Lecture #19 | Apoptosis and p53 Mutations

Simple: p53 role in apoptosis

p53 induces programmed cell death when the DNA damage is too great to repair

• p53 +/+ cells have a much lower percent survival after X-ray that p53 -/-; normal die off more after X-ray then -/-

• apoptosis is induced by wildtype p53 in response to DNA damage

Where are the major players located?

Mitochondria, cytochrome c, APAF-1, Bax/Bcl2, Caspases

What are the common features in cancer?

Deregulation of cell proliferation and suppression of apoptosis

Apoptosis overview

1. Programmed cell death

   1. normal developmental process

2. Characterized by discrete steps

   1. Tidy verses necrosis, which is messy

   2. changes in cell morphology

   3. cascade of caspases

3. Induced by variety of internal and external signals

   1. Intrinsic and extrinsic pathways

Role of apoptosis of normal development

1. Proper limb differentiation

   1. ex: well formed digits

   2. ex: well formed tails

2. immune system and mammary tissue

3. cell death in nervous system

   1. Dysregulation of apoptosis is associated with neurodegenerative diseases

Discrete steps in apoptosis

1. Cell shrinkage

   1. Condensation of cytoplasm

2. Nuclear disintegration

   1. DNA fragmentation

   2. Increased fragmentation as apoptosis increases

3. Blebbing

   1. Apoptotic bodies

4. Phagocytosis

Necrosis characteristics

Messy, Unplanned cell death

• cell swelling

• membrane disruption

• lysis of nucleus

• inflammation

• group of cells

messy homicide

Apoptosis characteristics

Programmed cell death

• cell compaction

• apoptotic body

• no inflammation

• surrounding cells are not affected

• all evidence removed by phagocytosis

Neat, tidy suicide

Executioners of apoptosis

Called capsases

• carry out killing

• identify DEVD “dead box” on N-terminus for cleavage site

target of caspases:

• actin in cytoskeleton → blebs

• lamins → condensation of chromatin

• ICAD → release CAD _ digest DNA

• release Dnase 1 from actin

Regulation of caspases

Regulated by caspases

• synthesized in an inactive from as procaspases

• activated by protease cleavage

• initiator caspases lead to the amplification of executioner caspases

Pathways to activate apoptosis

1. Extrinsic pathway: signal from outside

2. Intrinsic pathway: signal from inside

General: Extrinsic pathway

Initiator: Caspase 8

executors: Caspase 3,6,7,12

General: Intrinsic pathway

Initiator: Caspase 9

executors: Caspase 3,6,7,12

Intrinsic pathway overview

1. Signal: release of cytochrome c from mitochondria

2. Cytochrome c binds Apaf-1 (which is typically individual but becomes a flower structure), resulting in aggregation and activation of caspase 9 (initiator)

3. This binding forms the apoptosome= Apaf-1, cytochrome c and caspase 9

4. Activated caspase 9 initiates the apoptotic response by activating executioner caspases

5. These then cleave targets

6. Results in cell death

   

Smac/DIABLO

Inhibitors of apoptosis of executioner caspases 3,6,7

How is cytochrome c released from mitochondria?

Under stress conditions, pro-apoptotic proteins (Bad, Bim) activate Bax/Bak (other proteins founder in the outer mitochondrial membranes)

• forms pores in the outer mitochondrial membrane that releases cytochrome c

• open pores: release of cytochrome c

Under normal conditions, where is cytochrome c normally kept?

Anti-apoptotic proteins (Bcl) block the pores made by the Bax/ Bak proteins and sequester pro-apoptotic Bim and Bad

Bcl-2, Bax, Bak, Bim, Bad

Family of related proteins with different function

• Bcl-2 has the Bh4 domain, making it pro-survival

Bax, Bak, Bim, Bad all lack BH4, making them pro-apoptotic

Since Bcl is a life signal, it must be what?

An oncogene, meaning that it will try and increase proliferation

• identified as a translocation in 80% of patients with follicular b-cell lymphoma

• Translocation or Bcl-2 over expression suppresses lymphocyte apoptosis

Link between Bax and Bcl and release to cytochrome from the mitochondria to cancer

p53 can upregulate Bax (tumor suppressor) and down regulate Bcl2 (oncogene)

• increases Bax, increased cytochrome c

Why is p53 the most commonly mutated gene in human cancer?

1. p53 is the guardian of the genome

2. Single pt mutations can inactive p53

3. Mutations are dominant negative

   1. Both alleles need to be inactivated

4. Mutations are seldom lethal and can actually help select for the transformed phenotype

5. Mutations in p53 lead to genome instability which aids cancer progression

6. Mutation in p53 can act at several different points in cancer progression

How can single pt mutation inactivate p53?

can prevent p53 from regulating the transcript of gene involved with guardian processes

• mutations can occur at a relatively high frequency

• target site is large (200 aa)

• most mutations occur in the DBD (DNA binding domain) hot spots

Structure of p53 relation to dominant negative mutations?

If there is a mutation in the tetramerization domain, it will allow for dominant negative mutations

Mutations are dominant negative

Missense mutations are the most common type of mutations in p53

• one amino acid is converted to a different amino acid

• impacts function but not degraded

• tetramerization region is typically intact and full length mutant will be formed

  • Mutant is not targeted by MDM2, so is available in larger concentration

What does it mean that p53 mutations are seldom lethal and can actually help select for transformed phenotype

Rarely cause death but can help select for the transformed phenotype

• ex: sun burn → skin cell apoptosis

  • wt p53 will peel skin

  • mutant p53: some apoptosis but not all at same level which allows replication of mutant p53

  • Further UV damage will cause increase in mutation

Sp1 mutations leads to increased cancer risk

Sp1 is a transcription factor for MDM2

• leads to increased MDM2

• decreased p53