Inhibition of STEP61 ameliorates deficits in mouse and hiPSC-based schizophrenia models.

The brain-specific tyrosine phosphatase, STEP (STriatal-Enriched protein tyrosine Phosphatase) is an important regulator of synaptic function. STEP normally opposes synaptic strengthening by increasing N-methyl D-aspartate glutamate receptor (NMDAR) internalization through dephosphorylation of GluN2B and inactivation of the kinases extracellular signal-regulated kinase 1/2 and Fyn. Here we show that STEP61 is elevated in the cortex in the Nrg1+/- knockout mouse model of schizophrenia (SZ). Genetic reduction or pharmacological inhibition of STEP prevents the loss of NMDARs from synaptic membranes and reverses behavioral deficits in Nrg1+/- mice. STEP61 protein is also increased in cortical lysates from the central nervous system-specific ErbB2/4 mouse model of SZ, as well as in human induced pluripotent stem cell (hiPSC)-derived forebrain neurons and Ngn2-induced excitatory neurons, from two independent SZ patient cohorts. In these selected SZ models, increased STEP61 protein levels likely reflect reduced ubiquitination and degradation. These convergent findings from mouse and hiPSC SZ models provide evidence for STEP61 dysfunction in SZ.

Measurement of GSTP1 promoter methylation in body fluids may complement PSA screening: a meta-analysis.

BACKGROUND: Prostate-specific antigen (PSA) screening has low specificity. Assessment of methylation status in body fluids may complement PSA screening if the test has high specificity. METHOD: The purpose of this study was to conduct a meta-analysis of the sensitivity and specificity for prostate cancer detection of glutathione-s-transferase-π (GSTP1) methylation in body fluids (plasma, serum, whole blood, urine, ejaculate, and prostatic secretions). We conducted a comprehensive literature search on Medline (Pubmed). We included studies if they met all four of the following criteria: (1) measurement of DNA methylation in body fluids; (2) a case-control or case-only design; (3) publication in an English journal; and (4) adult subjects. Reviewers conducted data extraction independently using a standardised protocol. Twenty-two studies were finally included in this paper. Primer sequences and methylation method in each study were summarised and evaluated using meta-analyses. This paper represents a unique cross-disciplinary approach to molecular epidemiology. RESULTS: The pooled specificity of GSTP1 promoter methylation measured in plasma, serum, and urine samples from negative-biopsy controls was 0.89 (95% CI, 0.80-0.95). Stratified analyses consistently showed a high specificity across different sample types and methylation methods (include both primer sequences and location). The pooled sensitivity was 0.52 (95% CI, 0.40-0.64). CONCLUSIONS: The pooled specificity of GSTP1 promoter methylation measures in plasma, serum, and urine was excellent and much higher than the specificity of PSA. The sensitivity of GSTP1 was modest, no higher than that of PSA. These results suggest that measurement of GSTP1 promoter methylation in plasma, serum, or urine samples may complement PSA screening for prostate cancer diagnosis.

Biochemical alterations in sex hormone-induced hyperplasia and dysplasia of the dorsolateral prostates of Noble rats.

Simultaneous implantation of intact Noble (Nb) rats with testosterone and 17 beta-estradiol (E2)-filled silastic capsules for 16 weeks caused atypical hyperplasia (dysplasia) and striking enlargement exclusively in the dorsolateral prostates (DLPs) of all animals. The dysplastic lesion may be preneoplastic since long-term administration of these steroids to Nb rats is known to induce a high incidence of adenocarcinoma in the DLP. Treatment of rats with nonaromatizable 5 alpha-dihydrotestosterone (DHT) for 16 weeks caused enlargement but not dysplasia, implicating estrogen as a key factor in the genesis of the proliferative lesion. Compared with controls, the testosterone plus E2 treatment caused a 2.5-fold increase in nuclear type II estrogen binding sites which were confined to the DLP. Neither treatment significantly altered androgen content or levels of androgen receptor in the ventral prostate or DLP. Organ cultures of enlarged DLP containing foci of dysplasia metabolized more [3H]DHT than control tissue, which resulted in increased formation of the 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-androstanediol) metabolite by these explants. Because 3 beta-androstanediol has previously been shown to displace [3H]E2 from cytosolic type I estrogen binding sites, the dysplasia may be caused by hyperstimulation of the DLP by the hormones and their normal metabolites produced in abnormal amounts.

