细胞凋亡及周期阻滞基本信号通路.docx

CELL DEATH AND CELL-CYCLE CHECKPOINT DURING DNA DAMAGE细胞死亡及周期阻滞基本信号通路有哪些因素可引起DNA损伤？DNA损伤的结局如何？（课件）(一)DNA损伤的原因环境因素，化学因素，生物因素例如： UV ,离子辐射，基因毒性化学试剂引起ssDNA/dsDNA损伤，DNA两条链交联或链内交联。正常细胞线粒体的一些代谢物（ROS）活泼氧类过多引起损伤。(二) DNA损伤结局：急性效应：干扰核酸代谢，触发细胞周期阻滞或死亡长期效应：不可逆转突变导致肿瘤细胞周期阻滞，衰老，肿瘤/癌症，有丝分裂危象 (一)DNA损伤的原因1.DNA分子的自发性损伤(1)DNA复制中的错误。(2)DNA的自发性化学变化a.碱基的异构互变性损伤b.碱基的脱氨基作用c.脱嘌呤与脱嘧啶d.碱基修饰与链断裂2.物理因素引起的DNA损伤(1)紫外线引起的DNA损伤 (2)电离辐射引起的DNA损伤a.碱基变化b.脱氧核糖变化c.DNA链断裂d.交联3.化学因素引起的DNA损伤(1)烷化剂对DNA的损伤a.碱基烷基化b.碱基脱落c.断链d.交联(2)碱基类似物、修饰剂对DNA的损伤DNA损伤的后果 1.点突变(point mutation)指DNA上单一碱基的变异。嘌呤替代嘌呤(A与G之间的相互替代)、嘧啶替代嘧啶(C与T之间的替代)称为转换(transition)；嘌呤变嘧啶或嘧啶变嘌呤则称为颠换(transvertion)。2.缺失(deletion)指DNA链上一个或一段核苷酸的消失。3.插入(insertion)指一个或一段核苷酸插入到DNA链中。在为蛋白质编码的序列中如缺失及插入的核苷酸数不是3的整倍数，则发生读框移动(reading frame shift)，使其后所译读的氨基酸序列全部混乱，称为移码突变(frameshift mutaion)。4.倒位或转位(transposition)指DNA链重组使其中一段核苷酸链方向倒置、或从一处迁移到另一处。5.双链断裂已如前述，对单倍体细胞一个双链断裂就是致死性事件。（2） THE CONSEQUENCES OF DNA INJURY The outcome of DNA damage is diverse and generally adverse（有害的）. Acute effects arise from disturbed DNA metabolism（新陈代谢）, triggering（启动，控制） cell-cycle arrest or cell death. Long term effects result from irreversible mutations（转变，突变，变异） contributing to oncogenesis（）. Many Lesions（损伤） Block（阻碍） Transcription（转录） an outcome directly related to gene length. This has elicited（引出） the development of a dedicated repair system, transcription-coupled repair (TCR), which displaces or removes the stalled RNA polymerase and assures high priority repair. TRANSCRIPTIONAL STRESS, arising from persistent blockage of RNA synthesis, constitutes an efficient trigger for p53-dependent apoptosis, which may be a significant anti-cancer mechanism. Lesions Also Interfere With DNA Replication（复制） Recently, a growing class of DNA polymerases（聚合酶）, numbered to , was discovered which seems devoted specifically to overcoming damage-induced REPLICATIONAL STRESS. These special polymerases take over temporarily from the blocked replicative DNA polymerase-/, and possibly from pol . But this solution generally comes at the expense of a higher error rate. In fact, this process is responsible for most of damage-induced point mutations and is thus particularly relevant for oncogenesis. Nevertheless, translesion polymerases still protect the genome. Double-strand DNA breaks (DSBs) induced by X-rays, chemicals or during replication