Impact of targeted therapies in metastatic renal cell carcinoma on patient-reported outcomes: Methodology of clinical trials and clinical benefit.

BACKGROUND: Molecular targeted therapies have improved progression-free survival (PFS) without translating systematically into overall survival (OS) for patients with metastatic renal cell carcinoma (mRCC). In this population, patient-reported outcomes (PROs) have become a significant outcome. We evaluated the methodological quality of the assessment of PROs in randomized controlled trials (RCTs) and the clinical benefit of the different treatments including survival and quality of life (QoL). METHODS: A systematic review identified RCTs published between January 2005 and July 2014. They were evaluated according to 11 items derived from the 2013 CONSORT PROs reporting guidelines. Survival outcomes and PROs main results were analyzed and the magnitude of clinical benefit was assessed with the European Society for Medical Oncology Magnitude of Clinical Benefit Scale (ESMO-MCBS). RESULTS: 12 RCTs were included with a total of 22 publications. The mean CONSORT score for all items was 4.5 on an 11-point scale. No publication reported the power of the PROs analysis and only one reported a PRO hypothesis. 50% of studies did not interpret PROs in relation to clinical outcomes and only 18% discussed specific limitations of PROs and their implications for generalizability. By adding the QoL criterion to PFS, 4 trials (36.4%) obtained a high level of proven clinical benefit according to the ESMO-MCBS. CONCLUSION: The methodology for assessing PROs in mRCC is not optimal. Efforts should focus on defining PROs endpoint and increasing the quality of reporting of QoL. New-generation therapies in mRCC should demonstrate a gain not only in survival but also in QoL to be included in the therapeutic arsenal.

Cell Therapy for Parkinson's Disease: A Translational Approach to Assess the Role of Local and Systemic Immunosuppression.

Neural transplantation is a promising therapeutic approach for neurodegenerative diseases; however, many patients receiving intracerebral fetal allografts exhibit signs of immunization to donor antigens that could compromise the graft. In this context, we intracerebrally transplanted mesencephalic pig xenografts into primates to identify a suitable strategy to enable long-term cell survival, maturation, and differentiation. Parkinsonian primates received WT or CTLA4-Ig transgenic porcine xenografts and different durations of peripheral immunosuppression to test whether systemic plus graft-mediated local immunosuppression might avoid rejection. A striking recovery of spontaneous locomotion was observed in primates receiving systemic plus local immunosuppression for 6 mo. Recovery was associated with restoration of dopaminergic activity detected both by positron emission tomography imaging and histological examination. Local infiltration by T cells and CD80/86+ microglial cells expressing indoleamine 2,3-dioxigenase were observed only in CTLA4-Ig recipients. Results suggest that in this primate neurotransplantation model, peripheral immunosuppression is indispensable to achieve the long-term survival of porcine neuronal xenografts that is required to study the beneficial immunomodulatory effect of local blockade of T cell costimulation.

[Hard palate B cell lymphoma with spontaneous regression]. / Régression spontanée d'un lymphome B à grandes cellules du palais.

INTRODUCTION: Non-Hodgkin lymphomas are common cancers that can develop in the upper aero-digestive tract. We describe a case of a large B-cell palatine lymphoma with spontaneous clinical regression. CASE: A 58-year-old female patient presented with a sub-mucosal lesion of the hard palate. CT scan and magnetic resonance imaging revealed a lesion invading the right posterior palatine canal. At the second consultation, 15 days after performing the biopsy, the lesion had disappeared. PET scan proved the absence of lesion. Lymph node biopsy supported the diagnosis of large B-cell lymphoma. DISCUSSION: Large B-cell lymphoma of the hard palate is a rare disease. Only 27 cases have been described in the international literature. The anatomopathological analysis is often difficult to perform. The final diagnosis is often made by immunochemistry. The usual treatment is R-CHOP chemotherapy (cyclophosphamide, adriamycin, vincristine, prednisone combined to rituximab) with a 5-year survival rate at 55%.

Modification of mechanical properties of recycled polypropylene from post-consumer containers.

