Elevated levels of FOXA1 facilitate androgen receptor chromatin binding resulting in a CRPC-like phenotype.

Castration-resistant prostate cancer (CRPC) continues to pose a significant clinical challenge with new generation second-line hormonal therapies affording limited improvement in disease outcome. As the androgen receptor (AR) remains a critical driver in CRPC, understanding the determinants of its transcriptional activity is important for developing new AR-targeted therapies. FOXA1 is a key component of the AR transcriptional complex yet its role in prostate cancer progression and the relationship between AR and FOXA1 are not completely resolved. It is well established that FOXA1 levels are elevated in advanced prostate cancer and metastases. We mimicked these conditions by overexpressing FOXA1 in the androgen-responsive LNCaP prostate cancer cell line and observed a significant increase in AR genomic binding at novel regions that possess increased chromatin accessibility. High levels of FOXA1 resulted in increased proliferation at both sub-optimal and high 5α-dihydrotestosterone (DHT) concentrations. Immunohistochemical staining for FOXA1 in a clinical prostate cancer cohort revealed that high FOXA1 expression is associated with shorter time to biochemical recurrence after radical prostatectomy (hazard ratio (HR) 5.0, 95% confidence interval (CI) 1.2-21.1, P=0.028), positive surgical margins and higher stage disease at diagnosis. The gene expression program that results from FOXA1 overexpression is enriched for PTEN, Wnt and other pathways typically represented in CRPC gene signatures. Together, these results suggest that in an androgen-depleted state, elevated levels of FOXA1 enhance AR binding at genomic regions not normally occupied by AR, which in turn facilitates prostate cancer cell growth.

Minireview: The androgen receptor in breast tissues: growth inhibitor, tumor suppressor, oncogene?

Androgen receptor (AR) signaling exerts an antiestrogenic, growth-inhibitory influence in normal breast tissue, and this role may be sustained in estrogen receptor α (ERα)-positive luminal breast cancers. Conversely, AR signaling may promote growth of a subset of ERα-negative, AR-positive breast cancers with a molecular apocrine phenotype. Understanding the molecular mechanisms whereby androgens can elicit distinct gene expression programs and opposing proliferative responses in these two breast cancer phenotypes is critical to the development of new therapeutic strategies to target the AR in breast cancer.

Epigenetic modification of the X chromosome influences susceptibility to polycystic ovary syndrome.

CONTEXT: The cause of polycystic ovary syndrome (PCOS) is unknown, although genetic and environmental influences are clearly implicated. Some genetic studies have suggested the involvement of X-linked genes in PCOS, but the influence of X chromosome inactivation (XCI) on manifestation of this disorder has not previously been examined. OBJECTIVE: The objective of the study was to test the null hypothesis that XCI has no influence on clinical presentation of PCOS. DESIGN: We examined patterns of XCI between sister pairs with the same genotype at a polymorphic locus on the X chromosome in families with PCOS. SETTING: The study was conducted at a private practice. PARTICIPANTS: PCOS was defined as hyperandrogenemia with chronic anovulation. Forty families were studied in which DNA was obtained from at least one parent, the proband, and one sister that could be accurately diagnosed as being affected or unaffected. MAIN OUTCOME MEASURE(S): Relative expression of two X-linked alleles was determined, and the ratio of one to the other represented the pattern of XCI. RESULTS: The statistical odds on a different clinical presentation between sisters was approximately 29 times higher in sister pairs with different patterns of XCI, compared with sister pairs with the same pattern of XCI (odds ratio 28.9; 95% confidence interval 4.0-206; P = 0.0008). CONCLUSIONS: This study provides evidence to refute the null hypothesis and propose a closer inspection of X-linked genes in PCOS, one in which both genotype and epigenotype are considered. Environmental determinants of PCOS may alter clinical presentation via epigenetic modifications, which currently remain undetected in traditional genetic analyses.

Androgens augment the mitogenic effects of oocyte-secreted factors and growth differentiation factor 9 on porcine granulosa cells.

