MT1 and MT2 melatonin receptors play opposite roles in brain cancer progression.

Primary brain tumors remain among the deadliest of all cancers. Glioma grade IV (glioblastoma), the most common and malignant type of brain cancer, is associated with a 5-year survival rate of < 5%. Melatonin has been widely reported as an anticancer molecule, and we have recently demonstrated that the ability of gliomas to synthesize and accumulate this indolamine in the surrounding microenvironment negatively correlates with tumor malignancy. However, our understanding of the specific effects mediated through the activation of melatonin membrane receptors remains limited. Thus, here we investigated the specific roles of MT1 and MT2 in gliomas and medulloblastomas. Using the MT2 antagonist DH97, we showed that MT1 activation has a negative impact on the proliferation of human glioma and medulloblastoma cell lines, while MT2 activation has an opposite effect. Accordingly, gliomas have a decreased mRNA expression of MT1 (also known as MTNR1A) and an increased mRNA expression of MT2 (also known as MTNR1B) compared to the normal brain cortex. The MT1/MT2 expression ratio negatively correlates with the expression of cell cycle-related genes and is a positive prognostic factor in gliomas. Notably, we showed that functional selective drugs that simultaneously activate MT1 and inhibit MT2 exert robust anti-tumor effects in vitro and in vivo, downregulating the expression of cell cycle and energy metabolism genes in glioma stem-like cells. Overall, we provided the first evidence regarding the differential roles of MT1 and MT2 in brain tumor progression, highlighting their relevance as druggable targets. KEY MESSAGES: • MT1 impairs while MT2 promotes the proliferation of glioma and medulloblastoma cell lines. • Gliomas have a decreased expression of MT1 and an increased expression of MT2 compared to normal brain cortex. • Tumors with a high MT1/MT2 expression ratio have significantly better survival rates. • Functional selective drugs that simultaneously activate MT1 and inhibit MT2 downregulate the expression of cell cycle and energy metabolism genes in glioma stem-like cells and exert robust anti-tumor effects in vivo.

Level of circulating concentrations of progesterone during ovulatory follicle development affects timing of pregnancy loss in lactating dairy cows.

The objective of this experiment was to determine the effect of high versus low progesterone (P4) during the pre-dominance or dominance phase (or both) of ovulatory follicle development on follicular dynamics and fertility of lactating dairy cows. Progesterone (P4) was manipulated to reach high (H) or low (L) serum concentrations during the pre-dominance phase (d 0 to 4 of the wave) and dominance phase (d 5 to 7 of the wave) of a second follicular wave ovulatory follicle, creating 4 treatments: H/H, H/L, L/H, and L/L. Luteolysis was induced with PGF2α on d 7 of the wave and ovulation was induced with GnRH 56 h after PGF2α. Cows (n = 558) received artificial insemination (AI) 16 h following GnRH. Pregnancy was determined at 6 intervals during gestation and at calving to quantify pregnancy loss beginning at d 23 post-AI utilizing pregnancy-specific protein B (PSPB) in novel within-cow comparisons. Cows with single ovulations assigned to the L/L treatment had greater pre-ovulatory follicle diameter compared with cows assigned to the L/H or H/L treatments. Cows with single ovulations had greater pre-ovulatory follicle diameter compared with cows with double ovulations. Low P4 in H/L, L/H, and L/L increased double ovulation rate compared with H/H. Cows with double ovulations had greater pregnancies per AI (P/AI) on d 23 post-AI compared with cows with single ovulations but had greater losses if ovulations were unilateral. Cows with low P4 during the entire period of the ovulatory follicle development also had greater P/AI on d 23 post-AI compared with cows with high P4 during both phases. However, full-term P/AI was not different between treatments. This was a result of the greater incidence of pregnancy losses between d 35 and 56 of gestation for cows with unilateral double ovulations compared with bilateral double ovulations and single ovulatory cows. Cows with single ovulation and low circulating P4 during the dominance period of follicle development had increased pregnancy losses between d 35 and 56 of gestation compared with cows with single ovulations and high P4. The PSPB measurements on d 16 and 23 post-AI were highly accurate in the prediction of pregnancy at d 28. The PSPB differed on d 23 and 28 between cows that had versus cows that did not have pregnancy losses between d 28 and 35 of gestation. In summary, circulating concentrations of P4 during ovulatory follicle development affected numbers of follicles ovulated and timing of subsequent pregnancy losses.

Disruption of prion protein-HOP engagement impairs glioblastoma growth and cognitive decline and improves overall survival.