This study is conducted to look at the modification of mechanical properties of recycled polypropylene (PP) from post-consumer containers with the addition of stabilizers, elastomer (ethylene-octene rubber, EOR) and calcium carbonate (CaCO(3)). The mechanical and thermal properties of the blends were evaluated. The results showed limited changes with the addition of elastomer and calcium carbonate on the mechanical properties of the recycled polypropylene. Some differences were observed, but the trends were not reproducible over the different compositions. DSC analysis confirmed the presence of polyethylene (PE) in the recycled polypropylene. The polyethylene impurity and the presence of many different qualities of polypropylene in the recycled material may have prevented any possible improvement in the mechanical properties by the addition of EOR and CaCO(3), improvements seen in previous studies on virgin polypropylene. The compatibility of the different homopolymers and copolymers of PP used in consumer packaging is not known, while polyethylene and polypropylene are known not to be miscible with each other. The mixture of qualities and materials may explain such a poor blending. Reusing and upgrading of recycled PP from post-consumer containers would therefore first require a better sorting of the post-consumer waste. The use of an adequate compatibilizer that would allow a uniform and homogeneous blending of the raw material mixture might enhance the mechanical properties.

Decreased plasma folate concentration in young and elderly healthy subjects after a short-term supplementation with isotretinoin.

BACKGROUND: In the last two decades, there has been an increasing use of isotretinoin (13-cis-retinoic acid or 13-CRA) for treatment of severe, and recently mild and moderate, acne in Westernized populations. Recent human and animal studies emphasized alterations caused by 13-CRA administration on folate-dependent, one-carbon metabolism. Folate deficiency and subsequent hyperhomocysteinemia increase the risk of degenerative diseases. OBJECTIVES: We determine whether a short-term supplementation with 13-CRA alters folate status and homocysteinemia in young and elderly healthy human subjects. METHODS: Twenty young and 20 elderly (age mean, 26.1 and 65.4 years, respectively) healthy male volunteers were supplemented with approximately 0.5 mg/kg/day of 13-CRA for 28 days. Fasting plasma concentrations of 13-CRA, 5-methyltetrahydrofolate (5-mTHF) as the main circulating form of folate, and homocysteine (Hcy), as well as haematologic parameters and biochemical markers of liver and renal function, were measured at baseline and at the end of supplementation. Statistical analyses were carried out using two-way anova and standard tests. RESULTS: In both groups, isotretinoin supplementation caused a dramatic increase in the circulating concentration of 13-CRA and its derivatives. It also led to significant increases in serum triglyceride (P < 0.0001) and creatinine (P = 0.002) concentrations and gamma-glutamyltranspeptidase activity (P = 0.0001) and decrease in serum level of urea (P = 0.027). However, the latter four parameters remained within normal ranges. These changes were accompanied by a 17.7% and 13.5% decrease in the plasma level of 5-mTHF (P = 0.001) in the young and elderly volunteers, respectively. Supplementation with 13-CRA did not cause significant variations in their plasma Hcy concentration. However, the latter parameter seemed to respond differently in each group of age (P = 0.046). CONCLUSIONS: Our data indicate that a 28-day supplementation with isotretinoin alters the plasma folate in young and old healthy individuals. This stresses the necessity of studying the long-term effects of retinoid therapy on folate status and homocysteinemia in acne patients, given that alteration in the latter parameters is known to increase the risk of degenerative diseases.

Pig xenografts to the immunocompetent rat brain: Survival rates using distinct neurotoxic lesions in the nigrostriatal pathway and two rat strains.

Porcine foetal neurons for xenotransplantation in Parkinson's disease (PD) is an alternative source to human fetuses. One of the obstacles facing brain xenotransplantation is the existence of an immune response, which prevents long-term graft survival. Experimental results concerning the survival time of porcine foetal neurons implanted into the brain of immunocompetent rats have been quite different from one study to another, suggesting an effect on graft survival of uncontrolled experimental parameters. To identify such parameters, we have first analyzed the survival of porcine foetal nigral neurons at 5 and 10 weeks after implantation into the striatum of immunocompetent rats having different types of brain lesion affecting cells (quinolinic acid) or projections to the striatum (MPP+, 6-OHDA). In a second experiment, graft survival was analyzed in two strains of recipient rats (female Sprague-Dawley and male Lewis rats) in conditions of ipsilateral dopaminergic denervation using 6-OHDA. The characteristics of surviving grafts were assessed by measuring the graft volume, the number of TH+ neurons, the size of TH+ neurons soma, and CD5+ cell infiltration. Long-term survival (> or = 10 weeks) of porcine neurons could be observed in all experimental models. However, there was no significant difference in graft survival rates and characteristics of the surviving grafts between the lesioned groups, or between Sprague-Dawley and Lewis rats. Altogether, results were highly variable within groups of grafts exposed to similar experimental procedures at both 5 and 10 weeks post-grafting. We conclude that the distinct neurotoxins and host rat strains used in our experimental design are not major factors influencing the rejection time-course of primary neural xenografts.

