Cell Biology: Cell cycle, signalling, cell death and stem cells

🔬 Cell cycle, signalling, cell death and stem cells. Cells must divide, communicate, specialise and die in a controlled way. When control fails, the results are cancer, autoimmunity, degenerative disease and abnormal development. Many modern drugs - targeted cancer therapies, biologics, immunosuppressants and receptor agonists and antagonists - act on these pathways. This chapter covers the cell cycle and its checkpoints, receptor signalling, apoptosis and other forms of cell death, and stem cells and regeneration.
Your four questions: Which checkpoint or signal has been lost or overactivated? Is the effect on proliferation, survival, differentiation or death? Which drug targets that pathway? What are the expected toxicities of interfering with it in normal tissues?

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The Cell Cycle

• Phases: G1 (growth, preparing to replicate) → S (DNA synthesis) → G2 (preparation for division) → M (mitosis and cytokinesis). Interphase = G1 + S + G2.
• G0 - quiescent, non-dividing state; cells may re-enter the cycle.

Cell type	Behaviour	Examples
Labile	Continuously dividing	Skin, gut epithelium, bone marrow - most sensitive to chemotherapy and radiotherapy (mucositis, hair loss, myelosuppression)
Stable (quiescent)	G0 but can re-enter the cycle when stimulated	Hepatocytes (liver regeneration), renal tubular cells, fibroblasts, smooth muscle
Permanent	Cannot divide	Neurons, cardiac myocytes (very limited), skeletal muscle (satellite cells allow limited repair) - injury heals by scarring

• Cell-cycle-specific chemotherapy: antimetabolites (methotrexate, 5-FU, cytarabine) - S phase; vinca alkaloids and taxanes - M phase; bleomycin - G2; etoposide - S/G2 (topoisomerase II). Cell-cycle non-specific: alkylating agents (cyclophosphamide), platinum compounds, anthracyclines.

Checkpoints, Cyclins and Tumour Suppressors

• Cyclins bind cyclin-dependent kinases (CDKs) to drive progression; CDK inhibitors (p21, p16, p27) restrain them. CDK4/6 inhibitors (palbociclib, ribociclib, abemaciclib) treat hormone receptor-positive breast cancer.
• G1/S checkpoint (restriction point) - the retinoblastoma protein (Rb) holds the E2F transcription factor; CDK4/6–cyclin D phosphorylates Rb to release E2F and allow S phase. Loss of Rb → uncontrolled entry (retinoblastoma, osteosarcoma); HPV E7 protein inactivates Rb.
• p53 ("guardian of the genome") - activated by DNA damage → induces p21 (arrests the cycle for repair) or triggers apoptosis if damage is irreparable. Mutated in about half of all cancers; germline TP53 mutation → Li–Fraumeni syndrome (sarcomas, breast cancer, brain tumours, leukaemia, adrenocortical carcinoma); HPV E6 protein degrades p53.
• G2/M checkpoint - checks DNA replication is complete and undamaged.
• Spindle (metaphase) checkpoint - ensures chromosomes are attached to the spindle before separation; failure → aneuploidy.
• Knudson's two-hit hypothesis - tumour suppressor genes need loss of both alleles: hereditary cancers inherit the first hit (earlier, bilateral or multiple tumours - e.g. hereditary retinoblastoma).

Mitosis and Meiosis

• Mitosis - one division producing two genetically identical diploid daughter cells: prophase, metaphase (chromosomes aligned - karyotyping uses metaphase arrest), anaphase (sister chromatids separate), telophase, then cytokinesis.
• Meiosis - two divisions producing four haploid gametes; crossing over (recombination) in prophase I creates genetic diversity; meiosis I separates homologous chromosomes, meiosis II separates sister chromatids.
• Non-disjunction - failure of chromosomes to separate → aneuploidy (trisomy 21, 18, 13; Turner's 45,X; Klinefelter's 47,XXY). Maternal age increases meiosis I non-disjunction (oocytes arrested in prophase I from fetal life until ovulation).
• Spermatogenesis - continuous from puberty (about 74 days per cycle); oogenesis - all primary oocytes present before birth.

