P21Waf1/Cip1 is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21Waf1/cip1involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidencef...P21Waf1/Cip1 is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21Waf1/cip1involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidencefor a link between p21Waf1/cip1 and cellular senescence. While in murine cells, the role of p21Waf1/Cip1is indefinite. We explored this issue using NIH3T3 cells with inducible p21Waf1/cip1 expression. Induc-tion of p21Waf1/Cip1 triggered G1 growth arrest, and NIH3T3-p21 cells exhibited morphologic features,such as enlarged and flattened cellular shape, specific to the senescence phenotype. We also showed thatp21Waf1/Cip1-transduced NIH3T3 cells expressedβ-galactosidase activity at pH 6.0, which is known to bea marker of senescence. Our results suggest that p21Waf1/cipx can also induce senescence-like changes inmurine cells.展开更多
Objective: To investigate the post-transcriptional regulation of p21WAF1/CIP1 by p53. Methods: The MDA-MB-468 cells have endogenous mutant p53 and the MCF7 cells lines have wtp53. Recombinant p53 expression and p21WAF...Objective: To investigate the post-transcriptional regulation of p21WAF1/CIP1 by p53. Methods: The MDA-MB-468 cells have endogenous mutant p53 and the MCF7 cells lines have wtp53. Recombinant p53 expression and p21WAF1/CIP1 induction were detected by Western blot analysis. Northern blot analysis was carried out to examine whether changes in p21WAF1/CIP1 protein levels in MCF7 cells treated with AdCMVp53 are reflected at the mRNA level. Flow cytometric analysis of MCF7 cells following overexpression of recombination. Results: The ratio of p53: p21WAF1/CIP1 was below 1 at the early stages of AdCMVp53 infection, but increased to 1.6 by day 3 and to 9.7 by day 5 post-infection. As expected, p21WAF1/CIP1 expression was not detectable in MDA-MB-468 cells despite the presence of high levels of mutant p53 protein. The G1/S ratios in untreated controls and AdCMVβgal infected MCF7 cells were 1.10 and 1.35, respectively. By Northern blot analyzing the p21WAF1/CIP1: GAPDH ratios at different time points against the ratio at time point 0, a maximum 3-fold induction of p21WAF1/CIP1 mRNA expression relative to untreated control was observed on day 1 post-infection. The flow cytometric analysis indicated that MCF7 cells infected with AdCMVp53 undergo G1 arrest at both time points studied, with G1/S ratios ranging from 5.54 at day 1 to 5.65 at day 7. The G1/S ratios in untreated controls and AdCMVβgal infected MCF7 cells were 1.10 and 1.35, respectively. Conclusion: This study demonstrated that p53 could regulate p21WAF1/CIP1 gene expression at both the transcriptional and post-transcriptional levels in MCF7 cells. The latter mechanism may be involved in or be responsible for, the induction of cell cycle arrest by transcription-defective mutants of p53.展开更多
Neonatal rat cardiomyocytes were subjected to 24 h of hypoxia 95%N2/5%CO2 and 24 h of hypoxia plus 4 h of reoxygenation 95%O2/5%CO2. 24 h of hypoxia increased the levels of NO, --23NO/NO, TBARS and LDH. 24 h of hypoxi...Neonatal rat cardiomyocytes were subjected to 24 h of hypoxia 95%N2/5%CO2 and 24 h of hypoxia plus 4 h of reoxygenation 95%O2/5%CO2. 24 h of hypoxia increased the levels of NO, --23NO/NO, TBARS and LDH. 24 h of hypoxia plus 4 h of reoxygenation decreased the levels of NO, --23NO/NO, but further increased TBARS and LDH. The hypoxia up-regulated the expression of bcl-2, p53 and p21/waf1/cip1 but the reoxygenation down-regulated the expression of bcl-2, and further up-regulated p53 and p21/waf1/cip1. The hypoxia increased cell apoptosis and reoxygena-tion further increased both apoptotic and necrotic cell death. NO, -23NO/NO, TBARS, DNA frag-mentation and cell apoptosis were enhanced by SNP and inhibited by L-NAME respectively. In addition, SOD/catalase down-regulated the expression of p53, p21/wafl/cipl and TBARS but up-regulated bcl-2 and increased indirectly the level of NO, --23NO/NO, and inhibited DNA frag-mentation. The results suggest that hypoxia-induced cell death is associated with the activation of NO, bcl-2 and p53 pathway, while hypoxia-reoxygenation induced cell death via the generation of reactive oxygen species and activation of p53 pathway. The present study clarified that NO may be an initiative signal to apoptotic cell death and the activation of bcl-2, p53 and p21/waf1/cip1 path-way in hypoxic and hypoxia-reoxygenated cardiomyocytes.展开更多
Primary hepatocellular carcinoma (HCC) is one of the most common cancers occurring in human, and there is strong epidemiological evidence suggesting that persistent hepatitis B virus (HBV) infection is the most import...Primary hepatocellular carcinoma (HCC) is one of the most common cancers occurring in human, and there is strong epidemiological evidence suggesting that persistent hepatitis B virus (HBV) infection is the most important risk factor for its development. HBx gene was found to be a transactivator recently. Its continuous expression in hepatocytes may transactivate cellular genes which can play a certain role in development of HCC. The HBx gene fragment was used to construct a recombinant eukaryotic expression vector pCEP4 and introduced into HepG2 cells. The effect of HBx gene on HCC cells growth and its molecular mechanism in HCC cells regulation were investigated.展开更多
基金We thank Dr. Gordon Peters of Imperial CancerResearch Fund for providing p21(Waf1/Cip1) cDNA,Dr. Hong-Bing Shu of National Jewish Medica1and Reearch Center for pRetro-on-Bax and 293-10A1 packaging line. We also thank Drs. WeiTAO and Zhi Gang LU
文摘P21Waf1/Cip1 is a potent cyclin-dependent kinase inhibitor. As a downstream mediator of p53, p21Waf1/cip1involves in cell cycle arrest, differentiation and apoptosis. Previous studies in human cells provided evidencefor a link between p21Waf1/cip1 and cellular senescence. While in murine cells, the role of p21Waf1/Cip1is indefinite. We explored this issue using NIH3T3 cells with inducible p21Waf1/cip1 expression. Induc-tion of p21Waf1/Cip1 triggered G1 growth arrest, and NIH3T3-p21 cells exhibited morphologic features,such as enlarged and flattened cellular shape, specific to the senescence phenotype. We also showed thatp21Waf1/Cip1-transduced NIH3T3 cells expressedβ-galactosidase activity at pH 6.0, which is known to bea marker of senescence. Our results suggest that p21Waf1/cipx can also induce senescence-like changes inmurine cells.
