# Lecture 20: The Cell Cycle and Its Regulation

## Cell Biology

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## Learning Objectives

By the end of this lecture, students will be able to:

1. Describe the phases of the eukaryotic cell cycle and the key events in each phase
2. Explain the roles of cyclins and cyclin-dependent kinases (CDKs) in cell cycle progression
3. Describe the three major cell cycle checkpoints and their molecular mechanisms
4. Explain the role of the retinoblastoma protein (Rb) and E2F in G1/S transition
5. Discuss how external signals (growth factors, contact inhibition) regulate the cell cycle

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## Lecture Content

### I. Overview of the Cell Cycle

The cell cycle is an ordered series of events that leads to cell growth and division. It consists of **four phases**. **G1 (Gap 1)** is a period of cell growth, organelle duplication, and preparation for DNA synthesis. It is the most variable phase in duration and the stage where most regulatory decisions occur. Cells can exit the cycle from G1 and enter **G0**, a quiescent state from which they may be either reversibly quiescent (re-entering the cycle upon stimulation) or terminally differentiated. **S (Synthesis) phase** is when DNA replication occurs, with each chromosome replicated exactly once to produce sister chromatids held together by cohesin. The centrosome also duplicates during S phase. In a typical mammalian cell, S phase lasts approximately 6 to 8 hours. **G2 (Gap 2)** is a period of continued growth and DNA damage repair before mitosis, lasting approximately 3 to 5 hours. **M (Mitosis) phase**, lasting approximately 1 hour, includes both nuclear division (mitosis) and cytoplasmic division (cytokinesis). The total cell cycle time for a typical mammalian cell in culture is approximately 24 hours, though this varies enormously -- embryonic cells can divide in less than 30 minutes, while many adult cells rarely divide at all. The term **interphase** refers collectively to G1, S, and G2 (everything except M phase).

### II. Cyclins and Cyclin-Dependent Kinases (CDKs)

The cell cycle is driven by the sequential activation of **CDKs**, serine/threonine kinases that are catalytically inactive without their cyclin partners. CDK protein levels remain relatively constant, but **cyclins**, their regulatory subunits, oscillate in level through the cell cycle, being synthesized and degraded at specific times. Degradation occurs via the ubiquitin-proteasome system and is critical for ensuring the cycle moves forward irreversibly.

The major cyclin-CDK complexes in mammalian cells activate sequentially. **Cyclin D-CDK4/6** is active in early to mid G1 and is dependent on mitogenic signaling. **Cyclin E-CDK2** is active in late G1 and drives the G1/S transition. **Cyclin A-CDK2** is active during S phase and drives DNA replication. **Cyclin A-CDK1** is active in late S and G2. **Cyclin B-CDK1** (historically known as MPF, maturation-promoting factor) is active at the G2/M boundary and triggers entry into mitosis.

Full **CDK activation** requires three events: cyclin binding (which induces a conformational change exposing the active site), activating phosphorylation by CAK (CDK-activating kinase) on the T-loop (Thr-160 in CDK2), and removal of inhibitory phosphorylations on Thr-14 and Tyr-15 that are added by **Wee1** kinase and removed by **Cdc25** phosphatase. The interplay between Wee1 and Cdc25 is a key regulatory step for CDK1 activation at the G2/M transition.

**CDK inhibitors (CKIs)** provide negative regulation. The **INK4 family** (p16INK4a, p15, p18, p19) specifically inhibits CDK4/6 by blocking cyclin D binding. The **Cip/Kip family** (p21Cip1, p27Kip1, p57) has broader specificity, inhibiting cyclin E-CDK2 and cyclin A-CDK2 complexes. p21 is a critical target of p53 in the DNA damage response, while p27 levels are high in quiescent cells and decrease upon mitogenic stimulation.

<image>Cyclin oscillations and CDK activity during the cell cycle. Panel A: Graph showing cyclin protein levels across the cell cycle — Cyclin D rises in G1 (and remains elevated as long as mitogens are present), Cyclin E peaks at the G1/S boundary, Cyclin A rises during S phase and peaks in G2, Cyclin B peaks at G2/M and drops sharply during mitosis (anaphase). Below the graph, bars indicating the periods of activity for CDK4/6, CDK2, and CDK1. Panel B: CDK activation mechanism — inactive CDK monomer; cyclin binding induces partial activation; CAK phosphorylation of the T-loop (full activation); Wee1 adds inhibitory phosphorylation (inactive); Cdc25 removes inhibitory phosphorylation (reactivated). Panel C: CKI inhibition — p16INK4a preventing cyclin D from binding CDK4; p21/p27 binding and inhibiting the cyclin E-CDK2 complex.</image>

### III. The Restriction Point and G1/S Transition

The **restriction point (R)** is the critical decision point in late G1. Before the restriction point, cell cycle progression requires continuous mitogenic signaling. After passing R, the cell is committed to entering S phase regardless of whether external signals persist. The molecular basis of this commitment lies in the Rb-E2F pathway.

**Hypophosphorylated Rb** binds and inhibits **E2F** transcription factors, which control genes essential for S phase entry (cyclin E, cyclin A, DNA polymerase, thymidine kinase, DHFR). Mitogenic signaling through the Ras-MAPK pathway induces cyclin D expression. **Cyclin D-CDK4/6** partially phosphorylates Rb (mono-phosphorylation), and **cyclin E-CDK2** then further hyperphosphorylates Rb, fully inactivating it. Hyperphosphorylated Rb releases E2F, which activates transcription of S-phase genes. A crucial **positive feedback loop** locks in this decision: E2F drives cyclin E expression, which produces more cyclin E-CDK2, which hyperphosphorylates more Rb, which releases more E2F. This creates a switch-like, irreversible commitment to S phase.

