Download CELL PROLIFERATION AND APOPTOSIS

CELL PROLIFERATION AND
APOPTOSIS
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Overview
• In this session we will deal with cell proliferation,
apoptosis, repair, regeneration and how these
relate to the actions of drugs.
• About 10 billion new cells are manufactured in
the body daily through cell division-an output
that must be counterbalanced by the elimination
of a similar number of cells.
• We will first deal with the changes that occur
within an individual cell when, after stimulation
by growth factors, it gears up to divide into two
daughter cells.
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Overview…
• We then consider the interaction of cells, growth factors
and the extracellular matrix in cell proliferation
• We describe the phenomenon of apoptosis (the
programmed series of events that lead to cell death),
outlining the changes that occur in a cell that is preparing
to die, and the intracellular pathways that lead to its
demise.
• We consider how these processes relate to the repair of
damaged tissue and the possibility of its regeneration.
• Lastly, we consider the pathophysiological significance of
these events, and implications for the potential
development of clinically useful drugs
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CELL PROLIFERATION:
• Cell proliferation (cell division) is involved in
many physiological and pathological
processes including:
 Growth,
 Healing,
 repair,
 hypertrophy,
 hyperplasia
 and the development of tumours.
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• Angiogenesis (the development of new
blood vessels) necessarily occurs during
many of these processes.
• Proliferating cells go through what is
termed the cell cycle, during which the
cell replicates all its components and
then bisects itself into two identical
daughter cells.
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• Important components of the signaling
pathways in proliferating cells are receptor
tyrosine kinases or receptor-linked kinases
and the mitogen-activated kinase (MAP
kinase) cascade.
• In all cases, the pathways eventually lead to
transcription of the genes that control the cell
cycle.
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THE CELL CYCLE
• The cycle is an ordered series of events
consisting of several sequential phases: G1; S;
G2 and M.
• G1; preparation for DNA synthesis
• S phase is the phase of DNA synthesis &
chromosome duplication.
• G2: preparation for division
• M is the phase of mitosis: division into two
daughter cells
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• G1 is the gap between the mitosis that gave rise to the
cell and the S phase;
• During G1 the cell is preparing for DNA synthesis.
• G2 is the gap between S phase and the mitosis
that will give rise to two daughter cells: during G2
the cell is preparing for DNA synthesis.
• In cells that are dividing continuosly, G1 S and G2
comprise interphase-the phase between one mitosis
and the next
• Cell division requires the controlled timing of two
critical events of the cell cycle: S phase ( DNA
replication ) and M phase (mitosis).
• Entry into each of these phases is carefully regulated
and this gives rise to two ‘check points’ (restriction
points) in the cycle: one at the start of S and one at
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the start of M.
Figure 5-1 The main phases of the cell cycle
of dividing cells.
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• DNA damage results in the cycle being stopped at
one or other of these. The integrity of the
checkpoints is critical for the maintenance of genetic
stability; and failure of the checkpoints to stop the
cycle when it is appropriate to do so is a hallmark of
cancer.
• In an adult; most cells do not constantly divide; most
spend a varying amount of time in a quiescent phase
outside the cycle as it were, in the phase termed G0
(‘G nought’ not the word ‘Go’).
• Neurones and skeletal muscle cells spend all their
time in the G0; bone marrow cells and the lining cells
of the gastro-intestinal tract divide daily.
• Quiescent cells can be activated into G1 by chemical
stimuli associated with damage; for example, a
quiescent skin cell can be stimulated by a wound
into dividing and repairing the lesion.
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• The impetus for a cell to start off on the cell
cycle ( i.e. to move from G0 into G1 ) can be
provided by several stimuli, the most
important being growth factor action. (Note:
G-protein-coupled receptors can also stimulate cell
proliferation)
• Growth factors stimulate the production of
signal transducers of two types:
Positive regulators of the cell cycle that
control the changes necessary for cell division.
Negative regulators that control the positive
regulators.
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• The maintenance of normal cell numbers
in tissues and organs requires that there
be a balance between the positive
regulatory forces and the negative
regulatory forces.
• Apoptosis also has a role in the control of
cell numbers.
