RESUMO
Restriction landmark genomic scanning applied to a broad variety of cancer types can disclose tumour-specific and tumour-type-specific global methylation profiles. This and other genome-scanning approaches allows the rapid analysis of methylation profiles of thousands of genes in parallel-and promises to identify new genes critical to carcinogenesis and other biological processes.
Assuntos
Metilação de DNA , Genes Supressores de Tumor , Mutação , Neoplasias/genética , Mutação em Linhagem Germinativa , Humanos , Mapeamento por RestriçãoRESUMO
Densely methylated DNA associates with transcriptionally repressive chromatin characterized by the presence of underacetylated histones. Recently, these two epigenetic processes have been dynamically linked. The methyl-CpG-binding protein MeCP2 appears to reside in a complex with histone deacetylase activity. MeCP2 can mediate formation of transcriptionally repressive chromatin on methylated promoter templates in vitro, and this process can be reversed by trichostatin A (TSA), a specific inhibitor of histone deacetylase. Little is known, however, about the relative roles of methylation and histone deacetylase activity in the stable inhibition of transcription on densely methylated endogenous promoters, such as those for silenced alleles of imprinted genes, genes on the female inactive X chromosome and tumour-suppressor genes inactivated in cancer cells. We show here that the hypermethylated genes MLH1, TIMP3 (TIMP3), CDKN2B (INK4B, p15) and CDKN2A (INK4, p16) cannot be transcriptionally reactivated with TSA alone in tumour cells in which we have shown that TSA alone can upregulate the expression of non-methylated genes. Following minimal demethylation and slight gene reactivation in the presence of low dose 5-aza-2'deoxycytidine (5Aza-dC), however, TSA treatment results in robust re-expression of each gene. TSA does not contribute to demethylation of the genes, and none of the treatments alter the chromatin structure associated with the hypermethylated promoters. Thus, although DNA methylation and histone deacetylation appear to act as synergistic layers for the silencing of genes in cancer, dense CpG island methylation is dominant for the stable maintenance of a silent state at these loci.
Assuntos
Proteínas de Ciclo Celular , Metilação de DNA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Proteínas Supressoras de Tumor , Proteínas Adaptadoras de Transdução de Sinal , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Proteínas de Transporte/genética , Ilhas de CpG , Inibidor de Quinase Dependente de Ciclina p15 , Inibidor p16 de Quinase Dependente de Ciclina/genética , Metilases de Modificação do DNA/antagonistas & inibidores , Decitabina , Inibidores Enzimáticos/farmacologia , Humanos , Proteína 1 Homóloga a MutL , Proteínas de Neoplasias/genética , Neoplasias/genética , Proteínas Nucleares , Regiões Promotoras Genéticas , Inibidor Tecidual de Metaloproteinase-3/genética , Ativação Transcricional/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Hepatocellular carcinoma (HCC) is a major cause of cancer death, but the molecular mechanism for its development beyond its initiation has not been well characterized. Suppressor of cytokine signaling (SOCS-1; also known as JAB and SSI-1) switches cytokine signaling 'off' by means of its direct interaction with Janus kinase (JAK). We identified aberrant methylation in the CpG island of SOCS-1 that correlated with its transcription silencing in HCC cell lines. The incidence of aberrant methylation was 65% in the 26 human primary HCC tumor samples analyzed. Moreover, the restoration of SOCS-1 suppressed both growth rate and anchorage-independent growth of cells in which SOCS-1 was methylation-silenced and JAK2 was constitutively activated. This growth suppression was caused by apoptosis and was reproduced by AG490, a specific, chemical JAK2 inhibitor that reversed constitutive phosphorylation of STAT3 in SOCS-1 inactivated cells. The high prevalence of the aberrant SOCS-1 methylation and its growth suppression activity demonstrated the importance of the constitutive activation of the JAK/STAT pathway in the development of HCC. Our results also indicate therapeutic strategies for the treatment of HCC including use of SOCS-1 in gene therapy and inhibition of JAK2 by small molecules, such as AG490.
