Gene expression profile of prostate cancer cell lines: effect of nerve growth factor treatment.

A dysregulation of the nerve growth factor (NGF)-mediated control of prostate cell growth is associated with the malignant progression of prostate epithelial cells. Exogenous NGF induced in prostate cancer (PCa) cell lines DU145 and PC3 the expression of p75(NGFR), accompanied by a reduction of the cell malignancy. The aim of this study was to analyze the profile of NGF-regulated genes the PCa cell line DU145 by using the cDNA microarray technique. NGF treatment of DU145 cells decreased the expression of 52 known genes, while the expression of 40 known genes was increased. NGF treatment of the DU145 cell line modified the expression profile of clusters of genes involved in invasion and metastasis, in cell proliferation and apoptosis, inflammation, cell metabolism and transcriptional activity. Interestingly, NGF induced the same pattern of gene modifications in both PCa cell lines. Data presented here may help to identify gene/proteins that dispose to PCa progression and to assess future markers that could allow the development of new clinic diagnostic and therapeutical approaches.

Oligomeric assembly of dopamine D1 and glutamate NMDA receptors: molecular mechanisms and functional implications.

In the striatum, dopamine D(1)R (D(1) receptor) activation potentiates NMDA (N-methyl-D-aspartate) transmission and is required for NMDA-mediated long-term potentiation at corticostriatal synapses. By using a combination of co-immunoprecipitation, pull-out with glutathione S-transferase-fusion proteins and bioluminescence resonance energy transfer, we have reported that the D(1)R forms a heteromeric complex with the NMDAR (NMDA receptor) and that this mechanism is crucial to recruit the D(1)R to the postsynaptic density. By using confocal and radioligand-binding assay, we also demonstrated that the interaction with NMDAR abolishes agonist-mediated D(1)R sequestration, indicating that oligomerization with NMDAR could represent a novel regulatory mechanism modulating D(1)R cellular trafficking and desensitization.

Sex-related variations in serum nerve growth factor concentration in humans.

A role of nerve growth factor (NGF) in the neuro-endocrine-immune interactions has been recently suggested by the presence of NGF and its receptors in cells of the immune and endocrine systems. The improvement in the comprehension of the role played by NGF in humans is linked to the availability of a sensitive and reliable method to quantify NGF concentrations in body fluids and tissues. As a consequence of different methods used, normal levels of human serum NGF reported in the literature show wide differences. The present results indicate that ELISA appears very sensitive (detection limit 1.4pg/ml) and allows the discrimination of subtle variations of serum NGF concentrations. ELISA performed in serum obtained from men indicated that NGF concentration was 40.8+/-10.8pg/ml, whereas women showed significantly lower levels that were influenced by the menstrual cycle. In particular, the mean value of this neurotrophin during the follicular phase was 8.2+/-1.4pg/ml; the luteal phase, in turn, showed levels up to 14.4+/-2.9pg/ml. The difference of serum NGF concentrations between the follicular and luteal phase in each woman was statistically significant. Differences in NGF concentrations between men and women (in both phases of the menstrual cycles) were also statistically significant. In conclusion, a possible role of sex steroids as modulators of NGF secretion in humans is strongly supported by the present paper. However, mechanisms underlying this phenomenon are still unknown. The evidence indicating physiological sex hormone-related variations in NGF levels would be of interest in view of the possible use of circulating NGF modifications as a laboratory biomarker in different diseases.

Retinoic acid prevents experimental Cushing syndrome.

Cushing syndrome is caused by an excess of adrenocorticotropic hormone (ACTH) production by neuroendocrine tumors, which subsequently results in chronic glucocorticoid excess. We found that retinoic acid inhibits the transcriptional activity of AP-1 and the orphan receptors Nur77 and Nurr1 in ACTH-secreting tumor cells. Retinoic acid treatment resulted in reduced pro-opiomelanocortin transcription and ACTH production. ACTH inhibition was also observed in human pituitary ACTH-secreting tumor cells and a small-cell lung cancer cell line, but not in normal cells. This correlated with the expression of the orphan receptor COUP-TFI, which was found in normal corticotrophs but not in pituitary Cushing tumors. COUP-TFI expression in ACTH-secreting tumor cells blocked retinoic acid action. Retinoic acid also inhibited cell proliferation and, after prolonged treatment, increased caspase-3 activity and induced cell death in ACTH-secreting cells. In adrenal cortex cells, retinoic acid inhibited corticosterone production and cell proliferation. The antiproliferative action and the inhibition of ACTH and corticosterone produced by retinoic acid were confirmed in vivo in experimental ACTH-secreting tumors in nude mice. Thus, we conclude that the effects of retinoic acid combine in vivo to reverse the endocrine alterations and symptoms observed in experimental Cushing syndrome.

