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1.
Proc Natl Acad Sci U S A ; 112(4): 1137-42, 2015 Jan 27.
Artículo en Inglés | MEDLINE | ID: mdl-25583479

RESUMEN

Gaucher disease is caused by mutations of the GBA1 gene, which encodes the lysosomal anchored gluococerebrosidase (GCase). GBA1 mutations commonly result in protein misfolding, abnormal chaperone recognition, and premature degradation, but are less likely to affect catalytic activity. In the present study, we demonstrate that the Hsp90/HOP/Cdc37 complex recruits Hsp27 after recognition of GCase mutants with subsequent targeting of GCase mutant peptides to degradation mechanisms such as VCP and the 26S proteasome. Inhibition of Hsp27 not only increased the quantity of enzyme but also enhanced GCase activity in fibroblasts derived from patients with Gaucher disease. These findings provide insight into a possible therapeutic strategy for protein misfolding diseases by correcting chaperone binding and altering subsequent downstream patterns of protein degradation.


Asunto(s)
Enfermedad de Gaucher/metabolismo , Glucosilceramidasa/metabolismo , Proteínas de Choque Térmico HSP27/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Proteolisis , Deficiencias en la Proteostasis/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chaperoninas/genética , Chaperoninas/metabolismo , Fibroblastos/metabolismo , Enfermedad de Gaucher/genética , Glucosilceramidasa/genética , Proteínas de Choque Térmico HSP27/genética , Proteínas HSP90 de Choque Térmico/genética , Células HeLa , Proteínas de Choque Térmico , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Chaperonas Moleculares , Mutación , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Pliegue de Proteína , Deficiencias en la Proteostasis/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo
2.
Proc Natl Acad Sci U S A ; 110(3): 966-71, 2013 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-23277556

RESUMEN

Gaucher disease is caused by mutations of the GBA gene that encodes the lysosomal enzyme glucocerebrosidase (GCase). GBA mutations often result in protein misfolding and premature degradation, but usually exert less effect on catalytic activity. In this study, we identified the molecular mechanism by which histone deacetylase inhibitors increase the quantity and activity of GCase. Specifically, these inhibitors limit the deacetylation of heat shock protein 90, resulting in less recognition of the mutant peptide and GCase degradation. These findings provide insight into a possible therapeutic strategy for Gaucher disease and other genetic disorders by modifying molecular chaperone and protein degradation pathways.


Asunto(s)
Enfermedad de Gaucher/tratamiento farmacológico , Enfermedad de Gaucher/enzimología , Glucosilceramidasa/metabolismo , Inhibidores de Histona Desacetilasas/farmacología , Chaperonas Moleculares/metabolismo , Acetilación , Degradación Asociada con el Retículo Endoplásmico , Enfermedad de Gaucher/genética , Enfermedad de Gaucher/metabolismo , Glucosilceramidasa/genética , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/genética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Modelos Biológicos , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
3.
Proc Natl Acad Sci U S A ; 109(18): 6963-8, 2012 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-22505738

RESUMEN

Astrocytes are the most abundant cell of the CNS and demonstrate contact inhibition in which a nonproliferative, nonmotile cellular state is achieved once stable intercellular contacts are formed between mature cells. Cellular injury disrupts these intercellular contacts, causing a loss of contact inhibition and the rapid initiation of healing. Dysregulation of the molecular pathways involved in this process is thought to lead to an aggressive cellular state associated with neoplasia. We investigated whether a comparable correlation exists between the response of astrocytes to injury and the malignant phenotype of astrocytomas. We discovered that the loss of contact inhibition plays a critical role in the initiation and regulation of reactive astrocytes in the healing of wounds. In particular, injury of the astrocytes interrupts and destabilizes the cadherin-catenin complexes at the cell membrane leading to nuclear translocation of ß-catenin and characteristic changes associated with the activation of astrocytes. Similar signaling pathways are found to be active--but dysregulated--in astrocytomas. Inhibition of ß-catenin signaling diminished both the response of astrocytes to injury and induction of the malignant phenotype of astrocytomas. The findings shed light on a unique mechanism associated with the pathogenesis of astrocytomas and provide a model for the loss of contact inhibition that may broadly apply to understanding the mechanisms of tissue repair and tumorigenesis in the brain.