Glioblastomas (GBMs) are resistant to current therapy protocols and identification of molecules that target these tumors is crucial. Interaction of secreted heat-shock protein 70 (Hsp70)-Hsp90-organizing protein (HOP) with cellular prion protein (PrP(C)) triggers a large number of trophic effects in the nervous system. We found that both PrP(C) and HOP are highly expressed in human GBM samples relative to non-tumoral tissue or astrocytoma grades I-III. High levels of PrP(C) and HOP were associated with greater GBM proliferation and lower patient survival. HOP-PrP(C) binding increased GBM proliferation in vitro via phosphatidylinositide 3-kinase and extracellular-signal-regulated kinase pathways, and a HOP peptide mimicking the PrP(C) binding site (HOP230-245) abrogates this effect. PrP(C) knockdown impaired tumor growth and increased survival of mice with tumors. In mice, intratumor delivery of HOP230-245 peptide impaired proliferation and promoted apoptosis of GBM cells. In addition, treatment with HOP230-245 peptide inhibited tumor growth, maintained cognitive performance and improved survival. Thus, together, the present results indicate that interfering with PrP(C)-HOP engagement is a promising approach for GBM therapy.

Surgical outcome in mesial temporal sclerosis correlates with prion protein gene variant.

BACKGROUND: Mesial temporal lobe epilepsy related to hippocampal sclerosis (MTLE-HS) is the most common surgically remediable epileptic syndrome. Ablation of the cellular prion protein (PrP(c)) gene (PRNP) enhances neuronal excitability of the hippocampus in vitro and sensitivity to seizure in vivo, indicating that PrP(c) might be related to epilepsy. OBJECTIVE: To evaluate the genetic contribution of PRNP to MTLE-HS. METHODS: The PRNP coding sequence of DNA from peripheral blood cells of 100 consecutive patients with surgically treated MTLE-HS was compared to that from a group of healthy controls adjusted for sex, age, and ethnicity (n = 180). The presence of PRNP variant alleles was correlated with clinical and presurgical parameters as well as surgical outcome. RESULTS: A variant allele at position 171 (Asn-->Ser), absent in controls, was found in heterozygosis (Asn171Ser) in 23% of patients (p < 0.0001). The PRNP genotypes were not correlated with any clinical or presurgical data investigated. However, patients carrying the Asn171Ser variant had a five times higher chance of continuing to have seizures after temporal lobectomy (95% CI 1.65 to 17.33, p = 0.005) than those carrying the normal allele. At 18 months after surgery, 91.8% of patients with the normal allele at codon 171 were seizure free, in comparison to 68.2% of those carrying Asn171Ser (p = 0.005). CONCLUSIONS: The PRNP variant allele Asn171Ser is highly prevalent in patients with medically untreatable MTLE-HS and influences their surgical outcome. The results suggest that the PRNP variant allele at codon 171 (Asn171Ser) is associated with epileptogenesis in MTLE-HS.

Insights into the physiological function of cellular prion protein

Prions have been extensively studied since they represent a new class of infectious agents in which a protein, PrPsc (prion scrapie), appears to be the sole component of the infectious particle. They are responsible for transmissible spongiform encephalopathies, which affect both humans and animals. The mechanism of disease propagation is well understood and involves the interaction of PrPsc with its cellular isoform (PrPc) and subsequently abnormal structural conversion of the latter. PrPc is a glycoprotein anchored on the cell surface by a glycosylphosphatidylinositol moiety and expressed in most cell types but mainly in neurons. Prion diseases have been associated with the accumulation of the abnormally folded protein and its neurotoxic effects; however, it is not known if PrPc loss of function is an important component. New efforts are addressing this question and trying to characterize the physiological function of PrPc. At least four different mouse strains in which the PrP gene was ablated were generated and the results regarding their phenotype are controversial. Localization of PrPc on the cell membrane makes it a potential candidate for a ligand uptake, cell adhesion and recognition molecule or a membrane signaling molecule. Recent data have shown a potential role for PrPc in the metabolism of copper and moreover that this metal stimulates PrPc endocytosis. Our group has recently demonstrated that PrPc is a high affinity laminin ligand and that this interaction mediates neuronal cell adhesion and neurite extension and maintenance. Moreover, PrPc-caveolin-1 dependent coupling seems to trigger the tyrosine kinase Fyn activation. These data provide the first evidence for PrPc involvement in signal transduction

Repression of glucocorticoid receptor gene transcription by c-Jun.

