[Frontiers in Bioscience 1, d59-71, March 1, 1996]
Reprints
PubMed
CAVEAT LECTOR



THE ANDROGEN RECEPTOR: A MEDIATOR OF DIVERSE RESPONSES

Evan T. Keller, William B. Ershler, and Chawnshang Chang

The Institute on Aging and the Department of Human Oncology, University of Wisconsin, Madison, WI 53706, USA.

Received 01/16/96; Accepted 02/22/96; On-line 03/01/96

TABLE OF CONTENTS
1. Abstract
2. Introduction
3. An overview of the androgen receptor
3.1 Androgen Receptor Structure
3.2 Distribution of the Androgen Receptor
3.3 Regulation of Androgen Receptor Expression
3.4 The Androgen Receptor Gene 5'-flanking Region
4. How do androgens achieve distinct effects on various epithelial populations?
5. Why are there two androgens (testosterone and dihydrotestosterone) active on one androgen receptor?
6. Why are both consensus and non-consensus androgen response elements present ?
7. How does a nonspecific steroid response element confer androgen specific action?
8. How does prostate cancer acquire androgen independence?
9. Summary
10. Acknowledgments
11. References

1. ABSTRACT

Androgens mediate a number of diverse responses through the androgen receptor, a 110 kD ligand-activated nuclear receptor. Androgen receptor expression, which is found in a variety of tissues, changes throughout development, aging, and malignant transformation. The androgen receptor can be activated by two ligands, testosterone and dihydrotestosterone, which bind to the androgen receptor with different affinities. This difference in binding affinity results in different levels of activation of the androgen receptor by the two ligands. The androgen receptor acts as a transcriptional modifier of a variety of genes by binding to an androgen response element. The ability to confer androgen specific actions by the androgen response element may depend on other cell-specific transcription factors and cis-acting DNA elements in close proximity to it.

Testosterone and dihydrotestosterone appear to act upon an identical nuclear receptor. However, in certain instances, they mediate different physiologic responses. For example, dihydrotestosterone, but not testosterone, is capable of mediating full sexual development of the male external genitalia. In some cases, the androgen receptor may induce opposite physiologic responses in similar tissue types depending on their location. For example, in male pattern baldness, activated androgen receptors may suppress the growth of distinct hair follicle populations through initiating stromal-epithelial actions, whereas other hair follicles continue to proliferate. In other cases, altered androgen receptor activity due to its mutation or altered expression may lead to pathology such as recurrence of prostate cancer due to development of androgen independence allowing tumor cell proliferation under androgen deprivation.

2. INTRODUCTION

Two important androgens, testosterone (T) and its metabolite dihydrotestosterone (DHT), affect a diversity of responses in a variety of tissues. They mediate their effects through a ligand-dependent nuclear transcription factor, the androgen receptor (AR). Cloning of the AR cDNA (1-4) has allowed for cellular and molecular characterization of many different aspects of androgen physiology and pathophysiology. However, in spite of the intensity of research activity in this area, many interesting questions remain regarding the basic functions of androgen and AR on gene regulation. In this review, we will provide a brief overview of the AR and its regulation and then explore several intriguing aspects of the cellular and molecular biology of androgens and their receptor.

3. AN OVERVIEW OF THE ANDROGEN RECEPTOR.

3.1 Androgen Receptor Structure

The AR is a member of the steroid hormone receptor family. These receptors regulate gene transcription by interacting with a specific DNA sequence in a ligand-dependent manner. The AR is a 110 kD nuclear protein which consists of approximately 918 amino acid residues (1, 3, 5). Similar to other steroid hormone receptors, the AR consists of transactivation (6-10), DNA binding (11-16), nuclear localization (17-19), dimerization (20-22), and ligand binding domains (6, 23-27) (Fig. 1). Of these domains, the N-terminus is the most variable while the other regions are highly conserved. Within the N-terminus, the region of amino acids from 141 to 338 consist of polyglutamine and polyproline residues (3) which appear important in transcriptional activation (18, 28). The DNA-binding domain consists of 68 amino acids which fold into two zinc fingers capable of binding DNA. Four cysteine residues non-covalently bind the zinc ion within each finger. The remaining 295 amino acids form the C-terminus and encode the hinge region/nuclear localization signal and the ligand-binding domain.

Figure 1. Schematic illustration of the functional domains of the human androgen receptor. The exons are labeled A-H. The numbers above the box indicate the amino acid number at the junction between the exon and the intron. The domains responsible for their specific function are also indicated by lines with the numbers of amino acid residues.

3.2 Distribution of the androgen receptor

As demonstrated by a variety of methods, AR is present in most tissues. 3H-androgen binding assays were the initial approach used to determine tissue distribution of AR. By injecting (3H)-testosterone into rats followed by quantitation of (3H)-androgen uptake in various tissues, Gustafsson and Pousette (29) were able to identify target organs of androgens. The development of anti-AR antibodies has complemented the autoradiographic methods of AR detection (30-33). Using anti-AR antibodies, Takeda et al. (32) evaluated AR distribution in various rat tissues. They were able to demonstrate that all male sexual organs in the rat showed a strong positive nuclear staining for AR, whereas several other tissues, including hepatic, renal, neuronal, muscular and female reproductive organs had weak, albeit positive, nuclear staining. In fact, the only tissue which did not demonstrate staining for AR was the spleen. The use of microwave-based antigen retrieval for AR (34) enhanced the immunohistochemical detection of AR in paraffin sections allowing for evaluation of archival tissue sections.

In addition to identification of AR protein, detection of AR mRNA in tissues has been accomplished by several methods. Cloning of human AR (hAR) and rat AR (rAR) cDNA (1, 35) allowed for development of probes which were used to detect in various tissues the AR mRNA by Northern blotting or by in situ hybridization. In addition to confirming previous immunohistochemical staining data, the analysis of AR mRNA by Northern blot resulted in identification of two isoforms in the A/B domain of AR mRNA in the vocal organ of Xenopus (36) and the brain of rodents (37). The sensitivity of detection of AR mRNA was greatly improved by competitive RT-PCR (38). Table 1. summarizes the relative expression (compared to prostate) of AR mRNA in various tissues of male and female Sprague-Dawley rats based on competitive RT-PCR (38). These results demonstrate agreement between mRNA levels and immunohistochemical staining intensity (39). One notable exception was the ability to demonstrate AR mRNA in the spleen using RT-PCR (38) as opposed to the inability to detect AR protein by immunohistochemical staining (32). This result is most likely attributable to the extreme sensitivity of RT-PCR as compared to immunohistochemistry.

3.3 Regulation of androgen receptor expression

AR expression is modified during fetal development, sexual development, aging, and malignant transformation. Regulation of AR levels may occur anywhere along the path from AR gene transcription to post-translational modification. A variety of factors, including androgens, have been implicated in modulating the AR protein and mRNA expression.

In the case of mouse fetal development, AR mRNA, based on in situ hybridization, was not found in the urogenital sinus at 13.5 days of gestation, whereas at 15.5 days of gestation both AR mRNA and protein levels were detectable (39).

