A C619Y mutation in the human androgen receptor causes inactivation and mislocalization of the receptor with concomitant sequestration of SRC-1 (steroid receptor coactivator 1)

Androgen ablation therapy is a primary treatment for advanced prostate cancer, but tumors become refractive to therapy. Consequently, the role of the androgen receptors (ARs) and of mutations in the AR in prostate cancer has been a subject of much concern. In the course of analyzing tumors for mutations, we identified a somatic mutation that substitutes tyrosine for a cysteine at amino acid 619 (C619Y), which is near the cysteines that coordinate zinc in the DNA binding domain in the AR. The mutation was re-created in a wild-type expression vector and functional analyses carried out using transfection assays with androgen-responsive reporters. The mutant is transcriptionally inactive and unable to bind DNA. In response to ligand treatment, AR619Y localizes abnormally in numerous, well circumscribed predominantly nuclear aggregates in the nucleus and cytoplasm. Interestingly, these aggregates also contain the bulk of the coexpressed steroid receptor coactivator SRC-1, suggesting, in analogy to AR in spinal bulbar muscular atrophy, that this mutant may alter cellular physiology through sequestration of critical proteins. Although many inactivating mutations have been identified in androgen insensitivity syndrome patients, to our knowledge, this is the first characterization of an inactivating mutation identified in human prostate cancer.

7alpha-Iodo and 7alpha-fluoro steroids as androgen receptor-mediated imaging agents.

We have synthesized several 7alpha-fluoro (F) and 7alpha-iodo (I) analogues of 5alpha-dihydrotestosterone (5alpha-DHT) and 19-nor-5alpha-dihydrotestosterone (5alpha-NDHT) and tested them for binding to the androgen receptor and for their biological activity in an in vitro assay with cells that have been engineered to respond to androgens. The relative binding affinity to the androgen receptor determined in competition assays showed that in the androstane series the fluoro steroids have the highest affinity and that F-17alpha-CH3-DHT (4) has a higher affinity than 5alpha-DHT. All other steroids were somewhat less potent than 5alpha-DHT with F-DHT (2) = I-17alpha-CH3-DHT (3) >/= F-NDHT (6) > F-17alpha-CH3-NDHT (8) = I-DHT (1) >/= I-NDHT (5) > I-17alpha-CH3-NDHT (7). The relative biological activity in cells transfected with the androgen receptor and an androgen responsive reporter gene is 4 >> 5alpha-DHT > 2 > 6 > 3 >/= 1 >/= 8 >/= 5 > 7. The iodinated compound, I-17alpha-CH3-DHT (3), with the highest binding activity was synthesized labeled with 125I and was shown to bind with high affinity, Ka = 1.9 x 10(10) L/mol, and low nonspecific binding to the androgen receptor in rat prostatic cytosol. However, when radiolabeled [125I]-17alpha-CH3-DHT ([125I]3) was injected into castrated male rats, it showed very poor androgen receptor-mediated uptake into the rat prostate. This was unexpected in light of its superior receptor binding properties and its protection by the 17alpha-methyl group from metabolic oxidation at C-17. However, the biological potency of I-17alpha-CH3-DHT (3) was not as high as would have been expected. When I-DHT (1) and I-17alpha-CH3-DHT (3) were incubated in aqueous media at 37 degrees C they rapidly decomposed, but they were stable at 0 degrees C. The fluorinated analogue 4 treated similarly at 37 degrees C was completely stable. The products of the decomposition reaction of I-DHT (1) at 37 degrees C were identified as iodide and principally 17beta-hydroxy-5alpha-androst-7-en-3-one. The temperature dependence of this elimination reaction explains the inconsistency between the high binding to the androgen receptor (measured at 0 degrees C) and the low biological activity, as well as the poor androgen receptor mediated concentration in vivo. The fluorinated analogue F-17alpha-CH3-DHT (4) has both high affinity for the androgen receptor and high stability in aqueous media. Of the compounds tested, 4 has the highest affinity for the androgen receptor as well as the highest androgenic activity. Thus it is likely that F-17alpha-CH3-DHT 4 labeled with 18F will be an excellent receptor-mediated diagnostic imaging agent.

Transfected glucocorticoid receptor and certain GR fragments evoke cell death in malignant lymphoid, not myeloid cell lines.