Activation of c-Jun N-terminal kinase 1 (JNK-1) after amino acid deficiency in HeLa cells.

Long-term amino acid starvation represents a form of metabolic stress which stimulates gene expression. Here we report that depriving HeLa cells for any one of a series of amino acids activates c-Jun N-terminal kinase-1 (JNK-1). In contrast, the other mitogen-activated protein kinases (MAPKs) ERK-1 and, to a lesser extent, p38 activities decreased under such conditions. In methionine- or leucine-deprived cells, JNK-1 activation occurred after 4 or 6 h, respectively, and reached a steady maximum of 5- to 7-fold over control cells afterwards. This activation was dependent on the amino acid concentration and it could be reversed by resupplying the complete medium. Limitation for all amino acids also augmented JNK-1 activity, whereas increased amino acid concentrations had an opposite effect. The free radical scavenging thiol antioxidant N-acetylcysteine (NAC) alleviated partially JNK-1 activation in amino acid-deprived cells. The data indicate that activation of JNK-1 by long-term amino acid deprivation may be mediated in part by oxidative stress.

Different mechanisms mediate the rejection of porcine neurons and endothelial cells transplanted into the rat brain.

In order to investigate the early cellular responses mediating xenograft rejection in the brain, porcine aortic endothelial cells (PAEC) or porcine fetal mesencephalic neurons (PNEU) were transplanted into the striatum of LEW.1A rats. PAEC were detected with a specific anti-beta1 integrin antibody, and PNEU with an anti-porcine neurofilament antibody, or an antibody recognizing the NeuN antigen. PAEC grafts were massively infiltrated within 24 h by OX42-positive cells, which may correspond to polymorphonuclear (PMN) cells or macrophages. At that moment, the graft contained numerous cells expressing the inducible isoform of NO-synthase (iNOS). Infiltration by ED1-positive macrophages was effective after three days. The beta1-integrin labeling decreased from that time-point to day 7 post-implantation, and vanished after 11 days. Although some OX8-positive cells were present around the graft as soon as 3 days after transplantation, cells expressing the T-cell receptor (TCR)-beta chain infiltrated the graft after 7 days and their number remained low. A strong, diffuse OX8-and ED1-positive immunoreactive material remained in the scar up to the third week. In striking contrast, PNEU grafts remained poorly infiltrated by OX42- or ED1-positive cells during the first two weeks. A massive infiltration by macrophages and TCRbeta-positive lymphocytes occurred after 3 weeks. Natural killer (NK) cells were more scarce. The inflammation territory enlarged, and blood vessels were overloaded with macrophages or lymphocytes. Nevertheless, the graft contained NeuN-positive nuclei and neurites harbouring the porcine neurofilament protein. Hence, rejection was not completed at this time-point. These results suggest that the rapid rejection of PAEC is mainly driven by macrophages and possibly PMN cells, unlike PNEU, whose rejection is delayed and also involves lymphocytes. Differences in immunogenicity of grafted cells and/or patterns of production of pro-inflammatory cytokines may account for these contrasted rejection kinetics.

Epidermal growth factor modulates the expression of vascular endothelial growth factor in the human prostate.