Table 1. Relative abundance of androgen receptor mRNA in various organs to the level in the prostate gland
OrganMaleFemale
Hypothalamus42217
Adrenal gland141186
Epididymis115ND
Thyroid gland68ND
Harderian gland58ND
Pituitary gland569
Preputial gland4438
Quadriceps muscle35ND
Levator ani muscle30ND
Kidney277
Coagulating gland25ND
Seminal vesicle25ND
Testis20ND
Liver189
Submaxillary gland17ND
Bulbocavernosus muscle16ND
VaginaND9
Heart87
OvaryND4
UterusND2

Table1: The relative abundance of androgen receptor (AR) mRNA in rat tissues (38). Total RNA from the indicated tissues was subjected to competitive reverse transcription-polymerase chain reaction for quantitation of AR mRNA levels. The data are reported as percentage of AR mRNA relative to the prostate AR mRNA levels (averaged from 5 male rats). The data represent mean values from 3-5 rats. ND = not determined.

In the rat neonate, castration at 3 days of age did not result in altered AR expression in the rat prostate (40). In contrast, castration in the adult rat altered AR mRNA and protein levels (41-43). These findings suggest that one or more developmentally regulated factor(s) influence the AR expression.

An age-dependent decline in hepatic rAR expression has been shown to be associated with the expression of an Age-dependent factor (ADF) which is ubiquitously expressed in tissues (44). ADF binds to a rAR fragment between -310 to -330. Rat hepatic ADF binding activity on the rAR promoter in vitro was shown to decrease with age, When the ADF binding site was mutated in a reporter construct, a decreased rAR promoter activity was observed.

As discussed below, in prostate cancer, development of an androgen independent state is associated with a heterogeneous AR expression. In addition to prostate cancer, the AR protein or mRNA has been detected in other forms of cancer such as hepatomas (45), germ-cell neoplasias (46), and ovarian tumors (47). These findings suggest that dedifferentiation may result in expression of factors which modulate the AR expression.

Several factors have been reported to modify the AR expression. These factors include androgens which were shown to decrease the AR mRNA expression in the rat ventral prostate (42, 48), a human androgen-responsive prostate carcinoma cell line (LNCaP) (42, 49), and a hepatoma cell line (HepG2) (48). However, these observations are controversial because up-regulation of AR by androgens has been demonstrated in the rat and mouse prostate (43), genital skin fibroblasts (50), rat penile smooth muscle (51), and male rat fat-pad adipose precursor cells (52). Some of these conflicting observations may be due to different methodologies used such as moderately sensitive Northern blots versus highly sensitive in situ hybridization. However, the presence of tissue-specific transcription factors may also account for the opposite effects androgens play on AR expression.

The regulation of the AR also takes place at the protein level. For example, in the androgen-dependent prostate cancer cell line, PC-82, although the AR mRNA level was stable AR protein was up-regulated by androgen (53). On the other hand, it was reported that the down-regulation of AR mRNA by androgen in the LNCaP line was associated with an increased AR protein expression (42, 54, 55). Mizokami et al. showed that the AR protein up-regulation by androgen resulted from enhanced stability of AR protein (55) providing a clue for these discrepant observations.

In addition to androgen, several other hormones and growth factors can regulate the AR expression. Follicle-stimulating hormone increases the level of mRNA of AR in the Sertoli cells (56). Growth hormone, prolactin (57) and epidermal growth factor (55, 58) increase the AR mRNA levels in prostatic cells. Interestingly, trans-retinoic acid down-regulated AR mRNA in T47D breast cancer cells but up-regulated the expression of AR mRNA in MDA-MB-453 breast cancer cells (59) demonstrating that the effect of the signal depends on the cell type.

In conclusion, AR expression can be modified by a variety of factors which appear to act in a tissue and cell specific fashion. Close examination of the AR gene control elements may provide clues as to how this regulation is achieved.

3.4 The androgen receptor gene 5'-flanking region

Transcriptional control of the AR gene has been examined in a variety of conditions. The AR promoter lacks TATA and CCAAT boxes, but appears to have a GC box (a Sp1 binding site) which may play an important role in transcriptional initiation (60, 61). Additionally, it appears that there are two transcription initiation sites located 1127 base pairs (bp) and 1116 bp upstream of the translation initiation site (61, 62). A variety of putative cis-acting elements are present on the AR promoter (63). A cAMP response element (CRE) has been shown to stimulate AR transcription in both the human and murine AR (63-65). Though not reported for the human AR, the rAR promoter contains a NF-kB site which down-regulates transcription of the AR (66). In addition to the above studies, a cis-acting region between +57 and +575 of the 5'-untranslated region (5'-UTR) of the AR gene was identified which up-regulated an AR reporter gene's protein expression (62). However, this occurred without increasing the steady state mRNA levels suggesting that this region of the 5'-UTR functions by inducing translation (67).

4. How do androgens achieve distinct effects on various epithelial populations?

Perhaps the most dramatic example of the difference of T and DHT actions are observed during male sexual development. It has long been recognized that T is required for the development of the internal accessory sexual organs, whereas DHT is required for the development of external sex organs. In humans, these events appear to be triggered by T expression which begins at the 8th week of gestation (68). T, secreted by the Leydig cells, stimulates the Wolffian duct to differentiate into the epididymis, vas deferens, and seminal vesicles. The observation that unilateral orchiectomy results only in ipsilateral loss of Wolffian duct differentiation suggests that high concentrations of T are required for this event. The action of 5a-reductase on T results in the production of DHT which stimulates the differentiation of the urogenital sinus into the prostate, penis, and scrotum. The importance of DHT in this role is demonstrated in individuals with congenital 5a-reductase deficiency. Even though genotypically male (46, X, Y), these individuals cannot convert T to DHT and thus develop feminized external genitalia (69). However, because T is present during development, Wollfian duct differentiation proceeds normally resulting in formation of a pseudohermaphrodite. When coincident with puberty the level of T increases some of these individuals develop male characteristics such as increased skeletal muscle bulk, axillary and pubic hair growth, and maturation of the penis (70). This observation suggests that high levels of T may replace the loss of DHT. In agreement with this hypothesis are the following observations made by Imperato-McGinley et al (71): 1) flutamide, an androgen receptor blocker, administered to male rats in utero during the period of sexual differentiation completely inhibited the development prostate; and 2) administration of finasteride, a 5-a reductase inhibitor, resulted only in partial inhibition of development of prostate. Taken together, these results suggest that T, via the AR, can compensate for lack of DHT.

If as discussed below one subscribes to the idea that both T and DHT mediate their effects by interacting with a single nuclear receptor protein, then how can we account for the requirement of both T and DHT for sexual development? The answer may be found by examining the ability of these androgens to interact with the AR. Several investigators have demonstrated that T has approximately 3 times faster association and dissociation rates than DHT on both the rat (72) and human (73, 74) AR. In agreement with these binding kinetics, Zhou et al. have demonstrated that T is less effective at stabilizing AR than DHT (74). These observations suggest that DHT, by enhancing the stabilization of AR and its action, amplifies the T signal in those tissues which contain 5a-reductase. Perhaps in these tissues, the AR expression is not sufficient for T to mediate a physiologic response, but due to the ability of DHT to enhance AR activity, a response is observed.

