PB, mutagenic activation was of the same order of magnitude as that observed when induction was brought about by PB alone. The absence of additive effect, taken together with the observations already mentioned, indicate that steroids induce the same cytochrome isozymes that are induced by PB. The implications of sex-hormonal regulation of the metabolic activation of mutagens are briefly discussed.
KMBT_363
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