Introduction: Prostate cancer is the most common type of cancer in Sweden and is often treated using antiandrogenic drug therapy. Two substances belonging to this class of pharmaceuticals are bicalutamide and flutamide. After excretion from the human body, the drug molecules enter the wastewater treatment plant (WWTP). The WWTPs are not effective enough to completely remove pharmaceutical residues, why presence of both bicalutamide and flutamide can be detected in WWTP effluent water. Previous findings: Antiandrogens have been reported to affect reproduction in adult fish, but studies regarding possible effects on the embryonic development of fish are few. Aim: The present study sought to investigate if exposure to bicalutamide or flutamide cause toxicity in the early developmental stages of zebrafish embryos, and whether negative effects occur within concentrations relevant to measured environmental levels. Method: A modified OECD FET-test was used, where additional sublethal endpoints were included and the time period for assessment extended to 144 hours post fertilization (hpf). In addition, a locomotor activity assay was performed at 144 hpf in order to observe any sub-lethal swimming behavioral effects. Results: High doses (10 mg/L) of flutamide led to 100% lethality of the zebrafish embryos but the results suggest no acute toxic effects in the high dose treatment group of bicalutamide, or of either flutamide or bicalutamide within in the low (0.1 mg/L) or intermediate (1 mg/L) treatment groups. Neither did the locomotor activity assay result in statistically significant results, although the pattern of swimming activity in the low dose groups suggests that behavioral developmental effects could be present. Conclusions: High doses of flutamide caused mortality of the embryos, but no lethal or sublethal effects were present at environmentally relevant concentrations. The modest outcome of present study however suggests that further investigation of behavioral developmental effects of antiandrogens could be of future relevance. Analysis of the expression of genes related to neuronal growth, memory and other cognitive behaviors associated with behavioral changes, would then be of interest for further studies.

No description available.

No / Red deer stags produce an androgen-dependent mane of long hairs only in the breeding season; in the non-breeding season, when circulating androgen levels are low, the neck hair resembles the rest of the coat. This study was designed to determine whether androgen receptors are present in deer follicles throughout the year or only in the mane (neck) follicles when circulating testosterone levels are high in the breeding season. Although androgens regulate much human hair growth the mechanisms are not well understood; they are believed to act on the hair follicle epithelium via the mesenchyme-derived dermal papilla. The location of androgen receptors in the follicle was investigated by immunohistochemistry and androgen binding was measured biochemically in cultured dermal papilla cells derived from mane and flank follicles during the breeding season and from neck follicles during the non-breeding season. Immunohistochemistry of frozen skin sections using a polyclonal antibody to the androgen receptor localised nuclear staining only in the dermal papilla cells of mane follicles. Saturation analysis assays of 14 primary dermal papilla cell lines using [(3)H]-mibolerone demonstrated high-affinity, low-capacity androgen receptors were present only in mane (breeding season neck) cells; competition studies with other steroids confirmed the specificity of the receptors. Androgen receptors were not detectable in cells from either the breeding season flank nor the non-breeding season neck follicles. The unusual biological model offered by red deer of androgen-dependent hair being produced on the neck in the breeding, but not the non-breeding season, has allowed confirmation that androgen receptors are required in follicle dermal papilla cells for an androgen response; this concurs with previous human studies. In addition, the absence of receptors in the non-breeding season follicles demonstrates that receptors are not expressed unless the follicle is responding to androgens. Androgen receptors may be induced in mane follicles by seasonal changes in circulating hormone(s).

