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1、Apoptosis and cancer,To die or not to die, this is a question.,蝼蚁尚且贪生。,舍生取义。,Destiny.,“for their discoveries concerning genetic regulation of organ development and programmed cell death”,1090 131= 959(cells),Robert Horvitz,John Sulston,Sydney Brenner,Physiology or Medicine Nobel Prize 2002,Sydney Br
2、enner,20世纪60年代末，英国科学家悉尼布雷内希望能找到一种适于基因水平研究，并能确定完整的神经系统的实验生物模型。最终布雷内选择了一种较为简单的生物秀丽隐杆线虫。,“He chose the nematode because of its short life cycle, genetic simplicity and tiny size less than 1 mm long”,布雷内慧眼识线虫,John Sulston,苏尔斯顿提出“细胞程序性死亡” 利用布雷内建立的突变的线虫株，发现并分离了一系列影响细胞凋亡的基因，包括ced-1、ced-2 和 nuc-1，提出生物发育过程中细
3、胞的凋亡是由一系列基因控制的。确定了线虫发育过程中每一个细胞的分裂和分化及其最终的命运。,Caenorhabditis elegans,1090,959,131,Robert Horvitz,1986年，罗伯特霍维茨及其学生发现了线虫中在细胞凋亡过程中起关键作用的两种基因：ced-3和ced-4。这两种基因的缺失将改变细胞的命运，使线虫中原本正常情况下会死亡的细胞存活并继续分化下去。,霍维茨等人确认“死亡基因”,细胞凋亡的发现以及对细胞凋亡发生机制的研究，为科学开拓出一个神奇的新领域，意义非常深远。 它加深了人类对于生命的认识。 对细胞凋亡及其机制的研究将为癌症、糖尿病等许多疾病的治疗提供新的
4、可能的方法。对人类健康事业产生重大影响。,蝌蚪尾的消失,脊椎动物的神经系统的发育,发育过程中手和足的成形过程,细胞凋亡(Apoptosis),细胞凋亡由体内外因素触发细胞内预存的死亡程序而导致的细胞死亡过程。是一个由基因调控的细胞主动结束生命的过程。 早在1972年Kerr等已发现从细胞形态、超微结构和生化变化等方面来分析，细胞有二种死亡形式,一种是早被熟知的细胞坏死（Necrosis），另一种是新提出的程序性细胞死亡（Programmed cell death, PCD）学说。但该学说到九十年代初才进入研究高潮，现在普遍称之为细胞凋亡（Apoptosis）。,Apoptosis: 细胞凋亡,
5、Necrosis: 细胞坏死,细胞凋亡与细胞坏死的区别,坏死 凋亡,1.性质,2.诱导因素,强烈刺激，随机发生,较弱刺激，非随机发生,3.生化特点,4.形态变化,6.DNA电泳,5.炎症反应,7.凋亡小体,8.基因调控,被动过程，无新蛋白合成，不耗能,主动过程，有新蛋白合成，耗能,细胞结构全面溶解、破坏、细胞肿胀,胞膜及细胞器相对完整，细胞皱缩，核固缩,弥散性降解，电泳呈均一DNA片状,DNA片段化（180-200bp）， 电泳呈“梯”状条带,溶酶体破裂，局部炎症反应,溶酶体相对完整，局部无炎症反应,有,病理性，非特异性,生理性或病理性，特异性,无,有,无,Basic Features of
6、Apoptosis,形态学特征,凋亡的起始：细胞器、染色质等开始变化 凋亡小体的形成：质膜包裹染色质和细胞器 凋亡小体的消化：被吞噬细胞消化,Nuclear morphological changes of HeLa cells in apoptosis process （488nm 400）,凋亡的生物化学特征,非随机性DNA降解 细胞膜磷脂酰丝氨酸外翻,DNA断裂与“梯状条带”(1),DNA断裂与“梯状条带”(2),典型凋亡细胞DNA琼脂糖凝胶电泳（呈现梯状条带）,Sub-G1,DNA断裂的原位末端标记法（TUNEL测定法） 凋亡细胞DNA降解产生缺口和末端，故可用原位缺口翻译或末端标记法
7、显示凋亡细胞。,细胞膜磷脂酰丝氨酸外翻,Annexin V-FITC Fluorescence,PI Fluorescence,Time course of apoptosis in TF-1 cells cultured in GM-CSF-free medium,Camptothecin(喜树硷), an alkaloid isolated from the Chinese tree Camptotheca acuminata, represents a promising new class of anticancer drugs that target the intranuclear
8、 enzyme topoisomerase I (topo-I).Initial clinical studies of camptothecin were halted because of severe and unpredictable adverse effects. Supported by detailed understanding of their mechanism of action and facilitated by chemical manipulations that have amplified their solubility, camptothecins ha
9、ve advanced to the forefront of several areas of therapeutic and developmental chemotherapy. In the present study, we study the potential in vitro effect of NSC606985, a rarely studied water-soluble camptothecin ester derivative, on a cancer cell line called NB4. For this purpose, the experiments in
10、 Figure 2 were done.,根据以上结果，作者认为NSC606985能够诱导NB4细胞发生凋亡。请回答： 作者得出该结论的依据是什么？（10分）； 在得出如上结论后，作者应该如何开展更深入的研究（10分）。,How does Apoptosis occur?,细胞的凋亡至少受14个基因的控制,CED-9,CED-4,CED-3,APOPTOSIS,具严格底物特异性的半胱氨酸蛋白酶 通常以酶原（前体）的形式存在于细胞内 活化的Ced 3特异地降解蛋白底物,CED-9,CED-4,CED-3,EGL-1,EGL-1,1993年，袁钧英等发现Ced-3基因与哺乳细胞白细胞介素1转化酶(
