BIOL 3020 Exam 4 — Master Study Guide

G-actin (globular, 43 kDa) is the monomeric form; it binds ATP and polymerizes into F-actin (filamentous) — a two-stranded helical polymer, ~7 nm diameter. ATP hydrolysis occurs after incorporation; the filament interior is predominantly ADP-actin.

F-actin is polar: the barbed (+) end has faster polymerization kinetics (lower critical concentration, C_c ≈ 0.1 µM); the pointed (−) end has slower kinetics (C_c ≈ 0.7 µM).

Treadmilling

At steady state (when [G-actin] is between the two C_c values), net polymerization occurs at the barbed (+) end and net depolymerization at the pointed (−) end — the filament appears to "treadmill" without changing total length. This requires continuous ATP hydrolysis.

Cell crawling involves three steps: (1) protrusion of leading edge (lamellipodia/filopodia via actin polymerization), (2) adhesion to substratum (focal adhesions via integrins), (3) retraction of trailing edge (actomyosin contraction).

Rho Family GTPases

Focal adhesions are integrin-based structures linking F-actin stress fibers to ECM (fibronectin, laminin). Contain: talin, vinculin, paxillin, FAK (focal adhesion kinase).

Adherens junctions link actin cytoskeleton between cells via E-cadherin → catenins (α-catenin, β-catenin) → F-actin.

Sarcomere Structure

Sliding Filament Mechanism & Cross-Bridge Cycle

1. ATP binds myosin head → myosin releases actin (anti-rigor state)
2. ATP hydrolysis (→ ADP + Pi) → myosin head cocks to high-energy 90° position
3. Myosin binds actin (weak binding)
4. Pi release → power stroke (myosin head pivots to 45°, moves actin ~5–10 nm)
5. ADP release → rigor state (tight actin–myosin bond)
6. New ATP binds → cycle repeats

Ca²⁺ Regulation of Thin Filaments

Skeletal muscle: Tropomyosin lies in the groove of F-actin, blocking myosin-binding sites. Troponin complex (TnT: tropomyosin-binding; TnI: inhibitory; TnC: Ca²⁺-binding) controls position of tropomyosin. Ca²⁺ released from SR → binds troponin C → TnI releases actin → tropomyosin shifts → myosin binding sites exposed → contraction.

Smooth muscle: Lacks troponin. Ca²⁺–calmodulin complex activates myosin light-chain kinase (MLCK) → phosphorylates myosin II regulatory light chain (RLC) → activates myosin II.

Microtubules are hollow cylinders, 25 nm diameter, composed of 13 protofilaments of α/β-tubulin heterodimers arranged head-to-tail. β-tubulin is exposed at the plus (+) end; α-tubulin at the minus (−) end. GTP bound to β-tubulin is hydrolyzed after polymerization; GTP on α-tubulin is non-exchangeable.

The centrosome is the main microtubule-organizing center (MTOC) in animal cells. It consists of two centrioles (9-triplet barrel structure, 9+0) surrounded by pericentriolar material (PCM).

The γ-tubulin ring complex (γ-TuRC), located in the PCM (NOT the centrioles), nucleates the minus (−) end of new microtubules. Centrioles organize and recruit PCM; PCM actually does the nucleating.

MAPs — Microtubule-Associated Proteins

Motor mechanism: Both kinesins and dyneins are ATPases. They undergo conformational changes coupled to ATP hydrolysis → "walking" along MTs. Kinesin-1 takes 8 nm steps per ATP; highly processive (stays on track for many steps).

Intermediate filaments (IFs) are 10–12 nm in diameter — intermediate between actin (~7 nm) and microtubules (~25 nm). Unlike actin and MTs, IFs are NOT involved in cell motility; they provide mechanical strength and structural scaffolding.

Assembly

Central α-helical rod domain (~310 aa) forms coiled-coil dimers → staggered antiparallel tetramers → protofilaments → ~8 protofilaments wound into ropelike filament. Because tetramers are antiparallel, both ends are equivalent → IFs are APOLAR.

Key Functions

• Desmosomes: Cell–cell junctions. Keratin IFs → desmoplakin → desmoglein/desmocollin (cadherins) → adjacent cell.
• Hemidesmosomes: Cell–ECM junctions. Keratin IFs → plectin → α6β4 integrin → laminin.
• Desmin: Links Z-discs in muscle and anchors contractile elements to plasma membrane. Desmin mutations → cardiomyopathy.
• Neurofilaments: Abundant in motor neuron axons; support long, thin processes >1 m in length.
• Nuclear lamins: Phosphorylated by Cdk1 at mitosis → lamin disassembly → nuclear envelope breakdown.

The eukaryotic cell cycle is divided into four sequential phases:

• G1 (Gap 1): Cell grows, synthesizes proteins and organelles; monitors environmental cues. Duration is highly variable — the key regulatory phase for deciding whether to divide.
• S (Synthesis): DNA replication; each chromosome is duplicated to produce sister chromatids held together by cohesin. Centrosome duplication also begins.
• G2 (Gap 2): Further growth; repair of any replication errors; assembly of mitotic machinery begins. CDK1 activity builds toward M-phase entry.
• M (Mitosis + Cytokinesis): Chromosomes condense, segregate, and two daughter cells form. This phase is the shortest (~1 h in typical mammalian cells).

G0 quiescence: Cells that exit the cell cycle reversibly enter G0 (e.g., hepatocytes, lymphocytes awaiting stimulation). G0 cells lack cyclin D expression and have hypophosphorylated Rb. Terminally differentiated cells (neurons, muscle fibers) exit permanently. The relationship between proliferation and differentiation is inverse: as cells commit to a differentiated fate they typically downregulate cyclin D and upregulate CKIs, permanently (or stably) withdrawing from the cycle.

