The androgen receptor (AR), a nuclear transcription factor, mediates male sexual differentiation, and its excessive action is associated with prostate cancer. We have characterized a negative regulatory domain in the AR hinge region, which interacted with filamin A (FLNa), an actin-binding cytoskeletal protein. FLNa interfered with AR interdomain interactions and competed with the coactivator transcriptional intermediary factor 2 to specifically down-regulate AR function. Although full-length FLNa was predominantly cytoplasmic, a C-terminal 100-kDa fragment of FLNa colocalized with AR to the nucleus. This naturally occurring FLNa fragment repressed AR transactivation and disrupted AR interdomain interactions and transcriptional intermediary factor 2-activated AR function in a manner reminiscent of full-length FLNa, raising the possibility that the inhibitory effects of cytoplasmic FLNa may be transduced through this fragment, which can localize to the nucleus and form part of the pre-initiation complex. This unanticipated role of FLNa adds to the growing evidence for the involvement of cytoskeletal proteins in transcription regulation. T he androgen receptor (AR), a member of the steroid͞ nuclear receptor superfamily, mediates male morphogenesis in utero, gametogenesis and prostate growth at puberty, and the development of prostatic cancer in older men. The AR has four principal domains: a large N-terminal transactivation domain (ARTAD), a DNA-binding domain (ARDBD), and a hinge domain, followed by the C-terminal ligand-binding domain (ARLBD). In the absence of androgen, it is generally accepted that AR is cytoplasmic. Androgens bind specifically to a ligandbinding pocket in the lower half of the LBD, causing a conformation change (1), the release of heat-shock proteins, and translocation of the ligand-AR complex to the nucleus, where the DBD interacts with specific response elements on the promoters of target genes. Transactivation functions reside mainly in the ARTAD, and very minimal activity can be demonstrated in the ARLBD (2, 3). Unlike other steroid receptors, transactivity depends on ligand-induced interactions between the ARTAD and ARLBD, and mutations that reduce TAD-LBD interactions affect AR activity, causing androgen insensitivity syndromes (4-6). Like other transcription regulators, the promoter-bound AR serves as a nidus to recruit cofactors that up-regulate (coactivators) or down-regulate (corepressors) AR activity. The most clearly defined class of coactivators is the p160͞steroid receptor coactivator (SRC) family. They include SRC1, transcriptional intermediary factor 2 (SRC2͞TIF2), and SRC3͞TRAM1͞A IB1͞pCIP͞ACTR͞ RAC3, who bind hydrophobic grooves in the LBDs of steroid receptors via LXXLL motifs in their nuclear-receptorinteracting boxes, draw in cAMP-response element-binding protein (CBP͞p300), pCAF, and other cofactors to modulate chromatin and initiate transcription by RNA polymerase II (7). Of the p160͞SRC proteins, TIF2 interacts most strongly with the AR (8) and, in concert with CBP͞p30...

cdc28-1N mutants progress through the G1 and S phases normally at the restrictive temperature but fail to undergo nuclear division. We have isolated a gene, NDD1, which at a high dosage suppresses the nuclear-division defect of cdc28-1N. NDD1 (nuclear division defective) is an essential gene. Its expression during the cell cycle is tightly regulated such that NDD1 RNA is most abundant during the S phase. Cells lacking the NDD1 gene arrest with an elongated bud, a short mitotic spindle, 2N DNA content, and an undivided nucleus, suggesting that its function is required for some aspect of nuclear division. We show that overexpression of Ndd1 results in the upregulation of both CLB1 and CLB2 transcription, suggesting that the suppression of cdc28-1N by NDD1 may be due to an accumulation of these cyclins. Overproduction of Ndd1 also enhances the expression of SWI5, whose transcription, like that of CLB1 and CLB2, is activated in the late S phase. Ndd1 is essential for the expression of CLB1, CLB2, and SWI5, since none of these genes are transcribed in its absence. Both CLB2 expression and its upregulation by NDD1 are mediated by a 240-bp promoter sequence that contains four MCM1-binding sites. However, Ndd1 does not appear to be a component of any of the protein complexes assembled on this DNA fragment, as indicated by gel mobility shift assays. Instead, overexpression of NDD1 prevents the formation of one of the complexes whose appearance correlates with the termination of CLB2 expression in G1. The inability of GAL1 promoter-driven CLB2 to suppress the lethality of NDD1 null mutant suggests that, in addition to CLB1 and CLB2, NDD1 may also be required for the transcription of other genes whose functions are necessary for G2/M transition.

Androgens are critical steroid hormones that determine the expression of the male phenotype. Their actions are mediated by a single androgen receptor (AR) which, upon ligand binding, translocates to the nucleus to regulate the expression of androgen-responsive genes. Mutations that disrupt AR function totally result in the complete feminization of 46 XY individuals and the complete androgen insensitivity syndrome. Studies have revealed that AR mutations that do not lead to complete abrogation of its activity can cause a wide spectrum of milder androgen insensitivity syndromes, from ambiguous genitalia in newborn infants to 'idiopathic' male infertility. Recent studies indicate that missense amino-acid substitutions in the ligand-binding domain of the AR result in infertility through a novel mechanism that involves defective protein-protein interactions between receptor domains and coactivator proteins. Independent of missense mutations, studies involving Singaporean, Australian, North American and Japanese subjects indicate that increases in length of a trinucleotide repeat (CAG) tract, encoding a polyglutamine stretch in the transactivation domain of the AR, are associated with increased risk of defective spermatogenesis and undermasculinization. This association was however not observed in European populations, suggesting that the genetic background may play a significant role in the expression of the AR defects.

Estrogens maintain female sexual health. The hormone also drives the growth of estrogen receptor (ER) positive breast tumors, and ER modulators, like tamoxifen, are used to reduce tumor recurrence. To identify phytoestrogens with possible health benefits, we screened several Traditional Chinese Medicines and encountered an extract from the leaves of Epimedium brevicornum (EB), with strong (EC50: 1.3 microg/mL) and specific ER-stimulatory activity. It increased estrogen-responsive human breast cancer cell proliferation at low doses, but paradoxically caused profound inhibition of growth at higher doses. Using bioassay-guided fractionation, we isolated and characterized a new prenylflavone, breviflavone B, which exerted biphasic stimulatory and inhibitory effects on breast cancer cell proliferation, mimicking the effects of EB. In contrast to estradiol and genistein, high doses (> 2 microM) of breviflavone B almost eliminated ERalpha protein; a process that may be mediated through increased proteasome degradation. Pre-clinical studies are needed to explore whether these prenylflavones are of value in estrogen-deficiency states and for prophylaxis of breast cancer.