1. 细胞增生和凋亡的分子机制

2. Fate of cells
Undergoing cell cycle －proliferation
differentiating to specific cell
Death

3. 细胞分裂增生的研究
20世纪60年代 细胞周期分子机制的研究
Hartwell L, Nurse P, Hunt T
2001 Nobel prize for physiology and medicine

4. 细胞增生（proliferation）
细胞在严密调控下有序进入细胞周期而分裂繁殖。
细胞增殖的意义
细胞增殖为细胞分化提供来源
补充因死亡而消失的细胞

5. 细胞凋亡的研究始于上世纪60年代，
上世纪80年代在线虫首次阐明
2002年诺贝尔医学和生理学奖

6. Nobel Prize for Physiology and Medicine 2002
For “genetic regulation of organ development and programmed cell death”
Sydney Brenner (English)
H. Robert Horvitz (American)
John Sulston (English)

7. Sydney Brenner H. Robert Horvitz John Sulston

8. 细胞凋亡的概念
机体细胞在生理或病理状态下发生的自发性的程序性死亡
细胞凋亡的意义
清除错误细胞
清除多余细胞，使各组织的细胞达到平衡

9. 第一节 生长因子信号转导活化细胞周期是细胞增生的分子机制
第一节 生长因子信号转导活化细胞周期是细胞增生的分子机制
一、细胞经历细胞周期而增生

10. The 4 phases of a typical cell cycle and the events occurring during each phase are outlined
M phase is the period when cells prepare for and then undergo cytokinesis. During mitosis the chromosomes are paired and then divided prior to cell division.
G1phase corresponds to the gap in the cell cycle that occurs following cytokinesis. During this phase cells make a decision to either exit the cell cycle and become quiescent or to continue dividing.
G0 phase Quiescent and terminally differentiated cells are identified as being in G0 phase.
S phase is the phase of the cell cycle during which the DNA is replicated.
G2 phase is reached following completion of DNA replication. During G2 the chromosomes begin condensing, the nucleoli disappear and two microtubule organizing centers begin polymerizing tubulins for eventual production of the spindle poles.

11. G1-S transition G2-M transition G1 晚期的限制点 G1-S转折的DNA损伤关卡
◆Two transitions (两个转折点)：
G1-S transition
G2-M transition
◆ Four checkpoints (细胞周期中的四个关卡)
G1 晚期的限制点
G1-S转折的DNA损伤关卡
G2-M转折的DNA损伤关卡
有丝分裂中期的关卡

12. 二、参与调控细胞周期进程的蛋白质 细胞周期蛋白 周期蛋白依赖性激酶 周期蛋白-周期蛋白依赖性激酶抑制因子 RB-DP1转录因子
调节CDK的蛋白激酶和磷酸酶
泛素和使蛋白质泛素化的酶

13. 三、调控蛋白协同作用调控细胞周期 Cdk4/6和Cdk2的活化－－限制点 Cdk1活化－－G2M checkpoint
APC介导的多泛素化蛋白降解－－有丝分裂中期checkpoint
DNA损伤关卡与G1及G2期停滞相关

14. 四、生长因子等通过信号转导调控细胞周期
1. G0期进入细胞周期
2. G1期细胞也需要生长因子

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

16. Programmed Cell Death in Eukaryotes

17. 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

18. 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?

19. The C. elegans Organism

20. The Fundamental Genes Being Examined
Egl Ced Ced Ced 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

21. 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

22. Major Players in Apoptosis---caspase
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
Procaspase:
N—prodomain---p20 ---p10 domain-C

23. Initiator caspases (e. g
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

25. Major Players in Apoptosis---Bcl-2 family
Plays an integral role in regulating mitochondrial outer membrane permeabilization, and thus the release of key effector proteins including cyto c and Smac/DIABLO from the mit 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-xL, 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-Bak – examples of pro-apoptotic multidomain proteins