In contrast to the inability of T to mediate various androgen-dependent events during sexual development, the biology of hair growth offers an example of the different effects that androgens exert on the proliferation of similar populations of epithelial cells. Specifically, T can stimulate facial hair growth, but causes the regression of scalp hair in aging individuals (75). Hair follicles are intimately associated with the mesenchymally-derived dermal papilla which is believed to provide an important influence on the follicular proliferation. Several lines of evidence support the role of androgens in controlling growth of hair follicles through modulation of the dermal papilla activity: 1) AR has been identified by both ligand binding assay (76) and by immunohistochemical staining in the dermal papilla (77); 2) dermal papillae from androgen-dependent hair follicles contain a greater number of AR than those in non-balding areas (78); 3) in primary dermal papilla cell lines from either androgen-dependent (i.e., beard) or -independent (i.e., non-balding scalp) hair follicles, only the beard dermal papilla-derived cells were able to synthesize DHT when T was added to the media (75); 4) the level of 5a-reductase varied between hair follicles from frontal and occipital sites (79); and 5) dermal papilla can produce extracellular matrix components and mitogenic factors (75). Taken together, these findings suggest that androgen, via the AR, can indirectly mediate an effect on hair follicle proliferation through modulating dermal papilla activity. Though these data do not explain how T modulates cell proliferation in hair follicles, the differences in androgen metabolism and AR expression may, in part, account for the opposite proliferative responses observed in various epithelial tissues. The effect of androgens on cell proliferation in hair follicles may be regulated through regulation of expression of growth factors. Androgens can modulate expression of a variety of growth factors in the prostate stroma.

Mesenchymal-epithelial interactions are critical in the development of prostate tissue (reviewed in (80)). Prostate mesenchyme, when coincubated with Sertoli cells, synthesizes extracellular matrix in an androgen-independent manner; however, when exposed to androgen it produces a diffusible substance similar in action to P-Mod-S (81). P-Mod-S, a protein secreted by peritubular cells, can activate a variety of responses such as inhibin production and aromatase activity in Sertoli cells. Its effect on the prostate epithelia is currently unknown; however, it may also stimulate the activity similar to that observed in Sertoli cells thus accounting for one mechanism of mesenchymal-epithelial interaction within the prostate. Gleave et al. demonstrated that prostate fibroblasts secrete a diffusible substance which can stimulate the growth of LNCaP prostate carcinoma cells (82). Androgen deprivation mediated by castration resulted in expression of tenascin in the rat prostate (83).

5. Why are there two androgens (Testosterone and Dihydrotestosterone) active on one androgen receptor?

T and DHT mediate their effects by altering gene expression in target tissues via a single receptor, the AR. Despite the binding of T and DHT to a single receptor, the effect of these hormones on a single gene may be quite distinct. For example, differential regulatory effects of T and DHT on expression of several genes including Far-17a (84), and the cytokines interleukin-4 (IL-4), IL-5 and g-interferon (gIFN) has been reported (85). The observation that both T and DHT can differentially regulate the expression of the androgen responsive genes has led to the controversial idea that more than one AR may exist. This hypothesis is supported by the following observations (reviewed in (86)) : (1) (3H)T, when injected into rats, concentrates in the hypothalamic nuclei and 100x unlabeled DHT does not inhibit this localization; (2) hypothalamic localization is not observed after injection of (3H)DHT; and (3) the ability of T, but not DHT to induce neuronal proliferation or male sexual behavior in castrated male rats. These data are consistent with two different possibilities. One possibility is that two different androgen receptors indeed exist. On the other hand, these data may be due to different metabolic interactions of the androgens on a single androgen receptor. As discussed above, T dissociates from the AR 3 times faster than DHT and is less effective in stabilizing the AR (74). This difference in the dissociation rate has been directly related to the androgens' ability to stimulate transcription of an androgen responsive gene (87). These observations could account for the differential effects of these androgens mediated by one receptor.

Perhaps the strongest evidence for the presence of one androgen receptor is derived from the observation that genotypic XY mice and rats with testicular feminization syndrome (Tfm) do not have a fully functional AR (87-90) and even though they express both T and DHT they develop into phenotypic females. This experiment of nature demonstrates that loss of one AR can result in loss of action of both androgens.

The presence of two androgens acting on one receptor may serve several possible functions. Cells which contain steroid 5a-reductase can convert T into DHT. Thus, the overall effect of this system may be to amplify the action of T via conversion to DHT within these cells, thus providing a mechanism of local regulation.

6. Why are both consensus and non-consensus present?

As mentioned above, AR binds to the consensus ARE to induce the androgens' effects. However, several non-consensus ARE have also been reported in rat genes. The rat probasin gene contains a 17 bp non-consensus ARE (ARE-2) consisting of 5'-GTAAAGTACTCCAAGAA-3' (91). This element is located within the 5'-UTR and is approximately 100 bp downstream of a consensus ARE. Even though both the non-consensus and consensus elements bind AR, neither is an effective independent enhancer when tested with a heterologous thymidine kinase promoter. However, when they are both present in the same construct, a marked response to androgen is observed (91). The androgen-regulated 20-kD protein gene, which consists of four exons that code for a major secretory protein of rat ventral prostate, contains both consensus and non-consensus ARE (92). These are present in the first intron (In-1) and when CV1 cells were cotransfected with either GR or AR, In-1 and its most active subfragment, In-1c functioned as AREs but not as GRE (92). This effect was diminished in PC-3 and HeLa cells suggesting that other transcriptional factors may confer AR specificity to the non-consensus ARE. The human glandular kallikrein-1 gene (hKLK-2) contains an ARE in its 5' promoter region consisting of 5'-GGAACAGCAAGTGCT-3' (93). Deletion of the promoter immediately 5' to the non-consensus ARE resulted in a decreased induction of CAT activity (93). This suggests that the non-consensus ARE activity may be dependent on the interaction of AR with transcription factors which may bind to these upstream sequences.

In summary, it is likely that AR interaction with non-consensus ARE may depend on the interaction with other transcription factors which bind to cis-acting DNA elements in close proximity to the ARE. The existence of non-consensus ARE may allow androgen:AR complexes to expand their transactivating action. Thus, in conjunction with the classical AREs of androgen responsive genes, the non-consensus ARE may provide a degree of variability for the control of hormonal responses. Further identification and characterization of non-consensus ARE will provide additional clues as to how these elements participate in gene regulation.

7. How does a nonspecific steroid response element confer androgen specific action?

Characterization of the consensus ARE using the AR DNA binding domain (DBD) in a DNA binding-site selection assay revealed the consensus ARE to be identical to the consensus GRE (94). This finding, taken together with the observation that GR is nearly ubiquitous in cells elicits the question as how androgen specific action is mediated by the ARE. Several observations provide clues as to how specific action can be mediated from these apparently non-specific HREs. Many androgen responsive genes have promoters which consist of several regulatory elements which suggests that an androgen specific response may be due to a combination of interacting transcription factors. This control mechanism would allow for developmental and cell specific control of androgen response as is observed with the 20 kDa protein gene (92). That the receptor itself may interact differently at the same response element is suggested by the observation that even though the DBDs of members of the GR subfamily are highly conserved, the transactivation domains are not. Thus, even though GR and AR may bind to the same DNA element, they may invoke different activity on the promoter region. This is the case for the sex-limited protein (Slp) of the mouse (95). Slp is a murine serum protein of unknown function which is synthesized primarily in the liver of mature males or T-treated females. It is encoded by a gene (C4-slp) in the S region of the major histocompatability complex class III gene complex (95). Slp's androgen dependence has been attributed to an androgen-responsive promoter in the C4-slp gene. Hemenway et al. identified several DNAse I hypersensitive sites which appeared in the C4-slp promoter upon androgen treatment (96). An androgen responsive region was identified within a 750 bp fragment found 2-kb upstream of the transcription initiation site in the C4-slp promoter by Loreni et al. (97). This androgen-responsive enhancer was identified as a 5' long terminal repeat of an ancient provirus which is most likely a retrotransposon. Adler et al. delineated the androgen-responsive enhancer to a 160 bp fragment (98).

