APOPTOSIS

APOPTOSISMUHAMMAD SHOAIB

Discussion headingsIntroductionEtiopathogenesis Morphological, Biochemical changesMechanism Intrinsic & Extrinsic pathwayDisorders of apoptosisConclusion

Introduction

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.

The word "apoptosis is used in Greek to describe the "dropping off" or "falling off" of petals from flowers, or leaves from trees. Cell death mechanismsDeath by suicide Death by injury

APOPTOSISNECROSISNATURALYESNOEFFECTSBENEFICIAL DETRIMENTALPhysiological or pathologicalAlways pathologicalSingle cellsSheets of cellsEnergy dependentEnergy independentCell shrinkageCell swellingMembrane integrity maintainedMembrane integrity lost

APOPTOSISNECROSISRole for mitochondria and cytochrome CNo role for mitochondriaNo leak of lysosomal enzymesLeak of lysosomal enzymesCharacteristic nuclear changesNuclei lostApoptotic bodies formDo not formDNA cleavageNo DNA cleavageActivation of specific proteasesNo activationRegulatable processNot regulatedEvolutionarily conservedNot conservedDead cells ingested by neighboring cellsDead cells ingested by neutrophils and macrophagesSignificance of apoptosisDuring development many cells are produced in excess which eventually undergo programmed cell death and thereby contribute to sculpturing many organs and tissues

In human body about one lac cells are produced every second by mitosis and a similar number die by apoptosis

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

EtiopathogenesisWhy should a cell commit suicide?

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

Examples: The resorption of the tadpole tail in frog .

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

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

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

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

Examples: Cells infected with viruses

Cells of the immune system

Cells with DNA damage

Cancer cells (Uncontrolled proliferated cells)

Apoptosis in physiologic situations Programmed destruction during embryogenesis

Involution of hormone dependent tissues

Cell loss in proliferating cell populations

Elimination of harmful self- reactive lymphocytes

Death of host cells

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

Apoptosis: in embryogenesisMorphogenesis (eliminates excess cells):Selection (eliminates non-functional cells):15Apoptosis: in embryogenesisImmunity (eliminates dangerous cells):Self antigenrecognizing cellOrgan size (eliminates excess cells):16Apoptosis: importantance in adultsTissue remodeling (eliminates cells no longer needed):Virgin mammary glandLate pregnancy, lactationInvolution(non-pregnant, non-lactating)ApoptosisApoptosis- TestosteroneProstate gland 17Apoptosis: importantance in adultsTissue remodeling (eliminates cells no longer needed):Resting lymphocytes+ antigen (e.g. infection) - antigen (e.g. recovery) Apoptosis18 Apoptosis in pathological conditions - DNA damage

- Accumulation of misfolded proteins

- Cell death in certain infections

- Pathological atrophy in parenchymal organs

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.

Cells infected with viruses One of the methods by which cytotoxic T lymphocytes (CTLs) kill virus-infected cells is by inducing apoptosis

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.

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

Morphological & Biochemical changes

Classic changesCell shrinkage Nuclear fragmentation Chromatin condensationChromosomal DNA fragmentationFormation of cytoplasmic blebs& apoptotic bodiesPhagocytosis

HistologyApoptotic bodies Round oval mass of intensely eosinophillic cytoplasmFragments of dense nuclear chromatin

Bio chemical changesMechanisms of apoptosis

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- 9acts as an initiator of the caspase activation cascade.

Caspase-3key effector in the apoptosis pathway, amplifying the signal from initiator caspases and signifying full commitment to cellular disassembly.InitiationAbsence of stimuli - hormones, growth factorsActivation of receptors TNF familyHeat ,radiation, chemicalsGenetically programmed events

STAGES OF CLASSIC APOPTOSISHealthy cellDEATH SIGNAL / STIMULI (extrinsic or intrinsic)Commitment to die (reversible)EXECUTION (irreversible)Dead cell (condensed, crosslinked)ENGULFMENT (macrophages, neighboring cells)DEGRADATIONReceptor pathway (physiological):Death receptors:(FAS, TNF-R, etc)FAS ligandTNFDeathdomainsAdaptor proteinsPro-caspase 8 (inactive)Caspase 8 (active)Pro-execution caspase (inactive)Execution caspase (active)DeathMITOCHONDRIA33Apoptosis triggered by external signals: the extrinsic or death receptor pathway

Apoptosis triggered by external signals: the extrinsic or death receptor pathwayRegulation 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.Intrinsic pathway (damage):MitochondriaCytochrome c releasePro-caspase 9 cleavagePro-execution caspase (3) cleavageCaspase (3) cleavage of cellular proteins,nuclease activation, etc. DeathBAXBAKBOKBCL-XsBADBIDB IKBIMNIP3BNIP3BCL-2BCL-XLBCL-WMCL1BFL1DIVANR-13Several viral proteins37

Intracellular signalsOxidative damage from free radicals, Radiation, Virus infection, Nutrient deprivation, Pro-apoptotic FactorsDamage to the mitochondrial membrane increasing permeabilityEntry of Cytochrome C into the cytoplasmCytochrome C binds to Apaf-1 forming an apoptosomeApoptosome activates procaspase-9 to caspase-9Caspase-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 permeability

Physiological Intrinsicreceptor pathway damage pathway MITOCHONDRIAL SIGNALSCaspase cleavage cascadeOrderly cleavage of proteins and DNACROSSLINKING OF CELL CORPSES; ENGULFMENT(no inflammation)42Steps Disorders of apoptosisApoptosis: Role in Disease TOO MUCH: Tissue atrophyTOO LITTLE: HyperplasiaNeurodegenerationThin skinetcCancerAthersclerosisetc45Apoptosis: Role in DiseaseNeurodegenerationNeurons are post-mitotic (cannot replace themselves; neuronal stem cell replacement is inefficient)

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

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

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

PARKINSON'S DISEASE

ALZHEIMER'S DISEASE

HUNTINGTON'S DISEASE etc.46Apoptosis: Role in DiseaseCancerApoptosis eliminates damaged cells (damage => mutations => cancer

Tumor suppressor p53 controls senescence and apoptosis responses to damage.

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

High levels of anti-apoptotic proteins or Low levels of pro-apoptotic proteins ===> CANCER47Apoptosis: Role in DiseaseCancerVirus associated cancerSeveral 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).

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 DiseaseCancerOther 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 cellsApoptosis: Role in DiseaseCancerApoptosis: 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)51Apoptosis: Role in DiseaseApoptosis and AIDS Hallmark- the decline in the number of the patient's CD4+ T cells (normally about 1000 per microliter (l) of blood).

In Immune system

Very rarely humans are encountered with genetic defects in apoptosis. The most common one is a mutation in the gene for Fasmutations in the gene for FasL or even one of the caspases are occasionally seen. Autoimmune lymphoproliferative syndrome or ALPS.

ConclusionCells are balanced between life and deathDAMAGEPhysiological death signalsDEATH SIGNALPROAPOPTOTICPROTEINS(dozens!)ANTIAPOPTOTICPROTEINS(dozens!)DEATH55

Thank u.