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The history of Apoptosis

What Apoptosis means

Significance of Apoptosis.

Morphological changes

Major players in apoptosis.

Pathways to apoptosis

Therapeutic implications.

Pathological implication

Detection of apoptotic cells

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The History of Apoptosis

John Kerr:

a Brisbane pathologist in 1972

introduced a concept ‘Apoptosis’.

(Richardson, et al., 2000)

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Synonyms: - Antonym: -

Programmed cell death. Necrosis

Programmed cell cell death are of two types: - are of two types: -

Apoptosis : -(Type I)

Autophagic : -(Cytoplasmic, or Type II)

The formation of large vacuoles that eat away organelles

before the nucleus is destroyed.

(Offermanns & Rosenthal , 2004)

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Greek origin word known ‘Falling off or Dropping off.’ Self destruction programmed genetically sequenced in biomedical events.

It’s a kind of counterbalance by elimination of similar number of cells. Death by design.

Apoptosis: - (Rang & Dale, 2008)

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Significance of Apoptosis

Deletion of dangerous & damage cells. Development of organs Without apoptosis human

gut can grow 19 km,whole

epithelial lining changes every

23 days. Regulation of immune system(T-cells)

Incomplete differentiation due to lack of apoptosis

Cancer & Neurodegenerative diseases.

(Gewies, 2003)

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Apoptosis Necrosis

• Energy dependent PCD • Cell dies due to damage

• One cell • Group of cells

• No inflammation • Acute inflammation

• Role of mitochondria • No role of mitochondria

• Dead cells eaten by macrophages

• Dead cells removed by macrophages.

• Cell shrinkage • Cell swelling

(Haiat, 2006)

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The Apoptotic Cell

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Morphological changes in Apoptosis(Hengartner, 2000)

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Major players in Apoptosis

Apoptosis Initiating factor(AIF):-

Release from mitochondria enter in

nucleus & trigger cell suicide.

CaspasesBcl-2 Proteins Apoptosome p53 gene and p53 protein

(Rang & Dale, 2008)

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• It is family of cystein protease.

• present in cell in inactive form.

• Cleave protein bearing a amino acid

sequence located at the terminal.

(cystein aspartic acid specific protease)(Hill,et al., 2003)

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Caspases14 different Caspases

Initiator Caspases

Effectors Caspases

2,8,9,10.

3,6,7.

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Apoptosis carried out by Caspases(Susin, et al., 1999)

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CARD/DED Domain

Large subunit

Small subunit

Procaspase Structure 15

Caspase activation

Inactive Procaspase

Proteolysis

cleavage Activation of Caspase

Initiator Caspase

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Active Initiator Caspase

Cleavage by protein-protein interaction

Activation of effector caspase

Caspase activation

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The Bcl-2 Family

Regulator Protein of Mitochondria.

Isolated from gene involve in B-cell lymphoma.(Bcl-2)

All have BH3 domain (Bcl-2 Homology)

BH3:-It is Proapoptotic domain expose on activation.

(Dash, et al., 2001)

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Bcl-2 family members:-

BH1-4: - Anti-apoptotic proteins Bcl-2, Bcl-xL .

BH1-3: - Pro-apoptotic proteins Bax, Bok, Bak.

BH 3: - Only pro-apoptotic proteins Bad, Bik, Bid.

Bcl-2: -ß-cell Lymphoma, BH:-Bcl-2 Homolog, Bid: - Bcl-2 interacting domain, Bcl-xL :- x=Close homolog & L= inhibit apoptosis.

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Functions:-

Regulate the balance between Pro-Apoptotic &

anti-apoptotic.

Involved in Intrinsic pathway.

Induction of Bcl-2 is by death signals.

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Apoptosome

Multisubunit protease activation complex.

Form after induction of death signal in mitochondrial

pathway.

Discovery of Apoptosome by ‘prof. Wang’.

Wheel-like structure connected to seven radial spokes

(Adrain, 2001; Dash, 2003)

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Active caspase

Apoptic stimuli

Cytochrome-c

Binding

Apaf-1

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Apoptotic activation protease factor-1

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p53 gene & p53 protein

p53 is a tumor suppressor gene.