Even though both AR and GR can bind the HRE element in the context of the slp promoter, GR inhibits AR induction of the slp gene (99). Interestingly, the GR can activate a heterologous promoter (thymdine kinase promoter) when the slp HRE element itself is placed upstream of it. Additionally, when the HRE element is moved as little as 10 bases away from the enhancer region, GR can activate the promoter (100). These findings strongly support that the context of the HRE is important in determining the specificity of the steroid receptor response. In light of the multiple elements found in the promoter region, such specificity is most likely mediated by protein-protein interactions with accessory transcription factors. These factors either inhibit or are not sufficient for the GR-induced activation of the enhancer while they are permissive for the AR-induced activation. Differences in transactivation domains of the steroid receptors could account for the distinct interactions which occur with the accessory transcription factors.

Tissue specific expression of hormone receptors is another mechanism by which a steroid-specific response can be generated. Strahle et al. introduced a progesterone-receptor expression plasmid into the rat hepatoma cell line, Fto2B-3 which contains glucocorticoid receptor but is devoid of progesterone receptor (101). They observed that the expression of the progesterone receptor in Fto2B-3 cells rendered endogenous glucocorticoid-regulated genes inducible by progestins. This experiment demonstrated that the specificity of responsiveness by a HRE to a steroid receptor in cells can be reprogrammed by the expression of a different steroid receptor which is capable of binding the HRE in question.

In summary, steroid specific expression can be mediated at both the gene and cellular levels. At the gene level, the context of the HRE may be critical for orchestrating a specific steroid response. Response elements in the proximity of the HRE may provide binding sites for accessory transcription factors. These factors are required to generate activation of the enhancer and yet may only interact with a specific hormone receptor, generating the specificity of the steroid-induced gene activation. At the cellular level, tissue specific expression of steroid receptors will limit the steroid response only to those cognate receptors.

8. How does prostate cancer acquire androgen independence?

When initially diagnosed, the majority of prostate cancers respond to androgen deprivation; however, over time they lose their dependency on androgens and the tumor recurs. The molecular events responsible for development of androgen independence are currently unknown. Altered AR expression or function, loss of tumor suppressor genes, and overexpression of or altered response to growth factors may all be implicated in this phenomenon.

AR is an important mediator of early prostate cancer growth. This is underscored by the marked tumor regression associated with androgen deprivation. However, the importance of the AR during the late stages of prostate cancer is not very clear. Initial studies demonstrated that AR mRNA is present in androgen-sensitive prostate cancer cell lines but is absent or is expressed at low levels in androgen-independent lines (102). Sato et al. demonstrated that as prostate cancer progresses to a less differentiated state AR expression was decreased (103). Recent immunohistochemical studies have revealed that the total AR content of prostate cancer cannot be used for assessment of prognosis; rather the heterogeneity of AR expression throughout the tumor was significant as a predictor of response of tumor to hormonal therapy (104-106). Chodak et al. also demonstrated that AR was uniformly present in the nuclei of epithelial cells in normal prostate, whereas in prostatic adenocarcinoma only some of the epithelial nuclei stained for AR (104). Sadi and Barrack demonstrated that cancer cells in patients with advanced prostate cancer who responded poorly to therapy exhibited a significant heterogeneity in the staining intensity of the AR (105). These observations suggest that AR expression may be modified during prostate cancer progression and may somehow be associated with the development of androgen independence. One must bear in mind that these studies do not eliminate the possibility that a mutated form of the AR may be expressed that no longer reacts with the antibodies to AR.

AR mutation may confer androgen independence on prostate cancer cells. Although mutations of the AR in prostate cancer are not often identified, they have been reported in up to 25% of the patients in one study (107). A variety of unique mutations, typically occurring in the ligand-binding domain, have been found in prostate cancer (103, 108-111). The LNCaP AR has a single point mutation in codon 877 (ACT->GCT) changing Thr to Ala in the ligand binding domain (112). This mutation decreased dependency of AR activation on androgen because progestagens, estradiol and anti-androgens could compete with androgens for binding to the androgen receptor and could stimulate both cell growth and secretion of prostate specific acid phosphatase (113, 114). This mutation may have a biologic relevance in vivo since Gaddipati et al. reported that 6 of 24 prostatic tissue specimens derived from transurethral resections of patients with metastatic prostate cancer had AR mutations identical to those found in the LNCaP cell line (107). In addition to activation of mutated AR by a greater number of ligands, the AR can be mutated in such a fashion that it is constitutively active (6). In vitro induced deletion of the AR's ligand-binding domain resulted in an AR that in the absence of steroid possessed up to 90% of the activity a steroid-induced wildtype AR (6). Though not observed in prostate cancer, a constitutively activated AR would circumvent the need for androgen, resulting in a phenotype of androgen independence.

A variety of growth factors have been implicated in either autocrine or paracrine prostate cancer cell growth (reviewed in (115)). Culig et al. provided compelling evidence for the ability of various growth factors to activate androgen responsive genes via AR in prostate cancer cells (116). Insulin-like growth factor I (IGF-I), epidermal growth factor (EGF), or keratinocyte growth factor (KGF), stimulated an AR-responsive reporter gene used to cotransfect DU-145, an AR negative prostate cancer cell line, with an AR expression vector. In the absence of the AR expression vector, the reporter gene was not activated. Furthermore, exposure of LNCaP cells to IGF-1 resulted in expression of prostate-specific antigen, which is encoded by an androgen responsive gene (93, 117). These actions were blocked by casodex, a pure AR antagonist. These experiments suggest that androgen independence could arise as a result of overexpression of growth factors in the local environment of the prostate.

In summary, the molecular events which facilitate development of androgen independence are currently unknown. However, loss or mutation of androgen receptor may play a role in selection of androgen-independent tumors. Such mutations in the ligand binding domain of the AR may result in a promiscuous response to non-androgen ligands allowing prostate cancer proliferation in response to non-androgenic growth factors.

9. SUMMARY

We have reviewed several critical issues concerning the cellular and molecular biology of androgen regulation. Androgens, working via the AR, afford multiple levels of regulation. The levels of control include hormone metabolism, AR affinity for ligands, HRE, and mesenchymal-epithelial interactions. The overall effect of these phenomena may be the basis for the diverse response of tissues to androgen. Future studies on androgens and AR should elucidate the importance of protein-protein interactions and the role of ARE in imparting control over gene expression specificity. Other areas of research including analysis of receptor phosphorylation, cell cycle biology, and gene methylation will undoubtedly contribute to our understanding of the androgens' effect on expression of diverse androgen responsive genes.

10. ACKNOWLEDGMENTS

This work was supported by NIH grants AG00213 (WBE), AG00451 (WBE), and CA55639 (CC).

