APOPTOSIS

Dr.Dinesh T

Junior resident

Department of Physiology

JIPMERsclero dinesh

Discussion headings

• Introduction

• Etiopathogenesis

• Morphological, Biochemical changes

• Mechanism – Intrinsic & Extrinsic pathway

• Disorders of apoptosis

• Conclusion

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Introduction

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Apoptosis - Definition

• A pathway of cell death induced by a tightly

regulated suicidal program, in which the

cells destined to die activate enzymes that

degrade cells own nuclear DNA and nuclear,

cytoplasmic proteins.

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Kerr Wyllie and Currie paper, British Journal of Cancer, 1972 Aug;26(4):239-57

"We are most grateful to Professor James Cormack of the Department of Greek, University of Aberdeen, for suggesting this term. The word "apoptosis" (ἁπόπτωσισ) is used in Greek to describe the "dropping off" or "falling off" of petals from flowers, or leaves from trees. To show the derivation clearly, we propose that the stress should be on the penultimate syllable, the second half of the word being pronounced like "ptosis" (with the "p" silent), which comes from the same root "to fall", and is already used to describe the drooping of the upper eyelid

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Historical aspects

• German scientist Carl Vogt - Principle of apoptosis (1842).

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• Walther Flemming – Process of programmed cell death (1845).

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• John Foxton Ross Kerr – Distinguish apoptosis from traumatic cell death (1962).

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Nobel prize in 2002 – Sydney Brenner , Horvitz, John Buston.

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Cell death mechanisms

Death by suicide Death by suicide Death by injuryDeath by injury

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APOPTOSIS NECROSIS

NATURAL YES NO

EFFECTS BENEFICIAL DETRIMENTAL

Physiological or pathological

Always pathological

Single cells Sheets of cells

Energy dependent Energy independent

Cell shrinkage Cell swelling

Membrane integrity maintained

Membrane integrity lost

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APOPTOSIS NECROSIS

Role for mitochondria and cytochrome C No role for mitochondria

No leak of lysosomal enzymes Leak of lysosomal enzymes

Characteristic nuclear changes Nuclei lost

Apoptotic bodies form Do not form

DNA cleavage No DNA cleavage

Activation of specific proteases No activation

Regulatable process Not regulated

Evolutionarily conserved Not conserved

Dead cells ingested by neighboring cells Dead cells ingested by neutrophils and macrophages

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Significance of apoptosis

• During development many cells are produced in excess which eventually

undergo programmed cell death and thereby contribute to sculpturing many

organs and tissues [Meier, 2000]

• In human body about one lakh cells are produced every second by mitosis

and a similar number die by apoptosis (Vaux and Korsmayer ,1999, cell)

• Between 50 and 70 billion cells die each day due to apoptosis in the

average human adult. For an average child between the ages of 8 and 14,

approximately 20 billion to 30 billion cells die a day. ( Karam, Jose A. (2009).

Apoptosis in Carcinogenesis and Chemotherapy. Netherlands: Springer. ISBN 978-1-

4020-9597-9)

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Etiopathogenesis

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Why should a cell commit suicide?

• 1. Programmed cell death is as needed for proper normal development as mitosis is.

Examples: o The resorption of the tadpole tail in frog .

o The formation of the fingers and toes of the fetus requires the removal, by apoptosis.

o The sloughing off of the endometrium at the start of menstruation.

o The formation of the proper connections (synapses) between neurons in the brain.

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• 2. Programmed cell death is needed to destroy cells that represent a threat to the integrity of the organism.

• Examples: o Cells infected with viruses

o Cells of the immune system

o Cells with DNA damage

o Cancer cells (Uncontrolled proliferated cells)

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Apoptosis in physiologic situationso Programmed destruction during embryogenesis

o Involution of hormone dependent tissues

o Cell loss in proliferating cell populations

o Elimination of harmful self- reactive lymphocytes

o Death of host cells

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Apoptosis in bud formation during which many interdigital cells die.  They are stained black by a TUNEL method

  

Incomplete differentiation in two toes due to lack of apoptosis

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Apoptosis: in embryogenesis

Morphogenesis (eliminates excess cells):

Selection (eliminates non-functional cells):

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Apoptosis: in embryogenesis

Immunity (eliminates dangerous cells):

Self antigenrecognizing cell

Organ size (eliminates excess cells):