Asunto(s)
Astrocitos/metabolismo , Astrocitoma/etiología , Astrocitoma/metabolismo , beta Catenina/metabolismo , Animales , Astrocitoma/patología , Proliferación Celular , Transformación Celular Neoplásica , Células Cultivadas , Técnicas de Silenciamiento del Gen , Ratones , Modelos Neurológicos , Fenotipo , ARN Interferente Pequeño , Transducción de Señal , Células Tumorales Cultivadas , beta Catenina/antagonistas & inhibidores , beta Catenina/genética
4.
Neuro Oncol ; 10(1): 45-51, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18182627

RESUMEN

Dynamic changes in the expression of multiple genes appear to be common features that distinguish transformed cells from their normal counterparts. We compared the proteomic profiles of four glioblastoma multiforme (GBM) tissue samples and four normal brain cortex samples to examine the molecular basis of gliomagenesis. Trypsin-digested protein samples were separated by capillary isoelectric focusing with nano-reversed-phase liquid chromatography and were profiled by mass spectrometric sequencing. Wolf-Hirschhorn syndrome candidate 1 (WHSC1), along with 103 other proteins, was found only in the GBM proteomes. Western blot and immunohistochemistry verified our proteomic findings and demonstrated that 30-kDa WHSC1 expression increases with ascending tumor proliferation activity. RNA interference could suppress glioma cell growth by blocking WHSC1 expression. Our novel findings encourage the application of proteomic techniques in cancer research.


Asunto(s)
Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Glioma/metabolismo , Glioma/patología , N-Metiltransferasa de Histona-Lisina/biosíntesis , Proteínas Represoras/biosíntesis , Western Blotting , Proliferación Celular , Cromatografía Liquida , Humanos , Inmunohistoquímica , Proteómica , ARN Interferente Pequeño , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masas en Tándem , Transfección
5.
Cancer Res ; 66(20): 10199-204, 2006 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-17047085

RESUMEN

Meningiomas are classified into three groups (benign, atypical, and anaplastic) based on morphologic characteristics. Atypical meningiomas, which are WHO grade 2 tumors, and anaplastic meningiomas, which are WHO grade 3 tumors, exhibit an increased risk of recurrence and premature death compared with benign WHO grade 1 tumors. Although atypical and anaplastic meningiomas account for <10% of all of meningiomas, it can be difficult to distinguish them from benign meningiomas by morphologic criteria alone. We used selective tissue microdissection to examine 24 human meningiomas and did two-dimensional gel electrophoresis to determine protein expression patterns. Proteins expressed differentially by meningiomas of each WHO grade were identified and sequenced. Proteomic analysis revealed protein expression patterns unique to WHO grade 1, 2, and 3 meningiomas and identified 24 proteins that distinguish each subtype. Fifteen proteins showed significant changes in expression level between benign and atypical meningiomas, whereas nine distinguished atypical from anaplastic meningiomas. Differential protein expression was confirmed by Western blotting and immunohistochemistry. We established differential proteomic profiles that characterize and distinguish meningiomas of increasing grades. The proteins and proteomic profiles enhance understanding of the pathogenesis of meningiomas and have implications for diagnosis, prognosis, and treatment.


Asunto(s)
Neoplasias Meníngeas/clasificación , Meningioma/clasificación , Electroforesis en Gel Bidimensional , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Neoplasias Meníngeas/genética , Neoplasias Meníngeas/metabolismo , Neoplasias Meníngeas/patología , Meningioma/genética , Meningioma/metabolismo , Meningioma/patología , Proteómica , Regulación hacia Arriba
6.
Cancer Res ; 65(23): 10847-53, 2005 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-16322231