26. Bcl-2 family

27. Major Players in Apoptosis---adaptor protein
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

28. Major Players in Apoptosis---IAP
Suppress apoptosis triggered by wide variety of stimuli – e.g. viral infection, chemotherapeutic drugs and components of the TNF-a/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

29. 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

30. 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
Smac: second mitochondria-derived ativator of
caspase
DIABLO: direct IAP-binding protein with low pI

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

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

33. Two main apoptotic pathways
The activation of death receptors
（死亡受体途径）
Mitochondria pathway
（线粒体途径）
common pathway:
activation of caspase cascade

34. Major Apoptotic Pathways in Mammalian Cells
Mitochondrial Pathway
Death Receptor Pathway
FasL
Caspase 3 D
Fas/Apo1
/CD95
FADD
Procaspase 8
DISC
Caspase 8
BID
oxidants
ceramide
others
Bcl-2
Cytochrome c
dATP
Procaspase 9
Apaf -1
Caspase 9
Procaspase 3
apoptosome
DNA damage
Cellular targets
DISC: death inducing signal complex
FADD: Fas associated protein with death domain
Hengartner, M.O Nature. 407:770.
Green, D. and Kroemer, G Trends Cell Biol. 8:267.
Apoptosis Oxygen Society Education Program Tome & Briehl 3

35. 外源性的死亡受体途径 Fas：单跨膜受体，N端在胞外，DD位于胞内，分布广泛 FasL:单跨膜受体，在细胞表面形成三聚体，细胞毒T细胞表面
FADD：Fas-associated death domain
DD and DED (death effector domain)
DISC：FasL—Fas—FADD

36. Fas and Related Proteins with Death Domains
Death receptor: Fas, TNFR1, TNFR2, DR3, DR4, DR5,
DcR1,DcR2
TRADD: TNF receptor-associated death domain

37. Mechanisms of Apoptosis
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-a, 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 targets:
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

39. Cell death receptors
members of TNFR 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
4 TRAIL/APO-2L receptors identified – 2 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

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

41. Fas Signaling Pathway Fas Signaling Pathway
Fas/APO-1/CD95 (36 kDa) is a member of the tumor necrosis factor (TNF) receptor superfamily, a family of transmembrane receptors that also includes p75 neurotrophin receptor, TNFRI, and a variety of other cell surface receptors. Fas has been shown to be an important mediator of apoptotic cell death, as well as being involved in inflammation. Binding of the Fas ligand (Fas-L) induces trimerization of Fas in the target cell membrane. Activation of Fas causes the recruitment of Fas-associated protein with death domain (FADD) via interactions between the death domains of Fas and FADD. Pro-caspase-8 binds to Fas-bound FADD via interactions between the death effector domains (DED) of FADD and pro-caspase-8 leading to the activation of caspase-8. Activated caspase-8 cleaves (activates) nine other pro-caspases, in effect beginning a caspase cascade that ultimately leads to apoptosis. Caspases cleave nuclear lamins, causing the nucleus to break down and lose its normal structure. Fas-induced apoptosis can be effectively blocked at several stages by either FLICE-inhibitory protein (FLIP), by Bcl-2, or by the cytokine response modifier A (CrmA). Moreover, caspase-8 can activate Bid which is then able to associate with the mitochondria and promote leakage of cytochrome c. In the presence of dATP, cytochrome c complexes with and activates Apaf-1. Activated Apaf-1 binds to downstream caspases, such as pro-caspase-9, and processes them into proteolytically active forms. This begins a caspase cascade resulting in apoptosis. In addition, Smac/Diablo is released from the mitochondria and blocks IAP proteins that normally interact with caspase-9 to inhibit apoptosis.

