胡桃楸提取液诱导K562细胞凋亡机制的研究

目的：考察胡桃楸提取液是否具有诱导K562细胞p52蛋白及p21蛋白的作用,初步探讨胡桃楸提取液诱导K562细胞凋亡机制。方法：以Western-blot方法检测胡桃楸提取液对K562细胞p53蛋白及诱导K562细胞p53蛋白和p21蛋白的表达。     

p53转录非依赖活性介导细胞凋亡

p53主要通过两条途径诱导细胞凋亡：p53作为转录因子,促进细胞凋亡的靶基因的表达上调,如PUMA、NOXA、PIDD、p53AIP1、COP1等,并通过这些蛋白参与内源和外源凋亡途径;另一方面,胞浆中的p53能转位到线粒体,激活内源性的线粒体途径,促进凋亡。后者已成为研究p53促凋亡机制的热点。本文就p53对转录非依赖活性诱导细胞凋亡途径的研究进展作一概述。     

HPV16 E6通过阻碍ING4对p53作用而抑制细胞凋亡的实验研究

通过HPV16 E6干扰ING4对p53作用的实验研究,探讨HPV16 E6新的致癌机制。采用转染及免疫共沉淀实验证明HPV16 E6阻碍ING4和p53结合及其诱导的p53蛋白乙酰化的作用;将表达p53、ING4和p53报告基因与HPV16 E6或其突变体的质粒共转染p53蛋白阴性的SaoS2细胞系,荧光素酶报告基因检测HPV16 E6抑制ING4对p53基因在转录水平的影响;并采用细胞集落形成实验检测HPV16 E6对ING4所诱导p53途径所致细胞凋亡的抑制。HPV16 E6阻碍ING4和p53结合及其诱导的p53蛋白Lys-382的乙酰化;HPV16 E6减弱ING4在转录水平对p53基因的调控,HPV16 E6抑制ING4诱导的p53途径介导的细胞凋亡,且所有这些作用不依赖p53蛋白的降解。HPV16 E6阻碍ING4对p53的作用而抑制细胞凋亡可能是其引起癌变的途径之一。     

HPV16 E6通过阻碍ING4对p53作用而抑制细胞凋亡的研究

通过HPV16 E6干扰ING4对p53作用的实验研究,探讨HPV16 E6新的致癌机制。采用转染及免疫共沉淀实验证明HPV16 E6阻碍ING4和p53结合及其诱导的p53蛋白乙酰化的作用;将表达p53、ING4和p53报告基因与HPV16 E6或其突变体的质粒共转染p53蛋白阴性的SaoS2细胞系,荧光素酶报告基因检测HPV16 E6抑制ING4对p53基因在转录水平的影响;并采用细胞集落形成实验检测HPV16 E6对ING4所诱导p53途径所致细胞凋亡的抑制。HPV16 E6阻碍ING4和p53结合及其诱导的p53蛋白Lys-382的乙酰化;HPV16 E6减弱ING4在转录水平对p53基因的调控,HPV16 E6抑制ING4诱导的p53途径介导的细胞凋亡,且所有这些作用不依赖p53蛋白的降解。HPV16 E6阻碍ING4对p53的作用而抑制细胞凋亡可能是其引起癌变的途径之一。     

雷公藤甲素诱导HeLa细胞p53依赖的自噬和凋亡

自噬与凋亡被认为是细胞程序性死亡的两种重要途径,二者的交互联系对阐明药物的抗肿瘤机理有重要价值.众多的研究表明,雷公藤甲素对多种肿瘤细胞都具有显著的抑制作用.细胞凋亡与自噬可被相同的因素所诱导,p53蛋白可以同时对二者起调控作用,在自噬与凋亡的交互作用(crosstalk)中扮演着重要角色.本文以He La细胞为模型,研究雷公藤甲素诱导He La细胞发生自噬和凋亡的机制,并通过抑制p53依赖的转录,研究雷公藤甲素诱导He La细胞p53依赖的自噬和凋亡交互联系.     

