RESUMEN
Oncogenic gene fusions are hybrid genes that result from structural DNA rearrangements, leading to deregulated activity. Fusions involving the neuregulin-1 gene (NRG1) result in ErbB-mediated pathway activation and therefore present a rational candidate for targeted treatment. The most frequently reported NRG1 fusion is CD74-NRG1, which most commonly occurs in patients with invasive mucinous adenocarcinomas (IMAs) of the lung, although several other NRG1 fusion partners have been identified in patients with lung cancer, including ATP1B1, SDC4, and RBPMS. NRG1 fusions are also present in patients with other solid tumors, such as pancreatic ductal adenocarcinoma. In general, NRG1 fusions are rare across different types of cancer, with a reported incidence of <1%, with the notable exception of IMA, which represents ≈2%-10% of lung adenocarcinomas and has a reported incidence of ≈10%-30% for NRG1 fusions. A substantial proportion (≈20%) of NRG1 fusion-positive non-small-cell lung cancer cases are nonmucinous adenocarcinomas. ErbB-targeted treatments, such as afatinib, a pan-ErbB tyrosine kinase inhibitor, are potential therapeutic strategies to address unmet treatment needs in patients harboring NRG1 fusions.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Afatinib/uso terapéutico , Biología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neurregulina-1/genética , Proteínas de Fusión Oncogénica/genéticaRESUMEN
BACKGROUND: In the phase III LUX-Head & Neck 1 (LUX-H&N1) trial, second-line afatinib significantly improved progression-free survival (PFS) versus methotrexate in patients with recurrent/metastatic head and neck squamous cell carcinoma (R/M HNSCC). Here, we evaluated association of prespecified biomarkers with efficacy outcomes in LUX-H&N1. PATIENTS AND METHODS: Randomized patients with R/M HNSCC and progression following ≥2 cycles of platinum therapy received afatinib (40 mg/day) or methotrexate (40 mg/m2/week). Tumor/serum samples were collected at study entry for patients who volunteered for inclusion in biomarker analyses. Tumor biomarkers, including p16 (prespecified subgroup; all tumor subsites), EGFR, HER2, HER3, c-MET and PTEN, were assessed using tissue microarray cores and slides; serum protein was evaluated using the VeriStrat® test. Biomarkers were correlated with efficacy outcomes. RESULTS: Of 483 randomized patients, 326 (67%) were included in the biomarker analyses; baseline characteristics were consistent with the overall study population. Median PFS favored afatinib over methotrexate in patients with p16-negative [2.7 versus 1.6 months; HR 0.70 (95% CI 0.50-0.97)], EGFR-amplified [2.8 versus 1.5 months; HR 0.53 (0.33-0.85)], HER3-low [2.8 versus 1.8 months; HR 0.57 (0.37-0.88)], and PTEN-high [1.6 versus 1.4 months; HR 0.55 (0.29-1.05)] tumors. Afatinib also improved PFS in combined subsets of patients with p16-negative and EGFR-amplified tumors [2.7 versus 1.5 months; HR 0.47 (0.28-0.80)], and patients with p16-negative tumors who were EGFR therapy-naïve [4.0 versus 2.4 months; HR 0.55 (0.31-0.98)]. PFS was improved in afatinib-treated patients who were VeriStrat 'Good' versus 'Poor' [2.7 versus 1.5 months; HR 0.71 (0.49-0.94)], but no treatment interaction was observed. Afatinib improved tumor response versus methotrexate in all subsets analyzed except for those with p16-positive disease (n = 35). CONCLUSIONS: Subgroups of HNSCC patients who may achieve increased benefit from afatinib were identified based on prespecified tumor biomarkers (p16-negative, EGFR-amplified, HER3-low, PTEN-high). Future studies are warranted to validate these findings. CLINICAL TRIAL REGISTRATION: NCT01345682.
