2. molecular mechanism of cell death and cell proliferation Fate of cell  Undergoing cell cycle  differentiating to specific cell  death

3.  细胞分裂增生的研究 20 世纪 60 年代 分子机制的研究 Hartwell L, Nurse P, Hunt T Nobel prize for physiology and medicine

4. Why is dying so important? Physiologically: embyro stage, CNS development, thymus atrophy, endometrium desquamating Pathologically: tumor, Parkinson’s disease, Alzheimer’s disease

5. Nobel Prize for Physiology and Medicine 2002  Co-awarded on October 7, 2002  For “genetic regulation of organ development and programmed cell death”  Sydney Brenner (English)  H. Robert Horvitz (American)  John Sulston (English)

6. Sydney Brenner H. Robert Horvitz John Sulston

7. Caenorhabditis elegans: The Perfect Model C. elegans’s complexity but simplicity  A nematode approximately one mm long containing blood, muscle, heart, nervous, as well as other tissues  From fertilization to adult in three days  Life span of two to three weeks  Adult organism comprised of 959 cells  During embryological development will form 1090 cells

8.  Approximately 40 percent of the worm’s genes are also found in humans  Responds to taste, smell, temperature, touch, and possibly light  So, where did the other 131 cells go?

9. The C. elegans Organism

10. The Fundamental Genes Being Examined Egl-1 Ced9 Ced4 Ced3 apoptosis  EGL-1…initiates apoptosis by inhibiting the normal restraining action of CED-9 on CED-4  CED-3…triggered by CED-4 resulting in highly destructive proteases acting upon cell structure  CED-4…acted upon by EGL-1; required in cell death  CED-9… protects against cell death egl-1 egg laying defective-1 ced cell death abnormal

11. So what’s the big deal with studying worms?  EGL-1…has multiple mammalian killer gene counterparts  CED-3…human counterparts are called caspases which initiate apoptosis; protein ICE  CED-4…human counterpart called Apaf1 which promotes caspase activation  CED-9…comparable to the human oncogene BCL-2 which blocks cell suicide

12. Protein structure of Caspase family  Procaspase: N—prodomain---p20 ---p10 domain-C initiator caspase: 8, 9, 10 effector caspase: 3, 6, 7

14. How is this applied to human conditions?  Certain cancers…uncontrolled cell division versus uncontrolled cell destruction  ALS (Lou Gehrig’s disease)  Myocardial infarction  Cerebrovascular accident  Alzheimer’s disease  Embryological development

15. CED-4 translocates from mitochondria to nuclear envelope during programmed cell death All cells have been caused to initiate apoptosis. Red-CED-4 protein. Green-nuclear envelope protein lamin. In normal embryos, CED-4 is located in the mitochondria.

16. Summary  The 2002 Nobel Prize for Medicine or Physiology awarded for “genetic regulation of organ development and programmed cell death”  C. elegans used for its complexity but simplicity  Specific gene activation contributes to the programming of cells to die  This research can be applied to human gene control in development as well as certain health conditions

17. Programmed Cell Death in Eukaryotes

18. Cell death process three phases  Induction or initiation phase  Effector or decision phase activating hydrolase (protease and nuclease)  Degradation phase digestion of protein, fragmentation of DNA

19. Two main apoptotic pathways  The activation of death receptors  Mitochondria pathway common pathway: activation of caspase cascade

20. Major Apoptotic Pathways in Mammalian Cells Hengartner, M.O. 2000. Nature. 407:770. Green, D. and Kroemer, G. 1998. Trends Cell Biol. 8:267. Mitochondrial Pathway Death Receptor Pathway FasL Caspase 3 DD D D Fas/Apo1 /CD95 FADD Procaspase 8 DISC Caspase 8 BID oxidants ceramideothers Bcl-2 D Cytochrome c dATP Procaspase 9 Apaf -1 dATP Apaf -1 Caspase 9 Procaspase 3 apoptosome DNA damage Cellular targets ApoptosisOxygen Society Education Program Tome & Briehl 3

