Growth dynamics of human leiomyoma cells and inhibitory effects of the peroxisome proliferator-activated receptor-gamma ligand, pioglitazone.

Uterine leiomyomas (fibroids) are the most frequent tumour of the female reproductive tract and are the primary cause of hysterectomies in women worldwide. Effective treatment options are few. In a search for alternative treatments, we have established primary cultures of human leiomyoma cells and adjacent myometrial tissues, and documented their growth dynamics in response to estradiol (E2) and pioglitazone (PIO), a peroxisome proliferation-activated receptor-gamma (PPARgamma) ligand, currently in clinical use for type II diabetes mellitus. Human uterine primary cell cultures display morphology and desmin content consistent with their smooth muscle origin. Surprisingly, leiomyoma cells exhibited slower proliferation patterns relative to matched myometrial cells, both in the absence and presence of E2, suggesting that tumour genesis may not be because of increased growth potential but could be related to suppression of growth-inhibiting factors in vivo. PIO significantly inhibited the cell proliferation of both myometrial and leiomyoma cells in a dose-dependent manner. Our results suggest the possibility of using PPARgamma ligands, such as PIO, as therapeutic agents for the conservative management of uterine fibroids.

Filamin-A fragment localizes to the nucleus to regulate androgen receptor and coactivator functions.

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.

Androgen receptor gene and male infertility.

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.

Sex, infertility and the molecular biology of the androgen receptor.

Mutations that totally disrupt androgen receptor function cause the well known testicular feminizing syndrome or complete androgen insensitivity syndrome, wherein a 46 XY individual is completely feminized at birth. Recently it has been increasingly obvious that androgen receptor mutations not only result in the complete androgen insensitivity syndrome, but can cause a wide spectrum of milder insensitivity syndromes including ambiguous genitalia in newborn infants, and 'idiopathic' male infertility in otherwise normal males. Characterization of the molecular and structural mechanisms of androgen receptor dysfunction in these cases has led to directed hormonal therapy. Thus the differential response of a Met807Thr mutant androgen receptor to dihydrotestosterone but not testosterone, have been used to restore male genital development in an infant with partial AIS. Of greater significance, because they affect larger numbers of patients, are the mutations and polymorphisms that result in depressed spermatogenesis and male infertility in phenotypic males. Studies involving Singaporean, Australian, North American and Japanese subjects have established that increases in length of a trinucleotide repeat (CAG) tract, encoding a polyglutamine stretch in the transactivation domain of the androgen receptor, are associated with increased risk of defective spermatogenesis and undermasculinization. Independent of the CAG repeats, missense amino-acid substitutions in the ligand-binding domain, involving residues 727, 798 and 886 cause infertility through a novel mechanism. Pathogenicity is transmitted, not through defective ligand binding, but through defective protein-protein interactions between receptor domains and coactivator proteins that are essential for gene transcription. Elucidation of the molecular and structural basis of androgen receptor dysfunction in these cases allows precise genetic counseling and can lead to the design of rational hormonal therapy.

NDD1, a high-dosage suppressor of cdc28-1N, is essential for expression of a subset of late-S-phase-specific genes in Saccharomyces cerevisiae.

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.

Dephosphorylation of threonine 169 of Cdc28 is not required for exit from mitosis but may be necessary for start in Saccharomyces cerevisiae.

Entry into mitosis requires activation of cdc2 kinase brought on by its association with cyclin B, phosphorylation of the conserved threonine (Thr-167 in Schizosaccharomyces pombe) in the T loop, and dephosphorylation of the tyrosine residue at position 15. Exit from mitosis, on the other hand, is induced by inactivation of cdc2 activity via cyclin destruction. It has been suggested that in addition to cyclin degradation, dephosphorylation of Thr-167 may also be required for exit from the M phase. Here we show that Saccharomyces cerevisiae cells expressing cdc28-E169 (a CDC28 allele in which the equivalent threonine, Thr-169, has been replaced by glutamic acid) are able to degrade mitotic cyclin Clb2, inactivate the Cdc28/Clb2 kinase, and disassemble the anaphase spindles, suggesting that they exit mitosis normally. The cdc28-E169 allele is active with respect to its mitotic functions, since it complements the mitosis-defective cdc28-1N allele. Whereas replacement of Thr-169 with serine affects neither Start nor the mitotic activity of Cdc28, replacement with glutamic acid or alanine renders Cdc28 inactive for Start-related functions. Coimmunoprecipitation experiments show that although Cdc28-E169 associates with mitotic cyclin Clb2, it fails to associate with the G1 cyclin Cln2. Thus, an unmodified threonine at position 169 in Cdc28 is important for interaction with G1 cyclins. We propose that in S. cerevisiae, dephosphorylation of Thr-169 is not required for exit from mitosis but may be necessary for commitment to the subsequent division cycle.