Cell Cycle & Apoptosis

The Cell Cycle for the MCAT

Part 1 of 4 — Phases, Mitosis & Measuring the Cycle

The Four Phases (Plus One Exit)

• Interphase = G$_1$ + S + G$_2$ — about 90% of the cycle; chromosomes are decondensed chromatin, invisible as discrete bodies
• After S phase a chromosome consists of two sister chromatids joined at the centromere by cohesin — chromosome NUMBER (n) has not changed, DNA content (C) has doubled
• G$_0$ cells (most neurons, cardiac muscle) are metabolically active but non-dividing; hepatocytes can re-enter the cycle when stimulated; permanent G$_0$ tissue heals by scarring, not regeneration

Mitosis — PMAT in Mechanistic Terms

• Prophase condensation is driven by condensin complexes.

Anaphase is the moment ploidy bookkeeping changes: 46 chromosomes of 2 chromatids become (transiently) 92 chromosomes of 1 chromatid, then 46 per daughter.

Measuring the Cycle — Flow Cytometry

Stain DNA with a quantitative fluorescent dye and count cells by DNA content:

• 2C peak = G$_1$ (and G$_0$) cells
• 4C peak = G$_2$ and M cells
• Between the peaks = S phase (actively replicating)

A drug that arrests cells in mitosis grows the 4C peak; one that blocks replication initiation grows the 2C peak; one that stalls forks piles cells between the peaks. BrdU/EdU pulse labeling marks only cells synthesizing DNA during the pulse — the direct S-phase census. Mitotic cells are counted separately, by their condensed chromosomes (the mitotic index).

Phases & Mitosis 🎯

Checkpoints — Quality Control Gates (Preview of Part 2)

The Mitotic Spindle — Three Microtubule Jobs

• Kinetochore microtubules: attach to chromosomes and pull chromatids poleward (anaphase A)
• Polar (interpolar) microtubules: overlap at the midzone; motor proteins slide them apart, pushing poles away (anaphase B)
• Astral microtubules: anchor poles to the cell cortex and position the spindle — which determines the cleavage plane

Microtubule drugs both stop mitosis but oppositely:

• Vinca alkaloids / colchicine: prevent polymerization → no spindle
• Taxanes (paclitaxel): hyper-stabilize microtubules → spindle cannot remodel

Both leave kinetochores without proper tension → spindle checkpoint arrest → often apoptosis. Rapidly dividing normal tissues (marrow, gut, hair follicles) share the toxicity.

Karyokinesis Without Cytokinesis

• Cytokinesis is a separate, actin-based process; blocking it (or in normal liver/heart development) yields binucleate or polyploid cells
• Megakaryocytes deliberately replicate DNA without dividing (endomitosis) → giant polyploid platelet factories
• In plants, a cell plate (vesicle-built new wall) replaces the contractile ring — a favorite comparison question

Spindle & Cycle Measurement 🎯

Key Takeaways — Part 1

• Cycle order: G$_1$ → S → G$_2$ → M; interphase is ~90% of the time; G$_0$ = quiescent exit (neurons/cardiac permanent; hepatocytes facultative)
• Track n vs. C separately: S phase doubles DNA (2C → 4C) but not chromosome number; chromatids count as chromosomes only after anaphase separation
• Mitosis: condense (prophase) → envelope down/kinetochores capture (prometaphase) → align (metaphase) → cohesin cleaved, chromatids part (anaphase) → envelopes re-form (telophase) → actin ring pinches (cytokinesis)
• Three checkpoints: restriction point (G$_1$/S, growth-factor dependent), G$_2$/M (DNA integrity), spindle checkpoint (attachment/tension)
• Flow cytometry reads phase by DNA content (2C vs. 4C vs. between); EdU/BrdU pulse marks S phase directly
• Spindle poisons work oppositely (destabilize vs. hyper-stabilize) but both trigger checkpoint arrest — hence shared chemotherapy logic and toxicity
• Karyokinesis and cytokinesis are separable: endomitosis (megakaryocytes) and binucleate hepatocytes are normal outcomes

• Low-yield extras: condensin complexes drive prophase chromosome condensation

The Cell Cycle for the MCAT

Part 2 of 4 — Cyclins, CDKs & Checkpoint Machinery

The Engine: Cyclin-Dependent Kinases

The cycle is driven by CDKs — serine/threonine kinases that are inactive alone and switched on by binding a cyclin:

• CDK protein levels stay constant through the cycle; cyclin levels oscillate (synthesis then abrupt degradation) — the oscillation IS the clock
• Cyclin destruction is by ubiquitin-mediated proteolysis: an E3 ubiquitin ligase tags the cyclin, the proteasome destroys it. Degradation is irreversible, which makes cycle transitions one-way ratchets

The Restriction Point: Rb and E2F

1. Rb (retinoblastoma protein) binds and silences E2F transcription factors in early G$_1$
2. Growth factors → Ras-MAPK → cyclin D synthesis → CDK4/6 begins phosphorylating Rb
3. Hyperphosphorylated Rb releases E2F → E2F transcribes S-phase genes (cyclin E, DNA polymerase components, thymidylate synthase)
4. Cyclin E-CDK2 further phosphorylates Rb — a positive feedback loop that makes commitment all-or-none

Past the restriction point, the cycle runs to completion without growth factors. Cancer biology in one sentence: nearly all tumors break the Rb-E2F brake somehow (Rb loss, cyclin D overexpression, CDK4 amplification, or p16 loss).

CDK Inhibitors and Checkpoint Enforcement

• p21: transcribed by p53 after DNA damage; binds and inhibits G$_1$/S CDKs → G$_1$ arrest
• p16: blocks cyclin D-CDK4/6 specifically; lost in many cancers
• G$_2$/M enforcement: damage-activated checkpoint kinases inhibit Cdc25, the phosphatase that removes inhibitory phosphates from CDK1 — no Cdc25 activity, no mitosis

• The damage-sensing kinases are ATM and ATR, which act through the effector kinases Chk1/Chk2; p16 is also called INK4a.

The Spindle Checkpoint and APC/C

• Unattached kinetochores emit a "wait" signal that inhibits the APC/C (anaphase-promoting complex — an E3 ubiquitin ligase)
• When every kinetochore is attached under bipolar tension, APC/C activates and ubiquitinates:
  1. Securin → freed separase cleaves cohesin → chromatids separate (anaphase)
  2. Cyclin B → CDK1 turns off → mitotic exit (envelope re-forms, chromosomes decondense)

One ubiquitin ligase thus triggers both chromosome separation and the end of mitosis.

• The wait signal is generated by the Mad and Bub checkpoint proteins.

Cyclins & CDKs 🎯

The Experiments That Found the Engine

Cell-fusion experiments:

• Fuse an S-phase cell with a G$_1$ cell → the G$_1$ nucleus begins replication early — S-phase cytoplasm contains a diffusible activator
• Fuse an M-phase cell with any interphase cell → the interphase nucleus condenses its chromosomes prematurely — mitotic cytoplasm dominates
• Fuse a G$_2$ cell with an S-phase cell → the G$_2$ nucleus does not re-replicate — replicated DNA is licensed only once (see below)

MPF and frog eggs:

• Injecting cytoplasm from a mature (M-phase) frog egg into an immature oocyte drives it into M — the activity was named maturation-promoting factor (MPF)
• MPF = cyclin B-CDK1; sea urchin experiments showed cyclin protein rising each cycle and crashing at each anaphase — the first protein ever seen to oscillate with the cycle

• The researchers: Rao and Johnson (cell fusion), Masui (MPF in frog oocytes), Nurse (CDK genes in yeast), Hunt (cyclins in sea urchin embryos).

Licensing — Why DNA Replicates Exactly Once

• In G$_1$ (low CDK activity), origins are licensed: loader proteins place inactive replicative helicases on each origin
• S-phase CDK activity fires licensed origins AND simultaneously prevents new licensing (phosphorylates/degrades the loaders)
• Result: each origin can fire at most once per cycle; re-licensing requires passing through mitosis back to a low-CDK state
• This is the molecular answer to the fusion experiment: a G$_2$ nucleus has spent its licenses

• The loaders are ORC, Cdc6 and Cdt1; the helicase they load is MCM.

Reading a Cycle Mutant (cdc Logic)

Temperature-sensitive cdc (cell division cycle) yeast mutants arrest at a uniform stage at the restrictive temperature — the arrest point marks where the gene product acts. A mutant arresting with unreplicated DNA acts at G$_1$/S; one arresting as large budded cells with divided nuclei acts at cytokinesis. Uniform arrest morphology = a true cycle gene; random arrest = a general growth gene.