Age-related declines of androgens and libido in males have been observed for decades. This dissertation sought to elucidate the mechanisms by which hormones may act differentially upon their receptors in the hypothalamus of aging compared to young males. I also examined how sexual experience modulates the ability of hormones to facilitate sexual behavior with aging. Experiment one measured androgen receptors (AR) and estrogen receptor α (ERα) cells in male rats at young, middle-aged and old age. I found that AR cell numbers in hypothalamic regions studied underwent significant age-related increases. Numbers of heavily ERα labeled cells, but not total ERα cells, increased with age. This study demonstrates that the aging brain has the capacity to synthesize hormone receptors which is increased possibly due to decreased testosterone concentrations. Experiment two examined the effect of sexual experience on serum hormones and cells of AR and ERα in hypothalamic regions in young and middle-aged males. The results showed that AR cell numbers increased with aging but did not change with experience. No age- or experience-related alteration in ERα expression occurred. However, serum testosterone increased and estradiol decreased with age. Experience increased total and free testosterone. Interactions of age and experience on total testosterone, estradiol, and luteinizing hormone were found. These results show long-lasting effects of sexual experience on hormones, but not on their receptors in the hypothalamus. Experiment three investigated effects of exogenous testosterone on sexual behavior in young and middle-aged males. The results showed a decline in sexual behavior parameters with age. After castration with testosterone treatment, there were few differences in sexual behavior measures between young and middle-aged males. AR cell numbers were higher and ERα cell numbers lower in testosterone compared to vehicle-treated males of both ages, and few effects of age occurred. These findings indicate that testosterone and aging interact in a complex manner to control numbers of cells expressing hormone receptors in the brain and on the subsequent control of sexual behavior. This insight provides a better understanding of the relationship between molecular changes in the brain and behavior, and suggests new therapeutic targets to human testosterone treatment. / text

L'atrophie musculaire bulbo-spinale (SBMA) est une dégénérescence lente et progressive des motoneurones causée par l'élongation du triplet nucléotidique (CAG) dans le gène codant pour le récepteur aux androgènes (RA) localisé sur le chromosome X. Dans la SBMA, ce récepteur à extension polyglutaminique (polyQ) pathogène s'accumule de manière ligand dépendante dans le cytoplasme sous forme d'agrégats mais également dans le noyau y créant des corps d'inclusions nucléaires considérés comme la marque identitaire histologique, dont le caractère cytotoxique est aujourd'hui remis en question. Nous avons développé un modèle SBMA in vitro basé sur l'expression inductible d'un RA51Q dans la lignée hybride NSC34, qui est comparé au modèle normal NSC34 exprimant un RA contenant 20Q. Nous avons démontré que l'expression du RA51Q entraîne une diminution de la viabilité ainsi qu'une altération de la croissance neuritique sans formation d'agrégats insolubles dans le noyau ou le cytoplasme des cellules. Le RA en tant que membre de la superfamille des récepteurs nucléaires est un facteur de transcription mais peut également induire des voies de signalisation non génomiques via sa localisation membranaire. Après avoir montré une localisation du RA20Q et du RA51Q dans les « lipid rafts », nous avons corrélé la diminution de la viabilité et de la pousse neuritique induite par le RA51Q à une altération de la signalisation cellulaire non génomique. Les résultats obtenus mettent en évidence une dérégulation des voies de signalisation PI3K/Akt et JNK/c-jun induite par l'expression du RA muté dans notre modèle SBMA. / Spinal Bulbar Muscular Atrophy (SBMA) is a progressive inherited motoneuron disease caused by the expansion of a trinucleotide (CAG) repeat in the gene coding for the androgen receptor (AR) located on the X chromosome. This rare disease causes muscle weaknesses, hypotonia, hyporeflexia, fasciculations of facial muscles in male patients. The androgen-dependent formation of cytoplasmic aggregates and nuclear inclusions are pathological hallmarks of this polyglutamine disease but their potential neurotoxicity is still under debate. We developed a SBMA model based on a doxycycline-inducible AR51Q expression system in the NSC34 hybrid cell line. We have shown that the expression of the mutated AR leads to a reduced viability and to an alteration of neurite outgrowth compared to cells expressing the normal AR20Q. The AR belongs to the nuclear receptor superfamily of transcription factors. However, recent data have put in evidence a membrane localization of AR initiating non-genomic signaling pathways. Because we have not observed insoluble aggregates, reduced viability and neurite outgrowth could not be correlated to AR aggregation. We hypothesized that motoneuron death is not only due to aggregate formation but also to the alteration of AR signaling pathways. We focused on a correlation between the AR localization in lipid rafts and the observed phenotypes. Our results highlight the deregulation of PI3K/Akt and JNK/c-jun signaling pathways induced by the expression of AR51Q in our SBMA model.