Prevent completion of cell cycle.

If DNA damage occurs activation of gene p53 response to cell death.

Curable damage can be repaired

In curable damage of DNA leads to cell death

(Dlamini et al.,2005)

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Pathways to Apoptosis

1) Death receptor pathways /(Extrinsic Pathway)

2) Mitochondrial pathways /(Intrinsic Pathway)

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Death receptor pathways /(Extrinsic Pathway)

Induction is mediated through cell surface receptors

know to be ‘Death receptors’.

Death ligands are activating these receptors.

Further activation of Caspases leads to cell death

(Rang & Dale, 2008)

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Death Receptors

TNFR - Super family

TNFR-Tumor necrosis factor Receptor, CD: -Cluster differentiationTRAIL- Tumor necrosis factor –α related apoptosis inducing ligandFas:- Apoptosis stimulating fragment.

Death receptor Death ligands

TNFR-1 CD95 (Apo-1) (Fas) (TNFR-2) TRAIL-R1 (DR-4) TRAIL-R2 (DR-5)

TNF-α

CD95L (FasL)

TRAIL-R1

TRAIL-R2

(Ashkenazi, 2002)

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TNF Receptor signaling

Binding of Ligand to Death receptor

Binding of adapter protein with Activation of Another protein TRAF-2

TRAF-2: -TNF associated factor -2,TRADD:- TNF associated death domain,

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Fas:- Apoptosis stimulating fragment ., FADD:- Fas Associated death domain

Adapter Protein

Fas signaling pathway

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Death receptors signal transduction Death receptors signal transduction

Effectors domain

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The Extrinsic Apoptosis Pathway

FADD, Fas-associated death domain, DR:- Death receptors.

Pro-apoptotic ligands

Caspase 3, 6, 7

Apoptosis

FADD

DR5

DR4

Procaspase 8, 10Caspase 8, 10

Plasma membrane

(Ashkenazi A. Nat Rev Cancer 2002;2:420–430.)

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Macrophage release anti-inflammatory mediators like [transforming growth factor(TGF-ß] & IL-10]

Macrophage undergo phagocytosis of cell

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Mitochondrial pathways /(Intrinsic Pathway)

Release of protein from mitochondrial inner membrane

into cytosol.

DNA damage induce apoptosis.

Cellular stress, heat shock, oxidative stress, cellular

damage, that are also induced apoptosis.

(Martin, et al., 2003)

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Plasma membrane

Cytosol

DNA Damage

P53 protein

Pro-apoptic Bcl-2

Mitochondria

Apaf-1

Cytochrome-c

Procaspase-9 34

Caspase-9

Caspase-3

Activation

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APOPTOSIS

Video

(http://www.researchapoptosis.com) 36

Apoptosis By Injury

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Pathological Implications

Growth of tissue & organs in embryo. Replenishment of time-expire cells like gut-epithelium. Repair & healing after injury or inflammation. Regeneration of tissue. Hyperplasia.

(Barton, et al., 2004)

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Therapeutic implications

Neurodegenerative disease like Alzheimer’s disease.

Myocardial infraction.

Targets for anti-cancer drugs.

(Gerald, 2002)

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Neurodegenerative disease: -

Alzheimer’s disease show excessive Apoptosis.

Neuronal growth factor & brain derived neurotrophic factors. Secrets protein that maintain the balance Pro- Apoptotic & Anti-Apoptotic.

Blocking of these factor are new area of interest for Target drug delivery.

(Gerald, 2002)

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Apoptotic Neuron41

Myocardial Infraction: -

Occur due to blockage of coronary artery by thrombus.

Decrease in oxygen supply leads to (M.I.) by necrosis or Apoptosis

Two pathways of cell death 1) Necrosis 2) Apoptosis

(Rang & Dale, 2008)

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Current approach are finding to inhibit these pathway which are beneficial in clinical

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ICE:-interleukin -1 converting enzyme. PARP:- (poly-ADP-ribose-polymerase .