Selective increase in type II estrogen-binding sites in the dysplastic dorsolateral prostates of noble rats.

We demonstrated previously that simultaneous treatment of intact Noble (NBL) rats with testosterone and estradiol-17 beta (E2) for 16 weeks consistently induced a putative precancerous lesion, termed dysplasia, in the dorsolateral prostate (DLP) of all animals. Since treatment of rats with androgen alone did not elicit the same response, we concluded that estrogen played a critical role in the genesis of this proliferative lesion. In the present study, using radioligand binding assays, we investigated the properties and distributions of nuclear estrogen-binding sites in the two major prostatic lobes (DLP and ventral prostate) of the rat gland and examined the kinetics of alterations in estrogen-binding site levels following treatment of NBL rats with testosterone plus E2. Saturation analyses revealed two distinct types of nuclear [3H]E2-binding sites in the rat prostate. The high-affinity species or type I sites bound [3H]E2 with high affinity (KD 4-5 nM) and low capacity (0.4-0.6 pmol/mg DNA) and had a ligand specificity similar to that described for the classical estrogen receptor. The second estrogen-binding species or type II sites bound [3H]E2 with moderate affinity (KD 25-30 nM) and higher capacity (2-4 pmol/mg DNA) and had characteristics similar to those of type II estrogen-binding sites found in the rat uterus. Type I sites were found in the nuclei of both ventral prostate and DLP, and their levels in the two prostatic lobes did not change following testosterone plus E2 treatment of NBL rats. In contrast, type II sites were present exclusively in the nuclei of DLP. Treatment of NBL rats with testosterone plus E2 for a period of 16 weeks induced a gradual increase in the levels of DLP nuclear type II sites, which was accompanied by parallel increases in DLP wet weight and total DNA content. Since nuclear type II sites have been implicated as a proliferation regulator, our findings suggest that (a) the lobe-specific localization of type II sites in rat DLP may confer unique estrogenic susceptibility on this tissue and (b) elevation of nuclear type II sites in rat DLP following testosterone plus E2 stimulation may be the underlying cause of enhancement of cell proliferation and dysplasia induction in this prostatic lobe.

Expression of estrogen receptor (ER)-alpha and ER-beta in normal and malignant prostatic epithelial cells: regulation by methylation and involvement in growth regulation.

The aim of the current study is to demonstrate normal and malignant prostatic epithelial cells (PrECs) as targets for receptor-mediated estrogenic and antiestrogenic action. Using an improved protocol, we have successfully isolated and maintained highly enriched populations of normal PrECs from ultrasound-guided peripheral zone biopsies, individually determined to be morphologically normal. Semiquantitative reverse transcription-PCR analyses were used to determine whether transcripts of estrogen receptor (ER)-alpha and those of ER-beta were expressed in our normal PrEC primary cultures, in a commercially available PrEC preparation (PrEC; Clontech), in an immortalized PrEC line established from a benign prostatic hyperplasia specimen (BPH-1), and in three prostatic cancer cell lines (LNCaP, PC-3, and DU145). Expression levels of ER-alpha and ER-beta transcripts were related to those of two estrogen-responsive genes [progesterone receptor (PR) and pS2], at the message levels, to gain insights into the functionality of the ER subtypes in PrECs. Interestingly, only transcripts of ER-beta, but not those of ER-alpha, were found in our primary cultures of normal PrECs, along with both PR and pS2 mRNA. These data strongly suggest that estrogen action was signaled exclusively via ER-beta in normal human PrECs. In contrast, PrEC (Clontech) and BPH-1 cells expressed both ER-alpha and ER-beta transcripts and no PR nor pS2 mRNA in PrEC and only a minimal level of PR mRNA in BPH-1. Among the three prostate cancer cell lines, LNCaP expressed ER-beta mRNA along with transcripts of PR and pS2, DU145 expressed messages of ER-beta and PR, and PC-3 cells exhibited ER-alpha, ER-beta, and pS2 mRNA. Thus, unlike normal PrECs, expression patterns of these genes in malignant PrECs are more variable. Treatment of prostate cancer cells with demethylation agents effectively reactivated the expression of ER-alpha mRNA in LNCaP and DU145 and that of pS2 message in DU145. These findings provide experimental evidence that ER-alpha gene silencing in prostate cancer cells, and perhaps also in normal PrECs, are caused by DNA hypermethylation. To evaluate the potential of using antiestrogens as prostate cancer therapies, we have assessed the growth-inhibitory action of estrogens (estradiol and diethylstilbestrol) and antiestrogens (4-hydroxy-tamoxifen and ICI-182,780) on PC-3 and DU-145 cells. In PC-3 cells, which express both ER subtypes, estrogens as well as antiestrogens are effective inhibitors. In contrast, in DU145 cells, which express only ER-beta, antiestrogens, but not estrogens, are growth inhibitors. By comparison, ICI 182,780 is the more effective cell growth inhibitor. Importantly, the ICI 182,780-induced antiproliferative effects were reversed by cotreatment of DU145 cells with an ER-beta antisense oligonucleotide, hence lending additional support to a central role played by ER-beta in mediating growth-inhibitory action of antiestrogens.

