RESUMO
Aberrant methylation of 5'CpG islands is a key epigenetic event in many human cancers. A PCR-based technique of methylated CpG island amplification followed by representational difference analysis was used to identify genes methylated in cancer. Two of the CpG islands identified mapped to the 5' untranslated region of the PAX5 alpha and beta genes. These genes, located on chromosome 9p13, are transcribed from two distinct promoters and form two alternative first exons that are subsequently spliced to the common exons 2-10. The resulting splice variants encode two distinct transcription factors important in cell differentiation and embryonic development. Examination of the methylation status of each gene using methylation-specific PCR revealed that both genes are methylated in approximately 65% of breast and lung tumors. Bisulfite sequencing revealed dense methylation patterns within each 5'CpG island, strongly correlating with transcriptional silencing. Expression in cell lines with dense methylation of either the PAX5 alpha or beta promoter region was restored after treatment with the demethylating agent 5-Aza-2'-deoxycytidine. The PAX5 beta gene encodes for the transcription factor B cell-specific activating protein that, in turn, directly regulates CD19, a gene shown to negatively control cell growth. A strong association was observed between PAX5 beta methylation and loss of expression of the CD19 gene demonstrating that inactivation of the PAX5 beta gene likely contributes to neoplastic development by inhibiting growth regulation through effects on CD19 gene expression. Recent studies have demonstrated the importance of PAX5 gene alterations in human cancer. Our results are the first to identify aberrant promoter methylation as a common mechanism for dysregulation of these genes in solid tumors.
Assuntos
Azacitidina/análogos & derivados , Neoplasias da Mama/genética , Carcinoma de Células Escamosas/genética , Proteínas de Ligação a DNA/genética , Neoplasias Pulmonares/genética , Fatores de Transcrição/genética , Azacitidina/farmacologia , Neoplasias da Mama/metabolismo , Carcinoma de Células Escamosas/metabolismo , Ilhas de CpG , Metilação de DNA , Decitabina , Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/metabolismo , Repetições de Microssatélites/genética , Fator de Transcrição PAX5 , Regiões Promotoras Genéticas , Células Tumorais CultivadasRESUMO
Recent studies from our laboratory suggest that gene-specific methylation changes in sputum could be good intermediate markers for the early detection of lung cancer and defining the efficacy of chemopreventive interventions. The purpose of our study was to determine the prevalence for aberrant promoter methylation of the p16, O(6)-methylguanine-DNA methyltransferase (MGMT), death-associated protein (DAP) kinase, and Ras effector homologue (RASSFIA) genes in nonmalignant bronchial epithelial cells from current and former smokers in a hospital-based, case control study of lung cancer. The relationship between loss of heterozygosity, at 9p and p16 methylation in bronchial epithelium and the prevalence for methylation of these four genes in sputum from cancer-free, current and former smokers were also determined. Aberrant promoter methylation of p16 was seen in at least one bronchial epithelial site from 44% of cases and controls. Methylation of the DAP kinase gene was seen in only 1 site from 5 cases and 4 controls, whereas methylation of the RASSFIA was not detected in the bronchial epithelium. Promoter methylation for p16 and DAP kinase was seen as frequently in bronchial epithelium from current smokers as from former smokers. No promoter methylation of these genes was detected in bronchial epithelium from never-smokers. Methylation of the p16 gene was detected in sputum from 23 of 66 controls. DAP kinase gene promoter methylation was also seen in sputum from 16 controls, and 8 of these subjects were positive for p16 methylation. Methylation of the MGMT gene was seen in sputum from 9 controls, whereas RASSFIA promoter methylation was only seen in 2 controls. The correlation between p16 status in the bronchial epithelium obtained from lung lobes that did not contain the primary tumor and the tumor itself was examined. Seventeen of 18 tumors (94%) showed an absolute concordance, being either methylated in the tumor and at least 1 bronchial epithelial site, or unmethylated in both tumor and bronchial epithelium. These results indicate that aberrant promoter hypermethylation of the p16 gene, and to a lesser extent, DAP kinase, occurs frequently in the bronchial epithelium of lung cancer cases and cancer-free controls and persists after smoking cessation. The strong association seen between p16 methylation in the bronchial epithelium and corresponding primary tumor substantiates that inactivation of this gene, although not transforming by itself, is likely permissive for the acquisition of additional genetic and epigenetic changes leading to lung cancer.