Telomeres, Senescence and Ageing

• Telomeres - repetitive DNA (TTAGGG) at chromosome ends; shorten with each division because DNA polymerase cannot fully copy the ends.
• Critically short telomeres trigger replicative senescence (Hayflick limit - about 50–60 divisions in cultured cells) - a barrier against cancer.
• Telomerase (reverse transcriptase adding telomere repeats) is active in germ cells, stem cells and about 90% of cancers (immortality).
• Telomere disorders - dyskeratosis congenita (nail dystrophy, leukoplakia, skin pigmentation, bone marrow failure, pulmonary fibrosis).
• Senescent cells secrete inflammatory factors (senescence-associated secretory phenotype), contributing to ageing and age-related disease.

Receptor Types and Second Messengers

Receptor class	Mechanism	Examples
Ligand-gated ion channels	Milliseconds; ion flux	Nicotinic ACh, GABA-A, glutamate (NMDA, AMPA), 5-HT₃
G-protein-coupled receptors (GPCRs)	Seconds; second messengers	Adrenergic, muscarinic, opioid, dopamine, histamine, most hormones (PTH, glucagon, TSH, ADH); about a third of all drugs act here
Receptor tyrosine kinases	Minutes–hours; phosphorylation cascades	Insulin, EGFR, HER2, VEGF, PDGF receptors
Cytokine receptors (JAK-associated)	JAK–STAT	EPO, GH, prolactin, interleukins, interferons
Nuclear (intracellular) receptors	Hours–days; gene transcription	Steroids, thyroid hormone, vitamin D, retinoids, PPARs

• G-protein subtypes: Gs (↑ adenylyl cyclase → ↑cAMP - β₁, β₂, D1, H2, V2, glucagon), Gi (↓cAMP - α₂, M2, D2, opioid), Gq (phospholipase C → IP₃ + DAG → ↑Ca²⁺ and PKC - α₁, M1, M3, H1, V1, angiotensin II AT1).
• Toxins acting on G proteins: cholera toxin permanently activates Gs (↑cAMP in enterocytes → massive watery diarrhoea); pertussis toxin inactivates Gi.
• Agonists, partial agonists (buprenorphine, varenicline), antagonists, inverse agonists (antihistamines); receptor desensitisation and down-regulation explain tolerance (opioids, β-agonists, nitrates).

Growth Factor Pathways and Targeted Therapy

• RAS–RAF–MEK–ERK (MAPK) - growth factor signalling; KRAS mutations (pancreatic, colorectal, lung - predict resistance to anti-EGFR antibodies; KRAS G12C inhibitors now available), BRAF V600E (melanoma, hairy cell leukaemia, some colorectal and thyroid cancers - treated with BRAF + MEK inhibitors).
• PI3K–AKT–mTOR - growth and survival; mTOR inhibitors (sirolimus, everolimus) in transplantation and some cancers; PTEN (tumour suppressor) loss in Cowden syndrome.

Target	Drug	Use
HER2	Trastuzumab, pertuzumab	HER2-positive breast and gastric cancer (cardiotoxicity - echo monitoring)
EGFR	Erlotinib, gefitinib, osimertinib (small molecules); cetuximab (antibody)	EGFR-mutant lung cancer; RAS wild-type colorectal cancer
BCR-ABL	Imatinib, dasatinib, nilotinib	Chronic myeloid leukaemia (Philadelphia chromosome t(9;22))
ALK	Crizotinib, alectinib	ALK-rearranged lung cancer
VEGF	Bevacizumab; sunitinib (multi-kinase)	Colorectal, renal cancers (hypertension, proteinuria, poor wound healing)
BRAF/MEK	Dabrafenib + trametinib	BRAF V600E melanoma
PD-1 / PD-L1, CTLA-4	Pembrolizumab, nivolumab, ipilimumab (immune checkpoint inhibitors)	Many cancers; immune-related adverse events (colitis, hepatitis, pneumonitis, endocrinopathies)

• Naming: "-mab" monoclonal antibodies; "-tinib" tyrosine kinase inhibitors; "-ximab" chimeric, "-zumab" humanised, "-umab" human.

