Development of an androgen receptor-null model for identifying the initiation site for androgen stimulation of proliferation and suppression of programmed (apoptotic) death of PC-82 human prostate cancer cells

Whether androgen regulates the proliferation and survival of androgen-responsive prostate cancer cells directly or indirectly via a paracrine pathway initiated in androgen receptor (AR)-expressing stromal cells is unknown. To resolve this issue, female mice heterozygous for the testicular feminized male loss of function mutation in their X-linked AR genes were cross-bred to T cell-defective homozygous male nude mice. Using a PCR-based restriction enzyme digestion method, the resulting AR/tfm, Nu/nu F1 hybrid females were identified and back-crossed to homozygous male nude mice to produce AR-null male nude mice lacking both AR and T-cell function. Androgen-responsive PC-82 human prostate cancers were xenografted into these AR-null versus AR-wild-type male nude mice. In both backgrounds, the cancer cells did not grow in nonandrogenized hosts. In contrast, PC-82 prostate cancer cells grew with identical characteristics (i.e., take rate, morphology, PSA expression, growth rate, and percentage of cell proliferating or dying) in androgenized hosts of both backgrounds. Likewise, in both backgrounds, androgen ablation of mice bearing growing PC-82 cancers resulted in the inhibition of proliferation and activation of programmed (apoptotic) cell death of the cancer cells. These results demonstrate that both the androgen-stimulated proliferation and the suppression of programmed cell death of PC-82 human prostate cancer cells are initiated by the AR pathway directly within these cancer cells themselves and do not involve initiation by AR-expressing stromal cells in a paracrine manner.     

Different functions for the thyroid hormone receptors TRalpha and TRbeta in the control of thyroid hormone production and post-natal development

Par-4 is a widely expressed protein that sensitizes both prostatic and non-prostatic cells to apoptosis. Constitutive- or regulated- overexpression of Par-4 caused a reduction in the levels of the anti-apoptotic protein Bcl-2. Replenishment of Bcl-2 levels abrogated susceptibility to Par-4-dependent apoptosis, suggesting that Par-4-mediated apoptosis requires downmodulation of Bcl-2 levels. The inverse correlation between Par-4 and Bcl-2 expression was recapitulated in human prostate tumors. Par-4 but not Bcl-2 was detected in the secretory epithelium of benign prostatic tumors and in primary and metastatic prostate cancers that are apt to undergo apoptosis. Moreover, xenografts of human, androgen-dependent CWR22 tumors showed Par-4 but not Bcl-2 expression. By contrast, androgen-independent CWR22R tumors derived from the CWR22 xenografts showed mutually exclusive expression patterns of Par-4 and Bcl-2. These findings suggest a mechanism by which Par-4 may sensitize prostate tumor cells to apoptosis.     

Differential inhibition by hyperammonemia of the electron transport chain enzymes in synaptosomes and non-synaptic mitochondria in ornithine transcarbamylase-deficient spf-mice: restoration by acetyl-L-carnitine

These studies were undertaken to assess the relative expression and autocrine activation of the epidermal growth factor receptor (EGFR) in normal and transformed prostatic epithelial cells and to determine whether EGFR activation plays a functional role in androgen-stimulated growth of prostate cancer cells in vitro. EGFR expression was determined by Western blot analysis and ELISA immunoassays. Immunoprecipitation of radiophosphorylated EGFR and evaluation of tyrosine phosphorylation was used to assess EGFR activation. The human androgen-independent prostate cancer cell lines PC3 and DU145 exhibited higher levels of EGFR expression and autocrine phosphorylation than normal human prostatic epithelial cells or the human androgen-responsive prostate cancer cell line LNCaP. PC3 and DU145 cells also showed higher levels of autonomous growth under serum-free defined conditions. Normal prostatic epithelial cells expressed EGFR but did not exhibit detectable levels of EGFR phosphorylation when cultured in the absence of exogenous EGF. Addition of EGF stimulated EGFR phosphorylation and induced proliferation of normal cells. LNCaP cells exhibited autocrine phosphorylation of EGFR but did not undergo significant proliferation when cultured in the absence of exogenous growth factors. A biphasic growth curve was observed when LNCaP cells were cultured with dihydrotestosterone (DHT). Maximum proliferation occurred at 1 nM DHT with regression of the growth response at DHT concentrations greater than 1 nM. However, neither EGFR expression nor phosphorylation was altered in LNCaP cells after androgen stimulation. In addition, DHT-stimulated growth of LNCaP cells was not inhibited by anti-EGFR. These studies show that autocrine activation of EGFR is a common feature of prostatic carcinoma cells in contrast to normal epithelial cells. However, EGFR activation does not appear to play a functional role in androgen-stimulated growth of LNCaP cells in vitro.     

