Module 11: Programmed Cell Death

Why does apoptosis occur?

Maintains balance between cell growth and death

• 50–70 billion cells die daily via apoptosis

• Prevents diseases like cancer (too little cell loss)

• Dr. Horvitz won Nobel Prize for apoptosis research in C. elegans

What regulates apoptosis in cells?

A network of signaling proteins

• Just like mitosis, apoptosis is highly regulated

• Triggered by internal/external signals

How is apoptosis important during development?

Shapes body structures by removing excess cells

• Example: webbing between fingers/toes (week 6) removed by apoptosis (week 11)

• Without apoptosis → webbing remains

What does the TUNEL assay detect?

Cells undergoing apoptosis

• Stains DNA breaks with fluorescent dUTP by terminal deoxynucleotidyl transferase

• Bright green dots = apoptotic cells

• Shows cell death between developing mouse digits

How does apoptosis affect tadpole development?

Causes tail loss during metamorphosis

• Triggered by thyroid hormone

• Similar tail loss happens in human embryo

Why is apoptosis important in brain development?

Removes neurons that don’t make proper connections

• Up to 50% of neurons die

• Ensures correct matching of nerve and target cells

How can apoptosis be harmful?

Excessive apoptosis leads to disease

• Alzheimer’s: hippocampus neuron death and certain parts of cerebral cortex

• Huntington’s: striatum neuron death

• Parkinson’s: dopamine neuron loss in substantia nigra

• Duchenne muscular dystrophy: muscle degeneration from cell death

What are the two types of cell death?

Necrosis and apoptosis

• Necrosis = unregulated, causes inflammation

• Apoptosis = controlled, clean, and safe cell removal

• Apoptosis prevents damage to nearby cells

What are the three stages of apoptosis according to Dr. Horvitz?

1. Cell execution

2. Engulfment

3. Clearance

   • Dr. Horvitz studied cell execution in C. elegans

   • Apoptosis is an active, stepwise process

What changes occur in a HeLa cell during apoptosis?

• Cell shrinks

• Membrane blebbing (protrusions or bulges occur)

• Mitochondrial permeability changes

• Nucleus and DNA degrade

• Ends in formation of small, recyclable vesicles

What structural changes occur in an apoptotic cell?

• Nucleus:

  • Chromatin condenses

  • Nuclear envelope breaks down

  • DNA fragmented

  • Proteins degraded

• Cytoplasm:

  • Condenses as components aggregate

• Mitochondria:

  • Membrane becomes permeable

  • Proteins released into cytosol

• Cell membrane:

  • Changes shape → forms blebs (protrusions)

  • Cell fragments into vesicles

• Outcome:

  • Vesicles (apoptotic bodies) phagocytized

  • Cell contents recycled

• SEM image comparison:

  • Normal cell: intact membranes, visible chromatin

  • Apoptotic cell: condensed DNA and far from nuclear membrane, altered membrane shape

Why is C. elegans a good model for apoptosis studies?

• 947 somatic cells traced from 1 zygote

• 131 cells consistently undergo apoptosis

• Predictable, well-mapped development

• Easy to study effects of gene mutations

What is the significance of the ced-1 mutation in C. elegans?

• ced-1 mutation: cells die but aren’t engulfed by phagocytosis

• ced-1 + ced-3 mutation: cells don’t undergo apoptosis

• Screen used ced-1 mutants to identify essential apoptosis genes: ced-3, ced-4, ced-9, egl-1

  • Mammalian homologs exist for all four

  • Two homologs linked to human tumor formation due to failure in apoptosis

What are the key genes and protein interactions in the C. elegans and mammalian apoptotic pathways?

In C. elegans

• ced-3 & ced-4: essential for apoptosis

  • Form the caspase holoenzyme (a protease)

• ced-9: inhibits apoptosis by blocking caspase activation

  • Loss-of-function → all cells die

• egl-1: initiates apoptosis by inhibiting ced-9

In Mammals

• EGL-1 homologs: Bid and Bim (BH3 family)

• CED-9 homolog: Bcl-2 on mitochondrial membrane

  • Inhibits apoptosis; controls Bak/Bax (pro-apoptotic proteins)

• CED-4 homolog: Apaf-1

• CED-3 homolog: Caspase-9

  • Together form the apoptosome (mammalian version of caspase holoenzyme)

• Caspase holoenzyme/apoptosome = protease → degrades proteins → cell death

• Loss of ced-3 or ced-4: no apoptosis

• Loss of ced-9: uncontrolled cell death

How does EGL-1 activate apoptosis in C. elegans?

• CED-9 binds CED-4 dimers → keeps them inactive (prevents apoptosis)

• EGL-1 binds CED-9 → releases CED-4

• CED-4 joins CED-3 → forms caspase holoenzyme

• Caspase activation → degrades cytosolic & nuclear proteins

• Good model for mammalian apoptosome formation

What is the role of Bcl-2 in mammalian apoptosis?

