PROGRAMMED CELL DEATH (APOPTOSIS )

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PROGRAMMED CELL DEATH
(APOPTOSIS )
◘ Introduction:
– Apoptosis is a naturally occurring process by which a cell is
directed to programmed cell death.
– The name Apoptosis is a Greek name describing falling of leaves.
– The course apoptosis is accompanied by characteristic changes in
the cell morphology.
 Apoptosis is genetically programmed cell death which leads to tidy
breakdown and disposal of cells.
 Morphologically, apoptosis is characterized by changes in the cell
membrane (with formation of small blebs known as apoptotic bodies,
shrinking of nucleus, chromatin condensation, and fragmentation of
DNA.
 Macrophages and other phagocytic cells recognize apoptotic cells and
remove them by phagocytosis without inflammatory phenomena
developing.

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♦ Characteristic Changes Occur during Apoptosis in the Cell Morphology
♦ Morphological Comparison between Necrosis and Apoptosis

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◘ Basic function of Apoptosis
1- Tissue Homeostasis.
2- Development and Differentiation.
3- Immune System.
4- Cell Damage.
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1- Tissue Homeostasis.
2- Development and Differentiation
• Apoptosis has an indispensable role in development and differentiation
processes especially in the embryo.
• Here, it provides a means to switch off cells no longer needed during
embryonal morphogenesis and synaptogenesis

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3- Immune System.
• In the immune system, apoptotic programs are activated in various
situations.
• Examples include:
1– Elimination of target cells (e.g., virus-infected cells) by cytotoxic T
lymphocytes.
2– Elimination of auto-reactive B- or T-lymphocytes, natural selection and
elimination of cells in the thymus: 95% of T cells that migrate to the thymus
are eliminated by apoptosis.
4- Cell Damage.

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Cell Cycle
 At the center of the apoptotic program is a family of proteases named
Caspases.
 They are involved in the initiation and execution of the program and can
be activated by a large number of stimuli via two central pathways, one
involving mitochondria, the other using trans-membrane receptors of
the tumor necrosis factor α (TNF α) class.
♣ The major pathways of apoptosis
The intrinsic pathway:
- Activation of apoptosis via mitochondria is an intrinsic pathway
where stress signals, DNA damage signals, and defects in signaling
pathways are processed.
- The intrinsic pathway uses the mitochondria as a central
component for activation of apoptosis.
- In this pathway, a multitude of intracellular signals including
various stresses, DNA damage and inappropriate cell signaling
lead to activation of the pro-apoptotic protein Bax which induces
release of cytochrome C from mitochondria, formation of the
apoptosome and activation of the initiator caspase 9.

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- Finally, the executioner caspases are activated and cells are
destructed by proteolysis.
- Apoptosis via this pathway can be controlled by various
antiapoptotic proteins including the Bcl-2 protein and inhibitors of
apoptosis.
The extrinsic pathway
- Uses extracellular death ligands ( Fas ligand, tumor necrosis factor
TNF) to activate ‘death receptors’ which pass the apoptotic signal to
initiator caspases (e. g. capsase 8 ) and to the executioner caspases e. g.
caspase 3; caspase 7).
- In the execution phase of apoptosis, various cellular substrates are
degraded leading to cellular collapse.
- As a consequence of caspase activation, a number of key enzymes and
structural proteins of the cell are degraded, leading to cell death.

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- The stimuli that induce apoptosis are very diverse and include, for
example, DNA damage, stress conditions, and malfunction of pathways
regulating cell proliferation.

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Major pathways of apoptosis.
‫مهمة‬ ‫رسمة‬‫في‬ ‫ذكرهم‬ ‫لو‬‫نرغي‬ ‫ما‬ ‫بدل‬ ‫نرسم‬ ‫االمتحان‬
 The major part of the apoptotic program exists in the cell in a latent,
inactive form, and it only requires an apoptotic stimulus to activate the
program and to initiate apoptosis.
 Thus, apoptotic processes can also take place without activation of
transcription.
 There are also forms of apoptosis that are dependent on transcription.