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Biomarcadores de Tumor/metabolismo , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/metabolismo , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/metabolismo , Metotrexato/administración & dosificación , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/metabolismo , Quinazolinas/administración & dosificación , Administración Intravenosa , Administración Oral , Afatinib , Antimetabolitos Antineoplásicos/administración & dosificación , Biomarcadores de Tumor/sangre , Biopsia , Carcinoma de Células Escamosas/sangre , Carcinoma de Células Escamosas/patología , Supervivencia sin Enfermedad , Neoplasias de Cabeza y Cuello/sangre , Neoplasias de Cabeza y Cuello/patología , Humanos , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia/sangre , Recurrencia Local de Neoplasia/patología , Valor Predictivo de las Pruebas , Carcinoma de Células Escamosas de Cabeza y CuelloRESUMEN
BACKGROUND: The combination of the reversible epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) erlotinib with gemcitabine obtained FDA approval for treating patients with pancreatic cancer. However, duration of response is often limited and there is currently no reliable predictive marker. METHODS: We determined the sensitivity of a panel of human pancreatic tumour cell lines to treatment with afatinib, erlotinib, monoclonal antibody (mAb) ICR62, and gemcitabine, using the Sulforhodamine B colorimetric assay. The effect of these agents on cell signalling and cell-cycle distribution was determined by western blot and flow cytometry, respectively. RESULTS: At 200 nM, ICR62 had no effect on growth of these tumour cells with the exception of BxPC-3 cells. BxPC-3 cells were also sensitive to treatment with afatinib and erlotinib with respective IC(50) values of 11 and 1200 nM. Compared with erlotinib, afatinib was also more effective in inhibiting the growth of the other human pancreatic tumour cell lines and in blocking the EGF-induced phosphorylation of tyrosine, EGFR, MAPK, and AKT. When tested in BxPC-3 xenografts, afatinib induced significant delay in tumour growth. CONCLUSION: The superiority of afatinib in this study encourages further investigation on the therapeutic potential of afatinib as a single agent or in combination with gemcitabine in pancreatic cancer.
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Antineoplásicos/farmacología , Proteínas Oncogénicas v-erbB/antagonistas & inhibidores , Neoplasias Pancreáticas/patología , Quinazolinas/farmacología , Afatinib , Animales , Western Blotting , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular Tumoral , Colorimetría , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Objective: Angiogenin (ANG) is a pro-angiogenic and neurotrophic factor with an important role in stress-induced injury, by promoting neovascularization and neuronal survival. Identification of loss-of-function mutations and evidence of beneficial effect of ANG administration in transgenic SOD1G93A mice have linked ANG to the pathogenesis of Amyotrophic Lateral Sclerosis (ALS), stimulating interest in considering circulating ANG levels as an ALS disease biomarker although robust evidence is still lacking. Aim of our study was to assess differences of ANG levels in the cerebrospinal fluid (CSF) of a large cohort of patients with ALS and frontotemporal dementia (FTD) compared to controls and to explore correlations between ANG content and disease-related clinical variables. Methods: ANG levels were measured in CSF samples using a commercially available ELISA kit in 88 patients affected with ALS and/or FTD and 46 unrelated individuals (control group). Results: ANG levels didn't differ significantly between cases and controls. Patients with FTD or ALS-FTD showed significantly increased CSF concentration of ANG compared to ALS patients without dementia and controls in a multivariate regression model (p < 0.001). No correlations were found in ALS/FTD patients between ANG levels and clinical parameters, including age, presence of C9orf72 repeat expansion, body mass index (BMI). Conclusions: our findings highlight a role of ANG as CSF biomarker useful to identify ALS patients with concurrent FTD and suggest that it should be further explored as potential biomarker for FTD.