Testosterone-mediated increase in 5 alpha-dihydrotestosterone content, nuclear androgen receptor levels, and cell division in an androgen-independent prostate carcinoma of Noble rats.

An androgen-independent, transplantable prostate carcinoma line (AIT), originally derived from the dorsolateral prostate (DLP) of Noble rat, was implanted into orchiectomized Noble rats and its response to androgen stimulation was studied and compared to that of the regenerating DLP tissue in sexually ablated rats. AIT tumors carried in castrated hosts displayed a high basal level of proliferative activity (mitotic index (MI), 15.0 +/- 0.5) while DLP tissue in untreated castrates exhibited no proliferative activity. Following androgen stimulation by testosterone capsule implantation into host rats, the AIT responded with a marked increase in cell proliferation; MI values doubled to 30.0 +/- 2.9 on Day 5 following androgen stimulation. This androgen-induced increase in MI values was coincident with elevations in nuclear androgen receptor (20-fold increase) and 5 alpha-dihydrotestosterone content (3-fold increase) in the tumor. However, by Day 10 following androgen treatment, indices of cell proliferation in the AIT declined to pre-androgen-stimulated levels (MI, 14.8 +/- 1.9) despite the continued elevations in nuclear androgen receptor and tissue 5 alpha-dihydrotestosterone contents. Parallel changes in MI were also observed in the normal regenerating DLP following androgen stimulation. MI values in this tissue increased from nondetectable levels to 38.1 +/- 4.7 on Day 5 but declined to relatively low levels (4.5 +/- 0.9) by Day 10 following androgen replacement. Taken together these findings led us to conclude that the AIT carried in castrates is capable of responding to testosterone in a manner similar to that observed for androgen-stimulated DLP of sexually ablated rats. Thus, in both the neoplastic and regenerating tissues, the initial response to androgen is characterized by a marked enhancement of cell proliferation which was correlated with an increase in androgen receptor and 5 alpha-dihydrotestosterone content. However, like its tissue of origin, the AIT possesses mechanisms which act to limit androgen-induced cell division despite continued elevations in key parameters of androgen activation.

Expression of gonadotropin receptor and growth responses to key reproductive hormones in normal and malignant human ovarian surface epithelial cells.

Epidemiological data have implicated reproductive hormones as probable risk factors for ovarian cancer (OCa) development. Although pituitary and sex hormones have been reported to regulate OCa cell growth, no information is available regarding whether and how they influence normal ovarian surface epithelial (OSE) cell proliferation. To fill this data gap, this study has compared cell growth responses to gonadotropins and sex steroids in primary cultures of human OSE (HOSE) cells with those observed in immortalized, nontumorigenic HOSE cells and in OCa cell lines. Both malignant and normal cell lines/cultures responded equally well to the stimulatory actions of luteinizing hormone and follicle-stimulating hormone and to 17beta-estradiol and estrone, although the latter estrogen has a much lower affinity for estrogen receptor than does the former estrogen. In normal HOSE cell cultures/lines, 5alpha-dihydrotestosterone was found to be more effective than testosterone in stimulating cell growth, but in OCa cell lines, 5alpha-dihydrotestosterone and testosterone are equally potent. One OCa cell line, OVCA 433, was found to be nonresponsive to androgen stimulation. In general, primary cultures of normal HOSE cells exhibited the greatest hormone-stimulated growth responses (>10-fold enhancement), followed by immortalized HOSE cell lines (4-5-fold enhancement) and by OCa cell lines (2-4-fold enhancement). Interestingly, progesterone (P4), at low concentrations (10(-11) to 10(-10) M), was stimulatory to HOSE and OCa cell growth, but at high doses (10(-8) to 10(-6) M), P4 exerted marked inhibitory effects. In all cases, cotreatment of a cell culture/line with a hormone and its specific antagonist blocked the effect of the hormone, confirming specificity of the hormonal action. Taken together, these data support the hypothesis that reproductive states associated with rising levels of gonadotropins, estrogen, and/or androgen promote cell proliferation in the normal OSE, which favors neoplastic transformation. Conversely, those states attended by high levels of circulating P4, such as that seen during pregnancy, induce OSE cell loss and offer protection against ovarian carcinogenesis.