11、interleukin-1b-converting enzyme, ICE)存在功能和序列相似性。和Ced-3一样，ICE的高表达可导致啮齿动物成纤维细胞凋亡。,Caspases: cysteine-containing aspartate-specific protease（含半胱氨酸的天冬氨酸特异水解酶）,是一组对底物的天冬氨酸部位有特异水解作用，其活性中心富含半胱氨酸的蛋白酶。,Caspase家族的结构与分类 ICE亚类Caspase蛋白酶：ICE是单核细胞来源的参与成熟的一种蛋白酶，参与凋亡但不起主要作用； Caspase1，Caspase4， Caspase5， Caspase11
12、Caspase12， Caspase13， Caspase14。 凋亡启动亚类Caspase蛋白酶：Caspase8， Caspase2， Caspase 9，Caspase10。 凋亡效应亚类Caspase蛋白酶：Caspase3， Caspase6， Caspase7,prodomain,大亚基（20KD）,小亚基 （10KD）,Caspase前体,大亚基（20KD）,小亚基 （10KD）,(p20/p10)2,Caspase属于半胱氨酸蛋白酶。 具有严格的底物特异性，即特异地剪切天冬氨酸残基后的肽键。但是，邻近剪切位点N端的四肽序列及其结构对于caspases的有效酶解也是非常重要的。
13、不同caspases识别的四肽序列明显不同，这可能是造成它们的功能差异的重要基础。,使抑制凋亡因子失活，如ICAD 破坏细胞结构，如Lamin A 将调节区与催化区分离，使蛋白失活，如PARP,CAD激活方式,Molecular pathways of apoptosis,Two principle pathways,Extrinsic,Intrinsic pathway,参与死亡受体信号转导的接头蛋白： （死亡结构域蛋白death domain protein） TRADD：TNF受体相关死亡结构域蛋白 TNF+TNFRI（死亡结构域）+ TRADD细胞凋亡 FADD：Fas相关死亡结构域蛋
14、白 FasL+Fas （死亡结构域） +FADD 细胞凋亡 RIP：受体相互作用蛋白 CRADD：含有死亡结构域的caspase和RIP接头蛋白 MADD：活化MAP激酶的死亡结构域蛋白,Receptor-mediated pathway of apoptosis,FASL+FAS +FADD 死亡诱导信号复合物（DISC） 胞质中游离的caspase8聚集到这个复合物上 细胞有足够caspase8 细胞caspase8浓度不够 死亡受体活化， 切割Bid tBid从胞质到线粒体 细胞凋亡 Cty C 释放,The mitochondria-mediated pathway of apopto
15、sis,线粒体参与细胞凋亡或坏死的途径 释放caspase活化因子 细胞色素C：细胞色素C+Apaf-1+ caspase9凋亡体 AIF（凋亡诱导因子） Caspase3 Apaf-1：凋亡蛋白酶活化因子,凋亡诱导因子（apoptosis-inducing factor, AIF） Smac/DIABLO （second mitochondria-derived activators of caspase/direct IAP-binding protein with a low isoelectric points） 核酸内切酶G（endonuclease G，Endo G）,57KD黄素
16、蛋白AIF (Mit),Nucleus,Chr 浓缩 DNA 降解,Independent of caspase activation and oxidoreductases activity of AIF,Guido Kroemer,Early work on the role of mitochondria in apoptosis,Work on peripheral T-cell tolerance in Madrid in1989,We treated mice with SEB, a surperantigen whose injection can induce T-cell t
17、olerance through activation-induced cell death.,DNA ladder,Apoptosis of peripheral T cells was an in vitro artefact,J Exp Med, 1993,I decided to look for biochemical alterations that would distinguish normal T cells and preapoptotic T lymphocytes.,pH, cation concentrations, membrane permeability, ly
18、sosome change and so on.,DiOC6, a dye that indirectly measures the mitochondrial transmembrane potential(JEM,1995),Loss of MTP marked a irreversible early step of the apoptotic process(JEM,1995),A cell free system, in which we confronted purified mitochondrial with purifed nuclei.,Upon induction of
19、permeability transbility transition, we found that mitochondria released an apoptosis inducing factor that cause chromatin condensation and fragmentation when added to purified nulclei.(JEM,1996),The mitochondrial release of these apoptogenic factors but not their action on the nucleus was found to
20、be controlled by Bcl-2.(JEM,1996),Mitochondrial would decide the fate of cells and serves as a central integrator and coordinator of the apoptotic process.(FASEB J,1995),Mitochondrial are bull shit !?,Without this help, we would have been forced to abandon our work,Xiaodong Wang,Douglas Green,Cell f