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Pathways of Apoptosis

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◘ Components of Apoptosis
♣ Caspases: Death by Proteolysis
 A family of specialized proteases, named caspases, is central to the
apoptotic program.
 With respect to function, caspases are grouped into two biologically
distinct subfamilies.
 One subfamily mediates initiation (initiator caspases, caspases 8 and 9)
or execution (executioner or effector caspases, caspases 3, 6 and 7) of
the apoptotic program.
♣ Activation and Regulation
 The first level of caspase regulation involves the conversion of the
caspase precursors, the zymogens, to the active forms in response to
inflammatory or apoptotic stimuli.
 The second level of caspase control involves the specific inhibition by
binding of natural inhibitors.
♣ Control by Proteolytic Activation
 Proteolytic activation of procaspases uses two mechanisms, depending
on whether the caspase functions as an effector caspase (caspases 3, 6
and 7) or as an initiator caspase (caspases 8 and 9).
 Initiator caspases are the first to be activated in response to a
proapoptotic stimulus and are responsible for activating the effector
caspases by limited proteolysis.
 The effector caspases are thought to be responsible for most of the
substrate proteolysis observed during apoptosis.
 By digesting central proteins, the effector caspases direct the cell to
death.

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 The initiator caspases receive proapoptotic signals and initiate the
activation of a caspase cascade.
 They are activated by assembly into a multiprotein complex, and they
contain large prodomains responsible for this interaction.
♣ The Family of Bcl-2 Proteins: Gatekeepers of Apoptosis
 Bcl-2 family of proteins derives its name from B-cell
Lymphoma 2.
 At present, more than 2 members of the Bcl-2 family are known,
which have a negative or a positive effect on the initiation of the
apoptotic program.
 On the basis of structural and functional criteria, the Bcl-2
family has been divided into three groups.

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♦ Group I
 All members of this group have antiapoptotic functions.
 It is assumed that the members of this family prevent cell death
by binding and sequestering the pro-apoptotic Bcl-2 family
members of groups II and III.
 Over expression of group I members can prevent initiation of the
apoptotic program in various cell types.
 The oncogenic function of Bcl-2 protein, observed in association
with its over expression, can be explained by its antiapoptotic
effect:
- the high level of the Bcl-2 proteins suppresses initiation of the
apoptotic program and an important requirement for further
tumor progression is fulfilled.
 In this situation, damaged cells would have been in the normal
situation, can survive.
 An example of this group is the Bcl-XL protein.
♦ Group II
 This group includes the pro-apoptotic Bax and Bak proteins.
 Their activity is necessary for the induction of mitochondria-
mediated apoptosis.

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♦ Group III
 They act by binding to group I and/or group II family members
via their BH3 domain.
 These pro-apoptotic polypeptides include the Bid, Bad, Bim,
Noxa and Puma and proteins, which are thought to be sensors
of pro- and anti-apoptotic stimuli, integrating these into a life-or-
death decision.
♣ The Mitochondrial Pathway of Apoptosis
 The mitochondria have emerged as a central component of the
intrinsic apoptotic signaling pathways and are now known to
control apoptosis via the release of apoptogenic proteins.
 The apoptotic signals that are channeled through the
mitochondrial pathway of apoptosis include various stresses like
DNA damage, oxidative stress, UV radiation, protein kinase
inhibition, and growth factor deprivation.

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 Apoptotic stimuli are relayed to mitochondria via the pro-
apoptotic proteins of the Bax/Bak group, which translocate,
upon receipt of the appropriate stimuli, from the cytosol to the
outer membrane of the mitochondria and thereby allow the
release of different mitochondrial proteins that are normally
present in the intermembrane space of these organelles.
 Of these proteins, cytochrome c is most important for triggering
further downstream apoptotic events.
 In the unstimulated situation, cytochrome c is trapped at the
inner mitochondrial membrane by interaction with the lipid
cardiolipin.
 Furthermore, the pro-apoptotic Bax/Bak proteins are kept in
check by heterodimerization with the anti-apoptotic Bcl-2/Bcl-
XL proteins.
 The mechanism which initiate the release of cytochrome c upon
receipt of an apoptotic stimulus remain elusive.
 The driving force for transport of the Bax/Bak proteins to the
outer mitochondrial membrane is unclear, and it is not well
understood how cytochrome c is released.
 According to one model of the release of cytochrome c,
oligomerization of Bax/Bak proteins leads to the formation of
pore in the outer mitochondrial membrane through which
cyctochrome C can translocate into the cytosol and subsequently
become integrated into the apoptosome