High affinity binding of [3H]R5020 and [3H]progesterone by putative progesterone receptors in cytosol and nuclear extract of turtle oviduct.

The purpose of this investigation was to identify and characterize putative cytosolic and nuclear forms of progesterone receptor in the female reproductive tract of a turtle, Chrysemys picta. A dextran-coated charcoal adsorption assay and DNA-cellulose affinity chromatography were used as the primary methodologies with [3H]R5020 [3H-labeled 17 alpha-dimethyl-19-norpregna-4,9-diene-3,20-dione) and [3H]progesterone (P4) as the ligands. The receptor was of high affinity (Kd = 4.7 X 10(-10) M for [3H]P4; 2.2 X 10(-10) M for [3H]R5020) and limited capacity (500-6000 fmol/g tissue wet wt). Association was rapid (apparent equilibrium being reached in 30-40 min), as was dissociation (t1/2 = 45 min for [3H]P4 and 180 min for [3H] R5020). The putative receptor demonstrated strict steroid specificity, binding progestins but not estrogens, androgens, or glucocorticoids. Heterogeneity of the cytosolic receptor was demonstrated as two forms eluting off DNA-cellulose columns at 0.2- and 0.3-M salt concentrations. Binding of cytosolic receptor to DNA-cellulose was not increased by preexposure of cytosol to 25 C for 30 min. Some variations in cytosolic, but not nuclear, receptor were associated with different stages of the reproductive cycle and were positively correlated with body weight. Preliminary studies using an explant culture system suggest that the progesterone receptor in turtle oviduct may be maintained by estrogen and translocated from the cytosol to the nucleus by P4. In summary, we have partially characterized a putative P4 receptor in the oviduct of the turtle that is similar to mammalian and avian P4 receptors in specificity, affinity, and other physicochemical properties, supporting the idea that steroid receptor proteins have been highly conserved in vertebrate evolution. However, temperature sensitivity of activation and DNA affinity are different in the turtle and suggest modifications that may be related to physiological adaptation in such a poikilothermic species.

Rat estrogen receptor-alpha and -beta, and progesterone receptor mRNA expression in various prostatic lobes and microdissected normal and dysplastic epithelial tissues of the Noble rats.

Semiquantitative RT-PCR was used to determine if transcripts of the two estrogen receptor (ER) subtypes, ER alpha and ER beta, and the progesterone receptor (PR) are differentially expressed and/or regulated in the various normal lobes of the Noble (NBL) rat prostate. We found that ER beta mRNA was present at comparable, high levels in all three major prostatic lobes: dorsal (DP), lateral (LP) and ventral (VP) prostate. ER alpha mRNA was, however, expressed at low levels among the various lobes in the following descending order of abundance: LP>DP>VP. Expression of PR transcript was low and paralleled the expression pattern of ER alpha mRNA. Treatments of rats with testosterone (T) plus estradiol-17beta (E2) (T+E2) or T alone induced no discernible alterations in ER alpha, ER beta, and PR mRNA levels in the VP, DP and LP, while those with E2 caused a general decline in the expression of all three transcripts. We then studied the expression of the three receptors in the normal and dysplastic epithelium of the dorsolateral prostates (DLPs) of rats treated with T+E2. Comparable levels of ER beta mRNA were found in microdissected dysplastic and normal epithelia. In contrast, significantly higher levels of PR mRNA were present in epithelial samples from dysplastic acini. ER alpha mRNA was not detected in any of the microdissected epithelial samples. Results from this study suggest that upregulation of PR mRNA expression, likely mediated via ER beta action, is involved in the genesis of T+E2-induced dysplasia in this animal model.

