A human gene encoding diazepam-binding inhibitor/acy1-CoA-binding protein: transcription and hormonal regulation in the androgen-sensitive human prostatic adenocarcinoma cell line LNCaP.

Diazepam-binding inhibitor (DBI)/acyl-CoA-binding protein (ACBP) is a highly conserved 10-kD polypeptide expressed in various organs and implicated in the regulation of multiple biological processes such as GABAA/benzodiazepine receptor modulation, acyl-CoA metabolism, steroidogenesis, and insulin secretion. To extend our knowledge about the biology of DBI/ACBP and to elucidate the molecular mechanisms responsible for regulating DBI/ACBP gene expression, we have studied the androgen-regulated expression of DBI/ACBP transcripts in the human prostatic adenocarcinoma cell line LNCaP and have cloned and characterized a human gene encoding DBI/ACBP. Northern blotting, reverse transcription-assisted polymerase chain reaction (RT-PCR), ribonuclease protection, and 5' RACE analysis (rapid amplification of cDNA ends) of DBI/ACBP transcripts in LNCaP cells revealed androgen-regulated expression of multiple transcripts originating from multiple transcription start sites and alternative processing.

Control of LNCaP proliferation and differentiation: actions and interactions of androgens, 1alpha,25-dihydroxycholecalciferol, all-trans retinoic acid, 9-cis retinoic acid, and phenylacetate.

There is increasing evidence that growth and differentiation of prostatic carcinoma cells may be modulated not only by androgens and growth factors but also by vitamin D, retinoids, and phenylacetate (PA). The latter agonists may have a role in the prevention and therapy of prostate cancer but their exact therapeutic potential remains unclear. Since both retinoids and vitamin D act via nuclear receptors, the same way androgens do, we studied the interactions of these compounds with androgen-induced proliferation and differentiation using LNCaP cells as a model of androgen-responsive tumor cells.

Cytokines derived from activated human mononuclear cells markedly stimulate transferrin secretion by cultured Sertoli cells.

There is considerable evidence that Sertoli cell function is controlled not only by hormones, but also by locally produced growth factors and cytokines. To gain more insight into the nature and effects of cytokines potentially involved in the control of Sertoli cell function, we incubated rat Sertoli cells with media conditioned by activated human peripheral blood mononuclear cells. Such media (PBMC-CM) are known to be an extremely rich source of a variety of cytokines.

Characterization of a hormone-inducible, high affinity adenosine 3'-5'-cyclic monophosphate phosphodiesterase from the rat Sertoli cell.

In previous reports we have shown that FSH and beta-adrenergic agonists regulate the levels of mRNA and increase the activity of a high affinity cAMP phosphodiesterase (cAMP-PDE) in the immature rat Sertoli cell in culture. To identify and characterize the hormone-inducible form(s), the cAMP-PDE activity of the Sertoli cell was partially purified and its properties were determined using biochemical and immunological tools. The cAMP-PDE activity present in the 100,000g supernatant of Sertoli cell extracts was purified more than 2000-fold by four HPLC chromatographic steps.

Triiodothyronine modulates growth, secretory function and androgen receptor concentration in the prostatic carcinoma cell line LNCaP.

There is increasing evidence that the course of prostatic carcinoma is determined by a complex interplay between genetic events, paracrine interactions, and hormonal and dietary factors. These latter factors include several ligands of the nuclear receptor family such as androgens, vitamin D3 and retinoids. To test whether thyroid hormones also influence the growth and differentiated function of prostatic carcinoma cells, LNCaP cells were treated with or without triiodothyronine (T3) in the absence or in the presence of other regulatory factors.

Effect of androgens on the germ cell-depleted testes of prenatally irradiated rats.

