Biomedical subjects

W Heyns

Publications and source records attributed to W Heyns.

At least 37 records · Page 2Linked to original sources

PubMed1997

Androgens stimulate fatty acid synthase in the human prostate cancer cell line LNCaP.

In addition to modulation of cell proliferation and stimulation of prostate-specific antigen secretion, one of the most striking effects of androgens on the human prostate cancer cell line LNCaP is the accumulation of neutral lipids. These lipids are synthesized de novo, suggesting that LNCaP cells express all enzymes required for endogenous lipogenesis and that the expression and/or activity of some of these enzymes is affected by androgens. One of the key enzymes involved in lipogenesis is fatty acid synthase (FAS), a potential prognostic enzyme and therapeutic target that is found to be frequently overexpressed in a variety of cancers including prostate cancer. Here, using Northern blot analysis, the gene encoding FAS is shown to be abundantly expressed in LNCaP cells and in two other prostate cancer cell lines tested (PC-3 and DU-145). In LNCaP cells, androgen treatment (10(-8) M R1881) causes a 3-4-fold increase in FAS mRNA levels. Concomitantly with the increase in FAS gene expression, androgens induce a 10-12-fold stimulation of FAS activity. Effects are dose- and time-dependent and follow courses similar to those of the androgen induction of lipid accumulation. In support of the involvement of the androgen receptor, steroid specificity of regulation of FAS activity is in agreement with the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells. Stimulation of FAS activity is inhibited by the antiandrogen Casodex (bicalutamide) and is absent in the androgen receptor-negative cell lines PC-3 and DU-145. Taken together, these data demonstrate that androgens, mediated by the androgen receptor, stimulate the expression and activity of FAS and suggest that stimulation of FAS activity represents at least part of the mechanism by which androgens induce the accumulation of neutral lipids in LNCaP cells.

Adenocarcinoma↗

PubMed1997

LNCaP prostatic adenocarcinoma cells derived from low and high passage numbers display divergent responses not only to androgens but also to retinoids.

In the present paper, two strains of LNCaP cells derived from the same source (American Type Culture Collection), but studied either at a low passage number (LP) or at a high passage number (HP), were compared in their response to R1881 (a synthetic androgen), all-trans-retinoic acid (atRA), and 1alpha,25-dihydroxycholecalciferol (VD3). [3H]Thymidine incorporation and epidermal growth factor receptor (EGF-R) binding were measured as parameters related to the proliferative response of the cells. The secretion of prostate-specific antigen (PSA) and the mRNA expression of PSA, prostatic acid phosphatase (PAP), and diazepam-binding inhibitor (DBI) were used as parameters reflecting differentiated function. Marked differences were noted in the response of LP and HP cells to androgens. [3H]Thymidine incorporation displayed a bell-shaped dose-response curve in both strains. The amplitude of the response, however, was much higher in HP cells and growth inhibition at high levels of R1881 was only observed in LP cells. On the contrary, androgen induction of PSA secretion and PSA mRNA expression, as well as the expression of PAP was much more pronounced in LP cells, whereas DBI expression was not altered according to passage number. LP cells and HP cells also displayed striking differences in their response to atRA. An up to 6-fold stimulation of [3H]thymidine incorporation was observed in LP cells, whereas in HP cells the only significant effect was growth inhibition. VD3, on the contrary, inhibited [3H]thymidine incorporation to a comparable degree in LP and HP cells. Only marginal effects of atRA and VD3 were observed on PSA secretion. In both LP and HP cells EGF-R levels were increased by androgens and to a slight extent also by atRA and VD3. It is concluded that LP and HP LNCaP cells display markedly divergent responses not only to androgens but also to atRA. The proliferative rather than antiproliferative effects of atRA in some strains of LNCaP should caution against the uncontrolled use of these agents, or of drugs affecting their metabolism, in patients with prostate cancer.

Acid Phosphatase↗

PubMed1997

Identification of a functional androgen-response element in the exon 1-coding sequence of the cystatin-related protein gene crp2.