22. Mechanisms of Apoptosis

23.  The extrinsic or death receptor pathway  Initiated by binding of a death-inducing ligand to a Cys-rich repeat region in the extracellular domain of a death receptor  Death receptors such as Fas and the TNF receptor are integral membrane proteins with their receptor domains exposed at the surface of the cell  Binding of the complementary death activator (FasL and TNF- , respectively) transmits a signal (via an adaptor protein) to the cytoplasm that leads to  activation of caspase-8  Caspase-8 (like caspase-9) initiates a cascade of caspase activation leading to cell death  Example: when cytotoxic T-cells recognize (bind to) their taget:  they produce more FasL at their surface  this binds with Fas on the surface of the target cells and starts the cascade that leads to its death by apoptosis

24. Schematic for death receptor TNF or Fas ligand interact with death receptor Recruitment of adaptor molecules (FADD) Activating caspase 8 cleave Bid directly activating caspase 3 cleave Bid and caspase 7 translocate to mitochondria bcl-2 cyto C release

25. Fas and Related Proteins with Death Domains

26. Fas Signaling Pathway

27. TNF Signaling Pathway

28. Mitochondria pathway 1. The stimuli leading to cell death (growth factor deprivation,ionizing radiation and several chemical agents) 2. mitochondrial membrane permeabilization release of cytochrome C fromation of apoptosome(Apaf-1,cyto C, dATP) 3. Activating caspase 9 4. Activating caspase-3, -7,-6, cleave 45KD subunit of the DFF 5. Release DFF40 (CAD mouse homolog) with nuclease activity

29. Mechanisms of Apoptosis  The intrinsic or mitochondrial pathway  In a healthy cell, the outer membrane of mitochondria express the protein Bcl-2 on its surface  Bcl-2 is bound to Apaf-1 ( “ apoptotic protease activating factor-1)  Internal damage to cells (e.g. from reactive oxygen species) causes:  Bcl-2 to release Apaf-1  a related protein, Bax, to penetrate mitochondrial membranes causing  cytochrome c and other proteins such as Smac/DIABLO and AIF to leak out  The released cytochrome c and Apaf-1 binds to molecules of caspase-9 forming an aggregate called the apoptosome  This stimulates/amplifies activation of caspase-9 and downstream apoptotic events  Smac/DIABLO neutralizes IAP ( “ inhibitor of apoptosis ” ) proteins and allows caspase activation to proceed  AIF induces chromatin condensation and DNA fragmentation

30. Mitochondria in Apoptosis

31. Major Players in Apoptosis  Caspases  Cysteine proteases  Recognize tetrapeptide motifs and cleaves at the carboxyl side of an aspartate reside (caspase = cysteine aspartate-specific protease)  Synthesized as zymogens ( “ procaspases ” ) that are activated by caspase-mediated cleavage  Initiator caspases (e.g. caspase-8 and caspase-9) start a cascade of increasing caspase activity by processing and activating downstream effector caspases (e.g. caspase-3, -6 and -7)  activated effector caspases cleave and inactivate vital cellular proteins and induces morphological changes that are characteristic of cells undergoing apoptosis

32. Major Players in Apoptosis  Cell death receptors  members of the tumor necrosis factor (TNF) receptor family  can have pleiotropic action depending on cell type and signals received – i.e., can trigger cell proliferation, differentiation or death  Activated by structurally-related ligands of the TNF ligand family  e.g. CD95 (also called Fas or APO-1) – contains a cytoplasmic region called the death domain which transmits the signals via an adaptor protein to initiator caspases  Four TRAIL/APO-2L receptors identified – however, two of them, DcR1 and DcR2 lack the death domain and cannot induce apoptosis  acts as decoys to inhibit TRAIL/APO-2L-mediated apoptosis  Decoy receptor for FasL (DcR3) – found overexpressed in lung and colon tumors