MDA-MB-453, an androgen-responsive human breast carcinoma cell line with high level androgen receptor expression

The role of androgens and the androgen receptor (AR) in the development and progression of breast cancer is poorly understood. To further define a potential model for androgen action in breast cancer, MDA-MB-453 cells, which express AR in the absence of oestrogen receptors and progesterone receptors, were further characterised in terms of AR expression and androgen responsiveness. High level expression of AR was confirmed by northern blot analysis, radioligand binding and immunocytochemistry, and could not be accounted for by AR gene amplification. Three endogenous androgen-responsive genes (fatty acid synthetase, gross cystic disease fluid protein of 15 kDa and prolactin receptor) and a transfected reporter gene, containing an androgen-responsive element, were induced following androgen administration. A synthetic androgen, mibolerone, induced moderate (27% above control) stimulation of MDA-MB-453 cell proliferation, which was abrogated by the simultaneous administration of the synthetic androgen antagonist, anandron, demonstrating that the effect was AR-mediated. In summary, MDA-MB-453 cells express high levels of functional AR, and thus provide a valuable in vitro model for further studies on androgen regulation of gene expression, and perhaps cell proliferation in breast cancer.     

Identification of genes expressed in the rat prostate that are modulated differently by castration and Finasteride treatment

In mammals, testosterone and 5alpha-dihydrotestosterone (DHT) are the principal male hormones (androgens). Testosterone is the most abundant circulating androgen, and is converted in specific tissues to DHT by the 5alpha-reductase enzymes. Although each of these androgens binds to the same receptor protein (androgen receptor, AR), each exerts biologically distinct effects. Theories to explain the specific effects of testosterone and DHT have centered on kinetic differences of binding of androgens to the receptor or differences in the metabolic fates of the two hormones. In the current experiments, differential display PCR (ddPCR) was used to identify genes regulated differently by testosterone and DHT. Adult male rats were treated as follows: castrated, treated with Finasteride (an inhibitor of 5alpha-reductase) or left intact for ten days. RNA was prepared from the dissected prostates of these animals and used for ddPCR. Genes exhibiting four distinct patterns of regulation were observed among the mRNAs. Class 1 genes showed equivalent expression in intact and Finasteride-treated animals, but were absent in castrated animals (mRNAs D1, D2, D6, D10). Class 2 genes showed higher expression in intact animals, intermediate levels following Finasteride treatment, but were absent in castrated animals (mRNA D8). Two classes of gene were particularly intriguing: class 3 showed gene expression only in the intact animal (mRNA D7, D9) and class 4 showed increased gene expression following Finasteride treatment (mRNA D3). While the patterns observed for some of these genes (e.g. D8) suggest that the different biological effects of testosterone and DHT may be due to the lower affinity of the AR for testosterone and limiting tissue concentrations of androgen, our results also suggest that some genes expressed in the rat prostate may be regulated in fundamentally different ways in response to testosterone and DHT.     

Regulation of immunoreactive androgen receptor in the adrenal gland of the adult rat

The androgen receptor (AR) was measured by an immunoblot assay in adult tissues of both male and female rats. Relatively high levels of AR were detected in tissues of the male urogenital tract and in the adrenal glands and gonads of both sexes. Another group of tissues, including the male levator ani/bulbocavernosus muscles, preputial gland, scrotal skin, and vagina, had low, but detectable, levels of AR. In a third group of tissues, including the uterus, kidney, spleen, liver, gut, heart, lung, pituitary, and hypothalamus, AR was undetectable. In some androgen target tissues, such as the penis, androgens cause an apparent disappearance of AR from the tissue, and in other tissues, such as the ventral prostate, androgen therapy increases the amount of detectable AR. We compared the effect of androgen on AR levels in the adrenal gland and ventral prostate, tissues that differ markedly in their trophic responses to androgen. Castration appeared to have no effect on the amount of detectable AR in the adrenal gland, whereas it caused a profound decrease in AR levels in the ventral prostate. By contrast, 7 days after hypophysectomy, AR levels declined in both the adrenal gland and the ventral prostate. The effects of hypophysectomy plus castration were similar to those of hypophysectomy alone. Administration of ACTH to hypophysectomized rats for 7 days did not reverse the effects of hypophysectomy on adrenal AR, nor did treatment with levothyroxine, dexamethasone, rat GH, or rat PRL. Treatment of hypophysectomized rats with 5alpha-dihydrotestosterone for 7 days caused a dramatic increase in the amount of detectable AR in both the ventral prostate and the adrenal gland, but had a trophic effect only in the ventral prostate. These findings suggest that the amount of immunoreactive AR detected in both the adrenal gland and the ventral prostate is enhanced by androgens: testicular androgens in the case of the ventral prostate and adrenal androgen in the case of the adrenal glands.     

