Apo: Greek: from, away

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APOPTOSIS
Apo Greek from, away Ptosis Greek fall,
falling A specific type of cell death first
described in 1972 by Kerr, et al in the British
Journal of Cancer Programmed Cell Death
(Lockshin and Williams, 1964) Developmental
Event, may or may not be apoptotic
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• Essential for proper development of the
  multicellular organism
• "Webbed" tissue between the digits of developing
  human embryos is removed by apoptosis
• Tadpole tail removed by apoptosis during
  development

• Neuronal cell death by apoptosis is fundamental
  for CNS development

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• Essential for the proper functioning of the
  mature organism

•          Cells of the intestinal wall die by
  apoptosis to be
• replaced by new cells
•            Skin cells (keratinocytes) undergo
  apoptosis and
• migrate to the surface where they form the
• protective outer layer of skin
•          

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• Essential for the removal of cells that threaten
  homeostasis

 

• Virus-infected cells

.Cells whose
DNA has been damaged by
UV light, exposure to radiation,
chemotherapy

• Autoreactive T cells with the
• potential to attack "self" are
• removed by apoptosis
• At the termination of an immune
• response when they are no
• longer needed
•  

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Apoptosis or Programmed Cell Death (PCD)

• A genetically controlled process for cells to
  commit suicide.
• Distinct morphological and biochemical
  signatures of apoptosis are
• DNA fragmentation
• Chromatin condensation
• Cell shrinkage
• Plasma membrane blebbing.
• The term apoptosis was coined by Kerr, Wyllie
  and Currie (1972) to distinguish
• between the cell deaths that occur in
  homeostasis and pathological cell deaths such as
• Trauma and ischemia.
• Necrosis occurs when cells are exposed to extreme
  physiological Conditions
• result in damage to the plasma membrane.
• Usually triggered by agents like complement and
  lytic viruses, hypothermia, hypoxia

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Apoptosis in Development and Disease

• Role in animal development
• Sculpting structures e.g. formation of digits,
  neural tubes.
• Deleting unneeded structures e.g. vestigial
  structures like pronephric tubes
• Controlling cell numbers like neurons and
  oligodendrocytes.
• Function as quality control, eliminating harmful
  cells e.g self reacting lymphocytes
• Producing differentiated cells without
  organelles. E.g formation of RBC,
• differentiated keratinocytes.
• Role in Diseases
• Over-active apoptosis examples are
  neurodegenerative diseases (eg Alzheimer's),
• immunodeficiency diseases (eg. AIDS), Stroke
  and coronary heart disease.
• Under-active apoptosis examples are cancer,
  auto-immune and chronic
• inflammatory diseases (eg rheumatatoid
  arthritis). The latter may be caused by
• defects in the mechanisms of apoptotic-cell
  clearance.

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Apoptosis is an Essential Process

• Apoptosis (programmed cell death) plays an
  important rolein normal development and
  homeostasis
• Apoptosis is activated through two principal
  signaling pathways intrinsic and extrinsic
• Cancer is often initiated by DNA damage
• Normal cells undergo apoptosis in response to
  stress-inducing events in the cell, such as DNA
  damage
• Dysregulation of apoptosis is critical for cancer
  development and tumor cell survival