Assuntos
Carcinoma Hepatocelular/genética , Proteínas de Transporte/genética , Metilação de DNA , Inativação Gênica , Genes Supressores de Tumor , Peptídeos e Proteínas de Sinalização Intracelular , Neoplasias Hepáticas/genética , Proteínas Proto-Oncogênicas , Proteínas Repressoras , Antineoplásicos/uso terapêutico , Carcinoma Hepatocelular/terapia , Proteínas de Transporte/metabolismo , Ilhas de CpG , Proteínas de Ligação a DNA/metabolismo , Terapia Genética , Humanos , Janus Quinase 2 , Neoplasias Hepáticas/terapia , Dados de Sequência Molecular , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/metabolismo , Fator de Transcrição STAT3 , Transdução de Sinais , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina , Transativadores/metabolismo , Tirfostinas/uso terapêuticoRESUMO
Loss of heterozygosity on chromosome 9p21 is one of the most frequent genetic alterations identified in human cancer. The rate of point mutations of p16, a candidate suppressor gene of this area, is low in most primary tumours with allelic loss of 9p21. Monosomic cell lines with structurally unaltered p16 show methylation of the 5' CpG island of p16. This distinct methylation pattern was associated with a complete transcriptional block that was reversible upon treatment with 5-deoxyazacytidine. Moreover, de novo methylation of the 5' CpG island of p16 was also found in approximately 20% of different primary neoplasms, but not in normal tissues, potentially representing a common pathway of tumour suppressor gene inactivation in human cancers.
Assuntos
Proteínas de Transporte/genética , Ilhas de CpG , Citosina/análogos & derivados , DNA de Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Proteínas de Neoplasias/genética , 5-Metilcitosina , Alelos , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Sequência de Bases , Neoplasias Encefálicas/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma de Células Escamosas/genética , Proteínas de Transporte/biossíntese , Proteínas de Transporte/fisiologia , Cromossomos Humanos Par 9 , Inibidor p16 de Quinase Dependente de Ciclina , Citosina/fisiologia , Decitabina , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioma/genética , Neoplasias de Cabeça e Pescoço/genética , Humanos , Neoplasias Pulmonares/genética , Metilação , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/fisiologia , Neoplasias/genética , Neoplasias/patologia , Deleção de Sequência , Transcrição Gênica/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
BACKGROUND: To study how caretaker gene silencing relates to gatekeeper mutations in colorectal cancer (CRC), we investigated whether O6-methylguanine DNA methyltransferase (MGMT) and Human Mut-L Homologue 1 (MLH1) promoter hypermethylation are associated with APC, KRAS and BRAF mutations among 734 CRC patients. METHODS: We compared MGMT hypermethylation with G:C > A:T mutations in APC and KRAS and with the occurrence of such mutations in CpG or non-CpG dinucleotides in APC. We also compared MLH1 hypermethylation with truncating APC mutations and activating KRAS and BRAF mutations. RESULTS: Only 10% of the tumors showed both MGMT and MLH1 hypermethylation. MGMT hypermethylation occurred more frequently in tumors with G:C > A:T KRAS mutations (55%) compared with those without these mutations (38%, P < 0.001). No such difference was observed for G:C > A:T mutations in APC, regardless of whether mutations occurred in CpG or non-CpG dinucleotides. MLH1 hypermethylation was less common in tumors with APC mutations (P = 0.006) or KRAS mutations (P = 0.001), but was positively associated with BRAF mutations (P < 0.001). CONCLUSIONS: MGMT hypermethylation is associated with G:C > A:T mutations in KRAS, but not in APC, suggesting that MGMT hypermethylation may succeed APC mutations but precedes KRAS mutations in colorectal carcinogenesis. MLH1-hypermethylated tumors harbor fewer APC and KRAS mutations and more BRAF mutations, suggesting that they develop distinctly from an MGMT methylator pathway.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Neoplasias Colorretais/genética , Metilação de DNA/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Proteínas Nucleares/genética , Mutação Puntual , Proteínas Supressoras de Tumor/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Idoso , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Ilhas de CpG/genética , Metilases de Modificação do DNA/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Feminino , Inativação Gênica , Genes APC , Genes ras/genética , Humanos , Masculino , Pessoa de Meia-Idade , Proteína 1 Homóloga a MutL , Países Baixos , Proteínas Nucleares/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Estudos Prospectivos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas p21(ras) , Proteínas Supressoras de Tumor/metabolismo , Proteínas ras/genéticaRESUMO