苦参碱对鼻咽癌细胞凋亡及凋亡相关基因p53 的表达研究

目的：探讨苦参碱对体外培养的人鼻咽癌细胞增殖、凋亡及凋亡相关基因p53 mRNA 和蛋白表达的影响,初步探讨苦参碱 诱导人鼻咽癌细胞凋亡的可能机制。方法：采用MTT 法检测不同浓度苦参碱（0、0.25、0.5、1、1.5、2 mg/ml）对CNE1、CNE2 细胞增 殖的影响;采用荧光定量PCR 法检测这些浓度的苦参碱处理48 h后CNE2 细胞p53 mRNA的变化; Western Blot 检测其蛋白的 变化情况。结果：MTT结果显示苦参碱具有抑制CNE1、CNE2 细胞体外增殖作用,其抑制率存在浓度、时间依赖性。荧光定量 PCR及Western Blot 检测结果显示,苦参碱抑制CNE2细胞p53 mRNA 和蛋白的表达,且亦呈浓度依赖性。结论：苦参碱抑制 CNE2 细胞的增殖,诱导细胞凋亡,呈现浓度、时间依赖性,其作用与抑制CNE2 细胞中p53 基因和蛋白的表达密切相关。     

苦参碱对鼻咽癌细胞凋亡及凋亡相关基因p53的表达研究

目的：探讨苦参碱对体外培养的人鼻咽癌细胞增殖、凋亡及凋亡相关基因p53 mRNA和蛋白表达的影响,初步探讨苦参碱诱导人鼻咽癌细胞凋亡的可能机制。方法：采用MTT法检测不同浓度苦参碱（0、0.25、0.5、1、1.5、2 mg/ml）对CNE1、CNE2细胞增殖的影响;采用荧光定量PCR法检测这些浓度的苦参碱处理48 h后CNE2细胞p53 mRNA的变化;Western Blot检测其蛋白的变化情况。结果：MTT结果显示苦参碱具有抑制CNE1、CNE2细胞体外增殖作用,其抑制率存在浓度、时间依赖性。荧光定量PCR及Western Blot检测结果显示,苦参碱抑制CNE2细胞p53 mRNA和蛋白的表达,且亦呈浓度依赖性。结论：苦参碱抑制CNE2细胞的增殖,诱导细胞凋亡,呈现浓度、时间依赖性,其作用与抑制CNE2细胞中p53基因和蛋白的表达密切相关。     

米非司酮对恒河猴母胎界面细胞凋亡和凋亡分子p53表达的影响

胎盘形成过程中发生活跃的细胞增殖、迁移和凋亡等活动。p53蛋白是参与调节细胞周期和凋亡过程的原癌基因。本实验用原位末端标记、蛋白印迹和免疫组织化学方法研究正常和米非司酮(RU486)处理后恒河猴母胎界面绒毛和蜕膜组织细胞凋亡及p53蛋白表达。在正常妊娠的恒河猴母胎界面,凋亡信号主要集中在合体滋养层和细胞柱内的一些滋养层细胞;p53蛋白主要定位于细胞滋养层。在母体蜕膜中,也在部分基质细胞中检测到细胞凋亡和p53蛋白表达。经过RU486处理2d后,胎盘绒毛和母体蜕膜中凋亡细胞数都显著增加,绒毛中增加的凋亡信号集中于细胞滋养层。同时,RU486处理也导致绒毛细胞滋养层和蜕膜基质细胞中p53表达明显增加。以上结果提示,在正常妊娠中,生理性的细胞凋亡和p53表达可能是控制细胞滋养层细胞增殖、保持胎盘组织动态平衡的一个重要机制;RU486终止早孕的可能途径之一是促进母胎界面细胞凋亡,推测p53参与这一过程。     

蓝莓花青素通过下调p53基因DNA甲基化抑制口腔癌KB细胞增殖及诱导细胞凋亡

文章从内蒙古野生蓝莓(Vaccinium uliginosum Lim)中提取花青素, 观察其对口腔癌细胞株KB的增殖及凋亡的作用, 探讨其作用机制与p53基因甲基化的相关性。利用含0.1%盐酸的甲醇提取花青素, 用高效液相色谱-质谱(High performance liquid chromatography-mass spectrometry, HPLC-MS )鉴定花青素的成分。利用四甲基偶氮唑蓝(Methylthiazolyl-tetrazolium, MTT)比色法、流式细胞术、免疫荧光法、免疫细胞化学法和Western blot法分析蓝莓花青素对KB细胞增殖、细胞周期、细胞凋亡和p53蛋白表达的影响; 利用甲基化特异性PCR法(Methylation-specific PCR, MSP)分析蓝莓花青素诱导细胞凋亡与p53基因甲基化的关系。结果显示, 内蒙古自治区的野生蓝莓中至少存在14种花青素成分; 蓝莓花青素呈剂量依赖的方式抑制KB细胞增殖, 诱导细胞周期阻滞在G₂/M期, 而且能诱导细胞凋亡; 蓝莓花青素处理后Caspase-9蛋白和细胞色素C的表达明显增加; Western blot结果表明蓝莓花青素可以诱导KB细胞中p53蛋白表达上调; MSP结果表明随蓝莓花青素浓度增加, 未甲基化的p53的比例增加, 说明p53的甲基化状态有所下调。     

