Regulation of cell cycle and apoptosis — Comprehensive study notes

Regulation of the cell cycle

Purpose: The cell cycle is tightly controlled to ensure cells divide only when appropriate. If regulation is lost, it can lead to disease or cell death.
Key concept: Cell division is regulated by specific transitions between phases and by signals that respond to growth needs and DNA health.

Four phases of the eukaryotic cell cycle

Interphase = $G1 + S + G2$
- G1: The cell grows and prepares for DNA synthesis. The decision to divide often happens here.
- S: DNA replication occurs, duplicating the chromosomes.
- G2: The cell prepares for mitosis, checking that DNA replication is complete and error-free.
- M phase: Mitosis (nuclear division) and cytokinesis (cytoplasm division) result in two daughter cells.
Interphase is the growth and preparation period, while M phase is the actual division.

G0 and differentiation

G0: A temporary pause in the cell cycle (quiescence).
Terminal differentiation: A permanent exit from the cell cycle (e.g., mature nerve cells).
This regulation ensures division only when needed and allows time for DNA repair or triggers cell death (apoptosis) if damage is too severe.

Checkpoints and major regulatory transitions

Three main checkpoints ensure proper progression:
- G1/S checkpoint (start): Commits the cell to DNA replication.
- G2/M checkpoint: Checks if DNA replication is complete and the genome is healthy before entering mitosis.
- M phase checkpoint (spindle/mitotic checkpoint): Ensures all chromosomes are correctly attached to the spindle before they separate.
These checkpoints are controlled by cyclin-dependent kinases (Cdks), which, when active, phosphorylate targets to drive cell cycle progression.

Cdks and cyclins control of cell cycle transitions

Cdks are enzymes (kinases) that promote cell cycle progression by adding phosphate groups to other molecules.
They are activated when bound to proteins called cyclins, and their activity depends on signals and environmental conditions.
Cells only divide when activated by growth signals and when DNA is undamaged.

Activation steps for Cdks

To become active, Cdks need two things:
1. Binding to a cyclin to form a Cdk/cyclin complex.
2. Removal of inhibitory phosphate groups (dephosphorylation).
An active Cdk can then phosphorylate target proteins to advance the cell cycle. Cdks are only active when bound to cyclins.

How Cdks activate downstream molecules

Activated Cdks phosphorylate specific target proteins, acting like an "intracellular switch" that turns on events needed for cell cycle progression.
For example, G1 checkpoint Cdks are activated by signals (mitogens) that promote growth.

Examples and consequences of constitutive activation

If signaling molecules are constitutively (always-on) active, it can lead to uncontrolled cell division, as seen in cancer.
Example: Constitutively active Ras can continually signal downstream (e.g., through MAPKKK $o$ MAPKK $o$ MAPK cascade). Drugs can target these downstream parts to block the signal.

Cyclin availability and APC/C-mediated regulation

Cyclin levels fluctuate: they rise to activate Cdks then fall as they are degraded to allow the cell to move to the next phase.
APC/C (anaphase-promoting complex/cyclosome) is a protein complex that attaches ubiquitin to cyclins, marking them for destruction.
Ubiquitination targets cyclins for degradation by the proteasome, an enzyme complex that breaks down ubiquitinated proteins. This process inactivates Cdks and allows the cell cycle to progress or exit a phase.

Checkpoint regulation: summary of the molecular control

Active Cdks are needed to pass the G1 and G2 checkpoints.
APC/C activity is required to pass the M phase checkpoint and exit mitosis.
Overall flow: G1/S-Cdk promotes activation of S-Cdk; S-Cdk activates M-Cdk; M-Cdk drives mitosis and activates APC/C; APC/C degrades cyclins and other proteins (like securins), allowing exit from mitosis.
Inhibitors (CKIs) like p27 and p21 can pause the cell cycle by binding to and inactivating cyclin-Cdk complexes.

Inhibitors and DNA damage response

CKIs (e.g., p27, p21) stop cell cycle progression by forming inactive complexes with cyclin-Cdk.
When DNA damage occurs, it triggers signals that activate CKIs like p21, pausing the cell cycle to allow for DNA repair. If the damage is too severe, it can lead to apoptosis.

Mechanisms to pause the cell cycle at checkpoints

G1: Pause by inhibiting Cdks.
G2: Pause by inhibiting Cdks.
M phase: Pause by inhibiting APC/C, which prevents premature chromosome separation.

Therapeutic and practical implications

Mistakes in cell cycle regulation contribute to cancer and resistance to treatments.
Drugs can target components like Cdks, cyclins, APC/C, proteasomes, or CKIs to control cell division or induce cell death in cancer cells.

Regulation of apoptosis

Apoptosis is a programmed process that removes unwanted or damaged cells without causing inflammation to surrounding tissues, crucial for development and tissue health.
It is different from necrosis, which is uncontrolled cell death that often causes inflammation.

Apoptosis versus necrosis

Apoptosis: Controlled cell death, involving cellular remodeling and efficient removal by immune cells (phagocytosis) without inflammation.
Necrosis: Uncontrolled cell death, usually causing inflammation and tissue damage.
A marker for apoptosis is the externalization of phosphatidylserine on the cell surface, which acts as an "eat-me" signal for macrophages.

