RESUMO
The earliest genetic alteration in human astrocytoma progression is mutation of the p53 tumour suppressor gene, while one of the earliest phenotypic changes is the stimulation of neovascularization. Here, we tested the role of p53 in the angiogenic process by introducing a tetracycline-regulated wild type p53 gene into null glioblastoma cells. The parental cells expressed strong angiogenic activity while upon induction of wild type, but not mutant, p53 expression, the cells secreted a factor able to neutralize the angiogenicity of the factors produced by the parental cells as well as of basic fibroblast growth factor.
Assuntos
Neoplasias Encefálicas/patologia , Regulação Neoplásica da Expressão Gênica , Glioblastoma/patologia , Proteínas de Neoplasias/biossíntese , Neovascularização Patológica , Biossíntese de Proteínas , Proteína Supressora de Tumor p53/fisiologia , Inibidores da Angiogênese , Animais , Movimento Celular , Córnea/irrigação sanguínea , Progressão da Doença , Endotélio Vascular/patologia , Feminino , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Neovascularização Patológica/fisiopatologia , Proteínas/farmacologia , Ratos , Ratos Endogâmicos F344 , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/biossíntese , Proteína Supressora de Tumor p53/genéticaRESUMO
CpG islands frequently contain gene promoters or exons and are usually unmethylated in normal cells. Methylation of CpG islands is associated with delayed replication, condensed chromatin and inhibition of transcription initiation. The investigation of aberrant CpG-island methylation in human cancer has primarily taken a candidate gene approach, and has focused on less than 15 of the estimated 45,000 CpG islands in the genome. Here we report a global analysis of the methylation status of 1,184 unselected CpG islands in each of 98 primary human tumours using restriction landmark genomic scanning (RLGS). We estimate that an average of 600 CpG islands (range of 0 to 4,500) of the 45,000 in the genome were aberrantly methylated in the tumours, including early stage tumours. We identified patterns of CpG-island methylation that were shared within each tumour type, together with patterns and targets that displayed distinct tumour-type specificity. The expression of many of these genes was reactivated by experimental demethylation in cultured tumour cells. Thus, the methylation of particular subsets of CpG islands may have consequences for specific tumour types.
Assuntos
Metilação de DNA , Fosfatos de Dinucleosídeos/análise , Neoplasias/genética , Adenocarcinoma/genética , Sequência de Bases , Neoplasias Encefálicas/genética , Neoplasias da Mama/genética , Carcinoma Intraductal não Infiltrante/genética , Carcinoma Lobular/genética , Neoplasias do Colo/genética , Fosfatos de Dinucleosídeos/genética , Feminino , Genoma Humano , Humanos , Masculino , Dados de Sequência Molecular , Mapeamento por RestriçãoRESUMO
DNA amplification is a common mechanism invoked by many human tumors to elicit overexpression of genes whose products are involved in drug resistance or cell proliferation. Although amplified regions in tumor DNA may exceed several megabases in size, segments of amplicons with a high probability of containing gene sequences may be amenable to detection by restriction landmark genomic scanning (RLGS), a high-resolution DNA analysis that separates labeled NotI fragments in two dimensions. Here, we tested this by applying RLGS to matched samples of glioma and normal brain DNA and found tumor-specific amplification of the gene encoding cyclin-dependent kinase 6 (CDK6), an observation not previously reported in human tumors. The CDK6 gene has been localized to chromosome 7q21-22, but in the gliomas studied here, it was not coamplified with either the syntenic MET (7q31) or epidermal growth factor receptor (7p11-p12) genes, suggesting that this may be part of a novel amplicon in gliomas. We then corroborated this finding by identifying both amplification-associated and amplification-independent increases in CDK6 protein levels in gliomas relative to matched normal brain samples. These data implicate the CDK6 gene in genomic amplification and illustrate the potential of RLGS for the more general identification and cloning of novel genes that are amplified in human cancer.
Assuntos
Neoplasias Encefálicas/genética , Quinases Ciclina-Dependentes , Eletroforese em Gel Bidimensional/métodos , Amplificação de Genes , Glioma/genética , Proteínas Serina-Treonina Quinases/genética , Sequência de Bases , Southern Blotting , Western Blotting , Cromossomos Humanos Par 7 , Clonagem Molecular , Quinase 6 Dependente de Ciclina , DNA/análise , Humanos , Dados de Sequência MolecularRESUMO
P16INK4 is a cell cycle regulator that specifically binds to and inactivates cyclin-dependent kinase 4 (CDK4). Its encoding gene (p16/CDKN2) maps to chromosome 9p21, a region that undergoes frequent loss of heterozygosity in a variety of human tumors. We have analyzed the p16/CDKN2 gene and its expression in a series of primary glioma samples. Although homozygous deletion or mutation of the p16/CDKN2 gene was uncommon in this series and P16INK4 protein was detectable in all grade II tumors, it was present in only 50% of grade III and grade IV samples. Conversely, in some grade IV tumors that level of P16INK4 protein was elevated; in these cases, its target, CDK4, was amplified and overexpressed. These results suggest: (a) the involvement of P16INK4 in glioma progression; (b) that mechanisms other than mutation or deletion can down-regulate expression of the p16/CDKN2 gene; and (c) that the balance between CDK4 and its cognate inhibitor, P16INK4, may confer a cell growth advantage and facilitate tumor progression.
Assuntos
Astrocitoma/genética , Astrocitoma/patologia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Proteínas de Transporte/genética , Glioblastoma/genética , Glioblastoma/patologia , Sequência de Bases , Western Blotting , Proteínas de Transporte/análise , Inibidor p16 de Quinase Dependente de Ciclina , Progressão da Doença , Deleção de Genes , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor/genética , Humanos , Dados de Sequência Molecular , Mutação , Inibidores de Proteínas Quinases , RNA Mensageiro/análise , RNA Mensageiro/genética , Transcrição GênicaRESUMO
The gene for the major angiogenic factor, vascular endothelial growth factor (VEGF), encodes several spliced isoforms. We reported previously that overexpression of two VEGF isoforms, VEGF(121) and VEGF(165), by human glioma U87 MG cells induced tumor-associated intracerebral hemorrhage, whereas expression of a third form, VEGF(189), did not cause vessel rupture. Here, we test whether these VEGF isoforms have distinct activities for enhancing vascularization and growth of gliomas in mice. U87 MG cells that overexpressed VEGF(165) or VEGF(189) grew more rapidly than the parental cells in both s.c. and intracranial (i.c.) locations. However, cells that overexpressed VEGF(121) only showed enhancement of i.c. tumor growth but had a minimal effect on s.c. glioma progression. At both anatomical sties, VEGF(165) and VEGF(189) strongly augmented neovascularization, whereas VEGF(121) only increased vessel density in brain tumors. In each type of glioma, expression of VEGF receptors -1 and -2 largely phenocopied the tumor vasculature, because increased VEGF/VEGF receptor-activated microvessel densities were strongly correlated with the angiogenicity and tumorigenicity elicited by the VEGF isoforms at both anatomical sites. One notable difference between the sites was the expression of vitronectin, a prototypic ligand of alpha(v)beta(3) and alpha(v)beta(5) integrins, detected in i.c. but not in s.c., gliomas. Endothelial cell migration stimulated by VEGF(121) was potentiated by vitronectin to a greater extent than that stimulated by VEGF(165). This data demonstrates that VEGF isoforms have distinct activities at different anatomical sites and suggest that the microenvironment of different tissues affects the function of VEGF isoforms.