RESUMO
BACKGROUND: Genetic aberrations in DNA repair genes are linked to cancer, but less is reported about epigenetic regulation of DNA repair and functional consequences. We investigated the intragenic methylation loss at the three prime repair exonuclease 2 (TREX2) locus in laryngeal (n = 256) and colorectal cancer cases (n = 95) and in pan-cancer data from The Cancer Genome Atlas (TCGA). RESULTS: Significant methylation loss at an intragenic site of TREX2 was a frequent trait in both patient cohorts (p = 0.016 and < 0.001, respectively) and in 15 out of 22 TCGA studies. Methylation loss correlated with immunohistochemically staining for TREX2 (p < 0.0001) in laryngeal tumors and improved overall survival of laryngeal cancer patients (p = 0.045). Chromatin immunoprecipitation, demethylation experiments, and reporter gene assays revealed that the region of methylation loss can function as a CCAAT/enhancer binding protein alpha (CEBPA)-responsive enhancer element regulating TREX2 expression. CONCLUSIONS: The data highlight a regulatory role of TREX2 DNA methylation for gene expression which might affect incidence and survival of laryngeal cancer. Altered TREX2 protein levels in tumors may affect drug-induced DNA damage repair and provide new tailored therapies.
Assuntos
Metilação de DNA , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Neoplasias Laríngeas/mortalidade , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regulação para Cima , Idoso , Linhagem Celular Tumoral , Reparo do DNA , Epigênese Genética , Exodesoxirribonucleases/química , Feminino , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Neoplasias Laríngeas/genética , Neoplasias Laríngeas/metabolismo , Masculino , Pessoa de Meia-Idade , Fosfoproteínas/química , Domínios Proteicos , Análise de SobrevidaRESUMO
Colorectal cancer (CRC) presents as a very heterogeneous disease which cannot sufficiently be characterized with the currently known genetic and epigenetic markers. To identify new markers for CRC we scrutinized the methylation status of 231 DNA repair-related genes by methyl-CpG immunoprecipitation followed by global methylation profiling on a CpG island microarray, as altered expression of these genes could drive genomic and chromosomal instability observed in these tumors. We show for the first time hypermethylation of MMP9, DNMT3A and LIG4 in CRC which was confirmed in two CRC patient groups with different ethnicity. DNA ligase IV (LIG4) showed strong differential promoter methylation (up to 60%) which coincided with downregulation of mRNA in 51% of cases. This functional association of LIG4 methylation and gene expression was supported by LIG4 re-expression in 5-aza-2'-deoxycytidine-treated colon cancer cell lines, and reduced ligase IV amounts and end-joining activity in extracts of tumors with hypermethylation. Methylation of LIG4 was not associated with other genetic and epigenetic markers of CRC in our study. As LIG4 is located on chromosome 13 which is frequently amplified in CRC, two loci were tested for gene amplification in a subset of 47 cases. Comparison of amplification, methylation and expression data revealed that, in 30% of samples, the LIG4 gene was amplified and methylated, but expression was not changed. In conclusion, hypermethylation of the LIG4 promoter is a new mechanism to control ligase IV expression. It may represent a new epigenetic marker for CRC independent of known markers.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , DNA Ligases/genética , Metilação de DNA , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas/genética , Western Blotting , Ciclo Celular , Proliferação de Células , Colo/metabolismo , Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA Ligase Dependente de ATP , DNA Ligases/metabolismo , DNA Metiltransferase 3A , Feminino , Inativação Gênica , Humanos , Masculino , Metaloproteinase 9 da Matriz/genética , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Targeting synthetic lethality in DNA repair pathways has become a promising anti-cancer strategy. However little is known about such interactions with regard to the nucleotide excision repair (NER) pathway. Therefore, cell lines with a defect in the NER genes ERCC6 or XPC and their normal counterparts were screened with 53 chemically defined phytochemicals isolated from plants used in traditional Chinese medicine for differential cytotoxic effects. The screening revealed 12 drugs that killed NER-deficient cells more efficiently than proficient cells. Five drugs were further analyzed for IC(50) values, effects on cell cycle distribution, and induction of DNA damage. Ascaridol was the most effective compound with a difference of >1000-fold in resistance between normal and NER-deficient cells (IC(50) values for cells with deficiency in ERCC6: 0.15µM, XPC: 0.18µM, and normal cells: >180µM). NER-deficiency combined with ascaridol treatment led to G2/M-phase arrest, an increased percentage of subG1 cells, and a substantially higher DNA damage induction. These results were confirmed in a second set of NER-deficient and -proficient cell lines with isogenic background. Finally, ascaridol was characterized for its ability to generate oxidative DNA damage. The drug led to a dose-dependent increase in intracellular levels of reactive oxygen species at cytotoxic concentrations, but only NER-deficient cells showed a strongly induced amount of 8-oxodG sites. In summary, ascaridol is a cytotoxic and DNA-damaging compound which generates intracellular reactive oxidative intermediates and which selectively affects NER-deficient cells. This could provide a new therapeutic option to treat cancer cells with mutations in NER genes.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Reparo do DNA/genética , Medicamentos de Ervas Chinesas/farmacologia , Monoterpenos/farmacologia , Peróxidos/farmacologia , Antineoplásicos Fitogênicos/administração & dosagem , Linhagem Celular , Monoterpenos Cicloexânicos , Dano ao DNA/efeitos dos fármacos , Relação Dose-Resposta a Droga , Medicamentos de Ervas Chinesas/administração & dosagem , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Medicina Tradicional Chinesa , Monoterpenos/administração & dosagem , Peróxidos/administração & dosagem , Espécies Reativas de Oxigênio/metabolismoRESUMO
BACKGROUND AND PURPOSE: Repeated exposure to ionizing radiation (IR) can result in adaptive reactions. While DNA methylation changes in adaption to repeated stress exposure are established for a variety of drugs, their role in fractioned ionizing radiation is largely unknown. MATERIAL AND METHODS: MCF7 breast cancer cells were treated 5 times a week with IR in fractions of 2 Gy, resulting in total doses of 10 and 20 Gy. Cells were harvested 48 and 72 h after the last irradiation, as well as after a recovery period of at least 14 d. To identify genes differentially methylated in irradiated versus non-irradiated cells, we used methyl-CpG immunoprecipitation (MCIp) followed by global methylation profiling on CpG island microarrays. RESULTS: MCIp profiling revealed methylation changes in several CpG islands 48 h after FIR with 10 and 20 Gy. Cells receiving a total dose of 10 Gy started regrowing after 14 d and exhibited similar radioresistance as mock-treated cells. Differential methylation of the CpG units associated with FOXC1 (p<0.001) and TRAPPC9 (p<0.001) could be confirmed by time-of-flight mass spectrometry (Sequenom). CONCLUSIONS: In summary, these data indicate that regrowth of MCF7 cells after 10 Gy FIR is associated with locus-specific alterations in DNA methylation.