Assuntos
Brônquios/fisiologia , Metilação de DNA , Genes Supressores de Tumor , Neoplasias Pulmonares/genética , Regiões Promotoras Genéticas , Fumar/genética , Escarro/metabolismo , Proteínas Supressoras de Tumor , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas Reguladoras de Apoptose , Brônquios/metabolismo , Brônquios/ultraestrutura , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Estudos de Casos e Controles , Células Cultivadas , Cromossomos Humanos Par 9 , Proteínas Quinases Associadas com Morte Celular , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Células Epiteliais/ultraestrutura , Feminino , Genes p16/fisiologia , Humanos , Perda de Heterozigosidade , Neoplasias Pulmonares/etiologia , Neoplasias Pulmonares/metabolismo , Masculino , Pessoa de Meia-Idade , Proteínas de Neoplasias/genética , O(6)-Metilguanina-DNA Metiltransferase/genética , Fumar/efeitos adversos , Fumar/metabolismo , Escarro/citologiaRESUMO
Recent studies by our laboratory indicate that the p16(INK4a) gene is frequently methylated in lung tumors induced by genotoxic carcinogens and that the frequency for methylation of the estrogen receptor alpha (ER) gene varies as a function of carcinogenic exposure. The purpose of the current investigation was to define the role of these two genes in lung tumors induced by the particulate carcinogens carbon black (CB), diesel exhaust (DE) or beryllium metal. Methylation of p16 was observed in 59 and 46% of DE and CB tumors, respectively. In contrast, the ER gene was inactivated in only 15% of DE or CB tumors. Methylation of the p16 and ER genes was very common (80 and 50%, respectively) in beryllium-induced lung tumors; both genes were methylated in 40% of the tumors. Bisulfite sequencing revealed dense methylation throughout exon 1 of the ER gene. The inhibitory effect of methylation on gene transcription was confirmed through RT-PCR expression studies in which p16 gene expression was 30-60-fold lower in methylated than unmethylated tumors. Residual expression in methylated tumors was consistent with contamination by stromal and inflammatory cells. Results indicate that tumors induced by these particulate carcinogens arise, in part, through inactivation of the p16 and ER genes. Furthermore, the inactivation of the p16 gene by these carcinogenic exposures supports a possible role for oxidative stress and inflammation in the etiology of human lung cancer.
Assuntos
Ilhas de CpG , Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilação de DNA , Neoplasias Pulmonares/induzido quimicamente , Neoplasias Pulmonares/genética , Receptores de Estrogênio/genética , Animais , Berílio , Carbono , Carcinógenos , Relação Dose-Resposta a Droga , Receptor alfa de Estrogênio , Éxons , Feminino , Humanos , Masculino , Ratos , Ratos Endogâmicos F344 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Emissões de VeículosRESUMO
The retinoic acid receptor beta (RAR-beta) gene encodes one of the primary receptors for retinoic acid, an important signaling molecule in lung growth, differentiation and carcinogenesis. RAR-beta has been shown to be down-regulated by methylation in human lung cancer. We have used previously lung tumors induced in mice to evaluate the timing and effect of specific carcinogen exposures on targeting genes altered in human lung cancer. These studies were extended to characterize the role of methylation of the RAR-beta gene in murine lung cancers. After treatment with the demethylating agent 5-aza-2'-deoxycytidine (DAC), RAR-beta was re-expressed in silenced cell lines or expressed at a higher rate than without DAC, supporting methylation as the inactivating mechanism. Bisulfite sequencing detected dense methylation in the area of the CpG island that contained the 5' untranslated region and the first translated exon in non-expressing cell lines, compared with minimal and heterogeneous methylation in normal mouse lung. Methylation-specific PCR revealed that this gene is targeted differentially by carcinogen exposures with the detection of methylated alleles in virtually all primary tumors associated with cigarette smoke or 4-methylnitrosamino-1-(3-pyridyl)-butanone (NNK) in contrast to half of tumors induced by methylene chloride or vinyl carbamate. RAR-beta methylation was also detected in 54% of preneoplastic hyperplasias induced by treatment with NNK. Bisulfite sequencing of both premalignant and malignant lesions detected dense methylation in the same area observed in cell lines, substantiating that this gene is functionally inactivated at the earliest histologic stage of adenocarcinoma development. These studies demonstrate that aberrant methylation of RAR-beta is an early and common alteration in murine lung tumors induced by several environmentally relevant exposures.