In-Depth Notes on Apoptosis and Cancer Characteristics

Apoptosis: Programmed Cell Death

  • Description: A normal, regulated process unique to animal cells; characterized by an organized series of events.

  • Functions:

    • Essential for development (e.g., T-cell maturation).

    • Maintains normal tissue homeostasis.

    • Responds to DNA damage.

Morphological Characteristics of Apoptosis

  • Biochemical Markers:

    • Loss of adhesion to neighboring cells.

    • Cell & nuclear shrinkage

    • Fragmentation of DNA.

    • Cell blebbing (bubbles forming on the cell surface).

    • Engulfment by phagocytes

    • Phosphatidylserine translocation to the outer leaflet of the plasma membrane signals macrophages for engulfment.

Comparison of Apoptosis and Necrosis

  • Apoptosis:

    • Highly organized and regulated.

    • cell changes: cell shrinkage, blebs, nuclear breakup

    • outcome: Phagocytosis, no inflammation

  • Necrosis:

    • Less orderly

    • cell changes: organelle swelling, cell lysis,

    • outcome: Spillage of contents, inflammation

Inducers of Apoptosis

  • Developmental Role: T-cell maturation.

  • Maintains normal tissue homeostasis.

  • Responds to DNA damage.

Major Components in Apoptosis Induction

  • Caspases:

  • Protease (cysteine) enzymes that are synthesized as inactive zymogens.

  • Exist as inactive procaspases that are activated by cleavage

    • Cleave over 100 substrates leading to cell death

  • Caspase Targets:

    • Protein kinases: disrupt signaling & adhesion (e.g., FAK, PKB)

      • rho kinase: activates mysosin II-based membrane blebbing

    • Nuclear lamins: lead to nuclear shrinkage

    • Cytoskeletal proteins: cause blebbing

    • CAD (Caspase-activated DNase): fragments DNA

    • MDM2: inhibitor p53 tumor suppressor, preventing its activation and contributing to uncontrolled cell growth in cancer.

    • DNA repair enzymes:

  • Adaptor Molecules and Bcl-2 Family Proteins:

    • Regulate induction of cell death.

  • Mitochondria: Play a crucial role in intrinsic pathway

  • Classes:

    • Initiator Caspases (e.g., 2, 8, 9, 10): Activated by specific signals or receptor pathways. trigger apoptosis.

      • caspase 8: recruited/activated by cell surface receptors (extrinsic)

      • caspase 9: activated by intrinsic pathway

    • Effector Caspases (e.g., 3, 6, 7): Cleave major apoptotic substrates. degrade cellular components.

      • activated by initiator caspases

  • Caspases drive apoptosis by disassembling cellular structures, including nuclear components.

Necroptosis

  • Programmed necrosis; caspase-independent

  • Triggered when:

    • TNF signaling is intact

    • Caspase-8 is inhibited

  • Pathway:

    1. RIPK1 + RIPK3 → necrosome

    2. MLKL phosphorylated, inserts into membrane

    3. Loss of membrane integrity → cell rupture

  • Plays roles in inflammation, pathogen defense, and autoimmune diseases.

Cell Survival vs. Apoptosis

  • TNF can trigger both apoptosis & survival:

    • Survival via NF-κB activation → transcription of anti-apoptotic genes

  • Balance of signals determines fate:

    • Pro-survival: NF-κB, Bcl-2

    • Pro-death: caspases, Bax, tBid, BH3-only proteins

Extrinsic Pathway of Apoptosis (Receptor-Mediated)

  • Initiated by external death signals, such as Fas or TNF-alpha (ligand molecules) leading to cell apoptosis.

  • Involves the activation of caspases through receptor binding, forming complexes with adaptor proteins (e.g., FADD, TRADD).

  • Oligomerization leads to the activation of initiator caspases which activates effector caspases.

🔹 Extrinsic Pathway (Death Receptor Pathway)

Trigger:

  • External signals such as ligands binding to death receptors (e.g., FasL → FasR/CD95, TNF-α → TNFR).