11. REFERENCES

1. C. Chang, J. Kokontis & S. Liao: Molecular cloning of human and rat cDNA encoding the androgen receptor. Science 240, 324-326 (1988)

2. D. B. Lubahn, T. R. Brown, J. A. Simental, H. N. Higgs, C. J. Migeon, W. M. Wilson & F. S. French: Sequence of the intron/exon junctions of the coding region of the human androgen receptor gene and identification of a point mutation in a family with complete androgen insensitivity. Proc Natl Acad Sci USA 86, 9534-9538 (1989)

3. C. Chang, J. Kokontis & S. T. Liao: Structural analysis of complementary DNA and amino acid sequences of human and rat androgen receptors. Proc Natl Acad Sci USA 85, 7211-7215 (1988)

4. W. D. Tilley, M. Marcelli, J. D. Wilson & M. J. McPhaul: Characterization and expression of a cDNA encoding the human androgen receptor. Proc Natl Acad Sci USA 86, 327-331 (1989)

5. C. Chang, C. Wang, H. F. DeLuca, T. K. Ross & C. C. Shih: Characterization of human androgen receptor overexpressed in the baculovirus system. Proc Natl Acad Sci USA 89, 5946-50 (1992)

6. G. Jenster, H. A. van der Korput, C. van Vroonhoven, T. H. van der Kwast, J. Trapman & A. O. Brinkmann: Domains of the human androgen receptor involved in steroid binding, transcriptional activation, and subcellular localization. Mol Endocrinol 5, 1396-404 (1991)

7. N. L. Chamberlain, E. D. Driver & R. L. Miesfeld: The length and location of CAG trinucleotide repeats in the androgen receptor N-terminal domain affect transactivation function. Nucleic Acids Research 22, 3181-6 (1994)

8. W. W. He, J. K. Lindzey, J. L. Prescott, M. V. Kumar & D. J. Tindall: The androgen receptor in the testicular feminized (Tfm) mouse may be a product of internal translation initiation. Receptor 4, 121-34 (1994)

9. M. J. McPhaul, M. Marcelli, W. D. Tilley, J. E. Griffin, R. F. Isidro-Gutierrez & J. D. Wilson: Molecular basis of androgen resistance in a family with a qualitative abnormality of the androgen receptor and responsive to high-dose androgen therapy. J Clin Invest 87, 1413-21 (1991)

10. J. A. Simental, M. Sar, M. V. Lane, F. S. French & E. M. Wilson: Transcriptional activation and nuclear targeting signals of the human androgen receptor. Journal of Biological Chemistry 266, 510-8 (1991)

11. P. De Vos, F. Claessens, L. Celis, B. Peeters, W. Rombauts, W. Heyns & G. Verhoeven: Nuclear extracts enhance the interaction of fusion proteins containing the DNA-binding domain of the androgen and glucocorticoid receptor with androgen and glucocorticoid response elements. J Steroid Biochem Mol Biol 48, 317-23 (1994)

12. P. De Vos, F. Claessens, J. Winderickx, P. Van Dijck, L. Celis, B. Peeters, W. Rombauts, W. Heyns & G. Verhoeven: Interaction of androgen response elements with the DNA-binding domain of the rat androgen receptor expressed in Escherichia coli. Journal of Biological Chemistry 266, 3439-43 (1991)

13. M. Marcelli, S. Zoppi, P. B. Grino, J. E. Griffin, J. D. Wilson & M. J. McPhaul: A mutation in the DNA-binding domain of the androgen receptor gene causes complete testicular feminization in a patient with receptor-positive androgen resistance. J Clin Invest 87, 1123-6 (1991)

14. I. Mowszowicz, H. J. Lee, H. T. Chen, C. Mestayer, M. C. Portois, S. Cabrol, P. Mauvais-Jarvis & C. Chang: A point mutation in the second zinc finger of the DNA-binding domain of the androgen receptor gene causes complete androgen insensitivity in two siblings with receptor-positive androgen resistance. Mol Endocrinol 7, 861-9 (1993)

15. C. A. Quigley, B. A. J. Evans, J. A. Simental, K. B. Marscheke, S. M., D. B. Lubahn, P. Davies, L. A. Hughes, E. M. Wilson & F. S. French: Complete androgen insensitivity due to deletion of exon c of the androgen receptor gene highlights the functional importance of the second zinc finger of the androgen receptor in vivo. Mol Endocrinol 6, 1103-1112 (1992)

16. S. Zoppi, M. Marcelli, J. P. Deslypere, J. E. Griffin, J. D. Wilson & M. J. McPhaul: Amino acid substitutions in the DNA-binding domain of the human androgen receptor are a frequent cause of receptor-binding positive androgen resistance. Mol Endocrinol 6, 409-15 (1992)

17. Z. X. Zhou, M. Sar, J. A. Simental, M. V. Lane & E. M. Wilson: A ligand-dependent bipartite nuclear targeting signal in the human androgen receptor. Requirement for the DNA-binding domain and modulation by NH2-terminal and carboxyl-terminal sequences. Journal of Biological Chemistry 269, 13115-23 (1994)

18. J. A. Simental, M. Sar & E. M. Wilson: Domain functions of the androgen receptor. J Steroid Biochem Mol Biol 43, 37-41 (1992)

19. G. Jenster, J. Trapman & A. O. Brinkmann: Nuclear import of the human androgen receptor. Biochem J 293, 761-8 (1993)

20. T. Nemoto, Y. Ohara-Nemoto, S. Shimazaki & M. Ota: Dimerization characteristics of the DNA- and steroid-binding domains of the androgen receptor. J Steroid Biochem Mol Biol 50, 225-33 (1994)

21. C. I. Wong, Z. X. Zhou, M. Sar & E. M. Wilson: Steroid requirement for androgen receptor dimerization and DNA binding. Modulation by intramolecular interactions between the NH2-terminal and steroid-binding domains. Journal of Biological Chemistry 268, 19004-12 (1993)

22. P. J. Kallio, J. J. Palvimo, M. Mehto & O. A. Janne: Analysis of androgen receptor-DNA interactions with receptor proteins produced in insect cells. Journal of Biological Chemistry 269, 11514-22 (1994)

23. A. O. Brinkmann & J. Trapman: Androgen receptor mutants that affect normal growth and development. Cancer Surveys 14, 95-111 (1992)

24. M. J. McPhaul, M. Marcelli, S. Zoppi, J. E. Griffin & J. D. Wilson: Genetic basis of endocrine disease. 4. The spectrum of mutations in the androgen receptor gene that causes androgen resistance. J Clinical Endocrinol Metab 76, 17-23 (1993)

25. J. Veldscholte, C. Ris-Stalpers, G. G. Kuiper, G. Jenster, C. Berrevoets, E. Claassen, H. C. van Rooij, J. Trapman, A. O. Brinkmann & E. Mulder: A mutation in the ligand binding domain of the androgen receptor of human LNCaP cells affects steroid binding characteristics and response to anti-androgens. Biochemical & Biophysical Research Communications 173, 534-40 (1990)

26. C. Ris-Stalpers, M. C. T. Verleun-Mooijman, J. Trapman & A. O. Brinkmann: Threonine on amino acid postion 868 in the human androgen receptor is essential for androgen binding specificity and functional activity. Biochem Biophys Res Commun 196, 173-180 (1993)

27. M. Marcelli, W. D. Tilley, S. Zoppi, J. E. Griffin, J. D. Wilson & M. J. McPhaul: Androgen resistance associated with a mutation of the androgen receptor at amino acid 772 (Arg----Cys) results from a combination of decreased messenger ribonucleic acid levels and impairment of receptor function. J Clinical Endocrinol Metab 73, 318-25 (1991)

28. H. P. Gerber, K. Seipel, O. Georgiev, M. Hofferer, M. Hug & S. Rusconi: Transcriptional activation modulated by homopolymeric glutamine and proline stretches. Science 263, 808-811 (1994)

29. J. A. Gustafsson & A. Pousette: Demonstration and partial characterization of cytosol receptors for testosterone. Biochemistry 14, 3094-3101 (1975)