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Esclerosis Amiotrófica Lateral/líquido cefalorraquídeo , Proteína C9orf72/líquido cefalorraquídeo , Demencia Frontotemporal/líquido cefalorraquídeo , Ribonucleasa Pancreática/líquido cefalorraquídeo , Esclerosis Amiotrófica Lateral/diagnóstico , Esclerosis Amiotrófica Lateral/genética , Animales , Proteína C9orf72/genética , Estudios de Cohortes , Demencia Frontotemporal/diagnóstico , Demencia Frontotemporal/genética , Humanos , Masculino , Ratones TransgénicosRESUMEN
Receptors for alpha-melanocyte-stimulating hormone (alpha-MSH) on human malignant melanoma cell lines were investigated with a specific binding assay and characterized with structural analogues of alpha-MSH and adrenocorticotropic hormone and by photoaffinity cross-linking of the hormone-receptor complex. Specific binding of high-performance liquid chromatography-purified, monoiodinated alpha-MSH in the presence of 1 mM 1,10-phenanthroline as protease inhibitor was highest after a 2-h incubation at 37 degrees C. The nonspecific binding was less than 20% and dissociation of the ligand-receptor complex was relatively slow. Ten out of 12 human cell lines showed specific binding sites for alpha-MSH with Kp values ranging from 0.195 to 2.87 nM and the sites/cell being approximately 400 to approximately 1600. Virtually identical results were obtained in an assay where the cells remained attached to the culture dishes during the entire experiment. The study of hormone analogues with the D10 cell line showed that oxidized alpha-MSH had an approximately 40-fold lower affinity than alpha-MSH whereas [Nle4,D-Phe7]-alpha-MSH displayed a threefold and the adrenocorticotropic hormone fragments (1-17) and (1-24) a 20- and 8-fold higher affinity. Cross-linking of the alpha-MSH-receptor complex of three cell lines using monoiodinated [Nle4,D-Phe7,Trp(2-nitro-4-azidophenylsulfenyl)9]-alpha-MSH as photoaffinity label revealed a major Mr 45,000 protein band on sodium dodecyl sulfate-polyacrylamide gels, analogous to the MSH receptor of mouse B16 melanoma cells.
Asunto(s)
Hormonas Estimuladoras de los Melanocitos/metabolismo , Melanoma/metabolismo , Receptores de la Hormona Hipofisaria/metabolismo , Células Tumorales Cultivadas/metabolismo , Animales , Línea Celular , Perros , Glioma/metabolismo , Humanos , Cinética , Melanoma Experimental/metabolismo , Ratones , Neuroblastoma/metabolismoRESUMEN
BACKGROUND: This phase I study evaluated afatinib, an irreversible ErbB family blocker, plus paclitaxel in patients with advanced solid tumours likely to express human epidermal growth factor receptor (HER1/EGFR) or HER2. METHODS: Oral afatinib was combined with intravenous paclitaxel (80mg/m(2); days 1, 8 and 15 every four weeks) starting at 20mg once daily and escalated to 40 and 50mg in successive cohorts of ⩾3 patients. The primary objective was to determine the maximum tolerated dose (MTD) of afatinib combined with paclitaxel. Secondary objectives included safety, pharmacokinetics and antitumour activity. RESULTS: Sixteen patients were treated. Dose-limiting toxicities with afatinib 50mg were fatigue and mucositis. The MTD was determined as afatinib 40mg with paclitaxel 80mg/m(2), which proved tolerable with repeated dosing. Frequent adverse events (AEs) included diarrhoea (94%), fatigue (81%), rash/acne (81%), decreased appetite (69%) and inflammation of mucosal membranes (69%); no grade 4 treatment-related AEs were observed. Five (31%) confirmed partial responses were observed in patients with non-small cell lung cancer (n=3), oesophageal cancer and cholangiocarcinoma; eight (50%) patients remained on study for ⩾6months. Pharmacokinetic parameters of afatinib and paclitaxel were similar for single administration or in combination. CONCLUSIONS: The MTD and recommended phase II dose of once-daily afatinib combined with paclitaxel 80mg/m(2) (days 1, 8 and 15 every four weeks) was 40mg. AEs at or below this dose were generally manageable with repeated dosing. No pharmacokinetic interactions were observed. This combination demonstrated promising antitumour activity. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00809133.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Neoplasias/tratamiento farmacológico , Paclitaxel/administración & dosificación , Inhibidores de Proteínas Quinasas/administración & dosificación , Quinazolinas/administración & dosificación , Administración Intravenosa , Administración Oral , Adulto , Afatinib , Anciano , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacocinética , Esquema de Medicación , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Femenino , Humanos , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Neoplasias/enzimología , Neoplasias/patología , Paclitaxel/efectos adversos , Inhibidores de Proteínas Quinasas/efectos adversos , Inhibidores de Proteínas Quinasas/farmacocinética , Quinazolinas/efectos adversos , Quinazolinas/farmacocinética , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/metabolismo , Receptor ErbB-3/antagonistas & inhibidores , Receptor ErbB-3/metabolismo , Receptor ErbB-4/antagonistas & inhibidores , Receptor ErbB-4/metabolismo , Factores de Tiempo , Resultado del Tratamiento , Reino UnidoRESUMEN
The two mouse melanoma cell lines B16-F1 and B16-G4F retain their melanogenic capacity when cultured in vitro. Melanotropic peptides such as alpha-melanocyte-stimulating hormone (alpha-MSH) induce formation and release of melanin pigment in B16-F1 cells. In contrast, B16-G4F cells do not respond to alpha-MSH. Using receptor-binding analysis and photoaffinity crosslinking we demonstrate that the lack of response of B16-G4F cells to alpha-MSH is due to the absence of functional MSH receptors from the cell surface. Northern blot analysis of receptor mRNA revealed that MSH receptor mRNA is not expressed in B16-G4F cells. These cells represent a new tool for the study of signal pathways related to the control of melanogenesis in melanoma cells.