Cyclin-dependent kinases (CDKs) are the catalytic engines of the cell cycle. Their activity requires binding a cyclin regulatory subunit. CDK protein levels are constitutive (constant throughout the cycle); it is the cyclin levels that oscillate — rising and falling in a cell-cycle-dependent manner by regulated synthesis and proteasomal degradation.

Three Layers of CDK Regulation

1. Cyclin binding required: CDK alone is inactive. Cyclin binding induces a conformational change (T-loop repositioning) that opens the catalytic cleft.
2. Inhibitory phosphorylation: Wee1 kinase phosphorylates Tyr15 (and Thr14 by Myt1) on CDK1 (and CDK2), inactivating it. Cdc25 phosphatase removes these phosphates to activate the CDK. The balance of Wee1 vs. Cdc25 controls G2/M timing.
3. CKI binding: CDK inhibitory proteins (CKIs) block CDK activity:
   • Ink4 family (p15^INK4b, p16^INK4a): specifically inhibit Cdk4 and Cdk6; compete with cyclin D; tumor suppressors (p16 mutated in melanoma).
   • Cip/Kip family (p21^CIP1, p27^KIP1, p57^KIP2): inhibit Cdk2 complexes; p21 is a p53 transcriptional target (DNA damage checkpoint); p27 is downregulated by mitogens to allow S-phase entry.

Animal cells pass a Restriction Point in late G1 (equivalent to START in yeast) beyond which they are committed to S phase regardless of extracellular mitogen signals. Before the restriction point, mitogen withdrawal causes return to G0.

Rb/E2F Axis

This axis is the molecular switch for S-phase commitment:

1. G0 / early G1: Rb is unphosphorylated; it directly sequesters the transcription factor E2F (bound to its partner DP) and recruits HDACs (histone deacetylases) to repress E2F target genes (e.g., cyclin E, cyclin A, DNA polymerase, DHFR).
2. Mid G1: Mitogen-induced Cyclin D–Cdk4/6 phosphorylates Rb at multiple sites → partial phosphorylation → partial E2F de-repression → some cyclin E transcription.
3. Late G1 (commitment): Rising Cyclin E–Cdk2 hyperphosphorylates Rb → E2F fully released → transcribes S-phase genes → positive feedback (more cyclin E → more Cdk2 activity → more Rb phosphorylation) → switch-like, irreversible G1/S transition.

Ras → Cyclin D Induction

Growth factor receptors (RTKs) activate Ras (via Grb2/Sos GEF) → RAF → MEK → ERK → transcription factors (Fos/Jun/Myc) → cyclin D transcription. This links extracellular mitogen signals to G1 Cdk4/6 activity and thus to Rb phosphorylation.

Cdc25 and the Wee1/Cdc25 Antagonism

Wee1 kinase phosphorylates Tyr15 on Cdk1 (inhibitory). Cdc25 phosphatase removes Tyr15-P (activating). At G2/M, rising Cyclin B–Cdk1 activity phosphorylates and activates Cdc25 while simultaneously phosphorylating and inactivating Wee1 — a bistable positive feedback loop that drives rapid, switch-like M-phase entry.

MPF (Maturation-Promoting Factor) = Cyclin B + Cdk1. It was discovered biochemically by Masui and Markert (1971) from frog (Xenopus) oocytes: cytoplasm from mitotic cells injected into G2-arrested oocytes triggered entry into meiosis II, demonstrating a diffusible mitosis-promoting activity.

Activation — Bistable Switch

MPF activation at G2/M is switch-like due to mutual positive feedback:

• Active Cdk1 phosphorylates Cdc25 → Cdc25 more active → removes more Tyr15-P from Cdk1 → more active Cdk1 (positive feedback loop 1).
• Active Cdk1 phosphorylates Wee1 → Wee1 inhibited → less inhibitory phosphorylation on Cdk1 → more active Cdk1 (positive feedback loop 2).

This double-positive feedback creates an ultrasensitive, bistable switch ensuring rapid, complete commitment to M phase (no partial mitosis).

MPF Substrates During Mitosis

• Nuclear lamins (Lamin A/B/C): phosphorylation causes lamin depolymerization → nuclear envelope (NE) breakdown (NEBD) in prometaphase.
• Condensin complexes: phosphorylation activates condensin → chromosome compaction in prophase.
• Golgi matrix proteins: phosphorylation → Golgi fragmentation (for inheritance by daughter cells).
• Cdc25 and Wee1: as above (positive feedback).

MPF Inactivation — Exit from Mitosis

At metaphase → anaphase transition: APC/C–Cdc20 (Anaphase-Promoting Complex/Cyclosome with its co-activator Cdc20) ubiquitinates Cyclin B → proteasomal degradation → Cdk1 loses its activating subunit → MPF inactivated → mitotic exit. Phosphatases (PP2A-B55, PP1) then dephosphorylate MPF substrates, reversing mitotic events (NE reform, chromosome decondensation).

Two Sensor Kinases: ATM and ATR

• ATM (Ataxia-Telangiectasia Mutated): activated by double-strand breaks (DSBs); recruits and phosphorylates Chk2 kinase.
• ATR (ATM and Rad3-related): activated by single-stranded DNA (ssDNA) coated with RPA (from stalled replication forks or resected DSBs); phosphorylates Chk1 kinase.

G1/S Arrest via p53

ATM → Chk2 → phosphorylates p53 (on Ser20) preventing MDM2 binding → p53 accumulates → transcribes p21 (Cip/Kip CKI) → p21 inhibits Cyclin E/Cdk2 and Cyclin A/Cdk2 → G1 arrest. ATM/Chk2 also phosphorylate MDM2 directly, reducing its ability to ubiquitinate p53.

p53 additionally transcribes: Bax and PUMA (pro-apoptotic), GADD45 (DNA repair), and — as a negative feedback — MDM2 itself (which then resumes p53 degradation when damage is resolved).