RESUMEN

The von Hippel-Lindau (VHL) disease is caused by VHL germ line mutation. Inactivation of the wild-type copy of the VHL gene leads to up-regulation of hypoxic response and tumor formation within central nervous system (CNS), kidneys, pancreas, adrenal glands, epididymis, broad ligament, and the endolymphatic sac/petrous bone. Endolymphatic sac tumors (ELST) have been proposed to be derived from endolymphatic sac epithelium, but other possible structures of origin have been implicated. To clarify the anatomic and cellular origin of ELSTs, we did a morphologic and molecular pathologic analysis of 16 tumors. In addition, we investigated effects of VHL deficiency on "tumor-free" endolymphatic duct and sac of VHL patients. Several tumors included in this study were <1 cm in size, and their origin could be placed in the intraosseous portion of the endolymphatic duct/sac. Furthermore, by analysis of clinically uninvolved "tumor-free" endolymphatic duct and sac tissues of VHL patients, we discovered a variety of VHL-deficient microscopic abnormalities with morphologic similarities to ELSTs. We conclude that most, if not all, ELSTs arise within the intraosseous portion of the endolymphatic duct/sac, the vestibular aqueduct. In analogy to renal parenchyma and selected topographical sites within the CNS, endolymphatic duct/sac epithelia are preferentially and multifocally targeted in VHL disease. The primary effect of VHL deficiency on human endolymphatic duct/sac epithelium seems to be the generation of multifocal sites of VHL-deficient cell proliferations from which tumorigenesis may or may not occur. Therefore, inactivation of the VHL wild-type allele seems necessary but not sufficient for the formation of tumor.


Asunto(s)
Neoplasias del Oído/patología , Conducto Endolinfático/patología , Saco Endolinfático/patología , Lesiones Precancerosas/patología , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/metabolismo , Neoplasias del Oído/metabolismo , Conducto Endolinfático/metabolismo , Saco Endolinfático/metabolismo , Humanos , Inmunohistoquímica , Lesiones Precancerosas/metabolismo , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genética , Enfermedad de von Hippel-Lindau/complicaciones , Enfermedad de von Hippel-Lindau/genética
7.
Clin Cancer Res ; 11(3): 1059-64, 2005 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-15709172

RESUMEN

Von Hippel-Lindau (VHL) disease is characterized by multiple tumors in specific target organs. The tumors at different sites share distinct morphologic and genetic characteristics but their cell of origin is unknown. We show that VHL disease-associated renal clear cell carcinomas (RCC) consistently coexpress erythropoietin (Epo) and Epo receptor (EpoR). In addition, coexpression of Epo and EpoR is detected in many renal cysts, providing further evidence that renal cysts are potential precursors for RCC. In conjunction with VHL gene deficiency, coexpression of Epo and EpoR in renal cysts and tumors may reflect a developmental arrest in immature mesenchymal cells. Such arrest may lead to autocrine stimulation, cell proliferation, and renal tumor development, similar to tumorigenesis of VHL disease-associated hemangioblastomas.


Asunto(s)
Carcinoma de Células Renales/patología , Eritropoyetina/genética , Enfermedades Renales Quísticas/patología , Neoplasias Renales/patología , Receptores de Eritropoyetina/genética , Enfermedad de von Hippel-Lindau/complicaciones , Western Blotting , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Eritropoyetina/metabolismo , Femenino , Expresión Génica , Humanos , Inmunohistoquímica , Enfermedades Renales Quísticas/genética , Enfermedades Renales Quísticas/metabolismo , Neoplasias Renales/genética , Masculino , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Receptores de Eritropoyetina/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa
8.
Cancer Res ; 63(21): 7051-5, 2003 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-14612494

RESUMEN

The nature of the cell responsible for von Hippel-Lindau (VHL) disease-associated tumor formation has been controversial for decades. We demonstrate that VHL disease-associated central nervous system tumors are composed of developmentally arrested angioblasts that coexpress erythropoietin (Epo) and Epo receptor. The angioblasts are capable of differentiating into RBCs via formation of blood islands with extramedullary hematopoiesis. Because of VHL deficiency, Epo receptor-expressing, developmentally arrested angioblasts simultaneously coexpress Epo, which may represent a crucial pathogenetic step in tumor formation.