Asunto(s)
Hormonas Estimuladoras de los Melanocitos/metabolismo , Melanoma Experimental/metabolismo , Receptores de la Hormona Hipofisaria/metabolismo , Células Tumorales Cultivadas/metabolismo , Animales , Secuencia de Bases , Células Clonales , Ratones , Datos de Secuencia MolecularRESUMEN
Proliferation of astrocytes is a dramatic response of the central nervous system (CNS) to injury and disease. Such proliferation results in the formation of the neural/glial scar and the reconstitution of the glial limitans. However, not all astrocytes enter the proliferative cycle following injury, and for those that do, the period of cell division is limited. Little attention has focused on the events that regulate the duration and extent of astrocyte proliferation following damage, but clearly control mechanisms are in place as CNS injury does not result in the continuous astrocyte proliferation seen in glial tumorigenesis. Protein tyrosine phosphorylation has been implicated in both astrocyte proliferation and differentiation and plays an important role in the regulation of the cell cycle in a number of different systems. We have found a small subset of astrocytes in the chick auditory brainstem that are immunopositive for the protein tyrosine phosphatase SHP-1. SHP-1 appears to negatively regulate cellular division in the hematopoietic system and is involved in the mitogenic response to various growth factors. Following cochlea removal, there is a marked increase within the auditory brainstem nucleus, nucleus magnocellularis (NM), in both in the number of SHP-1-positive astrocytes and the length of their immunopositive fibers. Significantly, those animals showing the greatest increases in SHP-1 immunoreactivity do not exhibit large amounts of astrocyte proliferation. We hypothesize that the expression of SHP-1 plays a role in negatively regulating the mitotic behavior of astrocytes following deafferentation.
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Astrocitos/metabolismo , Tronco Encefálico/metabolismo , Pollos/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Astrocitos/citología , División Celular , Cóclea/lesiones , Desnervación , Péptidos y Proteínas de Señalización Intracelular , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteína Tirosina Fosfatasa no Receptora Tipo 6RESUMEN
Endogenous sphingolipid metabolites such as ceramides and sphingosines have been increasingly recognized as lipid mediators of cell growth, differentiation and apoptosis. We have previously studied the ability of sphingosine (Sph) and N,N-dimethylsphingosine (DMS) to induce apoptosis in a variety of solid tumor cell lines. Here we report that in tumor cell lines displaying high mitogen-activated protein kinase activity (MAPK), treatment with 5 mu M of these sphingolipids significantly inhibited MAPK activity within 2-5 min (p < 0.005-0.01 as compared to controls) and induced apoptosis within hours. In contrast, untransformed cells and those tumor cell lines with low MAPK activity showed no significant change in activity and no apoptosis. High concentrations of C2-ceramide (50-100 mM), which induced apoptosis in the solid tumor cells, did not show significant effect on MAPK activity. MAPK activity was not directly inhibited in vitro, but tyrosine phosphatase activity was increased 2-4 fold in solid tumor cells by Sph or DMS (p < 0.01-0.05), suggesting that a phosphatase may play an important role in sphingolipid-directed MAPK regulation. Sph/DMS-induced apoptosis, but not MAPK inhibition, was blocked by protease inhibitors, indicating that MAPK inhibition is an earlier step of Sph/DMS-induced apoptosis than proteolysis. Furthermore, in human breast carcinoma MDA468 cells and human epidermal carcinoma A431 cells, both of which overexpress the epidermal growth factor (EGF) receptor, 20-200 nM EGF inhibited MAPK (p < 0.005-0.01) and induced apoptosis. These observations suggest that inhibition of the MAPK cascade may be involved in apoptotic signaling by Sph/DMS in some solid tumor cells, or by EGF in some cancer cells which overexpress the EGF receptor. Finally, the PKC-specific inhibitor, calphostin C, under conditions in which PKC is completely suppressed, inhibited MAPK activity and induced apoptosis only weakly in these solid tumor cells, whereas the non-specific PKC inhibitor staurosporine induced both apoptosis and MAPK inhibition significantly, suggesting that MAPK inhibition and apoptosis by Sph/DMS occurs independently of PKC in these cell lines, although these pathways may act cooperatively in other cell types. This study provides insight into possible mechanisms involved in sphingolipid-induced apoptosis in solid cancer tumor cell lines.