The growth and dissemination of tumors in the body has been associated with angiogenesis. Vascular endothelial growth factor (VEGF) is an angiogenic factor that stimulates endothelial cell growth and enhances vascular permeability. VEGF exerts its action by binding to specific cell surface receptors. Three receptors, VEGFR-1 (flt-1), VEGFR-2 (flk-1), and VEGFR-3 (flt-4) have been identified. Very little information on the coordinated expression of VEGF and its receptors in normal prostate, benign prostatic hyperplasia (BPH), and prostate carcinoma is available. Therefore, we examined the immunohistochemical localization of VEGF and its receptors in tissues derived from normal human prostate, BPH, and prostatic carcinoma. Immunostaining for VEGF was absent in the normal prostate. Epithelium lining the glands of prostate derived from patients with BPH exhibited strong immunostaining. The intensity of staining was relatively less in prostate carcinoma. It is interesting that VEGFR-1 and VEGFR-3 were strongly expressed in both stromal and epithelial tissues in normal prostate, BPH, and carcinoma. In comparison, VEGFR-2 was not localized to normal prostate and its expression in the stroma of BPH and epithelium of carcinoma was very weak. Because progression of prostate cancer is accompanied by altered expression of epidermal growth factor (EGF) and its receptor (EGFR) in malignant cells, we investigated the effect of EGF on VEGF gene expression by Northern blot analysis in 2 human prostate cancer cell lines that express EGFR. EGF greatly enhanced the expression of VEGF messenger RNA in DU145 and PC3 cell lines in a dose-dependent manner. The EGF induction of VEGF gene expression suggests a mechanism by which angiogenesis could be accelerated in BPH and prostate carcinoma.

Differential regulation of GDNF, neurturin, and their receptors in primary cultures of rat glial cells.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) bind to GFR alpha-1 and GFR alpha-2 receptors, respectively, and their neurotrophic activity is mediated by the tyrosine kinase receptor, Ret. All these molecules were found to be expressed in primary cultures of rat glial cells, which were largely composed of astrocytes and maintained in serum-free medium. Although GDNF, NTN and Ret mRNA levels were at the limit of detection, RNase protection assays revealed relatively high amounts of GFR alpha-1 and GFR alpha transcripts. To characterize signals controlling their expression, glial cells were exposed to serum or treated with hormones acting through nuclear receptors and by activators of the cAMP or protein kinase C (PKC)-dependent pathways. Retinoic acid or 1,25-dihydroxyvitamin D3 appeared ineffective. In contrast, the 5-fold increase in GFR alpha-2 mRNA after 24 hr of treatment with 10(-10) M of tri-iodothyronine, suggests a physiological role of thyroid hormone in the regulation of this receptor in vivo. The serum induced a 7-fold increase in GFR alpha-1 mRNA levels. These changes may be mediated by the cAMP or PKC pathways because both forskolin and TPA up-regulated the GFR alpha-1 gene. Interestingly, only TPA led to a coordinated increase in the levels of GDNF, GFR alpha-1 and GFR alpha-2 mRNAs. On the other hand, NTN transcripts remained constant, irrespective of the culture conditions. Taken together, these results indicate that GDNF family ligands and their receptors are regulated in glial cells by common or independent transductional pathways, which could modulate their specific expression during brain development or in the case of trauma.

Increased translation efficiency and antizyme-dependent stabilization of ornithine decarboxylase in amino acid-supplemented human colon adenocarcinoma cells, Caco-2.

The mechanisms of the response of ornithine decarboxylase(ODC), the rate-limiting enzyme in polyamine biosynthesis, to amino acid supplementation were studied in the human colon adenocarcinoma cell line, Caco-2. Supplementation of serum-deprived, subconfluent Caco-2 cells with any one of a series of amino acids (10 mM) resultedin increased ODC activity, reaching a maximum of approx. 12.5-fold after approx. 4 h, over control cells either not supplemented or supplemented with iso-osmolar D-mannitol. Glycine, L-asparagine and L-serine, as well as their D-enantiomers, were the strongest effectors and acted in a concentration-dependent manner; millimolar concentrations of most of these amino acids being sufficient to significantly increase ODC activity. In contrast, supplementation with D-methionine, L-lysine, L-aspartate or L-glutamate had little or no effect on ODC activity, whereas supplemental L-methionine, L-arginine, L-ornithine or L-cysteine was inhibitory. Polyamine assays showed that the putrescine content of cells varied in accordance with the changes in ODC activity. Western-blot and Northern-blot analyses revealed specifically increased levels of ODC protein but not mRNA,respectively, in response to supplementation with an ODC-inducing amino acid. Suppression of the increase in cycloheximide-treated cellsconfirmed a requirement for protein synthesis. Pulse-labelling of cellswith [(35)S]methionine showed a 3-fold increase in thesynthesis of ODC protein after 4 h of supplementation with glycineor L-serine. Supplemental glycine also augmented, reversibly, the half-life of ODC by almost 4-fold and simultaneously decreased the activity of putrescine-induced free antizyme. These results suggest that translational, but not transcriptional, regulation of ODC takes part in ODC induction by amino acids in Caco-2 cells. However, it also appears to occur in concert with decreased enzyme in activation and/or degradation.