Plasma levels of vitellogenin in Chrysemys picta during the annual gonadal cycle: measurement by specific radioimmunoassay.

A RIA for turtle (Chrysemys picta) vitellogenin is described. After dimethylformamide precipitation of vitellogenin from the plasma of estrogen-treated female turtles, antibodies were developed in rabbits. The dimethylformamide precipitate was further purified by o-triethylaminoethyl cellulose column chromatography; the vitellogenin component eluted as a single peak. This material was used for iodination by a mild chloramine method. Antibodies to turtle vitellogenin did not cross-react with plasma from male turtles or vitellogenic females of other vertebrate groups, including lizards and snakes. Limited cross-reactivity exists among the chelonians, however. Using a 1:5000 dilution of antiserum, the limit of detection was 15 ng, and the midrange was 320 +/- 45 ng. For an antiserum dilution of 1:1000, these figures were 30 and 600 +/- 37 ng, respectively. Using this assay, the seasonal pattern of plasma vitellogenin in the turtle has been described, and preliminary studies on in vitro hepatic vitellogenesis have been performed.

A comparative study of hormonal regulation of three secretory proteins (prostatic secretory protein-PSP94, probasin, and seminal vesicle secretion II) in rat lateral prostate.

The rat dorsolateral prostate secretes several major known proteins, although their physiological and reproductive functions are largely undefined. In the present study we examined and compared the in vivo hormonal regulation of the messenger RNA (mRNA) expression of three major secretory proteins, including prostatic secretory protein of 94 amino acids (PSP94 or beta-microseminoprotein), probasin, and seminal vesicle secretion II (SVSII), in long-term castrated lateral prostates (LP) by in situ hybridization and semiquantitative RT-PCR. The protein levels of PSP94 in the castrated LPs were also examined by Western blotting. PSP94 is a small protein newly isolated from the rat prostate gland and demonstrates highly specific expression in the LP. The results of in situ hybridization showed that PSP94, probasin, and SVSII were highly expressed in the intact LP. The hybridization signals of probasin and PSP94 disappeared in the 60-day postcastrated LPs, whereas the signals of SVSII dropped sharply in the 14-day postcastrated LPs. Similar patterns of decreasing mRNA levels of the three proteins in the castrated LPs were observed by RT-PCR analysis. Their mRNA transcripts were restored to normal levels after replacement with testosterone. The results indicate that these secretory proteins are all under androgen regulation in the rat LP. Interestingly, we also observed that their degrees of sensitivity or responsiveness to androgen withdrawal are different. Their mRNA levels dropped in response to duration of castration in the following decreasing order: SVSII, PSP94, and probasin. Besides androgen [dihydrotestosterone (DHT)], we also examined the effects of glucocorticoid [dexamethasone (DEX)], progestin [medroxyprogesterone acetate (MPA)], and zinc on their gene expressions in castrated LPs. We observed that the mRNA transcripts of both PSP94 and probasin were increased after treatments with DHT, DEX, and MPA, suggesting that these two proteins could also be regulated by glucocorticoid and progestin. In contrast with probasin, PSP94 and SVSII were not induced by ZnSO4 treatment. On the other hand, SVSII expression was only increased significantly by DHT and moderately by MPA, but not by DEX, suggesting that SVSII is under strict control by androgen.

Morphological development of afferent segregation and onset of synaptic transmission in the trigeminothalamic pathway of the wallaby (Macropus eugenii).

