Search

Here, we show that three enzymes belonging to the 1B group of the aldo-keto reductase (AKR) superfamily, i.e., human placental aldose reductase (AKR1B1), mouse kidney aldose reductase (AKR1B3) and mouse vas deferens protein (AKR1B7), catalyse the reduction of prostaglandin (PG) H(2), a common intermediate of various prostanoids, to form PGF(2alpha) in the presence of NADPH. AKR1B1, AKR1B3 and AKR1B7 displayed higher affinities for PGH(2) (K(m) = 1.9, 9.3 and 3.8 microM, respectively) and V(max) values (26, 53 and 44 nmol/min/mg protein, respectively) than did the human lung PGF(2alpha) synthase (AKR1C3; 18 microM and 4 nmol/min/mg protein, respectively). The PGF(2alpha) synthase activity of AKR1B1 and AKR1B3 was efficiently inhibited by two AKR inhibitors, tolrestat (K(i) = 3.6 and 0.26 microM, respectively) and sorbinil (K(i) = 21.7 and 0.89 microM, respectively), in a non-competitive or mixed-type manner, whereas that of AKR1B7 was not sensitive to these inhibitors (K(i) = 9.2 and 18 mM, respectively). These data provide a molecular basis for investigating novel functional roles for AKR1B members and PGF(2alpha) as mediators of physiological and pathological processes in mammalian organisms.

The promotion and progression of prostate cancer (PCa) are associated with androgen receptor (AR) signalling. AR functions are modulated by a variety of co-factors amongst which we identified the nucleophosmin (NPM/B23), a member of the histone chaperone family. Here, we show that NPM is overexpressed in PCa compared to normal adjacent tissues. AR and NPM interact in vitro and in vivo, and NPM is critical for androgen-dependent transcriptional activation in LNCaP cells as an anti-NPM siRNA downregulates transcription of a transfected androgen response element (ARE)-containing reporter promoter as well as expression of the endogenous androgen responsive prostate-specific antigen (PSA) gene. By investigating the effect of NPM on AR, we have also observed that NPM enhances AR binding to an ARE in vitro in electrophoretic gel mobility-shift assay experiments. Chromatin immunoprecipitation studies further demonstrated that both AR and NPM associate with AREs of the PSA gene in vivo. Altogether, our data suggest that the molecular histone chaperone NPM could regulate AR functions by promoting assembly of AR-containing regulatory complexes and that high levels of NPM might alter AR functions in PCa.

The androgen receptor (AR) is a ligand-responsive transcription factor known to play a central role in the pathogenesis of prostate cancer. However, the regulation of AR gene expression in the normal and pathological prostate remains poorly understood. This study focuses on the effect of the phosphoinositide 3-kinase (PI 3-kinase)/Akt axis on AR expression in vas deferens epithelial cells (VDEC), a suitable model to study androgen regulation of gene expression, and LNCaP cells (derived from a metastasis at the left supraclavicular lymph node from a 50-year-old patient with a confirmed diagnosis of metastatic prostate carcinoma). Taken together, our data show for the first time that the PI 3-kinase/Akt pathway is required for basal and dihydrotestosterone-induced AR protein expression in both VDEC and LNCaP. Inhibition of the PI 3-kinase/Akt pathway reduced AR expression and the decline in AR protein level correlated with a decrease in AR mRNA in VDEC but not in LNCaP. Since PI 3-kinase/Akt axis is active in prostate cancer, cross-talk between PI 3-kinase/Akt and AR signalling pathways may have implications for endocrine therapy.