来源于鸡贫血病毒的凋亡素研究进展

肿瘤化疗药物多以p53依赖性途径诱导细胞凋亡,而许多肿瘤在发生过程中丧失了D53功能;此外,在肿瘤发生过程中往往伴随着抑制细胞凋亡的bcl-2蛋白的高水平表达。而来源于鸡贫血病毒的凋亡素能够选择性地诱导肿瘤细胞和转化细胞发生凋亡,并以非p53依赖性途径诱导细胞凋亡,不受bcl-2蛋白的抑制作用。因此,凋亡素作为1种抗肿瘤制剂用于肿瘤治疗能够选择性地诱导肿瘤细胞和转化细胞发生凋亡,而并不杀伤人和鼠的正常二倍体细胞,且无毒副作用…,具有很好的临床应用前景。     

The death domain kinase RIP has an important role in DNA damage-induced, p53-independent cell death

Tumor suppressor p53 plays a critical role in cellular responses, such as cell cycle arrest and apoptosis following DNA damage. DNA damage-induced cell death can be mediated by a p53-dependent or p53-independent pathway. Although p53-mediated apoptosis has been well documented, little is known about the signaling components of p53-independent cell death. Here we report that the death domain kinase, RIP (receptor-interacting protein), is important for DNA damage-induced, p53-independent cell death. DNA damage induces cell death in both wild-type and p53-/- mouse embryonic fibroblast cells. We found that RIP-/- mouse embryonic fibroblast cells, which have a mutant form of the p53 protein, are resistant to DNA damage-induced cell death. The reconstitution of RIP protein expression in RIP-/- cells restored the sensitivity of cells to DNA damage-induced cell death. We also found that RIP mediates this process through activating mitogen-activated protein kinase, JNK1. Furthermore, knocking down the expression of RIP blocked DNA damage-induced cell death in the human colon cancer cell line, p53 null HCT 116. Taken together, our study demonstrates that RIP is one of the critical components involved in mediating DNA damage-induced, p53-independent cell death.     

The Tyrosine Kinase c-Abl Promotes Homeodomain-interacting Protein Kinase 2 (HIPK2) Accumulation and Activation in Response to DNA Damage

The non-receptor tyrosine kinase c-Abl is activated in response to DNA damage and induces p73-dependent apoptosis. Here, we investigated c-Abl regulation of the homeodomain-interacting protein kinase 2 (HIPK2), an important regulator of p53-dependent apoptosis. c-Abl phosphorylated HIPK2 at several sites, and phosphorylation by c-Abl protected HIPK2 from degradation mediated by the ubiquitin E3 ligase Siah-1. c-Abl and HIPK2 synergized in activating p53 on apoptotic promoters in a reporter assay, and c-Abl was required for endogenous HIPK2 accumulation and phosphorylation of p53 at Ser⁴⁶ in response to DNA damage by γ- and UV radiation. Accumulation of HIPK2 in nuclear speckles and association with promyelocytic leukemia protein (PML) in response to DNA damage were also dependent on c-Abl activity. At high cell density, the Hippo pathway inhibits DNA damage-induced c-Abl activation. Under this condition, DNA damage-induced HIPK2 accumulation, phosphorylation of p53 at Ser⁴⁶, and apoptosis were attenuated. These data demonstrate a new mechanism for the induction of DNA damage-induced apoptosis by c-Abl and illustrate network interactions between serine/threonine and tyrosine kinases that dictate cell fate.     

p53- and Bax-Mediated Apoptosis in Injured Rat Spinal Cord

Spinal cord injury (SCI) induces a series of endogenous biochemical changes that lead to secondary degeneration, including apoptosis. p53-mediated mitochondrial apoptosis is likely to be an important mechanism of cell death in spinal cord injury. However, the signaling cascades that are activated before DNA fragmentation have not yet been determined. DNA damage-induced, p53-activated neuronal cell death has already been identified in several neurodegenerative diseases. To determine DNA damage-induced, p53-mediated apoptosis in spinal cord injury, we performed RT-PCR microarray and analyzed 84 DNA damaging and apoptotic genes. Genes involved in DNA damage and apoptosis were upregulated whereas anti-apoptotic genes were downregulated in injured spinal cords. Western blot analysis showed the upregulation of DNA damage-inducing protein such as ATM, cell cycle checkpoint kinases, 8-hydroxy-2′-deoxyguanosine (8-OHdG), BRCA2 and H2AX in injured spinal cord tissues. Detection of phospho-H2AX in the nucleus and release of 8-OHdG in cytosol were demonstrated by immunohistochemistry. Expression of p53 was observed in the neurons, oligodendrocytes and astrocytes after spinal cord injury. Upregulation of phospho-p53, Bax and downregulation of Bcl2 were detected after spinal cord injury. Sub-cellular distribution of Bax and cytochrome c indicated mitochondrial-mediated apoptosis taking place after spinal cord injury. In addition, we carried out immunohistochemical analysis to confirm Bax translocation into the mitochondria and activated p53 at Ser³⁹². Expression of APAF1, caspase 9 and caspase 3 activities confirmed the intrinsic apoptotic pathway after SCI. Activated p53 and Bax mitochondrial translocation were detected in injured spinal neurons. Taken together, the in vitro data strengthened the in vivo observations of DNA damage-induced p53-mediated mitochondrial apoptosis in the injured spinal cord.     