Rapid Response via Cdc25A Degradation

Both Chk1 and Chk2 phosphorylate Cdc25A phosphatase → Cdc25A is targeted for SCF-mediated ubiquitination and degradation → Cdk2 remains Tyr15-phosphorylated (inactive) → no firing of new replication origins and no progression through S phase. This is the fast checkpoint response (minutes), whereas p53-dependent transcription of p21 is slower (hours).

Prophase

Chromosomes begin condensing as condensin complexes (activated by MPF phosphorylation) introduce positive supercoils and compact chromatin into rod-like structures. Centrosomes separate and move to opposite poles driven by bipolar kinesins (Eg5/kinesin-5). Aurora A kinase phosphorylates TACC/ch-TOG to stabilize astral MTs; Aurora B kinase (chromosomal passenger complex) localizes to inner centromeres. Polo-like kinase 1 (Plk1) phosphorylates multiple mitotic substrates including Cdc25C (activating it) and is required for bipolar spindle formation.

Prometaphase

MPF-phosphorylated nuclear lamins (A, B, C) depolymerize → nuclear envelope breaks down (NEBD). Spindle microtubules emanating from centrosomes capture kinetochores (trilaminar protein structures on centromeric chromatin). Each sister chromatid has one kinetochore; correct attachment requires amphitelic (bi-orientation) — one kinetochore attached to each pole. Unattached kinetochores generate the "wait-anaphase" signal (SAC, see 18.7).

Metaphase

All chromosomes achieve bi-orientation and align at the metaphase plate (equatorial plane) under balanced kinetochore tension. Chromosomes are held at the plate by equal poleward forces on both kinetochores. SAC is satisfied when all kinetochores are under tension from bipolar attachment.

Anaphase

APC/C–Cdc20 ubiquitinates securin → securin degraded → separase (a protease previously inhibited by securin) is activated → separase cleaves cohesin subunit Scc1/Rad21 → sister chromatids disjoin and move to opposite poles.

• Anaphase A: Chromosomes move toward poles primarily via kinetochore MT depolymerization (kinesin-13/MCAK depolymerizes plus ends at kinetochore; poleward flux also contributes).
• Anaphase B: Poles move further apart — antiparallel overlap MTs pushed apart by kinesin-5 (Eg5) in the spindle midzone; cytoplasmic dynein at the cortex pulls poles outward.

Telophase

Chromosomes arrive at poles, begin decondensing. APC/C–Cdh1 (late-mitosis APC/C variant) ubiquitinates remaining cyclin B → complete MPF inactivation → phosphatases dephosphorylate lamin B → nuclear envelope re-forms (ER-derived membranes reseal around chromatin). Nucleolus reappears.

The SAC is the surveillance mechanism that prevents anaphase until every kinetochore is correctly attached to the spindle under tension. Even a single unattached kinetochore is sufficient to delay anaphase.

Molecular Mechanism

1. Unattached kinetochore recruits Mad1 (checkpoint scaffold) → Mad1 catalyzes conversion of cytosolic open-Mad2 → closed-Mad2.
2. Closed-Mad2 associates with BubR1 + Bub3 + Cdc20 → forms the MCC (Mitotic Checkpoint Complex).
3. MCC sequesters Cdc20, preventing it from activating APC/C.
4. Without APC/C–Cdc20 activity, securin and cyclin B are not ubiquitinated → separase remains inhibited → cohesin not cleaved → no anaphase.

Once all kinetochores achieve amphitelic attachment under tension, Mad1/Mad2 are stripped from kinetochores → MCC disassembles → free Cdc20 activates APC/C → securin and cyclin B degraded → anaphase proceeds.

Aurora B and Error Correction

Aurora B kinase (CPC — chromosomal passenger complex) detects and corrects erroneous attachments (syntelic: both kinetochores to same pole; merotelic: one kinetochore to both poles). Aurora B phosphorylates outer kinetochore proteins (Hec1/Ndc80) reducing MT affinity, allowing MT release and re-capture. Under correct amphitelic tension, Aurora B is spatially separated from Hec1, so correct attachments are stabilized.

Animal Cells — Contractile Ring

A contractile ring of actin and myosin II assembles at the equatorial cortex and constricts like a purse-string. Key signals:

• RhoA GTPase (activated by the centralspindlin complex on the central spindle): activates ROCK kinase → phosphorylates myosin II regulatory light chain (MLC) → activates myosin II ATPase → ring contraction. RhoA also activates formins (mDia) → actin polymerization in the ring.
• Central spindle midzone (between separating chromosomes) contains PRC1 (MT bundler) and kinesin-4 (MKLP1/kinesin-6 in centralspindlin), which positionally signals cleavage plane to the cortex.

After ring constriction, membrane fusion completes cytokinesis at the midbody (remnant of central spindle) through ESCRT-III machinery.

Plant Cells — Phragmoplast

Plant cells have a rigid cell wall and no cortical contractile ring. Instead, a phragmoplast (an array of antiparallel MTs, remnant of anaphase spindle midzone) directs Golgi-derived vesicles (carrying cell wall components: pectin, cellulose synthase) to the cell midplane. Vesicle fusion forms the cell plate, which expands centrifugally until it meets the parental cell wall, dividing the cell. No RhoA/myosin II-based contractile ring is used.

