Biomedical subjects

G Verhoeven

Publications and source records attributed to G Verhoeven.

At least 73 records · Page 4Linked to original sources

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

Inhibin and activin have antagonistic paracrine effects on gonadal steroidogenesis during the development of the chicken embryo.

Primary cultures of gonadal cells from chicken embryos were used to explore the paracrine role of inhibin and activin during gonadal development. Fetal testicular and ovarian cells secreted high amounts of immunoactive inhibin. FSH caused a dose-related increase of cAMP and immunoactive inhibin concentrations in testicular cell cultures. Postreceptor signalling through the protein kinase A (PKA) pathway was confirmed by additional experiments with 8-bromo-cAMP, 3-isobutyl-1-methyl-xanthine (MIX), prostaglandins, forskolin, and choleratoxin. The relative ability of these agonists to stimulate cAMP production did not always correlate with their ability to stimulate inhibin secretion. Experiments with phorbol 12-myristate 13-acetate suggested that the regulation of immunoactive inhibin secretion also involves the protein kinase C (PKC) pathway. In addition, it was shown that recombinant human (rh)-inhibin increases the conversion of pregnenolone to androgens whereas rh-activin has the opposite effect. Recombinant human follistatin, an activin-binding protein, antagonized the actions of rh-activin and to a lesser extent those of rh-inhibin. In conclusion, these results show that during the development of the chicken embryo, gonadal inhibin secretion may be regulated by hormones and by local factors such as prostaglandins. Cross talk between the PKA and PKC pathways may be involved in this regulation. Recombinant human inhibin and rh-activin may have antagonistic roles in the paracrine control of gonadal steroidogenesis.

Activins↗

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↗

PubMed1996

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. It was demonstrated that PMBC-CM and protein fractions derived from them stimulate Sertoli cell transferrin secretion and messenger RNA production more potently then peritubular cell-conditioned medium or FIRT (a combination of FSH, insulin, retinol, and testosterone). Transferrin secretion expressed per mg cell DNA was stimulated approximately 5-fold by peritubular cell-conditioned medium or FIRT and nearly 20-fold by PBMC-CM. The effects of PBMC-CM were accompanied by a limited increase in cAMP and a noticeable rise in cGMP. Affinity chromatography on a column coated with an antiserum directed against interleukin-1 beta (IL-1 beta) showed that part of the activity in the PBMC-CM was related to IL-1 beta. The remainder of the activity was largely retained by an affinity column coated with an antiserum that recognizes IL-6 and a number of other known and unknown cytokines. Purified IL-1 beta provoked a 2- to 3-fold stimulation of Sertoli cell transferrin secretion. More limited stimulatory effects were observed with IL-6. Neither of these cytokines or their combination approached the degree of stimulation observed with crude PBMC-CM, suggesting that other cytokines are involved. It is concluded that the mixture of cytokines present in PBMC-CM is a more powerful stimulator of Sertoli cell transferrin secretion than any other agonist known at the present time. IL-1 and IL-6 may be responsible for part of the observed effects, but one or more other cytokines are probably involved.

Animals↗

PubMed1996

Dimeric inhibin A and alpha-subunit immunoreactive material in maternal serum during spontaneous and in vitro fertilization pregnancies.

To increase our understanding of the nature and source of immunoreactive inhibin-related material during pregnancy, we studied inhibin secretion in women pregnant after spontaneous conception and after in vitro fertilization (IVF). Two solid phase enzyme-amplified immunoassays (EASIAs) were used to measure the inhibin A dimer and inhibin alpha-subunit immunoreactivities (alpha-inhibin). In spontaneous pregnancies, levels of both inhibin A and alpha-inhibin were low during the first two trimesters of pregnancy, but a significant increase was observed toward the end of gestation. Both assays confirmed that inhibin concentrations in IVF pregnancies exceeded those in spontaneous pregnancies during the entire first trimester. Moreover, the two assays displayed discordant profiles. The alpha alpha-EASIA, which detects all alpha-inhibin immunoactivity, displayed a major peak during the follicular phase and a second broader peak during the luteal phase and corpus luteum rescue. A progressive decline was observed during the subsequent weeks. EASIA measurements for inhibin A revealed distinct peaks during the follicular phase, the luteal phase, and the hCG peak. A marked fall, however, was seen at the time of corpus luteum rescue. In summary, these data indicate that the nature of the immunoreactive material changes considerably during the different phases of pregnancy. The available evidence further points to an ovarian source of dimeric inhibin in early pregnancy and a placental source toward the end of pregnancy.

Adolescent↗

PubMed1995

Gonadotrophin-regulated production of immunoactive inhibin and androgen by cultured testicular cells from chicken embryos.

