RESUMO
<p><b>OBJECTIVE</b>To construct pNTAP-MK2 eukaryotic expression plasmid and establish a HEK293 cell line stably expressing tandem affinity purification (TAP)-tagged MK2.</p><p><b>METHODS</b>The MK2-encoding region was subcloned into the vector pNTAP to construct the recombinant plasmid pNTAP-MK2, which was subsequently transformed into DH5 alpha.E.coli. After identification by PCR, digestion with restriction endonuclease and sequencing, the recombinant expression plasmid was transfected into HEK293 cells via liposome, and the cell line with stable expression of exogenous TAP tag-MK2 gene was selected by antibiotic G418. The expression and localization of the fusion protein TAP tag-MK2 were detected by Western blotting and immunofluorescence assay.</p><p><b>RESULTS</b>The results of PCR, restriction endonuclease digestion and sequencing all confirmed the correct construction of the recombinant eukaryotic expression plasmid pNTAP-MK2. Western blotting showed that the recombinant plasmid was expressed stably in HEK293 cells after transfection with G418 selection. Immunofluorescence assay identified the expression product TAP tag-MK2 mainly in the cell nuclei.</p><p><b>CONCLUSION</b>The eukaryotic expression vector pNTAP-MK2 has been successfully constructed, and in the established cell line with stable expression of TAP tag-MK2, TAP tag does not influence the localization of exogenous MK2.</p>
Assuntos
Humanos , Expressão Gênica , Vetores Genéticos , Células HEK293 , Peptídeos e Proteínas de Sinalização Intracelular , Genética , Plasmídeos , Proteínas Serina-Treonina Quinases , GenéticaRESUMO
The promyelocytic leukemia (PML) can selectively and dynamically recruit a number of proteins including p53 to form a sub-nuclear multiprotein chamber named PML-NBs. In DNA damage response, p53 is recruited into PML-NBs and modified by phosphorylations and acetylations, which in turn potentiate its transcriptional and pro-apoptotic activities. In contrast, in carcinoma cells, the role of PML in the irradiation induced p53-mediated apoptosis is not precisely understood. In this study, we have used the breast carcinoma cell line, MCF-7, and stably suppressed the expression of PML. Inhibition of PML expression had no detectable effect on the expression of endogenous p53 at the mRNA level; however, a significant decrease of p53 protein was observed. There was also an increase in the p53-MDM2 complexes, which may facilitate p53 protein degradation by the ubiquitin-proteasome pathway, also in irradiation treated cells. The p53 transcriptional activity was attenuated both in unstressed and 10 Gy irradiation treated cells. Moreover, inhibition of PML expression in MCF-7 cells significantly reduced p53 downstream genes, cell cycle arrest gene p21(WAF/cip-1) and pro-apoptotic gene Bax expression, then irradiation-induced apoptosis. These results suggest that PML is a key regulator in the irradiation activated p53 apoptotic pathway in breast carcinoma cells.
Assuntos
Apoptose/efeitos da radiação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas Nucleares/antagonistas & inibidores , Transdução de Sinais , Fatores de Transcrição/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores , Radioisótopos de Cobalto , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Regulação para Baixo , Feminino , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Luciferases/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína da Leucemia Promielocítica , Complexo de Endopeptidases do Proteassoma , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
To investigate the function and molecular mechanism of p21(WAF1/Cip-1) expression in MOLT-4 cells induced by HDAC inhibitor TSA, the expression pattern of p21(WAF1/Cip-1) and the distribution of cell cycle in TSA treated cells were analyzed. The results showed that TSA could effectively induce G(2)/M arrest and apoptosis of MOLT-4 cells. Kinetic experiments demonstrated that p21(WAF1/Cip-1) were upregulated quickly before cell arrested in G(2)/M and began decreasing at the early stage of apoptosis. Meanwhile, the proteasome inhibitor MG-132 could inhibit the decrease of p21(WAF1/Cip-1) at the early stage of apoptosis, which showed that proteasome pathway involved in p21(WAF1/Cip-1) degradation during the TSA induced G(2)/M arrest and apoptosis responses. This study also identified that the protein level of p21(WAF1/Cip-1) was highly associated with the cell cycle change induced by TSA. Compared to cells treated by TSA only, exposure MOLT-4 cells to TSA meanwhile treatment with MG-132 increased the protein level of p21(WAF1/Cip-1) and increased the numbers of cell in G(2)/M-phase, whereas the cell apoptosis were delayed. It is concluded that p21(WAF1/Cip-1) plays a significant role in G(2)/M arrest and apoptosis signaling induced by TSA in MOLT-4 cells.