Two hormone-responsive segments, one in the region of the promoter and one in intron 1, are identified in two homologous androgen-regulated and differentially expressed rat genes encoding the cystatin-related proteins (CRPs). Footprint analysis with the androgen receptor (AR) DNA-binding domain on the promoter-containing fragments reveals an AR-binding site downstream of the transcription start point in the crp2 gene (ARBSd/crp2, +40/+63). It displays an androgen response element-like sequence motif 5'-AGAAGAaaaTGTACA-3' and overlaps with the ATG translation start codon. A double-stranded oligonucleotide containing this sequence forms a DNA-protein complex with the full-length AR synthesized by vaccinia, as seen in band shift assays. Additional AR-binding sites, ARBSu/crp1 and ARBSu/crp2, occur 5' upstream of the transcription start point and are located at an identical position (-142/ -120) in crp1 and crp2. The AR affinity for these two slightly different sequence motifs is relatively weak. The biological function of all three AR-binding sites as transcription control elements has been studied. The ARBSd/crp2 element clearly shows androgen-response element characteristics. The contribution of the common upstream element to the androgen-dependent control of reporter gene transcription is less clear. The transcription of a reporter gene construct containing the crp2 footprint fragment crp2F (-273/+88) is hormonally regulated as determined by transfection into the human breast cancer cell line T-47D. Androgens, but also glucocorticoids, efficiently stimulate steroid-dependent transcription of the chloramphenicol acetyltransferase gene. Mutation of the 5'-TGTACA-3' sequence in ARBSd/crp2 destroys the AR binding and abolishes the androgen-dependent synthesis of chloramphenicol acetyltransferase. A large fragment derived from intron 1 of the crp1 and crp2 gene can also provide the androgen-dependent transcription of chimeric constructs in T-47D cells. However, the induction measured is less than the one observed with crp2F (-273/+88), and this activity seems to reside in several subfragments that each display a low but consistent androgen responsiveness.

Androgens↗

PubMed1996

Androgenic induction of cystatin-related protein and the C3 component of prostatic binding protein in primary cultures from the rat lacrimal gland.

Cystatin-related protein (CRP) and the C3 component of prostatic binding protein (C3) are synthesized in vivo under androgen control in the lacrimal gland and ventral prostate of adult male rats [1,2]. Androgen administration to female or 7-day castrated male rats, which do not express CRP, can induce its synthesis [3]. In this study, we show androgen-dependent expression of CRP1 and C3 in primary cultures of acinar cells of the lacrimal gland of female rats. Addition of androgens to the culture medium results in the synthesis and secretion of both proteins in a time- and dose-dependent way. Estradiol or progesterone are unable to induce their expression. Dexamethasone in low concentrations and present as a basal component of the serum free defined medium, is needed to sensitize the culture system for androgens. In high concentrations, this synthetic glucocorticoid seems to play a similar role as androgens in CRP1 and C3 regulation.

Androgen-Binding Protein↗

PubMed1996

Androgen regulation of the messenger RNA encoding diazepam-binding inhibitor/acyl-CoA-binding protein in the rat.

Our recent finding that diazepam-binding inhibitor/acyl-CoA-binding protein (DBI/ACBP) expression is regulated by androgens in the human prostatic adenocarcinoma cell line LNCaP, prompted us to study whether androgen regulation of DBI/ACBP also occurs in vivo in the prostate and in other organs of the rat. Northern blot analysis demonstrated that DBI/ACBP transcripts were expressed in male accessory sex organs such as ventral prostate, dorsolateral prostate, seminal vesicles and coagulating glands. Castration caused a 1.7- to 2.7-fold reduction in the levels of DBI/ACBP transcripts over a period of 6 days. Readministration of androgens during the last 3 days led to 4.2- to 7.5- fold higher levels of DBI/ACBP transcripts than in untreated castrates. In situ hybridization revealed that in the ventral prostate, DBI/ACBP transcripts were expressed predominantly in epithelial cells and that the observed effects of androgens were due both to modulation of gene expression per cell and to changes in cell composition. Androgen regulation of DBI/ACBP mRNA expression was also observed in the lacrimal glands, the adrenals, and the submandibular glands, but not in the liver and the kidney. These findings demonstrate that DBI/ACBP is androgen-regulated in vivo in various organs of the rat. In view of the proposed role of DBI/ACBP in the control of multiple biological processes, DBI/ACBP may be one of the target genes by which androgens affect a variety of physiological processes.

