Down-regulation of p21 contributes to apoptosis induced by HPV E6 in human mammary epithelial cells

Infection with human papillomaviruses (HPV) is strongly associated with the development of cervical cancer. The HPV E6 gene is essential for the oncogenic potential of HPV. E6 induces cell proliferation and apoptosis in cervical cancer precursor lesions and in cultured cells. Although induction of telomerase and inactivation of the tumor suppressor p53 play important roles for E6 to promote cell growth, the molecular basis of E6-induced apoptosis is poorly understood. While it is expected that inactivation of p53 by E6 should lead to a reduction in cellular apoptosis, numerous studies demonstrated that E6 could in fact sensitize cells to apoptosis. Understanding the mechanism of p53-independent apoptosis is of clinical significance. In the present study, we investigated the mechanism of apoptosis during E6-mediated immortalization of primary human mammary epithelial cell (HMEC). E6 by itself is sufficient to immortalize HMECs and is believed to do so at least in part by activation of telomerase. During the process of E6-mediated HMEC immortalization, an increased apoptosis was observed. Mutational analysis demonstrated that E6-induced apoptosis was distinct from its ability to promote cell proliferation, activate telomerase, or degrade p53. While the known pro-apoptotic E6 target proteins such as Bak or c-Myc did not appear to play an important role, down-regulation of the cyclin-dependent kinase inhibitor p21^Waf1/Cip1 (p21) by E6 correlated with its ability to induce apoptosis. Ectopic expression of p21 inhibited E6-induced apoptosis. Moreover, a p53 degradation defective E6 mutant was competent for p21 down-regulation and apoptosis induction. The anti-apoptotic function of p21 may not simply be the result of p21-induced growth arrest. These studies demonstrate an E6 activity to down-regulate p21 that is important for induction of apoptosis.     

Induction of cell death by adenoviruses

Adenoviruses have proved to be excellent tools for gaining insight into the regulation, and deregulation, of the mammalian cell cycle. With the widespread clinical use of gene therapy fast approaching, there comes a need for a better understanding of how the cell death process is regulated. A greater understanding will allow the development of therapeutic approaches that both maximise transgene expression while minimising cytotoxicity to the target cell. Consequently, much adenovirus research has centered on understanding the mechanisms governing adenovirus induced cell death or apoptosis. This review discusses recent advances in the field of adenovirus cell death regulation and evaluates the roles of implicated gene products and their respective data. The data suggest the existence of multiple virus gene products involved in cell death regulation and point towards several distinct, yet related, cell death pathways. A discussion of the shortcomings of current adenoviral research, along with a proposed model based upon the data is also given.     

促凋亡基因p53AIP1的研究进展

p53AIPl基因是近年发现的促凋亡基因,在p53依赖性的凋亡通路中起重要作用。p53AIPl介导线粒体凋亡途径,其表达依赖于p53蛋白的Ser^46的磷酸化。p53AIPl可直接促进凋亡,其促凋亡作用可能强于p53本身,并对p53抗性的肿瘤细胞也有作用。因此,对p53AIPl的深入研究可能会为对p53基因治疗有抗性的肿瘤患者带来新的希望。     

Apak competes with p53 for direct binding to intron 1 of p53AIP1 to regulate apoptosis

The KRAB-type zinc-finger protein Apak was recently identified as a negative regulator of p53-mediated apoptosis. However, the mechanism of this selective regulation is not fully understood. Here, we show that Apak recognizes the TCTTN₂₋₃₀TTGT consensus sequence through its zinc-fingers. This sequence is specifically found in intron 1 of the proapoptotic p53 target gene p53AIP1 and largely overlaps with the p53-binding sequence. Apak competes with p53 for binding to this site to inhibit p53AIP1 expression. Upon DNA damage, Apak dissociates from the DNA, which abolishes its inhibitory effect on p53-mediated apoptosis.     

p53调控骨关节炎软骨细胞凋亡

肿瘤抑制蛋白p53是一种可以有效调节哺乳动物细胞生长的核磷酸化蛋白质。p53表达增加能够激活一系列细胞基因,通过抑制多个细胞周期蛋白依赖性激酶导致细胞周期停滞并凋亡。有研究表明,骨关节炎(osteoarthritis,OA)软骨细胞中,p53的表达高于正常软骨细胞,通过下调p53表达能够减少软骨细胞凋亡,进而预防和缓解骨关节炎病变,这可能与线粒体凋亡途径密切相关,但是具体机制尚不明确。本文通过综述近年来p53调控骨关节炎软骨细胞凋亡的文献资料,为骨关节炎机制和治疗有关研究提供理论基础。     

Green tea constituent (−)-Epigallocatechin-3-gallate inhibits hep G2 cell proliferation and induces apoptosis through p53-dependent and fas-mediated pathways

