Cell Cycle 4 – Apoptosis

Anil Chopra

  1. Explain the difference between necrosis and apoptosis; describe how they may be distinguished.
  1. Discuss whether necrosis and apoptosis are the only forms of cell death.
  1. Describe the proteolytic caspase cascades which execute the apoptotic response.
  1. Discuss how apoptosis may be mediated through death receptors and/or mitochondria.
  1. Discuss how Bcl-2 family proteins may modulate apoptosis.

Apoptosis (cell death) occurs in the following cells:

1. Harmful cells (e.g. cells with viral infection, DNA damage)

2. Developmentally defective cells (e.g. B lymphocytes expressing antibodies against self antigens)

3. Excess / unnecessary cells:

(embryonic development – e.g. brain to eliminate

excess neurons; sculpting of digits and organs)

4. Obsolete cells (e.g. mammary epithelium at the end of lactation)

5. Chemotherapeutic killing of cells

Necrosis - unregulated cell death associated with trauma, cellular disruption and an inflammatory response. In necrosis the plasma membrane becomes permeable, the cells swells and all membranes in the cell rupture. This is results in proteases being released and autodigestion of the cell to occur. Leads to localized inflammation.

Apoptosis (programmed cell death) - regulatedcell death; controlled disassembly of cellular contents; no inflammatory response (e.g. haematopoietic cells). Apoptosis takes place in two phases:

Latent Phase: this is when the death pathways are activated but the cell appears morphologically the same

Execution phase:

  • Loss of microvilli and intercellular junctions
  • Cell shrinkage
  • Loss of plasma membrane asymmetry (phosphatidylserine lipid appears in outer membrane)
  • Chromatin and nuclear condensation
  • DNA fragmentation
  • Formation of membrane blebs
  • Fragmentation into membrane-enclosed apoptotic bodies
  • These are then phagocytosed by macrophages.
  • The plasma membrane remains intact throughout.

DNA Modification in Apoptosis

Diagram shows DNA ladders and shows the process of fragmentation in apoptosis. The DNA chain sizes get smaller through apoptosis:

This is determined by “TUNEL assays” (Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick EndLabelling):

•DNA fragmentation leads to more “ends” which are labelled by transferring a biotinylated U onto the end by TdT

• this is stained with fluorescent avidin that recognises biotin.

Apoptosis-like PCD - some, but not all, features of apoptosis. Display of phagocytic recognition molecules before plasma membrane lysis

Necrosis-like PCD - Variable features of apoptosis before cell lysis; “Aborted apoptosis

There is a graded response

Mechanisms of Cell Death

Caspases

Caspases are known as the “executioners”. They stand for Cysteine-dependentaspartate-directed proteases.They are activated by proteolysis and cause a cascade of activation.

There are 2 different types:

Initiator caspases: these initiate apoptosis by cleaving “pro-domains” off effector caspases, activating them. (proteolytic cleavage). Some contain a CARD – CAspase Recruitment Domain, and others contain a DED

(Death Effector Domain)

Effector caspases: these activate other proteins that initiate apoptosis.E.g. Caspase-ActivatedDNase, CAD either by direct cleavage or cleavage of inhibitory molecules.

Caspase activation can happen by two different means:

Death by design – Receptor-mediated (extrinsic) pathways

Death by default – Mitochondrial (intrinsic) death pathway

Death receptors span the membrane, which, when acted on by ligands, initiate apoptosis. E.g. the Fas receptor: when it is acted on by the Fas-ligand causes the FAS-associating death domain FADD, containing procaspase 8, to travel to the membrane. This results in theoligomerisation of procaspase 8 through DED forming a Death-InducingSignallingComplex (DISC).Initiator caspases are released.

Inhibitors

Caspases can be inhibited by FLIP via (NFkappaB). These cause caspase homology in DED domain, but no proteolytic activity. They are competitive inhibitors as they compete and bind to FADD (or other receptor tails) which interferes with cleavage.

Mitochondrial Control of Apoptosis

  1. Cellular stresses are placed on the mitochondria.
  2. The mitochondrial membrane loses its membrane potential.
  3. Cytochrome C is released.
  4. Other apoptotic factors are released.
  5. Apoptosome is formed (Apaf-1, caspase 9).

Apoptosome: cytochrome C binds to the WD-40 part of the Apaf-1 causing 7 Apaf-1 monomers to form a heptamer – the Apoptosome to which caspase 8 can bind. Caspase 8 is the main initiator caspase.Each Apaf-1 in the heptameric apoptosome can also bind a caspase 9, which in turn cleaves it – activating it and therefore resulting in its entering the caspase cascade and causing apoptosis.

BID

The two pathways are linked together by a molecule called BID. When caspase 8 is released at the receptor, it cleaves BID which in turn enhances the release of proteins from the mitochondria and is therefore an important initiator of the mitochondrial death pathway.

The apoptosome requires ATP.Energy levels in the cell may determine whether death is by necrosis or apoptosis.

Modulators of apoptosis: Bcl-2 family proteins

Bcl-2 is a family of proteins that are either

Pro-apoptotic: can be cytosolic or mitochondrial

  • Bad
  • Bax
  • Bcl-xS

Anti-apoptotic: usually mitochondrial

  • Bcl-2
  • Bcl-xL

In cell survival, the Bad proteins are inactive due to the presence of growth factors. Without these, the Bax and Bak proteins are bound to the Bcl-2 and Bcl-xL proteins on the mitochondrial membrane and are inactive.

In apoptosis, the Bad proteins are dephosphorylated and therefore released so that they can bind to the Bcl-2 and Bcl-xL on the mitochondrial membrane. Therefore allowing Bax and Bak to be active and allow cytochrome C to be released.

Inhibitor of Apoptosis Proteins

Inhibitor of Apoptosis Proteins (IAPs) regulate Programmed Cell Death. They do this either by inhibiting procaspase activation or inhibiting the activity of caspases themselves. The IAPs themselves can be inhibited by proteins that leak out of the mitochondria.

Growth Factors

Growth factors also inhibit programmed cell death (above) e.g. phosphatidylinositol 3’-kinase (PI3K) – a lipid kinase. It:

Phosphorylates and inactivates Bad

Inactivates FOXO transcription factors (FOXOs promote expression of apoptosis-promoting genes)

Phosphorylates and inactivates caspase 9

Other, e.g. stimulates protein synthesis.

Is counteracted by PTEN (lipid phosphatase)