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Cell Death Pathways
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The Nobel Prize in Physiology or Medicine 2002
Sydney Brenner of the Salk Institute used the
nematode Caenorhabditis elegans, which became a
multicellular model experimental system, to
follow cell division and differentiation from the
fertilized egg to the adult via microscopic
observation.  He demonstrated that a specific
gene mutation, induced by ethyl methane
sulfonate, could be linked to a specific effect
in nematode organ development. His work on
nematodes created an experimental system that
laid the foundation for the study of
apoptosis. John Sulston of the Wellcome Trust
Institute in England mapped cell lineages, where
every cell division and differentiation could be
followed in the development of C. elegans.  
There are only 959 cells in an adult nematode. He
showed that specific cells lineages (nerves)
undergo programmed cell death, as an integral
part of the normal differentiation
process. Robert Horvitz of MIT discovered and
characterized key genes controlling cell death in
C. elegans. He identified the first two bona fide
"death genes", ced-3 and ced-4. Functional ced-3
ced-4 genes are a prerequisite for cell death
to be executed. Another gene, ced-9, protects
against cell death by interacting with ced-3 and
ced-4.   He has shown how these genes interact
with each other in the cell death process and
that corresponding genes (a ced-3-like gene)
exists in humans.  
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Role of Apoptosis in development of
Caenorhabditis elegans                          
              The C. elegans genome is 9.7 x
107 bp and is now fully sequenced (24 x E. coli,
1/38 of human). The 19099 genes include 790
seven-pass transmembrane receptors, 480 zinc
finger proteins, and 410 protein kinases see
News and Views, Nature 396 620-621 (1998). The
life cycle of C. elegans from egg to sexual
maturity (and new eggs) is about 3 days. The
adult hermaphrodite consists of exactly 959
somatic cells of precisely determined lineage and
function. Individual cells are named and their
relationships to their neighbours are known.
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Overall the 959 cells of adult C.elegans arise
from 1090 original cells exactly 131 cells
undergo programmed cell death in the wild type
worm. Of the 1090, 302 are neurons, and many of
the programmed deaths also lie in the neuronal
lineage. Identification of Cell Death
genes ced-3, ced-4 and ced-9 (C.elegans cell
death genes) Robert Horvitz egl-1 (C.elegans
egg laying defective). The egl-1 gene was the
first gene discovered in the programmed cell
death system, as a gain of function mutation
causing unscheduled death of two neurons
innervating the vulva, and hence the egg laying
defect. Ced-4 was then found as the extragenic
suppressor of egl-1. Ced-3 suppressed the
persistence of cell corpses in phagocytosis
defective animals. Ced-1,-2,-5,-6,-7,-10 all turn
out to be phagocytosis or clean-up genes rather
than acting in the causative pathway of cell
death. Ced-3 has a mammalian counterpart,
originally known as ICE (Interleukin 1ß
Converting Enzyme), now termed Caspase 1 (Cys
catalytic Asp targeting protease). Thirteen
caspases are known in mammalian systems, and have
conserved sequence and subunit structure of
these four play key effector roles in apoptosis
and four are initiators in the activation
process. Ced-4 acts as an adapter for caspase
activation the mammalian counterpart Apoptosis
activating factor Apaf-1.
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Genetic Mechanism of Apoptosis
The apoptotic system in C.elegans.
Meier et.al Nat Reviews, Vol 407, 2000.

• Molecular nature of the PCD came from genetic
  studies done on C.elegans.
• 131 cells out of 1090 cells undergo PCD genetic
  screens of mutants identified Egl-1, CED3, CED4
  and CED 9 to be involved.
• Egl-1, CED3, CED4 are death promoters since
  their loss of function results in survival of all
  131 doomed cells.
• CED 9 is death inhibitor since its loss of
  function causes embryonic lethality by massive
  ectopic cell deaths.

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Molecular Identities of apoptotic genes
Kauffmann and Vaux. Oncogene,22,2003

• Fundamental components of apoptotic pathways are
  conserved across the species.
• Ced -9 is similar to human oncogene Bcl2.
• Ced-3 has a mammalian counterpart, originally
  known as ICE (Interleukin 1ß
• Converting Enzyme), now termed Caspase 1.
• Ced-4 acts as an adapter for caspase activation
  the mammalian counterpart
• Apoptosis activating factor Apaf-1.

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Caspases Cysteine Aspartate Specific ProteASEs

• Caspases more visible "hallmarks" of
• apoptosis.
• Highly specific proteases that cleave
• proteins exclusively after aspartate
• residues.
• Sequence of 3 amino acids before
• aspartate determines substrate
• specificity.
• Function Regulate proteolysis during
• apoptotic cell death.
• Synthesized as inactive zymogens.
• Upon activation, twice cleavage at Asp-
• X site releases large, small subunits.
• Active caspases are tetramers two large

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• Caspase types Based on sequence of activation
• 1. Initiator (Activator) caspases
• First to be activated on commitment of cell to
  die
• Cleave activate effector caspases
• prodomains Long Contain regulatory sequences
• Effector (Executioner) caspases
• Cleave activate cellular substrates
• Prodomains Short. No known
• regulatory sequences
• Cytokine processors (Inflammatory)
• Prodomains Long Contain regulatory sequences

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• Expressed widely in cells in inactive proenzyme
  form (pro-caspase)
• Must be activated for proteolysis

