RESUMO
In the melanoma M14 cell line, we found that the antimetastatic protein NM23/nucleoside diphosphate kinase binds to the promoters of the oncogene cMYC and of P53, a gene often mutated in human cancer (Cervoni et al. [2006] J. Cell. Biochem. 98:421-428). In a further study, we find now that IFI16, a transcriptional repressor, in both promoters binds to the G-rich fragment that also binds NM23/NDPK. These fragments possess non-B DNA structures. Moreover, by sequential chromatin immunoprecipitation (re-ChIP) we show that the two proteins (IFI16 and NM23/NDPK) are simultaneously bound in vivo to the same DNA fragments. Since P53 stimulates apoptosis and inhibits cellular growth, and cMYC promotes cell growth and, in several instances, also apoptosis, the presence of NM23 and IFI16 on the same DNA fragments suggests their common involvement in the reduced development of some tumors.
Assuntos
DNA/metabolismo , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Melanoma/patologia , Nucleosídeo NM23 Difosfato Quinases/fisiologia , Proteínas Nucleares/fisiologia , Oligodesoxirribonucleotídeos/metabolismo , Fosfoproteínas/fisiologia , Regiões Promotoras Genéticas , Proteínas Repressoras/metabolismoRESUMO
We isolated and analyzed by chromatin immunoprecipitation (ChIP) in viable M14 cells DNA sequences bound to the antimetastatic protein nucleoside diphosphate kinase (NM23/NDPK) to shed some light on the nuclear functions of this protein and on the mechanism by which it acts in development and cancer. We assessed the presence of selected sequences from promoters of platelet-derived growth factor A (PDGF-A), c-myc, myeloperoxidase (MPO), CD11b, p53, WT1, CCR5, ING1, and NM23-H1 genes in the cross-linked complexes. Quantitative PCR (Q-PCR) showed a substantial enrichment of the correlated oncosuppressor genes p53, WT1, ING1, and NM23-H1 in the immunoprecipitated (IP) DNA. This suggests that NM23/NDPK binding is involved in the transcription regulation of these genes. These results reveal new interactions that should help us to disclose the antimetastatic mechanism of NM23.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/genética , Genes Supressores de Tumor/fisiologia , Melanoma/genética , Compostos Organometálicos/química , Fatores de Transcrição/metabolismo , Sequência de Bases/genética , Sítios de Ligação , Biomarcadores Tumorais/metabolismo , Cromatina/química , Cromatina/isolamento & purificação , Imunoprecipitação da Cromatina/métodos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Genes myc/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Melanoma/metabolismo , Melanoma/secundário , Dados de Sequência Molecular , Nucleosídeo NM23 Difosfato Quinases , Núcleosídeo-Difosfato Quinase/metabolismo , Fator de Crescimento Derivado de Plaquetas/genética , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição/genética , Células Tumorais CultivadasRESUMO
Human isoforms A and B of nm23/nucleoside diphosphate (NDP) kinase, functionally important in development and cancer, have been reported to bind to DNA, and in particular isoform A to the PDGF-A promoter and isoform B to the c-myc promoter and to telomeric repeats. However, no direct proof of the binding in vivo has yet been obtained. To demonstrate this interaction, human erythroleukemic K562 cells were incubated with two different cross-linking reagents, formaldehyde or cis-diammine dichloro platinum H. The DNA-protein covalent complexes were isolated and analyzed by Western blotting. The positive immunochemical staining showed that in both conditions NDP kinase isoforms A and B were efficiently cross-linked to DNA in vivo. NDP kinase-linked DNA fragments obtained by immunoprecipitation, subjected to hybridization with different probes, showed a definite enrichment in the nuclease-hypersensitive silencer element of the PDGF-A promoter. No conclusive evidence was found by this technique of preferential hybridization with a nuclease-hypersensitive element of the c-myc promoter and with the telomeric TTAGGG repeats. The immunoprecipitated NDP kinase-DNA complexes are a promising material for the detection of other specific DNA sequences interacting with NDP kinase.