Introduction Prostate cancer (PCa) is the most common cancer among men in Sweden. The clinical course varies considerably, which makes it difficult to predict the prognosis in the individual case. In order to explore the early as well as the late course of the disease, large study groups and population-based cohorts are necessary. Aims To explore factors that influence the long-term outcome of men with low-risk tumours in a population-based register, to predict the long-term course, and to assess the mortality rate for men with prostate cancer (Paper I) To analyse long-term outcome and to investigate factors associated with long-term survival in patients with metastases to the skeleton (Paper II) To analyse early androgen deprivation treatment (ADT) failure and to define clinical predictors associated with short survival due to early ADT failure in prostate cancer patients with bone metastases (Paper III) To analyse the prognostic significance of the extent of bone metastases in relation to other pretreatment variables in prostate cancer patients, and to explore the impact of bone metastases on quality-of-life (Paper IV) Material and methods The study groups were assembled from The South East Region Prostate Cancer Register (SERPCR), and The Scandinavian Prostate Cancer Group (SPCG) Trial No. 5. In the first study, prognostic factors and long-term disease-specific mortality rates of low-risk prostate cancer patients from the early PSA era were analysed. In the second study, patient-related factors, quality-of-life (QoL) and long-term survival in 915 PCa patients with bone metastases (M1b) under ADT, were analysed. In Study III factors predicting primary failure to respond to ADT were identified. Study IV explored the impact of the extent of bone metastases on survival and QoL for these men. Result and conclusions The long-term disease-specific mortality of low-risk localised PCa is low, but the annual mortality rate gradually increases. This indicates that some tumours slowly develop into lethal cancer, particularly in men 70 years or older and with a PSA level ≥ 4 μg/L. From the SPCG Trial No. 5, a subgroup of patients with M1b disease and favourable set of predictive factors survived more than 10 years under ADT with an acceptable QoL. Independent predictors of long-term survival were identified as performance status (PS) < 2, limited extent of bone metastases, and a PSA level < 231 μg/L at the time of enrolment in the trial. However, four independent clinical predictors of early ADT failure could be defined. Men exhibiting these features should be considered for an alternative treatment. Patient grouping based on three categories of extent of bone metastases related to PS, haemoglobin, and QoL at presentation, as independent predictors of mortality, may provide improved accuracy of prognosis.

La voie de signalisation des androgènes, à travers le récepteur aux androgènes (AR), joue un rôle important dans le développement et la fonction de la prostate, ainsi que dans l’initiation et la progression du cancer de la prostate. La découverte de nouveaux effecteurs de la signalisation androgènes-AR permettra une meilleure compréhension de ces mécanismes. MiR-135a a été identifié comme un gène cible de la voie de signalisation androgènes-AR. Après stimulation androgénique, AR active directement la transcription du gène miR-135a2, en se fixant sur un élément de réponse aux androgènes dans la région promotrice.Une surexpression de miR-135a inhibe la migration et l’invasion de cellules prostatiques cancéreuses, en régulant négativement l’expression des protéines ROCK1 et ROCK2, deux gènes cibles de miR-135a nouvellement identifiés.De plus, miR-135a cible et régule négativement l’expression du facteur de transcription FOXN3, capable de moduler l’activité transcriptionnelle de AR et la prolifération cellulaire dépendante des androgènes.L’étude fonctionnelle de miR-135a suggère donc qu’il puisse être impliqué dans la progression du cancer de la prostate, en régulant la formation des métastases et la signalisation androgénique. L’expression de miR-135a, dans le tissu tumoral par rapport au tissu sain adjacent, de prostatectomies de patients, est inversement corrélée aux paramètres d’agressivité de la maladie, suggérant qu’il puisse être utilisé comme marqueur de pronostic du cancer de la prostate.Ces résultats font de miR-135a un nouvel effecteur de la voie de signalisation de AR, pouvant contribuer à la progression du cancer de la prostate. / Androgens signaling through the androgen receptor (AR) is critical for normal prostate development and function, as well as prostate cancer initiation and progression. The discovery of new effectors of androgens-AR pathway will allow a better understanding of these mechanisms.MiR-135a has been identified as a target gene in androgen-AR signaling pathway. After androgen stimulation, AR directly activates the transcription of miR-135a2 gene by binding to an androgen response element in the promoter region.Ectopic expression of miR-135a was found to induce morphological modification leading to an inhibition of migration and invasion in prostate cancer cells, by down-regulating ROCK1 and ROCK 2 expression, two newly identified miR-135a target genes.Moreover, miR-135a targets and downregulates the expression of the transcription factor FOXN3, able to modulate AR transcriptional activity and androgen-mediated cell proliferation.Thus, functional study of miR-135a suggests that it could be implicated in prostate cancer progression, by regulating metastases formation and androgen signaling.MiR-135a expression level in surgical cancerous speciments normalized to pair-matched normal counterpart tissues was inversely correlated with aggressivity parameters of the disease, suggesting that it could be used as a candidate prognostic marker in human prostate cancer.These results define miR-135a as a novel effector in androgens-AR signaling, which may contribute to prostate cancer progression.