NMDA receptor subunits are phosphorylated by activation of neurotrophin receptors in PSD of rat spinal cord.

We have investigated the distribution of NMDA and neurotrophin receptor systems and their reciprocal interactions in post-synaptic densities (PSD) purified from spinal cord. NMDA receptor subunits, trkA and trkB, but not trkC, were present in spinal cord PSD. The incubation of PSD with BDNF and NGF induced the phosphorylation of NR2A and B subunits. This phosphorylation was counteracted by antibodies directed against the catalytic domain of trkA and trkB receptors and by genistein. These results suggest the existence of a previously unexplored cross-talk between neurotrophins and NMDA receptors in rat spinal cord neurons.

Nerve growth factor and retinoic acid inhibit proliferation and invasion in thyroid tumor cells.

NGF has anti-proliferative and anti-invasive effects in neuroendocrine tumors. In the present work we examined the effects of NGF and retinoic acid on cell proliferation and invasion in thyroid carcinoma cells. We found that NGF and retinoic acid do not affect cell proliferation on their own but in combination they produce a strong inhibition. We also found that retinoic acid regulates the matrix metalloproteinase 2 activity and invasion. In contrast, NGF inhibited invasion and reverted the effect of retinoic acid. This effect of NGF is likely mediated by an increase in adhesion to laminin and collagen IV and the inhibition of cell migration. NGF also induced the expression of the p75 NGF receptor. In conclusion, NGF and retinoic acid in combination inhibit proliferation and invasion of thyroid papillary carcinoma cells. These data open the possibility of a potential combined therapy for thyroid papillary carcinomas.

Differential gene expression of dopamine D-2 receptor subtypes in rat chromaffin cells and sympathetic neurons in culture.

Chromaffin cells and sympathetic neurons arise from a common bipotential progenitor which, if exposed to nerve growth factor (NGF), matures into a sympathetic neuron, but if exposed to glucocorticoids (GCs), differentiates into a mature chromaffin cell. Pharmacological evidence indicates that, in adrenal medulla and sympathetic neurons, dopamine (DA) receptors belonging to the D-2 family inhibit catecholamine secretion. The molecular characterization of these receptors, however, is not been yet described. Our data suggest that bipotential cells obtained from newborn rat adrenal medulla express both isoforms of the D-2 receptor, while D-3 receptor and D-4 receptor messenger RNAs (mRNAs) are not present. GC-mediated maturation induces the expression of D-4 receptors, without modification of D-2 isoforms. Sympathetic neurons differentiated in vitro selectively express the D-2short mRNA. Taken together, present results suggest that NGF and GCs play a role in regulating D-2 family receptor expression in neural crest-derived cells.

Retinoic acid stimulates meningioma cell adhesion to the extracellular matrix and inhibits invasion.

Meningiomas are tumours derived from the arachnoid and pia mater. During embryogenesis, these membranes develop from the migrating craniofacial neural crest. We have previously demonstrated that meningiomas have characteristic features of embryonic meninges. Craniofacial neural crest derivatives are affected during normal development and migration by retinoic acid. We speculated, therefore, that meningioma cell migration and invasion would be affected in a similar way. In this study we investigated the mechanisms of invasion and migration in meningiomas and the effects of retinoic acid (RA). We found that low doses of RA inhibit in vitro invasion in meningioma cells, without affecting cell proliferation or viability. The matrix metalloproteinases MMP-2 (72 kDa gelatinase) and MMP-9 (92 kDa gelatinase), which play a key role in invasion in other tumours, are not affected by RA. RA inhibits cell migration on collagen I and fibronectin. A possible mechanism for these effects is provided by the fact that RA strongly stimulates adhesion of meningioma cells to extracellular matrix substrates. As in vitro invasion, migration and decreased adhesion to the extracellular matrix correlate with the clinical manifestation of tumour invasion, we conclude that RA induces a non-invasive phenotype in meningioma cells.

Suppression of telomerase, reexpression of KAI1, and abrogation of tumorigenicity by nerve growth factor in prostate cancer cell lines.