Tissues maintain cell number through a balance of cell production and cell death. Three broad categories of tissue renewal exist:

1. Differentiated cells that retain proliferative capacity: Most cells in liver (hepatocytes) and kidney tubule cells are quiescent (G0) but can re-enter the cycle after injury (e.g., partial hepatectomy → 2/3 of liver resected → regeneration within weeks). These are not true stem cells but can act as stem cells in emergency contexts.
2. Tissues continuously renewed from adult stem cells:
   • Intestinal epithelium: turns over every 3–5 days. Lgr5+ intestinal stem cells (Barker et al. 2007) reside at the base of crypts of Lieberkühn; they divide rapidly to produce transit-amplifying cells that differentiate as they migrate up villi toward the lumen. Wnt signaling (from Paneth cells in the crypt niche) maintains Lgr5+ stem cell identity.
   • Hematopoietic system: all blood cells (red cells, platelets, lymphocytes, myeloid cells) are continuously replenished from hematopoietic stem cells (HSCs) in bone marrow. HSCs are Lin⁻ Sca1⁺ cKit⁺ (LSK) in mouse. HSC niche signals include Notch, Wnt, CXCL12/CXCR4 retention, Thrombopoietin.
   • Skin: basal layer (stratum basale) contains proliferating progenitors; daughter cells move outward, terminally differentiate (cornification), and are shed. Hair follicle bulge contains slow-cycling stem cells activated in each hair cycle.
3. Tissues renewed from dormant adult stem cells activated by injury:
   • Skeletal muscle: satellite cells (Pax7+) lie quiescent beneath the basal lamina of muscle fibers; injury activates them → myoblasts → fusion → repair. Limited but real regenerative capacity.
   • Central nervous system: very limited; adult neurogenesis restricted to hippocampal subgranular zone (dentate gyrus) and olfactory bulb (from subventricular zone) in rodents; extent in humans is debated. Most CNS neurons are post-mitotic and irreplaceable.

Stem Cell Properties and Niche Signals

All stem cells share two defining properties: (1) self-renewal — ability to divide to produce at least one daughter with identical stem cell potential; (2) differentiation potential — ability to produce one or more specialized cell types (totipotent, pluripotent, multipotent, unipotent). The stem cell niche is the local tissue microenvironment that maintains stem cell identity via:

• Wnt (β-catenin pathway): promotes stemness and proliferation (intestinal crypts, HSCs)
• Notch (lateral inhibition and fate specification: Delta-Notch signaling)
• Hedgehog (Shh): hair follicle stem cells, neural stem cells
• Cell-cell and cell-matrix contacts, low O₂ (HSC niche hypoxia)

The relationship between proliferation and differentiation is fundamentally inverse: as cells move from the stem cell niche and commit to a differentiated fate, they downregulate Cyclin D, upregulate CKIs (p21, p27), and typically exit the cell cycle permanently (G0 or senescence).

Embryonic Stem (ES) Cells

Derived from the inner cell mass (ICM) of the preimplantation blastocyst (day 4–5 human). ES cells are pluripotent — they can give rise to all three germ layers and all embryonic cell types (except extraembryonic). Gail Martin (1981) first established mouse ES cell lines in culture (independently with Evans & Kaufman). Human ES cells: Thomson et al., 1998. Key limitation: requires destruction of a viable embryo, raising ethical concerns.

ES cell pluripotency is maintained by an autoregulatory transcription factor circuit: OCT4 + SOX2 + NANOG form a cross-regulatory network (each activates the others and activates pluripotency genes; each represses differentiation genes). Oct4 protein level matters: too low → trophectoderm; too high → endoderm/mesoderm; intermediate → pluripotent ICM.

Somatic Cell Nuclear Transfer (SCNT) and Therapeutic Cloning

Dolly the sheep (1997, Ian Wilmut's group): nucleus from an adult somatic cell (mammary epithelial) transferred into an enucleated egg → cloned lamb — proved that differentiated nuclei could be reprogrammed to totipotency by egg cytoplasm. The concept of therapeutic cloning: take a patient's somatic cell nucleus → SCNT → blastocyst → harvest ICM → patient-matched ES cells (genetically identical to patient, no immune rejection) → differentiate to desired cell type for transplantation. Ethically controversial (still produces and destroys an embryo); technically challenging in humans.

Induced Pluripotent Stem Cells (iPSCs)

Shinya Yamanaka (2006): introduced four transcription factors — Oct4, Sox2, Klf4, c-Myc (OSKM) — into mouse skin fibroblasts → reprogrammed to pluripotent stem cells virtually identical to ES cells. Human iPSCs: Yamanaka & Thomson 2007. Nobel Prize in Physiology or Medicine 2012 (Yamanaka & Gurdon).

iPSC advantages over ES cells:

• Patient-specific → no immune rejection for transplantation
• No embryo destruction → no ethical controversy
• Disease modeling in a dish (iPSCs from patients with monogenic disease)

Limitations: c-Myc is an oncogene (risk of tumor formation); early iPSC lines used viral vectors; epigenetic memory; efficiency is low (~0.01–1%). Current methods use non-integrating vectors (Sendai virus, mRNA, small molecules).

Transdifferentiation (direct reprogramming) is the conversion of one differentiated cell type directly into another without passing through a pluripotent stem cell intermediate. This demonstrates that cell identity is controlled by master transcription factors, not irreversible epigenetic lock-in.

• Fibroblasts → Myoblasts: Overexpression of a single transcription factor, MyoD (Harold Weintraub, 1987), converts mouse fibroblasts into skeletal muscle cells (myoblasts). MyoD is a bHLH master regulator of skeletal muscle identity.
• Fibroblasts → Cardiomyocytes: Gata4 + Mef2c + Tbx5 (GMT, Srivastava lab 2010) directly convert cardiac fibroblasts to cardiomyocyte-like cells in vitro and in the infarcted mouse heart (cardiac repair).
• Fibroblasts → Neurons: Ascl1 + Brn2 + Myt1l (BAM factors, Wernig lab 2010) directly convert mouse fibroblasts into functional induced neurons (iNs); NEUROD1 added for human cells.