The morphology of the mouse vas deferens still undergoes major changes from birth to 40 days of age, such as differentiation of the mesenchymal cells into fibroblasts and muscle cells, differentiation of the epithelium into basal and columnar epithelial cells, development of stereocilia, and the appearance of smooth endoplasmic reticulum organised in fingerprint-like structures or parallel, flattened saccules. In mutant homozygous ΔF508 (ΔF/ΔF) and knock-out (cf/cf) CFTR mice, strain 129/FvB and 129/C57BL-6, respectively, a similar development occurred until the age of 20 days. At 40 days, however, the lumen was filled with eosinophilic secretions, and sperm cells were absent in the majority of the animals examined, although sperm production in testis and epididymis appeared to be normal. CFTR was localised in the apical membrane and cytoplasm of the vas deferens epithelium from 40 days on but could not be detected in the vas deferens before 20 days or in mutant adult CFTR mice as expected. Western blots of membrane preparations showed that the mature form of CFTR was present in vas deferens and testis but absent in seminal vesicles. Our results suggest that the function of CFTR is probably essential after 20 days in the vas deferens and that its absence or dysfunction may result in a vas deferens with a differentiated epithelium but a collapsed lumen, which could at least temporarily delay the transport of spermatozoa. These observations contrast with those made in the overall majority of CF patients. Mol. Reprod. Dev. 55:125–135, 2000. © 2000 Wiley-Liss, Inc.

19 NO UV EL LE S M AG AZ IN E NOUVELLE C. Beaudoin, S. Maquaire, J. Allemand, C. Lours-Calet, G. Veyssiere, M. Manin, L. Morel : CNRS UMR 6247/ Clermont Universite/INSERM U931, Campus Universitaire des Cezeaux, 24, avenue des Landais, 63177 Aubiere Cedex, France. Laurent.MOREL@univ-bpclermont.fr L. Leotoing : Departement d’Hematologie, Inserm U567, Institut Cochin, 24, rue du Faubourg Saint-Jacques, 75014 Paris, France. NPM : un nouvel acteur dans le cancer de la prostate ?

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.

The MVDP (mouse vas deferens protein) gene, which encodes an aldose reductase-like enzyme, is mainly expressed in vas deferens epithelium and adrenal cortex. Vas deferens MVDP gene transcription was known to be under androgenic control, we now have evidence for androgen and probable ACTH responsiveness of the MVDP gene in the adrenal. To analyze the role of potential regulatory regions in hormonal, developmental, and tissue-specific aspects of MVDP regulation, we generated transgenic mice harboring MVDP-CAT fusion genes. The constructs carried either -1.8 or -0.5 kb 5'-flanking sequence attached to the chloramphenicol acetyltransferase gene in presence or absence of a 3.5-kb intragenic fragment in a downstream position. We show that at least two regions ensure proper gene regulation in vivo. The first, located within the 1.8-kb promoter fragment, directs tissue specificity; positive elements necessary for vas deferens and adrenal expression lay within positions -1804 to -510 and -510 to +41, respectively. The second, located within the 3.5-kb intragenic fragment spanning intron 1 to intron 2, increases percentage of expressing lines and behaves as a vas deferens-specific enhancer. Hormonal and developmental control of transgenes closely parallel endogenous gene regulation. Androgen and ACTH responsiveness in adrenals is conferred by 0.5-kb promoter, whereas in vas deferens, full androgenic response of the 1.8-kb promoter required the 3.5-kb intragenic fragment. Thus, vas deferens and adrenals use distinct cis-acting elements to direct and regulate the expression of the MVDP gene.

Although NHC-complexes are renowned for their catalytic properties, studies of their anti-cancer properties are scarce. In our search for new potent metal-based drugs, we envisioned that copper(I)-NHC could serve as a stable analogue of copper(I)-phenantroline. Herein, we report the results of the biological evaluation of the anticancer properties of [(SIMes)CuCl] 5. This complex showed impressive cytotoxicity against a panel of human tumor cell lines. Regarding IC50, up to 150 fold decrease is observed compared to cisplatin. Cellular effects of [(SIMes)CuCl] were evaluated at the cell cycle and the apoptotic levels. 5 causes cell cycle arrest at the G1 phase as demonstrated by the concomitant accumulation of protein p21 and the strong down-regulation of cyclin D1. Monitoring of poly-(ADP-ribose)-polymerase, implied in apoptosis response of cells, showed that [(SIMes)CuCl] induces apoptosis at low concentration. Finally, we showed that [(SIMes)CuCl] targets DNA by in vitro studies. When inducing aerobic DNA cleavage, [(SIMes)CuCl] behaves analogously to copper(I)-phenantroline. Thus, our experiments evidenced that employing NHC to deliver active copper(I) species in cellulo is a valuable strategy in anticancer research.