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POSITIVE REGULATORS OF THE
CELL CYCLE:
• The cell cycle is initiated when a growth factor
acts on a quiescent cell provoking it to divide.
• One of the main actions of a growth factors is to
stimulate production of the cell cycle
regulators; which are coded for by the
delayed response genes.
• The main components of the control system that
determines progress through the cycle are two
families of proteins:
 cyclins and
 cyclin-dependent kinases (cdks ).
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• [ The name cyclin comes from the fact that
these proteins undergo a cycle of synthesis
and breakdown during each cell division ].
• The cdks phosphorylate various proteins (
e.g enzymes ) activating some and inhibiting
others – to coordinate their activities.
• Sequential functioning of several different cdks
activates the process that promote progress
through the phase of the cycle.
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• Each cdks is inactive until it binds to a
cyclin, the binding enabling the cdk to
phosphorylate the protein(s) necessary for a
particular step in the cycle. (Fig 5.2)
• It is the cyclin that determines which
protein(s) are phosphorylated.
• After the phoshorylation event has taken place;
the cyclin is degraded (Fig 5.2) by the
ubiquitin/protease system.
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Figure 5-2 Schematic representation of the activation of a cyclindependent kinase. An inactive cdk. The inactive cdk is activated by
being bound to a cyclin; it can now phosphorylate a protein substrate
(e.g. an enzyme). After the phosphorylating event, the cyclin is
degraded.
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• There are 8 main groups of cyclins. Those
important in the control of the cell cycle are
cyclins A, B, D and E.
• Each cyclin is associated with and activates
particular cdk(s).
• Cyclin A activates cdks 1 and 2; cyclin B;
cdk 1; cyclin D cdks 4 and 6 and cyclin E;
cdk 2.
• Precise timing of each activity is essential and
many cycle proteins are degraded after they
have carried out their functions.
• The actions of the cyclin/cdk complexes in the
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cell cycle are depicted in Fig 5.3 (see next slide)
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Figure 5-3 Schematic diagram of the cell cycle,
showing the role of the cyclin/cyclin-dependent
kinase complexes. The processes outlined in the cycle occur
inside a cell such as the one shown in Figure 5.4. A quiescent cell
(in G0 phase), when stimulated to divide by growth factors, is
propelled into G1 phase and prepares for DNA synthesis. Progress
through the cycle is determined by sequential action of the
cyclin/cdk complexes-depicted here by coloured arrows, the arrows
being given the names of the relevant cyclins: D, E, A and B. The
cdks (cyclin-dependent kinases) are given next to the relevant
cyclins. The thickness of each arrow represents the intensity of
action of the cdk at that point in the cycle. The activity of the cdks is
regulated by cdk inhibitors. If there is DNA damage, the products of
the tumour suppressor gene p53 stop the cycle at check point 1,
allowing for repair. If repair fails, apoptosis (see fig. 5.5) is initiated.
The state of the chromosomes is shown schematically in each G
phase-as a single pair in G1, and each duplicated and forming two
daughter chromatids in G2. Some changes that occur during
mitosis (metaphase, anaphase) are shown in a subsidiary circle.
After the mitotic division, the daughter cells may enter G1 or G0
phase. Rb, retinoblastoma gene.
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CELLS IN G0
• In quiescent G0 cells, cyclin D is present in
low concentration and important regulatory
protein- Rb protein is hypophosphorylated.
• (Note: the Rb protein is coded for by the Rb gene. The
Rb gene is so named because mutations of this gene
are associated with retinoblastoma tumours)
• Hypophosphorylated Rb holds the cell cycle in
check at check point 1 by inhibiting the
expression of several proteins critical for cell
cycle progression.
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• The Rb protein accomplishes this by binding to
the E2F transcription factors, which control the
expression of the genes that code for cyclin
E and A, for DNA polymerase, for thymidine
kinase, for dihydrofolate reductase etc.- all
essential for DNA replication during S phase.
• Growth factor action on a cell in G0 propels it into
G1
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PHASE G1
• G1 is the phase in which the cell is preparing
for S phase by synthesizing the messenger
RNAs (mRNAs) and proteins needed for DNA
replication.