Methylation-specific polymerase chain reaction (PCR) (MSP) is frequently used to study gene silencing by promoter hypermethylation. However, non-specific primer design can lead to false-positive detection of methylation. We present a novel, web-based algorithm for the design of primers for bisulfite-PCRs (MSP, sequencing, COBRA and multiplex-MSP), allowing the determination of a specificity score, which is based on the thermodynamic characteristics of the primer 3'-end. PCR amplification with primers not reaching a high specificity score can result in false-positive findings. We used MSPprimer to design MSP primers for analysis of the ATM promoter. In 37 non-small cell lung cancer (NSCLC) samples and 43 breast cancer samples no promoter methylation was detected. Conversely, published MSP primers not reaching the required specificity score led to non-specific amplification of DNA not converted by bisulfite. The result was a false-positive incidence of ATM promoter methylation of 24% in NSCLC and 48% in breast cancers, similar to published studies. This highlights the critical need for specific primer design for MSP. MSPprimer is a convenient tool to achieve this goal, which is available free of charge to the scientific community.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Biologia Computacional , Metilação de DNA , Primers do DNA/síntese química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Software , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Algoritmos , Proteínas Mutadas de Ataxia Telangiectasia , Sequência de Bases , Linhagem Celular Tumoral , Células HCT116 , Humanos , Dados de Sequência MolecularRESUMO
The Wnt signalling pathway has a key function in stem cell maintenance and differentiation of haematopoietic progenitors. Secreted Frizzled-related protein genes (SFRPs), functioning as Wnt signalling antagonists, have been found to be downregulated by promoter hypermethylation in many tumours. To analyse epigenetic dysregulation of SFRPs in acute myeloid leukaemia (AML), we examined the promoter methylation status of SFRP1, -2, -4 and -5 in AML cell lines by methylation-specific polymerase chain reaction (MSP). Aberrant CpG island methylation was found for all four SFRP genes. By real-time reverse transcription-PCR, corresponding transcriptional silencing for SFRP1 and -2 was demonstrated and treatment of cell lines with 5-aza-2'-deoxycytidine resulted in re-expression. The methylation status of the SFRP genes was analysed in 100 specimens obtained from AML patients at diagnosis. The frequencies of aberrant methylation among the patient samples were 29% for SFRP1, 19% for SFRP2, 0% for SFRP4 and 9% for SFRP5. For SFRP2, a correlation between promoter hypermethylation and transcriptional downregulation was found in primary AML samples. Among AML cases with a favourable karyotype, hypermethylation of SFRP genes was restricted to patients with core binding factor (CBF) leukaemia, and aberrant methylation of the SFRP2 promoter was an adverse risk factor for survival in CBF leukaemia.
Assuntos
Epigênese Genética , Leucemia Mieloide Aguda/genética , Proteínas Wnt/genética , Proteínas Adaptadoras de Transdução de Sinal , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Ilhas de CpG , Proteínas do Olho/biossíntese , Proteínas do Olho/genética , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Leucemia Mieloide Aguda/patologia , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Metilação , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Transdução de SinaisRESUMO
An acquired autoactivating mutation with a V617F amino-acid substitution in the JAK2 tyrosine kinase is frequently found in BCR/ABL-negative myeloproliferative disorders (MPD). Hypermethylation of CpG islands within gene promoter regions is associated with transcriptional inactivation and represents an important mechanism of gene silencing in the pathogenesis of hematopoietic malignancies. In this study, we determined the DNA methylation status of 13 cancer-related genes in the context of JAK2 mutations in 39 patients with MPD. Genes analyzed for hypermethylation were SOCS-1, SHP-1, E-cadherin, MGMT, TIMP-2, TIMP-3, p15, p16, p73, DAPK1, RASSF1A, RARbeta2 and hMLH1. We found at least one hypermethylated gene in 15/39 MPD patient specimens, and in 6/39 samples aberrant methylation of the negative cytokine regulator SOCS-1 was present. The JAK2V617F mutation was found in 21/39 patients as determined by allele-specific polymerase chain reaction. Hypermethylation of SOCS-1 was observed in 3/21 patients with an autoactivating JAK2 mutation and in 3/18 patients with wild-type JAK2. Our results suggest that epigenetic inactivation of SOCS-1 may be a complementary mechanism to the JAK2V617F mutation in the pathogenesis of MPD that leads to dysregulation of JAK-STAT signal transduction and thus contributes to growth factor hypersensitivity.