Significance for regenerative medicine: transdifferentiation avoids the pluripotent intermediate (lower tumor risk), can be performed in situ (e.g., converting cardiac fibroblasts to cardiomyocytes directly in the infarct zone), and bypasses ethical concerns of ES cell derivation.

Apoptosis = programmed cell death — an active, energy-requiring self-destruction program. It is essential for development and tissue homeostasis:

1. Interdigital webbing removal: in limb development, cells between digit primordia undergo apoptosis (BMP signals drive apoptosis); failure → syndactyly.
2. T-cell deletion: autoreactive T cells in the thymus that recognize self-peptides strongly are eliminated by apoptosis (negative selection), preventing autoimmunity.
3. C. elegans development: exactly 131 of the 1090 cells born during hermaphrodite development undergo programmed cell death — a fixed, predictable cell fate. This invariance enabled genetic dissection of the apoptosis machinery.

Morphological Features of Apoptosis

• Cell shrinkage and cytoplasmic condensation
• Chromatin condensation (pyknosis) and margination to nuclear periphery
• Plasma membrane blebbing (plasma membrane integrity preserved)
• DNA ladder: internucleosomal DNA fragmentation at ~180–200 bp intervals (CAD nuclease cleaves between nucleosomes → discrete fragments visible on gel)
• Phosphatidylserine (PS) flip: PS normally on inner leaflet; scramblase (caspase-activated) flips PS to outer leaflet → "eat-me" signal for macrophage phagocytosis → apoptotic bodies cleared without inflammation
• Apoptotic bodies: cell fragments enclosed in membrane, phagocytosed by macrophages/neighboring cells

Necrosis vs. Apoptosis

C. elegans Apoptosis Genetics

Genetic analysis by Ellis, Horvitz, and colleagues identified the core death machinery:

• ced-3 (caspase homolog): required for cell death; loss-of-function → 131 cells survive that normally die.
• ced-4 (Apaf-1 homolog): required for cell death; activates CED-3.
• ced-9 (Bcl-2 homolog): prevents death; gain-of-function mutations → no cells die; loss-of-function → excess death. CED-9 prevents cell death by binding and inhibiting CED-4, preventing CED-4 from activating CED-3.

Caspases (cysteine-dependent aspartate-directed proteases): cysteine nucleophile in active site; cleave substrates after Asp residues. They exist as inactive procaspases (zymogens) activated by proteolytic cleavage or induced proximity dimerization.

Two Classes

• Initiator caspases (long prodomain — DED or CARD): caspase-8 (extrinsic pathway, DED), caspase-10 (extrinsic, DED), caspase-9 (intrinsic pathway, CARD). Activated by dimerization via adaptor platforms (DISC or apoptosome), not by direct cleavage.
• Effector/executioner caspases (short prodomain): caspase-3, -6, -7. Activated by proteolytic cleavage by initiator caspases. Execute the death program.

Key Caspase Substrates

• ICAD (Inhibitor of Caspase-Activated DNase): caspase-3 cleaves ICAD → releases CAD (caspase-activated DNase) → CAD enters nucleus → cleaves DNA at internucleosomal sites → DNA ladder.
• Nuclear lamins: caspase-6 cleavage → nuclear fragmentation.
• Cytoskeletal proteins (gelsolin, fodrin): caspase-3 cleavage → membrane blebbing.
• Scramblase: activation → PS externalization ("eat-me" signal).
• PARP (poly-ADP-ribose polymerase): inactivated by caspase-3 cleavage (PARP cleavage is a classic marker of apoptosis in Western blots — 116 kDa → 89 kDa fragment).

IAPs — Caspase Inhibitors

IAPs (Inhibitor of Apoptosis Proteins, e.g., XIAP, survivin) contain BIR (Baculovirus IAP Repeat) domains that directly bind and inhibit active caspase-3, -7, and -9. XIAP is the most potent endogenous caspase inhibitor.

Smac/DIABLO is released from the mitochondrial intermembrane space upon MOMP; it binds IAPs (BIR2/BIR3 domain) and displaces caspases, neutralizing IAP inhibition and permitting full caspase activation.

The Bcl-2 protein family controls the critical decision point of the intrinsic pathway: whether to permeabilize the outer mitochondrial membrane (OMM). All members share at least one BH (Bcl-2 Homology) domain.

Mechanism at the OMM

Anti-apoptotic Bcl-2/Bcl-X_L bind Bax/Bak in a "BH3-in-groove" interaction — the BH3 domain of Bax/Bak inserts into a hydrophobic groove on Bcl-2/Bcl-X_L, keeping Bax/Bak in inactive monomeric form. BH3-only proteins (Bad, Noxa) compete for this groove, displacing Bax/Bak → free Bax/Bak oligomerize → pore formation. Activator BH3-only proteins (Bim, tBid) can also directly contact and activate Bax/Bak, inducing a conformational change that exposes Bax hydrophobic helix α5 for membrane insertion.

BH3-Only Proteins as Stress Sensors

• Bim: sequestered to dynein motors in healthy cells; released during cytoskeletal stress, growth factor withdrawal
• PUMA: transcribed by p53 (DNA damage); potent activator
• Noxa: transcribed by p53; preferentially inhibits Mcl-1
• Bad: phosphorylated by Akt (survival) → binds 14-3-3 protein → sequestered in cytoplasm → Bcl-2/Bcl-X_L free to inhibit Bax/Bak; dephosphorylated Bad → goes to mitochondria → inhibits Bcl-2/Bcl-X_L → apoptosis
• Bid: cleaved by caspase-8 → truncated Bid (tBid) → inserts into OMM → activates Bax/Bak (cross-talk between extrinsic and intrinsic)

The intrinsic (mitochondrial) pathway is activated by intracellular stress signals: DNA damage, oxidative stress, ER stress, growth factor withdrawal, oncogene activation, cytotoxic drugs.