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• Responsible for the more visible "hallmarks" of
  apoptosis
• More Notable Members
• Caspases 3, 6, 7 Important downstream effector
  caspases
• Caspase 8 initiator in death receptor pathway
• Caspase 9 Initiator in intrinsic pathway.
  Activated by conformational change

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Effector Caspases Activate downstream caspases
and act on Various cellular substrates
In the first example, the effector caspases
cleave an inhibitor or an effector protein. An
example of this would be CAD (Caspase-Activated
Deoxyribonuclease), and ICAD (Inhibitor of CAD).
When ICAD binds to CAD, CAD is kept inactivated.
However, active effector caspases cleave ICAD
which then releases CAD. CAD can then cleave the
DNA into fragments (forming the characteristic
DNA laddering of apoptotic cells). The second
example illustrates that the effector caspases
can also cleave structural proteins, such as the
nuclear lamins. Nuclear lamins maintain the
integrity of the nucleus, but when they are
cleaved by the effector caspases, the nucleus
condenses (another characteristic of apoptotic
cells). Finally, in the third example, effector
caspases can cleave off the auto-inhibitory
domains of certain proteins. A good example of
this would be PAK2. When the effector caspase
cleaves off the auto-inhibitory domain of PAK2,
PAK2 now becomes constitutively active, playing a
role in the membrane blebbing that is
characteristic of apoptotic cells.
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BCL-2 BCL-2 is a human proto-oncogene located on
chromosome 18. Its product is an integral
membrane protein (called Bcl-2) located in the
membranes of the endoplasmic reticulum (ER),
nuclear envelope, and in the outer membranes of
the mitochondria.
The gene was discovered as the translocated locus
in a B-cell lymphoma In the cancerous B cells,
the portion of chromosome 18 containing the BCL-2
locus has undergone a reciprocal translocation
with the portion of chromosome 14 containing the
antibody heavy chain locus. This t(1418)
translocation places the BCL-2 gene close to the
heavy chain gene enhancer.
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Bcl-2 Members

• Bcl-2 first protooncogene gene to be
• discovered from the family cloned from
• t(1418) breakpoint in follicular lymphoma
• Presence of Bcl-2 homology domains BH
• domains.
• BH1 and BH2 in death antagonists, allow
• heterodimerization with Bax to repress
• apoptosis
• BH3 in death agonists (eg. Bax, Bak) allows
• heterodimerization with Bcl-XL and Bcl-2
• to promote apoptosis
• BH4 conserved in apoptosis antagonist
• members (eg. Bcl-XL) but absent in
• apoptosis agonists (except Bcl-Xs), this
• domain allows interaction with death

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The pro-survival family

• Their hydrophobic carboxy-terminal domain helps
  target them to the cytoplasmic face of three
  intracellular membranes.
• Bcl2 is an integral membrane protein, even in
  healthy cells, whereas Bcl-w and Bcl-xL only
  become tightly associated with the membrane after
  a cytotoxic signal.
• A hydrophobic groove, formed by residues from
  BH1, BH2 and BH3, can bind the BH3 a-helix of an
  interacting BH3-only relative.

The BH3 only family

• BH3-only proteins seem to be sentinels that are
  charged with triggering apoptosis in response to
  developmental cues or intracellular damage.
• They are thought to act by binding to and
  neutralizing their pro-survival relatives.
• They act upstream of Bax sub-family of proteins.

The Bax family

• Bax is a cytosolic monomer in healthy cells, but
  it changes conformation during apoptosis,
  integrates into the outer mitochondrial membrane
  and oligomerizes.
• Whereas, Bak is an oligomeric integral
  mitochondrial membrane protein, but it too
  changes conformation during apoptosis and might
  form larger aggregates.
• Bax and Bak oligomers are believed to cause
  permeabilization of the outer mitochondrial
  membrane, allowing efflux of apoptogenic
  proteins, leading to caspase activation.

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Pro-survival proteins can also act by inhibiting
Bax/Bak oligomerization

• Heterodimerization of Bcl-2 and Bax inhibits Bax
  function.
• Bcl-XS binding to Bcl-2 prevents Bcl-2 from
  binding to and neutralizing Bax.
• Also, heterodimerization with Bax and other
  pro-apoptotic members of the Bcl-2 family results
  in the the release of Apaf-1 from Bcl-xL and
  further activation of caspases.