• During G1, the concentration of cyclin D
increases and the cyclin D/cdk complex
phosphorylates and activates the necessary
proteins.
• In mid- G1, the cyclin D/cdk complex
phosphorylates the Rb protein, releasing
transcription factor E2F;
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• This then activates the genes for the
components specified above that are essential
for the next phase – DNA synthesis- namely
cyclins E and A, DNA polymerase and so on.
• The action of cyclin E/cdk complex is
necessary for transmission from G0 to S
phase, i.e past check point 1.
• Once past check point 1, into the S-phase, the
processes that have been set in motion
cannot be reversed and the cell is
committed to continue with DNA replication
an mitosis
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S PHASE:
• Cyclin E/cdk and cyclin A/cdk regulates
progress through S phase,
phosphorylating and thus activating
proteins/ enzymes involved in DNA
synthesis.
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G2 PHASE:
• In G2 phase; the cell, which now has doubled
the number of chromosomes; must duplicate
all other cellular components for allocation
to the two daughter cells.
• Synthesis of the necessary mRNAs and protein
occurs.
• Cyclin A/cdk and cyclin B/cdk complexes are
active during G2 phase and are necessary for
entry into M phase, i.e. for passing check point
2.
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• The presence of cyclin B/cdk complexes in
the nucleus is required for mitosis to
commence.
• Unlike cyclins C, D, and E, which are short
lived, cyclin A and B remain stable
throughout interphase but undergo
proteolysis by a ubiquitin-dependent
pathway during mitosis.
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MITOSIS:
• Revise processes involved in mitosis
• Mitosis is a continuous process but can be
considered to consist of 4 stages.
• Prophase: The duplicated chromosomes (
which have up to this point formed a tangled
mass filling the nucleus ) condense, each now
consisting of 2 daughter chromatids, ( the
original chromosome and a copy ). These are
released into the cytoplasm as the nuclear
membrane disintegrates.
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• Metaphase:The chromosomes are aligned at
the equator.
• Anaphase: A special device, the mitotic
apparatus, captures the chromosomes and
draws them to opposite poles of dividing
cell.
• Telophase: A nuclear membrane forms
round each set of chromosomes.
• Finally the cytoplasm divides between the two
forming daughter cells. Each daughter cell will
be in G0 phase and will remain there unless
stimulated into G1 phase.
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NEGATIVE REGULATORS OF CELL
CYCLE:
• One of the main negative regulators is the
Rb protein that holds the cycle in check
while it is hypophosphorylated.
• Another negative regulatory mechanism is
the action of inhibitors of the cdks.
These bind to and inhibit the action of
the complexes, the main action being at
check point 1.
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There are two families of inhibitors-:
• The CIP family (cdk inhibitory proteins; also
termed KIP or kinase inhibitory proteins-proteins
p21, p27, and p57
• The Ink family ( inhibitors of kinases )-proteins
p16, p19 and p15.
• The action of p21 serves as an example of the
role of a cyclin/cdk inhibitor.
• Protein p21 is under control of p53 gene- a
particularly important negative regulator that
operates at check point 1-which is relevant in
carcinogenesis.
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INHIBITION OF THE CYCLE AT
CHECK POINT 1:
• The p53 gene has been called the ‘
guardian of the genome’ . It codes for a
protein transcription factor- the p53
protein. In normal health cells, the steadystate concentration of p53 protein is low.
• But when there is DNA damage, the
protein accumulates and activates the
transcription of several genes, one of
which codes for p21.
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• Protein p21 inactivates cyclin/cdk complexes,
thus preventing Rb phosphorylation, which
means the cycle is arrested at check point 1.
This allows for DNA repair. If the repair is
successful, the cycle proceeds past check point
1 into S phase. If the repair is unsuccessful; the
p53 gene triggers apoptosis- cell suicide.
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INHIBITION OF THE CYCLE AT
CHECK POINT 2:
• There is evidence that DNA damage can
result in the cycle being stopped at check
point 2 but the mechanisms involved are
less clear than those at check point 1.