Signal Flow

1. Apoptotic signal → BH3-only proteins activated (Bim de-sequestered; PUMA/Noxa transcribed by p53)
2. BH3-only proteins neutralize Bcl-2/Bcl-X_L and/or directly activate Bax/Bak
3. Bax/Bak oligomerize → insert into OMM → form proteolipid pores → MOMP
4. Cytochrome c released from IMS (intermembrane space) into cytoplasm
5. Cytoplasmic cytochrome c binds Apaf-1 (WD40 domain) + dATP/ATP → Apaf-1 undergoes conformational change → 7 Apaf-1 molecules oligomerize into the apoptosome (heptameric wheel structure)
6. Apoptosome CARD domain recruits and dimerizes procaspase-9 via CARD-CARD interaction → caspase-9 activated (proximity/induced conformational change)
7. Active caspase-9 cleaves procaspase-3 and procaspase-7 → effector caspases execute death program
8. Also released: Smac/DIABLO (neutralizes XIAP) and AIF (apoptosis-inducing factor, causes caspase-independent chromatin condensation)

p53 and the intrinsic pathway: DNA damage → ATM/ATR → p53 stabilized → p53 transcribes Bax and PUMA (pro-apoptotic BH3-only) → amplifies intrinsic pathway. Cancers with p53 mutations are therefore resistant to genotoxic chemotherapy and radiation, which kill cells largely through p53-dependent apoptosis.

Extrinsic (Death Receptor) Pathway

1. Death ligand (FasL, TRAIL, TNF) binds its cognate receptor: Fas/CD95, DR4/DR5, TNFR1 respectively → receptor trimerization.
2. Receptor cytoplasmic death domain (DD) recruits adaptor FADD (Fas-Associated Death Domain) via DD–DD interaction.
3. FADD DED (death effector domain) recruits procaspase-8 (and procaspase-10) → forms the DISC (Death-Inducing Signaling Complex).
4. Proximity at DISC drives procaspase-8 dimerization → caspase-8 autoactivation → released into cytoplasm.
5. Active caspase-8 cleaves procaspase-3/7 directly → apoptosis (type I cells, e.g., thymocytes); or in cells with high XIAP (type II cells, e.g., hepatocytes): proceeds via cross-talk.

Cross-talk: Extrinsic → Intrinsic via Bid

Caspase-8 cleaves Bid (BH3-only protein) → tBid (truncated Bid) → tBid inserts into OMM → activates Bax/Bak → MOMP → cytochrome c → apoptosome → caspase-9 → caspase-3/7. This amplification loop is critical in type II cells where direct caspase-8 → caspase-3 activation is insufficient to overcome high IAP levels.

Extracellular Survival Signaling: PI3K/Akt

Growth factors (e.g., IGF-1, insulin, EGF) → RTK → PI3K → PIP3 → recruits and activates Akt (PKB) at the membrane (via PDK1 Thr308 and mTORC2 Ser473 phosphorylation). Active Akt promotes survival by:

• Phospho-Bad: Akt phosphorylates Bad on Ser136 → phospho-Bad binds 14-3-3 proteins → sequestered in cytoplasm → cannot inhibit Bcl-2/Bcl-X_L → mitochondria protected.
• Phospho-FOXO: Akt phosphorylates FOXO transcription factors → nuclear export (cytoplasmic) → cannot transcribe Bim and FasL → less BH3-only protein.
• MDM2 phosphorylation: Akt phosphorylates MDM2 → nuclear entry → promotes p53 degradation → less apoptotic transcription.

Growth factor withdrawal → Akt inactive → Bad dephosphorylated → Bad migrates to mitochondria → inhibits Bcl-2/Bcl-X_L → Bax/Bak free → MOMP → apoptosis.

Benign vs. Malignant

Tumor Classification by Tissue of Origin

• Carcinomas (~85–90%): from epithelial cells. Sub-types: adenocarcinoma (glandular epithelium: breast, colon, prostate, lung adenocarcinoma), squamous cell carcinoma (squamous epithelium: lung, skin, cervix). Most common cancers: breast, colon, lung, prostate.
• Sarcomas: from connective tissue (bone = osteosarcoma; muscle = rhabdomyosarcoma; fat = liposarcoma). Rarer; common in children/young adults.
• Leukemias and Lymphomas (~8%): from hematopoietic cells. Leukemia = cancer cells in blood/bone marrow; lymphoma = solid tumor in lymphoid tissue.
• Gliomas/CNS tumors: from glial cells (glioblastoma multiforme = most aggressive brain tumor).

Multi-step Colon Carcinoma Model (Vogelstein)

Colon cancer progression is the best-characterized example of multi-hit clonal selection:

1. Normal epithelium → Early adenoma: Loss of APC tumor suppressor (both alleles; ~80% of colon cancers). APC = Wnt pathway regulator; loss → constitutive β-catenin signaling → cyclin D ↑, cell proliferation ↑.
2. Early adenoma → Intermediate adenoma: Activating mutation in RAS (Gly12Val blocks GTPase activity → constitutively GTP-bound = active).
3. Intermediate adenoma → Late adenoma: Loss of SMAD4 (TGF-β signaling pathway tumor suppressor) and/or other TGF-β pathway components.
4. Late adenoma → Carcinoma: Loss of p53 (TP53 tumor suppressor) → no apoptosis or senescence in response to accumulated damage.
5. Carcinoma → Metastasis: Loss of E-cadherin expression (EMT — epithelial-mesenchymal transition) → reduced cell-cell adhesion → cells detach and invade.

This model illustrates that cancer is a multi-step, clonal evolution process: each mutation provides a growth/survival advantage, allowing that clone to outcompete others. Typically 4–6 rate-limiting driver mutations accumulate over years to decades.

