Apoptosis and Cancer

What is Apoptosis?
Apoptosis is programmed cell death.

Apoptosis is a normal physiological process:
Foetal development: finger and toe development requires loss of tissue
between.
Removal of endometrium at menstruation.
Formation of synapses in brain requires loss of surplus cells.

Apoptosis is need to remove cells that pose a threat:
Cytotoxic T lymphocytes kill viral infected cells by inducing apoptosis.
Effector cells of the immune system must be removed after the
response.
Damaged cells must be removed if they cannot be repaired.


Apoptosis
Apoptosis from Greek, meaning to fall away
from.

The release of apoptotic bodies from cells is
supposed to be analogous to leaves falling
from trees.

Pronounced A-po-toe-sis

Discovery of Apoptosis
Kerr, J.F.R, Wyllie, A.H. Currie, A.R., 1972. Apoptosis: a basic biological
phenomenon with wide ranging implications in tissue kinetics. British
Journal Cancer 26, 239-257.

They observed a necrosis-like process in the livers of rats where the
portal supply of blood had been interrupted.

No inflammation was observed (typical of necrosis).

Histologically, the cells appeared as small particles of chromatin
surrounded by cytoplasm.
Apoptosis in Cells
Cells that undergo apoptosis:
Shrink.
Develop bubble-like blebs on their surface.
Chromatin (DNA + protein) begins to degrade.
Mitochondria break up releasing cytochrome c
Cells break into small membrane wrapped fragment
The phospholipid, phosphatidylserine is exposed on surface
Cell fragments are removed by phagocytosis.

The process is very orderly and hence the name programmed cell
death

An intrinsic property of normal cellular metabolism.
What Does it Look Like?
Defects in Apoptosis
Defects in the regulation of apoptosis contributes to many
diseases.

Diseases where cell accumulation occurs
Cancer

Excessive cell loss
Stroke
Heart failure
Neuro-degeneration
Aids
What Causes these Changes?
The changes in cell morphology are caused by the action of proteases.

These are cysteine proteases which cleave proteins at aspartic acid residues.
These are known as caspases.

Cysteine Aspartate Specific ProteASEs

These proteins are present as inactive zymogens in essentially all cells.

Proteolytic cleavage of the caspases at conserved aspartic acid residues activates
enzymes of 10 and 20kDa subunits.
Mechanisms of Apoptosis
Three different mechanisms by which a cell commits suicide by
apoptosis.

1. Generated by signals arising within the cell (Intrinsic).
2. Triggered by death activators binding to receptors at the cell surface
(extrinsic):
Tumour necrosis factor- (TNF)
TNF- (Lymphotoxin)
A molecule that binds to the Fas cell surface receptor (Fas ligand: FasL)
3. Triggered by cell damage by, for example, reactive oxygen species
(extrinsic).
Intrinsic (Mitochondrial) Apoptosis
Outer membrane of mitochondria express the proto-oncogene Bcl-2

Bcl-2 is normally bound to a protein called Apaf1
Apaf1: apoptotic protease activating factor 1

Internal cell damage causes Bcl-2 to release Apaf1 which allows a related
protein Bax to penetrate mitochondria.
Causes release of cytochrome c

Cytochrome c, Apaf1 and caspase 9 form a complex
Apoptosome
Intrinsic (Mitochondrial) Apoptosis
Apoptosome complexes aggregate in the cytoplasm

Caspase 9 cleaves and activates other caspases
Digestion of structural proteins in cells
Degradation of chromosomal DNA

Phagocytosis of cells.
Pathway Summary
Bcl-2
Apaf1
Internal Death Signal
Bcl-2
Bax
Apaf1
Cytochrome c
Caspase 9
Apoptosome
Caspase cascade
Apoptosis
Mitochondria
Extrinsic Mechanisms of apoptosis
(Death Receptor Pathway)
The Fas and TNF receptors are integral cell membrane proteins

The binding of TNF or FasL (death activators) to the cell membrane
causes up-regulation and activation of caspase 8.

Caspase 8, like caspase 9 initiates a cascade of caspase activation leading
to phagocytosis of the cell undergoing apoptosis.
Death Receptor Pathway
DNA damage and Apoptosis
P53 and Apoptosis
Bcl-2 and Bax genes regulate the release of cytochrome c.

The promoter for the Bax gene contains P53 binding sites.
Upregulated in response to DNA damage.
P53 can also repress Bcl-2

Reactive oxygen species are powerful inducers of apoptosis.

Fas:FasL interaction causes the caspase cascade leading to apoptosis.
Fas expression is thought to be P53 dependent.
P53 summary
Apoptosis and Cancer
Loss of P53 activity in some 50% of all human tumours can disrupt
the normal apoptosis pathway.

HPV virus and cervical cancer E6 oncoprotein can also disrupt
apoptosis by degradation of P53.

Epstein Barr virus associated with some lymphomas produces a Bcl-2
homolog and stimulates Bcl-2 production in infected cells
Bcl-2 is an anti-apoptotic protein.
Translocation (t14:18)

Some B-cell leukaemias express high levels of Bcl-2
Oncogenic translocation of Bcl-2 gene to region for antibody production


Apoptosis and Cancer
Melanomas avoid apoptosis by inhibiting the expression of Apaf1.

Some lung and colon cancer cells express a protein that binds to
FasL.
Cytotoxic T cells cannot bind and induce apoptosis.

There are examples of cancer cells expressing high levels of FasL.
Can kill cytotoxic T cells since T cells express their own Fas)


Summary
Define apoptosis

Discovery of apoptosis

What happens to apoptotic cells

Routes to apoptosis
Intrinsic (Mitochondrial)
Extrinsic (death signal)
Cytotoxic T cells mediated

Role of P53 in Apoptosis

Apoptosis and cancer
Mechanisms of avoiding apoptosis