• Bcl-2 = mammalian homolog of CED-9

• Location: Outer mitochondrial membrane

• Function: Maintains low permeability → prevents apoptosis

• Inactivation of Bcl-2:

  • Inactivation = pore formation → apoptosis

  • Leads to mitochondrial permeabilization

  • Pores release apoptotic factors (e.g., cytochrome c)

How is apoptosis regulated by Bcl-2, Bad, and Bax in mammalian cells?

• Bad (pro-apoptotic signal)

  • Inactive when phosphorylated and bound to 14-3-3

  • Dephosphorylation → released from 14-3-3 → binds to Bcl-2 → blocks anti-apoptotic action

• Bax (CED-9 family)

  • Activated when Bcl-2 is inhibited

  • Forms pores in mitochondrial membrane

  • Releases cytochrome c into cytosol

• Cytochrome c

  • Triggers apoptosome formation

  • Essential for caspase activation → cell death

• Triggers of apoptosis

  • Intrinsic: DNA damage, cell stress

  • Extrinsic: External death signals

How do trophic factors affect Bad and apoptosis?

• Trophic factors → phosphorylate Bad → inhibit apoptosis

• No trophic factors → Bad dephosphorylates → apoptosis

• Kinase cascade keeps cell alive

• Removal = death signal

What are the key steps in the apoptotic pathway in response to the absence of trophic factors?

1. No trophic factor → Bad dephosphorylates

2. Bad inhibits Bcl-2/Bcl-XL→ Bax activates

3. Cytochrome c released

4. Apaf-1 + cytochrome c → caspase activation

   • Caspase = cysteine protease which targets proteins in nuclear lamina + cytoskeleton

5. Final apoptotic events

   • Chromatin condenses

   • Cytoplasm condenses

   • Nucleus fragments

   • Cell blebs → apoptotic bodies form

   • Apoptotic bodies phagocytosed by neighbors

Applied Lecture

Cancer

What is p53 and what does it do?

• Tumor suppressor protein and transcription factor

• Stops damaged cells from dividing → triggers apoptosis

• Activates DNA repair and stress response pathways

• Coordinates: cell cycle arrest, DNA repair, apoptosis, metabolism, anti-oxidant effects, etc.

What are the key domains of p53 protein?

• Tetramer of 4 chains (393 amino acids each)

• N-terminal:

  • TAD I & II → bind transcription machinery & MDM2

  • DNA-binding domain (DBD)

  • Nuclear export signal (NES)

• C-terminal:

  • Oligomerization domain (OD) → forms tetramer

  • Nuclear localization signals (NLS x3)

  • Second NES

  • NLS + NES → regulate nuclear-cytosolic shuttling

How does p53 protect the genome?

• Cell cycle arrest:

  • Activates p21 → inhibits cyclin E/Cdk2 and cyclin D/Cdk4 → G1 arrest

  • Regulates G2/M via 14-3-3σ and cdc25C

• DNA repair:

  • Activates multiple repair pathways: NER (nucleotide excision repair), BER (base excision repair), MMR (mismatch repair), NHEJ (non-homologous end joining)

  • Halts cycle to allow DNA repair

  • Constantly surveys for damage

What happens when p53 or other tumor suppressors are mutated?

• Checkpoint failure → cells divide with DNA damage

• Loss of mitotic control → indefinite division

• p53 normally halts cycle or induces apoptosis

• Mutated p53 found in >50% of cancers

How is p53 involved in cancer development?

• Mutated/deleted in ~50% of cancers

• Remaining cancers → p53 pathway disrupted

• Mutations let cells bypass checkpoints, resist apoptosis, proliferate unchecked

• Targeting p53 is difficult but under study

• Future therapies may combine p53 targeting + immunotherapy

What are challenges in targeting mutant p53?

• Hard to find low-molecular-weight drug to bind mutant p53

• Mutant p53 is nuclear → inaccessible to many drugs

  • Monoclonal antibodies don’t easily reach nucleus

• Many different p53 mutations → unclear if one or multiple drugs needed

What is PC14586 and how does it work?

• Small molecule corrector for Y220C p53 mutation

• Restores wild-type shape and function

• Selectively binds crevice formed by Y220C mutation

• Found in ~1–2% of all p53 mutations across many tumors

• Shows promise in early (PYNNACLE) trials

How do cancer cells spread (metastasize)?

• Use invadopodia to breach basement membranes

• Migrate to distant body sites

• Example: breast carcinoma cells

How does apoptosis relate to cancer?

• Cancer = uncontrolled cell growth + failure of apoptosis

• Damaged cells avoid death → continue dividing

• Leads to tumor formation (benign or malignant)