Assuntos
Ilhas de CpG , Metilação de DNA , Epigênese Genética , Inativação Gênica , Janus Quinase 2/genética , Transtornos Mieloproliferativos/genética , Proteínas Supressoras da Sinalização de Citocina/genética , Substituição de Aminoácidos , Análise Mutacional de DNA , Progressão da Doença , Feminino , Proteínas de Fusão bcr-abl , Regulação da Expressão Gênica , Genes Neoplásicos , Humanos , Janus Quinase 2/fisiologia , Leucemia Mieloide/genética , Masculino , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos/patologia , Mutação Puntual , Receptores do Ácido Retinoico/genética , Transdução de Sinais/genética , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/fisiologiaAssuntos
Síndrome de Angelman/genética , Metilação de DNA , Impressão Genômica , Reação em Cadeia da Polimerase/métodos , Síndrome de Prader-Willi/genética , Ribonucleoproteínas Nucleares Pequenas , Líquido Amniótico/fisiologia , Síndrome de Angelman/diagnóstico , Autoantígenos/genética , Autoantígenos/metabolismo , Southern Blotting , Vilosidades Coriônicas/fisiologia , Primers do DNA , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Síndrome de Prader-Willi/diagnóstico , Estudos Prospectivos , Proteínas Centrais de snRNPRESUMO
Although cyclin D1 is overexpressed in a significant number of human cancers, overexpression alone is insufficient to promote tumorigenesis. In vitro studies have revealed that inhibition of cyclin D1 nuclear export unmasks its neoplastic potential. Cyclin D1 nuclear export depends upon phosphorylation of a C-terminal residue, threonine 286, (Thr-286) which in turn promotes association with the nuclear exportin, CRM1. Mutation of Thr-286 to a non-phosphorylatable residue results in a constitutively nuclear cyclin D1 protein with significantly increased oncogenic potential. To determine whether cyclin D1 is subject to mutations that inhibit its nuclear export in human cancer, we have sequenced exon 5 of cyclin D1 in primary esophageal carcinoma samples and in cell lines derived from esophageal cancer. Our work reveals that cyclin D1 is subject to mutations in primary human cancer. The mutations identified specifically disrupt phosphorylation of cyclin D1 at Thr-286, thereby enforcing nuclear accumulation of cyclin D1. Through characterization of these mutants, we also define an acidic residue within the C-terminus of cyclin D1 that is necessary for recognition and phosphorylation of cyclin D1 by glycogen synthase kinase-3 beta. Finally, through construction of compound mutants, we demonstrate that cell transformation by the cancer-derived cyclin D1 alleles correlates with their ability to associate with and activate CDK4. Our data reveal that cyclin D1 is subject to mutations in primary human cancer that specifically disrupt phosphorylation-dependent nuclear export of cyclin D1 and suggest that such mutations contribute to the genesis and progression of neoplastic growth.
Assuntos
Carcinoma/metabolismo , Núcleo Celular/metabolismo , Ciclina D1/genética , Ciclinas/genética , Neoplasias Esofágicas/metabolismo , Proteínas de Neoplasias/genética , Processamento de Proteína Pós-Traducional , Alelos , Substituição de Aminoácidos , Animais , Carcinoma/genética , Carcinoma/patologia , Linhagem Celular , Linhagem Celular Tumoral/química , Transformação Celular Neoplásica/genética , Ciclina D , Ciclina D1/metabolismo , Quinase 4 Dependente de Ciclina/metabolismo , Ciclinas/metabolismo , Análise Mutacional de DNA , DNA de Neoplasias/genética , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patologia , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Camundongos , Mutação de Sentido Incorreto , Células NIH 3T3 , Proteínas de Neoplasias/metabolismo , Fosforilação , Fosfotreonina/metabolismo , Mutação Puntual , Transporte Proteico/genética , Proteínas Recombinantes de Fusão/metabolismo , Deleção de Sequência , SpodopteraRESUMO
Recently, the concept that epigenetic, as well as genetic, events might be central to the evolution of human cancer is re-emerging. Cancers often exhibit an aberrant methylation of gene promoter regions that is associated with loss of gene function. This DNA change constitutes a heritable state, not mediated by altered nucleotide sequence, that appears to be tightly linked to the formation of transcriptionally repressive chromatin. This epigenetic process acts as an alternative to mutations to disrupt tumor-suppressor gene function and can predispose to genetic alterations through inactivating DNA-repair genes. Dissecting the molecular processes that mediate these methylation changes will enhance our understanding of chromatin modeling and gene regulation and might present novel possibilities for cancer therapy. Methylation changes constitute potentially sensitive molecular markers to define risk states, monitor prevention strategies, achieve early diagnosis, and track the prognosis of cancer.