Rat oligodendrocytes express the vitamin D(3) receptor and respond to 1,25-dihydroxyvitamin D(3).

The present study investigates the presence of vitamin D receptor (VDR) in cells of the rat oligodendrocyte (OL) lineage. VDR transcripts were detected by in situ hybridization in a fraction of rat OL in secondary cultures. The VDR protein was shown to be co-localized in cells that are also recognized by an anti-myelin basic protein (MBP) antibody. Likewise, in vivo, VDR-positive cells were found in the brain white matter, such as the internal capsule of the striatum or the corpus callosum but also in the spinal cord. At least part of these positive cells in vivo correspond to OL, since they were co-stained by an anti-carbonic anhydrase II antiserum. Northern blot analyses of the CG-4 OL cell line demonstrated that the VDR transcripts are already found in the O-2A precursors. There was a two-fold increase in the relative abundance of these transcripts in differentiated OL or in type-2 astrocytes. 1, 25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)] increased the pool of transcripts encoding its own receptor, the VDR. The hormone also enhanced the abundance of the mRNA of the nerve growth factor (NGF) and of its low-affinity receptor, the p75(NTR) protein. By contrast, the hormone had no effect on the levels of MBP or proteolipid protein (PLP) mRNA. This finding suggests that unlike retinoic acid (RA) or thyroid hormone, 1,25-(OH)(2)D(3) has no regulatory action on the synthesis of myelin proteins.

Intracerebral implantation of NGF-releasing biodegradable microspheres protects striatum against excitotoxic damage.

Intrastriatal implantation of genetically modified cells synthesizing nerve growth factor (NGF) constitutes one way to obtain a long-term supply of this neurotrophic factor and a neuronal protection against an excitotoxic lesion. We have investigated if NGF-loaded poly(d,l-lactide-co-glycolide) microspheres could represent an alternative to cell transplantations. These microspheres can be implanted stereotaxically and locally release the protein in a controlled and sustained way. In order to test this paradigm, the NGF release kinetics were characterized in vitro using radiolabeled NGF, immunoenzymatic assay, and PC-12 cells bioassay and then in vivo after implantation in the intact rat striatum. These microspheres were thus implanted into the rat striatum 7 days prior to infusing quinolinic acid. Control animals were either not treated or implanted with blank microspheres. The extent of the lesion and the survival of ChAT-, NADPH-d-, and DARPP-32-containing neurons were analyzed. In vitro studies showed that microspheres allowed a sustained release of bioactive NGF for at least 1 month. Microspheres implanted in the intact striatum still contained NGF after 2.5 months and they were totally degraded after 3 months. After quinolinic acid infusion, the lesion size in the group treated with NGF-releasing microspheres was reduced by 40% when compared with the control groups. A marked neuronal sparing was noted, principally concerning the cholinergic interneurons, but also neuropeptide Y/somatostatin interneurons and GABAergic striatofuge neurons. These results indicate that implantation of biodegradable NGF-releasing microspheres can be used to protect neurons from a local excitotoxic lesion and that this strategy may ultimately prove to be relevant for the treatment of various neurological diseases.

1,25-dihydroxyvitamin D3 regulates the synthesis of gamma-glutamyl transpeptidase and glutathione levels in rat primary astrocytes.