• These mutants were Hartwell's budding-yeast screen.

Checkpoint Machinery 🎯

Key Takeaways — Part 2

• CDK levels constant, cyclin levels oscillate; cyclin binding (plus phosphatase Cdc25 removing inhibitory phosphates) switches kinase activity on; ubiquitin-proteasome destruction of cyclins makes transitions irreversible
• Pairs to know: cyclin D-CDK4/6 (G$_1$, growth-factor input), cyclin E-CDK2 (restriction point), cyclin A-CDK2 (S), cyclin B-CDK1 = MPF (M)
• Restriction point = Rb-E2F: Rb phosphorylation releases E2F → S-phase genes; positive feedback makes commitment all-or-none; virtually all cancers break this brake
• CDK inhibitors enforce arrest: p21 (p53-induced, G$_1$ damage arrest), p16 (blocks CDK4/6); G$_2$/M arrest works by inhibiting Cdc25
• APC/C at anaphase: degrades securin (freeing separase to cut cohesin) and cyclin B (ending mitosis)
• Spindle checkpoint: one unattached kinetochore inhibits APC/C — the anti-aneuploidy gate
• Licensing (helicase loading only at low CDK) explains once-per-cycle replication and the fusion-experiment results; cdc mutants map genes by uniform arrest stage

• Low-yield extras: ATM/ATR are the damage-sensing kinases and Chk1/Chk2 their effectors; p16 = INK4a; the spindle-checkpoint wait signal comes from Mad/Bub proteins; ORC, Cdc6 and Cdt1 load the MCM helicase at licensing; the discoverers (Rao and Johnson, Masui, Nurse, Hunt, Hartwell)

The Cell Cycle for the MCAT

Part 3 of 4 — p53, Apoptosis & Cancer

p53 — Guardian of the Genome

p53 is a transcription factor kept scarce in healthy cells: the E3 ubiquitin ligase MDM2 continuously tags it for proteasomal destruction. DNA damage signaling (checkpoint kinases) phosphorylates p53, blocking MDM2 binding — p53 stabilizes and accumulates (regulation by degradation, not synthesis).

• The damage-sensing kinases are ATM and ATR.

Accumulated p53 transcribes a triage program:

• PUMA is the other classic pro-apoptotic p53 target.

• TP53 is mutated in over half of human cancers — the single most commonly mutated cancer gene
• Li-Fraumeni syndrome: inherited one bad TP53 allele → early cancers of many tissues (the second allele is lost somatically)

Apoptosis — Ordered Self-Destruction

Apoptosis is ATP-dependent, non-inflammatory, and executed by caspases (cysteine proteases cleaving after aspartate, made as inactive zymogens):

Intrinsic (mitochondrial) pathway — the cell-cycle-relevant one:

1. Stress/damage shifts the balance of Bcl-2 family proteins: pro-apoptotic BAX/BAK overcome anti-apoptotic Bcl-2
2. BAX/BAK permeabilize the outer mitochondrial membrane → cytochrome c escapes to the cytosol
3. Cytochrome c assembles the apoptosome, activating initiator caspase-9
4. Caspase-9 activates executioner caspases-3/7 → controlled demolition

• Bcl-xL is a second anti-apoptotic family member, and the adaptor that cytochrome c binds to build the apoptosome is Apaf-1.

Extrinsic (death receptor) pathway: Fas ligand or TNF binds surface death receptors → initiator caspase-8 → executioners. Used by cytotoxic T cells and immune homeostasis.

Morphology (vs. necrosis): cell shrinkage, chromatin condensation, DNA laddering (internucleosomal cleavage), membrane blebbing, phosphatidylserine flipped to the outer leaflet ("eat me"), packaging into apoptotic bodies eaten by macrophages — no lysis, no inflammation. Necrosis = swelling, rupture, contents spill, inflammation.

The Two-Gene-Class Logic of Cancer

Knudson's two-hit insight: hereditary retinoblastoma (one RB hit inherited, one somatic) is early and bilateral; sporadic (two somatic hits in one cell) is late and unilateral. Statistics of hits, not different genes.

p53 & Apoptosis 🎯

Hallmarks of the Transformed Cell (Culture Signatures)

Soft-agar colony formation is the standard in-vitro transformation assay — expect it in passages testing whether a manipulated gene is oncogenic.