To study the potential intragonadal role of inhibin and inhibin-related proteins in the developing gonad, a method was developed to culture testicular cells of chicken embryos. A single-step collagenase/DNase digestion was used to disperse the cells. Except for the primordial germ cells and the erythrocytes, the cells attached well to plastic culture dishes. Moreover, they could easily be grown in the absence of serum or other additives. Inhibin secretion was measured using a heterologous radioimmunoassay validated for use in this species. The fetal testicular cells secreted high amounts of immunoactive inhibin and remained responsive to gonadotrophins. Two different cell populations could be recognized in monolayers of testicular cells: the first population had a fibroblast-like stromal appearance, resembling interstitial cells; the second had an epitheloid appearance and contained large numbers of refractile vacuoles, resembling Sertoli cells. Both cell populations were enriched using a Percoll density gradient. The epitheloid cells displayed a higher capacity to secrete immunoactive inhibin, while the stromal cells were responsible for the bulk of androgen secretion. FSH, but also LH, stimulated inhibin secretion in the epitheloid cells. Although ovine LH was the most potent stimulus for androgen secretion by the stromal cells, ovine FSH was also capable of increasing androgen output in stromal cells and to a lesser extent in epitheloid cells. As the two enriched cell populations were still contaminated by other cell types, this may indicate, as in other species, that FSH-induced paracrine factors are involved in the regulation of androgen secretion in the developing gonad.(ABSTRACT TRUNCATED AT 250 WORDS)

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

Developmental changes in immunoreactive inhibin and FSH in plasma of chickens from hatch to sexual maturity.

1. The relationship between immunoreactive inhibin and follicle-stimulating hormone (FSH) was studied in male and female chickens from hatch to sexual maturity. Plasma inhibin was estimated by a heterologous radioimmunoassay validated for use in the chicken. FSH was measured by a recently developed homologous radioimmunoassay. 2. In a cross-sectional study, blood samples and gonads were collected from chickens of both sexes at 1, 3, 5, 7, 14, 21 and 28 d after hatching and subsequently at 14-day intervals until 182 d of age. 3. In the female, plasma progesterone concentration (P4) progressively increased during sexual development. The plasma luteinising hormone (LH) concentration rose during the first week after hatching, and fluctuated thereafter, with troughs at 6 and 14 weeks and peaks at weeks 10 and 18. The plasma inhibin and FSH concentrations remained low until the start of puberty and increased simultaneously thereafter. However, from week 18 on, plasma inhibin continued to rise while plasma FSH fell. Hence, FSH and inhibin were positively correlated before puberty, but developed a negative correlation during sexual maturation. 4. In the male, plasma testosterone and LH concentrations increased 38- and 3.7-fold respectively over the period studied. Inhibin and FSH followed similar time courses and were consequently positively correlated. 5. These results suggest sex differences in the role of inhibin in regulating FSH secretion during development. The FSH-inhibin feedback loop may become operational at the onset of sexual maturity in the hens. In male chickens, the similar pattern of inhibin and FSH secretion suggests that inhibin secretion is driven by FSH.

Aging↗

PubMed1995

Inhibin and steroid responses to testicular stimulation in normal men.

Static measurements of immunoreactive inhibin have proved to be of little relevance in the diagnosis of testicular disorders. To explore whether a dynamic evaluation of inhibin secretion might yield a more useful parameter of testicular function we compared the responses of inhibin with steroids to i.v. injections of pure follicle-stimulating hormone (FSH; 300 IU) or human chorionic gonadotrophin (HCG; 1500 IU) and oral administration of the anti-oestrogen Tamoxifen (20 mg/day for 7 days) in four normal fertile men. Blood was aspirated between 1 and 72 h after the injections and daily during Tamoxifen intake. Four controls were injected with physiological saline solution. An additional four men were injected with pure FSH, and blood was taken after 24, 48 and 72 h. Injection of FSH was accompanied by nycthemeral variations of testosterone comparable with those observed in the controls. The concentration of inhibin showed similar nycthemeral variations but a significant increase was observed in all eight cases at 12 noon on days 2 and 3 after FSH injection. HCG injection resulted in the expected biphasic response of testosterone. Inhibin displayed a pronounced increase 18 h after injection but the delayed response after 48 and 72 h was not observed. Tamoxifen intake increased testosterone but not inhibin, and caused a moderate and temporary increase of luteinizing hormone and FSH. It was concluded that primary stimulation both of Leydig cells by HCG and Sertoli cells by FSH increase circulating inhibin.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗

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

Production of transferrin-like immunoreactivity by rat anterior pituitary and intermediate lobe.

