RESUMEN
OBJECTIVE: To observe the inhibitory effect of cyclin-dependent kinase inhibitor p21 on regulation of survivin transcription in human liver cancer HepG cells, and explore the related mechanisms. METHODS: Doxorubicin (DOX) was used to treat HepG cells. Eukaryotic vector pEGFP-C2-p21 was transfected into HepG cells by lipofectamine and positive clones were screened out by G418. The mRNA expression of p21, p53 and survivin were detected by real-time fluorescent quantitative polymerase chain reaction (RQ-PCR). Flow cytometry was used to determine the cell cycle phases, and reverse transcription polymerase chain reaction (RT-PCR) was used to measure the levels of E2F-1 or p300. RESULTS: After treatment with DOX, the expression of p53 and p21 was increased, whereas that of survivin was reduced during 24 hours of the treatment. After transfection the p21 level was 2100.1-fold or 980.9-fold enhanced in comparison with that in HepG2 cells or HepG2-pEGFP cells. Survivin level was markedly down-regulated to 0.5% or 0.6% relative to that in the other two groups, nevertheless, significant p53 changes were not observed. Overexpression of p21 resulted in G1/G0 phase arrest (F = 31.59, P < 0.01), meanwhile, E2F-1 mRNA or p300 mRNA were less expressed compared with that in the other controls (F(E2F-1) = 125.28, P < 0.05; Fp300 = 46.01, P < 0.01). CONCLUSION: p21 could be a potential mediator of survivin suppression at transcription level in HepG2 cells, which might be through the block at G1/G0 phase and down-regulation of transcription factors E2F-1 and p300.
Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Doxorrubicina/farmacología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Antibióticos Antineoplásicos/farmacología , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Factor de Transcripción E2F1/genética , Factor de Transcripción E2F1/metabolismo , Fase G1 , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Humanos , Proteínas Inhibidoras de la Apoptosis , Proteínas Asociadas a Microtúbulos/genética , ARN Mensajero/metabolismo , Fase de Descanso del Ciclo Celular , Survivin , Transfección , Proteína p53 Supresora de Tumor/genética , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
The effect of cyclin-dependent kinase inhibitors Cip1/Waf1 (p21) on regulatory expression of survivin transcription in human hepatocellular carcinoma cell HepG2 was observed and the related mechanisms explored. Doxorubicin (DOX) was used to treat HepG2. Eukaryotic vector pEGFP-C2-p21 was transfected into HepG2 by lipofectamine and positive clones were screened out by G418. The mRNA expression of p21 and survivin was detected by real-time fluorescent quantitative polymerase chain reaction (RQ-PCR). Flow cytometry was used to examine the cell cycle, and reverse transcription polymerase chain reaction (RT-PCR) was used to measure the levels of E2F-1 and p300. The results showed that: (1) After treatment with DOX, the expression of p21 was increased, whereas that of survivin was reduced during 24 h of treatment; (2) After transfection of pEGFP-C2-p21 into HepG2, p21 level was significantly enhanced to 2100.11-folds or 980.89-folds in comparison to HepG2 or HepG2-C2 group, and survivin level was markedly down-regulated to 0.54% or 0.59% relative to the control groups; (3) Overexpressed p21 resulted in G1/G0 phase arrest (F=31.59, P<0.01), meanwhile E2F-1 mRNA and p300 mRNA were reduced as compared with those of controls (F(E2F-1)=125.28, P<0.05; F(p300)=46.01, P<0.01). It was suggested that p21 could be a potential mediator of survivin suppression at transcription level in HepG2 cell, which might be through the block at G1/G0 phase and down-regulation of transcription factors E2F-1 and p300.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Regulación Neoplásica de la Expresión Génica , Neoplasias Hepáticas/metabolismo , Proteínas Asociadas a Microtúbulos/biosíntesis , Proteínas Asociadas a Microtúbulos/fisiología , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/fisiología , Transcripción Genética , Antineoplásicos/farmacología , Ciclo Celular , Línea Celular Tumoral , Doxorrubicina/farmacología , Perfilación de la Expresión Génica , Humanos , Proteínas Inhibidoras de la Apoptosis , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Survivin , TransfecciónRESUMEN
In order to investigate peptide mimics of carbohydrate blood group A antigen, a phage display 12-mer peptide library was screened with a monoclonal antibody against blood group A antigen, NaM87-1F6. The antibody-binding properties of the selected phage peptides were evaluated by phage ELISA and phage capture assay. The peptides were co-expressed as glutathione S-transferase (GST) fusion proteins. RBC agglutination inhibition assay was performed to assess the natural blood group A antigen-mimicking ability of the fusion proteins. The results showed that seven phage clones selected bound to NaM87-1F6 specifically, among which, 6 clones bore the same peptide sequence, EYWYCGMNRTGC and another harbored a different one QIWYERTLPFTF. The two peptides were successfully expressed at the N terminal of GST protein. Both of the fusion proteins inhibited the RBC agglutination mediated by anti-A serum in a concentration-dependent manner. These results suggested that the fusion proteins based on the selected peptides could mimic the blood group A antigen and might be used as anti-A antibody-adsorbing materials when immunoabsorption was applied in ABO incompatible transplantation.
