2.2 Cell Cycle Control, Apoptosis & Cell Signalling

Cell Cycle Regulation, Checkpoints & Apoptosis

The eukaryotic cell cycle progresses through four discrete phases (G1 -> S -> G2 -> M), driven by cyclic assemblies of cyclins and Cyclin-Dependent Kinases (CDKs).

      G1 Phase ───────────────────> S Phase ───────────────────> G2 Phase ─────────────> M Phase
         │                             │                            │                        │
   [Cyclin D-CDK4/6]             [Cyclin E-CDK2]              [Cyclin A-CDK2]          [Cyclin B-CDK1]
         │                             │                            │                        │
         ▼                             ▼                            ▼                        ▼
  Rb Phosphorylation            DNA Replication             DNA Damage Check        Spindle Assembly
  (E2F Factor Release)         Initiation & Progress       (ATM/ATR -> Chk1/2)          Checkpoint

Checkpoints and Tumour Suppressor Kinetics

1. G1/S Restriction Point: Commits the cell to cell division. Governed by the retinoblastoma tumour suppressor protein (Rb):
   • Hypophosphorylated Rb (active form) binds and sequesters the E2F transcription factor, repressing genes required for DNA synthesis.
   • Mitogenic signals induce Cyclin D, which complexes with CDK4 and CDK6 to partially phosphorylate Rb.
   • Cyclin E-CDK2 complexes complete the hyperphosphorylation of Rb.
   • Hyperphosphorylated Rb (inactive form) dissociates from E2F. Free E2F enters the nucleus, driving transcription of thymidine kinase, DNA polymerase alpha, and Cyclin A, propelling the cell into S phase.
2. CDK Inhibitors (CDKIs):
   • INK4 family (p15, p16, p18, p19): Selectively bind and inhibit CDK4 and CDK6. Loss of p16INK4a (via CDKN2A deletion) is frequent in melanoma, pancreatic cancer, and glioblastoma.
   • CIP/KIP family (p21, p27, p57): Inhibit all cyclin-CDK complexes. p21Cip1 is directly transactivated by p53 in response to DNA damage.
3. p53 "Guardian of the Genome" Cascade: DNA double-strand breaks activate ATM (ataxia-telangiectasia mutated) and ATR kinases, which phosphorylate p53, preventing its degradation by the E3 ubiquitin ligase MDM2. Stable p53 accumulates as a tetrameric transcription factor that:
   • Transactivates p21, inducing G1 arrest to allow enzymatic DNA repair.
   • Transactivates GADD45, promoting nucleotide excision repair.
   • If repair fails, transactivates pro-apoptotic effectors Bax, Bak, PUMA, and Noxa, triggering apoptosis.
   • Li-Fraumeni Syndrome: Autosomal dominant germline mutation in TP53 on chromosome 17p13. Characterized by early-onset sarcomas (osteosarcoma, rhabdomyosarcoma), breast carcinoma, brain tumors (glioma), adrenocortical carcinoma, and leukemias (the SBLA cancer complex).