Assuntos
Metilação de DNA , DNA de Neoplasias , Neoplasias/genética , Humanos , Neoplasias/fisiopatologiaRESUMO
The p16INK4a (p16) tumor suppressor gene is frequently inactivated by homozygous deletion or methylation of the 5' CpG island in cell lines derived from human non-small-cell lung cancers. However, the frequency of dysfunction in primary tumors appears to be significantly lower than that in cell lines. This discordance could result from the occurrence or selection of p16 dysfunction during cell culture. Alternatively, techniques commonly used to examine tumors for genetic and epigenetic alterations may not be sensitive enough to detect all dysfunctions within the heterogeneous cell population present in primary tumors. If p16 inactivation plays a central role in development of non-small-cell lung cancer, then the frequency of gene inactivation in primary tumors should parallel that observed in cell lines. The present investigation addressed this issue in primary rat lung tumors and corresponding derived cell lines. A further goal was to determine whether the aberrant p16 gene methylation seen in human tumors is a conserved event in this animal model. The rat p16 gene was cloned and sequenced, and the predicted amino acid sequence of its product found to be 62% homologous to the amino acid sequence of the human analog. Homozygous deletion accounted for loss of p16 expression in 8 of 20 cell lines, while methylation of the CpG island extending throughout exon 1 was observed in 9 of 20 cell lines. 2-Deoxy-5-azacytidine treatment of cell lines with aberrant methylation restored gene expression. The methylated phenotype seen in cell lines showed an absolute correlation with detection of methylation in primary tumors. Aberrant methylation was also detected in four of eight primary tumors in which the derived cell line contained a deletion in p16. These results substantiate the primary tumor as the origin for dysfunction of the p16 gene and implicate CpG island methylation as the major mechanism for inactivating this gene in the rat lung tumors examined. Furthermore, rat lung cancer appears to be an excellent model in which to investigate the mechanisms of de novo gene methylation and the role of p16 dysfunction in the progression of neoplasia.
Assuntos
Proteínas de Transporte/genética , Metilação de DNA , Genes Supressores de Tumor/genética , Neoplasias Pulmonares/genética , Sequência de Aminoácidos , Animais , Azacitidina/análogos & derivados , Azacitidina/farmacologia , Sequência de Bases , Clonagem Molecular , Ilhas de CpG/genética , Inibidor p16 de Quinase Dependente de Ciclina , Decitabina , Modelos Animais de Doenças , Éxons , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Dados de Sequência Molecular , Proteínas/genética , Ratos , Ratos Endogâmicos F344 , Análise de Sequência de DNA , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Células Tumorais Cultivadas , Proteína Supressora de Tumor p14ARFRESUMO
BACKGROUND: Inherited mutations in the BRCA1 gene may be responsible for almost half of inherited breast carcinomas. However, somatic (acquired) mutations in BRCA1 have not been reported, despite frequent loss of heterozygosity (LOH or loss of one copy of the gene) at the BRCA1 locus and loss of BRCA1 protein in tumors. To address whether BRCA1 may be inactivated by pathways other than mutations in sporadic tumors, we analyzed the role of hypermethylation of the gene's promoter region. METHODS: Methylation patterns in the BRCA1 promoter were assessed in breast cancer cell lines, xenografts, and 215 primary breast and ovarian carcinomas by methylation-specific polymerase chain reaction (PCR). BRCA1 RNA expression was determined in cell lines and seven xenografts by reverse transcription-PCR. P values are two-sided. RESULTS: The BRCA1 promoter was found to be unmethylated in all normal tissues and cancer cell lines tested. However, BRCA1 promoter hypermethylation was present in two breast cancer xenografts, both of which had loss of the BRCA1 transcript. BRCA1 promoter hypermethylation was present in 11 (13%) of 84 unselected primary breast carcinomas. BRCA1 methylation was strikingly associated with the medullary (67% methylated; P =.0002 versus ductal) and mucinous (55% methylated; P =.0033 versus ductal) subtypes, which are overrepresented in BRCA1 families. In a second series of 66 ductal breast tumors informative for LOH, nine (20%) of 45 tumors with LOH had BRCA1 hypermethylation, while one (5%) of 21 without LOH was methylated (P =.15). In ovarian neoplasms, BRCA1 methylation was found only in tumors with LOH, four (31%) of 13 versus none of 18 without LOH (P =.02). The BRCA1 promoter was unmethylated in other tumor types. CONCLUSION: Silencing of the BRCA1 gene by promoter hypermethylation occurs in primary breast and ovarian carcinomas, especially in the presence of LOH and in specific histopathologic subgroups. These findings support a role for this tumor suppressor gene in sporadic breast and ovarian tumorigenesis.
