Martin Luther University Halle-Wittenberg
Lecture 8:Apoptosis -
Controlled Death of Cells
Prof. Thomas Groth
Biomedical Materials Group
Martin Luther University Halle-Wittenberg
Martin Luther University Halle-Wittenberg
Content
• Definition and overview of Apoptosis
• Apoptosis in Embryognesis and adult-tissue remodeling
• Necrosis vs. Apoptosis
• Apoptotic cell removal
• Detection and control of apoptosis
• Role of integrins in apoptosis
• Apoptosis in Neurodegeneration & Cancer
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Apoptosis
• Meaning of greek word “falling of” leaves from trees
• Dying cell shrinks and fragments release of apoptotic bodies
• Specific process of DNA fragmentation
• Engulfment by phagocytes
• Occurs with single cells
• Programme critical for homeostasis embryonic and adult development
• Maintenance of normal cell number
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Apoptosis during the metamorphosis of a tadpole into a frog.
Role of Apoptosis During Development
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Apoptosis and normal morphogenesis.
TUNEL assay (immunodetection) detects breaks in DNA as cells undergo apoptosis
1 day later
Apoptosis of Limb Tissue During Morphogenesis
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Apoptosis in Embryogenesis
Selection to eliminate non-functional cells e.g. neurons that do not contact muscle
Regulation of size and shape of tissue – removal of excess of cells
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Apoptosis in Embryogenesis
Development of immune system removes lymphocytes with auto immunity
Self-antigen recognizing lymphocytes (auto immunity)
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Apoptosis in Adult – Tissue Remodeling
Virgin mammary gland Late pregnancy, lactation Involution(non-pregnant, non-lactating)
Apoptosis
Apoptosis
- Testosterone
Non-activated lymphocytes (red - competent lymphocytes
+ antigen (e.g. infection, red e.g. plasma cells)
- antigen (e.g. recovery)
Apoptosis
Prostate gland
Proliferation
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Necrosis vs. Apoptosis
www.imm.ki.se/sft/text/enews4.htm
• Swelling and burst of cells
• Cause of cell death by hypoxia, intoxication, trauma, inflammation, etc.
• Uncontrolled release of intracellular contents release of enzymes etc.
• Large groups of cells
• Inflammation of surrounding tissue !
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Necrosis vs. Apoptosis
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Morphology of Apoptotic Cells in Culture
Collins JA, et al. 1997
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Phagocytic Macrophage Removes Apoptotic Cell
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“Eat me” Signals Expressed on Surface of Apoptotic Cells
Lauber et al.(2004)
CD – 31 PECAM – Platelet-Endothelial CAM
Eat-me –signals: Phosphatidylserin, degraded ICAMs
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Three Steps of Apoptotic Cell Removal
Lauber et al.(2004)
Find me – signals : many different degradation products of cells
Eat-me –signals: Phosphatidylserin, degraded ICAMs
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The Apoptotic Sequence
Death signals
Modulators Effectors Substrates Death
. FADD
. TRADD
. FLIP
. Bcl-2 family
. Cytochrome c
. p53
. Mdm2
. Caspases . Many cellularproteins
. DNA
. Growth factor Deprivation
. Hypoxia
. Loss of adhesion
. Death receptors
. Radiation
. Chemotherapy
Normal white blood cell
Lodish 6th
Apoptotic white blood cell
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Key Players of Apoptosis
• Extrinsic pathway External transducers such as TNF-a or loss of contact to ECM (Anoikis)
Activation of intracellular Caspase 8 (proteolytic enzyme) Activation of Caspase pathway
• Intrinsic pathway Release of proapoptotic factors from mitochondria (e.g. cytochrom C) due to DNA damage
Activation of Caspase 9 – onset of Caspase pathway
Destruction of DNA and proteolysis
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Maniati et al. 2008
Two Major Pathways of Apoptosis –Intrinsic & Extrinsic Pathway
DISC – death inducing signaling complex
FasLigand – belongs to TNF family found on cytotoxic T lymphocytes
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Apoptosis - Survival and Damage Signals
• Signal transducers - DNA damage, (lack of) growth factor receptor (GFR) signalling, extracellular death ligands such as FasL and TNFR (DR) and lack of adhesion (integrins)
• Control of expression or activity of BH3-only proteins
• BH3 proteins as sensors Signals to multidomain proteins such as Bax and Bcl-XL,
• Permeabilization of the outer mitochondrial membrane release of factors activating caspases Execution phase of apoptosis.