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Interactions of Bcl-2 members

• Bcl2 family proteins regulate apoptosis
• via their effect on mitochondria.
• Antideath prodeath molecules
• e.g Bcl-2/Bax
• Prodeath prodeath molecules
• e.g Bid/Bax
• Multimerization of same molecules.
• e.g Bcl2/Bcl2, Bax/Bax.
• Either localised in the mitochondria
• or are induced by death signals.
• Translocation to mitochondria is
• facilitated by post translational
• modification
• Conformation change
• Caspase cleavage

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Bcl-2 family members can regulate apoptosis
related mitochondrial changes such as the
permeability transition pore (PT)

• The PT pore is a poly protein channel, comprising
  of VDAC and PBR on the outer membrane and ANT and
  cychlophilin D on the inner membrane.
• Cytochrome c release may occur through outer
  membrane rupture resulting from mitochondrial
  swelling caused by PT pore perturbation.
• Bcl-2, Bcl-xL and Bax have been shown to form ion
  channels in synthetic lipid membranes.
• Thus, Bcl-2 members having the BH1 and BH2
  domains may function by forming pores in
  organelles such as mitochondria or rather,
  stabilize or perturb the pre-existing channel, PT.

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Models for release of cyt c from mitochondria

• Involves closure of voltage
• dependent ion channel (VDAC) and
• impairment of ATP-ADP exchange.
• Opening of permeability transition
• pore (PTP).
• Channel formed in the outer
• mitochondrial membrane
• By Bax only
• By Bax and VDAC in combination
• By lipid or lipid protein complex

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The Role of Mitochondria in Apoptosis The
mitochondrion has been identified as playing a
central role in apoptosis 1. Bcl-2 and Bcl-XL
localize to the mitochondrial membrane.. 2. Bcl-2
can recruit kinases such as Raf-1 which are
involved in mediating Bcl-2's death antagonizing
action. 3. Bcl-2/-XL can recruit ced-4 and its
mammalian homolg, Apaf-1 to the mitochondrial
membrane. This may prevent ced-4/Apaf-1 from
activating caspases, thereby inhibiting
apoptosis. 4. Mitochondial proteins, when leaked
into the cytosol, are capable of inducing
apoptosis. During apoptosis, cytochrome c and
SMAC are released from the mitochonria and with
other factors, such as Apaf-1 (apoptosis protease
activating factor-1) and Apaf-3, lead to caspase
activation and apoptosis. Increased levels of
Bcl-2 can prevent the release of these molecules,
whereas, caspase inhibitors cannot. This
indicates the release of cytochrome c and SMAC is
downstream of Bcl-2 function but upstream of the
caspases. 5. Apoptosis is associated with a
change in the mitochondrial membrane potential, a
phenomenon known as permeability transition (PT).
The PT can be blocked by excess Bcl-2 but not by
inhibitors of caspases, indicating the PT is
downstream of Bcl-2 but upstream of caspase
activation. 6. Bcl-2, Bcl-XL, and Bax are capable
of forming selective ion pores in membranes.
Thus, they may form channels in the mitochondrial
membrane that could regulate the PT and the
release of molecules such as cytochrome c and
AIF.  
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BAD is phosphorylated by Akt, binds to 14-3-3 and
is degraded
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The BAX gene, the promoter of apoptosis, is
mutated in genetically unstable cancers of the
colorectum, stomach, and endometrium (1998)
Inactivating mutation of the pro-apoptotic gene
BID in gastric cancer (2004) (6) Inactivating
mutations of proapoptotic Bad gene in human colon
cancers. (4.3) 2004




Caspase-8 gene is frequently inactivated by the
frameshift somatic mutation 1225_1226delTG in
hepatocellular carcinomas (2005) 10 Somatic
mutations of CASP3 gene in human cancers 2004
occasional
Deletion and aberrant CpG island methylation of
Caspase 8 gene in medulloblastoma.2004
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The Death Receptor Family

• Cell surface cytokine receptors belonging
• to TNF/NGF receptor superfamily.
• Receptors are Type I transmembrane
• proteins intracellular C terminal tail,
• membrane spanning region, an
• extracellular ligand binding domain.
• Significant homology in 60-80 aa cytopl.
• sequence Death domain (DD).
• Death receptors are activated by their
• natural ligands group of cytokines
• belonging to TNF family.

