molecular mechanism of cell death and cell proliferation

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molecular mechanism of cell death and cell
proliferation

• Fate of cell
• Undergoing cell cycle
• differentiating to specific cell
• death

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

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Apoptosis
Why is dying so important?

• Physiologically embyro stage, CNS development,
  
• thymus atrophy,
  endometrium
  
• desquamating
• Pathologically tumor, Parkinsons disease,
  Alzheimers disease

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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)

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Sydney Brenner H. Robert Horvitz John
Sulston
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Caenorhabditis elegansThe Perfect Model

• C. eleganss 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

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• 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?

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The C. elegans Organism
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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

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So whats the big deal with studying worms?

• EGL-1has multiple mammalian killer gene
  counterparts
• 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

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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

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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.
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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

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Programmed Cell Death in Eukaryotes
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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

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Two main apoptotic pathways

• The activation of death receptors
• Mitochondria pathway
• common pathway
• activation of caspase cascade

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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
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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 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

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Schematic for death receptor

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

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Fas and Related Proteins with Death Domains
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Fas Signaling Pathway
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TNF Signaling Pathway
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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
  activity

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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

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Mitochondria in Apoptosis
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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

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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

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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

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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-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

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Major Players in Apoptosis

• The Bcl-2 family (contd)

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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
  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

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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

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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.
• Mechanismbinding to IAP

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Other molecules of Apoptosis

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

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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

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