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Overview Apoptosis
Survival
Survival
Death
Group of proteins
www.biochemsoctrans.org/.../0421/bst0320421.htm
GFR – Growth factor receptorsDR – Death receptorsDISC – Death-inducing signaling complex
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Integrin-relatedGrowth factor-related
BH3-only Proteins as Regulators of Apoptosis
Group of proteins stopping anti-apoptotic proteins Bcl-2 promoting pro-apoptotic protein Bax
Caspases: Death proteins !
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Detection of Apoptotic & Necrotic Cells by Annexin-5
A heterogeneous mixture of macrophages after 48 h IFNgamma+LPS treatment: healthy cells with mitochondrial membrane potential (stained red with TMRM), early stage apoptotic cells (stained green with annexin V-FITC and red with TMRM), and late stage apoptotic cells (stained green with annexin V-FITC).http://www.ucl.ac.uk/wibr/research/mito/sm/garedew/figure2.htm
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Apoptosis – DNA Fragmentation
Oxford Textbook of Pathology, Volume 1, Oxford University Press, 1992
• Key feature of apoptosis due to activation of endonucleases
• Cleavage of chromatin into internucleosomal fragment of roughly 180 base pairs (bps) and multiples thereof DNA laddering
• DNA fragmentation by Caspase-Activated DNAse (CAD)
• Apoptosis DNA ladder (right lane)
• Necrosis – DNA smear (left lane)
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Detection of Apoptosis by TUNEL AssayTerminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)
dUTP – desoxy uracil triphosphate
http://www.phnxflow.com/apobrdu.html
TdT - terminale Desoxyribonukleotidyltransferase adds Nukleotides to 3'-terminal end of DNA after activity of endonucleases
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Control of Apoptosis - Extrinsic Pathway
Death receptors:
FAS-R, TNF-R,
T-LymphocytesFAS ligand
Macrophages TNF – Tumor necrosis factor
Deathdomains
Adaptor proteins
Pro-caspase 8 (inactive) Caspase 8 (active)
Pro-execution caspase (inactive)Execution caspase (active)
Cell deathMitochondria
Cytotoxic T-lymphocyte
Target cells
Apoptosis can occur via cell-surface-death-receptor- ormitochondrial-dependent pathways. Cytotoxic Tlymphocytes (CTLs) express Fas ligand (FasL), whichbinds to the death receptor Fas on the target cell.Regulation of lymphocyte proliferation and death by flipMargot Thome & Jürg TschoppNature Reviews Immunology 1, 50-58 (October 2001)
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Control of Apoptosis - Intrinsic Pathway
Mitochondria
Cytochrome C release
Pro-caspase 9 cleavage
Pro-execution caspase (3) cleavage
Caspase (3) cleavage of cellular proteins,Nuclease activation,
Etc.
Cell Death
BAXBAKBOKBCL-XsBADBIDB IKBIMNIP3BNIP3
BCL-2BCL-XLBCL-WMCL1BFL1DIVANR-13Severalviralproteins
Pro-apoptotic proteins
Anti-apoptotic proteins
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Integrin-Ligation and Apoptosis
• FAK major role in transfer of survival signals FAK activates phosphatidylinositol -3 kinase (PI-3K) activates further kinases Inactivation of proapoptotic proteins (Bad und Caspase-9)
• Integrin-specific survival signals generated by a5b1-integrin Increased expression of anti-apoptotic protein Bcl-2.
Fibronectin
b1
Caspase-9
FAK
PI-3 K
a5Bcl-2
FN
Bad
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0
20
40
60
80
100
0
0,5
1
1,5
2
2,5
3
CH3 PEG NH2
C11
NH2 C3 COOH OH
Growth index
Viability and Growth of Fibroblasts
Viability(%)
Viability and growth of cells reduced on surfaces with low tyrosine phosphorylation (see lecture on signal transduction)
Lower ligation of integrins on extremely hydrophilic surfaces (low protein adsorption) reduced survival of cells indicated by lower viablilty
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Jay S. Desgrosellier and David A. Cheresh
Role of Integrins in Apoptosis• Integrins pro-survival as well as pro-
apoptotic signals. dependent on ligation status of integrins expressed by a given cell.
