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Programmed Cell death
Saeb AliwainiApril/2013
Introduction • Human Body makes 10 billion cells every day.
• Cell death makes balance :
• There are various forms of cell death. Amongst these, two well-known pathways are necrosis and apoptosis. Other less described cell death pathways are mitotic catastrophe ,autophagy and necroptosis.
Apoptosis
• Apo = from , ptosis = falling ( dropping off)
• Apoptosis is characterized by specific biochemical and morphological features that culminate in shrinkage of the cell to apoptotic bodies that are engulfed by neighboring macrophages.
A- Apoptosis occurs during normal cell turnover, tissue homeostasis, embryogenesis, induction and maintenance of immune tolerance.
Major inducers of apoptosis
- Irreparable DNA damage- Cell cycle perturbation- Death ligands, e.g., Fas ligand- Growth factor withdrawal- Calcium influx-Free radicals- Radiation therapy- Chemotherapy drugs
Other inducers
Mechanisms of Apoptosis
• The molecular mechanisms of apoptosis were highly conserved through evolution
Mechanisms of Apoptosis
• Caspases:• The "c" of "caspase" refers to a cysteine protease, while the
"aspase" refers to the enzyme's unique property to cleave after aspartic acid residues
• Their proteolytic activity cleaves their substrate after Asp residues.
• Caspases are synthesized in cells as catalytically inactive zymogens
• Their activity is irreversible• Tow types of caspases in Apoptosis: Initiator and effector
Caspases and Caspases dependent Apoptosis
- Is typically activated in the early stages of apoptosis.- It cleaves key cellular components: Such cytoskeleton proteins and nuclear proteins such as DNA repair enzymes. - Nuclear condensation and cell shrinkage. - The cells express (translocation of phosphatidyl serine from
the inside of the cell to the outer surface).
Caspases and Caspases dependent Apoptosis
A- Extrinsic pathway
- Is mediated by death receptors- Apoptosis via this mechanism is very rapid
Induction of apoptosis by TNF receptor
- Cell membrane receptors, which belong to tumor necrosis factor (TNF) family.
- Including TNF-receptor 1, Fas, DR3, DR4, DR5, and DR6
- Specific ligands, such as TNF-α, lymphotoxin, Fas ligand
- Conformational changes to form death inducing signaling complex. ( DISC)
- Followed by recruitment of one of the caspases, typically caspase 8, to the DISC
Caspases and Caspases dependent Apoptosis
B- Intrinsic pathway
- Also called mitochondrial-mediated apoptosis.
- Main inducer : DNA damage
- The Bcl2 families of proteins are the main mediators of this process.
This family has up to 4 conserved domains, known as the Bcl2 homology (BH) domains
• The Bcl2 family contains both pro-apoptotic members and anti-apoptotic members.
• The pro-apoptotic members :- Bax family that consist of Bax, Bok and Bak. - BH-3 only proteins, such as Bid, Bad, and Bim.
- The anti-apoptotic members of the Bcl2 family contain all 4 conserved domains, such as Bcl2 and Bclxl .
- Bcl2 are found exclusively within intracellular membranes and within the cytosol.
How they work?
• Pro apoptotic proteins : to release apoptotic-signaling factors from the mitochondria through mitochondrial outer membrane permeabilization (MOMP) via opening of the membrane permeability transition pore.
• Anti-apoptotic: function by preventing apoptosis through heterodimerization with pro-apoptotic proteins and self over-expression.
- cytochrome c, Smac/DIABLO, and HtrA2/Omi are capsases dependent
- The apoptosome formation and effector caspase activation that causes the token apoptotic events, such as chromatin condensation, plasma membrane asymmetry, and cellular blebbing
Caspase-independent apoptosis
• Various factors are involved: 1- Apoptosis-inducing factor: - The lysosomal protease cathepsin D trigger AIF .- AIF becomes an active cell killer when it is released to the
cytosol.
- With factor # 2 (endonuclease G) they induce peripheral chromatin condensation.
- The lethal effects of AIF are controlled by the anti-apoptotic protein heat shock protein 70 that interacts with AIF and protects against its apoptogenic effects.
3- Endoplasmic reticulum• Calcium release and activation of caspase12
Inhibitor of apoptosis proteins:
- The IAP proteins function as endogenous caspase inhibitors
- NAIP, c-IAP1, c-IAP2, XIAP, and Survivin
- Survivin prevent caspase-9 activation within a functional apoptosome
- Control of mitosis- Survivin can enhance microtubule stability in metaphase spindle formation.