In this study, we test the hypothesis that the growth-promoting action of androgens on granulosa cells requires paracrine signaling from the oocyte. Mural granulosa cells (MGCs) from small antral (1-3 mm) prepubertal pig follicles were cultured in the presence or absence of denuded oocytes (DO) from the same follicles to determine whether mitogenic and/or steroidogenic responses, to combinations of FSH, insulin-like growth factor 1 (IGF1), and dihydrotestosterone (DHT) were influenced by oocyte-secreted factors (OSFs). To further explore the identity of such factors we performed the same experiments, substituting growth differentiation factor 9 (GDF9), a known OSF, for the DO. OSFs and GDF9 both potently enhanced IGF1-stimulated proliferation, and inhibited FSH-stimulated progesterone secretion. Alone, DHT had little effect on DNA synthesis, but significantly enhanced the mitogenic effects of OSFs or GDF9 in the presence of IGF1. Denuded oocytes, GDF9, and DHT independently inhibited FSH-stimulated progesterone secretion, and androgen, together with DO or GDF9, caused the most potent steroidogenic inhibition. Focusing on mitogenic effects, we demonstrate that both natural androgen receptor (AR) agonists, testosterone and DHT, dose-dependently augmented the mitogenic activity of DO or GDF9. Antiandrogen (hydroxyflutamide) treatment, which is used to block androgen receptor activity, opposed the interaction between androgen and GDF9. In conclusion, androgens stimulate porcine MGC proliferation in vitro by potentiating the growth-promoting effects of oocytes or GDF9, via a mechanism that involves the AR. These signaling pathways are likely to be important regulators of folliculogenesis in vivo, and may contribute to the excess follicle growth that is observed in androgen-treated female animals.

Interactions between androgen and growth factors in granulosa cell subtypes of porcine antral follicles.

Androgens acting via the androgen receptor (AR) have been implicated in regulation of folliculogenesis in many animal species. These effects are possibly mediated via enhancement of FSH and/or insulin-like growth factor (IGF)-I activity in granulosa cells, which contain high levels of AR protein. We examined the in vitro effect of dihydrotestosterone (DHT) on DNA synthesis and progesterone secretion by follicular cells in response to FSH and IGF-I, alone or in combination. Cells from separate pools of 1- to 3-mm and 3- to 5-mm antral follicles were aspirated from gilt ovaries and fractioned into mural granulosa cells (MGCs) and cumulus-oocyte complexes (COCs) for subsequent cell culture. Androgen alone or with any combination of mitogen had minimal effect on proliferative and no effect on steroidogenic responses of MGCs from 3- to 5-mm antral follicles. Conversely, in MGCs from 1- to 3-mm follicles, DHT significantly enhanced IFG-I-stimulated proliferation and had variable influence on progesterone secretion. The effects of DHT on proliferative responses of COCs were also dependent on follicle size: DHT significantly augmented either IGF-I-stimulated proliferation (1- to 3-mm follicles) or FSH-stimulated proliferation (3- to 5-mm follicles). However, the steroidogenic responses of all COCs were identical, whereby DHT significantly suppressed progesterone secretion, predominantly in the presence of FSH. Addition of an AR antagonist, hydroxyflutamide, generally reversed the proliferative responses invoked by DHT but not the steroidogenic responses. We conclude that androgen-receptor-mediated activity in granulosa cells of antral follicles is dependent on follicle size, is influenced by proximity of cells to the oocyte, and possibly involves both classic and nonclassic steroid mechanisms.

Campylobacter jejuni cytolethal distending toxin mediates release of interleukin-8 from intestinal epithelial cells.

Live cells of Campylobacter jejuni and Campylobacter coli can induce release of interleukin-8 (IL-8) from INT407 cells. Additionally, membrane fractions of C. jejuni 81-176, but not membrane fractions of C. coli strains, can also induce release of IL-8. Membrane preparations from 81-176 mutants defective in any of the three membrane-associated protein subunits of cytolethal distending toxin (CDT) were unable to induce IL-8. The presence of the three cdt genes on a shuttle plasmid in trans restored both CDT activity and the ability to release IL-8 to membrane fractions. However, CDT mutations did not affect the ability of 81-176 to induce IL-8 during adherence to or invasion of INT407 cells. When C. jejuni cdt genes were transferred on a shuttle plasmid into a C. coli strain lacking CDT, membrane preparations became positive in both CDT and IL-8 assays. Growth of C. jejuni in physiological levels of sodium deoxycholate released all three CDT proteins, as well as CDT activity and IL-8 activity, from membranes into supernatants. Antibodies against recombinant forms of each of the three CDT subunit proteins neutralized both CDT activity and the activity responsible for IL-8 release. The data suggest that C. jejuni can induce IL-8 release from INT407 cells by two independent mechanisms, one of which requires adherence and/or invasion and the second of which requires CDT.

Sialylation of lipooligosaccharide cores affects immunogenicity and serum resistance of Campylobacter jejuni.