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Apoptosis: importantance in adults

Tissue remodeling (eliminates cells no longer needed):

Virgin mammary gland Late pregnancy, lactation Involution(non-pregnant, non-lactating)

Apoptosis

Apoptosis

- Testosterone

Prostate gland

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Apoptosis: importantance in adultsTissue remodeling (eliminates cells no longer needed):

Resting lymphocytes + antigen (e.g. infection) - antigen (e.g. recovery)

Apoptosis

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Apoptosis in pathological conditions - DNA damage

- Accumulation of misfolded proteins

- Cell death in certain infections

- Pathological atrophy in parenchymal organs

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Cells of the immune system • CTLs induce apoptosis in each other and even in

themselves. • Defects in the apoptotic machinery is associated with

autoimmune diseases such as lupus erythematosus and rheumatoid arthritis.

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Cells infected with viruses • One of the methods by which cytotoxic T lymphocytes

(CTLs) kill virus-infected cells is by inducing apoptosis

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Cells with DNA damage • Damage to its genome

can cause a cell » to disrupt proper

embryonic development leading to birth defects

» to become cancerous.

• Cells respond to DNA damage by increasing their production of p53. p53 is a potent inducer of apoptosis.

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– Cancer cells • Radiation and chemicals used in cancer therapy induce apoptosis

in some types of cancer cells.

Fig. 1: SC-1 induced apoptosis in stomach carcinoma cells Left: Before induction Middle: 24h after induction Right: 48h after induction sclero dinesh

Morphological & Biochemical changes

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Classic changes

Cell shrinkage

Nuclear fragmentation

Chromatin condensation

Chromosomal DNA fragmentation

Formation of cytoplasmic blebs& apoptotic bodies

Phagocytosis

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Histology

Apoptotic bodies • Round oval mass

of intensely eosinophillic cytoplasm

• Fragments of dense nuclear chromatin

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Bio chemical changes

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Mechanisms of apoptosis

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Caspases

• Caspase are Cysteine- Aspartic acid specific proteases that mediates the events that are associated with programmed cell death.

• Their catalytical activity depends on a critical cysteine-residue within a highly conserved active-site pentapeptide QACRG,

• Caspases specifically cleave their substrates after Asp residues.• Caspase- 8, Caspase- 9

• acts as an initiator of the caspase activation cascade.

• Caspase-3• key effector in the apoptosis pathway, amplifying the signal from

initiator caspases and signifying full commitment to cellular disassembly.

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Initiation

• Absence of stimuli - hormones, growth factors

• Activation of receptors – TNF family

• Heat ,radiation, chemicals

• Genetically programmed events

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STAGES OF CLASSIC APOPTOSISHealthy cell

DEATH SIGNAL / STIMULI (extrinsic or intrinsic)

Commitment to die (reversible)

EXECUTION (irreversible)

Dead cell (condensed, crosslinked)

ENGULFMENT (macrophages, neighboring cells)

DEGRADATION

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Receptor pathway (physiological):

Death receptors:(FAS, TNF-R, etc)

FAS ligand TNF

Deathdomains

Adaptor proteins

Pro-caspase 8 (inactive) Caspase 8 (active)

Pro-execution caspase (inactive)Execution caspase (active)

DeathMITOCHONDRIAsclero dinesh

Apoptosis triggered by external signals: the extrinsic or death receptor pathway

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Regulation of apoptosis

• Regulatory proteins – BCL -2, equivalent to CED -9

• Apoptosis depends on binding of BCL -2 with pro apoptotic and anti apoptotic proteins.

• Situated in the outer mitochondrial membrane.

• Apaf -1 equivalent to CED -4. • Tp 53, caspases, BAX, viruses such as adeno,

papilloma , hepatitis B.

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Intrinsic pathway (damage):

Mitochondria

Cytochrome c release

Pro-caspase 9 cleavage

Pro-execution caspase (3) cleavage

Caspase (3) cleavage of cellular proteins,nuclease activation,

etc.

Death

BAXBAKBOKBCL-XsBADBIDB IKBIMNIP3BNIP3

BCL-2BCL-XLBCL-WMCL1BFL1DIVANR-13Several viral proteins

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Intracellular signalsOxidative damage from free radicals, Radiation, Virus infection, Nutrient

deprivation, Pro-apoptotic Factors

Damage to the mitochondrial membrane increasing permeability

Entry of Cytochrome C into the cytoplasm

Cytochrome C binds to Apaf-1 forming an apoptosome

Apoptosome activates procaspase-9 to caspase-9

Caspase-9 cleaves and activates caspase-3 and caspase-7.