Cytokine Signalling and JAK–STAT

• Cytokines - small proteins coordinating immunity and inflammation: IL-1, IL-6, TNF-α (fever, acute phase response), IL-2 (T-cell proliferation), IL-4/IL-5/IL-13 (allergy, eosinophils), IL-17 and IL-23 (psoriasis, spondyloarthritis), interferons (antiviral), IL-10 and TGF-β (anti-inflammatory).
• JAK–STAT pathway - cytokine receptors activate Janus kinases → STAT transcription factors. JAK2 V617F mutation drives polycythaemia vera, essential thrombocythaemia and myelofibrosis (ruxolitinib).
• Biologic and small-molecule targets: anti-TNF (infliximab, adalimumab, etanercept - screen for TB and hepatitis B), anti-IL-6R (tocilizumab - GCA, RA, cytokine release syndrome), anti-IL-17 (secukinumab), anti-IL-12/23 (ustekinumab), anti-IL-5 (mepolizumab - eosinophilic asthma), anti-IL-4R (dupilumab - eczema, asthma), JAK inhibitors (tofacitinib, baricitinib, upadacitinib - VTE, MACE and malignancy warnings in older or high-risk patients; MHRA 2023).
• Cytokine storm / cytokine release syndrome - CAR-T therapy, severe COVID-19, HLH, sepsis.

Apoptosis

• Apoptosis - programmed, energy-dependent cell death without inflammation: cell shrinkage, chromatin condensation (pyknosis), nuclear fragmentation (karyorrhexis), membrane blebbing, apoptotic bodies phagocytosed by macrophages.
• Physiological roles: embryogenesis (separation of fingers), removal of autoreactive lymphocytes (thymic selection), involution of hormone-dependent tissues (endometrium in menstruation), turnover of epithelial cells and neutrophils, ending immune responses.
• Intrinsic (mitochondrial) pathway - triggered by DNA damage, growth factor withdrawal, cellular stress; regulated by the BCL-2 family (BCL-2 anti-apoptotic; BAX/BAK pro-apoptotic) → cytochrome c release → apoptosome → caspase-9 → executioner caspases (3, 6, 7). BCL-2 overexpression - t(14;18) in follicular lymphoma; venetoclax (BCL-2 inhibitor) for CLL and AML.
• Extrinsic (death receptor) pathway - Fas ligand/Fas, TNF/TNF receptor → caspase-8. Cytotoxic T cells use Fas ligand and perforin/granzyme.
• Too little apoptosis - cancer, autoimmunity (Fas mutations - autoimmune lymphoproliferative syndrome), persistent viral infection. Too much - neurodegeneration, ischaemic injury (penumbra), HIV (CD4 depletion), graft-versus-host disease.

Necrosis, Necroptosis, Pyroptosis and Autophagy

Feature	Necrosis	Apoptosis
Cause	Always pathological - ischaemia, toxins, trauma, infection	Physiological or pathological
Cell size	Swelling	Shrinkage
Membrane	Ruptures - contents leak	Intact; blebs
Inflammation	Yes	No
Extent	Groups of cells	Single cells

• Types of necrosis (see the Pathology chapters): coagulative (infarcts), liquefactive (brain, abscess), caseous (TB), fat (pancreatitis, breast trauma), fibrinoid (vasculitis, malignant hypertension), gangrenous.
• Necroptosis - regulated necrosis (RIPK1/RIPK3/MLKL) when caspases are blocked; inflammatory.
• Pyroptosis - inflammatory death of infected immune cells via the inflammasome and caspase-1 (IL-1β release); gasdermin pores.
• Ferroptosis - iron-dependent lipid peroxidation.
• Autophagy - "self-eating" of organelles during starvation or stress; a survival mechanism; defects in Crohn's disease (ATG16L1) and neurodegeneration.