Androgens↗

PubMed1996

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. PA was included because of its suspected different mode of action [H3]-thymidine incorporation was used as a measure of proliferative activity, secretion of prostate-specific antigen (PSA) as a measure of differentiated function. The present data show that 1alpha,25-dihydroxycholecalciferol (VD3), all-trans retinoic acid (atRA), 9-cis retinoic acid (9cRA), and PA stimulated LNCaP cell-differentiated function in the presence or absence of androgens. The effects on cell growth were more complicated. In the absence of androgens growth stimulatory effects were observed for the retinoids and under some conditions for VD3. These effects were limited, however, and tended to be more pronounced at low cell densities. In the presence of androgens nearly exclusively growth inhibitory effects were observed. On a molar basis VD3 was the most effective antiproliferative agonist (ED50 = 10(-9) M). It completely neutralized the stimulatory effects of androgens. Growth inhibition was not due to a decrease in the concentration of androgen receptor: whereas atRA, 9cRA, and PA did not alter androgen receptor levels, VD3 provoked a twofold increase. Neither in the presence nor in the absence of androgens did we observe any cooperativity in the growth stimulatory or inhibitory effects of VD3, atRA, or 9cRA. To test whether treatment with any of the studied agonists resulted in a phenotypic reversion and sustained growth arrest, LNCaP cells were pretreated with VD3, atRA, 9cRA, or PA for 6-12 days and reseeded at equal densities as untreated cells. In all cases tested [3H]-thymidine incorporation was restored within 6 days suggesting that none of these compounds caused irreversible growth inhibition.

Androgens↗

PubMed1996

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. The most abundant type of transcripts (referred to as type 1 transcripts) encodes genuine DBI/ACBP of 86 amino acids, while the minor type (type 2 transcripts) harbors an insertion of 86 bases and might encode an unrelated protein of 67 amino acids. Examination of a cloned DBI/ACBP gene revealed a structural organization of four exons present in all transcripts and one alternatively used exon present only in type 2 transcripts. The promoter region is located in a CpG island and lacks a canonical TATA box. Transient transfection of DBI/ACBP promoter fragments into LNCaP cells demonstrated that a region of 1.1 kb upstream of the translation start site is able to drive high-level expression of luciferase in LNCaP cells in an androgen-regulated fashion. Taken together these data indicate that the isolated human gene encoding DBI/ACBP is functional, has a high degree of structural similarity with the corresponding rat gene, exhibits hallmarks of a typical housekeeping gene, and harbors cis-acting elements that are at least partially responsible for androgen-regulated transcription in LNCaP cells.

Adenocarcinoma↗

PubMed1996

Androgens markedly stimulate the accumulation of neutral lipids in the human prostatic adenocarcinoma cell line LNCaP.

Microscopic evaluation of LNCaP cells stained with the lipophilic dye Oil red O revealed that androgens induce a marked stimulation of lipid droplet accumulation. As determined by quantitative analysis of the Oil red O extracted from the stained cells, stimulatory effects of the synthetic androgen R1881 became apparent at concentrations as low as 10(-11) M. Maximal induction (15-fold) was reached at 10(-8) M. Increases were observed 2 days after hormone addition and were maximal 1 day later. Accumulation of lipid droplets was also induced by mibolerone (another synthetic androgen) and by the natural androgens testosterone and dihydrotestosterone. In agreement with the aberrant ligand specificity of the mutated androgen receptor in LNCaP cells, stimulation of lipid accumulation was also apparent after treatment with progesterone and estradiol. Cortisol and the synthetic glucocorticoid dexamethasone were ineffective. The androgen antagonist Casodex (bicalutamide) abolished the stimulatory effect of R1881, further supporting the involvement of the androgen receptor. In agreement with this conclusion, no changes in lipid accumulation were observed after androgen treatment of the androgen receptor-negative prostate tumor lines PC-3 and DU-145. To investigate the nature of the lipids affected by androgens, lipid extracts were analyzed by TLC, complemented with enzymatic lipid analyses. Androgens were shown to have major effects on the content of triglycerides and cholesterol esters (33- and 7-fold stimulation, respectively), the two main classes of lipids stained by Oil red O. Phospholipid and cholesterol contents were increased by a factor of 2. Incorporation studies with [2-14C]acetate revealed that androgens caused a major stimulation of 2-14C incorporation into triglycerides and cholesterol esters (11- and 13-fold, respectively), suggesting that androgens act at least in part at the level of lipid synthesis. Taken together, these findings indicate that androgens, besides affecting proliferation and protein secretion, also markedly stimulate the production and accumulation of neutral lipids, revealing a novel interesting aspect of androgen regulation of LNCaP cells.

Adenocarcinoma↗

PubMed1996

Androgens transcriptionally regulate the expression of cystatin-related protein and the C3 component of prostatic binding protein in rat ventral prostate and lacrimal gland.