of single-strand breaks (SSBs) and presumably during repair of interstrand crosslinks are particularly relevant for the recombination machinery. Eg：Cells with specialized DNA recombination activities, such as B- and T-cells, may be very sensitive to DSBs when they are rearranging their immunoglobin or T-cell-receptor genes. This explains the frequent involvement of these genetic loci in oncogenic translocations in leukaemia(白血病) and lymphomas（淋巴瘤） and the preferential induction of these cancers by ionizing irradiation. Eg：DSBs also pose problems during mitosis（有丝分裂）, as intact（未受损的） chromosomes are a prerequisite（先决条件） for proper chromosome segregation（分离） during cell division. Thus, these lesions（损伤） frequently induce various sorts of chromosomal aberrations（染色体病）, including aneuploidy（）, deletions（缺失） (loss of heterozygosity, LOH) and chromosomal translocations（迁移，置换） events which are all intimately associated with carcinogenesis（癌变）.The cell-cycle machinery somehow senses genome injury and arrests（阻滞） at specific checkpoints in G1, S, G2 and M to allow repair of lesions before they are converted into permanent mutations. Lesion detection may occur by blocked transcription, replication or specialized sensors. When damage is too significant, a cell may opt for the ultimate mode of rescue by initiating（开始） apoptosis（凋亡） at the expense of a whole cell什么分子可作为DNA双链断裂/损伤的标志？用什么方法测定？ （1）Senescence(衰老), can be triggered when telomeres（端粒）the ends of linear chromosomescannot fulfil（执行） their normal protective functions. Here we show that senescent human fibroblasts（纤维原细胞） display molecular markers characteristic of cells bearing DNA double-strand breaks. These markers include nuclear foci of phosphorylated histone H2AX and their co-localization with DNA repair and DNA damage checkpoint factors such as 53BP1, MDC1 and NBS1. We also show that senescent cells contain activated forms of the DNA damage checkpoint kinases（激酶，致活酶） CHK1 and CHK2.（2）fluorescence（荧光）DNA发生双链断裂后最早反应之一是位于断裂点附近的组蛋白H2AX的C末端第139位丝氨酸残基被快速磷酸化形成-H2AX。磷酸化的-H2AX快速转导DNA损伤信号，导致下游分子磷酸化的激活，引发一系列的生物级联反应和和细胞学反应。-H2AX是迄今所研究的最重要的DNA损伤感应分子。免疫印记法检测。基于双链断裂/损伤，常在有丝分裂过程出现问题，引起各种类型染色体畸变，包括非整倍体，缺失和染色体异位的发生，也用流式细胞仪做细胞倍体检查。采用PFGE法（脉冲场凝胶电泳法）测定链断裂量，以孔外进入凝胶的DNA占孔外和孔内总DNA比例FAR（DSB FAR（%）。用FITC-结合抗生素蛋白 检测DNA损伤。写出G1、G2/M checkpoint 基本信号通路主要分子成分。 