Stem Cells, Regeneration and Stem Cell Therapy

• Stem cells - self-renew and differentiate. Totipotent (zygote - all tissues including placenta), pluripotent (embryonic stem cells, induced pluripotent stem cells reprogrammed from adult cells by Yamanaka factors - all three germ layers), multipotent (adult/tissue stem cells - haematopoietic, mesenchymal, intestinal crypt, skin basal layer, limbal), unipotent (spermatogonia).
• Niches - specialised microenvironments maintaining stem cells (bone marrow, intestinal crypts, hair follicle bulge, limbus of the cornea).
• Regeneration versus repair: labile and stable tissues regenerate if the framework (basement membrane, connective tissue) is intact; otherwise repair occurs by fibrosis (scar). The liver can regenerate after losing up to about two-thirds of its mass (living donor transplant).
• Clinical uses: haematopoietic stem cell transplantation (autologous - myeloma, lymphoma; allogeneic - leukaemia, aplastic anaemia, inherited disorders; graft-versus-leukaemia effect versus graft-versus-host disease), limbal stem cell grafts, skin grafts and cultured epidermis for burns, CAR-T cells (gene-modified T cells - not stem cells but cell therapy), gene therapy of autologous stem cells (sickle cell disease and β-thalassaemia - CRISPR-edited exa-cel licensed in the UK in 2023).
• Unproven "stem cell" clinics - risks of infection, tumour formation and harm; advise patients accordingly.

At a Glance

Concept	Remember
G1/S checkpoint	Rb holds E2F; CDK4/6 phosphorylates Rb
p53	Arrest or apoptosis after DNA damage; Li–Fraumeni
Two-hit hypothesis	Tumour suppressors need both alleles lost
Gs / Gi / Gq	↑cAMP / ↓cAMP / IP₃–Ca²⁺
Cholera / pertussis toxin	Activates Gs / inactivates Gi
BCL-2	Anti-apoptotic; t(14;18) follicular lymphoma; venetoclax
Apoptosis vs necrosis	No inflammation vs inflammation
JAK2 V617F	Myeloproliferative neoplasms

Spot the Answer

• A child with bilateral retinoblastoma later develops osteosarcoma. → Germline RB1 mutation (two-hit hypothesis).
• A woman whose mother, brother and cousin had sarcoma, brain tumour and breast cancer at young ages. → Li–Fraumeni syndrome (TP53).
• Profuse "rice-water" diarrhoea after travel. → Cholera toxin activating Gs → ↑cAMP → chloride secretion.
• Painless lymphadenopathy; biopsy shows t(14;18). → Follicular lymphoma (BCL-2 overexpression).
• Diarrhoea, hepatitis and hypophysitis in a patient receiving pembrolizumab. → Immune-related adverse events from checkpoint inhibition.

Exam-style Scenarios

• Scenario. Why does chemotherapy cause mucositis, alopecia and neutropenia but rarely acute damage to neurons or cardiac muscle (apart from specific drugs)?
  Answer: Cytotoxic drugs mainly kill rapidly dividing (labile) cells - gut epithelium, hair follicles, bone marrow. Neurons and myocytes are permanent (non-dividing) cells, though specific drugs damage them by other mechanisms (vincristine neuropathy, anthracycline cardiotoxicity).
• Scenario. A patient with rheumatoid arthritis starting adalimumab asks what tests are needed. Why?
  Answer: TNF is essential for granuloma maintenance - screen for latent TB (IGRA, CXR) and hepatitis B and C (and HIV) before starting, and update vaccinations (avoid live vaccines during treatment).
• Scenario. A 70-year-old smoker with rheumatoid arthritis is considered for a JAK inhibitor. What are the safety concerns?
  Answer: MHRA advice (2023): in people over 65, current or past long-term smokers, or those with cardiovascular or malignancy risk factors, JAK inhibitors should be used only if no suitable alternatives - increased risk of MACE, VTE, malignancy and serious infections.

Key Points

• Labile cells divide constantly; permanent cells heal by scarring.
• Rb and p53 are the key tumour suppressors controlling the cell cycle.
• Know the G-protein subtypes and their classic receptors and toxins.
• Targeted therapies follow the pathways - know the main drug–target pairs and their toxicities.
• Apoptosis is non-inflammatory; necrosis is inflammatory.

Further Reading

• Alberts B et al. Molecular Biology of the Cell
• Weinberg RA. The Biology of Cancer
• Rang and Dale's Pharmacology (receptors and signalling)
• MHRA (2023): Janus kinase (JAK) inhibitors - new measures to reduce risks of major cardiovascular events, malignancy, VTE and serious infections