Cell cycle and apoptosis- Singh lecture exam 5

Result of too LITTLE cell death

leads to cancer and autoimmunity

Result of too MUCH cell death

leads to neurodegeneration, immunodeficiency, and some blood disorders.

How many cells die due to programed cell death daily?

over 50 billion cells

How is programed cell death important to development?

sculpts tissues (EX: removing the webbing between fingers.)

In adults, it balances proliferation to keep tissue size constant.

How is programed cell death important to the immune system?

- removes damaged, mutated, or virus-infected cells

- protects against cancer and spreading infection.

- deletes self-reactive lymphocytes

- allows killer lymphocytes to destroy targets via Fas ligand.

How do killer lymphocytes destroy target cells

Fas ligand

What are the main mechanisms of cell death?

- necrosis (accidental injury from heat, pressure, or poor blood supply)

- autophagy (self-digestion during starvation)

- terminal differentiation (e.g., keratinocytes, red cells)

- apoptosis (programmed death).

What drives apoptosis

caspases

What is a caspase?

cysteine proteases made as inactive zymogens

What are initiator caspases (the numbers)

Caspases 2, 8, 9, 10

What are effector caspases (the numbers)

Caspases 3, 6, 7

How are initiator caspases activated

Dimerization on adaptor platforms

What action is performed by activated caspases?

Cleavage of effector caspases (3,6,7) in an amplifying cascade

In the EXTRINSIC pathway, what molecules bind to the trimerized death receptor?

FasL, TNF-α, or TRAIL binds a trimerized death receptor.

morphological characteristics of apoptotic cell death

- shrinkage

- Intact, blebbed membrane

- nucleus fragments and chromatin condenses

- break into membrane bound apoptotic bodies

- intact lysosomes, leaky mitochondria

- effects single cells that are phagocytosed without inflammation

morphological characteristics of necrotic cell death

- cell swelling and mitochondrial swelling

- ruptured membrane

- randomized nucleic breakdown

- lysed completely

- leaky lysosomes

- effects group of cells and promotes inflammation

What is the energetic demands of apoptosis and necrosis different?

- Apoptosis is ATP- DEPENDENT

- Necrosis is ATP- INDEPENDENT

How does apoptosis appear on gel electrophoresis? Why?

- appears as a ladder

- DNA is cut into oligonucleosomal fragments

How does necrosis appear on gel electrophoresis? why?

- Looks like a smear

- DNA is degraded randomly (array of size variations)

How can you confirm apoptosis?

- Release of cytochrome c (cleaved caspases )

- PARP on Western blot

-TUNEL staining.

What is different between apoptosis and necrosis genetic regulation?

Apoptosis is genetically regulated (Bcl-2 family, p53, IAPs)

- Necrosis is unregulated, accidental death.

How can you test a cell membrane to confirm method of cell death?

- Apoptotic cells flip phosphatidylserine to outer leaflet, can be detected by Annexin V

- necrotic membranes maintain phosphatidylserine asymmetry but become permeable to propidium iodide.

How long is the typical human cell cycle?

24 hours

How long is the G1 phase?

8-10 hours

What occurs during G1 phase?

growth and synthesis of enzymes needed for DNA replication and essential organelles

How long is S phase?

6-8 hours

What occurs during S phase?

DNA replication and histone synthesis

How long is G2 phase?

4-6 hours

What occurs during G2 phase?

further growth and preparation for division

How long is M phase?

1 hour

What occurs during M phase?

mitosis and cytokinesis

What happens after cytokinesis?

Cell may re-enter G1 phase to keep dividing or may go into a resting/functioning state (G0)

What is the purpose of the G1 checkpoint?

- checks for size, nutrients, growth factors, and DNA damage before proceeding

What is the purpose of the G2 checkpoint?

- checks for damaged or incompletely replicated DNA before proceeding

What is the purpose of the M checkpoint?

- holds anaphase until all kinetochores are attached

What happens if there is DNA damage detected in the G1 phase?

ATM/ATR → Chk1/2 → p53 → p21, which blocks cyclin E-Cdk2.

What happens if there is DNA damage detected in the G2 checkpoint?

Chk1/2 inactivate Cdc25C, keeping cyclin B-Cdk1 off until repair or apoptosis.

What happens if the criteria for M phase are not yet met?

Mad2 inhibits Cdc20/APC/C, so securing persists and separase cannot cleave cohesion.

Predominate CDKs during early G1 phase?

D-CDK 4/6

Predominate CDKs during late G1 and S phase?

E-CDK 2

Predominate CDKs during Late S early G2?

A- CDK 2/1

Predominate CDKs during M phase?

B-CDK 1

How are cyclins degraded?

ubiquitin ligases (SCF, APC/C)

Effect of KIP (p21, p27, p57) on CDKs?

block Cdk2/Cdk1.

Effect of INK4 (p15, p16, p18, p19) on CDKs?

block Cdk4/6.

Effect of CAK on CDKs?

Activates CDKs

Effect of Wee1/Myt1 on CDKs?

inhibit them

How are CDKs reactivated?

Cdc25

How can you turn on S-phase genes?

G1 Cdks phosphorylate Rb, releasing E2F

Examples of positive tumor regulators

cyclin D1 or Cdk4 amplification, My, mutant RAS

Examples of negative tumor regulators

Rb, p53, p16, p27