Three genes involved in biosynthesis of the lipooligosaccharide (LOS) core of Campylobacter jejuni MSC57360, the type strain of the HS:1 serotype, whose structure mimics GM(2) ganglioside, have been cloned and characterized. Mutation of genes encoding proteins with homology to a sialyl transferase (cstII) and a putative N-acetylmannosamine synthetase (neuC1), part of the biosynthetic pathway of N-acetylneuraminic acid (NeuNAc), have identical phenotypes. The LOS cores of these mutants display identical changes in electrophoretic mobility, loss of reactivity with cholera toxin (CT), and enhanced immunoreactivity with a hyperimmune polyclonal antiserum generated against whole cells of C. jejuni MSC57360. Loss of sialic acid in the core of the neuC1 mutant was confirmed by fast atom bombardment mass spectrometry. Mutation of a gene encoding a putative beta-1,4-N-acetylgalactosaminyltransferase (Cgt) resulted in LOS cores intermediate in electrophoretic mobility between that of wild type and the mutants lacking NeuNAc, loss of reactivity with CT, and a reduced immunoreactivity with hyperimmune antiserum. Chemical analyses confirmed the loss of N-acetylgalactosamine (GalNAc) and the presence of NeuNAc in the cgt mutant. These data suggest that the Cgt enzyme is capable of transferring GalNAc to an acceptor with or without NeuNAc and that the Cst enzyme is capable of transferring NeuNAc to an acceptor with or without GalNAc. A mutant with a nonsialylated LOS core is more sensitive to the bactericidal effects of human sera than the wild type or the mutant lacking GalNAc.

Campylobacter jejuni-stimulated secretion of interleukin-8 by INT407 cells.

Incubation of INT407 cells with various clinical isolates of Campylobacter jejuni resulted in secretion of interleukin-8 (IL-8) at levels ranging from 96 to 554 pg/ml at 24 h. The strains which produced the highest levels of IL-8 secretion were 81-176 and BT44. Induction of IL-8 secretion required live cells of 81-176 and was dependent on de novo protein synthesis. Site-specific mutants of 81-176, which were previously shown to be defective in adherence and invasion, resulted in reduced levels of secretion of IL-8, and cheY mutants of strains 81-176 and 749, which are hyperadherent and hyperinvasive, resulted in higher levels of IL-8 secretion. Another mutant of 81-176, which adheres at about 43% of the wild-type levels but is noninvasive, also showed marked reduction in IL-8 levels, suggesting that invasion is necessary for high levels of IL-8 secretion. When gentamicin was added to INT407 cells at 2 h after infection with 81-176, IL-8 secretion 22 h later was equivalent to that of controls without gentamicin, suggesting that the events which trigger induction and release of IL-8 occur early in the interactions of bacteria and eukaryotic cells.

Measurement of CD69 induction in the assessment of immune function in asymptomatic HIV-infected individuals.

Peripheral blood mononuclear cells from many asymptomatic HIV-infected patients exhibit defects in cytokine production and impaired proliferative responses in vitro but the mechanisms underlying this subclinical immune deficiency are controversial. To determine whether abnormalities in the earliest events following receptor engagement may help to explain the in vitro immune dysfunction, we measured the inducibility of the early activation marker CD69 in T cells from asymptomatic HIV-infected individuals in response to stimulation with anti-CD2 or anti-CD3 mAb. In a whole blood assay, we found that induction of CD69 was markedly impaired in CD4+ T cells from later-stage HIV-infected patients (CD4 counts 200-400/mm3) compared to uninfected controls. Among early stage patients (CD4 > 400/mm3), a subset (29%) had impaired CD69 induction. CD69 responses were equally depressed after stimulation through the CD3 or CD2 receptor pathways. Survey of a panel of immunophenotypic markers and propensity for apoptosis demonstrated a significant association between depressed induction of CD69 and decreased percentages of CD4+CD26+ and CD4+CD95+ cells but no association with the level of apoptosis. These data indicate that defects in T lymphocyte activation through CD3 and CD2 can be measured within hours of receptor stimulation in asymptomatic HIV-infected individuals and might be useful to monitor as an indicator of immune function in these patients.

Intact antigen receptor-mediated calcium signals in patients with early stage HIV-1 infection.

A variety of deficiencies in T cell activation have been described in HIV-1 infection. To determine whether one component of Ag receptor signal transduction might be impaired and contribute to the immunopathology of HIV infection, we tested CD4 cells from patients with early to mid-stage HIV infection for TCR-induced calcium mobilization. There was no detectable difference between patients and controls in the mean CD4 cell calcium response or in the fraction of responding CD4 cells after cross-linking the TCR with OKT3 Ab. In addition, in HIV-infected patients, there was no correlation between calcium mobilization and the CD4 cell count. These results indicate that there are no intrinsic impairments of Ag receptor calcium signaling in circulating CD4 cells from HIV-infected patients with more than 400 CD4 cells/mm3, although abnormalities in patients with later stage infections cannot be excluded.

Mutations in the androgen receptor gene are associated with progression of human prostate cancer to androgen independence.