In the male, androgens promote growth and differentiation of sex reproductive organs through ligand activation of the androgen receptor (AR). Here, we show that androgens are not major actors of the cell cycle arrest associated with the differentiation process, and that the epidermal growth factor (EGF)-mediated signalling interferes with AR activities to regulate androgen response when epithelial cells are differentiated. Higher AR expression and enhanced androgen responsiveness correlate with reduction of phosphorylated ERK1/2 over differentiation. These modifications are associated with recruitment of cells in phase G(0)/G(1), up-regulation of p27(kip1), down-regulation of p21(Cip1) and p53 proteins, and accumulation of hypo-phosphorylated Rb. Exposure to EGF reduces AR expression levels and blocks androgen-dependent transcription in differentiated cells. It also restores p53 and p21(Cip1) levels, Rb hyper-phosphorylation, ERK1/2 activation and promotes cell cycle re-entry as p27(kip1) protein levels are decreased. Treatment with a MEK inhibitor reverses the EGF-mediated AR down-regulation in differentiated cells, thus suggesting the existence of an inverse correlation between EGF and androgen signalling in non-tumoural epithelia. Interestingly, when androgen signalling is set in differentiated cells, dihydrotestosterone exerts an inhibitory effect on ERK activity but paradoxically does not modify EGFR (ErbB1) phosphorylation, indicating that androgens are able to disrupt the EGFR-ERK cascade. Overall, our data demonstrate the existence of a balance between AR and mitogen-activated protein kinase activities that favours either the maintenance of differentiated conditions or the enhancement of cell proliferation capacities.

International audience; We report here on the characterization of tissue-culture cell lines derived from primary cultures of the mouse caput epididymidis epithelium. The cell lines were spontaneously immortalized without the use of transforming oncogenes. In defined conditions, our epididymal cells adopted various morphological features that resembles that of the in vivo epididymis epithelium such as a polarized organization and the presence of junctional structures at their apical/lateral membranes as revealed by electron microscopy analyses. Flow cytometry analysis revealed that we were dealing with homogenous cell populations that had reached a near-tetraploid state. RT-PCR assays were used in order to show that several genes that can be considered as markers of in vivo caput epididymidis epithelium activity were expressed in our cell lines confirming that these cells were indeed in a differentiated state close to their endogenous state.

Androgens are known to modulate many cellular processes such as cell growth and survival by binding to the androgen receptor (AR) and activating the transcription of target genes. Recent data suggested that AR can also mediate non-transcriptional actions outside the nucleus in addition to its ligand-inducible transcription factor function. Here, we describe a transcription-independent activation of the phosphatidylinositol 3-OH kinase (PI3-K) signaling pathway by androgens. Using non-transformed androgen-sensitive epithelial cells, we show that androgens enhance the PI3-K activity by promoting accumulation of phosphoinositide-3-P phospholipids in vitro. This activation is found in conjunction with an increased time-dependent phosphorylation of the downstream kinase AKT/protein kinase B on both Ser(473) and Thr(308) residues. Hormone-stimulated phosphorylation of AKT requires AR since incubation with the anti-androgen bicalutamide completely abolishes the androgen-stimulated AKT phosphorylation. Accordingly, we show that androgens increase AKT phosphorylation level in prostatic carcinoma PC3 cells only once they have been transfected with AR. Downstream, androgens enhance phosphorylation of transcription factor FKHR (Forkhead in rhabdomyosarcoma)-L1 and proapoptotic Bad protein and promote cell survival as they can counteract an apoptotic process. We also report that non-genomic effects of androgens are based on direct interaction between AR and the p85alpha regulatory subunit of class I(A) PI3-K. Together, these novel findings point out an important and physiologically relevant link between androgens and the PI3-K/AKT signaling pathway in governing cell survival.