Cancer cells acquire multiple properties that distinguish them from normal cells:

1. Loss of density-dependent growth arrest (contact inhibition): Normal cells stop dividing when they contact each other (E-cadherin → p27 CKI upregulation, Hippo pathway YAP/TAZ inhibition). Cancer cells grow past confluence — they lose contact inhibition due to E-cadherin loss and Hippo pathway dysregulation.
2. Anchorage-independent growth: Normal epithelial cells require integrin-mediated attachment to extracellular matrix (ECM) for survival (detachment → anoikis, an integrin-withdrawal apoptosis). Cancer cells resist anoikis (Bcl-2 upregulation, integrin signaling decoupled from survival). This is tested by the soft-agar assay — cells are plated in soft agar (semi-solid medium preventing attachment); only transformed cells form colonies.
3. Autocrine growth factor loops: Cancer cells secrete their own growth factors (e.g., TGF-α, EGF, PDGF) that activate their own receptors → self-stimulated proliferation independent of exogenous mitogens.
4. Reduced adhesion / E-cadherin downregulation: E-cadherin (epithelial adherens junction) is lost in late-stage carcinomas — either by mutation, promoter methylation, or transcriptional repression by Snail/Twist (EMT factors). Loss of E-cadherin enables cell detachment and metastasis.
5. Protease secretion: Cancer cells upregulate matrix metalloproteinases (MMPs) and other proteases (uPA, cathepsins) that degrade basement membrane and ECM, enabling local invasion and intravasation.
6. Angiogenesis (VEGF): Tumors >1–2 mm require new vasculature; cancer cells upregulate VEGF (Vascular Endothelial Growth Factor), often via HIF-1α (hypoxia-inducible factor) due to tumor hypoxia, or via oncogenic Ras → VEGF transcription. Anti-VEGF therapy (bevacizumab) exploits this.
7. Apoptosis resistance: Upregulation of Bcl-2, Bcl-X_L, Mcl-1 (e.g., t(14;18) in follicular lymphoma juxtaposes BCL2 gene with IgH enhancer → BCL2 overexpression); downregulation of Bax/PUMA; mutation of p53; loss of PTEN → constitutive Akt survival signaling.
8. Telomerase upregulation: Normal somatic cells lack telomerase → telomeres shorten with each division (~50–70 divisions, "Hayflick limit") → replicative senescence. Cancer cells reactivate telomerase (TERT) → telomeres maintained → unlimited replicative potential (immortalization). ~90% of human cancers express telomerase.
9. Differentiation block: Cancer cells often fail to terminally differentiate, remaining in a proliferative progenitor-like state. This is especially apparent in leukemias (e.g., AML, where myeloid progenitors fail to mature).
10. Warburg effect (aerobic glycolysis): Cancer cells preferentially use glycolysis even in the presence of O₂ (rather than oxidative phosphorylation) — generating lactate. This provides biosynthetic precursors (pentose phosphate pathway, nucleotides, lipids) for rapid proliferation. Basis for PET scanning (FDG-PET): tumors avidly take up [¹⁸F]-fluorodeoxyglucose.

Chemical and Physical Carcinogens

• UV radiation: UVB (280–320 nm) causes pyrimidine dimers (cyclobutane pyrimidine dimers, CPDs) between adjacent Thy or Cyt residues on the same strand → if unrepaired by nucleotide excision repair → C→T (and CC→TT) signature mutations → BRAF, CDKN2A, TP53 mutations in melanoma and squamous cell skin cancer.
• Tobacco smoke: Polycyclic aromatic hydrocarbons (PAHs, e.g., benzo[a]pyrene) form bulky DNA adducts after metabolic activation by CYP enzymes → G→T transversions preferentially at TP53 codon 248/249. Nitrosamines in tobacco → alkylating adducts. ~85% of lung cancers attributed to smoking.
• Aflatoxin B1 (from Aspergillus fungi on stored grains/peanuts): metabolized by CYP3A4 → aflatoxin-8,9-epoxide → G adduct → G→T transversion specifically at codon 249 of TP53 → hepatocellular carcinoma.
• Tumor initiators vs. promoters: Initiators (mutagens) cause irreversible DNA mutation (e.g., DMBA). Promoters (e.g., phorbol esters/TPA, 12-O-tetradecanoylphorbol-13-acetate) are not mutagenic but stimulate proliferation (activate PKC → Ras/MAPK pathway) of initiated cells, enabling clonal expansion. Promoters have no effect on uninitiated cells and are reversible.

Tumor Viruses

RSV and the Discovery of Proto-Oncogenes

Rous Sarcoma Virus (RSV): RNA retrovirus isolated by Peyton Rous (1911); causes sarcomas in chickens. Its transforming gene is v-src — encodes a constitutively active tyrosine kinase (Src). v-Src lacks the C-terminal Tyr527 regulatory tail present on the normal cellular c-Src, which when phosphorylated by Csk binds the SH2 domain of Src, autoinhibiting it. Loss of Tyr527 regulation → constitutive kinase activity.

Avian Leukosis Virus (ALV) lacks v-src and does not acutely transform cells (no captured oncogene); it transforms cells by insertional activation (long latency: provirus integrates near c-MYC → enhances c-MYC transcription).

Harold Varmus and J. Michael Bishop (1976): used low-stringency Southern hybridization to show that sequences homologous to v-src exist in the genomes of all vertebrates examined — the c-src proto-oncogene (also present in fruit fly, sea urchin). This established that viral oncogenes are captured and mutated versions of conserved host genes (proto-oncogenes) with normal roles in cell growth regulation. Nobel Prize in Physiology or Medicine, 1989.

