lecture controlled cell death - apoptosis · •necrosis vs. apoptosis •apoptotic cell removal...

Martin Luther University Halle-Wittenberg

Lecture 8:Apoptosis -

Controlled Death of Cells

Prof. Thomas Groth

Biomedical Materials Group



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


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


Apoptosis during the metamorphosis of a tadpole into a frog.

Role of Apoptosis During Development


Apoptosis and normal morphogenesis.

TUNEL assay (immunodetection) detects breaks in DNA as cells undergo apoptosis

1 day later

Apoptosis of Limb Tissue During Morphogenesis


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


Apoptosis in Embryogenesis

Development of immune system removes lymphocytes with auto immunity

Self-antigen recognizing lymphocytes (auto immunity)


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


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 !

http://www.imm.ki.se/sft/text/enews4.htm


Necrosis vs. Apoptosis


Morphology of Apoptotic Cells in Culture

Collins JA, et al. 1997


Phagocytic Macrophage Removes Apoptotic Cell


“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


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


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


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


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


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.


Overview Apoptosis

Survival

Survival

Death

Group of proteins

www.biochemsoctrans.org/.../0421/bst0320421.htm

GFR – Growth factor receptorsDR – Death receptorsDISC – Death-inducing signaling complex

http://www.biochemsoctrans.org/.../0421/bst0320421.htm


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 !


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


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)


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


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)


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


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


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


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

http://en.wikipedia.org/wiki/Protein

http://en.wikipedia.org/wiki/Gene


• 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


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


• 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


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


- 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


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


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

lecture controlled cell death - apoptosis · •necrosis vs. apoptosis •apoptotic cell removal...

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