**DNA replication licensing** ensures that each origin fires exactly once per cell cycle. Pre-replication complexes (pre-RC) are assembled during G1: ORC proteins mark origins, Cdc6 and Cdt1 load the MCM helicase. During S phase, S-CDK and DDK (Dbf4-dependent kinase) fire origins while simultaneously preventing re-licensing, enforcing the "once and only once" rule of DNA replication.

### IV. Cell Cycle Checkpoints

**Checkpoints** are surveillance mechanisms that ensure each phase of the cell cycle is properly completed before the next phase begins. The **G1/S DNA damage checkpoint** uses ATM/ATR kinases to detect DNA damage. These kinases activate Chk1/Chk2, which stabilize **p53**. p53 activates transcription of **p21** (a CKI that inhibits cyclin E-CDK2), causing G1 arrest. p53 also activates DNA repair genes, and if damage is irreparable, it induces apoptosis through Bax, PUMA, and NOXA. Under normal conditions, p53 levels are kept low by the E3 ubiquitin ligase **MDM2**, which targets p53 for proteasomal degradation. DNA damage activates ATM, which phosphorylates both p53 and MDM2, disrupting their interaction and stabilizing p53. p53 in turn induces MDM2 expression, creating a negative feedback loop.

The **intra-S checkpoint** slows replication fork progression and inhibits late origin firing in response to DNA damage, primarily through the ATR-Chk1 pathway responding to stalled forks and single-stranded DNA. The **G2/M checkpoint** prevents entry into mitosis with damaged DNA. ATM/ATR activate Chk1/Chk2, which phosphorylate and inactivate Cdc25. Without Cdc25 activity, CDK1 remains phosphorylated by Wee1 and cannot drive mitotic entry. The **spindle assembly checkpoint (SAC)** operates during mitosis to ensure all chromosomes are properly attached to the spindle before anaphase (detailed in Lecture 21).

<image>Cell cycle checkpoints and the p53 pathway. Panel A: Overview of the cell cycle with three checkpoint positions marked — G1/S checkpoint (DNA damage before replication), intra-S checkpoint (during replication), G2/M checkpoint (DNA damage before mitosis), and spindle assembly checkpoint (during M phase). Panel B: The p53-MDM2 pathway — in normal conditions, MDM2 ubiquitinates p53 for proteasomal degradation (low p53 levels); DNA damage activates ATM kinase, which phosphorylates both p53 and MDM2, disrupting their interaction; p53 accumulates and activates target genes: p21 (cell cycle arrest), DNA repair genes, and pro-apoptotic genes (Bax, PUMA) if damage is irreparable. Panel C: G2/M checkpoint — DNA damage activates ATM/ATR -> Chk1/Chk2 -> inhibitory phosphorylation of Cdc25 (exported from nucleus by 14-3-3 proteins) -> CDK1 remains phosphorylated and inactive -> no mitotic entry.</image>

### V. External Regulation of the Cell Cycle

**Growth factors and mitogens** stimulate entry into the cell cycle from G0 or G1. When EGF, PDGF, or FGF binds RTKs, the resulting Ras-MAPK and PI3K-Akt signaling induces cyclin D transcription, stabilizes cyclin D protein, and reduces p27 levels. Mitogens must be present continuously from early G1 to the restriction point to drive commitment to division.

**Growth inhibitory signals** oppose mitogenic stimulation. **TGF-beta** induces expression of p15INK4b and p21, inhibiting CDK4/6 and CDK2 to enforce G1 arrest. This growth-inhibitory response is frequently lost in cancer.

**Contact inhibition** causes cells to stop dividing when they reach confluency. This phenomenon is mediated by cell-cell adhesion molecules (cadherins) and the **Hippo signaling pathway**. Merlin (NF2) activates the Hippo kinase cascade, in which LATS1/2 kinases phosphorylate the transcriptional coactivators YAP/TAZ, excluding them from the nucleus and silencing pro-proliferative gene expression.

**Cell size control** ensures that cells reach a critical size before passing the restriction point. The mTOR pathway integrates nutrient and growth factor signals to promote cell growth (ribosome biogenesis and protein synthesis). Growth (increase in cell mass) and division (progression through the cell cycle) are coordinated but fundamentally distinct processes.

### VI. Senescence and Quiescence

**Cellular senescence** is an irreversible cell cycle arrest triggered by telomere shortening (replicative senescence), oncogene activation (oncogene-induced senescence), DNA damage, or oxidative stress. Senescent cells are characterized by flat, enlarged morphology, senescence-associated beta-galactosidase activity, and the SASP (senescence-associated secretory phenotype), which involves secretion of inflammatory cytokines (IL-6, IL-8) and matrix metalloproteinases. Both the p53/p21 and p16INK4a/Rb pathways enforce senescence. Senescence is tumor suppressive because it prevents the proliferation of damaged cells, but it also contributes to aging: the accumulation of senescent cells in tissues causes chronic inflammation through the SASP and progressive tissue dysfunction. **Senolytics**, drugs that selectively kill senescent cells, are an active area of aging research.

**Quiescence (G0)** is a reversible cell cycle exit maintained by high p27 levels, low E2F activity, and low mTOR signaling. Quiescent cells can re-enter the cycle upon mitogenic stimulation.

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