A light and electron microscopic study has been made of the time of formation of whisker-related patterns in trigeminothalamic afferents and the onset of synapse formation between afferents and cells in the ventroposteromedial nucleus (VPM) of the marsupial mammal, the wallaby, by labelling afferents with a carbocyanine dye. A parallel in vitro study was made of the functional development of the trigeminothalamic pathway to the VPM. Evoked synaptic responses could be recorded in the VPM from the time that afferents first reached the VPM at postnatal day 15 (P15). At all stages, the excitatory response comprised both N-methyl-D-aspartate- and non-N-methyl-D-aspartate-mediated components. At P40, the response decreased markedly in duration, coinciding with the onset of synaptogenesis. This implies that transmission is occurring prior to synapse formation, probably through transmitter release from growth cones. At P50, synaptic responses became dominated by a fast, non-N-methyl-D-aspartate potential, and this coincided with the first appearance of whisker-related patterns in the VPM. A gamma-aminobutyric acid (subtype A)-mediated, inhibitory component was also present from the time of afferent arrival. These findings support the idea that functional interactions between afferents and their targets may play a role in pattern formation in the somatosensory thalamus.

Sex hormone-induced nuclear DNA damage and lipid peroxidation in the dorsolateral prostates of Noble rats.

To date, few studies have been reported on any aspect of DNA damage in organs of intact animals. We and others demonstrated induction of high incidences of dysplasia, a precancerous proliferative lesion, and adenocarcinoma selectively in the dorsolateral prostates (DLPs) of Noble rats exposed to a long-term combined testosterone and estradiol-17 beta treatment (T + E2-treatment). We here report induction of a significant increase in DNA strand breakage and accumulation of lipid peroxidation fluorescent products in the DLPs, but not in the ventral prostates (VPs), of rats treated with T + E2 for 16 weeks. These results indicate that this dual hormone treatment has free radical-based DNA damaging effect on rat DLP and therefore may have tumor-initiating action. Since a similar effect was not observed in regenerating DLPs of castrated rats following testosterone replacement, we conclude that the genotoxic effect of the dual hormone treatment is a direct result of hormone action and not secondarily due to enhancement of cell proliferation.

Nuclear acceptor sites for estrogen-receptor complexes in the liver of the turtle, Chrysemys picta. I. Sexual differences, species specificity and hormonal dependency.

Hepatic estrogen receptors (ERs) of the female turtle, Chrysemys picta, when complexed with [3H]estradiol ([3H]E2), were shown to bind specifically to liver chromatin isolated from the same species. The binding of the [3H]E2 receptor complex to chromatin requires both the steroid ligand and the receptor protein. Maximal binding occurred within 60-70 min of incubation at 4 degrees C in a Tris buffer containing 0.1 M KCl. The binding of the [3H]E2 receptor complex to intact chromatin was saturable, whereas the binding to turtle or calf thymus DNA remained linear. Scatchard analyses revealed more estrogen receptor binding sites on hepatic chromatin isolated from female turtles than that prepared from the males (binding capacities: female chromatin = 67.9 +/- 6.8 fmol/mg DNA equivalent; male chromatin = 28.5 +/- 2.5 fmol/mg DNA equivalent). Furthermore, the [3H]E2 receptor complex was bound with a higher affinity to female chromatin than to male chromatin (association constants: female chromatin = 11.7 +/- 2.7 X 10(10) M-1; male chromatin = 2.5 +/- 0.7 X 10(10) M-1). In contrast to turtle hepatic [3H]E2 receptors, ERs in rat liver or mouse uterine cytosol exhibited little binding affinity for hepatic chromatin isolated from the turtle. Tissue specificity was demonstrated in the interaction of the [3H]E2 receptor complex and chromatin; high affinity, saturable binding of the [3H]E2 receptor complex was only observed on chromatin isolated from the liver but not on those prepared from the heart, kidney and muscle. A 3- to 4-fold increase in the number of hepatic chromatin [3H]E2 receptor binding sites was observed in 21-day ovariectomized or hypophysectomized female (capacities = 209.3 +/- 6.1 and 270 +/- 10.1 fmol/mg DNA equivalent, respectively). It is postulated that [3H]E2 receptor binding sites on the chromatin of intact females are partially 'masked', and removal of a gonadal and/or pituitary factor(s) unveils additional binding sites on the female chromatin. This paper is first to report the presence of high affinity, species- and tissue-specific acceptor sites on the liver chromatin of a reptilian species. The fact that the levels and properties of these acceptor sites are dependent on the sex and hormonal state of the animal suggests that they may play a role in the regulation of hepatic estrogen responsiveness and vitellogenesis in this species.

Induction of progesterone receptor by androgens in the mouse uterus.