To study the effect of androgens on somatic testicular cells, rats were rendered germ cell depleted by prenatal irradiation (RX). Adult RX rats were treated with a desensitizing dose of a GnRH agonist (GnRHa; Zoladex), combined with an antiandrogen (Nilutamide) to preclude all androgen effects, or combined with testosterone or hCG to restore androgen action. The effect of these treatments for 3 weeks on the weight of testes and accessory sex glands, hormones (LH, FSH, testosterone, inhibin), testicular proteins, the pattern of incorporation of [35S]-methionine into testicular proteins (studied by two dimensional gel electrophoresis) and steady state mRNA levels for transferrin and androgen-binding protein (ABP) were evaluated.

Chromosomal localization of the human and rat genes (PDE4D and PDE4B) encoding the cAMP-specific phosphodiesterases 3 and 4.

Through the use of somatic cell hybrids segregating either human or rat chromosomes, we determined the chromosome localizations of two genes encoding cAMP-specific phosphodiesterases (cAMP-PDEs). PDE4D, the gene encoding the cAMP-PDE isoform 3 (IVd), was assigned to human chromosome 5 and rat chromosome 2, and PDE4B, the gene encoding the cAMP-PDE isoform 4 (IVb), was assigned to human chromosome 1 and rat chromosome 5. These localizations extend the homology between rat chromosome 2 and human chromosome 5, on the one hand, and between rat chromosome 5 and human chromosome 1, on the other hand.

Androgen regulation of the messenger RNA encoding diazepam-binding inhibitor/acyl-CoA-binding protein in the human prostatic adenocarcinoma cell line LNCaP.

To study the mechanisms by which androgens intervene in the regulation of growth and differentiation of human prostatic epithelial cells, cDNA clones encoding putative prostate-secreted proteins were characterized and tested as potential markers for androgen action. One of the isolated cDNAs expressed diazepam-binding inhibitor/acyl-CoA-binding protein (DBI/ACBP), suggesting that this polypeptide, that has been implicated in a large number of biochemical processes, is expressed and secreted by prostate cells. As demonstrated by Northern blot analysis, the mRNA encoding DBI/ACBP was expressed in prostate tissue and in the three human prostatic adenocarcinoma cell lines tested: LNCaP, PC-3 and DU-145.

ADH resistance of LLC-pk1 cells caused by overexpression of cAMP-phosphodiesterase type-IV.

The studies of animal models of nephrogenic diabetes insipidus (NDI) suggest that abnormally high activity of cAMP phosphodiesterase (cAMP-PDE), may cause unresponsiveness to the diuretic effect of AVP. We explored whether overexpression of one of the cAMP-PDE type isozymes, PDE-IV, in [8-Arg]-vasopressin (AVP) sensitive renal epithelial LLC-PK1 cells can prevent the hormone-elicited cAMP increase. LLC-PK1 cells were stably transfected with ratPDE3.

Characterization of the structure of a low Km, rolipram-sensitive cAMP phosphodiesterase. Mapping of the catalytic domain.

Considerable structural similarities are present in a region of approximately 270 amino acids in most known cyclic nucleotide phosphodiesterase (PDE) sequences, opening the possibility that this region encodes the catalytic domain of the enzyme. To test this hypothesis, the structure of a high affinity cAMP PDE (cAMP-PDE) was analyzed by deletion mutations and site-directed mutagenesis. A ratPDE3 cDNA was mutated using a strategy based on fragment amplification by polymerase chain reaction.

A polymerase chain reaction strategy to identify and clone cyclic nucleotide phosphodiesterase cDNAs. Molecular cloning of the cDNA encoding the 63-kDa calmodulin-dependent phosphodiesterase.

Multiple isozymes of cyclic nucleotide phosphodiesterases (PDEs) are expressed simultaneously in mammalian tissues. To identify and clone these PDEs, a polymerase chain reaction (PCR) strategy was developed using degenerate oligonucleotide primers designed to hybridize with highly conserved PDE DNA domains. Both known and novel PDEs were cloned from rat liver, the mouse K30a-3.