Phospholipase D elevates the level of MDM2 and suppresses DNA damage-induced increases in p53

Phospholipase D (PLD) has been reported to generate survival signals that prevent apoptosis induced by serum withdrawal. We have now found that elevated expression of PLD also suppresses DNA damage-induced apoptosis. Since DNA damage-induced apoptosis is often mediated by p53, we examined the effect of elevated PLD expression on the regulation of p53 stabilization. We report here that PLD suppresses DNA damage-induced increases in p53 stabilization in cells where PLD has been shown to provide a survival signal. Elevated expression of PLD also led to increased expression of the p53 E3 ubiquitin ligase MDM2 and increased turnover of p53. PLD1-stimulated increases in MDM2 expression and suppression of p53 activation were blocked by inhibition of mTOR and the mitogen-activated protein kinase pathway. Although PLD did not activate the phosphatidylinositol 3-kinase (PI3K)/Akt survival pathway activate the basal levels of PI3K activity were partially required for PLD1-induced increases in MDM2. These data provide evidence that survival signals generated by PLD involve suppression of the p53 response pathway.     

MST1 promotes apoptosis through regulating Sirt1-dependent p53 deacetylation

Mammalian Sterile 20-like kinase 1 (MST1) protein kinase plays an important role in the apoptosis induced by a variety of stresses. The MST1 is a serine/threonine kinase that is activated upon apoptotic stimulation, which in turn activates its downstream targets, JNK/p38, histone H2B and FOXO. It has been reported that overexpression of MST1 initiates apoptosis by activating p53. However, the molecular mechanisms underlying MST1-p53 signaling during apoptosis are unclear. Here, we report that MST1 promotes genotoxic agent-induced apoptosis in a p53-dependent manner. We found that MST1 increases p53 acetylation and transactivation by inhibiting the deacetylation of Sirtuin 1 (Sirt1) and its interaction with p53 and that Sirt1 can be phosphorylated by MST1 leading to the inhibition of Sirt1 activity. Collectively, these findings define a novel regulatory mechanism involving the phosphorylation of Sirt1 by MST1 kinase which leads to p53 activation, with implications for our understanding of signaling mechanisms during DNA damage-induced apoptosis.     

Tip60 and p400 are both required for UV-induced apoptosis but play antagonistic roles in cell cycle progression

The histone acetyl transferase Tip60 (HTATIP) belongs to a multimolecular complex involved in the cellular response to DNA damage. Tip60 participates in cell cycle arrest following DNA damage by allowing p53 to activate p21CIP (p21) expression. We show here that Tip60 and the E1A-associated p400 protein (EP400), which belongs to the Tip60 complex, are also required for DNA damage-induced apoptosis. Tip60 favours the expression of some proapoptotic p53 target genes most likely through the stimulation of p53 DNA binding activity. In contrast, p400 represses p21 expression in unstressed cells, thereby allowing cell cycle progression and DNA damage-induced apoptosis. Tip60 and p400 have thus opposite effects on p21 expression in the absence of DNA damage. We further found that this antagonism relies on the inhibition of Tip60 function by p400, a property that is abolished following DNA damage. Therefore, taken together, our results indicate that Tip60 and p400 play distinct roles in DNA damage-induced apoptosis and underline the importance of the Tip60 complex and its regulation in the proper control of cell fate.     

DNA damage-induced MDMX degradation is mediated by MDM2

Although genetic studies have demonstrated that MDMX is essential to maintain p53 activity at low levels in non-stressed cells, it is unknown whether MDMX regulates p53 activation by DNA damage. We show here that DNA damage-induced p53 induction is associated with rapid down-regulation of the MDMX protein. Significantly, interference with MDMX down-regulation results in the suppression of p53 activation by genotoxic stress. We also demonstrate that DNA damage-induced MDMX reduction is mediated by MDM2, which targets MDMX for proteasomal degradation by a distinct mechanism that permits preferential MDMX degradation and therefore ensures optimal p53 activation.