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Death Receptor Signaling

• Ligand binding to the death receptors
• leads to oligomerization of these receptors.
• Recruitment of adaptor molecules through
• their Death Domains (DD)
• This protein protein interaction is restricted
• e.g Fas , DR4, DR5 recruits FADD whereas TNFR1
• recruits TRADD.
• Adaptor molecules recruit initiator caspases
• through interaction of their Death effector
• Domain (DED) and CARD domain in caspases.
• The resulting complex is called Death Inducing
• Signaling (DISC) complex.
• 
  

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• Role of Fas/FasL
• Deletion of activated T-cells at the termination
  of an immune response
• Cytotoxic T-cell mediated killing of cells
  (virus-infected, cancerous)
• Destruction of inflammatory or immune cells in
  immune-privileged sites (i.e, eyes, reproductive
  organs)

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Two pathways in TNFR signaling
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Pathways to Apoptosis
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Inhibitor of Apoptosis Family
1993 First member identified in
baculoviruses one or more BIR domains critical
for activity 1995 first mammalian IAP
identifiedNAIP positional cloning spinal
muscular atrophy 1997 XIAP, c-IAP1 and 2 shown
to inhibit caspase activity 1999 NMR structure
of XIAP, caspase binding active site 1999
yeast has IAPs but no caspases other
function Survivin cell cycle regulation
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The IAP proteins have been divided into three
classes (classes 1, 2, and 3) based on the
presence or absence of a RING finger and the
homology of their BIR domains.
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Inhibitors of Apoptosis (IAPs)

• IAPs function as intrinsic regulators of
• caspase cascade to apoptosis.
• Inhibit both initiator and
• effector caspases.
• First member identified in
• baculoviruses one or more
• BIR domains critical for activity

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• IAPs are characterized by 70-80
• aa Baculoviral IAP Repeat (BIR)
• domains.
• IAPs with multiple BIR domains
• use third BIR domain to inhibit
• caspase 9 and second BIR domain
• to inhibit caspase 3 ,7.
• BIR 1 domain has no caspase
• inhibiting activity and is least
• conserved.
• RING domain function as adaptors
• provide specificity for proteosomal
• degradation.
• NOD domain facilitates self association
• whereas coiled coil domain mediates
  interaction

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IAPs inhibit active caspases XIAP inhibits
caspases 3, 7 and caspase 9 through separate
domains. Its BIR2 domain (amino acids 163240)
with its NH2-terminal extension (amino acids
124162) inhibits caspases 3 and 7, whereas its
BIR3 domain (amino acids 241356) inhibits
caspase 9. These studies provided the basis for
developing IAP inhibitors that target the
caspase-binding pockets of the molecule.
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• IAP regulators
• Regulatory IAP-binding proteins were first
  identified in Drosophila. The proteins Reaper,
  Hid, Grim, and Sickle were shown to bind and
  inhibit the Drosophila IAP, DIAP1.
• Later, human IAP inhibitors identified called
  SMAC/DIABLO and Omi/HTRA2. These IAP inhibitors
  share a homologous sequence in their NH2 terminus
  that is responsible for binding and inhibiting
  IAPs.

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• SMAC and HTRA2
• Human SMAC and HTRA2 are mitochondrial proteins
  that are released along with cytochrome c during
  the disruption of the mitochondria. In their
  active state, SMAC and HTRA2 bind IAPs, thereby
  preventing their association with caspases.
• The IAP-inhibitory functions of the SMAC family
  of proteins are encoded in their NH2 terminus.
  Mutation of the NH2-terminal alanine to glycine
  abolishes the ability of the SMAC peptide to bind
  IAPs and exert its proapoptotic function. Similar
  results have been observed with HTRA2.
• Structural studies have demonstrated that SMAC
  binds XIAP at two distinct sites. The NH2
  terminus of active SMAC (residues 5659) binds
  the BIR3 pocket of XIAP and competitively
  inhibits the BIR3 domain from binding caspase 9.
• SMAC also bind the BIR2 domain of XIAP, but with
  lower affinity than that for BIR3. The mechanism
  by which SMAC disrupts the association of BIR2
  from caspase 3 is unclear.
• HTRA2 binds to the BIR3 domain of XIAP, but with
  weaker affinity than SMAC. In addition to
  inhibiting IAPs through binding the BIR3 pocket,
  HTRA2 can also cleave and inactivate multiple
  IAPs including XIAP,cIAP1, and cIAP2, but not
  survivin. Omi has serine protease activity

