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