Deregulated expression of the c-myc proto-oncogene can lead to apoptosis under certain physiological conditions. By introducing a conditionally active Myc allele into primary embryo fibroblasts null for p53, and into fibroblasts without endogenous p53 expression but ectopically expressing a temperature-sensitive p53 allele, we show that expression of wild-type p53 is required for susceptibility to Myc-mediated apoptosis. Although ectopic expression of wild-type p53 blocked cells in the G 1 phase of the cell cycle, G I arrest by isoleucine starvation, in a manner independent of p53, did not confer susceptibility to apoptosis. Thus, growth arrest per se is not sufficient to induce Myc-mediated apoptosis; instead, a property intrinsic to p53 is specifically required. Moreover, apoptosis did not require induction of p53 target proteins, including the cyclin-dependent kinase inhibitor p21 wa~l/cipl. Therefore, the role of p53 in apoptosis may be distinct from its role in cell cycle arrest.

Complementary DNAs (cDNAs) encoding androgen receptors were obtained from human testis and rat ventral prostate cDNA libraries. The amino acid sequence deduced from the nucleotide sequences of the cDNAs indicated the presence of a cysteine-rich DNA-binding domain that is highly conserved in all steroid receptors. The human cDNA was transcribed and the RNA product was translated in cell-free systems to yield a 76-kilodalton protein. The protein was immunoprecipitable by human autoimmune antibodies to the androgen receptor. The protein bound androgens specifically and with high affinity.

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.

Structural analysis of cDNAs for human and rat androgen receptors (ARs) indicates that the amino-terminal regions of ARs are rich in oligo-and poly(amino acid) motifs as in some homeotic genes. The human AR has a long stretch of repeated glycines, whereas rat AR has a long stretch of glutamines. There is a considerable sequence similarity among ARs and the receptors for glucocorticoids, progestins, and mineralocorticoids within the steroid-binding domains. The cysteine-rich DNA-binding domains are well conserved. Translation of mRNA transcribed from AR cDNAs yielded 94-and 76-kDa proteins and smaller forms that bind to DNA and have high affinity toward androgens. These rat or human ARs were recognized by human autoantibodies to natural ARs. Molecular hybridization studies, using AR cDNAs as probes, indicated that the ventral prostate and other male accessory organs are rich in AR mRNA and that the production of AR mRNA in the target organs may be autoregulated by androgens.

The cDNA for a member of the nuclear receptor family was cloned and named ubiquitous receptor (UR), since UR protein and mRNA are detected in many cell types. Rat UR/human retinoid X receptor a (hRXRa) heterodimers bound preferentially to double-stranded oligonucleotide direct repeats having the consensus half-site sequence AGGTCA and 4-nt spacing (DR-4). Coexpression of UR in COS-1 cells inhibited the stimulation of chloramphenicol acetyltransferase (CAT) reporter gene expression by hRXRa and human retinoic acid, receptor a in the presence of all-transretinoic acid when DR-4 (but not DR-5) was present upstream of the promoter of a CAT reporter gene (DR-4-CAT). UR expression also inhibited the activation of a DR-4-CAT reporter gene by hRXRa and 9-cis-retinoic acid or by thyroid hormone receptor a in the, presence of thyroid hormone. However, in the absence of 9-cis-retinoic acid, UR in combination with hRXRa stimulted DR-4-CAT expression. Coex (4,5). The nature of the DNA flanking these half-sites also is important in determining the specificity of a response element (6). Homodimers of the thyroid hormone/retinoid receptor family are also able to modulate transcriptional activity in different ways than their heterodimeric forms (7). The effect of these homo-and heterodimeric receptors on transcriptional activity also depends on their occupancy by specific ligands (8). The complexity of this network of interacting factors is increasing with the discovery of new members of this superfamily of nuclear receptors, many of which are called orphan receptors, since they lack known ligands. An interplay of receptors, ligands, response elements, and yet-to-be-discovered factors may ultimately control the activity of these transcriptional factors and ensure the appropriate cellular responses during development and in the adult.We report here our discovery of a member of the nuclear receptor family of transcription factors that we have named ubiquitous receptor (UR),t because of its wide tissue distribution. UR is not an isoform of any known receptor and interacts with the response elements and network of receptors in the thyroid receptor (TR)/retinoic acid receptor (RAR) subfamily.

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When the human prostate cancer cell line, LNCaP 104-S, the growth of which is stimulated by physiological levels of androgen, is cultured in androgen-depleted medium for >100 and androgen-independent LNCaP 104-R2t sublines were isolated as described (6). The characteristics of these cells in vitro were confirmed before injection into nude mice. Briefly, proliferation of LNCaP 104-S cells increased 10-to 13-fold in medium containing charcoal-treated fetal bovine serum (FBS) and 0.1 nM synthetic androgen R1881, compared with cells cultured in medium containing charcoal-treated FBS without added androgen. LNCaP 104-R2 cells grew in medium supplemented with charcoal-treated FBS without additional androgen. Their proliferation was not stimulated but was repressed by 0.1 nM R1881. LNCaP 104-S cells were maintained in DMEM (GIBCO) supplemented with 1 nM S5t-dihydrotestosterone and 10% FBS (Summit Biotechnology, Ft. Collins, CO), and LNCaP 104-R2 cells were maintained in DMEM supplemented with 10% FBS treated with charcoal to remove steroid (6). PC-3 and MCF-7 cell lines were obtained from the American Type Culture Collection (Rockville, MD) and were maintained in DMEM supplemented with 10% FBS.Animals. BALB/c athymic (nude) male (LNCaP, PC-3 cell lines) and female (MCF-7 cell line) mice (Taconic, Germantown, NY), 5 to 7 weeks old, were used. Mice were housed in a pathogen-free environment, four to five mice per cage. Cages (filter top), bedding, and water were autoclaved before use. Feed was irradiated Pico Lab Mouse Chow 20 5058 (Purina).Abbreviations: AR, androgen receptor; FBS, fetal bovine serum; PSA, prostate-specific antigen; TP, testosterone propionate.

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