• Inhibition of the accumulation of cyclin
B/cdk complex in the nucleus seems to
be a factor.
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INTERACTIONS BETWEEN CELLS, GROWTH
FACTORS AND EXTRACELLULAR MATRIX:
• During cell proliferation, there is integrated
interplay between growth factors, cells, the
extracellular matrix (ECM), and the matrix
metalloproteinases (MMPs).
• The extracellular matrix supplies the
supporting frame work for the cells of the
body and is secreted by cells themselves.
• Matrix expression is regulated by the action
on the cell of growth factors and cytokines.
• The activation status of some growth factors
is, in turn, determined by the matrix.
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• Since they are sequestered by interaction
with matrix components released by
enzymes ( e.g. metalloproteinases ) secreted
by the cells.
• It is clear that the action of growth factorswhich act through receptor tyrosine kinases or
receptor-coupled kinases initiating the cell cycle
is fundamental part of these processes.
• There are numerous growth factors; important
examples being fibroblast growth factor
(FGF), epidermal growth factor (EGF ) ,
platelet-dependent growth factor ( VEGF )
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and transformating growth factor- ( TGF-).
• The main components of the extracellular matrix are-:
• Fibre forming elements e.g. collagen species and
elastin. Collagens:These are main proteins of
extracellular matrix.
• Non-fibre-forming, e.g proteoglycans, glucoproteins
and adhesive proteins
• Proteoglycans: These have a growth regulating role,
in part by functioning as a reservoir of
sequestrated growth factors. Some proteoglycans
are associated with the cell surface, where they bind
cells to the matrix.
• Adhesive proteins (e.g. Fibronectin ):
These link the various elements of the matrix together
and also form links between the cells and the matrix
through integrins on the cells.
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• Integrins are transmembrane receptors
with alpha and -subunits.
• On interaction with elements of the
extracellular matrix (ECM) , cooperate with
growth factor signalling pathways (this is
necessary for the optimum cell division)
and also mediate cytoskeletal adjustments
within the cell)
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ANGIOGENESIS:
•
Angiogenesis, which normally accompanies
cell proliferation, Is the formation of new
capillaries from existing blood vessels,
• an important stimulus being vascular
endothelial growth factor (VEGF ).
• The sequence of events is as follows:
1. VEGF induces nitric oxide and also the
expression of proteases ( e.g
metalloproteinases ).
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•
Nitric oxide causes local vasodilatation and
the protease degrade the local basement
membrane and local matrix and
• also mobilize further growth factors from
matrix.
2. Endothelial cells migrate out forming a solid
capillary sprout.
3. The endothelial cells behind the leading cells
are activated by growth factors and start to
divide.
4. A lumen forms in the sprout
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5. Local fibroblasts; activated by growth factors;
proliferate and lay down matrix around
capillary sprout.
6. A process of maturation occurs in which
there is stabilization of the endothelial
layer through cell to cell binding by
adherence proteins and integrin binding of
the cells to the matrix.
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APOPTOSIS AND CELL REMOVAL:
• Apoptosis is cell suicide by a built in selfdestruct mechanism; it consists of a
genetically programmed sequence of
biochemical events.
• It is therefore, unlike necrosis, which is
disorganized disintegration of damage
cells resulting in products that trigger
the inflammatory response.
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• Apoptosis is the mechanism which each day
un-obtrusively removes 10 billion cells from the
adult human body.
• It is involved in the shedding of intestinal lining,
the regression of mammary gland cells after
lactation and the death of time expired
neutrophils.
• It is the basis for development of selftolerance in the immune system and is
implicated in the pathophysiology of cancer;
autoimmune disease, neurodegenarative
conditions, cardiovascular disease and the
acquired immunodeficiency syndrome
(AIDS ).
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• It plays an important role in embryogenesis,
helping to shape organs during development by
eliminating cells that have become redundant
• It has a role in the monitoring of cancerous
change because it acts as a first line defense
against mutations- purging cells with abnormal
DNA that could become malignant.
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MORPHOLOGICAL CHANGES IN
APOPTOSIS:
• As cell dies it rounds up, the chromatin in
the nucleus condenses into dense masses
and cytoplasm shrinks.