Three pathways of cell death (overview)

Intrinsic (mitochondrial) pathway
Extrinsic (death receptor) pathway
Autophagy-related processes (can lead to cell death or survival) and necrosis under certain conditions.

BH3-only and BH123 proteins; Bcl-2 family balance

The Bcl-2 family of proteins controls whether mitochondria release factors that trigger apoptosis.
- BH123 proteins (e.g., Bax, Bak) promote apoptosis by making the mitochondrial outer membrane permeable (MOMP), leading to the release of cytochrome c.
- Bcl-2 (anti-apoptotic) proteins prevent apoptosis by binding to and inhibiting BH3-only and/or BH123 proteins.
The balance between pro-apoptotic (BH3-only, BH123) and anti-apoptotic (Bcl-2) proteins determines if a cell undergoes apoptosis. An increase in BH3-only proteins usually promotes apoptosis.

Intrinsic pathway: mitochondrial signals to caspases

Trigger: Cellular stress causes MOMP.
Key steps:
1. Release of cytochrome c from mitochondria into the cytosol.
2. Cytochrome c, along with APAF1, forms the apoptosome.
3. The apoptosome activates initiator procaspase-9.
4. Activated caspase-9 then activates executioner caspases.
5. Executioner caspases break down cellular proteins, leading to cell death.
Cytochrome c release is a critical step in intrinsic apoptosis.

Extrinsic pathway: death receptors and DISC

Trigger: External signals, such as Fas ligand binding to its Fas receptor on the cell surface.
This binding leads to the formation of the DISC (death-inducing signaling complex), which involves FADD and death domains.
The DISC activates initiator caspases (e.g., caspase-8 or caspase-10).
These then activate executioner caspases, leading to apoptosis.

Caspases: activation and roles

Caspases are enzymes that are essential for apoptosis. They exist as inactive procaspases and are activated by cleavage (removal of a prodomain).
Initiator caspases (caspase-9 in intrinsic, caspase-8/10 in extrinsic) activate executioner caspases (caspase-3, -6, -7).
Executioner caspases dismantle the cell by degrading key cellular proteins.
Caspase activity can be regulated by IAPs (inhibitors of apoptosis proteins) and anti-IAPs.

Inhibitors of apoptosis and anti-IAPs

IAPs block caspase activity, preventing apoptosis.
Anti-IAPs promote caspase activation and apoptosis.
The balance between these two groups influences the cell's readiness to undergo apoptosis.

Survival signals and cellular checkpoints against apoptosis

Survival pathways (e.g., PI3K/Akt pathway) promote cell survival by inhibiting apoptotic pathways and supporting anti-apoptotic proteins like Bcl-2.
These signals help cells avoid unnecessary apoptosis but can contribute to cancer if they become overactive.

Phosphatidylserine externalization and phagocytosis

During apoptosis, phosphatidylserine moves from the inner to the outer surface of the cell membrane.
This acts as an "eat-me" signal, attracting macrophages to clear the dying cell, preventing inflammation.

Therapeutic implications and disease relevance

Understanding apoptosis allows for targeting it in diseases like cancer (promoting apoptosis) or neurodegenerative diseases (inhibiting excessive apoptosis).
For cancer therapy, strategies include restoring apoptotic responses or blocking survival pathways (e.g., PI3K/Akt, Bcl-2 family) to improve treatment.

Connections to the broader curriculum

Cell cycle regulation and apoptosis are linked: DNA damage can halt the cell cycle and, if irreparable, trigger apoptosis.
Both processes are influenced by growth factor signals (mitogens) and stress signals.
Therapeutic approaches often target both pathways (e.g., Cdks, cyclins, APC/C for cell cycle; caspases, Bcl-2 family, IAPs for apoptosis).

Practical takeaways for exams and clinical relevance

Key checkpoints: G1/S, G2/M, and M-phase, controlled by Cdks, cyclins, APC/C, and CKIs.
DNA damage: Responds by using CKIs (e.g., p21) to pause the cycle and attempt repair.
APC/C: Essential for degrading cyclins, allowing cells to exit mitosis.
Apoptosis: Can be intrinsic (mitochondrial) or extrinsic (death receptor-mediated). Caspases execute the process, regulated by Bcl-2 family proteins and IAPs.
Phosphatidylserine externalization: A key marker for apoptotic cell clearance.
Therapeutic relevance: Targeting these pathways can modulate cell fate in diseases involving dysregulated cell cycle or apoptosis.

Learning objectives

Describe the four phases of the eukaryotic cell division and the consequences of inhibiting each.
Explain how Cdks and cyclins control the transition between these phases.
Discuss how growth factors/mitogens and DNA damaging agents influence the cell cycle.
Describe the intrinsic and extrinsic pathways of apoptosis, and explain how they are regulated.
Utilize knowledge of cell cycle and apoptosis regulation to predict the impact of modulating cell cycle and apoptosis pathway components and regulators on cell physiology.
Utilize knowledge of cell cycle and apoptosis regulation to identify appropriate therapeutic targets and interventions for diseases that result from loss of regulation of cell cycle and apoptosis.