Key Players:

  • Death receptors (Fas, TNFR1, DR4/DR5)

  • Adaptor protein: FADD

  • Initiator caspase: Caspase-8 (or caspase-10)

  • Effector caspase: Caspase-3

Mechanism:

  1. Ligand binds death receptor → receptor trimerization

  2. Recruitment of FADD

  3. Formation of DISC (Death-Inducing Signaling Complex)

  4. Procaspase-8 → Caspase-8 activation

  5. Caspase-8 activates caspase-3 → cell death

Cross-talk:

  • Caspase-8 can cleave Bid (a Bcl-2 family protein), which connects to the intrinsic pathway

  • Key components TNF pathway:

    1. TNF signal binds TNFR1 (death receptor/DR) → conformational change in death domain

    2. Recruitment of TRADD, RIP1K, FADD adaptor proteins

    3. Procaspase-8 (inactive) binds and activates/cleaves → caspase-8

    4. Caspase-8 activates executioner/effector caspases → apoptosis

  • key components FAS pathway:

    1. FAS ligand (FasL): Binds to the FAS receptor on target cells, initiating the apoptotic signal

    2. FAS receptor (CD95): Triggers intracellular signaling cascade upon ligand binding

    3. Caspase-8 is recruited -> cleaved → activated

    4. DISC formed

    5. Caspase-3 is subsequently activated- → execution phase → degradation of cellular components (DNase, proteolytic enzymes) and ultimately cell death.

  • Necroptosis can occur if caspase-8 is inhibited:

    • RIP1K + RIP3K form necrosome

    • RIP3K phosphorylates MLKL

    • MLKL disrupts membrane → inflammatory cell death

Intrinsic Pathway of Apoptosis (Mitochondria-Mediated)

  • Triggered by internal stimuli: hypoxia, viral infection, ER stress, oxidative stress, and cell cycle arrest.

  • Bcl-2 Family Proteins: Balance between life and death signals.

  • Regulated by Bcl-2 protein family and p53:

    • Proapoptotic: Bax, Bak

    • Antiapoptotic: Bcl-2, Bcl-xL

    • BH3-only proteins: Bid, Bad, Bim, Puma

    • Activation of pro-apoptotic members like Bax and Bak oligomerization and insert into outer membrane → mitochondrial apoptosis-induced channel (MAC) forms → leads to mitochondria releasing cytochrome c into the cytoplasm, marking the point of no return.

🔹 Intrinsic Pathway (Mitochondrial Pathway)

Trigger:

  • Internal stress like DNA damage, oxidative stress, growth factor withdrawal, ER stress

Key Players:

  • Mitochondria

  • Bcl-2 family proteins: Pro-apoptotic (Bax, Bak), Anti-apoptotic (Bcl-2, Bcl-xL)

  • Cytochrome c

  • Apaf-1

  • Initiator caspase: Caspase-9

  • Effector caspase: Caspase-3

Mechanism:

  1. Internal stress activates Bax/Bak, which permeabilize the mitochondrial membrane

  2. Release of cytochrome c into cytosol

  3. Cytochrome c binds Apaf-1, forming apoptosome

  4. Apoptosome recruits procaspase-9 → caspase-9

  5. Caspase-9 activates caspase-3 → cell death

  • Mechanism:

    1. Stress → BH3-only proteins inhibit Bcl-2

    2. Bax/Bak oligomerize on outer mitochondrial membrane (OMM)

    3. Cytochrome c released into cytosol

    4. Forms apoptosome with Apaf-1 and procaspase-9

    5. Activates caspase-9 → executioner caspases → apoptosis

    • tBid, a cleaved form of Bid (by caspase-8), links extrinsic to intrinsic pathway.

Role of Mitochondria

  • Cytochrome C:

    • Essential for forming the apoptosome and activating caspases during apoptosis (serves as a cofactor)

    • links the intrinsic pathway to the execution of apoptotic processes.

    • transfers e- from cyto b/c1 complex → cytochrome oxidase

  • Bcl-2 Protein:

    • Functions to inhibit apoptosis by binding to pro-apoptotic proteins (Bax/Bak) and preventing their action.

    • in mitochondrial membrane

Differences Between Normal Cells and Cancer Cells

  • Growth and Division:

    • Normal cells stop dividing when enough cells are present.

    • Cancer cells continue dividing regardless, often due to mutations in growth factor genes (oncogenes).

  • Appearance:

    • Cancer cells show variability in size and shape, larger/darker nuclei, and disorganized chromosome arrangements compared to normal cells.

  • Invasiveness:

    • Normal cells respect boundaries and signals to stop growth (contact inhibition).

    • Cancer cells invade surrounding tissues, leading to tumor formation without boundaries.