Senescence — The Third Fate

Besides cycling and apoptosis, damaged or telomere-exhausted cells can enter senescence: permanent G$_1$ exit enforced by p16/Rb and p53/p21, cells remain metabolically alive. Senescence is a tumor-suppressive barrier: an activated oncogene can itself trigger senescence (oncogene-induced senescence), halting a lesion before it becomes a cancer.

• Benign moles are the classic example: they carry activating BRAF mutations, yet stop growing because of oncogene-induced senescence.

Metastasis in One Paragraph

Invasion requires loss of E-cadherin (epithelial cell-cell adhesion) — part of the epithelial-to-mesenchymal transition (EMT) — then basement-membrane degradation (matrix metalloproteinases), intravasation, survival in circulation, extravasation, and colonization. Tumors also secrete VEGF to recruit blood vessels (angiogenesis); without new vessels a tumor stalls at a small size, set by the diffusion limit for oxygen and nutrients.

• That avascular limit is roughly 1-2 mm.

Therapy Logic Preview

• Traditional chemo (spindle poisons, antimetabolites, alkylators) targets proliferation itself → toxic to gut/marrow/hair
• Targeted agents exploit specific lesions: imatinib (BCR-ABL), trastuzumab (HER2), CDK4/6 inhibitors (only useful when Rb is intact, a beautiful pathway-logic question)

• Palbociclib is the prototype CDK4/6 inhibitor.

• Synthetic lethality: PARP inhibitors kill BRCA-mutant cells because losing BOTH single-strand repair (drug) and homologous recombination (mutation) is lethal, while normal cells retain one route

Cancer Logic 🎯

Key Takeaways — Part 3

• p53 is regulated by degradation: MDM2 (an E3 ubiquitin ligase) destroys it continuously; damage signaling stabilizes it → p21 (arrest), repair genes, or BAX (apoptosis); mutated in >50% of cancers; Li-Fraumeni = inherited first hit
• Intrinsic apoptosis: Bcl-2 family balance → BAX/BAK pores → cytochrome c → apoptosome → caspase-9 → caspases-3/7; extrinsic: death receptors → caspase-8
• Apoptosis is tidy (shrinkage, laddering, PS exposure, no inflammation); necrosis is messy (swelling, lysis, inflammation)
• Oncogenes = stuck accelerator, one allele, gain-of-function (RAS, MYC, HER2, Bcl-2); tumor suppressors = cut brakes, two hits, loss-of-function (RB, TP53, PTEN, BRCA)
• Two-hit model: inherited first hit → early, multiple, bilateral tumors; sporadic → late, single
• Transformation signatures: no contact inhibition, anchorage independence (soft agar), immortality via telomerase, reduced growth-factor need
• Therapy logic: targeted drugs need their pathway node intact (CDK4/6 inhibitors need Rb); synthetic lethality (PARP + BRCA) kills only doubly-deficient cells

• Low-yield extras: ATM/ATR are the damage kinases; PUMA is a second pro-apoptotic p53 target; Bcl-xL is another anti-apoptotic Bcl-2 member; Apaf-1 is the apoptosome adaptor; BRAF-mutant moles illustrate oncogene-induced senescence; the avascular tumor limit is ~1-2 mm; palbociclib is the prototype CDK4/6 inhibitor

The Cell Cycle for the MCAT

Part 4 of 4 — Experimental Methods & MCAT Integration

The Cycle Experimentalist's Toolkit

• Mitotic cells can also be stained specifically with an antibody to phospho-histone H3.

Synchronization — Making a Population March in Step

Asynchronous cultures blur phase-specific events; synchronization sharpens them:

• Double thymidine block: excess thymidine stalls S phase; release, block again → all cells parked at the G$_1$/S border

• Excess thymidine works by feedback-inhibiting ribonucleotide reductase, unbalancing the dNTP pools.

• Spindle-poison block: checkpoint arrest in M; gently shake off the rounded mitotic cells (mitotic shake-off)
• Serum starvation: withdraw growth factors → cells retreat to G$_0$; re-adding serum starts a synchronized G$_1$ cohort — also THE assay for restriction-point timing

Restriction-point experiment: starve cells, re-feed with serum, then remove serum at increasing times. Cells given serum for less than ~R hours return to G$_0$; cells past R complete the entire cycle without serum. Commitment is all-or-none.