33. Major Players in Apoptosis  Adaptor proteins  Form bridges between cell death effectors (caspases) and the cell death regulators (death receptors and Bcl-2 family members)  Death receptors of the TNF-R family interact with adaptor proteins via the death domain (DD) of the receptor and the death effector domain (DED) of the adaptor.  e.g. the DD of the CD95 effector is associated with the adaptor molecule designated FADD (Fas-associating death domain protein)  interactions between the DD of CD95 and FADD results in pro-caspase 8 aggregation and activation

34. Major Players in Apoptosis  The Bcl-2 family  Plays an integral role in regulating mitochondrial outer membrane permeabilization, and thus the release of key effector proteins including cytochrome c and Smac/DIABLO from the mitochondrial intermembrane space  At least 20 Bcl-2 related proteins identified in mammalian cells  Bcl-2 family members share one or more Bcl-2 homology (BH) domains and are divided into two main groups – whether they promote or inhibit apoptosis  Anti-apoptotic members such as Bcl-x L, Bcl-w and Boo/Diva share at least three or four regions of extensive amino acid sequence similarity with the prototypical Bcl-2 (BH1 – BH4 regions)  Pro-apoptotic members usually posses only a BH3 region – e.g. Bad, Bik/Nbk/Blk, and Bid  Bax and Bak – examples of pro-apoptotic multidomain proteins

35. Major Players in Apoptosis  The Bcl-2 family (cont ’ d)

36. Major Players in Apoptosis  Inhibitor of apoptosis (IAP) proteins  Suppress apoptosis triggered by wide variety of stimuli – e.g. viral infection, chemotherapeutic drugs and components of the TNF-  /Fas signaling pathway  Characterized by one or more repeats of highly conserved ~70 amino acid domain termed baculoviral IAP repeat (BIR)  Currently six human IAP members – c-IAP1, c-IAP2, XIAP, NIAP, Livin and Survivin  Most of IAP family members have been shown to interact with caspases, inhibiting their activity  Play a role in pathological conditions – e.g. NIAP gene originally identified in patients with spinal muscular atrophy; XIAP and c- IAP1 are found in most cancer cell lines; Survivin is overexpressed in nearly all human tumors but is rarely present in adult tissues

37. Other molecules of Apoptosis  Apoptosis-inducing factor (AIF)  Flavoprotein that is normally located in the intermembrane space of mitochondria.  When cells receive a signal for apoptosis  AIF is released from the mitochondria  AIF translocates into the nucleus and causes  nuclear fragmentation and cell death  DNA destruction mediated by AIF is not blocked by caspase inhibitors and is thus considered a caspase- independent pathway

38. Other molecules of Apoptosis  Smac: The second mitochondria-derived activator of caspase, 239aa’, N-terminal 55aa’ as mitochondria signal. It normally resident in mitochondria but is released into the cytosol when cell undergo apoptosis. Mechanism:binding to IAP

39. Other molecules of Apoptosis  Omi: most recently discovered proapoptotic protein released from mitochondria and shows much similarity to Smac.  The localization of Omi protein

40. Methods of Apoptosis Analysis  Molecular expression and caspase mediated cleavage  Membrane associated changes (Annexin V and Ceramide-sphingomyelin pathways)  Nuclear morphology (condensation, apoptotic bodies, endonuclease activity)  Analysis: IHC, flow cytometry, DNA electrophoresis, cell staining, TUNEL assay

41. Apoptosis: Targets for Therapy 1) Anti-bcl-2 therapy with antisense technologies 2) Gene therapy with p53 and p27 or p21 vectors 3) Anti-p27 ubiquination therapies 4) Induce caspases or bax function

42. DNA Repair and Clinical Syndromes : Increased Sensitivity; Chromosomal Instability and Increased Cancer Risk

43. XP: Skin Cancer Incidence Rapid Secondary to NER Faulty Repair of UV-induced DNA Damage and Genetic NER Mutations