Le travail présenté a porté sur l’analyse in vivo du proto-oncogène c-cbl, dont la forme connue est c-Cbl (p120cbl). Il s’agit d’une E3-Ubiquitine ligase et un poly-adaptateur moléculaire. Nous avons montré l’androgéno-dépendance de l’expression de c-Cbl dans les cellules germinales testiculaires et les cellules épithéliales de la prostate de rats et de souris. Nous avons montré la régulation anti-apoptotique exercée in vivo par la c-Cbl dans la prostate par comparaison des souris c-cbl invalidées ou non pour c-cbl (KO ou WT). L’effet exercé par c-cbl dans le testicule est pro-apoptotique (J.Cell Biol, 2005), que nous avons ultérieurement attribué à une nouvelle isoforme testiculaire de c-Cbl (Δ-c-Cbl). La comparaison des MEF KO et WT après induction d’apoptose par l’étoposide, a conforté l’effet anti-apoptotique exercée in vivo par c-Cbl dans la prostate. Elle a aussi montré la forte apoptose des MEF KO au peroxyde d’hydrogène : c-Cbl peut être considérée comme un protecteur du stress oxydant. L’intensité du stress oxydant associé aux cancers et leur forte résistance à l’apoptose sont des propriétés qui pourraient être reliées à c-Cbl. L’analyse in situ effectuée à partir de tumeurs congelées et de Tissue Microarrays (TMA) a montré une expression élevée de c-Cbl dans certains cancers, dont l’intensité pourrait correspondre à la gravité de l’atteinte anatomo-pathologique. La protéine c-Cbl est apparue être un marqueur d’agressivité du cancer de la prostate, probablement de l’ovaire, de l’utérus, du cerveau, du poumon, du colon et du rectum. Nous la considérons aussi comme une cible thérapeutique car, protecteur du stress oxydant, elle prendrait part à la résistance à l’apoptose des cellules tumorales. Un brevet a été déposé (2009, co-inventeurs : S.Yakoub et al). Un article rapportant ces résultats est en cours de soumission (S. Yakoub et al) / This work has focused on the in vivo analysis of the proto-oncogene c-cbl, coding for c-Cbl (p120cbl). We demonstrated the androgen-dependency of c-Cbl in the testicular germ cells and the prostatic epithelial cells of rats and mice. We then identified the anti-apoptotic regulation exerted by p120cbl in the prostate, comparing mouse c-cbl KO and WT, unlike this exerted in the testis (J.Cell Biol, 2005). We reported this difference to the high expression in testis of a new c-Cbl isoform, Δc-Cbl. The comparison of MEF KO and WT allowed confirming the anti-apoptotic regulation to etoposide exerted by c-Cbl. A very high apoptotic effect was observed in MEF KO with H2O2: c-Cbl is a strong stress oxidative protector. Knowing the intensity of oxidative stress in several cancers and their particular resistance to apoptosis as well, the in situ analysis of these malignancies was made from frozen tumours and tissue microassays (TMA). c-Cbl was indeed highly expressed and its intensity appears to reflect the aggressiveness of the pathology. c-Cbl could then be considered as a marker of severity of prostate cancer but probably also ovary, uterus, brain, lung, colon and rectum. It can also be considered as a therapeutic target involved in resistance to apoptosis as a stress oxidative protector. A patent was filed in the United States (2009, co-inventors: S. Yakoub et al)

No description available.