Astrocytes play a pivotal role in CNS detoxification pathways, where glutathione (GSH) is involved in the elimination of oxygen and nitrogen reactive species such as nitric oxide. We have previously demonstrated that the specific activity of gamma-glutamyl transpeptidase (gamma-GT), an enzyme of central significance in GSH metabolism, is regulated in vivo in astrocytes by 1,25-dihydroxyvitamin D3 (1,25-D3). The aim of the present work was to investigate, in primary cultures of newborn rat astrocytes, the effects of this hormone on gamma-GT synthesis and on GSH and nitrite levels after lipopolysaccharide (LPS) treatment. This study demonstrates that both gamma-GT gene expression and specific activity, induced by LPS, are potentiated by 1,25-D3. In contrast, 1,25-D3 does not regulate the expression of other enzymes involved in astrocyte detoxification processes, such as superoxide dismutase or GSH peroxidase. In parallel, 1,25-D3 enhanced intracellular GSH pools and significantly reduced nitrite production induced by LPS. Taken together, these results suggest that gamma-GT, GSH, and 1,25-D3 play a fundamental role in astrocyte detoxification pathways.

Antizyme-dependent and -independent mechanisms are responsible for increased spermidine transport in amino acid-restricted human cancer cells.

Amino acid deprivation can inhibit tumour cell proliferation. Since polyamines are required for cell growth, we hypothesised that their regulatory pathways can respond to amino acid restriction. We report here that exposure of human colon adenocarcinoma Caco-2 cells to a medium restricted for a single amino acid, but not for D-glucose, activates spermidine transport. The increase was rapid and seemed transient with a maximum 4-6 hr after amino acid removal. Kinetics showed that the maximal velocity of transport was solely increased in L-methionine- or L-leucine-deprived cells, indicating increased number of transporters. The intracellular level of complex of ornithine decarboxylase (ODC) with antizyme, a negative regulator of polyamine transport, was decreased by 16-29% in amino acid-deprived cells. However, exposure to limited amounts of amino acid increased transport without altering the ODC-antizyme complex level. We propose that antizyme-independent mechanisms, sensitive to the amino acid concentration, also participate to the control of spermidine transport.

Selective sites for polyamine binding to rabbit intestinal brush-border membranes.

The intestinal polyamine transporters have not yet been identified. Our aim was to characterize specific polyamine binding sites in rabbit intestinal brush-border membranes (IBBM) as a starting step for identification of polyamine transporters. This was investigated at 4 degrees and at low membrane concentration. Saturation isotherms for [3H]putrescine (PUT) binding indicated a single population of sites (puT) with a dissociation equilibrium constant Kd of 3.8 microM and a density of sites Bmax of 58 pmol/mg of protein. [3H]spermidine (SPD) binding also involved only one class of sites (spD), albeit with a lower affinity (Kd = 106 microM) and higher abundance (Bmax = 1240 pmol/mg of protein) than puT. On the contrary, [14C]spermine (SPM) bound two classes of sites (spM1 and spM2) differing in their affinity (Kd = 2.5 and 31.4 microM) and abundance (Bmax = 467 and 1617 pmol/mg of protein, respectively). Membrane association of SPM at 4 degrees was much faster than that of SPD and PUT, both of which proceeded at a similar rate. In contrast to PUT and SPD dissociation, SPM dissociation at 23 degrees did not follow a first-order reaction. Specifically bound [3H]PUT, unlike [3H]SPD and [14C]SPM, dissociated at 23 degrees independently of the addition of nonradioactive polyamine. Methylglyoxal-bis-(guanylhydrazone) was an extremely potent inhibitor of PUT binding (Ki = 3.2 +/- 1.5 nM), but as with PUT and cadaverine (CAD), it did not alter [3H]SPD and [14C]SPM binding substantially. The intestinal brush-border membrane may contain at least three sites specific for polyamine binding and exhibiting different ligand selectivity. Site puT might be associated with the transport system already described for intestinal uptake of PUT.

Delta9-tetrahydrocannabinol induces apoptosis in C6 glioma cells.

delta9-Tetrahydrocannabinol (THC), the major active component of marijuana, induced apoptosis in C6.9 glioma cells, as determined by DNA fragmentation and loss of plasma membrane asymmetry. THC stimulated sphingomyelin hydrolysis in C6.9 glioma cells. THC and N-acetylsphingosine, a cell-permeable ceramide analog, induced apoptosis in several transformed neural cells but not in primary astrocytes or neurons. Although glioma C6.9 cells expressed the CBI cannabinoid receptor, neither THC-induced apoptosis nor THC-induced sphingomyelin breakdown were prevented by SR141716, a specific antagonist of that receptor. Results thus show that THC-induced apoptosis in glioma C6.9 cells may rely on a CBI receptor-independent stimulation of sphingomyelin breakdown.