• Most of integrins ligated pro-survival pathway is initiated through increased nuclear factor-κB (NF-κB) or PI3K–AKT activity, decreased p53 activation and increased expression of the pro-survival molecules BCL-2 and FLIP (also known as CFLAR).
• Cooperative signalling between growth factor receptors and integrins activates Raf leading to distinct mechanisms of cell survival.
• Signalling through integrin αvβ3 and the fibroblast growth factor receptor promotes phosphorylation of Ser338 and Ser339 of Raf, protecting cells from the intrinsic pathway of apoptosis,
• Integrinαvβ5 and vascular endothelial growth factor receptor 2 phosphorylate Tyr340 and Tyr341 of Raf, preventing apoptosis through the extrinsic pathway.
• In cells in which many of integrins are unligated, cleavage of caspase 8, triggering apoptosis through integrin-mediated death (IMD).
• On complete loss of adhesion, cell death is initiated through a process termed anoikis.
CASP8 and FADD-like apoptosis regulator is a protein that in humans is encoded by the CFLAR gene – also called FLIP
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• β1 integrins (that is, αxβ1) and integrin αvβ3 induce adhesion-dependent activation of focal adhesion kinase (FAK) and Src, in addition to phosphorylation of the adaptor protein p130 CRK-associated substrate (p130CAS).
• Signaling events result in invasion, proliferation and survival of tumour cells bound to the extracellular matrix (ECM).
• In suspended tumour cells unligated integrin αvβ3 signals directly through SRC and p130CAS increase cell survival independently of FAK.
• High expression of avb3 poor prognosis
Jay S. Desgrosellier and David A. Cheresh, Nature 2010
Effect of Integrins on Apoptosis in Cancer Cells
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Another Escape of Tumor Cells by Release of Small Integrin-Binding Proteins
http://www.nature.com/nrc/journal/v8/n3/full/nrc2345.html
DMPI, BSP, OPN –small integrinbinding proteins
uPA –plasminogen activator
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• Small integrin-binding ligand-N-linked glycoproteins (SIBLING) gene family includes bone sialo protein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), matrix extracellular phosphoglycoprotein (MEPE) and osteopontin (OPN)
• Expression level elavated in tumor tissue anti-apoptotic function
• but also found in serum of patients tumor marker
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Apoptosis in Neurodegeneration & Cancer
Increased apoptosis• Neurons are post-mitotic (cannot replace
themselves)
• Neuronal death caused by loss of proper connections,
• loss of proper growth factors (e.g. NGF),
• damage (especially oxidative damage)
• Neuronal dysfunction or damage results in loss of synapses
• (synaptosis; reversible)
• apoptosis (irreversible)
• PARKINSON'S DISEASE
• ALZHEIMER'S DISEASE
• HUNTINGTON'S DISEASE etc.
Inhibition of apoptosis
• Apoptosis eliminates damaged cells
• (damage => mutations => cancer
• Tumor suppressor p53 controls senescence and apoptosis responses to damage
• Most cancer cells defective in apoptotic response
• High levels of anti-apoptotic proteins
or
• Low levels of pro-apoptotic proteins
• ===> CANCER
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- Increased Bcl-2 Poor prognosis
- Increased FasL Decreased cytotoxic T lymphocyte number
- FasL induction (with Doxorubicin) Determines chemosensitivity
- Overexpression of BaxImproves the efficacy of chemotherapy
- p53 auto antibodies Resistance to chemotherapy with cisplatin + 5-Fluorouracil
Expression Levels of Apoptosis-RelatedProteins Determine Patient-Specific Malignancy
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Summary
• Apotosis highly regulated process to remove excess or defectivecells
• Extrinsic and intrinsic pathways switch on Caspasesdegradation of DNA, proteolysis and blebbing of membranes intovesicles
• Uptake and digestion by macrophages
• Opposite role of integrins in health and diseases
• Dysregulation of apoptosis in degenerative diseases and blockingof apoptosis in cancer
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Literature
• Molecular Biology of the Cell, 4th edition, Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter, New York: Garland Science
• Molecular Cell Biology,Harvey Lodish, Arnold Berk, Chris A. Kaiser, Monty Krieger, Matthew P. Scott, Anthony Bretscher
• The Biology of Cancer, Robert A. Weinberg, Garland Science