Assessment of androgen receptor function in genital skin fibroblasts using a recombinant adenovirus to deliver an androgen-responsive reporter gene

Mutations of the androgen receptor (AR) cause defects in virilization and can result in a spectrum of phenotypic abnormalities of male sexual development that includes patients with a completely female phenotype (complete testicular feminization) and individuals with less severe defects of virilization, such as Reifenstein syndrome. These phenotypes are not specific for mutations of the AR gene, however, and defects in other genes can also result in similar abnormalities of male development. For this reason, the diagnosis of an AR defect is laborious and requires data from endocrine studies, the family history, and in vitro binding experiments. To assist in the evaluation of patients with possible AR defects, we previously employed the use of a recombinant adenovirus to deliver an androgen-responsive gene into fibroblast cultures to assay AR function in normal subjects and patients with complete forms of androgen resistance. Although these studies demonstrated measurable differences between these two groups of subjects, we did not assay samples from patients with partial defects of androgen action. In the current study, we have modified this method to examine AR function in three groups of patients with known or suspected defects of AR function: patients with Reifenstein syndrome, patients with spinobulbar muscular atrophy, and patients with severe forms of isolated hypospadias. When assayed using this method, the AR function of patients with Reifenstein syndrome was intermediate between that of normal control subjects and that of patients with complete testicular feminization. Using the parameters established by the aforementioned experiments, we found that defective AR function can be detected in fibroblasts established from patients with spinobulbar muscular atrophy and in some patients with severe forms of isolated hypospadias, including two with a normal AR gene sequence. These results suggest that this method may have some utility in screening samples to detect defects of AR function, particularly when viewed in the context of other AR assays results.     

Clathrin gene expression is androgen regulated in the prostate

Androgens are required for the development and function of the prostate. In a normal human prostate, androgens control the synthesis of proteins such as prostate-specific antigen and human glandular kallikrein. The prostate secretes these proteins as well as a number of other compounds to form the prostatic fluid. Using differential display PCR to detect novel androgen-regulated genes, clathrin heavy chain expression was identified as potentially being up-regulated by androgens in the prostate cancer cell line LNCaP. We report here that the clathrin heavy chain and light chain genes are regulated by androgens. Clathrin heavy chain messenger RNA was up-regulated by androgens in a concentration- and time-specific manner in the LNCaP cell line. Translation of clathrin heavy chain messenger RNA was stimulated by androgens. Steady state levels of clathrin light chains a and b were up-regulated in the presence of androgen in LNCaP cells. Clathrin gene expression was examined in normal rat prostates, and similar results were found. Clathrin heavy chain protein levels in the rat prostate are also affected by the androgen status of the animal. We hypothesize that clathrin may be involved in the exocytosis of androgen-regulated secretory proteins such as prostate-specific antigen and human glandular kallikrein.     

Differential transactivation by the androgen receptor in prostate cancer cells

BACKGROUND: The purpose of this study was to determine the contribution of different transactivating regions of the androgen receptor (AR) to the induction of androgen-regulated promoters in poorly (PC3 cells) and well-differentiated (LNCaP cells) prostate cancer cell lines. METHODS: PC3 and LNCaP cells were co-transfected with plasmids expressing full-length AR or deletion mutants together with luciferase reporters linked to the probasin (PB) and PSA promoters; as well as to ARR3tk, a PB-derived recombinant promoter. RESULTS: Androgen induction of the ARR3tk promoter in the presence of AR was 8- to 10-fold higher than that seen with the PB promoter. Activation of ARR3tk was greatest with an androgen-independent construct in which the first 231 amino acids and the ligand binding domain had been removed, indicating that this promoter is more responsive to activating functions in the N-terminal domain than in the ligand binding domain. By comparison, induction of the PB promoter was greatest with the full-length AR, which suggests that the ligand binding domain also makes a major contribution to the activation of this promoter. In similar analyses with the PSA promoter, AR regions required for promoter induction was dependent on the host cell type. In PC3 cells, the predominant AR transactivation function was androgen-independent and resided in the N-terminal domain, whereas in LNCaP cells, the highest level of induction was androgen dependent and also required participation of the ligand binding domain. CONCLUSIONS: Our results indicate that the relative utilization of transactivating functions in N-terminal and ligand binding domains of the AR is promoter and cell specific.     