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Marcadores de Afinidad , Receptores de la Hormona Hipofisaria/metabolismo , Agonistas alfa-Adrenérgicos/farmacología , Antagonistas Adrenérgicos alfa/farmacología , Secuencia de Aminoácidos , Animales , Humanos , Melanoma/metabolismo , Datos de Secuencia Molecular , Receptores de la Hormona Hipofisaria/efectos de los fármacosAsunto(s)
Biomarcadores de Tumor/análisis , Melanoma Experimental/química , Melanoma/química , Proteínas de Neoplasias/análisis , Receptores de la Hormona Hipofisaria/análisis , Marcadores de Afinidad , Secuencia de Aminoácidos , Animales , Autorradiografía/métodos , Densitometría , Humanos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Ensayo de Unión Radioligante , Programas Informáticos , Células Tumorales Cultivadas , alfa-MSH/análogos & derivados , alfa-MSH/metabolismoAsunto(s)
Hormonas Estimuladoras de los Melanocitos/metabolismo , Melanoma Experimental/metabolismo , Melanoma/metabolismo , Receptores de la Hormona Hipofisaria/metabolismo , alfa-MSH/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Humanos , Cinética , Ratones , Datos de Secuencia Molecular , Monofenol Monooxigenasa/metabolismo , Estructura Secundaria de Proteína , Receptores de la Hormona Hipofisaria/química , Receptores de la Hormona Hipofisaria/genética , Células Tumorales Cultivadas , alfa-MSH/análogos & derivados , alfa-MSH/farmacologíaRESUMEN
Genetic alterations in the kinase domain of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) patients are associated with sensitivity to treatment with small molecule tyrosine kinase inhibitors. Although first-generation reversible, ATP-competitive inhibitors showed encouraging clinical responses in lung adenocarcinoma tumors harboring such EGFR mutations, almost all patients developed resistance to these inhibitors over time. Such resistance to first-generation EGFR inhibitors was frequently linked to an acquired T790M point mutation in the kinase domain of EGFR, or upregulation of signaling pathways downstream of HER3. Overcoming these mechanisms of resistance, as well as primary resistance to reversible EGFR inhibitors driven by a subset of EGFR mutations, will be necessary for development of an effective targeted therapy regimen. Here, we show that BIBW2992, an anilino-quinazoline designed to irreversibly bind EGFR and HER2, potently suppresses the kinase activity of wild-type and activated EGFR and HER2 mutants, including erlotinib-resistant isoforms. Consistent with this activity, BIBW2992 suppresses transformation in isogenic cell-based assays, inhibits survival of cancer cell lines and induces tumor regression in xenograft and transgenic lung cancer models, with superior activity over erlotinib. These findings encourage further testing of BIBW2992 in lung cancer patients harboring EGFR or HER2 oncogenes.
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Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Receptores ErbB/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Quinazolinas/uso terapéutico , Receptor ErbB-2/antagonistas & inhibidores , Afatinib , Animales , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/patología , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Receptores ErbB/genética , Receptores ErbB/metabolismo , Femenino , Humanos , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Ratones Transgénicos , Células 3T3 NIH , Fosforilación/efectos de los fármacos , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Resultado del Tratamiento , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The heterogeneity of melanotropin receptors on B16 sublines was tested by using photoaffinity crosslinking techniques and the superpotent alpha-MSH derivative [Nle4, D-Phe7, 1'-(2-nitro-4-azido-phenylsulfenyl)-Trp9]-alpha- MSH (NAPS-MSH). Specific crosslinking of this compound to B16-F1, B16-F10, B16-M2R or B16-W4 cells revealed three different subtypes of MSH receptor based on SDS-PAGE analysis. Binding of monoiodinated alpha-MSH to these different subclones is saturable and characteristic for a single class of complexes (0.9 nM less than KD less than 1.6 nM). In this article the nature of the different MSH receptor subtypes as well as their possible correlation to the melanogenic potential of a particular cell line is discussed.