BACKGROUND Primary cultures and subcultures of prostate epithelial cells (PEC) proliferate markedly, but rapidly loose secretory differentiated function and androgen responsiveness. Here, we investigated whether differentiation could be restored or preserved by using three-dimensional reaggregation cultures treated with retinoids and/or androgens. METHODS PEC were cultured as monolayers or as reaggregation cultures on a rotatory shaker. Reaggregation cultures were also developed from freshly isolated cells. Morphology was evaluated microscopically. Expression of cytokeratins (CKbasal for basal cells and CK18 for luminal cells), E-cadherin, α- and β-catenin, androgen receptor (AR), and prostate specific antigen (PSA) was evaluated by immunohistochemistry and/or Western blotting. Differentiated function was further evaluated by measurements of PSA in the medium and by reverse transcriptase-polymerase chain reactions for AR, PSA, prostate specific membrane antigen, β-microseminoprotein, and zinc-α2-glycoprotein. Proliferation was evaluated by immunohistochemical staining for Ki-67. RESULTS Monolayer cultures of PEC expressed CKbasal as well as CK18, a combination compatible with an intermediary amplifying population of epithelial cells. No expression of PSA could be detected, and all attempts to re-induce differentiation of PEC in classic two-dimensional culture systems failed. In reaggregation cultures of subcultured PEC, retinoids proved essential to maintain a compact three-dimensional structure. This effect was accompanied by increased levels of E-cadherin and of the catenins and by a shift in the cytokeratin expression pattern toward that typical for secretory differentiated cells (CK18 only). Even in the presence of androgens, however, PSA remained undetectable. Similar effects of retinoids were observed in reaggregation cultures of freshly prepared PEC, and in the latter cultures, the combination of androgens and retinoids maintained a low level of PSA secretion for at least 40 days. CONCLUSIONS A combination of retinoids and androgens is able to preserve, for a prolonged period of time, some degree of secretory differentiation in freshly isolated PEC maintained in reaggregation culture. The same combination is unable to restore secretory differentiation in subcultured PEC. Prostate 53: 34–49, 2002. © 2002 Wiley-Liss, Inc.

Vas deferens epithelial cell subcultures were used to study the sequential regulation of jun/fos proto-oncogene expression and AP1 activities during cell proliferation, polarization and androgen-induced expression of a terminal differentiation marker, i.e. the mvdp gene. Proliferation of epithelial cells is associated with a high expression in the nucleus of most Jun and Fos oncoproteins. After cell seeding on an extracellular matrix which allows polarization and expression of the mvdp gene in response to androgens, AP1 protein accumulation is greatly altered and consists in a loss of JunB, Fra1, FosB and a decrease in c-Fos, c-Jun and Fra2, while JunD remained at the same level. This was correlated with a drop in AP1 binding activity as evaluated by gel shift assay using either AP1 consensus sequence or AP1 binding sites of the mvdp gene promoter region, and in AP1 transactivating activity, as estimated by stable transfection experiments using an AP1 responsive promoter (TRE-TK-luc). Androgens did not significantly influence AP1 activities. On the contrary, stimulation of AP1 proteins by the tumor-promoting phorbol ester caused a decrease in androgen-induced mvdp mRNA accumulation, and this effect was reversed by staurosporine, a potent inhibitor of PKC. Our data suggest that a down-regulation of AP1 activities induced by epithelial cell differentiation is a prerequisite to androgen-induced mvdp gene expression. The high AP1 activities observed during proliferative state or induced in TPA-treated polarized cells, exert a repressive effect on androgen action.

Mouse vas deferens protein (MVDP), a member of the aldo-keto reductase superfamily, is exclusively produced in the vas deferens. To better understand androgen-regulated MVDP gene expression we have used RNA hybridization to study the effects of androgens on the steady-state levels of MVDP mRNA in vas deferens epithelial cell subcultures. Northern blot analysis revealed that these cells only express MVDP mRNA in the presence of androgens. There was a close relationship between MVDP mRNA levels and dihydrotestosterone concentrations. MVDP mRNA is induced over a period of 24h and maximal induction is about 25-fold. Treatment of cells with cycloheximide completely abolished the observed androgen effect suggesting that the induction of the MVDP gene by androgens depends on continuous protein synthesis. Transient transfection of vas deferens epithelial cells with MMTV-CAT vector showed that these cells contained functional androgen receptors and that they are a suitable system to study androgen effect on MVDP gene regulatory elements.