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Caspase-independent Apoptosis
Death associated with activation of lysosomal and
proteosomal Proteases and granzyme B and matrix
metalloproteases, calpains. Many programmed or
physiological deaths do not appear to depend on
caspase activation. Caspase 3 or 9 k. o. embryos
die only after embryonic day 10
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Techniques to detect Apoptosis

• Determination of Cell Viability Test
• Vital Dye exclusion assay
• Trypan blue, propidium iodide do not stain
  the viable cells
• Fluorescein diacetate, Calcein-AM stain the
  viable cells.
• 2) Measurement of cytosolic Leakage based on the
  fact that viable cells have intact
• cellular components.
• Lactate
  Dehydrogenase
• Pyruvate NADH
  Lactate NAD NADH
• NADH exhibits fluorescence at an excitation
  wavelength of 360 nm with emission at
• 450nm.
• Velocity of decrease of Ex 360nm/Em450 nm
  indicates conversion of NADH to NAD and
• hence activity of LDH.
• 3) Clonogenic assay Ability of cells to divide
  and form colonies is called clonogenic activity.
• However, integrity of pl memb is not compromised
  till late stage and hence not very useful

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Alterations in Plasma Membrane

• In normal cells, phosphoidylcholine and
  sphingomyelin are on external leaf and
• phosphotidylethnolamine and
  phosphatidylserine (PS) are in the inner leaflet
  .
• Redistribution of phospholipids in the plasma
  memb. is an early apoptotic change.
• Annexin V conjugated with flourophore like FITC
  can bind to exposed to PS in Ca
• dependent manner.
• Can be viewed microscopically or by flow
  cytometry.

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Annexin V staining
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Alterations in Cytosol caspase activation

• Activation of caspases hallmark of apoptosis,
  can be measured.
• Western blot for PARP, small subunits of
• caspase 3,7,8 and 9.
• Biochemical analysis of caspase activity
• Tetrapeptide sequence (recognition site
• of caspase) conjugated with report group
• like p-nitroanilide (pNA)
• 7- amino -4-methylcoumarin (AMC)
• 7-amino-4 triflouromethylcoumarin (AFC)
• Calorimetric/flourimetric measurements.
• Immunohistochemica
• Immunostaining/flourescent substrates in
  tissues/cells.
• l/immunoflorescent staining on paraffin sections.
• Cell permeable flourescent substrates PhiphiLux
  ( Oncogene research products)

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Bcl2 family Proteins

• Western blot with antibodies specific to
  individual members.
• If subcellular localisation is not relevant
  cells can be lysed in non-ionic detergent.
• If subcellular is needed, cellular components
  are subfractionated to obtain
• mitochondrial and cytosolic fractions.
• To determine Bax or Bak oligomers, crosslinking
  agents are added e.g
• disuccinimidyl suberate (DSS), Bis
  (Sulphosuccinimidyl) suberate (BS3) to the
• mitochondrial fraction suspended in isotonic
  buffer .
• The crosslinker is quenched with 1 M Tris-HCl
  ph7.5.
• Membranes are then lysed in radioimmunol
  precipitation assay (RIPA) buffer and
• cleared by centrifugation at 12000g analysis
  by SDS-PAGE and Western Blot.
• Oligomerisation is visualised using Western blot
  as a high molecular weight species.

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Mitochondrial Changes
Mitochondrial Changes
Mitochondrial release of Cyt C

• Cyt c release is the most common parameter of
  active mitochondrial pathway.
• Subcellular fractionation to yield mitochondrial
  fraction which is suspended in
• isotonic buffer with energising agents.
• Western Blot of both supernatant and pellet with
  cyt c antibody cyt c in sup and
• reduction in the pellet indicate cyt c
  release.
• ELISA Supernatant is prepared for ELISA for
  detection of Cyt c.
• Immunostaining
• cells undergoing apoptosis are washed and
  fixed Incubated with anti cyt c Ab
• Wash and incubation
  with sec Ab tagged with flourophore
  
• Diffused cytoplasmic staining indicates cyt c
  release.

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Mitochondrial Changes
Mitochondrial Transmembrane Potentials

• Mitochondria transmembrane potential is a
  functional parameter during apoptosis.
• Can be determined by lipophilic cations
  accumulation is potential dependent.
• Commonly used are Rhodamine 123 (Rh 123),
  DiOC6, Tetramethylrhodamine
• methyl ester (TMRM), JC-1.
• Probes can be directly added to cultured cells
  or isolated mitochondria, incubated
• for 15 min and harvested to be analyzed by
  flow or fluorescent microscopy.