The regulation of glucocorticoid receptor gene expression by members of the AP-1 family was examined in glucocorticoid-free NIH3T3 cells transfected with the human glucocorticoid receptor gene promoter driving expression of a CAT reporter gene. c-Jun inhibited the promoter activity by 80% and JunB by 30%, whereas c-Fos and JunD had no inhibitory effect. Electrophoretic mobility shift assays showed that c-Jun is unable to efficiently interact with the AP-1-like site present in the human glucocorticoid receptor promoter. Moreover, c-Jun was still able to repress promoter mutants in which the region containing the AP-1-like site was deleted. NIH3T3 cell clones overexpressing c-Jun exhibited lower glucocorticoid receptor mRNA levels, which suggests that the murine glucocorticoid receptor gene can also be regulated by AP-1. These results provide a new mechanism for cross-talk between the glucocorticoid receptor and the AP-1 family of transcription factors in the absence of glucocorticoid ligands.

Changes in cortical and hippocampal ectonucleotidase activities in mice lacking cellular prion protein.

Animals lacking cellular prion protein (PrP(c)) expression are more susceptible to seizures. Adenosine is an endogenous anticonvulsant agent and it levels in the synaptic cleft are regulated by ectonucleotidases. We evaluated ectonucleotidase activities in synaptosomes from hippocampus and cerebral cortex of adult PrP(c) null mice and wild-type mice (genetic background 129/Sv X C57BL/6J). There was an increase (47%) in adenosine triphosphate (ATP) hydrolysis in hippocampal synaptosomes of PrP(c) knockout mice as compared with the wild-type animals. In cortical synaptosomes, ATP hydrolysis was similar in both PrP(c) mice and controls. However, there was a significant decrease in adenosine diphosphate (ADP) hydrolysis in both hippocampal (-39%) and cortical (-25%) synaptosomes in PrP(c) null animals compared to wild-type mice. Changes in brain ectonucleotidases activities related to modifications in the PrP(c) expression may contribute, at least in part, to the higher sensitivity to seizures of PrP(c) null mice.

Laminin-induced PC-12 cell differentiation is inhibited following laser inactivation of cellular prion protein.

Prions, the etiological agents for infectious degenerative encephalopathies, act by inducing structural modifications in the cellular prion protein (PrPc). Recently, we demonstrated that PrPc binds laminin (LN) and that this interaction is important for the neuritogenesis of cultured hippocampal neurons. Here we have used the PC-12 cell model to explore the biological role of LN-PrPc interaction. Antibodies against PrPc inhibit cell adhesion to LN-coated culture plaques. Furthermore, chromophore-assisted laser inactivation of cell surface PrPc perturbs LN-induced differentiation and promotes retraction of mature neurites. These results point out to the importance of PrPc as a cell surface ligand for LN.

Cellular prion protein binds laminin and mediates neuritogenesis.

Laminin (LN) plays a major role in neuronal differentiation, migration and survival. Here, we show that the cellular prion protein (PrPc) is a saturable, specific, high-affinity receptor for LN. The PrPc-LN interaction is involved in the neuritogenesis induced by NGF plus LN in the PC-12 cell line and the binding site resides in a carboxy-terminal decapeptide from the gamma-1 LN chain. Neuritogenesis induced by LN or its gamma-1-derived peptide in primary cultures from rat or either wild type or PrP null mice hippocampal neurons, indicated that PrPc is the main cellular receptor for that particular LN domain. These results point out to the importance of the PrPc-LN interaction for the neuronal plasticity mechanism.

A receptor for infectious and cellular prion protein

Prions are an unconventional form of infectious agents composed only of protein and involved in transmissible spongiform encephalopathies in humans and animals. The infectious particle is composed by PrPsc which is an isoform of a normal cellular glycosyl-phosphatidylinositol (GPI) anchored protein, PrPc, of unknown function. The two proteins differ only in conformation, PrPc is composed of 40 percent helix while PrPsc has 60 percent ß-sheet and 20 percent helix structure. The infection mechanism is trigged by interaction of PrPsc with cellular prion protein causing conversion of the latter's conformation. Therefore, the infection spreads because new PrPsc molecules are generated exponentially from the normal PrPc. The accumulation of insoluble PrPsc is probably one of the events that lead to neuronal death. Conflicting data in the literature showed that PrPc internalization is mediated either by clathrin-coated pits or by caveolae-like membranous domains. However, both pathways seem to require a third protein (a receptor or a prion-binding protein) either to make the connection between the GPI-anchored molecule to clathrin or to convert PrPc into PrPsc. We have recently characterized a 66-kDa membrane receptor which binds PrPc in vitro and in vivo and mediates the neurotoxicity of a human prion peptide. Therefore, the receptor should have a role in the pathogenesis of prion-related diseases and in the normal cellular process. Further work is necessary to clarify the events triggered by the association of PrPc/PrPsc with the receptor

Complementary hydropathy identifies a cellular prion protein receptor.