Assuntos
Neoplasias da Mama/metabolismo , Genes BRCA1/genética , Perda de Heterozigosidade , Neoplasias Ovarianas/metabolismo , Regiões Promotoras Genéticas/genética , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metilação , Neoplasias Ovarianas/genética , RNA , RNA Neoplásico/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
BACKGROUND: Conventional cytologic analysis of sputum is an insensitive test for the diagnosis of non-small-cell lung cancer (NSCLC). We have recently demonstrated that polymerase chain reaction (PCR)-based molecular methods are more sensitive than cytologic analysis in diagnosing bladder cancer. In this study, we examined whether molecular assays could identify cancer cells in bronchoalveolar lavage (BAL) fluid. METHODS: Tumor-specific oncogene mutations, CpG-island methylation status, and microsatellite alterations in the DNA of cells in BAL fluid from 50 consecutive patients with resectable (stages I through IIIa) NSCLC were assessed by use of four PCR-based techniques. RESULTS: Of 50 tumors, 28 contained a p53 mutation, and the identical mutation was detected with a plaque hybridization assay in the BAL fluid of 39% (11 of 28) of the corresponding patients. Eight of 19 adenocarcinomas contained a K-ras mutation, and the identical mutation was detected with a mutation ligation assay in the BAL fluid of 50% (four of eight) of the corresponding patients. The p16 gene was methylated in 19 of 50 tumors, and methylated p16 alleles were detected in the BAL fluid of 63% (12 of 19) of the corresponding patients. Microsatellite instability in at least one marker was detected with a panel of 15 markers frequently altered in NSCLC in 23 of 50 tumors; the identical alteration was detected in the BAL fluid of 14% (three of 22) of the corresponding patients. When all four techniques were used, mutations or microsatellite instability was detected in the paired BAL fluid of 23 (53%) of the 43 patients with tumors carrying a genetic alteration. CONCLUSION: Although still limited by sensitivity, molecular diagnostic strategies can detect the presence of neoplastic cells in the proximal airway of patients with surgically resectable NSCLC.
Assuntos
Líquido da Lavagem Broncoalveolar , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/genética , Ilhas de CpG , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Repetições de Microssatélites/genética , Mutação , Oncogenes/genética , Alelos , Líquido da Lavagem Broncoalveolar/citologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Primers do DNA , Genes ras/genética , Humanos , Hibridização In Situ , Neoplasias Pulmonares/patologia , Metilação , Estadiamento de Neoplasias , Reação em Cadeia da Polimerase/métodos , Estudos Prospectivos , Sensibilidade e Especificidade , Proteína Supressora de Tumor p53/genéticaRESUMO
Inactivation of p16ink4A and other tumor suppressor genes has been associated with promoter region hypermethylation in neoplasia. However, direct proof for aberrant DNA methylation as an independent event for loss of gene function has been difficult to obtain. We addressed this question in the colon carcinoma cell line HCT116, which contains one allele of p16ink4A with a coding region frameshift mutation and one wild-type allele. Neither allele contains a mutation in the proximal promoter region. The promoter of the wild-type allele, but not the mutant allele, is hypermethylated, and only the mutant allele is expressed. Transcription from the methylated/wild-type allele was restored after cell treatment with the demethylating agent 5-aza-2'-deoxycytidine. Thus, in neoplastic cells, stable allele-specific loss of transcription may arise from aberrant methylation of a nonmutated promoter region, identifying hypermethylation as a direct mechanism for tumor suppressor gene inactivation.