Drug-Arrest Signatures (Combine With Flow Cytometry)

• Named lab and clinic examples: aphidicolin (DNA polymerase inhibitor), etoposide (topo II poison), nocodazole (the lab spindle poison used for mitotic shake-off), palbociclib (CDK4/6 inhibitor).

Cycle Arithmetic

For an asynchronous, steadily cycling population, the fraction of cells in a phase is proportional to that phase's duration:

$\frac{N_{phase}}{N_{total}} = \frac{t_{phase}}{t_{cycle}}$

Example: 5% mitotic cells with a 20-hour cycle → M lasts about $0.05 \times 20 = 1$ hour. Growth of the whole population: $N = N_0 \cdot 2^{t/t_{cycle}}$ — but only if every cell cycles (a tumor's growth fraction is usually well below 1, which is why doubling times exceed cycle times).

Cycle Experiments 🎯

Integrated Passage Reasoning — Worked Patterns

Pattern 1: Order two drugs. Cells treated with drug A arrest at 2C; drug B arrests at 4C with condensed chromosomes. Given sequentially, A-then-release-then-B synchronizes cells in M; B-then-release-then-A synchronizes at G$_1$/S. Arrest points tell you where in the cycle each drug's target acts, and release order becomes a scheduling tool.

Pattern 2: Checkpoint status as a diagnostic. Irradiate two cell lines; measure mitotic index over time.

• Line 1: mitotic index falls (cells arrest in G$_2$) → checkpoints intact
• Line 2: mitotic index unchanged, cells enter mitosis with broken chromosomes → checkpoint-deficient (loss of the damage-sensing kinases or p53)
• A drug that inhibits the damage-sensing kinases converts line 1 into line 2 — "checkpoint override"

• The classic override drug is caffeine, which inhibits ATM/ATR at high doses in vitro.

Pattern 3: Growth fraction vs. cycle speed. A tumor whose cells cycle every 2 days but with a growth fraction of 10% grows slower than a tissue cycling every 5 days with 100% participation. Chemotherapy that targets S or M phase spares the non-cycling 90% — the rationale for repeated dosing cycles, which catch cells as they re-enter.

Pattern 4: Cancer + virus. DNA tumor viruses disable the same two brakes cancers mutate: HPV E6 degrades p53, E7 binds and inactivates Rb. A passage describing a viral protein that binds a host protein and causes unscheduled S-phase entry is describing an Rb-binder; one that blocks damage-induced arrest and apoptosis is describing a p53 antagonist.

Mitosis vs. Meiosis — One-Table Contrast (Bookkeeping Guard)

The cell-cycle machinery (CDKs, APC/C, cohesin/separase) is shared; meiosis I protects centromeric cohesin so only arm cohesin is cleaved — chromatids stay paired until meiosis II.

• The protector of centromeric cohesin in meiosis I is shugoshin.

MCAT Integration 🎯

Key Takeaways — Part 4

• Phase census tools: flow cytometry (DNA content), EdU/BrdU pulse (S), mitotic index (M); pulse-chase lag to labeled mitoses = G$_2$ length
• Synchronization: double thymidine (G$_1$/S), spindle-poison shake-off (M), serum starvation (G$_0$) — the last doubles as the restriction-point assay (all-or-none serum commitment)
• Drug arrest profiles: hydroxyurea/DNA polymerase inhibitors ~2C (S entry), topo II poisons 4C (G$_2$ damage), spindle poisons 4C condensed (M), CDK4/6 inhibitors 2C only with intact Rb
• Cycle arithmetic: fraction in phase = phase time / cycle time; population doubling requires the growth fraction, not just cycle speed
• Checkpoint diagnostics: damage should drop the mitotic index; entering M with broken DNA = checkpoint loss (or a drug overriding the checkpoint)
• HPV E6 = p53 degradation, E7 = Rb inactivation — a virus that installs both classic cancer lesions
• Meiosis reuses the machinery: MI separates homologs (reductional, centromeric cohesin protected); MII separates chromatids like mitosis

• Low-yield extras: phospho-histone H3 staining marks mitotic cells; thymidine blocks act through ribonucleotide reductase; named drugs aphidicolin, etoposide, nocodazole and palbociclib; caffeine as the ATM/ATR checkpoint-override drug; shugoshin protects centromeric cohesin in meiosis I