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molecular mechanism of cell death and cell proliferation


molecular mechanism of cell death and cell proliferation Fate of cell Undergoing cell cycle differentiating to specific cell death Major Players in Apoptosis The Bcl ... – PowerPoint PPT presentation

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Title: molecular mechanism of cell death and cell proliferation

molecular mechanism of cell death and cell
  • Fate of cell
  • Undergoing cell cycle
  • differentiating to specific cell
  • death

  • ?????????
  • 20??60?? ???????
  • Hartwell L, Nurse P, Hunt T
  • Nobel prize for physiology and
  • medicine

Why is dying so important?
  • Physiologically embyro stage, CNS development,
  • thymus atrophy,
  • desquamating
  • Pathologically tumor, Parkinsons disease,
    Alzheimers disease

Nobel Prize for Physiologyand 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)

Sydney Brenner H. Robert Horvitz John
Caenorhabditis elegansThe Perfect Model
  • C. eleganss complexity but simplicity
  • A nematode approximately one mm long containing
    blood, muscle, heart, nervous, as well as other
  • 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

  • Approximately 40 percent of the worms genes are
    also found in humans
  • Responds to taste, smell, temperature, touch, and
    possibly light
  • So, where did the other 131 cells go?

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

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

Protein structure of Caspase family
  • Procaspase
  • Nprodomain---p20 ---p10 domain-C
  • initiator caspase 8, 9, 10
  • effector caspase 3, 6, 7

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How is this appliedto human conditions?
  • Certain cancersuncontrolled cell division versus
    uncontrolled cell destruction
  • ALS (Lou Gehrigs disease)
  • Myocardial infarction
  • Cerebrovascular accident
  • Alzheimers disease
  • Embryological development

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.
  • 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

Programmed Cell Death in Eukaryotes
Cell death processthree phases
  • Induction or initiation phase
  • Effector or decision phase
  • activating hydrolase (protease and nuclease)
  • Degradation phase
  • digestion of protein, fragmentation of DNA

Two main apoptotic pathways
  • The activation of death receptors
  • Mitochondria pathway
  • common pathway
  • activation of caspase cascade

Major Apoptotic Pathways in Mammalian Cells
Hengartner, M.O. 2000. Nature. 407770. Green, D.
and Kroemer, G. 1998. Trends Cell Biol. 8267.
Apoptosis Oxygen Society Education Program
Tome Briehl 3
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Mechanisms of Apoptosis
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
  • 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 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

Schematic for death receptor
  • TNF or Fas ligand interact with death
  • Recruitment of adaptor molecules
  • Activating caspase 8
  • directly activating caspase 3 cleave
  • and caspase 7
    translocate to mitochondria

  • bcl-2

  • cyto C release

Fas and Related Proteins with Death Domains
Fas Signaling Pathway
TNF Signaling Pathway
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
  • Release DFF40 (CAD mouse homolog) with nuclease

Mechanisms of Apoptosis
  • The intrinsic or mitochondrial pathway
  • In a healthy cell, the outer membrane of
    mitochondria express the protein Bcl-2 on its
  • 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

Mitochondria in Apoptosis
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

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

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

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
  • 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 and Bak examples of pro-apoptotic
    multidomain proteins

Major Players in Apoptosis
  • The Bcl-2 family (contd)

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-a/Fas signaling
  • 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

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

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
  • Mechanismbinding to IAP

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

Methods of Apoptosis Analysis
  • Molecular expression and caspase mediated
  • 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

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
DNA Repair and Clinical Syndromes Increased
Sensitivity Chromosomal Instability and
Increased Cancer Risk
XP Skin Cancer Incidence Rapid Secondary to NER
Faulty Repair of UV-induced DNA Damage and
Genetic NER Mutations
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