Assuntos
Neoplasias do Colo/genética , Quinases Ciclina-Dependentes/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Genes p16 , Regiões Promotoras Genéticas , Alelos , Sequência de Bases , Ilhas de CpG , Mutação da Fase de Leitura , Humanos , Células Tumorais CultivadasRESUMO
We are in an era where the potential exists for deriving comprehensive profiles of DNA alterations characterizing each form of human cancer. Such profiles would provide invaluable insight into mechanisms underlying the evolution of each tumor type and will provide molecular markers, which could radically improve cancer detection. To date, no one type of DNA change has been defined which accomplishes this purpose. Herein, by using a candidate gene approach, we show that one category of DNA alteration, aberrant methylation of gene promoter regions, can enormously contribute to the above goals. We have now analyzed a series of promoter hypermethylation changes in 12 genes (p16(INK4a), p15(INK4b), p14(ARF), p73, APC,(5) BRCA1, hMLH1, GSTP1, MGMT, CDH1, TIMP3, and DAPK), each rigorously characterized for association with abnormal gene silencing in cancer, in DNA from over 600 primary tumor samples representing 15 major tumor types. The genes play known important roles in processes encompassing tumor suppression, cell cycle regulation, apoptosis, DNA repair, and metastastic potential. A unique profile of promoter hypermethylation exists for each human cancer in which some gene changes are shared and others are cancer-type specific. The hypermethylation of the genes occurs independently to the extent that a panel of three to four markers defines an abnormality in 70-90% of each cancer type. Our results provide an unusual view of the pervasiveness of DNA alterations, in this case an epigenetic change, in human cancer and a powerful set of markers to outline the disruption of critical pathways in tumorigenesis and for derivation of sensitive molecular detection strategies for virtually every human tumor type.
Assuntos
Metilação de DNA , Neoplasias/genética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes Supressores de Tumor , Marcadores Genéticos , Humanos , Regiões Promotoras GenéticasRESUMO
The recently identified cyclin-dependent kinase inhibitor p15INK4B is localized to a region on chromosome 9p21 frequently deleted in human tumors. Previous evidence has pointed to a related gene, p16INK4A, as the principal target of this deletion. We report that in gliomas and, to a striking degree, in leukemias, the p15 gene is commonly inactivated in association with promoter region hypermethylation involving multiple sites in a 5'-CpG island. In some gliomas and all of the primary leukemias, this event occurs without alteration of the adjacent gene, p16INK4A. In other tumors, including lung, head and neck, breast, prostate, and colon cancer, inactivation of p15INK4B occurs only rarely and only with concomitant inactivation of p16. Aberrant methylation of p15INK4B is associated with transcriptional loss of this gene. Treatment with the demethylating agent 5-aza-2'-deoxycytidine leads to re-expression of p15 mRNA. In selected leukemia cell lines, p15 inactivation correlates with known resistance to the growth-suppressive effects of transforming growth factor-beta. These results suggest that p15INK4B is inactivated selectively in leukemias and gliomas and seems to constitute an important tumor suppressor gene loss in these neoplasms.
Assuntos
Proteínas de Transporte/genética , Proteínas de Ciclo Celular , Deleção Cromossômica , Cromossomos Humanos Par 9 , Inibidor p16 de Quinase Dependente de Ciclina , Genes Supressores de Tumor , Glioma/genética , Leucemia/genética , Proteínas Supressoras de Tumor , Adulto , Neoplasias da Mama/genética , Linhagem Celular , Criança , Mapeamento Cromossômico , Neoplasias do Colo/genética , Inibidor de Quinase Dependente de Ciclina p15 , DNA/sangue , DNA/química , DNA de Neoplasias/química , DNA de Neoplasias/isolamento & purificação , Fosfatos de Dinucleosídeos/análise , Feminino , Homozigoto , Humanos , Leucemia Mieloide Aguda/genética , Neoplasias Pulmonares/genética , Linfócitos , Masculino , Metilação , Reação em Cadeia da Polimerase , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Neoplasias da Próstata/genética , Valores de Referência , Mapeamento por Restrição , Células Tumorais CultivadasRESUMO
Formation of transcriptional repression complexes such as DNA methyltransferase (DNMT) 1/histone deacetylase (HDAC) or methyl-CpG binding protein/HDAC is emerging as an important mechanism in silencing a variety of methylated tissue-specific and imprinted genes. Our previous studies showed that treatment of estrogen receptor (ER)-alpha-negative human breast cancer cells with the DNMT inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) led to ER mRNA and protein re-expression. Also, the HDAC inhibitor trichostatin A (TSA) could induce ER transcript about 5-fold. Here we show that 5-aza-dC alone induced ER transcript about 30-40-fold, and the addition of TSA elevated ER mRNA expression about 10-fold more in the human ER-negative breast cancer cell lines MDA-MB-231 and MDA-MB-435. Overall, the combination of 5-aza-dC and TSA induced a 300-400-fold increase in ER transcript. Restoration of estrogen responsiveness was demonstrated by the ability of the induced ER protein to elicit estrogen response element-regulated reporter activity from an exogenous plasmid as well as induce expression of the ER target gene, progesterone receptor. The synergistic activation of ER occurs concomitantly with markedly reduced soluble DNMT1 expression and activity, partial demethylation of the ER CpG island, and increased acetylation of histones H(3) and H(4). These data suggest that the activities of both DNMT1 and HDAC are key regulators of methylation-mediated ER gene silencing.