30. D. A. Husmann, C. M. Wilson, M. J. McPhaul, W. D. Tilley & J. D. Wilson: Antipeptide antibodies to two distinct regions of the androgen receptor localize the receptor protein to the nuclei of target cells in the rat and human prostate. Endocrinology 126, 2359-68 (1990)

31. W. D. Tilley, S. S. Lim-Tio, D. J. Horsfall, J. O. Aspinall, V. R. Marshall & J. M. Skinner: Detection of discrete androgen receptor epitopes in prostate cancer by immunostaining: measurement by color video image analysis. Cancer Res 54, 4096-102 (1994)

32. H. Takeda, G. Chodak, S. Mutchnik, T. Nakamoto & C. Chang: Immunohistochemical localization of androgen receptors with mono- and polyclonal antibodies to androgen receptor. J Endocrinol 126, 17-25 (1990)

33. C. Chang, C. Whelen, T. Popovich, J. Kokontis & S. Liao: Fusion proteins containing androgen receptor sequences and their use in the production of poly- and monoclonal anti-androgen receptor antibodies. Endocrinology 125, 1097-1099 (1989)

34. P. J. Janssen, A. O. Brinkmann, W. J. Boersma & T. H. van der Kwast: Immunohistochemical detection of the androgen receptor with monoclonal antibody F39.4 in routinely processed, paraffin-embedded human tissues after microwave pre-treatment. J Histochem Cytochem 42, 1169-1175 (1994)

35. D. B. Lubahn, D. R. Joseph, P. M. Sullivan, H. F. Willard, F. S. French & E. M. Wilson: Cloning of the human androgen receptor complementary DNA and localization to the X chromosome. Science 240, 327-330 (1988)

36. L. Fischer, D. Catz & D. Kelley: An androgen receptor mRNA isoform associated with hormone-induced cell proliferation. Proc Natl Acad Sci USA 90, 8254-8258 (1993)

37. L. H. Burgess & R. J. Handa: Hormonal regulation of androgen receptor mRNA in the brain and anterior pituitary gland of the male rat. Brain Res Mol Brain Res 19, 31-38 (1993)

38. W. J. Young, E. B. Roecker, R. Weindruch & C. Chang: Quantitation of androgen receptor mRNA by competitive reverse transcription-polymerase chain reaction. Endocrine J 2, 321-329 (1994)

39. H. Takeda & C. Chang: Immunohistochemical and in-situ hybridization analysis of androgen receptor expression during the development of the mouse prostate gland. J Endocrinol 129, 83-9 (1991)

40. D. A. Husmann, M. J. McPhaul & J. D. Wilson: Androgen receptor expression in the developing rat prostate is not altered by castration, flutamide, or suppression of the adrenal axis. Endocrinology 128, 1902-6 (1991)

41. P. Henttu, S. S. Liao & P. Vihko: Androgens up-regulate the human prostate-specific antigen messenger ribonucleic acid (mRNA), but down-regulate the prostatic acid phosphatase mRNA in the LNCaP cell line. Endocrinol 130, 766-72 (1992)

42. V. E. Quarmby, W. G. Yarbrough, D. B. Lubahn, F. S. French & E. M. Wilson: Autologous down-regulation of androgen receptor messenger ribonucleic acid. Mol Endocrinol 4, 22-28 (1990)

43. H. Takeda, T. Nakamoto, J. Kokontis, G. W. Chodak & C. Chang: Autoregulation of androgen receptor expression in rodent prostate: Immunohistochemical and in situ hybridization analysis. Biochem Biophys Res Comm 177, 488-496 (1991)

44. P. C. Supakar, C. S. Song, M. H. Jung, M. A. Slomczynska, J. Kim, R. L. Vellanoweth, B. Chatterjee & A. K. Roy: A novel regulatory element associated with age-dependent expression of the rat androgen receptor gene. J Biol Chem 268, 26400-26408 (1993)

45. H. Nakagama, T. Gunji, S. Ohnishi, T. Kaneko, T. Ishikawa, R. Makino, J. Shiga, F. Takaku & M. Imawari: Expression of androgen receptor mRNA in human hepatocellular carcinomas and hepatoma cell lines. Hepatology 14, 99-102 (1991)

46. E. Rajpert-DeMeyts & N. E. Skakkebaek: Immunohistochemical identification of androgen receptors in germ cell neoplasia. J Endocrinol 135, R1-R4 (1992)

47. S. Chadha, B. R. Rao, B. J. Slotman, C. C. van Vroonhoven & T. H. van der Kwast: An immunohistochemical evaluation of androgen and progesterone receptors in ovarian tumors. Human Pathol 24, 90-95 (1993)

48. L. X. Shan, M. C. Rodriguez & O. A. Janne: Regulation of androgen receptor protein and mRNA concentrations by androgens in rat ventral prostate and seminal vesicles and in human hepatoma cells. Mol Endocrinol 4, 22-28 (1990)

49. D. A. Wolf, T. Herzinger, H. Hermeking, D. Blaschke & W. Horz: Transcriptional and posttranscriptional regulation of human androgen receptor expression by androgen. Mol Endocrinol 7, 924-936 (1993)

50. M. Kaufman, L. Pinsky & R. Feder-Hollander: Defective up-regulation of the androgen receptor in human androgen insensitivity. Nature 293, 735-737 (1981)

51. N. F. Gonzalez-Cadavid, R. S. Swerdloff, C. A. Lemmi & J. Rajfer: Expression of the androgen receptor gene in rat penile tissue and cells during sexual maturation. Endocrinol 129, 1671-1678 (1991)

52. G. Pergola, X. Xu, S. Yang, R. Giorgino & P. Bjorntorp: Up-regulation of androgen receptor binding in male rat fat pad adipose precursor cells exposed to testosterone: study in a whole cell binding assay system. J Steroid Biochem 37, 553-558 (1990)

53. J. A. Ruizeveld de Winter, W. M. van Weerden, P. W. Faber, G. J. van Steenbrugge, A. O. Brinkmann & T. H. van der Kwast: Regulation of androgen receptor expression in the human hetero-transplantable prostate carcinoma PC-82. Endocrinol 131, 3045-3050 (1992)

54. A. Krongrad, C. M. Wilson, J. D. Wilson, D. R. Allman & M. J. McPhaul: Androgen increases androgen receptor protein while decreasing receptor mRNA in LNCaP cells. Mol Cell Endocrinol 76, 79-88 (1991)

55. A. Mizokami, H. Saiga, T. Matsui, T. Mita & A. Sugita: Regulation of androgen receptor by androgen and epidermal growth factor in a human prostatic cancer cell line, LNCaP. Endocrinologia Japonica 39, 235-243 (1992)

56. B. M. Sanborn, L. A. Caston, C. Chang, S. Liao, R. Speller, L. D. Porter & C. Y. Ku: Regulation of androgen receptor mRNA in rat Sertoli and peritubular cells. Biol Repro 45, 634-41 (1991)

57. E. Reiter, P. Bonnet, B. Sente, D. Dombrowicz, J. Level, J. Closset & G. Hennen: Growth hormone and prolactin stimulate androgen receptor, insulin-like growth factor-I (IGF-I) receptor levels in the prostate of immature rats. Mol Cell Endocrinol 88, 77-87 (1992)

58. P. Henttu & P. Vihko: Growth factor regulation of gene expression in the human prostatic carcinoma cell line LNCaP. Cancer Res 53, 1051-8 (1993)