Signals involved in Apoptosis
Stimulus
Cisplatin mechanism of action
Necroptosis Autophagy Mitotic catastropheNecrosis
Tamoxifin, Doxorubicin, 5 FU and other chemotherapeutics
Apoptosis assays
• 1. Cytomorphological alterations• 2. DNA fragmentation• 3. Detection of caspases, cleaved substrates,
regulators andinhibitors• 4. Membrane alterations• 5. Mitochondrial assay
• Transmission electron microscopy (TEM)• Detection of Caspases, Cleaved Substrates. (PARP, H2AX,.. )• Apoptosis PCR microarray is a relatively new methodology
that uses real-time PCR to profile the expression of at least 112 genes involved in apoptosis
• Membrane Alterations externalization of phosphatidylserine residues on the outer plasma membrane of apoptotic cells allows detection via Annexin V
• Mitochondrial Assays mitochondrial assays and cytochrome c release allow the detection of changes in the early phase of the intrinsic pathway.
Cytomorphological alterations
1. Cytomorphological alterations and 2- DNA fragmentation
2. DNA fragmentation
2-DNA fragmentation
G0-G1
Typical Cell cycle profile
G2-M
S SubG1
G1
SG2M
2n
4n
2n
Cell cycle analysis
MDA-MB-231 cells
Plate 3 X 105 cells/well in 6 well plates
48 hours settle
Treat with 0.2 µm AJ-5 For 24 and 48 hours
Trypsinize and fix with cold ethanol for at least 30min
FACS process and analysis
A- Cell cycle analysis
A- Cell cycle analysis
AJ-5 induces a G1 arrest and apoptosis (sub-G1 peak) in breast cancer cells
MDA
-MB-
231
MCF
7
33
Treat cells with 0.2 µm AJ-5 for 24 and 48 hours
Annexin V staining
MDA-MB-231 cells
Plate 3 X 10 5cells/well in 6 well plates
48 hours settle
Trypsinize and stain cells with (Annexin V and PI)
Quadrant Statistics
File: Data.010 Log Data Units: Linear Values
Sample ID: 0.2 48 h MDA-mb Acquisition Date: 28-Jan-12
Gate: G1 Gated Events: 11236
Total Events: 20000 X Parameter: ANEXIN V FITC (Log)
Y Parameter: PI FL3 (Log)
Quad % Gated X Geo Mean Y Geo Mean
UL 1.20 106.74 609.88
UR 43.34 938.40 1023.18
LL 23.32 65.77 26.52
LR 32.14 713.67 94.40
Quadrant Statistics
File: Data.009 Log Data Units: Linear Values
Sample ID: 0.1 48 h MDA-mb Tube: Untitled
Panel: Untitled Acquisition Tube List Acquisition Date: 28-Jan-12
Gate: G1 Gated Events: 16408
Total Events: 20000 X Parameter: ANEXIN V FITC (Log)
Y Parameter: PI FL3 (Log)
Quad % Gated X Mean Y Mean
UL 1.58 149.10 696.57
UR 8.17 1453.71 2421.16
LL 87.66 85.89 13.68
LR 2.58 702.37 40.15
Quadrant Statistics
File: Data.005 Log Data Units: Linear Values
Sample ID: untreated) Acquisition Date: 28-Jan-12
Gate: G1 Gated Events: 17188
Total Events: 20000 X Parameter: ANEXIN V FITC (Log)
Y Parameter: PI FL3 (Log)
Quad % Gated X Mean Y Mean
UL 0.70 82.38 550.14
UR 6.37 1091.43 2972.33
LL 91.72 31.64 9.83
LR 1.21 456.55 41.69
0 200 400 600 800 1000Forward Scatter
Data.006
R1
100 101 102 103 104
ANEXIN V FITC
Data.005
100 101 102 103 104
ANEXIN V FITC
Data.009
100 101 102 103 104
ANEXIN V FITC
Data.010
Necrosis Late apoptosis
Early apoptosis Viable cells
FACS analysis
3- Membrane alterations
AJ-5 induces apoptosis in breast cancer cells in accordance with the cell cycle analysis.
Detection of caspases, cleaved substrates, regulators andinhibitors
0.0 µM AJ-5 0.2 µM AJ-5 0.0 µM AJ-5 0.2 µM AJ-5
- Nuclear fragmentation after treatment with 0.2 µM AJ-5 for 24 h followed by Hoescht staining.
MCF7 MDA-MB-231
AJ-5 treatment leads to an increase in PARP cleavage level and nuclear fragmentation confirming that it induces apoptosis.
• 5. Mitochondria assays
o Cytochrome C release
Methodologies: Apoptosis Assays
Gang et. al (2010)
38
Extrinsic pathway
Apoptosis
Intrinsic pathway
Bid, Bad, Puma, Noxa
p53
Effector Caspases 3,7 and 6
Caspase 8
Cyto C
MAPK
p21
Cell cycle arrest
BH3 proteins
Which apoptosis pathway is induced by AJ-5 ?
MCF7 MDA-MB-231
apoptosis pathway Markers?
Check for intrinsic and extrinsic apoptosis pathways markers
AJ-5 activates both intrinsic and extrinsic as early as 1h of the treatment
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