A proto-oncogene is a normal cellular gene encoding a growth-promoting protein (growth factors, RTKs, GTPases, kinases, transcription factors). An oncogene is a mutated version that is constitutively active or overexpressed → drives cell proliferation in a growth-factor-independent manner. Key features: dominant gain-of-function mutation — one mutant allele is sufficient (contrast with tumor suppressors: recessive).

Mechanisms of Proto-Oncogene Activation

1. Point mutation: Ras Gly12→Val (G12V) — glycine 12 is in the GTPase catalytic site. Val substitution sterically blocks GAP (GTPase Activating Protein) binding → Ras cannot hydrolyze GTP → permanently GTP-bound = constitutively active. Mutant Ras (K-Ras, N-Ras, H-Ras) found in ~30% of all human cancers (colorectal, pancreatic, lung NSCLC). RAF mutations (BRAF V600E: activating kinase domain mutation; ~60% of melanomas).
2. Gene amplification: MYC amplification (amplified in neuroblastoma — N-MYC; breast cancer — c-MYC); ERBB2/HER2 amplification (HER2-positive breast cancer, ~20%); EGFR amplification (GBM, ~40%).
3. Chromosomal translocation:
   • t(9;22) — Philadelphia chromosome (CML): BCR gene on chr 22 fused to ABL1 tyrosine kinase on chr 9 → BCR-ABL1 fusion oncoprotein → constitutively active cytoplasmic tyrosine kinase → activates RAS/MAPK, PI3K/Akt, JAK/STAT → chronic myelogenous leukemia. Target of imatinib (Gleevec).
   • t(8;14) — Burkitt lymphoma: c-MYC on chr 8 juxtaposed to IgH enhancer on chr 14 → c-MYC overexpression → constitutive proliferation stimulus.
   • t(14;18) — Follicular lymphoma: BCL2 from chr 18 juxtaposed to IgH locus → BCL2 overexpression → apoptosis resistance.

Ras/Raf/ERK Pathway and Receptor Tyrosine Kinases

The canonical RTK → Ras → MAPK pathway:

EGF → EGFR (ErbB1) dimerization → autophosphorylation of Tyr residues → Grb2 (SH2 domain) → Sos (GEF) → exchanges GDP for GTP on Ras → active Ras-GTP → binds Raf (MAP3K) → Raf phosphorylates MEK (MAP2K) → MEK phosphorylates ERK (MAPK) → ERK translocates to nucleus → phosphorylates transcription factors (Fos, Elk-1) → cyclin D, c-Myc transcription → G1/S progression.

HER2 (ErbB2): has no known ligand; functions by heterodimerization with other ErbB receptors; amplified in ~20% of breast cancers. Target of trastuzumab (Herceptin) (monoclonal antibody) and lapatinib (RTK inhibitor).

Myc — Transcription Factor Oncogene

c-Myc is a bHLH/LZ transcription factor (dimerizes with Max) that activates thousands of target genes promoting cell growth, ribosome biogenesis, cell cycle progression (cyclin D, CDK4, E2F), and also apoptosis (Myc → p14ARF → MDM2 inhibition → p53 stabilization → apoptosis as fail-safe). Therefore cancer cells with Myc overexpression must co-select for loss of p53 or overexpression of anti-apoptotic Bcl-2. Myc is amplified or overexpressed in ~50% of human cancers.

Rb — The Paradigmatic Tumor Suppressor

Alfred Knudson's two-hit hypothesis (1971): he compared retinoblastoma occurrence in familial vs. sporadic cases. Familial: one germline mutation in Rb (inherited) + one somatic mutation in second allele → tumor develops in childhood, often bilateral. Sporadic: two independent somatic mutations required → rare, unilateral, later onset. This established that tumor suppressors are recessive — both alleles must be inactivated.

Rb function: unphosphorylated Rb inhibits E2F → no S-phase gene transcription. Cyclin D/Cdk4/6 → hyperphospho-Rb → E2F released → S-phase genes. Rb mutated in retinoblastoma, osteosarcoma, and many other human cancers. Rb-null mice develop pituitary tumors.

p53 — Guardian of the Genome

p53 (TP53) is mutated in ~50% of all human cancers — the most frequently mutated gene in cancer. p53 is a sequence-specific transcription factor that is maintained at low levels in unstressed cells by MDM2-mediated ubiquitination (proteasomal degradation).

Activation: DNA damage (ATM/Chk2), oncogene activation (ARF), ribosomal stress → p53 stabilization (phosphorylation at Ser15/20 blocks MDM2 binding; ARF sequesters MDM2 in nucleolus). p53 then transcribes:

• p21 (CKI) → Cdk2 inhibition → G1/S arrest (buys time for repair)
• GADD45 → DNA repair
• Bax, PUMA, Noxa → apoptosis (if damage is irreparable)
• MDM2 → negative feedback (p53 self-limits after damage resolves)

Oncogene → ARF → p53 fail-safe: Activated oncogene (e.g., Ras mutation) → p14^ARF transcription (ARF = alternate reading frame product of the CDKN2A locus, which also encodes p16) → ARF binds MDM2 → MDM2 sequestered in nucleolus → p53 stabilized → senescence or apoptosis. This is why oncogene activation in a normal cell typically triggers senescence, not cancer — cancer requires loss of this fail-safe (p53 mutation, ARF deletion, or MDM2 amplification).

Mechanisms of Tumor Suppressor Inactivation

• LOH (Loss of Heterozygosity): deletion of chromosomal region containing the remaining wild-type allele (second hit); detected by microsatellite marker analysis or CGH
• Epigenetic silencing: promoter CpG methylation (e.g., BRCA1, p16, MLH1 silenced in sporadic cancers) by DNMT3A/3B; once methylated, maintained by DNMT1 during replication
• Point mutation: dominant-negative mutations in p53 (mutant p53 protein can sequester remaining WT p53 in inactive oligomers)

Cancer Treatment Overview