Prions, the etiological agents for infectious degenerative encephalopathies, act by entering the cell and inducing conformational changes in PrPC (a normal cell membrane sialoglycoprotein), which result in cell death. A specific cell-surface receptor to mediate PrPC and prion endocytosis has been predicted. Complementary hydropathy let us generate a hypothetical peptide mimicking the receptor binding site. Antibodies raised against this peptide stain the surface of mouse neurons and recognize a 66-kDa membrane protein that binds PrPC both in vitro and in vivo. Furthermore, both the complementary prion peptide and antiserum against it inhibit the toxicity of a prion-derived peptide toward neuronal cells in culture. Such reagents might therefore have therapeutic applications.

Attenuation of glucocorticoid receptor levels by the H-ras oncogene.

Certain oncogene products are known to affect the cellular response to glucocorticoids. In particular, glucocorticoid-induced transcription is impaired in H-ras-transformed cells. In this study, we examine the mechanism for this effect in NIH3T3 cells containing stably integrated H-ras genomic sequences. NIH3T3ras cells transfected with the MMTV-CAT reporter exhibit a pronounced reduction in the level of glucocorticoid-induced CAT activity, compared to normal NIH3T3 cells. As the response to glucocorticoids depends on the amount of glucocorticoid receptor protein, we have examined the cellular receptor content in both cell lines. The cytosolic and total cellular GR protein are both markedly lower in NIH3T3ras cells, suggesting that the reduced response is directly due to an attenuation of receptor levels. The steady-state level of glucocorticoid receptor mRNA is appreciably reduced in NIH3T3ras cells, which accounts for the attenuated level of glucocorticoid receptor protein. The rate of glucocorticoid receptor gene transcription is concomitantly decreased in NIH3T3ras cells. Theras effect maps to the proximal promoter of the glucocorticoid receptor gene. These results suggest that a target for activated H-Ras protein may be a transcription factor which partially represses transcription of the glucocorticoid receptor gene.

The effects of ras gene expression on glucocorticoid receptors in mouse fibroblasts.

Analysis of induction of glutamine synthetase activity by dexamethasone showed a 2-fold increase in NIH3T3 but no change in NIH3T3 ras (EJ-ras) cells. The observed increase could be abolished by the antagonist RU486. The lack of response in ras transformed cells might reflect oncoprotein effects on the glucocorticoid receptor (GR). Several GR parameters were studied in order to clarify this point. Total GR level was the same for both cells; cytoplasmic receptor level however, was 3 times lower in NIH3T3 ras than in NIH3T3 cells. Hormone-receptor binding affinity, specificity, thermostability, sedimentation coefficient, molecular weight as well as the cytoplasmic GR transformation ratio were similar for the two cell lines. On the other hand, the fraction of the total receptor pool involved with the recycling process was approximately 20% lower in NIH3T3 ras than in NIH3T3 cells. After 24 h of dexamethasone treatment, no GR down regulation was observed in NIH3T3 ras cells, whereas normal NIH3T3 cells exhibited a decrease of GR binding capacity around 80%. Further studies are necessary to define the mechanisms underlying the association between glucocorticoid insensitivity, and modifications in the GR nuclear/cytoplasmic ratio, in the recycling GR fraction and in the down-regulation process observed in ras transformed cells.

Regulaçäo dos receptores de glicocorticóide em leucócitos mononucleares humanos / Glucocorticoid receptores of human mononuclear leukocytes

O presente estudo foi realizado com a finalidade de analisar receptores de glicocorticóide em leucócitos mononucleares (MNL) de sangue periférico de indivíduos normais, näo tratados e de indivíduos normal aos quais foram administrados exogenamente os corticóides: prednisona e deflazacort. MNL de mulheres e homens normais apresentaram uma concentraçäo de receptores de glicocorticóide de 7,52 ñ 2,5fMol/10**6 cel e uma constante de dissociaçäo (Kd) de 9,5 ñ 2,3nM. Näo houve diferença em relaçäo ao sexo e as fases do ciclo menstrual. Em indivíduos tratados com predinisona, näo houve mudança nos parâmetros de ligaçäo. A administraçäo deflazacort determinou uma sensível reduçäo dos sítios de ligaçäo. Concluímos que certos glicocorticóides podem exercer um efeito regulatório sobre seus receptores

Steroid receptors in the myasthenic thymus.

