RESUMO
Glioblastomas often show activation of epidermal growth factor receptor (EGFR) and loss of PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor, but it is not known if these two genetic lesions act together to transform cells. To answer this question, we infected PTEN-/- neural precursor cells with a retrovirus encoding EGFRvIII, which is a constitutively activated receptor. EGFRvIII PTEN-/- cells formed highly mitotic tumors with nuclear pleomorphism, necrotic areas, and glioblastoma markers. The transformed cells showed increased cell proliferation, centrosome amplification, colony formation in soft agar, self-renewal, expression of the stem cell marker CD133, and resistance to oxidative stress and ionizing radiation. The RAS/mitogen-activated protein kinase (ERK) and phosphoinositide 3-kinase/protein kinase B (PI3K/Akt) pathways were activated, and checkpoint kinase 1 (Chk1), the DNA damage regulator, was phosphorylated at S280 by Akt, suppressing Chk1 phosphorylation at S345 in response to ionizing irradiation. The PTEN-/- cells showed low levels of DNA damage in the absence of irradiation, which was increased by EGFRvIII expression. Finally, secondary changes occurred during tumor growth in mice. Cells from these tumors showed decreased tumor latencies and additional chromosomal aberrations. Most of these tumor lines showed translocations of mouse chromosome 15. Intracranial injections of one of these lines led to invasive, glial fibrillary acidic protein-positive, nestin-positive tumors. These results provide a molecular basis for the occurrence of these two genetic lesions in brain tumors and point to a role in induction of genomic instability.
Assuntos
Neoplasias Encefálicas/genética , Instabilidade Cromossômica , Receptores ErbB/metabolismo , Glioma/genética , PTEN Fosfo-Hidrolase/fisiologia , Animais , Western Blotting , Encéfalo/metabolismo , Neoplasias Encefálicas/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células/efeitos da radiação , Transformação Celular Neoplásica/efeitos da radiação , Células Cultivadas , Centrossomo/metabolismo , Receptores ErbB/genética , Feminino , Glioma/metabolismo , Humanos , Integrases/metabolismo , Proteínas de Filamentos Intermediários/fisiologia , Cariotipagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Microscopia de Fluorescência , Células NIH 3T3 , Proteínas do Tecido Nervoso/fisiologia , Nestina , Fosforilação/efeitos da radiação , Radiação Ionizante , Retroviridae/genéticaRESUMO
PTEN is a lipid phosphatase, and PTEN mutations are associated with gliomas, macrocephaly, and mental deficiencies. We have used PTEN +/- and PTEN +/+ mice to prepare subventricular zone (SVZ) precursor cells. Using DNA microarrays, we compared the expression profiles of PTEN +/+ and PTEN +/- cells and identified 91 differentially expressed genes in PTEN +/- precursor cells. Many of the PTEN-regulated genes are involved with signaling, cytoskeleton, extracellular matrix, metabolism, and transcription factors. Some of these changes are likely mediated by the transcription factor, HIF-1. We confirmed a subset of these changes by real-time PCR. In addition, we examined protein levels for two of the PTEN-up-regulated genes, vascular endothelial growth factor (VEGF) and doublecortin (DCX). PTEN haploinsufficiency increases immunostaining for VEGF for both cultured precursor cells and sections of the SVZ. PTEN haploinsufficiency shifted most of the DCX-positive cells from the SVZ to the olfactory bulb. These observations indicate that even a small decrease in PTEN levels results in substantial changes in gene expression and precursor cell function.
Assuntos
Perfilação da Expressão Gênica/métodos , Neurônios/metabolismo , Monoéster Fosfórico Hidrolases/biossíntese , Células-Tronco/metabolismo , Proteínas Supressoras de Tumor/biossíntese , Animais , Proteína Duplacortina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , PTEN Fosfo-Hidrolase , Monoéster Fosfórico Hidrolases/deficiência , Monoéster Fosfórico Hidrolases/genética , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genéticaRESUMO
PTEN is a lipid phosphatase, and PTEN mutations are associated with gliomas, macrocephaly, and mental deficiencies. We have used PTEN +/- mice to assess PTEN's role in subventricular zone (SVZ) precursor cells. For cultured SVZ neurosphere cells, haploinsufficiency for PTEN increases phosphorylation of Akt and forkhead transcription factor and slightly enhances proliferation. Based on a filter penetration assay, PTEN +/- cells are substantially more migratory and invasive than +/+ cells. The +/- cells also are more resistant to H(2)O(2)-induced apoptosis. Analysis of PTEN +/- and +/+ mice by BrdU labeling reveals no difference in the rate of cell proliferation in the SVZ. Exit of BrdU-labeled cells from the SVZ and radial migration to the outer layers of the olfactory bulb are more rapid for +/- cells. These observations indicate that PTEN regulates SVZ precursor cell function and is particularly important for migration and apoptosis in response to oxidative stress.