Asunto(s)
Neoplasias Cerebelosas/etiología , Neoplasias Cerebelosas/patología , Hemangioblastoma/etiología , Hemangioblastoma/patología , Enfermedad de von Hippel-Lindau/complicaciones , Enfermedad de von Hippel-Lindau/patología , Neoplasias Cerebelosas/metabolismo , Eritropoyetina/biosíntesis , Eritropoyetina/metabolismo , Hemangioblastoma/metabolismo , Hematopoyesis Extramedular , Humanos , Inmunohistoquímica , Receptores de Eritropoyetina/biosíntesis , Receptores de Eritropoyetina/metabolismo , Enfermedad de von Hippel-Lindau/genética
9.
J Neurosurg ; 103(2): 284-8, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16175858

RESUMEN

OBJECT: Von Hippel-Lindau (VHL) disease is characterized by multiple tumors in specific organs. The cell of origin and the reason for the particular organ distribution of the tumors remains unknown. Endolymphatic sac tumor (ELST) is one of the lesions associated with VHL disease. Data from previous studies of VHL disease-associated hemangioblastomas (HBs) and renal cell carcinomas (RCCs) have indicated that VHL gene deficiency causes coexpression of erythropoietin (Epo) and its receptor (Epo-R), which facilitates tumor growth. METHODS: The authors studied ELSTs from five patients with VHL germline mutations. Analysis of the five ELST samples revealed loss of the wild-type allele, consistent with Knudson's two-hit hypothesis for tumorigenesis. All five ELST specimens were characterized microscopically and by immunohistochemical analysis. Coexpression of Epo and Epo-R was found in all five tumors on immunohistochemical studies and confirmed through reverse transcription-polymerase chain reaction and Western blot analysis. CONCLUSIONS: Expression of Epo appears to be a result of VHL gene deficiency, whereas the simultaneous coexpression of Epo-R may reflect a developmental mechanism of tumorigenesis. Coexpression of Epo and Epo-R in ELSTs together with the morphological and genetic similarities of these lesions with other VHL disease-associated tumors indicates that VHL disease-associated tumors in different organs share common pathogenetic pathways.


Asunto(s)
Neoplasias del Oído/genética , Neoplasias del Oído/patología , Saco Endolinfático/patología , Eritropoyetina/biosíntesis , Receptores de Eritropoyetina/biosíntesis , Adulto , Perfilación de la Expresión Génica , Mutación de Línea Germinal , Humanos , Inmunohistoquímica , Masculino , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Enfermedad de von Hippel-Lindau
10.
Mol Cancer Ther ; 14(7): 1540-1547, 2015 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-25939762

RESUMEN

Protein phosphatase 2A (PP2A) is a tumor suppressor whose function is lost in many cancers. An emerging, though counterintuitive, therapeutic approach is inhibition of PP2A to drive damaged cells through the cell cycle, sensitizing them to radiotherapy. We investigated the effects of PP2A inhibition on U251 glioblastoma cells following radiation treatment in vitro and in a xenograft mouse model in vivo. Radiotherapy alone augmented PP2A activity, though this was significantly attenuated with combination LB100 treatment. LB100 treatment yielded a radiation dose enhancement factor of 1.45 and increased the rate of postradiation mitotic catastrophe at 72 and 96 hours. Glioblastoma cells treated with combination LB100 and radiotherapy maintained increased γ-H2AX expression at 24 hours, diminishing cellular repair of radiation-induced DNA double-strand breaks. Combination therapy significantly enhanced tumor growth delay and mouse survival and decreased p53 expression 3.68-fold, compared with radiotherapy alone. LB100 treatment effectively inhibited PP2A activity and enhanced U251 glioblastoma radiosensitivity in vitro and in vivo. Combination treatment with LB100 and radiation significantly delayed tumor growth, prolonging survival. The mechanism of radiosensitization appears to be related to increased mitotic catastrophe, decreased capacity for repair of DNA double-strand breaks, and diminished p53 DNA-damage response pathway activity.


Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Glioblastoma/tratamiento farmacológico , Mitosis/efectos de los fármacos , Piperazinas/farmacología , Proteína Fosfatasa 2/antagonistas & inhibidores , Carga Tumoral/efectos de los fármacos , Animales , Western Blotting , División Celular/efectos de los fármacos , División Celular/efectos de la radiación , Línea Celular Tumoral , Terapia Combinada , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de la radiación , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Histonas/metabolismo , Humanos , Inmunohistoquímica , Ratones Desnudos , Mitosis/efectos de la radiación , Proteína Fosfatasa 2/metabolismo , Tolerancia a Radiación/efectos de los fármacos , Tolerancia a Radiación/efectos de la radiación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Carga Tumoral/efectos de la radiación , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
11.
J Neurosurg ; 113(2): 225-33, 2010 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-20001590