Pure epithelial cell cultures, obtained from primary culture of vas deferens tissue collected from 20- to 30-day-old mice, were amplified by subculturing the cells over 3T3 feeder layer in a serum-free defined medium. Adhesion and proliferation of epithelial cells did not require androgens, but a minimal concentration of 5.10(-7) M hydrocortisone. In that system, epithelial cells expressed cytokeratin but failed to produce the tissue specific mouse vas deferens protein (MVDP) in response to androgens. Various culture procedures and medium compositions were assayed for induction of MVDP expression. Culture onto microporous membrane inserts, which allow polarization of cells, is absolutely required for androgenic induction of MVDP. Androgen action did not require the presence of hydrocortisone, insulin, triiodothyronine, pituitary extracts, epidermal growth factor and acetylcholine. A minimal supplemented medium was then defined in which the expression of MVDP by epithelial cells in response to androgens was dose dependent. It has also been shown that this response at each concentration of dihydrotestosterone was heterogeneous at individual cell level. Highly reproducible results were obtained from epithelial cell cultures between 8th to 16th passages, showing that subcultured cells have maintained their ability to differentiate and express specialized functions.

New weapons to fight cancer are constantly needed. Among chemotherapeutics, anti-cancer metal-drugs have enjoyed a long and successful history since the discovery of the benchmark cisplatin. Advances in metal-drug discovery have motivated chemists to build plethora of complex structures. Among them, a novel area is emerging. This article presents a survey of the metal-N-Heterocyclic Carbenes (Ag(I), Au(I), Pd(II) and Cu(I)-NHCs) as potential anti-cancer agents. Most of the metal-NHCs considered display higher cytotoxicities than the reference metallo-drug cisplatin. Some of them are even selective for particular cell lines. Their mechanisms of action at the cellular level are further discussed, showing that the nature of the metal is of great importance. All these promising results demonstrate that this approach deserves more attention and work.

Insulin/glucagon control of hepatic metabolism, i.e. a endocrine-nervous system, is one of the general systems of integration in vertebrates. In this system, substrates coming from the digestive tract or from extrahepatic metabolism are important messenger molecules. Liver uptake of insulin and glucagon mainly accounts for high metabolic clearance rates of these hormones in both ruminants and non-ruminants. Glucagon infusion into ruminants results in an increase in the net hepatic uptake of glucose precursors and gluconeogenesis. Glucagon effects have also been demonstrated in isolated hepatocytes. Glucagon, through its effect on pyruvate carboxylase (EC 6.4.1.1.) may regulate gluconeogenesis. Insulin infusion induces hypoglycaemia. As a result, glucagon secretion increases and counterregulates insulin action. However, it has been shown that hepatic gluconeogenesis decreases during euglycaemic hyperinsulin clamp, mainly due to a decrease in the hepatic supply of glucose precursors following insulin action in extrahepatic tissues. Insulin fails to elicit any significant effect in vitro. Hepatocytes exhibit insulin and glucagon receptors. The apparent characteristics of hormone binding in vitro are similar in ruminants and non-ruminants, but the characteristics of postreceptor events are unknown in the former. Glucagon, which influences hepatic glucose synthesis, may be a major hormone in ruminants.

National audience

Insulin binding and tyrosine kinase activity of the insulin receptor have been measured in the liver and muscles of rats fed or submitted to a 72-h-fasting. In both tissues, insulin binding increased in fasting rats. In liver, the ability of insulin to simulate receptor tyrosine kinase activity greatly unpaired during fasting, but remained unchanged in muscle. The change during fasting of the insulin-stimulated tyrosine kinase activity of the insulin receptor is specific to certain tissue.