Nerve growth factor (NGF) is expressed in the prostate, where it appears to be involved in the control of epithelial cell growth and differentiation. NGF production is decreased in prostate tumors. However, the role of this neurotrophin in the control of proliferation and progression of prostate cancers is still a matter of investigation. Prostate adenocarcinomas are telomerase-positive tumors. Chronic exposure of DU145 and PC3 prostate tumor cell lines to NGF resulted in a dramatic down-regulation of telomerase activity. This effect was correlated in terms of concentrations and time with a remarkable down-regulation of cell proliferation both in vitro and in vivo but was not secondary to NGF-induced quiescence. No down-regulation of telomerase activity was, in fact, detectable during serum starvation-induced quiescence. LNCaP cells, which do not express NGF receptors, appear to be insensitive to the actions of NGF. DU145 and PC3 cells do not express the KAI1 metastasis suppressor gene, which is present in the prostate and is progressively lost during the progression of prostate cancers. Chronic NGF treatment strongly induced the reexpression of this gene in these cell lines, and this effect was correlated with the suppression of their invasive potential in vitro. The data presented here suggest that NGF reverts two metastatic prostate cancer cell lines to slowly proliferating, noninvasive phenotypes characterized by a very low telomerase activity and by the expression of the KAI1 metastasis suppressor gene.

Growth factors in pituitary tumors.

Prolactin-secreting tumors are the most frequently occurring neoplasms in the human pituitary. Although the clinical syndrome associated with prolactinomas is well recognized the molecular and cellular mechanisms leading to cell transformation and development of these tumors remain elusive. In this paper we summarize recent evidence suggesting that both hypothalamic and intrapituitary defects can be involved in the development of prolactinomas. In particular alterations of the hypothalamo-pituitary dopaminergic transmission result in the dysregulation of the proliferative activity of lactotrope cells leading to tumor development. Similarly changes in the expression and activity of resident growth factors also play a role in pituitary tumorigenesis. In particular both overexpression of TGF alpha and loss of NGF production appear to be involved in the development and progression of prolactin-secreting tumors.

Nerve growth factor abrogates the tumorigenicity of human small cell lung cancer cell lines.

Nerve growth factor (NGF) has antiproliferative and differentiating effects on adenomas of neuroendocrine origin. Cell lines derived from small-cell lung carcinoma (SCLC), a very aggressive neuroendocrine tumor, express NGF receptors. The role of NGF in the control of proliferation and progression of this carcinoma, however, has never been investigated. Chronic exposure of NCI-N-592 and GLC8 SCLC cell lines to NGF remarkably inhibited their proliferation rate both in vitro and in vivo, prevented their anchorage-independent clonal growth in soft agar, impaired their invasive capacity in vitro, and abolished their tumorigenic potential in nude mice. The proliferative response of SCLC cell lines to nicotine was also remarkably impaired by in vitro NGF treatment. Furthermore, NGF treatment activates in SCLC cell lines the expression and secretion of NGF. NGF thus reverts SCLC cell lines to a noninvasive, nontumorigenic phenotype that does not respond to nicotine and produces NGF.

Nerve growth factor in pituitary development and pituitary tumors.

Cells in the anterior pituitary originate from a common pluripotent precursor whose phenotypic development is determined by intrapituitary transcription factors as well as by hypothalamic and peripheral signals. A rapidly growing body of evidence revealed that essential to the differentiation and proliferation of pituitary cells are an array of growth factors that are produced within the pituitary and act mainly through autocrine mechanisms. Growth factors are polypeptides that are released in carefully measured amounts by some cells to regulate cell growth and differentiation by activating specific tyrosine kinase receptors in the plasma membrane of target cells. Both overproduction of mitogenic growth factors and loss of factors inhibiting cell proliferation result in uncontrolled cell growth and tumor development. There is now increasing evidence that disruption of the calibrated signalling network activated by pituitary growth factors plays a central role in pituitary tumorigenesis. This paper is focussed on the role of nerve growth factor (NGF) in pituitary physiology and pathology. In particular, we propose that NGF plays a dual role in the gland: a local one as a stimulator of differentiation and proliferation of lactotrope cells during pituitary development and a systemic one as a neurohormone which is cosecreted with prolactin into the bloodstream. Furthermore, we discuss the evidence that NGF is an autocrine differentiation factor for prolactin-secreting cells. Escape from NGF control appears to be one of the mechanisms involved in the development and progression of prolactinomas. Along the same line, exposure of prolactinomas refractory to dopaminergic therapy to exogenous NGF results in their differentiation into lactotrope-like cells reexpressing the D2 receptor protein. This observation may open the way to a sequential therapy with NGF and bromocriptine for patients refractory to the conventional therapy.