Apoptosis versus Necrosis

Molecular Cascades of Apoptosis

• Intrinsic (Mitochondrial) Pathway: Triggered by cellular stress, withdrawal of survival factors, or DNA damage.
  1. Pro-apoptotic BH3-only sensors (Bim, Bid, Bad, Puma, Noxa) neutralize anti-apoptotic Bcl-2, Bcl-xL, and Mcl-1.
  2. Unopposed pro-apoptotic effectors Bax and Bak oligomerise to form permeability transition pores in the outer mitochondrial membrane.
  3. Cytochrome c leaks from the intermembrane space into the cytosol.
  4. Cytosolic Cytochrome c binds Apaf-1 (apoptotic protease activating factor 1) in the presence of dATP, assembling a wheel-like heptameric apoptosome.
  5. The apoptosome recruits and cleaves pro-caspase-9 (initiator caspase).
  6. Active Caspase-9 cleaves downstream executioner caspases-3, -6, and -7.
  • Bcl-2 Overexpression: In follicular lymphoma, the reciprocal translocation t(14;18)(q32;q21) juxtaposes the BCL2 proto-oncogene with the immunoglobulin heavy chain (IGH) promoter, causing Bcl-2 overexpression, which sequesters Bax/Bak and makes germinal centre B cells resistant to apoptosis.
• Extrinsic (Death Receptor) Pathway: Triggered by extracellular death ligands.
  1. Fas ligand (FasL / CD95L) binds Fas (CD95), or TNF-alpha binds TNFR1.
  2. Trimerisation of receptor death domains recruits the adaptor protein FADD (Fas-Associated Death Domain).
  3. FADD recruits pro-caspase-8 or pro-caspase-10 to assemble the DISC (death-inducing signaling complex).
  4. Active Caspase-8 cleaves executioner Caspase-3 directly, and cleaves cytosolic Bid to truncated Bid (tBid), providing cross-talk that activates the intrinsic mitochondrial pathway.

Cell Signaling & Second Messenger Pathways

Intercellular communication relies on transmembrane receptors coupling extracellular ligands to downstream metabolic and transcriptional responses.

G-Protein Coupled Receptors (GPCRs)

GPCRs are 7-transmembrane alpha-helical receptors that couple to heterotrimeric G-proteins comprising alpha, beta, and gamma subunits. Ligand binding catalyzes GDP-to-GTP exchange on the alpha subunit, dissociating G-alpha-GTP from the G-beta-gamma dimer.

        ┌─────────────────────────────────── GPCR Subtypes ───────────────────────────────────┐
        │                                                      │                              │
        ▼                                                      ▼                              ▼
   Gs Pathway                                             Gi Pathway                     Gq Pathway
[G-alpha-s • GTP]                                     [G-alpha-i • GTP]              [G-alpha-q • GTP]
        │                                                      │                              │
        ▼                                                      ▼                              ▼
Stimulates Adenylyl Cyclase                           Inhibits Adenylyl Cyclase       Stimulates Phospholipase C beta
        │                                                      │                              │
        ▼                                                      ▼                              ▼
   cAMP Rises                                             cAMP Falls              PIP2 ──> IP3 + DAG
        │                                                      │                            │     │
        ▼                                                      ▼                            ▼     ▼
  Activates PKA                                           Suppresses PKA             Ca2+ Release  Activates PKC
(Phosphorylates targets)                              (Reduced phosphorylation)     (from ER/SR)        │
                                                                                                        ▼
                                                                                                 Smooth Muscle Contraction

GPCR Subunit Classes and Clinical Manifestations

• Gs-alpha Pathway: Ligand binding stimulates adenylyl cyclase, converting ATP to cyclic AMP (cAMP). cAMP binds regulatory subunits of Protein Kinase A (PKA), releasing active catalytic subunits that phosphorylate serine/threonine residues on metabolic enzymes and the transcription factor CREB.
  • Receptors: Beta-1, Beta-2, Beta-3, V2 (renal), H2 (gastric), Glucagon, PTH, TSH, ACTH, Calcitonin, GHRH.
  • Cholera Toxin: Produced by Vibrio cholerae; catalyzes ADP-ribosylation of Gs-alpha, abolishing its intrinsic GTPase activity. Gs-alpha is locked in an active state, causing uncontrolled cAMP production in intestinal enterocytes, massive PKA-mediated opening of CFTR channels, and voluminous watery "rice-water" diarrhea.
• Gi-alpha Pathway: Ligand binding inhibits adenylyl cyclase, decreasing cAMP production and suppressing PKA activity.
  • Receptors: Alpha-2, M2 (cardiac), D2 (dopamine).
  • Pertussis Toxin: Produced by Bordetella pertussis; catalyzes ADP-ribosylation of Gi-alpha, locking it in the inactive, GDP-bound state. Inactivated Gi can no longer inhibit adenylyl cyclase, causing high intracellular cAMP that impairs leukocyte chemotaxis, producing severe lymphocytosis.
• Gq-alpha Pathway: Ligand binding stimulates Phospholipase C beta (PLC-beta), which cleaves membrane phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers:
  1. Inositol 1,4,5-trisphosphate (IP3): Diffuses to the sarcoplasmic/endoplasmic reticulum, binding IP3-gated Ca2+ channels to release Ca2+ into the cytoplasm.
  2. Diacylglycerol (DAG): Remains membrane-bound and, together with released Ca2+, activates Protein Kinase C (PKC).
  • Receptors ("HAVe 1 M&M"): H1, Alpha-1, V1 (vascular), M1, M3.