Targets for Anti-cancer drugs: -

Bcl-2 family as a target for new drug.

Bcl-2 is Anti-apoptotic & increase resistance to

cancer chemotherapy.

Death receptor & there respective ligands are useful

in targeting the Anti-cancer drug.

(Fischer, et al., 2005)

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How do we recognize Programmed Cell Death?

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Detection of apoptic cells

Electron Microscopy Fluorescence Microscopy Staining with Eosin

Detection of PCD Based on Morphology

Detection of DNA Fragmentation

Agarose Gel Electrophoresis DNA Fragments by Cell Fractionation Detection of DNA Fragmentation by Filtration Assay.

(Kaufmann, 2000)

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Terminal Deoxyribonucleotidyl Transferase-Mediated dUTP Nick End Labeling.(TUNEL)

Flow Cytometry.

Enzyme Assay.

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Detection Based on Morphology: -Detection Based on Morphology: -

Shrinkage of the cell.

Detachment from its neighbors.

Fragmentation of nucleus.

Packaging of the cell into multiple plasma membrane.

Phagocytosis of these fragments.

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Control Apoptotic cells

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Electron Microscopy: -

A beam of electron using electric & magnetic field to form enlargement of image on photographic plate.

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Electron Microscopy: -

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Detection of DNA fragmentation by Gel Electrophoresis.

Agarose gel with suitable separation properties.

Application of total cellular DNA to an Agarose.

The fragments are spared throughout agarose gel.

Fragments will be detected by Ethidium bromide.

(Anders, 2002)

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1. DNA from apoptotic cells.

2. DNA isolated from necrotic

cells

3. No samples

4. DNA from normal cell

5. DNA from normal cell

6. No sample

Gel Electrophoresis

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Flow Cytometry

Well known as a ‘Flow Microfluriometer. To study Normal & Malignant Lymphocytes. Measures amount of fluorescence associated with cell. Photomultiplier tube is a detector. Analyzing the population of cell. Around 20,000 cells can be analyzed in few min.

(Kaufmann, 2000)

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http://www.kimmelcancercenter.orgFlow Cytometry

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Advance Flow Cytometry

http://www_biotech_missouri_edu

Adrain, C. Martin, S.J. 2001 The mitochondrial apoptosome: A killerunleashed by the cytochrome seas. Trends Biochem. Sci. 26, 390–397.

Ashkenazi, A.Dixit, V. M., 1999. Apoptosis control by death and decoy receptors. Current Opinion in Cell Biology 11, 255-60.

Dash, P., 2001. Apoptosis Basic Medical Sciences St.George’s, University of London Fischer, U.OsthofF, K. S., 2005. New Approaches and Therapeutics Targeting Apoptosis in Disease: Pharmacological reviews, 57 (2), p.187-215.

Hill, M.M. Adrain, C. Martin S.J., 2003.Molecular Cell Biology Laboratory, Department of Genetics Smurfit Institute, 3 (1) p.19-24.

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Gewies, A., 2003. Introduction to Apoptosis :Apo Review p.1-26

Hill, M. M.Adrain, C., 2003. Portrait of the killer the mitochondrial Apoptosome emerges from the shawdo:molecular intervention. 3 (1)p.19-24.

Kaufmann, S.2001. Apoptosis:Phaarmacological implication and therapeutic opportunites.New York: Academic Press

Li, L.Y., Luo, X. Wang, X., 2001 Endonuclease G is an apoptoticDNase when released from mitochondria. Nature 412, 95–99.

Margaret, K.T. Cohen, J., 2001. Standard Quantitative Assays for Apoptosis: Molecular Biotechnology, 19 p.305-315

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Offermanns, S. Rosentha, W., 2004. Encyclopedic Reference ofMolecular Pharmacology,Springer

Rang, H. P. Dale, M. M., 2008.Cell proliferation and Apoptosis: Pharmacology.6th ed.Churchill Livingstone Elsevier.p.72-88.

http://www.researchapoptosis.com

http://www.apoptosisinfo.org

http:// www.bitessizebio.com

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