Cell cycle inhibitors

• Two families of genes, the cip/kip family and the INK4a/ARF (Inhibitor of Kinase 4/Alternative Reading Frame) prevent the progression of the cell cycle. 
• Because these genes are instrumental in prevention of tumor formation, they are known as tumor suppressors.
• The cip/kip family includes the genes p21, p27 and p57. 
• They halt cell cycle in G₁ phase, by binding to, and inactivating, cyclin-CDK complexes. p21 is activated by p53 (which in turn is triggered by DNA damage e.g. due to radiation). p27 is activated by Transforming Growth Factor β (TGF β), a growth inhibitor. 
• The INK4a/ARF family includes p16INK4a, which binds to CDK4 and arrests the cell cycle in G₁ phase, and p14arf which prevents p53 degradation. And the amount of chromosomes are able to double at the same rate as in phase 2

Checkpoints

• Cell cycle checkpoints are used by the cell to monitor and regulate the progress of the cell cycle. 
• Checkpoints prevent cell cycle progression at specific points, allowing verification of necessary phase processes and repair of DNA damage. 
• The cell cannot proceed to the next phase until checkpoint requirements have been met.
• Several checkpoints are designed to ensure that damaged or incomplete DNA is not passed on to daughter cells. 

1. G1 Checkpoint

• The G1 checkpoint is the first checkpoint in the cell cycle of a mammalian cell and the start point in the yeast cell that determines whether the cell enters the cell cycle or not.
• The checkpoint is present between the G1 phase and S phase and is responsible for the entry of the cell in the division phase.
• Depending on the external and internal factors and stimuli, the decision of whether the cell enters the cell cycle or undergoes the G0 phase is determined.
• The checkpoints are essential in the cell cycle as they limit the chances of genomic instability arising due to DNA damage during the cycle.
• The G1 checkpoint is regulated by p53 which aids in the downregulation of tumors and cell lines.
• In order to cause G1 checkpoint arrest, the p53 regulates the transcription of CDK inhibitor p21.
• The arrest is stimulated by factors like a break in the DNA double-strand, which prevents the proliferation of irreparably damaged cells.
• The G1 checkpoint arrest is a positive feedback mechanism where the presence of breaks in the DNA strand enhances the expression of the p53 gene.
• Because of the proteins involved in the checkpoint, the G1 checkpoint is an important checkpoint during tumor suppression and prevention of excessive cell proliferation.
• Cells with reparable DNA damage are held at the checkpoint to provide time for repair while others are either signaled for apoptosis or moved to the G0 phase.

Type of Cells: 

a. Permanent Go Cells: Those cells that enter Go state and remain in ground state. Example;- neurons, skeletal muscle, RBC 

b. Stable Go Cells: Those Cells that enter Go state and leave ground state given the right stimuli. Example;- Hepatocytes and Lymphocytes 

c. Labile Go Cells: Those cells that never enter GO state. Example:- Skin, gut epithelium, bone marrow. 

• In non-diving cells, or cells at G0 the transcription activator E2F is bounded with repressor protein Retinoblastoma (pRB) also known as “master brake”. 
• The removal of pRb from E2F is the cue for coming out of Go state. 
• The removal of inhibition (Rb) is achieved by reducing the binding affinity through phosphorylation of pRb by cyclin-CDK. 
• The free E2F now becomes activates Genes that are required in S-phase. 

Depending on the involvement of transcription factor p53 DNA damage has two DNA damage Checkpoint mechanism

2. G2 Checkpoint

• The G2 checkpoint is the second checkpoint in the cell cycle where is present at the transition between G2 and S phase.
• The checkpoint prevents the entry of cells into the S phase of the cycle by preventing the activation of regulators like cyclins and CDKs.
• This checkpoint, like the G1 checkpoint, looks for DNA damage and breaks to prevent the proliferation of mutated or damaged cells.
• As the checkpoint helps maintain genomic stability, studies on the checkpoint help to understand the molecular mechanism of cancer.
• The target of the G2 checkpoint arrest is the CDK2 that usually drives the transition from G2 to the S phase.
• In the checkpoint, DNA damage triggers the activation of the ATM pathway, which causes phosphorylation of ATM and inactivation of checkpoint kinases.
• The checkpoint also involves the p53 genes which inactivate enzymes by the expression of p21 proteins.
• Additional pathways in the G2 checkpoint ensure the stability of the arrest by the expression of proteins like Rb and downregulation of several genes that code for proteins required for the S phase. 