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Changes in the Nucleus
Nuclear Condensation and Fragmentation

• Nuclear condensation and fragmentation can be
  visualised by staining with
• fluorescent dyes e.g Hoechst (bisbenzimide)
  and DAPI (46diamidino 2-phenylindole,
• dilactate).
• Intensity of staining in the nucleus is
  proportional to the extent of apoptosis due to
• increased permeability of the dyes.

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DNA content staining by Propidium Iodide
Control
Tamoxifen Treated
G1
G2
G0
S

• Degradation of nuclear DNA results in decrease
  in DNA content or hypoploidy.
• Cells are suspended in ice cold PBS, fixed with
  cold ethanol.
• Cells are permeabilised with Triton x100,
  stained with PI and analysed with flow.
• However, necrotic and other cellular debris can
  also get included.

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DNA Fragmentation

• Cleavage of DNA at nucleosomal sites
• results in 180-200 bp fragments which
• appears as DNA ladder.
• Quantitative measurement amount of
• fragmented DNA is proportional to
• frequency of apoptosis.

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Detection of DNA breaks by TUNEL assay
(Terminal deoxynucleotidyl transferase (TDT)
mediated dUTP nick end labeling)

• Based on the principle that TdT mediates
  incorporation of
• biotinylated dUTP into 3 OH ends of
  fragmented DNA .
• Cells are permeabilised using Triton X 100 or
  Proteinase K
• in case of formalin fixed tissue sections.
• Cells/Tissue sections are the incubatd with TdT
  and dUTP-
• biotin.
• After wash, cells /Tissues can be incubated
  either with
• FITC conjugated avidin for detection under
  fluoroscent
• microscope or avidin-HRP conjugate can be
  added followed
• by DAB.

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Compounds in development for targeting the BCL-2
family in vivo
Compound Class
Mechanism Academic
institution Developmental




/company
stage Genasense Antisense oligonucleotide
Antiapoptotic mRNA
Genta Clinical

downregulation (BCL-2) HA14-1 analogs
Small molecule Antiapoptotic
inhibition Raylight Chemokine


Pharmaceuticals
Preclinical Compound 6 Small molecule
Antiapoptotic inhibition
University of Michigan
Preclinical Antimycin A3 Small molecule
Antiapoptotic inhibition
University of Washington Preclinical BH3Is
Small molecule
Antiapoptotic inhibition Harvard
University Preclinical AT101 ()
Gossypol Small molecule
Antiapoptotic inhibition Ascenta
Therapeutics Clinical Apogossypol
Small molecule
Antiapoptotic inhibition The Burnham
Institute Preclinical Theaflavan
in Small molecule
Antiapoptotic inhibition The Burnham
Institute Preclinical Polyphenol
E Small molecule
Antiapoptotic inhibition Mayo Clinic
Preclinical GX15-070
Small molecule
Antiapoptotic inhibition Gemin X
Clinical ABT-737
Small molecule
Antiapoptotic inhibition Abbott
Laboratories/Pfizer


(Idun)
Preclinical IFI-983L, IFI-194
Small molecule Antiapoptotic
inhibition Infinity Pharmaceuticals/Novartis
Preclinical CPM-1285 analogs Lipidated peptide
Antiapoptotic inhibition
Raylight Chemokine


Pharmaceuticals
Preclinical Terphenyl derivative Peptidomimetic
Antiapoptotic inhibition
Yale University
Preclinical SAHBs Stapled
peptide Antiapoptotic inhibition
Dana-Farber Cancer


Institute/Harvard University
Preclinical 4-Phenylsulfanyl- phenylamineDerivativ
es Small molecule Proapoptotic inhibition
(BID) The Burnham Institute
Preclinical 3,6-Dibromocarbazole Piperazine
derivatives of 2-propanol Small
molecule Proapoptotic inhibition (BAX)
Serono
Preclinical Humanin peptides Peptide
Proapoptotic inhibition (BAX) The
Burnham Institute Preclinical Ku70
peptides Peptide
Proapoptotic inhibition (BAX) The Blood Center
of South

Eastern Wisconsin
Preclinical
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