Receptor Tyrosine Kinases (RTKs) and Intrinsic Kinase Cascades

• Structure and Activation: Single-pass transmembrane receptors with intrinsic cytosolic tyrosine kinase domains (insulin receptor, IGF-1, EGFR, PDGF, VEGF, FGF). Ligand binding induces receptor dimerisation, activating trans-autophosphorylation of tyrosine residues within the cytosolic tail.
• Downstream Pathways:
  • Ras-Raf-MEK-ERK (MAPK) Pathway: Phosphotyrosines recruit SH2-domain adaptor proteins (GRB2) complexed with Sos (guanine nucleotide exchange factor). Sos converts inactive Ras-GDP to active Ras-GTP. Active Ras recruits and activates Raf kinase, which phosphorylates MEK, which in turn phosphorylates ERK (extracellular signal-regulated kinase). Activated ERK translocates to the nucleus to induce transcription of c-Myc and c-Fos, driving cellular proliferation.
  • PI3K-Akt-mTOR Pathway: Phosphotyrosines recruit Phosphoinositide 3-kinase (PI3K), which converts PIP2 to PIP3. Membrane PIP3 recruits Akt (Protein Kinase B) and PDK1. Activated Akt phosphorylates Bad (inhibiting apoptosis) and stimulates mTOR (mammalian target of rapamycin), promoting protein synthesis, cell growth, and angiogenesis. The lipid phosphatase PTEN dephosphorylates PIP3 back to PIP2; loss-of-function PTEN mutations drive unrestrained Akt activation in Cowden syndrome and endometrial carcinoma.
• Non-Receptor Tyrosine Kinases (JAK-STAT): Receptors for cytokines, growth hormone, prolactin, erythropoietin, and thrombopoietin lack intrinsic kinase activity. Ligand binding induces receptor oligomerisation, cross-activating associated Janus kinases (JAK1, JAK2, JAK3, TYK2). Activated JAKs phosphorylate receptor tyrosine residues, creating docking sites for STATs (Signal Transducers and Activators of Transcription). Phosphorylated STATs dimerise, dissociate, and translocate directly to the nucleus as active transcription factors. The gain-of-function JAK2 V617F mutation produces autonomous STAT phosphorylation, driving BCR-ABL-negative myeloproliferative neoplasms (polycythaemia vera, essential thrombocythaemia, primary myelofibrosis).
• Cyclic GMP / Nitric Oxide Cascade: Endothelial nitric oxide synthase (eNOS) synthesizes nitric oxide (NO) from L-arginine. NO diffuses into adjacent vascular smooth muscle cells and binds the heme moiety of soluble guanylyl cyclase (sGC), converting GTP to cyclic GMP (cGMP). Elevated cGMP activates Protein Kinase G (PKG), which phosphorylates phospholamban and myosin light-chain phosphatase, promoting Ca2+ sequestration and smooth muscle relaxation. Atrial and B-type natriuretic peptides (ANP, BNP) stimulate a transmembrane, particulate guanylyl cyclase receptor (NPR-A). cGMP is degraded to 5'-GMP by phosphodiesterase-5 (PDE5), which is selectively inhibited by sildenafil and tadalafil to prolong vasodilation.