Asunto(s)
Marcadores de Afinidad/metabolismo , Melanoma Experimental/química , Receptores de la Hormona Hipofisaria/análisis , alfa-MSH/análogos & derivados , Animales , Bioensayo , Células Cultivadas , Células Clonales/química , Radioisótopos de Yodo , Ratones , Fotoquímica , Ensayo de Unión Radioligante , alfa-MSH/metabolismoRESUMEN
The melanotropin (MSH) receptor of mouse B16-F1 melanoma cells was characterized by photoaffinity cross-linking, using a potent alpha-MSH photolabel, [norleucine4, D-phenylalanine7, 1'-(2-nitro-4-azidophenylsulfenyl)-tryptophan9]-alpha-melanotropin (Naps-MSH). Its monoiodinated form, 125I-Naps-MSH, displayed a approximately 6.5-fold higher biological activity than alpha-MSH. Scatchard analysis of the saturation curves with 125I-Naps-MSH revealed approximately 20,000 receptors/B16-F1 cell and an apparent KD of approximately 0.3 nM. Analysis of the cross-linked MSH receptor by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that a photolabeled band of approximately 45 kDa occurs in B16-F1, B16-F10, and Cloudman S91 mouse melanoma, as well as in human D10 and 205 melanoma but not in non-melanoma cells. The labeled 45-kDa protein had an isoelectric point of 4.5-4.9 as determined by two-dimensional gel electrophoresis. Treatment of the labeled 45-kDa protein of B16-F1 cell membranes by neuraminidase shifted the band to approximately 42 kDa. A similar band of about 42 kDa was also observed after receptor labeling of B16-W4 cells, a cell line with a decreased number of terminal N-linked neuraminyl residues. These results indicate that the labeled 45-kDa glycoprotein contains terminal sialic acid residues, explaining the low pI of this protein, and that it is characteristic for melanoma cells and hence part of the MSH receptor.
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Marcadores de Afinidad , Melanoma/metabolismo , Receptores de la Hormona Hipofisaria/análisis , Células Tumorales Cultivadas/metabolismo , alfa-MSH/metabolismo , Animales , Línea Celular , Glicoproteínas/aislamiento & purificación , Glicoproteínas/metabolismo , Humanos , Punto Isoeléctrico , Melanoma/análisis , Ratones , Peso Molecular , Neuraminidasa , Fotoquímica , Ratas , Receptores de la Hormona Hipofisaria/aislamiento & purificación , Células Tumorales Cultivadas/análisisRESUMEN
Three different monoiodinated radioligands of alpha-MSH (alpha-melanocyte-stimulating hormone) were compared in a binding assay with human D10 melanoma cells: [Tyr(125I)2]-alpha-MSH, [Tyr(125I)2,NIe4]-alpha-MSH, and [Tyr(125I)2,NIe4,D-Phe7]-alpha-MSH. They were prepared either by the classical chloramine T method or by the Enzymobead method. A simple and rapid purification scheme was developed consisting of a primary separation on reversed-phase C18 silica cartridges immediately after the iodination, followed by HPLC purification before each binding experiment. Biological testing of the three radioligands showed that they all retained high melanotropic activity in the B16 melanin assay and the Anolis melanophore assay. However, in human D10 melanoma cells, [Tyr(125I)2,NIe4]-alpha-MSH led to a high degree of non-specific binding to the cells which could not be displaced by excess alpha-MSH and only partially by [NIe4]-alpha-MSH. The [Tyr(125I)2,NIe4,D-Phe7]-alpha-MSH tracer gave similar results but with a much lower proportion of non-specific binding. On the other hand, [Tyr(125I)2]-alpha-MSH proved to be an excellent radioligand whose non-specific binding to the D10 cells was not higher than 20% of the total binding.
