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
In all our tissues, the size of individual cell populations is governed by cell birth brought about by
division of cells - mitosis - and by a controlled cell deletion or 'suicide' process known as apoptosis.
The name apoptosis is derived from the ancient Greek term meaning 'the falling of leaves from
trees or petals from flowers'. During embryonic development, we are fashioned not only through
mitosis, cell differentiation and cell migration, but also through the 'useful cell death' that is
apoptosis. Numerous tissues within our adult bodies are maintained by a fine balance between
mitosis and apoptosis. For example, immune responses to infection are boosted by cell division and
finely tuned and curtailed by apoptosis; cells of the gut and skin are renewed through cycles of
mitosis and apoptosis; the lactating breast regresses post-lactation by apoptosis.
Apoptotic B Cells
•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
•
•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
•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
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
Necrosis
Apoptosis
Morphological changes
• Loss of membrane integrity.
• Begins with swelling of cytoplasm and
mitochondria.
• No vesicle formation, complete lysis
• Membrane blebbing, but no loss of integrity.
Aggregation of chromatin at nuclear memb.
• Begins with shrinking of cytoplasm and
condensation of nucleus.
• Formation of membrane bound vesicles
(apoptotic bodies)
Biochemical changes
• No energy requirement passive process
• Random digestion of DNA
• Postlytic DNA fragmentation
• Energy (ATP)-dependent
• Non-random mono- and oligonucleosomal
length fragmentation of DNA. Laddering pattern
• Prelytic DNA fragmentation
Physiological changes
• Phagocytosis by macrophages
• Phagocytosis by adjacent cells or macrophages
• Significant inflammatory response
• No inflammatory response
Apoptosis in Development and Disease :
Role in animal development:
1. Sculpting structures e.g. formation of digits, neural tubes.
2. Deleting unneeded structures e.g. vestigial structures like pronephric tubes
3. Controlling cell numbers like neurons and oligodendrocytes.
4. Function as quality control, eliminating harmful cells e.g self reacting lymphocytes
5. 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.
Apoptosis is an Essential Process
• Apoptosis (programmed cell death) plays an important role
in 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
Cell Death Pathways
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.
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: 620621 (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.
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.
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.
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.
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 AspX site releases large, small subunits.
• Active caspases are tetramers: two large
and two small subunits : two active
sites.
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
3. Cytokine processors
(Inflammatory)
• Prodomains: Long; Contain
regulatory sequences
•Expressed widely in cells in inactive proenzyme form (pro-caspase)
•Must be activated for proteolysis
•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
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.
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(14;18) translocation places the BCL-2
gene close to the heavy chain gene enhancer.
Bcl-2 Members
• Bcl-2 :first protooncogene gene to be
discovered from the family: cloned from
t(14;18) 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
regulatory proteins such as Raf-1, Bad,
and perhaps Ced-4.
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 α-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.
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 Bcl2 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.
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
Phosphorylation and dephosphorylaion.
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.
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
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.
BAD is phosphorylated by Akt, binds to 14-3-3 and is degraded
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 occasiona
Deletion and aberrant CpG island methylation of Caspase 8 gene in
medulloblastoma.2004
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.
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.
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 immuneprivileged sites (i.e, eyes, reproductive organs)
Two pathways in TNFR signaling
Pathways to Apoptosis
Inhibitor of Apoptosis Family
1993: First member identified in baculoviruses
one or more BIR domains critical for activity
1995: first mammalian IAP identified
NAIP: 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
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.
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
•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
with Beta Tubulin.
IAP inhibitors : Smac/Diablo, Omi and XAF1.
IAPs inhibit active caspases:
XIAP inhibits caspases 3, 7 and caspase 9 through separate domains. Its BIR2 domain
(amino acids 163–240) with its NH2-terminal extension (amino acids 124–162) inhibits
caspases 3 and 7, whereas its BIR3 domain (amino acids 241–356) inhibits caspase 9.
These studies provided the basis for developing IAP inhibitors that target the caspasebinding pockets of the molecule.
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.
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 56–59) 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
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
Techniques to detect Apoptosis
Determination of Cell Viability Test :
1) 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 activit
However, integrity of pl memb is not compromised till late stage and hence not very usefu
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.
Annexin V staining
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)
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.
MitochondrialChanges:
Changes:
Mitochondrial
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.
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.
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 (4’6’diamidino 2-phenylindole,
dilactate).
• Intensity of staining in the nucleus is proportional to the extent of apoptosis due to
increased permeability of the dyes.
DNA content staining by Propidium Iodide::
Control
Tamoxifen Treated
Tamoxifen
G1
Control
S
G2
G0
• 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.
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.
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 dUTPbiotin.
• 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.
Compounds in development for targeting the BCL-2 family in vivo
Compound
Genasense
Class
Antisense oligonucleotide
HA14-1 analogs
Small molecule
Compound 6
Antimycin A3
BH3Is
AT101:
(–) Gossypol
Apogossypol
Theaflavanin
Polyphenol E
GX15-070
ABT-737
Small molecule
Small molecule
Small molecule
Small molecule
Small molecule
Small molecule
Small molecule
Small molecule
Small molecule
IFI-983L,
IFI-194
Small molecule
CPM-1285 analogs Lipidated peptide
Terphenyl derivative Peptidomimetic
SAHBs
Stapled peptide
Mechanism
/company
Antiapoptotic mRNA
downregulation (BCL-2)
Antiapoptotic inhibition
Antiapoptotic inhibition
Antiapoptotic inhibition
Antiapoptotic inhibition
Antiapoptotic inhibition
Antiapoptotic inhibition
Antiapoptotic inhibition
Antiapoptotic inhibition
Antiapoptotic inhibition
Antiapoptotic inhibition
Antiapoptotic inhibition
Antiapoptotic inhibition
Antiapoptotic inhibition
Antiapoptotic inhibition
Academic institution
stage
Genta
Developmental
Clinical
Raylight Chemokine
Pharmaceuticals
University of Michigan
University of Washington
Harvard University
Ascenta Therapeutics
The Burnham Institute
The Burnham Institute
Mayo Clinic
Gemin X
Abbott Laboratories/Pfizer
(Idun)
Infinity Pharmaceuticals/Novartis
Raylight Chemokine
Pharmaceuticals
Yale University
Dana-Farber Cancer
Institute/Harvard University
4-PhenylsulfanylphenylamineDerivatives Small molecule Proapoptotic inhibition (BID) The Burnham Institute
3,6-Dibromocarbazole
Piperazine derivatives
of 2-propanol
Small molecule
Proapoptotic inhibition (BAX)
Serono
Humanin peptides
Peptide
Proapoptotic inhibition (BAX) The Burnham Institute
Ku70 peptides
Peptide
Proapoptotic inhibition (BAX) The Blood Center of South
Eastern Wisconsin
Preclinical
Preclinical
Preclinical
Preclinical
Clinical
Preclinical
Preclinical
Preclinical
Clinical
Preclinical
Preclinical
Preclinical
Preclinical
Preclinical
Preclinical
Preclinical
Preclinical
Preclinical
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