文摘Objective: To investigate the post-transcriptional regulation of p21WAF1/CIP1 by p53. Methods: The MDA-MB-468 cells have endogenous mutant p53 and the MCF7 cells lines have wtp53. Recombinant p53 expression and p21WAF1/CIP1 induction were detected by Western blot analysis. Northern blot analysis was carried out to examine whether changes in p21WAF1/CIP1 protein levels in MCF7 cells treated with AdCMVp53 are reflected at the mRNA level. Flow cytometric analysis of MCF7 cells following overexpression of recombination. Results: The ratio of p53: p21WAF1/CIP1 was below 1 at the early stages of AdCMVp53 infection, but increased to 1.6 by day 3 and to 9.7 by day 5 post-infection. As expected, p21WAF1/CIP1 expression was not detectable in MDA-MB-468 cells despite the presence of high levels of mutant p53 protein. The G1/S ratios in untreated controls and AdCMVβgal infected MCF7 cells were 1.10 and 1.35, respectively. By Northern blot analyzing the p21WAF1/CIP1: GAPDH ratios at different time points against the ratio at time point 0, a maximum 3-fold induction of p21WAF1/CIP1 mRNA expression relative to untreated control was observed on day 1 post-infection. The flow cytometric analysis indicated that MCF7 cells infected with AdCMVp53 undergo G1 arrest at both time points studied, with G1/S ratios ranging from 5.54 at day 1 to 5.65 at day 7. The G1/S ratios in untreated controls and AdCMVβgal infected MCF7 cells were 1.10 and 1.35, respectively. Conclusion: This study demonstrated that p53 could regulate p21WAF1/CIP1 gene expression at both the transcriptional and post-transcriptional levels in MCF7 cells. The latter mechanism may be involved in or be responsible for, the induction of cell cycle arrest by transcription-defective mutants of p53.
基金the Guangdong Natural Science Foundation and National Natural Science Foundation of China (Grant No. 29935080).
文摘Neonatal rat cardiomyocytes were subjected to 24 h of hypoxia 95%N2/5%CO2 and 24 h of hypoxia plus 4 h of reoxygenation 95%O2/5%CO2. 24 h of hypoxia increased the levels of NO, --23NO/NO, TBARS and LDH. 24 h of hypoxia plus 4 h of reoxygenation decreased the levels of NO, --23NO/NO, but further increased TBARS and LDH. The hypoxia up-regulated the expression of bcl-2, p53 and p21/waf1/cip1 but the reoxygenation down-regulated the expression of bcl-2, and further up-regulated p53 and p21/waf1/cip1. The hypoxia increased cell apoptosis and reoxygena-tion further increased both apoptotic and necrotic cell death. NO, -23NO/NO, TBARS, DNA frag-mentation and cell apoptosis were enhanced by SNP and inhibited by L-NAME respectively. In addition, SOD/catalase down-regulated the expression of p53, p21/wafl/cipl and TBARS but up-regulated bcl-2 and increased indirectly the level of NO, --23NO/NO, and inhibited DNA frag-mentation. The results suggest that hypoxia-induced cell death is associated with the activation of NO, bcl-2 and p53 pathway, while hypoxia-reoxygenation induced cell death via the generation of reactive oxygen species and activation of p53 pathway. The present study clarified that NO may be an initiative signal to apoptotic cell death and the activation of bcl-2, p53 and p21/waf1/cip1 path-way in hypoxic and hypoxia-reoxygenated cardiomyocytes.
文摘Primary hepatocellular carcinoma (HCC) is one of the most common cancers occurring in human, and there is strong epidemiological evidence suggesting that persistent hepatitis B virus (HBV) infection is the most important risk factor for its development. HBx gene was found to be a transactivator recently. Its continuous expression in hepatocytes may transactivate cellular genes which can play a certain role in development of HCC. The HBx gene fragment was used to construct a recombinant eukaryotic expression vector pCEP4 and introduced into HepG2 cells. The effect of HBx gene on HCC cells growth and its molecular mechanism in HCC cells regulation were investigated.