In this report, it is demonstrated that the C3 component of prostatic binding protein (PBP) is also expressed and androgen regulated in the exorbital lacrimal gland, as shown previously for cystatin-related protein (CRP), another abundant secretory protein from the ventral prostate. The presence of C3 messenger RNA (mRNA) could be demonstrated by both Northern blot hybridization and PCR amplification and sequencing. The mRNAs encoding the C1 and C2 components of PBP, however, were undetectable. At the protein level, the C3 component in the lacrimal gland is glycosylated and linked by disulfide bridges to a new 10-kDa component not reacting with the PBP antiserum. As shown previously for CRP, the expression of C3 in the lacrimal gland requires the simultaneous presence of androgens and a functional androgen receptor. The effects of castration and androgen treatment on CRP and C3 mRNA concentrations were studied by Northern blot and dot blot hybridization; effects on transcription rates were determined by nuclear run-on assay. Two days after castration, the relative abundance of CRP mRNA had declined significantly (P < 0.01) to 10.5 +/- 1.5% (+/-SEM) of precastration levels in the prostate and to 14.5 +/- 8.0% in the lacrimal gland; the transcription rates declined to 14.3% and 10.0%, respectively. The C3 mRNA level and transcription rate in the prostate showed a more moderate decrease (P < 0.05) to 40.6 +/- 8.5% and 41.7%, but were hardly measurable in the lacrimal gland. Androgen administration resulted in a rapid increase in the transcription rates, which reached or exceeded control levels after 6-9 h of treatment and clearly preceded the increase in mRNA levels. It is concluded that the lacrimal gland, which can be studied conveniently in female and long term androgen-depleted animals offers a suitable model for the study of androgen-regulated gene expression.

Analysis of Variance↗

PubMed1995

Immunocytochemical localization of seminal proteins in salivary and lacrimal glands of the rat.

Antibodies against 10 different secretory proteins from the accessory sex glands of the male rat were used for immunohistochemical studies of salivary and lacrimal glands from intact and castrated rats, at the light- and electron-microscopic levels. In the parotid gland, secretory acinar cells showed immunoreactivity with antibodies against prostatic binding protein, cystatin-related peptide and acid phosphatase (isoenzyme pI 8.0; 5.6) typical of ventral prostate, and seminal vesicle secretion VI. Western blotting analysis indicated that immunoreactivity against prostatic binding protein was attributable to a subunit, presumably C3. Acid phosphatase pI 5.6 showed a molecular weight of 66 kDa, which is at variance with the prostatic form. Immunoreactivity for secretory transglutaminase, derived from the coagulating gland, was restricted to myoepithelial and stromal cells. In castrated animals, the immunoreactivity of acinar cells was reduced to the background level, whereas stromal transglutaminase immunoreactivity was unaltered. The distribution pattern of immunoreactivity for the proteins mentioned was almost identical in the lacrimal gland. Significant differences were however observed in the immunoreactivity of the inframandibular gland, where serous glandular cells were non-immunoreactive for seminal proteins, with the exception of acid phosphatase isoenzyme pI 8.0. Granules present in the convoluted granular ducts were immunoreactive particularly for acid phosphatase (isoenzyme pI 5.6) but much less for cystatin-related peptide; immunoreactivity was reduced after castration. The straight portion of the inframandibular duct system was immunoreactive for transglutaminase, but no influence of castration was visible. The distribution of immunoreactivity for seminal proteins present in the salivary and lacrimal glands and the pronounced androgen-dependence of their expression point to functional relationships of the respective proteins at both glandular sites.

Androgens↗

PubMed1995

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. Exposure of LNCaP cells to T3 for 6 days in the absence of androgens caused a dose-dependent increase in [3H]-thymidine incorporation with a maximal stimulation of 2.5-fold at 10(-9) M T3. Secretion of prostate-specific antigen (PSA) was also stimulated 2-3-fold. The observed effects may well be mediated by a nuclear T3 receptor as evidenced by displaceable T3 binding studies. Combined treatment of LNCaP cells with androgens and T3 revealed intriguing interactions between these two pathways. Below and up to 10(-10) M of the synthetic androgen R1881, the concentration that evokes optimal proliferative responses, T3 stimulated [3H] thymidine incorporation. At higher concentrations of androgens, T3 displayed antiproliferative effects. No androgen-dependent effects on T3 receptor levels were observed. Conversely, T3 increased androgen receptor levels up to twofold. Androgen as well as T3 stimulation of proliferation was abolished by high concentrations of the retinoid 9-cis-retinoic acid. These data add T3 to the list of factors that influence growth and differentiation of prostatic tumor cells and contribute to our understanding of the intricate pathways that ultimately determine the course of prostatic carcinoma.