Using a specific anti-rat transferrin (Tf) antiserum, Tf-like immunoreactivity (Tf-lir) was detected by immunostaining in intact rat pituitaries and in reaggregated pituitary cells cultured in serum-free medium. Tf-lir cells were present in the anterior pituitary (AP), and in the intermediate (IL) and neural lobes (NL). In the AP, Tf-lir cells were oval or polygonal. An unusual topographical distribution was found. Tf-lir cells mainly occurred as dense clusters in the lateral wings. In the central part of the AP, Tf-lir was found in flattened perisinusoidal cells. Double immunostaining for Tf and the different pituitary hormones showed that Tf-lir co-localized with some gonadotrophs and somatotrophs (7% and 3% of Tf-lir cells, respectively, in typical sections). No co-localization was seen with PRL, ACTH, TSH, or alpha-MSH. The distribution of Tf-lir cells and their cell shape completely differed from that of S-100-positive cells in the AP. In the IL, clusters of large stellate Tf-lir cells were found. Again, their distribution completely differed from S-100-positive cells. In the NL, diffuse staining was found. Double immunostaining of paraffin-embedded sections of reaggregate cell cultures of the AP did not reveal any co-localization of Tf-lir with ACTH, alpha-MSH, LH, FSH, TSH, GH, or PRL. In aggregates consisting of NL + IL cells, Tf-lir was located in clusters: no co-localization with ACTH or alpha-MSH could be demonstrated. Reaggregate cell cultures of AP and NL + IL secreted Tf-lir as measured by radioimmunoassay, at least during 21 days of culture. After metabolic labeling with [35S]-methionine and immunoprecipitation of [35S]-methionine-labeled material present in the culture medium of both AP and NL + IL aggregates with anti-Tf antiserum, a 35S-labeled substance was found, which on SDS-PAGE showed an apparent M(r) of approximately 78 KD, corresponding to the M(r) of rat Tf. The present data show that a specific population of cells of rat anterior pituitary is capable of synthesizing, storing, and secreting transferrin or a substance closely related to it. Cells different from melanotrophs and S-100 cells in the IL, as well as pituicytes in the NL, also appear to produce this material. We suggest that transferrin or a transferrin-like substance may have a local role in the transport of iron or other metals or may play a role as growth factor in the three lobes of the pituitary gland.

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

Human androgen receptor expressed in HeLa cells activates transcription in vitro.

The androgen receptor (AR) is a ligand-responsive transcription factor, belonging to the class of steroid receptors. AR mutations have been associated with various X-linked diseases, characterized by complete or partial resistance to androgens. To further analyse the molecular mechanism of action of the AR, we have produced the human AR in HeLa cells with a Vaccinia virus expression system. Binding studies on infected HeLa cells demonstrate that the recombinant AR interacts specifically and with high affinity with natural and synthetic androgens. In a gel retardation assay the partially purified AR specifically recognizes an androgen response element of the rat prostatic binding protein gene. Moreover, the recombinant AR activates transcription in vitro from a synthetic promoter construct containing glucocorticoid response elements (GRE).

Amino Acid Sequence↗

PubMed1994

Changes of immunoreactive inhibin, follicle-stimulating hormone, luteinizing hormone, and progesterone in plasma after short-term food deprivation and during the ovulatory cycle of the domestic hen.

The interrelationship between immunoreactive inhibin and follicle-stimulating hormone (FSH) was measured in laying hens after short-term food deprivation and during the ovulatory cycle. Plasma inhibin was estimated by a heterologous radioimmunoassay validated for use in chicken. FSH was measured by a recently developed homologous radioimmunoassay. Ten hens were deprived of food for 10 days (a method to induce artificial moult or to stop egg laying). Blood samples were taken daily via the brachial vein for 10 consecutive days starting on the day of food withdrawal. The data are expressed in relation to the last day of oviposition. Short-term food deprivation, followed by ovarian atrophy, significantly depressed the plasma inhibin and progesterone (P4) concentration. Plasma FSH concentrations significantly increased, while luteinizing hormone (LH) concentrations slightly but not significantly decreased. In another experiment, 6 hens were induced to moult. The control hens (n = 6) were fed ad libitum. On Day 17, the hens were killed and follicular distribution was studied. The atrophy of the large yolky follicles in restricted hens was associated with a significant increase in the number of white follicles. In a third experiment, serial blood samples were taken from hens (n = 15) at 2-hr intervals for 36 hr via a brachial cannula. Plasma immunoreactive inhibin and FSH concentrations did not vary during the ovulatory cycle. LH and P4 increased simultaneously from basal levels to preovulatory peak values 6 hr before ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

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↗