Dopamine receptors: from structure to function.

The diverse physiological actions of dopamine are mediated by at least five distinct G protein-coupled receptor subtypes. Two D1-like receptor subtypes (D1 and D5) couple to the G protein Gs and activate adenylyl cyclase. The other receptor subtypes belong to the D2-like subfamily (D2, D3, and D4) and are prototypic of G protein-coupled receptors that inhibit adenylyl cyclase and activate K+ channels. The genes for the D1 and D5 receptors are intronless, but pseudogenes of the D5 exist. The D2 and D3 receptors vary in certain tissues and species as a result of alternative splicing, and the human D4 receptor gene exhibits extensive polymorphic variation. In the central nervous system, dopamine receptors are widely expressed because they are involved in the control of locomotion, cognition, emotion, and affect as well as neuroendocrine secretion. In the periphery, dopamine receptors are present more prominently in kidney, vasculature, and pituitary, where they affect mainly sodium homeostasis, vascular tone, and hormone secretion. Numerous genetic linkage analysis studies have failed so far to reveal unequivocal evidence for the involvement of one of these receptors in the etiology of various central nervous system disorders. However, targeted deletion of several of these dopamine receptor genes in mice should provide valuable information about their physiological functions.

Anterior pituitary hypoplasia and dwarfism in mice lacking the dopamine transporter.

Deletion of the dopamine transporter (DAT) results in increased dopaminergic tone, anterior pituitary hypoplasia, dwarfism, and an inability to lactate. DAT elimination alters the spatial distribution and dramatically reduces the numbers of lactotrophs and somatotrophs in the pituitary. Despite having normal circulating levels of growth hormone and prolactin in blood, hypoplastic glands from DAT-/- mice fail to respond to secretagog stimulation. The effects of DAT deletion on pituitary function result from elevated DA levels that down-regulate the lactotroph D2 DA receptors and depress hypothalamic growth hormone-releasing hormone content. These results reveal an unexpected and important role or DA in the control of developmental events in the pituitary gland and assign a critical role for hypothalamic DA reuptake in regulating these events.

Nerve growth factor suppresses the tumoral phenotype of human prolactinomas.

We summarize here our data showing that various phenotypical characteristics distinguish prolactinoma cell lines obtained from responder and nonresponder patients, as defined by their responses to bromocriptine administration. Nonresponder cell lines have a higher degree of malignancy than responder cells and do not express D2 receptors for dopamine. Both cell lines express NGF receptors. Exposure of the most malignant nonresponder cell lines to NGF, both in vitro and when transplanted in vivo in nude mice, results in their differentiation into the responder phenotype reexpressing D2 receptors. Sequential administration of NGF and bromocriptine thus may be a promising therapy for patients refractory to bromocriptine.

Opposite effects of dopamine D2 and D3 receptors on learning and memory in the rat.

Mesolimbocortical dopamine plays a role in learning and memory. The specific receptor subtypes mediating the effects of dopamine, however, are still unknown. Dopamine D2, D3 and D4 receptors are expressed in the hippocampus and dopamine D3 receptors are present in the septal area, suggesting that these receptor subtypes can contribute to the behavioral effects of dopamine D2-like receptor agonists. We now investigated the role of dopamine D2 and D3 receptors in learning and memory by using the transient amnesia induced by scopolamine in the passive avoidance test as experimental model. The data strongly suggest that both dopamine D2 and D3 receptors mediate the effects of dopamine on the integrative function of learning and memory. In particular, we show that the non-selective dopamine agonist apomorphine prevents the scopolamine-induced disruption of consolidation of the previously acquired passive avoidance behavior. This effect is mediated by receptors belonging to the dopamine D2 family since it was antagonized by (-)-sulpiride and mimicked by quinpirole. Nafadotride, a relatively selective antagonist for dopamine D3 receptors, antagonized scopolamine-induced memory disruption and potentiated the facilitatory effect of quinpirole. Taken together, these results suggest that the effects of dopamine on memory consolidation are the result of a balance between dopamine D2 receptor-mediated facilitation and dopamine D3 receptor-mediated inhibition, and that dopamine D2 and D3 receptors play opposite roles in the control of the mechanisms leading to memory consolidation.