59. R. E. Hall, W. D. Tilley, M. J. McPhaul & R. L. Sutherland: Regulation of androgen receptor gene expression by steroids and retinoic acid in human breast-cancer cells. Int J Cancer 52, 778-784 (1992)

60. P. W. Faber, H. C. J. van Rooij, H. A. G. M. van der Korput, W. M. Baarends, A. O. Brinkmann, J. A. Grootegoed & J. Trapman: Characterization of the human androgen receptor transcription unit. J Biol Chem 266, 10743-10749 (1991)

61. W. D. Tilley, M. Marcelli & M. J. McPhaul: Expression of the human androgen receptor gene utilizes a common promoter in diverse human tissues and cell lines. J Biol Chem 265, 13776-13781 (1990)

62. P. W. Faber, H. C. J. van Rooij, H. J. Schipper, A. O. Brinkmann & J. Trapman: Two different, overlapping pathways of transcription initiation are active on the TATA-less human androgen receptor promoter. J Biol Chem 268, 9296-9301 (1993)

63. A. Mizokami, S. Yeh & C. Chang: Identification of 3',5'-cyclic adenosine monophosphate response element and other cis-acting elements in the human androgen receptor gene promoter. Mol Endocrinol 8, 77-88 (1994)

64. L. J. Blok, A. P. N. Themmen, A. H. F. M. Peters, J. Trapman, W. M. Baarends, J. W. Hoogerbrugge & J. A. Grootegoed: Transcriptional regulation of androgen receptor gene expression in Sertoli cells and other cell types. Mol Cell Endocrinol 88, 153-164 (1992)

65. J. Lindzey, M. Grossmann, M. V. Kumar & D. J. Tindall: Regulation of the 5'-flanking region of the mouse androgen receptor gene by cAMP and androgen. Mol Endocrinol 7, 1530-1540 (1993)

66. P. C. Supakar, M. H. Jung, C. S. Song, B. Chatterjee & A. K. Roy: Nuclear factor kB functions as a negative regulator for the rat androgen receptor gene and NF-kB activity increases during the age-dependent desensitization of the liver. J Biol Chem 270, 837-842 (1995)

67. A. Mizokami & C. Chang: Induction of translation by the 5'-untranslated region of human androgen receptor mRNA. J Biol Chem 269, 25655-25659 (1994)

68. P. K. Siiteri & J. D. Wilson: Testosterone formation and metabolism during male sexual differentiation in the human embryo. J Clin Endocrinol Metab 38, 113-125 (1974)

69. J. Imperato-McGinley: 5-alpha-reductase deficiency. Current Therapy in Endocrinology & Metabolism 5, 351-4 (1994)

70. J. Imperato-McGinley, M. Miller, J. D. Wilson, R. E. Peterson, C. Shackleton & D. C. Gajdusek: A cluster of male pseudohermaphrodites with 5 alpha-reductase deficiency in Papua New Guinea. Clinical Endocrinology 34, 293-8 (1991)

71. J. Imperato-McGinley, R. S. Sanchez, J. R. Spencer, B. Yee & E. D. Vaughan: Comparison of the effects of the 5 alpha-reductase inhibitor finasteride and the antiandrogen flutamide on prostate and genital differentiation: dose-response studies. Endocrinology 131, 1149-56 (1992)

72. E. M. Wilson & F. S. French: Binding properties of androgen receptors: evidence for identical receptors in rat testis, epididymis and prostate. J Biol Chem 251, 5620-5629 (1976)

73. P. B. Grino, J. E. Griffin & J. D. Wilson: Testosterone at high concentrations interacts with the human androgen receptor similarly to dihydrotestosterone. Endocrinology 126, 1165-72 (1990)

74. Z.-X. Zhou, M. V. Lane, J. A. Kemppainen, F. S. French & E. M. Wilson: Specificity of ligand-dependent androgen receptor stabilization: Receptor domain interactions influence ligand dissociation and receptor stability. Mol Endocrinol 9, 208-218 (1995)

75. V. A. Randall: Androgens and human hair growth. Clin Endocrinol 40, 439-57 (1994)

76. M. B. Hodgins, R. Choudhry, G. Parker, R. F. Oliver, C. A. Jahoda, A. P. Withers, A. O. Brinkmann, T. H. van der Kwast, W. J. Boersma, K. M. Lammers & a. et: Androgen receptors in dermal papilla cells of scalp hair follicles in male pattern baldness. Annals New York Acad Sci 642, 448-51 (1991)

77. R. Choudhry, M. B. Hodgins, T. H. Van der Kwast, A. O. Brinkmann & W. J. Boersma: Localization of androgen receptors in human skin by immunohistochemistry: implications for the hormonal regulation of hair growth, sebaceous glands and sweat glands. J Endocrinol 133, 467-75 (1992)

78. V. A. Randall, M. J. Thornton & A. G. Messenger: Cultured dermal papilla cells from androgen-dependent human hair follicles (e.g. beard) contain more androgen receptors than those from non-balding areas of scalp. J Endocrinol 133, 141-7 (1992)

79. M. E. Sawaya: Biochemical mechanisms regulating human hair growth. Skin Pharmacol 7, 5-7 (1994)

80. G. R. Cunha, E. T. Alarid, T. Turner, A. A. Donjacour, E. L. Boutin & B. A. Foster: Normal and abnormal development of the male urogenital tract. Role of androgens, mesenchymal-epithelial interactions, and growth factors. J Andrology 13, 465-75 (1992)

81. G. Verhoeven, K. Swinnen, J. Cailleau, L. Deboel, L. Rombauts & W. Heyns: The role of cell-cell interactions in androgen action. J Steroid Biochem Mol Biol 41, 487-494 (1992)

82. M. E. Gleave, J. T. Hsieh, A. C. von Eschenbach & L. W. Chung: Prostate and bone fibroblasts induce human prostate cancer growth in vivo: implications for bidirectional tumor-stromal cell interaction in prostate carcinoma growth and metastasis. J Urol 147, 1151-9 (1992)

83. G. Vollmer, H. Michna, M. R. Schneider & R. Knuppen: Stromal expression of tenascin is inversely correlated to epithelial differentiation of hormone dependent tissues. J Steroid Biochem Mol Biol 48, 487-94 (1994)

84. T. Seki, R. Ideta, M. Shibuya & K. Adachi: Isolation and characterization of cDNA for an androgen-regulated mRNA in the flank organ of hamsters. J Invest Derm 96, 926-31 (1991)

85. B. A. Araneo, T. Dowell, M. Diegel & R. A. Daynes: Dihydrotestosterone exerts a depressive influence on the production of interleukin-4 (IL-4), IL-5, and g-interferon, but not IL-2 by activated murine T cells. Blood 78, 688-699 (1991)

86. P. J. Sheridan: Can a single androgen receptor fill the bill? Mol Cell Endocrinol 76, C39-C45 (1991)

87. J. P. Deslypere, M. Young, J. D. Wilson & M. J. McPhaul: Testosterone and 5 alpha-dihydrotestosterone interact differently with the androgen receptor to enhance transcription of the MMTV-CAT reporter gene. Mol Cell Endocrinol 88, 15-22 (1992)

88. T. Yamamoto, T. Nishida, N. Miyajima, S. Kawai, T. Ooi & K. Toyoshima: The v-erb B gene of Avian erythroblastosis virus is a member of the Src gene family. Cell 35, 71-78 (1983)