Estrogen (ER), progesterone (PR), androgen (AR) and glucocorticoid receptors (GR) were evaluated in thymus cytosols from 15 myasthenia gravis (MG) patients, using a dextran charcoal assay. ER was found in 40%, PR and AR in 53% and GR in 100% of MG thymi. Competition studies demonstrated steroid specificity for all receptors. Mean ER and AR levels in MG thymi were higher than those in the thymi of 4 control children. Female patients have higher AR titers. Myasthenic thymi presented lower GR values than in normal controls: Differences in steroid receptor content observed in the MG thymus as compared to the child thymus may reflect variations in cell populations.

Glucocorticoid receptors in acute leukemia.

We evaluated the applicability of glucocorticoid receptor (GR) determinations to predict clinical responsiveness to polychemotherapy in acute leukemias by measuring GR in leukemic cells from 20 patients as well as in lymphocytes from 20 normal volunteers. The whole-cell binding assay with [3H]-dexamethasone was used. The GR level (mean +/- SD) was 4583 +/- 1384 sites/cell (range: 2050-8140) for normal lymphocytes. A significant amount of GR (5300 to 17,000 sites/cell) was detected in the blasts from 9/12 patients with acute lymphoblastic leukemia (ALL). The concentration of GR sites in ALL cells greatly exceeded that found in normal mononuclear cells (P less than 0.01). The absence of GR in ALL patients correlated with poor response to polychemotherapy including glucocorticoids. High receptor levels were associated with complete remission (P less than 0.005). The GR concentration (7288 +/- 2345 sites/cell) found in acute non-lymphoblastic leukemias (ANLL) was in the same range as that found in ALL cases. All ANLL patients had a substantial number of GR, significantly higher than the sites/cell found in normal lymphocytes (P less than 0.05). No correlation between clinical responsiveness and receptor level was demonstrable for ANLL patients.

Steroid receptors in meningiomas.

Cytosolic estrogen (ER), progesterone (PR), androgen (AR) and glucocorticoid receptors (GR) were evaluated in 10 meningiomas using a dextran charcoal coated method. We consider as positive specific receptor values greater than or equal to 10 fMol/mg protein. In this study 20% of the meningiomas contained very low titers of specific ER. PR was detectable in 90% of the tumors, at high levels. The mean PR content of PR+ tumors was 60 +/- 38 fMol/mg prot. GR and AR were present in moderate levels, in 70% of the tumors. Competition studies demonstrated steroid specificity for these hormone-binding proteins. Female patients have a higher receptor incidence and titer. In conclusion, it can be hypothesized that the meningioma are a target tissue for steroids and that endocrine therapy may be relevant to unoperable and/or recurrent tumors.

Steroid receptors in meningiomas

Dados epidemiológicos sugerem uma associaçäo entre meningiomas e hormônios sexuais. Há preponderância da incidência da incidência destes tumores em mulheres, sendo diagnosticados mais frequentemente entre 35 e 55 anos. Progressäo tumoral pode ocorrer durante a gravidez e a associaçäo entre meningiomas e câncer de mama é estatisticamente significativa. O efeito benéfico positivo dos glicocorticóides no tratamento clínico de tumores intracranianos está bem documentado, embora este efeito seja relacionado com o edema cerebral e näo com proliferaçäo tumoral. A presença de receptores intracelulares específicos parece ser condiçäo necessaria embora näo suficiente para a expressäo dos efeitos celulares destes esteróides. O objetivo do nosso trabalho foi a determinaçäo de receptores de estrógeno (ER), progesterona (PR), andrógeno (AR) e glicocorticóide (GR) em 10 meningiomas através da metodologia do carväo-dextrana. Neste estudo verificamos que somente 20% dos meningiomas contém receptores de estrógeno e as concentraçöes determinadas säo baixas. Receptores de progesterona estäo presentes em 90% dos tumores, em alta concentraçäo (média + ou - dp = 60 + ou - 38 fMol/mg prot). Cerca de 70% dos meningiomas contém níveis intermediários de AR e GR. Testes de competiçäo demonstram que todos os receptores säo específicos. Incidência e concentraçäo de todos os receptores säo maiores em pacientes do sexo feminino. A ocorrência de receptores de progesterona, andrógeno e glicocorticóide pode indicar a possível utilidade de manipulaçäo endócrinas em meningiomas näo operáveis