Assuntos
Azacitidina/farmacologia , Neoplasias da Mama/enzimologia , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Receptores de Estrogênio/fisiologia , Acetilação/efeitos dos fármacos , Azacitidina/análogos & derivados , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferase 1 , Metilação de DNA/efeitos dos fármacos , Decitabina , Sinergismo Farmacológico , Estradiol/análogos & derivados , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Receptor alfa de Estrogênio , Fulvestranto , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histonas/metabolismo , Humanos , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Receptores de Estrogênio/biossíntese , Receptores de Estrogênio/genética , Células Tumorais CultivadasRESUMO
The INK4a/ARF locus encodes two cell cycle-regulatory proteins, p16INK4a andp14ARF, which share an exon using different reading frames. p14ARF antagonizes MDM2-dependent p53 degradation. However, no point mutations in p14ARF not altering p16INK4a have been described in primary tumors. We report that p14ARF is epigenetically inactivated in several colorectal cell lines, and its expression is restored by treatment with demethylating agents. In primary colorectal carcinomas, p14ARF promoter hypermethylation was found in 31 of 110 (28%) of the tumors and observed in 13 of 41 (32%) colorectal adenomas but was not present in any normal tissues. p14ARF methylation appears in the context of an adjacent unmethylated p16INK4a promoter in 16 of 31 (52%) of the carcinomas methylated at p14ARF. Although p14ARF hypermethylation was slightly overrepresented in tumors with wild-type p53 compared to tumors harboring p53 mutations [19 of 55 (34%) versus 12 of 55 (22%)], this difference did not reach statistical significance. p14ARF aberrant methylation was not related to the presence of K-ras mutations. Our results demonstrate that p14ARF promoter hypermethylation is frequent in colorectal cancer and occurs independently of the p16INK4a methylation status and only marginally in relation to the p53 mutational status.
Assuntos
Proteínas de Transporte/genética , Neoplasias Colorretais/genética , Inativação Gênica , Genes p53/genética , Proteínas/genética , Proteínas de Transporte/metabolismo , Neoplasias Colorretais/metabolismo , Ilhas de CpG , Inibidor p16 de Quinase Dependente de Ciclina , Metilação de DNA , Genes ras/genética , Células HL-60 , Humanos , Polimorfismo Conformacional de Fita Simples , Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas , Proteína Supressora de Tumor p14ARFRESUMO
The DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) removes alkyl adducts from the O6 position of guanine. MGMT expression is decreased in some tumor tissues, and lack of activity has been observed in some cell lines. Loss of expression is rarely due to deletion, mutation, or rearrangement of the MGMT gene, but methylation of discrete regions of the CpG island of MGMT has been associated with the silencing of the gene in cell lines. We used methylation-specific PCR to study the promoter methylation of the MGMT gene. All normal tissues and expressing cancer cell lines were unmethylated, whereas nonexpressing cancer cell lines were methylated. Among the more than 500 primary human tumors examined, MGMT hypermethylation was present in a subset of specific types of cancer. In gliomas and colorectal carcinomas, aberrant methylation was detected in 40% of the tumors, whereas in non-small cell lung carcinomas, lymphomas, and head and neck carcinomas, this alteration was found in 25% of the tumors. MGMT methylation was found rarely or not at all in other tumor types. We also analyzed MGMT expression by immunohistochemistry in relation to the methylation status in 31 primary tumors. The presence of aberrant hypermethylation was associated with loss of MGMT protein, in contrast to retention of protein in the majority of tumors without aberrant hypermethylation. Our results suggest that epigenetic inactivation of MGMT plays an important role in primary human neoplasia.