42. TNFR-TNFa 凋亡途径 TNFR1单跨膜受体，分布广泛 TNFa由活化的巨噬细胞和淋巴细胞产生
TNFR1胞内DD募集TRADD, 后者与TRAF2和RIP形成复合物，
RIP活化NFkB，通过FLIP抑制caspase8活化
DISC 复合物1
FLIP: Fas associated death domain-like interleukin beta converting enzyme inhibitory protein

43. TNF Signaling Pathway TNF Signaling Pathway
When bound to tumor necrosis factor (TNF), the TNF receptor (TNFR) (55 kDa) transduces growth regulatory signals into the cell. TNF is mitogenic for normal cells; however, TNF initiates programmed cell death (PCD) or apoptosis in transformed cells causing DNA fragmentation and cytolysis. Functional studies have identified a conserved region within the receptor, termed the death domain (DD), a protein-protein interaction motif that is necessary to transmit the apoptotic signal. The TNF-induced survival pathway is mediated by the transcription factor NF-B. Activation of NF-B occurs via phosphorylation of IB at Ser and Ser, resulting in the dissociation and subsequent nuclear localization of active NF-B. Recent studies have demonstrated that cells in which the NF-B signaling pathway is blocked are more likely to undergo apoptosis in response to TNF. Therefore, the availability of NF-B may play a critical role in the ability of TNF to act as an apoptosis-inducer and anti-tumor agent.

44. Mitochondria pathway 内源性凋亡的诱因： －－失去赖以生存的生长因子或激素支持 －－脱离原来的生长环境 －－DNA损伤等
1. The stimuli leading to cell death
(growth factor deprivation, ionizing radiation and several chemical agents)
2. mitochondrial membrane permeabilization
release of cytochrome C
formation of apoptosome(Apaf-1,cyto C, dATP)
（apoptotic proteonase activating factor）
3. Activating caspase 9 by Apaf1 CARD （caspase recruitment domain）
4. Activating caspase-3, -7,-6, cleave 45KD subunit of the DFF
5. Release DFF40 (CAD mouse homolog) with nuclease activity

45. Three models of translocation for CytC
1.Bax和Bak在mit外膜打孔
2.VDAC在外膜形成通道
3.外膜上ATP-ADP转运蛋白孔道保持开 放

46. The intrinsic or mitochondrial pathway
In a healthy cell, the outer membrane of mit 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 cyto c and other proteins such as Smac/DIABLO and AIF to leak out
The released cyt 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

48. Mitochondria in Apoptosis
Oxidative damage due to the endogenous production of reactive oxygen species by mitochondria can lead to cell death via apoptosis or necrosis. Increases in cytosolic Ca2+ levels due to ion channel-linked receptors, such as the excitatory amino acid neurotransmitter glutamic acid, can induce permeability transition (PT) in the mitochondrial membrane. PT constitutes the first rate-limiting event of the common pathway of apoptosis. Upon PT, apoptogenic factors leak into the cytoplasm from the mitochondrial intermembrane space. Two such factors, cytochrome c and apoptosis inducing factor (AIF), begin a cascade of proteolytic activity that ultimately leads to nuclear damage (DNA fragmentation, DNA mutations) and cell death. Cytochrome c, a key protein in electron transport, appears to act by forming multimeric complexes with Apaf-1, a protease, which in turn activates pro-caspase-9, and begins a cascade of activation of downstream caspases. Bcl-2 and Bcl-x can prevent pore formation and block the release of cytochrome c from the mitochondria and prevent activation of the caspase cascade and apoptosis. PT is also related to the mitochondrial generation of reactive oxygen species, which play a role in the degradation phase of apoptosis (i.e. plasma membrane alterations). In contrast, high levels of oxidative stress can lead to PARP activation. Activated PARP causes conformational changes in the chromatin structure allowing the recruitment of DNA repair proteins or other transcription factors. This process consumes large amounts of NAD+, effectively causing a depletion of ATP leading to necrosis.

49. Positive regulation：caspase 激活和 caspase级联放大作用
三、Positive and negative regulation for apoptosis and crosstalk (凋亡途径的正负调节和串话)
Positive regulation：caspase 激活和 caspase级联放大作用
Negative regulation：FLIP抑制caspase8活化
Bcl-2抑制凋亡
IAP含有BIR（baculovirus IAP repeat）
BIR2抑制caspase3 和7；
BIR3抑制caspase 9