21、ree system of xiaodong wang,Cell free system of kroemer,To detect caspase activation as the final readout,To look for a protein that would cause chromatin condensation, in the absence of cytosol, in a caspase-independent manner.,AIF (Nature,1999),Artefact-inducing factor?,It is probably wrong to con
22、centrate on just one or few proteins (ANT, VDAC,p53) as being crucial regulators, we should, at least sometimes, concentrated on processes, rather than on individual molecules.,Cyto C,How apoptosis is regulated,Tsujimoto和Crose发现极大多数非何杰金氏滤泡状B细胞淋巴瘤(B-cell lymphomas)存在染色体易位t(14;18)。,caspases,Apaf-1,?,1
23、4号染色体的IgH基因Bcl (B cell lymphoma) -2基因,基因转染实验显示Bcl-2高表达广泛抑制大多数刺激剂诱导的细胞凋亡。,Bcl2,含有1-4个Bcl-2同源结构域（BH1-4）。分两类：抗凋亡基因，如：Bcl-2；促凋亡基因，如：Bax。 促凋亡蛋白能引起线粒体外膜通透性改变，导致cyt c 释放，引起凋亡。 bcl-2相当于线虫的ced9。主要定位于线粒体膜、也存在于ER和外核膜，拮抗促凋亡蛋白的功能。,bcl-2基因家族,Bcl-2 gene family（Zimmermann 2001）,Early work on the function of Bcl-2,D
24、avid Vaux,Full length of bcl-2 cDNA was cloned in 1986 by both Tsujimoto in Philidelphia and Mike Cleary at Stanford,1987, received the bcl-2 cDNA from Mike but was asked not to make transgenic mice.,To figure out whether bcl-2 was an oncogene whose overexpression was responsible for causing follicu
25、lar lymphoma,To see if it could act like other oncogene to transform fibroblasts, as had been shown for ras, v-raf, or make growth factor dependent cells factor independent, as had been shown for v-abl, v-fms.,NIH-3T3,FDC-P1: IL-3 dependent,Infected with bcl-2,Infected with vector,Injected into syng
26、eneic mice,Growth factor withdraw,Growth factor withdraw,No colonies,No colonies,No tumors grow,Immunology today, 1989,Death DNA ladder,No death No DNA ladder,Whether bcl-2 and c-myc could synergize in the transformation of cells in vitro.,Normal bone marrow cells,E-myc bone marrow cells,Bcl-2,Soft
27、agar,Soft agar,?,lymphoma,Nature,1988,F1 cross (1989,5),Transgenic bcl-2 mice (1988,9),Transgenic E-myc mice,Pre- or pro-B cell leukemias,Nature,1990,Thinking that evolution might provide some clues as to how Bcl-2 functioned, I thought Id make a zoo blot, and probe it with the human bcl-2 cDNA.,Mou
28、se, Drosophila, Xenopus, herring, yeast, C. elegans DNA,Make trangenic worms with human bcl-2,The worms expressing human bcl-2 had only a third as many corpses as the control,Science,1992,细胞凋亡的途径,Apoptosis and cancers,Apoptosis-related cellular proteins involve in the progression of malignancies,. p
29、53 . pRb . Fas,. Mdm2 . c-myc . Bcl-2 family,Whats the big deal about imatinib?,Gleevec inhibits Abl by binding to the kinase domain This is a TARGETTED THERAPY based on our knowledge of the molecular defect in CML,Brian Druker, M.D. Oregon Health & Science University,Chronic Myelogenous Leukemia,ABL,Imatinib,BCR,t(9;22) translocation juxtaposes BCR and ABL genes BCR-ABL fusion kinase is constitutive active Imatinib directly inhibits BCR-ABL Long-term remissions observed in treated pts,THANK YOU,