Cell Cycle + Apoptosis (Lectures 7 + 8)

Question 1

Eukaryotic Cell Cycle Steps

Answer 1

G1 (Growth and metabolism )

S (DNA Replication)

G2 (Preparation for cell division)

M (Mitosis)

G0 (Non-dividing)

Question 2

Stages of Mitosis

Answer 2

• Prophase (condensing chromosomes)
• Prometaphase (spindle attaches to chromosome)
• Metaphase
• Anaphase
• Telophase

Question 3

Anaphase Promoting Complex (APC)

Answer 3

facilitates the ubiquitination of cyclins, such as A and B, and their degradation

Question 4

Cyclin D (D1, D2, D3)

Answer 4

• GO/G1 transition and G1 progression
• bound by CDK4,6

Question 5

Cyclin E (E1, E2)

Answer 5

• G1/S transition (check point)
• bound by CDK2

Question 6

Cyclin A

Answer 6

• S phase progression and transition to G2 (check point)
• bound by CDK2

Question 7

Cyclin B

Answer 7

• G2/M transition (check point) and early M phase
• bound by CDC2

Question 8

CDKs

Answer 8

• cyclin dependent kinase
• activated by binding a cyclin

Question 9

CKIs

Answer 9

• CDK inhibitors
• also referred to as tumor suppressors
• p27 or p21 binds and inactivates the cyclin E-CDK2 complex
• p16 binds and inactivates the cyclin D-CDK complex

Question 10

CDK2

Answer 10

• binds Cyclin E (G1/S transition) + A (S/G2 transition + checkpoint)
• can inhibited by p21 or p27

Question 11

CDK4/6

Answer 11

-binds Cyclin D (G0/G1 transition and G1 progression) -can be inhibited by p16

Question 12

CDC2

Answer 12

• binds Cyclin B (G2/M transition + check point, early M phase)
• this kinase complex activates proteins on the chromosomes that are responsible for chromosome segregation (disregulation of cyclin B is implicated in aneuploidy)

Question 13

Cyclin B

Answer 13

In addition to binding CDC2 (a CDK), cyclin B binds and activates CDK1 to phosphorylate proteins that mediate the changes in nuclear and cytoskeletal architecture required for chromosome segregation

Question 14

Retinoblastoma protein (Rb)

Answer 14

• tumor suppressor -binds to transcription factor E2F
• When Rb is phosphorylated and cannot bind with E2F, binds to S phase gene promoters and activates them (Cyclin E, CDK2, etc)

Question 15

p53

Answer 15

• first identified as a tumor supressor gene
• transcription factor upregulated with exposure to DNA-damaging agents, which induces apoptosis by:
  
  • increasing transcription of Bax and Noxa
    
    • (pro-apoptotic)
  • activating cytoplasmic Bax to bind to mito memb.
• also upregulates the expression of the p21 gene, which leads to cell cycle arrest

Question 16

Necrosis

Answer 16

Lysis of cell membrane leads to release of cell’s contents into extracellular space (ie, in trauma, bacterial or viral infection)

Question 17

Apoptosis

Answer 17

Initiation of intracellular biochemical pathways not associated with cell lysis. It is intiated by signals from damaged DNA, lack of necessary growth factors, etc, leading to activation of caspases (proteases). Caspases hydrolyze specifc peptide bonds in proteins and contribute to cell death by inactivation of important proteins.

Since caspase activity is tightly regulated, apoptosis is also referred to as: programmed cell death

Question 18

Caspases

Answer 18

All are cysteine proteases produced as inactive pro-caspases

Activation of autocatalytic pro-caspases 8, 9, 10 leads to proteolytic activation of downstream effectors (capases 3, 6, 7), which then cleave cellular proteins

Question 19

Characteristics of an apoptotic cell

Answer 19

• nuclear DNA starts fragmenting –> due to activation of endonucleases (caspase-activated DNase)
• cell shape changes--> due to degradation/changes in cytoskeletal proteins (actin, plectin, etc)
• plasma membrane changes –> causes the membrane to become blebbed and to shed membraneous particles that contain intracellular material

Question 20

Two main pathways to apoptosis

Answer 20

1. Death receptor pathway
2. Mitochondrial pathway of apoptosis

Question 21

Death receptor pathway

Answer 21

1. Triggered by members of death receptor superfamily (including CD95).
   * death ligands–> proteins that activate death receptor and downstream signaling (ie, TNF-a, FAS ligand)
2. Ligand binding to receptor promotes binding of FADD (intracellular adaptor death domain protein) to receptor
3. Once bound, it recruits procaspase 8 (degrades)–> caspase 8
4. Caspase 8 released–> initiates caspase cascade to create active caspases

Question 22

Activation Cascade of Caspase

Answer 22

TNF alpha –>

Fas death receptor –>

FADD –>

Procaspase -8,-9,-10 —>

(autocatalytic upon binding to FADD anchored at plasma membrane)

Caspase -8,-9,-10 – (proteolytic activation)–>

Caspase -3, -6, -7 – (cleavage) –>

Cellular proteins

Question 23

Mitochondrial Pathway of Apoptosis

(Cytochrome c Caspase Pathway)

Answer 23

1. Cytochrome c is translocated to cytosol (pores are generated when facilitators Bid, Bad, Nox dimerize/polymerize with pro-apoptotic proteins Bax + Bak)
2. Cytochrome c binds apoptotic protease-activating factor (Apaf1) and pro-caspase-9 –> forms apoptosome
   * (this leads to autocatalytic activation to caspase-9)*
3. Caspase-9 is released from the complex–> activates caspases 3, 6, 7
4. This leads to cleavage of the apoptotic substrate

Question 24

How does BCL-2 prevent apoptosis ?

Answer 24

(In the Cytochrome c apoptotic pathway)

• Anti-apoptotic Bcl-2 and Bcl-X bind to Bak and Bax, preventing self-association to form pores
• Ratio between anti/rpo apoptotic proteins determines cell death vs. survival

Question 25

Mitochondrial pathway of apoptosis

(Smac / DIABLO pathway)

Answer 25

XIAP = x-linked inhibitor of apoptosis

( caspase inhibitor protein)

The protein Smac/DIABLO inhibits XIAP, allowing activation of the caspase cascade

Question 26

MAPK

(mitogen-activated protein kinases)

Answer 26

• activated by growth factor signals, UV light, radiation, etc
• Raf ( a MAPK) phosphorylates Bad, leading to dissociation from mito memb, inhibiting apoptosis