Progression to androgen-independent growth of human prostate cancers may be mediated by alterations in the structure and/or expression of the androgen receptor (AR) gene. To date, mutations in the AR gene have largely been identified in hormone refractory tumors. In this study, single-strand conformational polymorphism analysis and DNA sequencing of the entire AR gene coding region was performed on 25 primary prostate tumors sampled prior to initiation of hormonal (i.e. , androgen ablation) therapy. Base changes leading to amino acid substitutions in the AR were identified in 11 (44%) tumors. The presence of AR amino acid substitutions was associated with decreased immunohistochemical staining for AR in tumor cells and the rapid failure of subsequent hormonal therapies. Single-strand conformational polymorphism analysis of exons 2, 3, and 8 of the X-linked hypoxanthine guanine phosphoribosyl transferase (HPRT) gene in the same samples revealed no bandshifts, suggesting that the high frequency of AR gene mutations detected was not a consequence of generalized genetic instability. These data indicate that AR gene mutations occur commonly in advanced prostate cancers prior to endocrine treatment of disease and may contribute to altered androgen responsiveness of the tumors.

Androgens induce divergent proliferative responses in human breast cancer cell lines.

Although the majority of primary human breast cancers express the androgen receptor (AR), the role of androgens in breast cancer growth and progression is poorly understood. We have investigated the effects of the naturally occurring androgen, dihydrotestosterone (DHT), and a synthetic non-metabolizable androgen, mibolerone, on the proliferation of six human breast cancer cell lines. The anti-proliferative and proliferative effects of androgens were only observed in cell lines that expressed the AR. Two of the AR-positive cell lines, T47-D and ZR-75-1 were growth inhibited in the presence of either DHT or mibolerone, while the proliferation of MCF-7 and MDA-MB-453 cells was increased by both androgens. Co-incubation of cultures with 1 nM DHT and a 100-fold excess of the androgen receptor antagonist, hydroxyflutamide, resulted in reversal of both inhibitory and stimulatory effects of DHT on T47-D, MCF-7 and MDA-MB-453 cell proliferation, indicating that DHT action is mediated by the AR in these lines. Hydroxyflutamide only partially reversed the DHT-induced growth inhibition of ZR-75-1 cultures, which suggests that growth inhibition of these cells may be mediated by non-AR pathways of DHT (or DHT metabolite) action. Mibolerone action on breast cancer cell growth was similar to that of DHT, with the exception that growth stimulation of MCF-7 and MDA-MB-453 cells was only partially reversed in the presence of a 100-fold excess of hydroxyflutamide. Anandron, another androgen receptor antagonist, was able to reverse all inhibitory and stimulatory actions of the androgens. AR antisense oligonucleotides reduced the level of immunoreactive AR expression in MDA-MB-453 and ZR-75-1 cells by more than 60%, but only reversed the growth inhibitory action of mibolerone in ZR-75-1 cultures. The results suggest that androgen action in breast cancer cell lines may not be solely mediated by binding of androgen to the AR. For example, metabolites of DHT with oestrogenic activity, or androgen binding to receptors other than the AR, may explain the divergent responses to androgens observed in different breast cancer cell lines.

Demodectic mange in a tamarin (Saguinus geoffroyi).

A young female tamarin (Saguinus geoffroyi) developed erythema, alopecia, and papule formation on the skin of the limbs, tail, and head. Examination of skin scrapings and a biopsy specimen revealed mites identified as Demodex sp. Histologically the mites were found in hair follicles, and they were associated with focal degenerative and inflammatory changes in the skin. Successful treatment included topical application of 1% ronnel solution and rotenone ointment.

Housing mice in a caging system with automatic flushing.

Male Har:(ICR)BR mice were housed 8 wk in wire bottom cages over paper, in wire bottom cages over an automatic cascade flushing system, or in solid-bottom plastic cages with bedding material. There were no substantial differences in general health or weight gain. Pentobarbital LD50 values were lower for the mice housed in wire bottom cages than for the mice in solid bottom cages with bedding. This difference was probably related to gastrointestinal content. It appears that the automatic cascade flushing system is suitable for housing mice for periods up to 8 wk.

Specific hypotensive and antihypertensive ocular effects of d-isoproterenol in rabbits.

d-Isoproterenol (ISO) applied topically to the rabbit eye specifically lowered intraocular pressure. Thus, it effectively reduced normal intraocular pressure and inhibited intraocular pressure elevation induced by water load without causing other obvious local or systemic pharmacologic effects. By comparison, dl-ISO was pharmacologically nonspecific in that amounts required to reduce intraocular pressure also produced significant and marked tachycardia. Furthermore, maximal intraocular pressure reduction was less with dl-than with d-ISO. Accordingly, and in consideration of reported clinical experience with dl-ISO in glaucomatous man, the d-isomer should be the preferred form of ISO for treating glaucoma. d-ISO should offer advantages over any other topical medication used currently in the treatment of this condition. Toxicity studies employing large, topical doses of drug in rabbit eyes showed that d-ISO was free from ocular irritation as well as systemic toxicologic effects and should be safe for controlled studies in man.