1,25-Dihydroxyvitamin D3 regulates the expression of VDR and NGF gene in Schwann cells in vitro.

The vitamin D receptor (VDR) is a nuclear receptor that mediates the effect of the active metabolite of vitamin D3, the 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). To investigate the potential role of this hormone in the peripheral nervous system, we have studied the VDR expression in Schwann cells. The VDR mRNA was detected by Northern blot analysis in rat primary cultures of Schwann cells, and its levels were strongly increased in the presence of 1,25-(OH)2D3. Using the mouse Schwann cell line, MSC80, we showed that concentrations as low as 10(-10) M of hormone stimulated the expression of the VDR gene and strongly increased the amounts of activated VDR, capable of binding to the specific vitamin D responsive element (VDRE). We also found that 1,25-(OH)2D3 stimulated the expression of the nerve growth factor gene in MSC80. These data suggest a role for the hormone in the peripheral nervous system, possibly as a mediator active in trauma.

Epigenetic control of programmed cell death: inhibition by 5-azacytidine of 1,25-dihydroxyvitamin D3-induced programmed cell death in C6.9 glioma cells.

In mammalian DNA cytosine methylation occurs specifically at CpG dinucleotide. Although the full array of function of DNA methylation is yet to be elucidated, it is well established that DNA methylation is an important mechanism involved in gene expression, DNA replication and cancer. Rat glioma C6.9 cells undergo programmed cell death (PCD) after treatment with 1,25-dihydroxyvitamin D3 (1,25-D3). Hence, these cells were used to study whether DNA methylation was involved in the control of PCD. We found that 1,25-D3-mediated PCD of C6.9 cells was suppressed by exposure of the cells to the DNA demethylating agents 5-azacytidine (5-AzaC) and 5-aza-2'-deoxycytidine. This effect remains detectable several cell divisions following removal of 5-AzaC and, therefore, involves DNA methylation as an epigenetic regulatory mechanism of PCD. Accordingly, internucleosomal fragmentation, a feature of apoptosis that is detected in 1,25-D3-treated cells, is no longer observable after treatment of these cells with 5-AzaC. However, 5-AzaC does not totally suppress the responsiveness of C6.9 cells to 1,25-D3 since the induction of the c-myc gene remains unaffected. These results suggest that a change in DNA methylation pattern could suppress 1,25-D3-mediated PCD through the expression of previously hypermethylated genes such as proto-oncogenes with death-repressor activity, endogenous virus sequences or even genes inducing change in the differentiated state of these cells.

Vitamin D receptor stable transfection restores the susceptibility to 1,25-dihydroxyvitamin D3 cytotoxicity in a rat glioma resistant clone.

Recently, 1,25-dihydroxyvitamin D3 (1,25-D3) and less hypercalcemic analogs were shown to exert a delayed cytotoxic effect on rat C6 glioma cells. 1,25-D3 induces in these cells a programmed cell death, accompanied by the induction of c-myc, p53 and gadd 45 genes. The involvement of the intracellular vitamin D receptor (VDR) remained to be determined. In this lethal process, we have investigated its role in a subclone of C6 cells, which was isolated on the basis of its resistance to 1,25-D3, and in which VDR expression was not detected either at the mRNA or protein levels. The stable transfection of a rat VDR cDNA into this clone restored its susceptibility to the cytotoxic effects of 1,25-D3. This phenomenon was accompanied by a dramatic upregulation of c-myc mRNA expression, as already described in a C6-sensitive clone. These results provide the first evidence that VDR expression, if not sufficient, is necessary to mediate 1,25-D3 cytotoxic effect in C6 glioma cells. Since VDR mRNA expression has been already reported in human brain tumors, our data imply that the identification of VDR expression could become a prerequisite in any strategy of glioma treatment with vitamin D analogs.