RESUMEN

OBJECT: Nuclear receptor corepressor (N-CoR) forms a complex that maintains neural stem cells in an undifferentiated state through transcriptional repression. Recently, it has been shown that N-CoR is overexpressed in glioblastoma multiforme (GBM) tumor stem cells and has a putative role in maintaining these cells in an undifferentiated immortal state. To determine the effects of disruption of N-CoR complex function by serine/threonine protein phosphatase 2A (PP2A) inhibition on GBM tumor cell differentiation and proliferation, the authors developed and investigated a competitive small molecule inhibitor (LB1) of PP2A in GBM. METHODS: The authors investigated the effects of LB1 on GBM proliferation and molecular differentiation pathways using in vitro and in vivo studies. RESULTS: The LB1 inhibited PP2A, leading to increased levels of phosphorylated Akt kinase and decreased NCoR expression, as well as dose-dependent antiproliferative activity in cultured U87 and U251 malignant glioma cells (dose range 1-10 microM). Systemic LB1 treatment (1.5 mg/kg/day for 21 days) had significant tumor antiproliferative effects in mice harboring U87 glioma xenografts (73% mean reduction in tumor volume compared with controls; p < 0.001). Moreover, a reduction in PP2A expression and activity after LB1 treatment in vivo correlated with increased Akt phosphorylation, reduced nuclear N-CoR expression and N-CoR cytoplasmic translocation, and increased accumulation of acetylated core histones, which coincided with the appearance of glial fibrillary acidic protein-expressing tumor cells. CONCLUSIONS: These findings indicate that PP2A inhibition effectively disrupts N-CoR complex function/expression and leads to cytoplasmic translocation of N-CoR with subsequent tumor cell differentiation and/or death. Therapeutic paradigms that target N-CoR function in the cancer stem cell component of malignant gliomas may have treatment utility.


Asunto(s)
Antineoplásicos/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Inhibidores Enzimáticos/farmacología , Glioma/tratamiento farmacológico , Proteína Fosfatasa 2/antagonistas & inhibidores , Acetilación/efectos de los fármacos , Animales , Antineoplásicos/química , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , División Celular/efectos de los fármacos , Línea Celular Tumoral , Inhibidores Enzimáticos/química , Glioma/metabolismo , Glioma/patología , Histonas/metabolismo , Humanos , Ratones , Ratones SCID , Co-Represor 1 de Receptor Nuclear/metabolismo , Fosforilación/efectos de los fármacos , Proteína Fosfatasa 1/metabolismo , Proteína Fosfatasa 2/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
12.
Cell Cycle ; 5(4): 452-6, 2006 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-16479164

RESUMEN

The pathogenesis of de novo glioblastoma multiforme (GBM) is poorly understood and precursor cells are not known. To gain insight into the pathogenesis of GBM we analyzed brains from primates that developed de novo tumors ten years after whole brain radiation. Four animals had clinical and radiological evidence of GBM, and two animals had no evidence of GBM at the time of euthanization. Tumor precursor cells were identified diffusely scattered in the grossly normal white matter of all animals including two monkeys without evidence of GBM by MR-imaging or on autopsy examination. Tumor precursors demonstrated cellular atypia and mitoses, and were negative for tumor-associated markers GFAP, EGFR and p53. The cells were positive for Ki67 and N-CoR, the nuclear corepressor of astroglial differentiation. These results suggest that radiation-induced nuclear damage to neural stem cells or early astrocytic precursor cells can prevent normal differentiation and lead to tumor development. The findings provide insight into the tumorigenesis of de novo GBMs and suggest a new strategy for treatment of these lethal tumors by targeting both inactivation of N-CoR and inhibition of EGFR.


Asunto(s)
Encéfalo/patología , Encéfalo/efectos de la radiación , Glioblastoma/patología , Células Madre Neoplásicas/patología , Primates , Animales , Línea Celular Tumoral , ADN Viral/análisis , Humanos , Inmunohistoquímica , Imagen por Resonancia Magnética , Masculino , Células Madre Neoplásicas/efectos de la radiación , Virus 40 de los Simios/química
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