(-)-Epigallocatechin-3-gallate (EGCG) is a polyphenolic compound found in green tea. It has been reported to possess a wide range of pharmacological properties, and is one of the most promising chemopreventive agents for cancer. To provide a better understanding of the preventive effect of EGCG on liver cancer, we examined EGCG for its effect on proliferation and cell cycle progression in a human liver cancer cell line, Hep G2. The results showed that EGCG inhibited the proliferation of Hep G2 by inducing apoptosis and blocking cell cycle progression in the G1 phase. ELISA showed that EGCG significantly increased the expression of p53 and p21/WAF1 protein, and this contributed to cell cycle arrest. An enhancement in Fas/APO-1 and its two form ligands, membrane-bound Fas ligand (mFasL) and soluble Fas ligand (sFasL), as well as Bax protein, was responsible for the apoptotic effect induced by EGCG. Taken together, our study suggests that the induction of p53 and the activity of the Fas/FasL apoptotic system play major roles in the antiproliferative activity of EGCG in Hep G2 cells.     

重组腺病毒介导人野生型P53和B7-1基因在喉癌细胞中的高效转移和高水平表达

为开展肿瘤的复合基因治疗,构建以串联方式携带人野生型p53和B7-1基因的重组腺病毒穿梭质粒pXC53/B7-1。将pXC53/B7-1与腺病毒包装质粒GT4050共转染293细胞,通过细胞内同源重组获得重组腺病毒Ad-p53/B7-1。在293细胞中扩增病毒,并通过氯化铯密度梯度超速离心纯化病毒,获得高滴度稿纯度的病毒,分别经免疫组织化学分析和流式细胞分析检测Ad-p53/B7-1介导的人野生型p53和B7-1基因在喉癌细胞ep-2中的表达。结果表明Ad-p53/B7-1能够有效地将其所携带的目的基因导入Hehp-2细胞并使其在细胞中高效表达。     

p53蛋白在周期调节蛋白A1变异引起的雄性小鼠生殖细胞凋亡中的作用

为研究p5 3蛋白在周期调节蛋白A1(cyclinA1)变异引起的雄性小鼠生殖细胞凋亡中的作用 ,以p5 3基因敲除的小鼠和周期调节蛋白A1基因敲除的小鼠杂交 ,获取同胎生单基因变异和双基因同时变异的雄性后代共 4组 12只 .比较它们的性腺和生殖细胞发育 ,并用TUNEL染色法观察和比较生殖细胞的凋亡情况 .在睾丸最大横切面上观察到 :周期调节蛋白A1变异组凋亡细胞最多 (348± 10 4个 ) ,明显高于p5 3 周期调节蛋白A1双基因变异组 (12 1± 38个 ) ,t=3 2 5 79,P =0 0 4 72 .p5 3变异组凋亡细胞最少 (45± 2 4个 ) ,配对t检验显示有非常显著性差异 ,t=8 4 0 13,P =0 0 0 35 .这一研究结果提示 ,p5 3基因可能在雄性生殖细胞的发育中起监视作用 ,并在周期调节蛋白A1变异引起发育异常时启动p5 3途径造成异常细胞的凋亡 .     

E1A激活基因阻遏子过表达抑制体外人血管平滑肌细胞凋亡

为探讨E1A激活基因阻遏子（cellular repressor of E1A-stimulated genes,CREG）对人血管平滑肌细胞（vascular smooth muscl ecells,VSMCs）凋亡的影响及调控机制,应用正、反义重组逆转录病毒表达载体pLNCX,（＋）／CREG及pLXSN（-）／CREG制备稳定感染人胸廓内动脉平滑肌细胞克隆株（human internal thoracic artery-Shenyang,HITASY）细胞模型,观察CREG蛋白过表达及表达抑制对平滑肌细胞凋亡的影响。荧光显微镜下观察DAPI染色后凋亡细胞核形态,AnnexinV／PI流式细胞术检测细胞凋亡率,RT-PCR技术分析凋亡相关基因caspase-9mRNA的表达,蛋白质印迹法分析p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase,p38MAPK）、磷酸化p38MAPK（phosphorylated p38 mitogen-activated proteinkinase,P-p38 MAPK）的表达变化。研究结果显示,CREG蛋白过表达明显抑制血清饥饿诱导的HITASY细胞凋亡的发生;同时细胞中p38MAPK、P-p38MAPK的表达增加。相反,抑制CREG蛋白表达则引起正常血清培养状态下VSMCs的自发凋亡明显增加,同时细胞内p38MAPK、P-p38MAPK表达显著下降。进一步研究发现,预先应用特异性抑制剂SB203580阻断p38MAPK信号转导通路后,CREG蛋白过表达引起的细胞凋亡抑制作用被明显减弱,血清饥饿后CREG蛋白过表达引起的HITASY细胞凋亡现象明显增加。上述结果提示,CREG蛋白过表达可以抑制体外培养的VSMCs凋亡,p38MAPK信号转导通路可能介导CREG蛋白对VSMCs凋亡的抑制作用。