Asunto(s)
Antígenos de Grupos Sanguíneos/química , Péptidos/química , Adsorción , Bacteriófagos , Ensayo de Inmunoadsorción Enzimática , Epítopos/química , Glutatión Transferasa/metabolismo , Humanos , Biblioteca de Péptidos , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/químicaRESUMEN
The effect of cyclin-dependent kinase inhibitors Cip1/Wafl (p21) on regulatory expression of survivin transcription in human hepatocellular carcinoma cell HepG2 was observed and the related mechanisms explored. Doxorubicin (DOX) was used to treat HepG2. Eukaryotic vector pEGFP-C2-p21 was transfected into HepG2 by lipofectamine and positive clones were screened out by G418. The mRNA expression of p21 and survivin was detected by real-time fluorescent quantitative polymerase chain reaction (RQ-PCR). Flow cytometry was used to examine the cell cycle, and reverse transcription polymerase chain reaction (RT-PCR) was used to measure the levels of E2F-1 and p300. The results showed that: (1) After treatment with DOX, the expression of p21 was increased, whereas that of survivin was reduced during 24h of treatment; (2) After transfection of pEGFP-C2-p21 into HepG2, p21 level was significantly enhanced to 2100.11-folds or 980.89-folds in comparison to HepG2 or HepG2-C2 group, and survivin level was markedly down-regulated to 0.54% or 0.59% relative to the control groups; (3) Overexpressed p21 resulted in G1/G0 phase arrest (F=31.59,P<0.01), meanwhile E2F-1 mRNA and p300 mRNA were reduced as compared with those of controls (FE2F-1=125.28,P<0.05;Fp300=46.01,P<0.01). It was suggested that p21 could be a potential mediator of survivin suppression at transcription level in HepG2 cell, which might be through the block at G1/G0 phase and down-regulation of transcription factors E2F-1 and p300.
RESUMEN
In order to investigate peptide mimics of carbohydrate blood group A antigen, a phage display 12-met peptide library was screened with a monoclonal antibody against blood group A antigen, NaM87-1F6. The antibody-binding properties of the selected phage peptides were evaluated by phage ELISA and phage capture assay. The peptides were co-expressed as glutathione S-transferase (GST) fusion proteins. RBC agglutination inhibition assay was performed to assess the natural blood group A antigen-mimicking ability of the fusion proteins. The results showed that seven phage clones selected bound to NaM87-1F6 specifically, among which, 6 clones bore the same peptide sequence, EYWYCGMNRTGC and another harbored a different one QIWYERTLPFrF. The two peptides were successfully expressed at the N terminal of GST protein. Both of the fusion proteins inhibited the RBC agglutination mediated by anti-A serum in a concentration-dependent manner. These results suggested that the fusion proteins based on the selected peptides could mimic the blood group A an- tigen and might be used as anti-A antibody-adsorbing materials when immunoabsorption was applied in ABO incompatible transplantation.