Unique adenosine 3',5' cyclic monophosphate phosphodiesterase messenger ribonucleic acids in rat spermatogenic cells: evidence for differential gene expression during spermatogenesis.

Four cAMP phosphodiesterase (cAMP-PDE) genes (ratPDE1, ratPDE2, ratPDE3, and ratPDE4) are expressed in the rat testis (Swinnen et al., PNAS USA 1989; 86:5325). Since multiple ratPDE1 and ratPDE2 mRNAs were present in male germ cells, their developmental expression was investigated by using purified spermatogenic cell populations.

Properties and hormonal regulation of two structurally related cAMP phosphodiesterases from the rat Sertoli cell.

Upon exposure to follicle-stimulating hormone (FSH), the gonadotropin-responsive Sertoli cell expresses increased rolipram-sensitive cAMP-specific phosphodiesterase (cAMP-PDE) activity. To understand the mechanisms leading to this activation, the cAMP-PDEs present in the Sertoli cell were characterized and their regulation studied. Comparison of the conceptual translates of two groups of PDE cDNA clones isolated from a Sertoli cell cDNA library (ratPDE3 and ratPDE4) showed that the encoded proteins were structurally similar, containing a core region of 455 amino acids with a sequence identity of 87%.

Attenuation of cAMP-mediated responses in MA-10 Leydig tumor cells by genetic manipulation of a cAMP-phosphodiesterase.

In order to assess the effect of increased cAMP degradation on the responsiveness on an endocrine cell, we have obtained stable transfectants of MA-10 Leydig tumor cells that overexpress a mammalian cAMP-phosphodiesterase. Two novel cell lines, designated MA-10(P+8) and MA-10(P+29), that express high levels of the transfected enzyme were characterized. Although the basal levels of cAMP in the mutant cell lines are comparable to those of the wild-type cells, the increase in cAMP accumulation elicited by human choriogonadotropin (hCG) is severely blunted.

[Value and limits of bypassing the distal ileum in the treatment of hypercholesterolemia].

Partial ileal bypass (PIB) was performed in 8 young adults (5 males and 3 females, mean age 37 +/- 5 years) with a history of vascular surgery (aorto-coronary bypass, ACB, n = 6; stroke, n = 2), presenting with hyperlipidemia (II B: n = 7; IIA: n = 1). None of the patients had diabetes, 2 had mild hypertension, and all were cigarette smokers. Hypolipidemic drugs were discontinued prior to PIB.

The mRNA encoding a high-affinity cAMP phosphodiesterase is regulated by hormones and cAMP.

To elucidate the mechanisms by which hormones regulate cAMP phosphodiesterases (PDEs), a group of cDNA clones that had been isolated from a rat Sertoli cell library were characterized. These cDNAs are derived from a single gene (ratPDE3). The deduced amino acid sequence of the ratPDE3 cDNA corresponds to a 66,200-Da protein homologous to other testicular PDEs, to the Drosophila melanogaster dunce-encoded cAMP PDE, and to bovine and yeast PDEs.

Molecular cloning of rat homologues of the Drosophila melanogaster dunce cAMP phosphodiesterase: evidence for a family of genes.

To study the structure and function of cyclic nucleotide phosphodiesterases (PDEs) involved in mammalian gametogenesis, a rat testis cDNA library was screened at low stringency with a cDNA clone coding for the Drosophila melanogaster dunce-encoded PDE as a probe. This screening resulted in the isolation of two groups of cDNA clones, differing in their nucleotide sequences (ratPDE1 and ratPDE2). In the rat testis, RNA transcripts corresponding to both groups of clones were expressed predominantly in germ cells.

Importance of functional vestibular examination even in cases with vague dizziness complaints.

The data of two recent cases prove that not only typical rotatory vertigo asks for vestibular investigation. In these cases with rather atypical vertigo and dizziness, diagnosis only is obtained by the ENG investigation, which at the same time indicates the central localisation.