The comparative abilities of 3 naturally occurring androgens to compete for [3H]estradiol-17 beta ([3H]E2)-binding sites in uterine cytosol and to induce uterotrophic responses in immature female mice have been investigated. 5 alpha-Androstane-3 beta,17 beta-diol (3 beta-diol), 5 alpha-androstane-3 alpha,17 beta-diol (3 alpha-diol) and 5-androstene-3 beta,17 beta-diol (delta 5-diol) are all effective at diminishing cytoplasmic [3H]E2 binding. Their relative effectiveness are in the order of 3 beta-diol greater than delta 5-diol = 3 alpha-diol. When these steroids were injected intramuscularly (two 100-200 micrograms injections) into immature female mice (21 days old), they induced similar elevations of uterine dry weight, water content, cytosolic estrogen (ER) and progesterone (PR) receptor contents, and nuclear ER content as did estradiol-17 beta (E2). 3 beta-Diol was the most effective steroid at inducing cytosolic and nuclear ER, and PR production in mouse uterus. Followed in effectiveness was delta 5-diol and 3 alpha-diol. Unexpectedly, 3 beta-diol was found to be more potent than E2 in stimulating uterine ER and PR increases. Nonetheless, the androgens are poorer stimulators of uterine dry weight increase and water retention than E2 is. These results indicate that the androgens may interact with the ER system in the uterus to bring about the uterotrophic responses and 3 beta-diol is a more estrogenic steroid than delta 5-diol and 3 alpha-diol in the mouse uterus.

Species, interindividual, and tissue specificity in endocrine signaling.

The activity of endocrine-active agents exhibits specificity at many levels. Differential responsiveness to these agents has been observed between different species and extends to interindividual differences within a species and between different tissues as well. In cases where they have been identified, the biologic and molecular mechanisms underlying this specificity are quite diverse. Determinants of species specificity include differences that exist in receptor binding, gene transcription, and cellular responses to endocrine-active compounds between species. Interindividual differences in responsiveness may be determined at the level of genetic polymorphisms in hormone-metabolizing enzymes, hormone receptors, and in those genes that are transactivated by these receptors, as well as during changing windows of susceptibility that occur as a function of age, such as prenatal and postmenopausal exposures. Extrinsic factors such as diet can also impact individual susceptibility to endocrine-active agents. Tissue-specific determinants of susceptibility are well documented, but little is known regarding the mechanisms underlying these different responses. Differences in the expression of accessory proteins for steroid hormone receptors and different patterns of receptor expression, estrogen receptor alpha and estrogen receptor beta; for example, may contribute to tissue specificity, as may differences in the pattern of expression of other genes such as hormone-metabolizing enzymes. The use of animal model systems and development of appropriate mathematical models has the potential to yield additional valuable information for elucidating the role of these determinants of specificity at low-dose exposures and for improved risk assessments for the adverse health effects of endocrine-active compounds.

Increased expression of estrogen receptor beta in chemically transformed human breast epithelial cells.

Recent molecular cloning of estrogen receptor beta (ERbeta) suggests alternative pathways of estrogen signaling, but little is known concerning the role of ERbeta in the development of human breast cancer. In the present study, expression of ERalpha and ERbeta mRNA was determined in a series of chemically transformed human breast epithelial cells as well as various normal and malignant breast cancer cell lines. We observed a very low level of ERbeta expression in the mortal S130 and the spontaneously immortalized MCF10-F human breast epithelial cell lines. As MCF-10F cells were treated with environmental chemical carcinogens, an elevated level of ERbeta expression was observed in the resultant transformed BP1, D3 and BP1-ras cells. An even higher level of ERbeta expression was detected in the more transformed BP1-E, D3-1 and D3-1-ras cell lines. Therefore, results from our study indicate that expression of ERbeta can be induced in chemical carcinogen-transformed human breast epithelial cells, and the more transformed cells showed higher levels of ERbeta expression, regardless of which chemical carcinogens were initially used for cell transformation. These results suggest that expression of ERbeta may contribute to the initiation and progression of chemical carcinogen-induced neoplastic transformation.

Prostatic androgen receptor and plasma testosterone levels in streptozotocin-induced diabetic rats.