• This is followed by blebbing of plasma
membrane and finally transformation of
the cell into cluster of membranebound entities, which are rapidly
phagocytosed by macrophages
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MAJOR PLAYERS IN APOPTOSIS:
• The major players are the caspases- a
family of cysteine proteases present in
inactive form.
• They do not perform generalized
proteolysis; they undertake delicate
protein surgery, selectively cleaving a
specific set of target proteins (
enzymes; structural components , etc. )
inactivating some and activating others.
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• A cascade of about 9 different caspases
take part in bringing about apoptosis,
some functioning as initiators that
transmit the initial apoptotic signals
and some being responsible for the
final effector phase of cell death.
• The caspases are not the only executors
of apoptotic change.
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• Various pathways that result in apoptosis without
the action of caspase fraternity have recently
been described.
• One involves a protein termed AIF (apoptotic
initiating factor), which is released from
mitochondria, enters the nucleus and
triggers cell suicide.
• NB:not all caspases are death-mediating enzymes;
some have a role in the processing and activating of
cytokines e.g caspase 8 is active in processing the
inflammatorycytokines IL-1 and IL-18
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PATHWAY TO APOPTOSIS:
• There are two pathways to activation of
effector caspases: the death receptor
pathway and the mitochondrial pathway
• The death receptor pathway involves
stimulation of members of tumour
necrosis factor ( TNFR ) family; and the
main initiator caspase is caspase 8.
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• The mitochondrial pathway is activated
by internal factors such as DNA
damage, which results in transcription of
gene p53.
• The p53 protein activates a subpathway
that results in release from mitochondrion
of cytochrome c and the end result is
activation of initiator caspase 9.
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• In undamaged cells, survival factors ( cytokines,
hormones, cell to cell contact factors )
continuously activates anti-apoptotic
mechanism. Withdrawal of survival factors
stimulation causes cell death through the
mitochondrial pathway.
• The effector caspases ( e.g. caspase 3 ) start a
pathway that results in cleavage of cell
constituents: DNA; cytoskeletal components,
enzymes ; etc. This reduces the cell to a
cluster of membrane- bound entities that
are eventually phagocytosed by
macrophages.
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TARGETS FOR NEW DRUG
DEVELOPMENT:
• Angiogenesis has a critical role in
numerous bodily processes, some
physiological ( e.g. growth, repair) some
pathological (e.g. tumour growth, chronic
inflammatory conditions)
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•
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ANGIOGENESIS INHIBITORS
these are being sought for use in
pathological angiogenesis and there are
currently 30 compounds in clinical trial.
The approaches being used include:
1. Interference with endothelial cell growth,
for example by the use of monoclonal
antibodies that prevent the interaction of
VEGF (vascular endothelial growth
factor) and FGF (fibroblast growth
factor) with their receptors.
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2. Interference with the necessary adherence of
endothelial cells in the endothelial sprout to
the matrix; an anti-integrin monoclonal
antibody has shown promise.
3. Interference with the necessary degradation of
matrix round the developing endothelial
sprout; inhibitors of metalloproteinases are
under test
• It should be noted that though antiangiogenesis drugs may be helpful in some
conditions ( e.g cancer ) they could be harmful
in others ( e.g heart disease ).
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ANGIOGENESIS STIMULATORS:
• Angiogenesis stimulators are also being
investigated for use in various ischaemic
conditions; for example coronary
disease, limb ischaemia, and gastrointestinal ulcers associated with
insufficient local perfusion.
• The main compound under investigation is
VEGF.
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APOPTOTIC MECHANISMS:
• Example of defective apoptosis include
cancer cell proliferation, resistance to
cancer chemotherapy and ineffective
eradication of virus-infected cells.
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• Examples of over-exuberant apoptosis
include depletion of T cells in human
deficiency virus ( HIV ) infection; allograft
rejection, loss of neurones in
neurodegenerative disease and loss of
chrondocytes in osteoarthritis
• Several anti-apoptosis compounds are in
clinical trials for neurodegenerative and
inflammatory disease.
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• PLEASE READ THOROUGHLY &
DISCUSS!
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