Apoptosis and Cancer

Apoptosis definition

• Programmed cell death

• Integral part of homeostasis, ensures balance of cell numbers

• E.g., fingers in mammals form due to controlled apoptosis between integers during embryogenesis

Control of apoptosis

• Extracellular (by immune surveillance)

• Intracellular (by DNA damage)

Can be blocked by anti-apoptotic signals

Pro and anti-apoptotic stimuli fight it out

In contrast to necrosis where cells die due to poisoning/damage/injury. Non programmed, cells often seem to explode

Classic apoptosis signs

• Normal cell intact cell membrane and nucleus intact

• Cell membrane blebbing, cell shrinking, nucleus shrinking and picnotic

• Nuclear fragmentation, cell disintegrates into vesicles (apoptotic bodies), which get engulfed by neighbours

Necrosis signs

Dying cells spew cellular contains into local cells/tissues, leading to toxicity and sometimes stimulation of proliferation

The caspase cascade

• Mitochondria central to apoptosis

• Cytochrome C drives caspase cascade

• Binds to APAF and Casp9 to form apoptosome

• Caspases are proteases

• Each activating the next

• Terminal caspases (casp 3) chew cell up

• Many signals in apoptosis, but caspases are the effectors

How to study apoptosis

• Caspase activation using antibodies to activate caspase 3 by Western blotting/immunohistochemistry

• Annexin V staining; membrane flipping occurs in vesicle formation during apoptosis. Stains detect flipped cell membrane components (phosphatidylserine and flippase)

• DNA laddering; detection of the chromosomal degradation occuring with apoptosis

• Histological; detecting apoptotic bodies through morphology using light microscopy

Immune surveillance

• Locates abnormal cells and targets them for death

• By doing so, keeps the organism free of cancer

• Macrophages secrete TNF and other apoptosis-inducing cytokines

• Natural killer cells and cytotoxic T cells kill targeted cells in a different way, but can force cells to display apoptosis inducing receptors

Extrinsic activation of apoptosis

• Death receptors on cell surface trigger apoptosis

• Converges on intrinsic pathway using caspase cascade

• Uses FADD (fas activated death domain protein)

• DISC (death inducing signalling complex)

Intrinsic activation of apoptosis

• Self policing aspect

• Cells bearing damage or under severe stress can elect to commit suicide

• The intrinsic pathway uses caspases and mitochondria

p53

• Tumour viruses induce cancer by limiting p53 function

• Blocks cell cycle progression

• Induces apoptosis

Apoptosis and cancer

• Oncogene activation can activate apoptosis

• Cancer cells circumvent the normal apoptotic process

  By inactivating pro-apoptotic pathways
  Or by activating anti-apoptotic pathways

Pro and anti-apoptotic members

• Stark similarities between protein structure of the opposing factors

• 24 such proteins in human genome

• 6 anti-apoptotic, 18 pro-apoptotic

• Bcl-2 up-regulated in many cancers

NF-kB

• An oncogenic transcription factor implicated in many cancers

• Plays a major role in inflammation too

• Is a ROS sensitive TF, activated by oxidative stress

• Anti-apoptotic factors needed to prevent excess cell death

• Mice without NF-kB die from massive liver apoptosis at birth

• Stressed cells must give a chance to recover, before consigning them to apoptosis

NF-kB upregulating genes

• Up-regulates the anti-apoptotic genes XIAP and BclXL (as well as others)

• NF-kB activated by cellular stress and keeps the cell alive during the crisis, allowing cells to recover

• Gets high-jacked by budding cancer cells

Measuring NF-kB

• As well as measuring the gene expression changes induced by NF-kB

• Can measure its cellular location in vitro and in vivo in tissue specimens