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♣ Formation of the Apoptosome and Triggering of a Caspase
Cascade
 In a further step of apoptosis, the cytochrome c released from
the mitochondria promotes the assembly of a multiprotein
complex, termed apoptosome, which contains cytochrome c, the
adaptor protein Apaf1, and procaspase-9.
 The apoptosome requires ATP for its formation and is able to
cleave and activate procaspase-3, an effector caspase.
 The adaptor protein Apaf1 appears to play a major structural
role in this assembly.
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♣ Death Receptor-triggered Apoptosis
 One major pathway of apoptosis is activated by external ligands
that bind to and activate receptor systems known as death
receptors.
 The death receptors are transmembrane receptors that belong to
the super family of tumor necrosis factor (TNF) receptors.
 The death receptor family includes a receptor termed Fas (also
known as CD95), tumor necrosis factor a (TNF-α) receptor 1
(TNF-R1), and two other receptors, DR4 and DR 5...
 Depending on the cellular context, the death receptors can
transmit pro-apoptotic, anti-apoptotic, anti-inflammatary, or
pro-inflammatory signals.

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Links of Apoptosis and Cellular Signaling Pathways
♣ The Protein p53
and Apoptosis:
 The tumor suppressor protein p53
has both growth-inhibiting and
pro-apoptotic properties that are essential to its tumor-
suppressing activity.
 These functions of p53 can be separated and are mediated by
distinct pathways.
 The growth-controlling activity is mediated mainly by the kinase
inhibitor p21, which is regulated by p53
at the level of
expression.
 In addition, p53
can exert a pro-apoptotic function which is
separate from the growth-inhibiting function.
 Apoptosis induced by p53
is especially important during
conditions of DNA damage and stress as shown in figure.
 It can be categorized in transcription-dependent and
transcription-independent reactions.

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♦ Cancer and apoptosis
 The importance of apoptotic pathway in cancer progression is
seen when there are mutations that alter the cell ability to
undergo apoptosis and allow transformed cell to keep
proliferation rather than die.
 Such genetic alterations include translocation of Bcl-2 gene in
lymphomas that prevents apoptosis and promotes resistance to
cytotoxic drugs.
 Various oncogene products can suppress apoptosis.
 This includes adenovirus proteins E1b, ras, and n-abl.
 It would be therapeutically advantageous to tip the balance in
favor of apoptosis over mitosis in tumors, if that could be done.
 It is clear that a number of anticancer drugs induce apoptosis in
cancer cells.
 The problem is that they usually do this in normal proliferation
cells as well.
 Therefore, the goal should be to manipulate selectively the
genes involved in inducing apoptosis in tumor cells.
 Understanding how genes work may go a long way to achieving
this goal.
 It would be a mistake to portray apoptosis as only a mechanism
to kill cells damaged by some exogenous insult such as DNA-
damaging toxins, drugs, or irradiation.

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 Apoptosis is, in fact, a normal mechanism used by all
multicellular organisms to facilitate normal development, of the
differentiated cells that the organism needs and control of tissue
size.
 The ability of the lymphoid progeny cells to avoid apoptosis
may lead to lymphatic leukemias or lymphomas.
 In addition, cancers develop multiple mechanisms evade
destruction by immune system such as decreased expression of
MHC molecules on cancer cell surfaces and production of
immunosuppressive cytokines.
 Several cell proliferation-promoting events take place cancer
cells as they evolve over time into growth dysregulated,
invasive, metastatic cell types.
 These events include activation of proliferation-promoting
oncogenes such as ras and myc, overexpression of cell cycle
regulatory factors as cyclin D, increased telomerase to overcome
cell senescence, and increased angiogenesis to enhance blood
supply to tumor tissue, In addition, a number of mutations in
apoptotic factors and up-regulation of anti-apoptotic factors
occur in cancer cells during progression.
- These include mutation or inactivation of p53 and over expression of
Bcl-2 and Bcl-XL.
- Over expression of inhibitors of apoptosis proteins (IAPs) and heat
shock proteins (Hsps 70).
- Which can inhibit caspase-9 activation, have also been observed in
human cancers.

