Murielle Esquenet

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. [3H]‐thymidine incorporation was used as a measure of proliferative activity, secretion of prostate‐specific antigen (PSA) as a measure of differentiated function.

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.

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.

Androgens decrease and retinoids increase the expression of insulin-like growth factor-binding protein-3 in LNcaP prostatic adenocarcinoma cells

Changes in circulating levels of insulin-like growth factors (IGF) and IGF-binding proteins (IGFBP) have been related to prostate cancer, but the nature and the significance of this relationship remains elusive. Recent reports suggest that modulation of the production of IGFBP-3 by retinoids may affect growth of breast and prostate tumor cells. In the present study we explored whether androgens (R1881), retinoids (all-trans- and 9-cis-retinoic acid: atRA and 9cRA), deltanoids (1alpha,25-dihydroxycholecalciferol: VD3) and thyroid hormone (triiodothyronine: T3) influence the production of IGFBPs by LNCaP prostatic adenocarcinoma cells and whether the observed changes affect tumor cell growth. Northern blot experiments demonstrated that LNCaP cells express IGFBP-2, -3, -4 and (to a small extent) -5. IGFBP-4 and -5 were not measurably affected by the mentioned agonists. At a growth promoting concentration (10(-10) M), R1881 increased IGFBP-2 transcript levels two- to three-fold and this effect was neutralized by atRA and VD3. Similar effects could not be demonstrated, however, at the protein level using Western ligand blotting. R1881 decreased and atRA increased the mRNA levels of IGFBP-3 and these effects were confirmed by Western ligand blotting and by radioimmunoassay. The effects of atRA were mimicked by 9cRA and by a specific RAR agonist but not by a RXR agonist. VD3 and T3 had no significant effect on IGFBP-3 secretion but respectively enhanced or decreased the effect of 9cRA. The effects of retinoids required high concentrations (10(-6)-10(-5) M) that also induced growth inhibition. R1881, however, decreased IGFBP-3 at growth promoting (10(-10) M) as well as at growth inhibitory (10(-8) M) concentrations. Moreover, under serum-free conditions, we were unable to demonstrate any growth modulating effect of IGFBP-3. It is concluded that several agonists acting by nuclear receptors affect IGFBP-3 secretion by LNCaP cells but that the functional significance of these changes warrants further investigation.

Lipase-based quantitation of triacylglycerols in cellular lipid extracts: requirement for presence of detergent and prior separation by thin-layer chromatography

A protocol, based on the use of Pseudomonas lipase, is presented to measure quantitatively the amount of triacylglycerols in extracts from cultured cells or tissues. Since the lipase also acts on di- and monoacylglycerols, separation of the extracts by thin-layer chromatography is recommended. In order to allow the lipase-catalyzed hydrolysis to proceed efficiently, lipid extracts or eluates from silica scraping were mixed with the detergent Thesit [dodecylpoly(ethylene glycol ether)], prior to drying. After dissolution of the dried residues in water, the amount of triacylglycerols was quantified using Pseudomonas sp. lipase, glycerol kinase, glycerol-phosphate oxidase, and peroxidase. The activity of the latter enzyme was followed either colorimetrically in the presence of 4-aminoantipyrine and 2,4,6-tribromo-3-hydroxybenzoic acid or fluorimetrically in the presence of homovanillic acid.

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.

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.

Retinoids stimulate lipid synthesis and accumulation in LNCaP prostatic adenocarcinoma cells.

In a previous report we demonstrated that androgens markedly stimulate accumulation of lipid droplets in LNCaP cells. The effects were already evident at low concentrations of androgens optimal for proliferation but became much more pronounced at high concentrations optimal for differentiation. In the present report we explored whether other agonists acting by nuclear receptors and modulating LNCaP growth and differentiation also affect lipid accumulation. The agonists investigated were 1alpha,25-dihydroxycholecalciferol (VD3), all-trans-retinoic acid (atRA), and triiodothyronine (T3). Lipid accumulation was evaluated by Oil Red O staining followed by image analysis of Oil Red O-stained cells or by extraction and measurement of absorbency. Only marginal effects were noted for VD3 and T3. The atRA, on the contrary, increased lipid staining 5-12-fold. This effect required high concentrations of retinoids (10[-6] M) and was accompanied by growth stimulation. Lipid accumulation was less pronounced than that observed with maximally effective concentrations of androgens (10[-3] M R1881). Thin layer chromatography (TLC) and enzymatic determination of the various lipid fractions demonstrated that retinoids increase triacylglycerides and an unidentified lipid fraction with a slightly higher mobility. In contrast with androgens, however, they did not stimulate the accumulation of cholesterol esters. Incorporation studies with [2-14C]acetate revealed that the increased accumulation of the mentioned lipids is related both to increased synthesis and to decreased secretion. Retinoid-induced lipid accumulation is accompanied by increased steady-state levels of the mRNA encoding fatty acid synthase (FAS), a key enzyme involved in lipid synthesis, while the expression of HMG-CoA-reductase, an enzyme controlling cholesterol synthesis is only marginally affected. It is concluded that retinoids share the ability of androgens to increase lipid accumulation in LNCaP cells. The nature of the lipids affected by both agonists, however, differs at least in part suggesting that the underlying mechanisms may also be different. For the studied compounds (androgens, VD3, atRA, and T3) no simple and consistent relationship could be observed between their ability to decrease proliferation and increase differentiation on the one hand and their ability to promote lipid accumulation on the other hand.