Androgens↗

PubMed1995

Propofol anesthesia does not inhibit stimulation of cortisol synthesis.

Recent data suggest a negative effect of propofol anesthesia on cortisol secretion. The present study was designed to evaluate the effect of propofol anesthesia on the steroidogenic potential of the adrenal glands. The response of cortisol secretion to stimulation with an adrenocorticotropic hormone (ACTH) analog during intravenous anesthesia with propofol has not been reported before. The response of the secretion of cortisol, 11-deoxycortisol, and 17 alpha-hydroxyprogesterone to tetracosactide stimulation was compared in patients anesthetized with propofol-nitrous oxide (n = 10) or thiopental-isoflurane-nitrous oxide (n = 10) and in normal volunteers (n = 10). The response to tetracosactide was similar in all three groups. An adequate increase in cortisol plasma concentration (more than 7.25 micrograms/dL) was obtained in all subjects except one volunteer. The increase in the plasma concentration of the cortisol precursors was also similar. We were unable to detect any influence of propofol anesthesia on the synthesis of cortisol in response to tetracosactide stimulation.

17-alpha-Hydroxyprogesterone↗

PubMed1995

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. Combined treatment with GnRHa and antiandrogen virtually eliminated gonadotrophins, androgens and androgen effects. Testicular weight was reduced to 50% of that observed in RX controls. Treatment with GnRHa and testosterone resulted in supraphysiological levels of testosterone and testicular weights comparable to those observed in RX controls. FSH levels in these animals, however, were in the normal range. A low dose of hCG also restored testicular weight in the presence of low concentrations of serum testosterone and low normal levels of FSH. Neither polyacrylamide gel electrophoresis of total testicular proteins nor two dimensional gel electrophoresis of [35S]-methionine labelled proteins revealed striking changes in distinct testicular proteins as a result of androgen withdrawal or androgen treatment. Dot blot hybridization showed a three-fold increase in the mRNA level for ABP (expressed per microgram total RNA) in the Sertoli cell enriched testes of RX rats. This level was barely influenced by androgen withdrawal or androgen administration. The mRNA level for transferrin was increased six-fold in RX rats. A 50% reduction of this level was observed after combined treatment with GnRHa and antiandrogen. It is concluded that, in the germ cell-depleted testis, the major effect of androgens is an overall increase in protein and RNA synthesis rather than a very important and selective increase of a few gene products.

Androgen Antagonists↗

PubMed1995

The genes coding for rat cystatin-related prostate protein (Cstrp) map to chromosome 3q41.

Two genes encoding rat cystatin-related prostate protein (Cstrp), previously called CRP (Devos et al., 1993), were mapped to chromosome 3q41 by fluorescent in situ hybridization. The results were confirmed using a panel of mouse-rat hybrids that segregate rat chromosomes. Analysis of genomic DNA indicates that the Cstrp locus comprises probably more than three very similar genes.

Animals↗

PubMed1995

Characterization of newly established testicular peritubular and prostatic stromal cell lines: potential use in the study of mesenchymal-epithelial interactions.

Testicular peritubular and prostatic stromal cells produce extracellular matrix elements and paracrine factors that modulate the cytodifferentiation and function of the corresponding epithelial cells. The present paper describes the establishment and characterization of five rat testicular cell lines with peritubular characteristics and one prostatic stromal cell line. Four peritubular cell lines were isolated after transfection of a mixed peritubular-Sertoli cell culture with a v-myc-containing plasmid. The same immortalization procedure applied to prostatic stromal cells yielded one cell line. An additional testicular cell line arose by spontaneous immortalization during serial subculture. Except for one testicular cell line (RTC-8T1), the morphology of all of the immortalized cell lines strongly resembled that of primary cultures of peritubular and stromal cells. Flow cytometric analysis demonstrated that all cell lines scored positive for alpha-smooth muscle isoactin and negative for cytokeratins, confirming their myofibroblast-like nature. None of the cell lines, however, stained positive for alkaline phosphatase, and androgen receptor expression was also lost. Typical Leydig cell characteristics, such as steroidogenesis, and Sertoli cell markers, such as transferrin secretion, were absent. Coculture of the cell lines with Sertoli cells resulted in the formation of tubular structures. A cell attachment assay and an enzyme-linked immunosorbent assay for fibronectin confirmed the production of extracellular matrix elements by all of the established cell lines. Media conditioned by the cell lines stimulated Sertoli cell transferrin production. The active principle was partially purified and resembled the P-MOD-S-like factors produced by primary cultures of peritubular and stromal cells. It is concluded that the immortalized cell lines have retained several of the characteristics of primary cultures of peritubular and stromal cells and may be useful for further studies on mesenchymal-epithelial interactions in testis and prostate.