G1-phase Checkpoint PathwayATM-CHK2-CDC25A-CDK2 axis forms a rapid response system;ATR-CHK1-CDC25A-CDK2;CDC25A-CDK4;ATM-CHK2/ATR-CHK1-P53-p21 pathway S-phase Checkpoint PathwayATM-CHK2-CDC25A-CDC45 axis forms a rapid response system G2/M-phase Checkpoint PathwayA key effector of G2 checkpoint is CDC2 (CDK1) :ATM-CHK2- CDC25C-CDC2 axisATR-CHK1- CDC25C-CDC2 axisATR-CHK1- CDC25A-CDC2 axisWeel-CDC2 inhibitionPLK1 (-) and PLK3 (+) (polo-like kinase family) play a crucial roles in initiation and exit from mitosisP21-PCNA-CDC2-Cyclin B complex excludes CDC25C(-)PCD(程序性细胞死亡)都有哪些形式？ PCD主要形态学特征如何？ I（凋亡Apoptosis）；Morphology: Chromatin condensation, fragmentation, apoptotic bodies II型（自噬Autophagic）Triggers: Serum/amino-acid starvation, protein aggregates (3)Type III PCDAtypica(非典型性)l forms of cell death: Paraptosis (Aponecrosis)Morphology: ER swelling, mitochondrial swelling(胞浆空泡化，线粒体，内质网肿胀，无核固缩) (4) Type IV PCD Calcium-mediated cell death（钙离子介导的细胞死亡）Morphology: Membrane whorls (5) Type V PCD AIF/PARP-dependent cell death（AIF/PARP依赖性细胞死亡）Morphology: Mild chromatin condensation (6)Type VI PCDOncosis （胀亡）Morphology: Cellular swelling形态学变化:首先出现的是细胞体积缩小，连接消失，与周围的细胞脱离，然后是细胞质密度增加，线粒体膜电位消失，通透性改变，释放细胞色素C到胞浆，核质浓缩，核膜核仁破碎，DNA降解成为约180bp-200bp片段；胞膜有小泡状形成，膜内侧磷脂酰丝氨酸外翻到膜表面，胞膜结构仍然完整，最终可将凋亡细胞遗骸分割包裹为几个凋亡小体，无内容物外溢，因此不引起周围的炎症反应，凋亡小体可迅速被周围专职或非专职吞噬细胞吞噬。其基本信号通路所涉及的主要激活分子是什么？如何检测或鉴定？各有何意义？凋亡是多基因严格控制的过程。这些基因在种属之间非常保守，如Bcl-2家族、caspase家族、癌基因如C-myc、抑癌基因P53等，1）细胞凋亡的膜受体通路Fas(CD95)是一种跨膜蛋白，属于肿瘤坏死因子受体超家族成员，它与FasL结合可以启动凋亡信号的转导引起细胞凋亡2）细胞色素C释放和Caspases激活的生物化学途经细胞色素C从线粒体释放是细胞凋亡的关键步骤, 线粒体还释放凋亡诱导因子，如AIF，参与激活caspase(半胱天冬蛋白酶)Fas是一种跨膜蛋白，属于肿瘤坏死因子受体超家族成员, Fas又称CD95，是由325个氨基酸组成的受体分子，Fas一旦和配体FasL结合，可通过Fas分子启动致死性信号转导，最终引起细胞一系列特征性变化，使细胞死亡。Fas作为一种普遍表达的受体分子，可出现于多种细胞表面Caspase是半胱氨酸家族蛋白酶，根据功能可把Caspase基本分为二类：一类参与细胞的加工，如Pro-IL-1和Pro-IL-1，形成有活性的IL-1和IL-1；第二类参与细胞凋亡，包括caspase2,3,6,7,8,9.10。细胞凋亡的过程实际上是Caspase不可逆有限水解底物的级联放大反应过程. 参与诱导凋亡的Caspase分成两大类：启动酶（inititaor）和效应酶（effector）它们分别在死亡信号转导的上游和下游发挥作用。细胞色素C在dATP存在的条件下能与凋亡相关因子1（Apaf-1）结合，使其形成多聚体，并促使caspase-9与其结合形成凋亡小体AIF凋亡诱导因子，参与激活caspase凋亡抑制因子：P35，CrmA,IAPs，FLIPs以及Bcl-2家族P35和CrmA是广谱凋亡抑制剂，体外研究结果表明P35以竞争性结合方式与靶分子形成稳定的具有空间位阻效应的复合体并且抑制Caspases活性FLIPs(FLICE-imhibirory proterins)能抑制Fas/TNFR1介导的细胞凋亡凋亡抑制蛋白（IAPs,inhibitors of Apoptosis protien）为一组具有抑制凋亡作用的蛋白质Bcl-2生理功能是阻遏细胞凋亡，延长细胞寿命1 早期检测：1） PS（磷脂酰丝氨酸）在细胞外膜上的检测： PS从细胞膜内侧转移到外侧在细胞受到凋亡诱导后不久发生，可能作为免疫系统的识别标志,通过简单的显色或发光系统进行检测2）细