RESUMO
Four tumour suppressor genes (Chk1, Chk2, Apaf1 and Rb1) in nine human malignant melanoma cell lines were screened for a loss of gene expression, point mutations and small deletions/insertions by cDNA-based DGGE/SCCP analysis. In two cell lines alterations of the investigated genes could be demonstrated. This result confirms our assumption of the participation of dysfunctional p53 inducer/effector genes in human melanoma aetiology. Furthermore, it points towards the probable principal role of diverse alternative p53-pathway disruption mechanisms in this highly therapy-resistant malignancy without affecting p53 itself. To our knowledge, this is the first CHK1/CHK2 mutation screening in human melanoma.
Assuntos
Fator Apoptótico 1 Ativador de Proteases/genética , Genes Supressores de Tumor , Melanoma/genética , Mutação , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteína do Retinoblastoma/genética , Sequência de Bases , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Códon , Análise Mutacional de DNA , Humanos , Dados de Sequência MolecularRESUMO
Eighteen congenital melanocytic naevi (CMN) from 17 patients and 18 dysplastic melanocytic naevi (DMN) from 18 patients were screened for mutations in the BRAF oncogene (present study) and the N-ras oncogene (in the course of two foregoing studies) by single-strand conformational polymorphism (SSCP)/sequencing analysis. BRAF mutations were demonstrated in both types of lesion. As a whole, 17 of 18 CMN (94.4%) and five of 18 DMN (27.7%) harboured either BRAF or N-ras mutations. As the BRAF oncogene is frequently found to be mutated in human cutaneous melanomas, it may constitute a risk factor for melanoma formation within CMN and DMN.
Assuntos
Nevo Pigmentado/congênito , Nevo Pigmentado/genética , Proteínas Proto-Oncogênicas B-raf/genética , Neoplasias Cutâneas/congênito , Neoplasias Cutâneas/genética , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Análise Mutacional de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Genes ras/genética , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita SimplesRESUMO
Seven tumour suppressor genes (Chk1, Chk2, Apaf1, Rb1, p53, p16(INK4a) and p14(ARF)) and two oncogenes (N-ras and BRAF) were screened in nine human malignant melanoma (HMM) cell lines for point mutations or small deletions/insertions by DGGE, TGGE and SCCP analysis. For the first time in human mesothelioma, Chk1 gene mutations were detected in two of the nine investigated HMM cell lines. P53 gene mutations were found in three cell lines and p16(INK4a) mutations in 5. Mutation of the Chk1 gene implies a novel disruption mechanism of the p53 pathway in HMM, without affecting p53 itself. According to our knowledge, this is the first mutation screening of Chk1, Chk2, Apaf1 and Rb1 in human malignant mesothelioma.
Assuntos
Mesotelioma/genética , Mutação , Proteínas Oncogênicas/genética , Proteínas Supressoras de Tumor/genética , Sequência de Bases , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem , Inibidor p16 de Quinase Dependente de Ciclina/genética , Análise Mutacional de DNA , Humanos , Mesotelioma/patologia , Proteínas Quinases/genética , Homologia de Sequência do Ácido Nucleico , Proteína Supressora de Tumor p53/genéticaRESUMO
Asbestos fibers are well known environmental carcinogen, however, the underlying mechanisms of their action have still not clearly been identified. Asbestos is capable of depleting glutathione and generating reactive oxygen species (ROS), which are important mediators of damage in biological system. Asbestos-induced mutagenecity, may be mediated by the generation. It is known that a number of scavengers and antioxidants attenuate asbestos-induced ROS release. Furthermore, it is known that garlic, contains numerous sulfur compounds and glutathione precursors which act as antioxidants and also demonstrate anticarcinogenic properties. The aim of this study was to investigate whether garlic extract has any influence on asbestos-mediated genotoxicity. As an assay system, we applied the micronucleus assay, sister chromatid exchanges, and chromosomal aberrations with human peripheral blood lymphocytes, which has already been used to analyze the genotoxicity of asbestos fibers. Our results indicate that garlic extract, when administered to the lymphocytes cell culture simultaneously with chrysotile reduced the rates of micronucleus formation, sister chromatid exchanges, and chromosomal aberrations significantly. We conclude that garlic extract may be an efficient, physiologically tolerable quencher of asbestos-mediated genotoxicity.
Assuntos
Asbestos Serpentinas/toxicidade , Carcinógenos/toxicidade , Alho/química , Linfócitos/efeitos dos fármacos , Mutagênicos/toxicidade , Aberrações Cromossômicas/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Testes para Micronúcleos , Extratos Vegetais/farmacologia , Troca de Cromátide Irmã/efeitos dos fármacosRESUMO
Fourteen primary human malignant mesothelioma (HMM) samples obtained from 14 patients were screened for point mutations and microdeletions/microinsertions in exons 1-16 of the chromosome 22q-located tumour suppressor gene neurofibromin 2 (nf2) by single strand conformation polymorphism (SSCP) analysis. In one tumour (7%) a 10 basepair microdeletion of exon 10 was detected by SSCP and subsequently characterised in detail by sequencing. Deletion of the second nf2 allele in laser-microdissected regions of the 10 bp mutation-harbouring tumour was demonstrated by denaturing gradient gel electrophoresis (DGGE) analysis. Simultaneous comparative genomic hybridisation (CGH) analysis also showed losses at chromosome 22q. Our data indicate that functional loss of the NF2 protein may be involved in the formation of a subset of HMMs.
Assuntos
Cromossomos Humanos Par 22 , Genes da Neurofibromatose 2 , Mesotelioma/genética , Idoso , Sequência de Bases , Mapeamento Cromossômico , Códon/genética , Análise Mutacional de DNA/métodos , Primers do DNA , Elementos de DNA Transponíveis , Éxons , Feminino , Humanos , Masculino , Mesotelioma/classificação , Pessoa de Meia-Idade , Mutação , Hibridização de Ácido Nucleico , Mutação Puntual , Polimorfismo Conformacional de Fita Simples , Deleção de SequênciaRESUMO
The epidemiologic association between asbestos exposure and human malignant mesothelioma is well established. However, the molecular mechanisms linking asbestos exposure of humans and the subsequent mesothelioma formation is not well understood. The most frequent genetic changes found so far in human malignant mesothelioma (HMM) are deletions and point mutations in the tumor suppressor genes p16INK4a and NF2. Whereas homozygous deletions appear to be the predominant mechanism leading to p16/CDKN2A inactivation, inactivating point mutations coupled with allelic loss mainly occur at the NF2 locus. In the present study, asbestos-treated human mesothelial cells (HMC), SV40-transformed human mesothelial cells (MeT-5A) and a human mesothelioma cell line (COLO) were investigated for genetic changes of cell cycle genes (cyclin D1, p16INK4a, RB1, CDK2) using multicolor fluorescence in situ hybridization (mFISH) in interphase cells. The results show that cyclin D1 is unaffected in all investigated cells. The p16INK4a gene locus was shown to be mutated in COLO cells but not in HMC. After labeling of CDK2 and RB1, hemizygous loss of one allele of each gene was observed in asbestos-treated HMC whereas gene amplification of these genes was detectable in MeT-5A and COLO cells. Our data indicate that disarrangement of the RB1 dependent pathway seems to be involved in mesothelioma formation.