Previously we have shown that glucocorticoid sensitivity could be restored to a clone of glucocorticoid-resistant leukemic T cells by transfecting them with an expression vector for the glucocorticoid receptor. Furthermore, transfection with plasmids expressing fragments of the receptor containing the DNA-binding domain resulted in constitutive loss of cells. In this paper, we report the results of transfecting both types of constructs into lines of glucocorticoid-resistant human leukemic cells of T cell, B cell, and myeloid origin. In all the lymphoid lines tested, transfection of the holoreceptor gene resulted in appearance of steroid-dependent cell death. In the same lines, transfection of glucocorticoid receptor fragments expressing amino acids 1-465* (465 residues of the normal sequence plus a novel 21 amino acid C-terminus) or expressing only 398-465* caused cell death without the addition of steroids. The amount of cell loss following transfection of these constitutively lethal fragments was in the same range as that following transfection of the holo glucocorticoid receptor plus administration of glucocorticoid. However, the cell loss due to the constitutively active fragments occurred more rapidly. Neither of the myeloid lines tested were sensitive to any of the transfected constructs, with or without added steroid.

Activation of the human androgen receptor through a protein kinase A signaling pathway.

Aberrant activation of the androgen receptor through signaling pathways independent of androgen may be responsible for the progression of prostate tumors to the rapidly proliferating androgen-independent state. In this study, the effects of protein kinase A modulators on human androgen receptor activity were tested. Using an adenoviral DNA delivery system, we demonstrate that the androgen receptor can be activated by a protein kinase A activator, forskolin, in the absence of androgen when androgen receptor is co-transfected into monkey kidney CV1 cells or human prostate PC-3 cells with androgen-responsive reporters. Immunoblotting reveals that there is no significant change in androgen receptor protein level following forskolin treatment, suggesting that the enhanced activity is due to activation of the receptor. This activation can be blocked by a protein kinase A inhibitor peptide. Two potent anti-androgens, casodex and flutamide, can significantly reduce this activation, confirming that the ligand-independent pathway is an androgen receptor-mediated phenomenon. An intact DNA binding domain of the receptor is critical for this alternate signaling pathway since mutants with reduced DNA binding ability are inactive. The phosphorylation status of the androgen receptor or associated proteins may critically modulate receptor activity and should be considered when designing improved approaches to prostate cancer therapy.

Glucocorticoids in malignant lymphoid cells: gene regulation and the minimum receptor fragment for lysis.

We have examined clones of human malignant lymphoid cells for markers that correlate with glucocorticoid-mediated cell lysis. In glucocorticoid-sensitive clones of CEM, a human T-cell lymphoblastic leukemia line, two genes correlate with glucocorticoid-induced cell lysis. The glucocorticoid receptor (GR) itself is induced by standard glucocorticoids in sensitive clones and not in insensitive clones. The phenylpyrazolo-glucocorticoid cortivazol (CVZ) is capable of lysing several clones resistant to high concentrations of standard potent glucocorticoids. When these clones were tested for cortivazol responses, they were not only lysed by cortivazol but also showed induction of GR mRNA. Thus receptor induction appears to correlate with the lysis function of receptor in these cells. To determine what parts of the GR are required for lysis, we have mapped this function by transfecting and expressing GR and GR fragment genes in a GR-deficient CEM clone. Our results indicate that none of the known trans-activation regions of the GR are required. Removal of the steroid binding domain gives a fragment that is fully constitutive. Only one and one-half "Zn fingers" of the DNA binding region are required. We also find in CEM cells rapid suppression of the c-myc protooncogene, preceding growth arrest and cell lysis by glucocorticoids. This occurs only in clones possessing both intact receptors and lysis function. Thus the simple presence of GR alone is not sufficient to guarantee c-myc down-regulation. Introduction into the cells of c-myc driven by a promoter that does not permit suppression by glucocorticoids confers resistance to steroids. Furthermore, suppression of c-myc by antisense oligonucleotides also kills the cells. Therefore, c-myc appears to be a pivotal gene related both to ability of steroid to kill and to cell viability.

Mapping the human glucocorticoid receptor for leukemic cell death.

We have mapped the regions of the glucocorticoid receptor important for killing the cells of a line of human leukemic lymphoblasts. The results show that the glucocorticoid response element-specific DNA binding domain is essential, and that only the sequence including the amino acids that subsume the first zinc finger through about half of the second zinc finger are absolutely necessary. Furthermore, in contrast to assays of receptor mutants for ability to increase gene transcription, deletion of the steroid binding domain results in a functionally constitutive receptor fully as active for cell kill as is the holoreceptor after addition of ligand. Deletion of most known transcription-activation, dimerization, ligand-binding, and nuclear translocation regions still leaves a receptor fragment highly potent for cell lethality. The results suggest that delivery systems for such a fragment could result in effective new therapies for certain malignancies.