Diabetes was induced in male Sprague-Dawley (S-D) rats by streptozotocin (STZ) administration. Following STZ injection, plasma glucose levels in the treated rats were significantly elevated from values of untreated controls. Over the experimental period (140 days) plasma testosterone (T) levels, prostatic nuclear androgen receptor (AR) contents and prostatic weights declined with increasing age in the rats. The declines in both STZ-treated and untreated rats were similar in manner and no notable differences were discerned in the data obtained from the two groups. On the contrary, prostatic cytosolic AR contents in untreated rats remained unchanged with advancing age, but was reduced to 50% of normal control values in diabetic rats following STZ treatment. Correlation analyses revealed that prostatic nuclear AR contents correlated positively with plasma T levels while prostatic cytosolic AR contents correlated negatively with plasma glucose levels. These data support former claims that prostatic nuclear AR content is dependent on circulating T level and suggest a possible link between prostatic cytosolic AR content and plasma glucose concentrations.

Hormonal regulation of nuclear type II estrogen binding sites in the dorsolateral prostate of noble rats.

We previously demonstrated that simultaneous treatment of Noble (NBL) rats with estradiol (E2) and testosterone (T) for 16 weeks induces a proliferative response selectively in the dorsolateral prostates (DLP) of all treated animals [1, 2]. The unique sensitivity of rat DLP to the conjoint androgen-estrogen-induced mitogenic action may be attributable to the presence of a moderate affinity, high capacity, nuclear estrogen binder (type II sites) found exclusively in this prostatic lobe [2, 3]. Little is known about whether prostatic type II site levels are under hormonal regulation. The aim of this study is to determine whether testicular steroids play a role in regulating the basal and/or induced levels of type II site expression in rat DLP. In the first experiment, rats were castrated and immediately treated with 5 alpha-dihydrotestosterone (DHT) and/or E2 for 6 weeks to determine whether these steroids, separately or jointly, could sustain DLP type II site levels in castrates. Treatments of castrated rats with DHT and DHT+E2 were found to be effective in maintaining DLP type II site levels and gland wet weights at values close to those found in intact untreated controls, while treatments with E2 failed to maintain these parameters at levels observed in intact animals. In the second experiment, intact rats were treated with an androgen (T or DHT) or E2, alone or in combination, for 16 weeks to ascertain which hormonal regimen could induce a higher level of type II site expression in the DLP. Treatments of rats with an androgen (T or DHT) or E2 alone did not change DLP type II site levels even though T treatment caused a slight increase in gland weight, while E2 treatment induced prostatic atrophy. Contrary to single hormone treatments, combined T + E2 and DHT+E2 treatments were effective in inducing a doubling of type II sites and increases in wet weight of the DLPs. These data indicate that testicular androgen is the primary factor responsible for maintaining a basal level of type II site expression in rat DLP, while conjoint androgenic-estrogenic action is needed for the induction of a higher level of type II site expression in the tissue.

In vitro and in vivo inhibition of nuclear type II estrogen binding sites in the dorsolateral prostate of noble rats.

Competition analyses with a number of known bioflavonoids and related compounds revealed that three of them competed effectively for type II [3H]estradiol- 17 beta ([3H]E2) binding sites (type II sites) in the nuclei of rat dorsolateral prostate (DLP). Amongst the bioflavonoids tested, quercetin was the most effect, exhibiting approx. a 50% inhibition at 3000-fold molar excess concentration. In contrast, rutin and hesperitin were both not effective. Methyl p-hydroxyphenyllactate (MeHPLA), a suspected "endogenous" ligand for uterine type II sites [1; J. Biol. Chem. 263, 1988, 7203-7210], competed as well as estradiol-17 beta (E2) for prostatic type II sites (50% inhibition at 30-fold molar excess), whereas its demethylated product, HPLA, did not. 4,4'Dihydroxybenzylidene acetophenone, an esterase-stable MeHPLA analog, was also found to be a good competitor, exhibiting a 50% inhibition at 100-fold molar excess concentration. In a preliminary in vivo study, quercetin, administered either orally or subcutaneously, was found to be effective in preventing a joint testosterone (T) and E2 treatment-induced elevation of type II sites in rat DLP. Quercetin treatments also caused a small but significant reduction (17-18%) in DLP relative gland weights (gland wt/body wt) in T + E2-treated animals. Taken together, these data suggest that bioflavonoids and related compounds may influence prostatic function via interactions with prostatic type II sites.