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Conclusion
 Apoptosis or programmed cell death is a series of genetically controlled
events that result in the removal of unwanted cells.
 Apoptosis is an important method of cellular control and any disruption
of this process leads to abnormal growth – cancer.
 Apoptotic cells show a very characteristic morphology as well as
specific molecular features.
 Induction of apoptosis in cancer cells or malignant tissues is recognized
as an efficient strategy for cancer chemotherapy.
 Apoptosis also seems to be a reliable marker for the evaluation of
potential agents for cancer prevention.
 A wide variety of natural compounds appear to possess significant
cytotoxic as well as chemopreventive activity.
 Many of these agents act via apoptosis.
 Extracts of plants used in traditional medicine also have a similar
property. Many more screening studies are necessary using plant
extracts and compounds isolated from them.
 Potential apoptotic inducers should not be cytotoxic to normal tissues
and the immune cell system.
 Naturally occurring compounds that are included in the diet are non-
toxic and may partially regulate programmed cell death in several
tissues and organs.
 Elaborate studies with such compounds with respect to their abilities to
induce apoptosis and understanding their mechanism of action may
provide valuable information for their possible application in cancer
therapy and prevention.

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◘ Tumor Markers
♣ What are they?
 Tumor markers are substances, usually proteins, that are produced by
the body in response to cancer growth or by the cancer tissue itself.
 Some tumor markers are specific for one type of cancer, while others
are seen in several cancer types.
 Many of the well-known markers are seen in non-cancerous
conditions as well as cancer.
 Consequently, they cannot be used to diagnose cancer.
 There are only a handful of well-established tumor markers that are
being routinely used by doctors.
 Many other potential markers are still being researched.
 Some marker tests cause great excitement when they are first
discovered but, upon further investigation, prove to be no more useful
than markers already in use.
 The goal is to be able to screen for and diagnose cancer early, when it
is the most treatable and before it has had a chance to grow and
spread.
 So far, no tumor marker has gained acceptance in the UK as a general
screen, including the Prostate Specific Antigen (PSA) for men.
 The markers are either not specific enough (too many false positives,
leading to expensive and unnecessary follow-up testing) or they are
not elevated early enough in the disease process.
 Medical support and treatment must be available and be more
beneficial if given at an early stage.

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 The health benefits must be greater than any harm done by the
screening process which itself must be cost effective.
 Some people are at a higher risk for particular cancers because they
have inherited a genetic mutation.
 While not considered tumor makers, there are tests that look for these
mutations in order to estimate the risk of developing a particular type
of cancer.
 BRCA1 and BRCA2 are examples of gene mutations related to an
inherited risk of breast cancer and ovarian cancer.
♣ Why are they done?
 Tumor markers are not diagnostic in themselves.
 A definitive diagnosis of cancer is made by looking at biopsy
specimens (e.g., of tissue ) under a microscope.
 However, tumor markers provide information that can be used to:
 Monitor
- While at present no markers are suited for general screening, some may
be used to monitor those with a strong family history of a particular
cancer.
- In the case of genetic markers, they may be used to help predict risk in
family members.
 Help diagnose
- In a patient that has symptoms, tumor markers may be used to help
identify the source of the cancer, such as CA-125 for
- ovarian cancer, and to help differentiate it from other conditions.
- Remember that tumor markers cannot diagnose cancer themselves but
aid in this process.

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 Stage
- If a patient does have cancer, tumor marker elevations can be used to help
determine how far the cancer has spread into other tissues and organs.
 Determine prognosis
- Some tumor markers can be used to help doctors determine how
aggressive a cancer is likely to be.
 Guide treatment
- Some tumor markers, such as Her2/neu, will give doctors information
about what treatments their patients may respond to (for instance,
breast cancer patients who are Her2/neu positive are more likely to
respond to Herceptin therapeutic drug treatment).
 Monitor treatment
- Tumor markers can be used to monitor the effectiveness of treatment, especially
in advanced cancers.
- If the marker level drops, the treatment is working; if it stays elevated,
adjustments are needed.
- The information must be used with care, however.
- CEA, for instance, is used to monitor bowel cancer, but not every bowel cancer
patient will have elevated levels of CEA.
- If the marker level is not initially elevated with the cancer, it cannot be used later
as a monitoring tool.
 Determine recurrence
– Currently, one of the biggest uses for tumor markers is to monitor for cancer
recurrence.
– If a tumor marker is elevated before treatment, low after treatment, and then
begins to rise over time, then it is likely that the cancer is returning.
– (If it remains elevated after surgery, then chances are that not all of the cancer
was removed).