This executioner caspases activate a cascade of proteolytic activity that leads to: Chromatin condensation, DNA fragmentation, Protein cleavage,

Membrane permeabilitysclero dinesh

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Physiological Intrinsicreceptor pathway damage pathway

MITOCHONDRIAL SIGNALS

Caspase cleavage cascade

Orderly cleavage of proteins and DNA

CROSSLINKING OF CELL CORPSES; ENGULFMENT(no inflammation)

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Steps

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Disorders of apoptosis

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Apoptosis: Role in Disease

TOO MUCH: Tissue atrophy

TOO LITTLE: Hyperplasia

NeurodegenerationThin skin

etc

CancerAthersclerosis

etc

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Apoptosis: Role in DiseaseNeurodegeneration

oNeurons are post-mitotic (cannot replace themselves; neuronal stem cell replacement is inefficient)

oNeuronal death caused by loss of proper connections, loss of proper growth

factors (e.g. NGF), and/or damage (especially oxidative damage).

oNeuronal dysfunction or damage results in loss of synapses or loss of cell bodies (synaptosis, can be reversible; apopsosis, irreversible)

oPARKINSON'S DISEASE

oALZHEIMER'S DISEASE

oHUNTINGTON'S DISEASE etc.

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Apoptosis: Role in DiseaseCancer

oApoptosis eliminates damaged cells (damage => mutations => cancer

oTumor suppressor p53 controls senescence and apoptosis responses to damage.

oMost cancer cells are defective in apoptotic response(damaged, mutant cells survive)

oHigh levels of anti-apoptotic proteins or oLow levels of pro-apoptotic proteins ===> CANCER

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Apoptosis: Role in DiseaseCancer

Virus associated cancer• Several human papilloma viruses (HPV) have been

implicated in causing cervical cancer. One of them produces a protein (E6) that binds and inactivates the apoptosis promoter p53.

• Epstein-Barr Virus (EBV), the cause of mononucleosis and associated with some lymphomas – produces a protein similar to Bcl-2 – produces another protein that causes the cell to

increase its own production of Bcl-2. Both these actions make the cell more resistant to apoptosis (thus enabling a cancer cell to continue to proliferate).

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• Some B-cell leukemia and lymphomas express high levels of Bcl-2, thus blocking apoptotic signals they may receive. The high levels result from a translocation of the BCL-2 gene into an enhancer region for antibody production.

• Melanoma (the most dangerous type of skin cancer) cells avoid apoptosis by inhibiting the expression of the gene encoding Apaf-1.

Apoptosis: Role in DiseaseCancer

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•Other cancer cells express high levels of FasL, and can kill any cytotoxic T cells (CTL) that try to kill them because CTL also express Fas (but are protected from their own FasL).

•Some cancer cells, especially lung and colon cancer cells, secrete elevated levels of a soluble "decoy" molecule that binds to FasL, plugging it up so it cannot bind Fas. Thus, cytotoxic T cells (CTL) cannot kill the cancer cells

Apoptosis: Role in DiseaseCancer

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Apoptosis: Role in DiseaseAging

Aging --> both too much and too little apoptosis(evidence for both)

Too much (accumulated oxidative damage?)---> tissue degeneration

Too little (defective sensors, signals?---> dysfunctional cells accumulatehyperplasia (precancerous lesions)

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Apoptosis: Role in Disease

• Apoptosis and AIDS

Hallmark- the decline in the number of the patient's CD4+ T cells (normally about 1000 per microliter (µl) of blood).

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In Immune system

o Very rarely humans are encountered with genetic defects in apoptosis.

o The most common one is a mutation in the gene for Fas

o mutations in the gene for FasL or even one of the caspases are occasionally seen.

Autoimmune lymphoproliferative syndrome or ALPS.

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Conclusion

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Cells are balanced between life and death

DAMAGE Physiological death signals

DEATH SIGNAL

PROAPOPTOTICPROTEINS(dozens!)

ANTIAPOPTOTICPROTEINS(dozens!)

DEATHsclero dinesh

Thank u….

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