Multiple G1 regulatory elements control the androgen-dependent proliferation of prostatic carcinoma cells

Prostatic epithelial cells and most primary prostate tumors are dependent on androgen for growth, but how androgen regulates cellular proliferation remains unsolved. Using poorly understood mechanisms, recurrent tumor cells evade the androgen requirement. We utilized androgen-dependent prostatic tumor cells to demonstrate that androgen exerts its effect on the cell cycle by influencing specific aspects of G1-S progression. Androgen depletion of these cells results in early G1 arrest, characterized by reduced cyclin-dependent kinase activity, and underphosphorylated retinoblastoma tumor suppressor protein (RB). The reduction in kinase activity was partially attributed to reduction of specific G1 cyclins and alternate regulation of cyclin-dependent kinase inhibitors. Using this information, we developed a reliable assay to assess the ability of specific G1 regulatory proteins to circumvent these controls and promote androgen-independent growth. As expected, inactivation of RB was required for progression through the cell cycle. Surprisingly, overexpression of G1 cyclins, which drives RB phosphorylation, was insufficient to promote androgen-independent cell cycle progression. Introduction of viral oncoproteins did promote G1-S progression in the absence of androgen, dependent on their ability to sequester RB and related proteins. These results provide the first evidence that multiple elements governing the G1-S transition dictate androgen-dependent growth, and the formation of androgen-independent prostatic tumors may be because of misregulation of these processes.     

Regulation of androgen receptor mRNA expression in hamster facial motoneurons: differential effects of non-aromatizable and aromatizable androgens

We have previously shown inherent sex differences in the levels of androgen receptor mRNA (AR mRNA) in hamster facial motor neurons (FMN). FMN of intact females contained approximately 50% less AR mRNA than their male counterparts. Gonadectomy in males down-regulated AR mRNA levels in FMN by approximately 50%, whereas no effects of gonadectomy were observed in females. Sex differences in the regulation of AR mRNA levels by exogenous testosterone propionate (TP) were also observed. In those studies, AR mRNA levels were up-regulated after 1 day of treatment with exogenous TP in FMN of gonadectomized (GDX) males and after 7 days in FMN of intact females, with no effects in GDX females. Since TP is aromatizable to estrogen, and given recent findings of transient expression of estrogen receptors (ER) in rodent FMN, the effects of dihydrotestosterone (DHT), a non-aromatizable form of the steroid, on AR mRNA expression in hamster FMN were examined in the present study. If testosterone (TES) were the active hormone regulating AR mRNA levels in FMN, DHT treatment should render a similar regulatory pattern as TP, but if metabolism of TES to estradiol plays a role in AR mRNA regulation, effects of the two treatments should differ. In situ hybridization and computerized image analysis were used to quantify the regulation of AR mRNA by DHT in individual FMN of hamsters of both sexes. Exogenous DHT was administered to intact and gonadectomized (GDX) male and female hamsters by implantation of one 10-mm Silastic capsule for 1, 2 or 7 days. AR mRNA levels were significantly up-regulated in intact females at all time points of DHT exposure, with no effects in GDX groups. These results differ from previous work using TP, in which a modest up-regulation in AR mRNA levels was observed in FMN of intact females only after 7 days. As with TP, DHT exposure gradually down-regulated AR mRNA levels in FMN of intact males. Thus, DHT only regulated AR mRNA levels in intact animals, with endogenous sources of estrogen available, but not in GDX animals, with endogenous estrogens reduced by gonadectomy. Taken together, these results substantiate our previous findings of sex differences in AR mRNA levels/regulation and suggest a synergism between estrogen and androgen in the regulation of AR mRNA levels in peripheral motor neurons.     

Progression of LNCaP prostate tumor cells during androgen deprivation: hormone-independent growth, repression of proliferation by androgen, and role for p27Kip1 in androgen-induced cell cycle arrest