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Melanoma/metabolismo , Receptores de la Hormona Hipofisaria/metabolismo , Compuestos de Tosilo , alfa-MSH/análogos & derivados , alfa-MSH/metabolismo , Bioensayo , Cloraminas , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Humanos , Radioisótopos de Yodo , Marcaje Isotópico/métodos , Ensayo de Unión Radioligante , Células Tumorales Cultivadas , alfa-MSH/aislamiento & purificaciónRESUMEN
The determination of protein tyrosine phosphatase (PTP) activity using different protein substrates such as modified lysozyme or myelin basic protein is greatly affected by the type of enzyme involved, the condition of the assay, and the presence of effectors. The purpose of this study was to develop a general substrate of wide applicability with which variations in enzymatic activity would be minimized. A nonapeptide ENDYINASL derived from a highly conserved region of the T-cell phosphatase TC.PTP (Cool et al. (1989) Proc. Natl. Acad. Sci. USA 86, 5257-5261) was phosphorylated with a recombinant tyrosine kinase domain of the EGF receptor and purified on a C18 cartridge. Phosphatase activities of intracellular and receptor-linked PTPs were as high as the highest values obtained with the protein substrates. The intracellular, low-molecular-weight PTPs exhibited Km values between 0.5 and 1.3 microM, whereas the receptor forms CD45 and RPTP alpha gave values of 14 and 35 microM, respectively. All PTPs displayed similar properties toward the peptide including a low pH optimum and inhibition by vanadate, divalent cations, and heparin. Following immunoprecipitation, 1 ng of TC.PTP could be detected with ENDY(P)INASL compared to 10 ng in presence of protein substrates.
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Oligopéptidos/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Secuencia de Aminoácidos , Cinética , Datos de Secuencia Molecular , Peso Molecular , Fosforilación , Pruebas de Precipitina , Especificidad por SustratoRESUMEN
The identification, purification, and biochemical characterization of specific markers for neuroglial cells in the central nervous system is an essential step toward a better understanding of the function of glial cells. This manuscript reports the identification and purification of a neuroglia-associated protein (NAP-185) with an apparent molecular mass of 185 kDa. While its expression is not restricted to the brain, it was first identified in a specific subpopulation of glial cells when chick brain stem sections were analyzed with an affinity-purified rabbit antiserum raised against the catalytic domain of the T-cell protein tyrosine phosphatase. This 185-kDa antigen was purified to apparent homogeneity and confirmed to be responsible for the neuroglial staining observed. In spite of its immunological relation to T-cell protein tyrosine phosphatase, purified NAP-185 failed to display tyrosine phosphatase activity. The primary sequence of five NAP-185-derived peptides shows that this protein has not yet been characterized and that it is possibly related to AP180, a clathrin-associated protein.
Asunto(s)
Astrocitos/química , Tronco Encefálico/química , Proteínas del Tejido Nervioso/química , Secuencia de Aminoácidos , Animales , Biomarcadores , Pollos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/genética , Distribución TisularRESUMEN
We have cloned from cultured vascular smooth muscle cells a protein tyrosine phosphatase, rat density-enhanced phosphatase-1 (rDEP-1), which is a probable rat homologue of DEP-1/HPTP eta. rDEP-1 is encoded by an 8.7-kb transcript and is expressed as a 180- to 220-kD protein. The rDEP-1 gene is located on human chromosome 11 (region p11.2) and on mouse chromosome 2 (region 2E). The cDNA sequence predicts a transmembrane protein consisting of a single phosphatase catalytic domain in the intracellular region, a single transmembrane domain, and eight fibronectin type III repeats in the extracellular region (GenBank accession number U40790). In situ hybridization analysis demonstrates that rDEP-1 is widely expressed in vivo but that expression is highest in cells that form epithelioid monolayers. In cultured cells with epitheliod morphology, including endothelial cells and newborn smooth muscle cells, but not in fibroblast-like cells, rDEP-1 transcript levels are dramatically upregulated as population density increases. In vivo, quiescent endothelial cells in normal arteries express relatively high levels of rDEP-1. During repair of vascular injury, expression of rDEP-1 is downregulated in migrating and proliferating endothelial cells. In vivo, rDEP-1 transcript levels are present in very high levels in megakaryocytes, and circulating plates have high levels of the rDEP-1 protein. In vitro, initiation of differentiation of the human megakaryoblastic cell line CHRF-288-11 with phorbol 12-myristate 13-acetate leads to a very strong upregulation of rDEP-1 transcripts. The deduced structure and the regulation of expression of rDEP-1 suggest that it may play a role in adhesion and/or signaling events involving cell-cell and cell-matrix contact.