89. L. A. Aarden: Hybridoma growth factor. Ann New York Acad Sci 557, 192-199 (1989)

90. W. G. Yarbrough, V. E. Quarmby, J. A. Simental, D. R. Joseph, M. Sar, D. B. Lubahn, K. L. Olsen, F. S. French & E. M. Wilson: A single base mutation in the androgen receptor gene causes androgen insensitivity in the testicular feminized rat. J Biol Chem 265, 8893-900 (1990)

91. P. S. Rennie, N. Bruchovsky, K. H. Leco, P. C. Sheppard, S. A. McQueen, H. Cheng, R. Snoek, A. Hamel, M. E. Bock, B. S. MacDonald, B. E. Nickel, C. Chang, S. Liao, P. A. Cattini & R. J. Matusik: Characterization of two cis-acting DNA elements involved in the androgen regulation of the probasin gene. Mol Endocrinol 7, 23-36 (1993)

92. K. C. Ho, K. B. Marschke, J. Tan, S. G. Power, E. M. Wilson & F. S. French: A complex response element in intron 1 of the androgen-regulated 20-kDa protein gene displays cell type-dependent androgen receptor specificity. J Biol Chem 268, 27226-35 (1993)

93. P. Murtha, D. J. Tindall & C. Y. F. Young: Androgen induction of a human prostate-specific kallikrein, hKLK2: Characterization of an androgen response element in the 5' promoter region of the gene. Biochemistry 32, 6459-6464 (1993)

94. P. J. Roche, S. A. Hoare & M. G. Parker: A consensus DNA-binding site for the androgen receptor. Mol Endocrinol 6, 2229-2235 (1992)

95. M. Nonaka, K. Nakayama, Y. D. Yeul, A. Shimizu & M. Takahashi: Molecular cloning and characterization of complementary and genomic DNA clones for mouse C4 and Slp. Immunological Rev 87, 81-99 (1985)

96. C. Hemenway & D. M. Robins: DNase I-hypersensitive sites associated with expression and hormonal regulation of mouse C4 and Slp genes. Proc Natl Acad Sci USA 84, 4816-20 (1987)

97. F. Loreni, J. Stavenhagen, M. Kalff & D. M. Robins: A complex androgen-responsive enhancer resides 2 kilobases upstream of the mouse Slp gene. Mol Cell Biol 8, 2350-60 (1988)

98. A. J. Adler, A. Scheller, Y. Hoffman & D. M. Robins: Multiple components of a complex androgen-dependent enhancer. Mol Endocrinol 5, 1587-96 (1991)

99. A. J. Adler, M. Danielsen & D. M. Robins: Androgen-specific gene activation via a consensus glucocorticoid response element is determined by interaction with nonreceptor factors. Proc Natl Acad Sci USA 89, 11660-3 (1992)

100. A. J. Adler, A. Scheller & D. M. Robins: The stringency and magnitude of androgen-specific gene activation are combinatorial functions of receptor and nonreceptor binding site sequences. Mol Cell Biol 13, 6326-35 (1993)

101. U. Strahle, M. Boshart, G. Klock, F. Stewart & G. Schutz: Glucocorticoid- and progesterone-specific effects are determined by differential expression of the respective hormone receptors. Nature 339, 629-32 (1989)

102. J. Trapman, C. Ris-Stalpers, J. A. G. M. van der Korput, F. F. J. M. Kuiper, P. W. Faber, J. C. Romijn, E. Mulder & A. O. Brinkmann: The androgen receptor: Functional structure and expression in transplanted human prostate tumors and prostate tumor cell lines. J Steroid Biochem Mol Biol 37, 837-842 (1990)

103. N. Sato, H. Suzuki & J. Shimazaki: Mechanism of loss of androgen dependency in androgen-dependent tumor (Japanese). Human Cell 6, 170-175 (1993)

104. G. W. Chodak, D. M. Kranc, L. A. Puy, H. Takeda, K. Johnson & C. Chang: Nuclear localization of androgen receptor in heterogeneous samples of normal, hyperplastic and neoplastic human prostate. J Urol 147, 798-803 (1992)

105. M. V. Sadi & E. R. Barrack: Image analysis of androgen receptor immunostaining in metastatic prostate cancer. Heterogeneity as a predictor of response to hormonal therapy. Cancer 71, 2574-2580 (1992)

106. T. van der Kwast H, J. Schalken, J. Ruizeveld de Winter A, C. van Vroonhoven C, J, E. Mulder, W. Boersma & J. Trapman: Androgen receptors in endocrine-therapy-resistant human prostate cancer. Int J Cancer 48, 189-193 (1991)

107. J. P. Gaddipati, D. G. McLeod, H. B. Heidenberg, I. A. Sesterhenn, M. J. Finger, J. W. Moul & S. Srivastava: Frequent detection of codon 877 mutation in the androgen receptor gene in advanced prostate cancers. Cancer Res 54, 2861-2864 (1994)

108. M. P. Schoenberg, J. M. Hakimi, S. Wang, G. S. Bova, J. I. Epstein, K. H. Fischbeck, W. B. Isaacs, P. C. Walsh & E. R. Barrack: Microsatellite mutation (CAG24 to 18) in the androgen receptor gene in human prostate cancer. Biochem Biophys Res Comm 198, 74-80 (1994)

109. J. R. Newmark, D. O. Hardy, D. C. Tonb, B. S. Carter, J. I. Epstein, W. B. Isaacs, T. R. Brown & E. R. Barrack: Androgen receptor gene mutations in human prostate cancer. Proc Natl Acad Sci USA 89, 6319-23 (1992)

110. H. Suzuki, N. Sato, Y. Watabe, M. Masai, S. Seino & J. Shimazaki: Androgen receptor gene mutations in human prostate cancer. J Steroid Biochem Mol Biol 46, 759-65 (1993)

111. Z. Culig, A. Hobisch, M. V. Cronauer, A. C. Cato, A. Hittmair, C. Radmayr, J. Eberle, G. Bartsch & H. Klocker: Mutant androgen receptor detected in an advanced-stage prostatic carcinoma is activated by adrenal androgens and progesterone. Mol Endocrinol 7, 1541-50 (1993)

112. S. E. Harris, Z. Rong, M. A. Harris & D. D. Lubahn: Androgen receptor in human prostate carcinoma LNCaP/ADEP cells contains a mutation which alters the specificity of the steroid-dependent transcriptional activation region. Endocrinology 126(Supp), 93-97 (1990)

113. G. Wilding, M. Chen & E. P. Gelmann: Aberrant response in vitro of hormone-responsive prostate cancer cells to antiandrogens. Prostate 14, 103-115 (1989)

114. J. Veldscholte, C. A. Berrevoets, A. O. Brinkmann, J. A. Grootegoed & E. Mulder: Anti-androgens and the mutated androgen receptor of LNCaP cells: differential effects on binding affinity, heat-shock protein interaction, and transcription activation. Biochemistry 31, 2393-9 (1992)

115. J. L. Ware: Growth factors and their receptors as determinants in the proliferation and metastasis of human prostate cancer. Cancer Met Rev 12, 287-301 (1993)

116. Z. Culig, A. Hobisch, M. V. Cronauer, C. Radmayr, J. Trapman, A. Hittmair, G. Bartsch & H. Klocker: Androgen receptor activation in prostatic tumor cell lines by insulin-like growth factor-I, keratinocyte growth factor, and epidermal growth factor. Cancer Res 54, 5474-8 (1994)

117. M. C. Luke & D. S. Coffey: Human androgen receptor binding to the androgen response element of prostate specific antigen. J Androl 15, 41-51 (1994)