The molecular mechanism of androgen-independent growth of prostate cancer after androgen ablation was explored in LNCaP cells. An androgen-dependent clonal subline of the LNCaP human prostate carcinoma cell line, LNCaP 104-S, progressed to a slow growing stage (104-R1) and then to a faster growing stage (104-R2) during more than 2 yr of continuous culture in the absence of androgen. Androgen-induced proliferation of 104-S cells is inhibited by the antiandrogen Casodex, while proliferation of 104-R1 and 104-R2 cells is unaffected by Casodex. This indicates that proliferation of 104-R1 and 104-R2 cells is not supported by low levels of androgen in the culture medium. Compared with LNCaP 104-S cells, both 104-R1 and 104-R2 cells express higher basal levels of androgen receptor (AR), and proliferation of these two cell lines is paradoxically repressed by androgen. After continuous passage in androgen-containing medium, 104-R1 cells reverted back to an androgen-dependent phenotype. The mechanism of androgenic repression of 104-R1 and 104-R2 sublines was further evaluated by examining the role of critical regulatory factors involved in the control of cell cycle progression. At concentrations that repressed growth, androgen transiently induced the expression of the cyclin-dependent kinase (cdk) inhibitor p21waf1/cip1 in 104-R1 cells, while expression of the cdk inhibitor p27Kip1 was persistently induced by androgen in both 104-R1 and 104-R2 cells. Induced expression of murine p27Kip1 in 104-R2 cells resulted in G1 arrest. Specific immunoprecipitates of Cdk2 but not Cdk4 from androgen-treated 104-R1 cells contained both p21waf1/cip1 and p27Kip1. This observation was confirmed by in vitro assay of histone H1 and Rb (retinoblastoma protein) phosphorylation by the proteins associated with the immune complex. Furthermore, inhibition of Cdk2 activity correlated with the accumulation of p27Kip1 and not p21waf1/cip1. From these results we conclude that androgenic repression of LNCaP 104-R1 and 104-R2 cell proliferation is due to the induction of p27Kip1, which in turn inhibits Cdk2, a factor critical for cell cycle progression and proliferation.     

Immunohistochemical localization of androgen receptor in mouse testicular germ cells during fetal and postnatal development

BACKGROUND: Determination of the cellular distribution of the androgen receptor (AR) in testicular cells is necessary for understanding the mode of AR action in the testis. We here investigated immunohistochemically the localization of AR by use of anti-human AR polyclonal antibody NH27, with special reference to the AR in germ cells in the developing mouse testis. METHODS: ICR mouse testes taken from day 14 post coitum (p.c.) to day 56 post partum (p.p) were used for AR immunohistochemistry by the routine immunoperoxidase method at the light microscopic level and the pre-embedding method at the electron microscopic level. RESULTS: On day 14 p.c., AR immunoreactivity was present in nuclei of prospermatogonia but not in those of Sertoli cells or interstitial cells. On day 14 p.p., the AR was detected in the nuclei of spermatogonia, Sertoli cells, and myoid cells. AR immunoreactivity in nuclei of Leydig cells appeared on day 21 p.p. In the mature mouse testis, the AR was present in the nuclei of spermatogonia, Sertoli cells, myoid cells, and Leydig cells. CONCLUSIONS: AR was present both in germ cells and in somatic cells during fetal and postnatal development of the mouse testis. In the fetal testis, AR was localized exclusively in prospermatogonia and spermatogonia, suggesting that androgen may act directly on germ cells during prespermatogenesis and the early stage of spermatogenesis. Based on the fact that AR is expressed in Sertoli cells, myoid cells, and Leydig cells around the onset of spermatogenesis, the regulation of AR expression in the germ cells seems to be different from that in the somatic cells. Furthermore, our present data suggest the ultrastructural localization in nuclei of mouse testicular cells is similar to that of some other steroid receptors, both in germ cells and somatic cells.     

Inhibition of androgen action by dehydroepiandrosterone sulfotransferase transfected in PC-3 prostate cancer cells

Age-dependent loss of androgen sensitivity of the rat liver is associated with a marked increase in dehydroepiandrosterone/hydroxysteroid sulfotransferase (rStd) activity. Sulfonated steroid hormones are known to be ineffective in binding receptor proteins. These observations suggest that intracellular androgen sulfonation can physiologically influence androgen action. We have examined the inhibitory effect of rStd on androgen action in the human prostate cancer-derived PC-3 cells transfected with the rat androgen receptor (AR) expression plasmid and two androgen-responsive promoter reporter constructs (murine mammary tumor long-terminal repeat ligated to chloramphenicol acetyltransferase (CAT) gene and rat probasin androgen response element (ARE) ligated to firefly luciferase (LUC) gene). These transfected cells were dependent on 5alpha-dihydrotestosterone (DHT) for the activation of both reporter genes and showed about a 200- and a 800-fold increase of CAT and LUC activity, respectively, at 10(-10) M DHT over the no-hormone control. Expression of the sulfonating enzyme in this cell transfection system via the rStd expression plasmid caused a dose-dependent decline in the reporter activity with approximately 90% inhibition of androgen action at a rStd:AR plasmid ratio of 100. From these results we conclude that irrespective of a high level of AR, changes in the Std expression can markedly alter the androgen sensitivity of target cells.