Actins↗

PubMed1994

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. In androgen-sensitive LNCaP cells, the synthetic androgen R1881 stimulated the DBI/ACBP steady state mRNA levels with half maximal effects at a concentration of 0.2 nM. Increases were a maximal 12 h after addition of the synthetic hormone. DBI/ACBP mRNA levels could also be stimulated by the synthetic androgen mibolerone and by the natural androgens testosterone and dihydrotestosterone. In agreement with the altered steroid specificity of the androgen receptor in LNCaP cells, estradiol and progesterone also exerted a stimulatory effect. Cortisol and the synthetic glucocorticoid dexamethasone were without effect. Androgen stimulation of DBI/ACBP mRNA levels was abolished in the presence of the protein synthesis inhibitor cycloheximide, implying a role for labile or androgen-induced proteins in this androgen stimulation. This is in contrast to the androgen stimulation of the mRNA encoding prostate-specific antigen (PSA), suggesting that different mechanisms are involved in the androgen regulation of these two genes. Although further experiments are required to confirm that DBI/ACBP is secreted by prostatic epithelial cells, these data demonstrate that the mRNA encoding DBI/ACBP is expressed in prostate cells and is affected by androgens in androgen-responsive LNCaP cells.

Adenocarcinoma↗

PubMed1994

Different cells and cell lines produce factors that modulate Sertoli cell function.

Peritubular myoid cells derived from immature rat testes produce factors that modulate Sertoli cell function (P-Mod-S). The secretion of these factors is controlled in part by androgens. Cultured prostatic stromal cells strongly resemble peritubular myoid cells and produce mediators with similar activity. Here we investigated whether myoid cell lines can be used as a source of P-Mod-S-like factors. Rat kidney fibroblast (NRK) and mouse fibroblast (3T3) cell lines were used as non-myoid controls. Surprisingly, serum-free media conditioned by all cell lines studied modulated Sertoli cell function in a similar fashion as media conditioned by peritubular cells (PTCM) or stromal cells (STCM). Using Sertoli cell transferrin secretion as an endpoint for P-Mod-S-like activity, the nature of the active principles involved was further explored. The observed activity could not be explained by residual contamination with fetal calf serum. Moreover, the effects of the conditioned media could not be mimicked by classical growth factors (IGF-I, bFGF, EGF, TGF-beta, NGF, PDGF-BB) added singly or in combination with submaximally effective concentrations of PTCM. Finally, the possibility that conditioned media might indirectly enhance Sertoli cell function by promoting the production or deposition of extracellular matrix elements was made unlikely by the demonstration that the observed effects were not mimicked by Matrigel and were unaffected when Sertoli cells were seeded on Matrigel. Superdex 75 chromatography after analytical reversed-phase chromatography indicates that the factors from different origin have a similar size (45-50 kDa). It is concluded that mediators with P-Mod-S-like activity are produced by various cells and cell lines both with and without smooth muscle cell characteristics. Whether the active principles involved are really identical requires further investigation.

Animals↗

PubMed1994

Nuclear extracts enhance the interaction of fusion proteins containing the DNA-binding domain of the androgen and glucocorticoid receptor with androgen and glucocorticoid response elements.

Comparable fragments of the androgen receptor (AR) (amino acids 540-607) and of the glucocorticoid receptor (GR) (amino acids 412-515) were expressed in E. coli as fusion proteins with protein A. Both fusion proteins, denoted ARF1 and GRF1, contain the DNA-binding domain and some flanking amino acids. In vitro binding assays have shown that both fusion proteins interact with androgen/glucocorticoid response elements (ARE/GREs) in an intron fragment of the C3(1) gene of the androgen-regulated rat prostatic binding protein and in the typically glucocorticoid-responsive long terminal repeat (LTR) promoter of mouse mammary tumour virus. Present results indicate that the interaction of both ARF1 and GRF1 with the C3(1) as well as the LTR fragments is enhanced in the presence of nuclear extract. The factor that gives rise to this enhancement appears to be ubiquitous and sensitive to trypsin and temperature treatment. In the C3(1) fragment, the enhancing effect requires the presence of an intact functional ARE/GRE (Core II) as well as a region spanning the ARE/GRE half-site Core I.

Androgens↗