Retinoide sind Steroide, Derivate des Vitamin A, die ihre Wirkung durch Interaktion mit Retinoid-Rezeptoren, lokalisiert im Zellkern, entfalten. Diese Retinoid-Rezeptoren, weisen funktionelle und strukturelle Ähnlichkeiten mit dem Androgenrezeptor auf. Untersuchungen an Zellkulturen zeigen, daß der Effekt von Retinoiden auf das Zellwachstum von Prostata-Epithelzellen und Prostatakarzinomzellen keiner einfachen Kinetik folgt, sondern neben der Abhängigkeit von der Retinoid-Dosis, auch von der Zellinie, insbesondere deren Androgenrezeptor abhängt. So zeigten LNCaP-Zellen Unterschiede in der Zellproliferation im Vergleich zu PC3 oder NRP154 (Prostatakarzinom Ratte) und NRP152 (Prostataepithel Ratte). Northern-blots mit Poly(A)RNA von verschiedenen Prostata-Zellinien (benigne und maligne) nach Behandlung mit Retinoic acid (RA) bestätigte dosisabhängige Unterschiede in der Expression der Androgenrezeptor (AR)-mRNA.Umgekehrt verursachte die Behandlung mit Testosteron in verschiedenen Prostata-Zellinien (benigne und maligne) Unterschiede in der Expression der Retinoid-Rezeptor-mRNA für RAR( und RAR(. Die Ergebnisse unterstützen somit die Hypothese einer Interaktion von Retinoiden und Androgenen mit deren respektiven Rezeptoren. Untersuchungen an humanem Prostatagewebe bestätigten Unterschiede in der Expression von RAR(mRNA und RAR(mRNA mittels RT-PCR. Durch immunhistochemische Untersuchungen an humanem Prostatagewebe mit Antikörpern gegen RAR ( und RAR( konnten deren Lokalisation und Expression nachgewiesen werden. Dabei zeigte sich wiederum eine erhöhte Immunreaktivität von RAR( beim Prostatakarzinom, im Gegensatz zu RAR(, das bei benignem Prostataepithelium eine deutlich stärkere Immunreaktivität aufwies. Zusammenfassend belegen unsere Untersuchungen, daß Retinoide einen meist wachstumshemmenden Effekt auf Prostatakarzinomzellinien verursachen, der wahrscheinlich neben Bindung an Retinoid-Rezeptoren (RARs`) durch Interaktion mit dem Androgenrezeptor (AR), vermittelt durch Hemmung verschiedener membrangebundener Zellproteine und Rezeptoren, wie EGF-R verursacht wird. / Retinoids are steroids, derivatives of vitamine A, that excert their activities by interaction with the retinoic acid receptors (RARs') located in the cell nucleus. The RARs possess structural and functional similarities to the androgen receptor (AR). Investigations in cell cultures demonstrated that the effect of retinoic acid (RA) on cell proliferation is dependent not only on the RA dosage, but also on the androgen receptor status of the cell line. LNCaP cells showed a difference in cell proliferation when treated with RA, as opposed to PC3 or NRP154 (rat prostate cancer cell line) and NRP152 (rat prostate epithelial cell line). Northern blots with Poly(A)RNA from different prostate cell lines (benigne and maligne, with different androgen dependency) when treated with different concentrations of RA, demonstrated a dose-dependent expression of the androgen receptor (AR)mRNA. Conversely resulted the treatment with different concentrations of testosterone to different expressions of the RAR-mRNAs. The results, therefore, support the hypothesis of an interaction of retinoids and androgen with their respective receptors. Investigations with human prostate tissue (malignant and benign) confirms differences in RAR-expression byRT-PCR and immunhistochemistry. Again, prostate cancer showed an overexpression of RAR(, whereas benign prostate tissue demonstrated an overexpression of RAR(. Our investigations, in summary, demonstrate the overall inhibitory effect of retinoids with respect to cell proliferation in prostate cancer cell lines. There is evidence , that this biologic effect is not only triggert by interaction of retinoids and androgens with their own receptors, but occurs by cross-interaction of retinoids with the androgen receptor and androgens with the RARs. Furthermore, the biologic effect of retinoids on cell growth is dependent on membrane bound receptors, such as EGF-R.