3. Metaphase Checkpoint (Spindle checkpoint)

• The metaphase checkpoint or M phase checkpoint or Spindle checkpoint is the checkpoint during mitosis which checks if all the sister chromatids are correctly attached to the spindle fibers.
• The checkpoint ensures that all the chromosomes of cells entering the anaphase are firmly attached to at least two spindle fibers from opposite poles of the cell.
• The separation of chromosomes in anaphase is an irreversible process, which is why this checkpoint is crucial in mitosis.
• The proteins in the checkpoint look for straggler chromosomes that can be detected in the cytoplasm.
• The checkpoint acts by negative regulation of CDC20 which prevents the activation of ubiquitin tag by the anaphase-promoting complex.
• There are different mechanisms to deactivate the checkpoint once all chromosomes are correctly attached.
• One of the important mechanisms is by transporting the motor complex proteins away from the kinetochores. The proteins are then redistributed to the spindle poles.

🔬 Cell Cycle Inhibitors:

1. Which of the following genes belongs to the cip/kip family?
   A) p16
   B) p21
   C) p14
   D) pRb
   Answer: B) p21
2. What is the role of p21 in the cell cycle?
   A) Activates cyclin-CDK complex
   B) Inhibits p53
   C) Binds and inactivates cyclin-CDK complexes
   D) Promotes chromosome duplication
   Answer: C) Binds and inactivates cyclin-CDK complexes
3. Which protein activates p21?
   A) p27
   B) TGF-β
   C) p53
   D) CDK4
   Answer: C) p53
4. What type of damage typically activates p53?
   A) Heat
   B) Radiation-induced DNA damage
   C) Protein misfolding
   D) Spindle misalignment
   Answer: B) Radiation-induced DNA damage
5. Which growth inhibitor activates p27?
   A) IL-2
   B) EGF
   C) TGF-β
   D) FGF
   Answer: C) TGF-β
6. Which gene prevents degradation of p53?
   A) p21
   B) p16
   C) p14ARF
   D) p27
   Answer: C) p14ARF
7. What is the function of p16INK4a?
   A) Activates p53
   B) Binds to CDK4 and arrests the cell cycle
   C) Stimulates DNA repair
   D) Triggers chromosome condensation
   Answer: B) Binds to CDK4 and arrests the cell cycle
8. Which gene family includes p16INK4a and p14ARF?
   A) Cip/Kip
   B) Cyclin-CDK
   C) INK4a/ARF
   D) ATM
   Answer: C) INK4a/ARF
9. In which phase does p21 halt the cell cycle?
   A) G2
   B) S
   C) G1
   D) M
   Answer: C) G1
10. Which of the following is NOT a cip/kip family gene?
    A) p21
    B) p27
    C) p57
    D) p16
    Answer: D) p16

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⏳ Checkpoints in Cell Cycle:

11. What is the main role of cell cycle checkpoints?
    A) Promote rapid cell division
    B) Regulate cytokinesis
    C) Monitor and regulate cell cycle progression
    D) Activate telomerase
    Answer: C) Monitor and regulate cell cycle progression
12. Where is the G1 checkpoint located?
    A) Between S and G2
    B) Between M and G1
    C) Between G1 and S
    D) After cytokinesis
    Answer: C) Between G1 and S
13. What is checked at the G1 checkpoint?
    A) Spindle formation
    B) DNA damage and external signals
    C) Chromosome alignment
    D) Chromatin condensation
    Answer: B) DNA damage and external signals
14. Which transcription factor is key in the G1 checkpoint?
    A) Rb
    B) E2F
    C) ATM
    D) p53
    Answer: D) p53
15. Which CDK inhibitor is upregulated by p53 at the G1 checkpoint?
    A) p16
    B) p21
    C) p27
    D) pRb
    Answer: B) p21
16. Which of these cells is an example of a ‘Permanent Go’ cell?
    A) Hepatocyte
    B) Neuron
    C) Skin epithelium
    D) Lymphocyte
    Answer: B) Neuron
17. Which type of cell never enters the G0 phase?
    A) Muscle cells
    B) Neurons
    C) Bone marrow cells
    D) Hepatocytes
    Answer: C) Bone marrow cells
18. What removes pRb from E2F to activate S phase genes?
    A) Ubiquitin
    B) CDK-cyclin phosphorylation
    C) ATM phosphorylation
    D) p53 binding
    Answer: B) CDK-cyclin phosphorylation
19. What is the role of pRb in non-dividing cells?
    A) Activates S-phase genes
    B) Represses E2F
    C) Repairs DNA
    D) Phosphorylates cyclin
    Answer: B) Represses E2F
20. Which phase follows the removal of pRb from E2F?
    A) G2
    B) S phase
    C) M phase
    D) Cytokinesis
    Answer: B) S phase

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🔬 G2 Checkpoint:

21. Where is the G2 checkpoint located?
    A) Between M and G1
    B) Between S and G2
    C) Between G2 and M
    D) Between S and G1
    Answer: C) Between G2 and M
22. What is the primary purpose of the G2 checkpoint?
    A) Check spindle attachment
    B) Ensure correct chromosome separation
    C) Check DNA damage before mitosis
    D) Prevent apoptosis
    Answer: C) Check DNA damage before mitosis
23. Which kinase pathway is triggered by DNA damage in G2?
    A) MAPK
    B) ATM
    C) PKA
    D) mTOR
    Answer: B) ATM
24. Which CDK is targeted for arrest at the G2 checkpoint?
    A) CDK1
    B) CDK2
    C) CDK4
    D) CDK6
    Answer: B) CDK2
25. What protein is responsible for inactivating enzymes during G2 arrest?
    A) p53
    B) pRb
    C) CDC20
    D) E2F
    Answer: A) p53
26. Which protein maintains stability of G2 arrest?
    A) APC
    B) p21
    C) p16
    D) Cdc25
    Answer: B) p21
27. What does the G2 checkpoint prevent?
    A) Cytokinesis
    B) Entry into S phase
    C) Entry into mitosis
    D) Entry into G1
    Answer: C) Entry into mitosis
28. Which protein is NOT involved in G2 checkpoint regulation?
    A) p21
    B) ATM
    C) CDK4
    D) p53
    Answer: C) CDK4
29. Downregulation of which gene class helps G2 arrest?
    A) Telomerase genes
    B) Spindle checkpoint genes
    C) S phase protein genes
    D) Ribosomal genes
    Answer: C) S phase protein genes
30. What phase is delayed by a functioning G2 checkpoint?
    A) G1
    B) G0
    C) M phase
    D) S phase
    Answer: C) M phase

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🔬 Spindle Checkpoint (Metaphase Checkpoint):

31. What is another name for the spindle checkpoint?
    A) G1 checkpoint
    B) S checkpoint
    C) M phase checkpoint
    D) G2 checkpoint
    Answer: C) M phase checkpoint
32. What is the function of the spindle checkpoint?
    A) Checks cell size
    B) Ensures proper attachment of chromatids to spindle
    C) Initiates cytokinesis
    D) Phosphorylates Rb
    Answer: B) Ensures proper attachment of chromatids to spindle
33. Why is the spindle checkpoint crucial?
    A) DNA replication is reversible
    B) Chromosome separation is irreversible
    C) Prevents mitotic slippage
    D) Activates telomerase
    Answer: B) Chromosome separation is irreversible
34. What prevents activation of the anaphase-promoting complex (APC)?
    A) Cyclin B
    B) p27
    C) Inactive CDC20
    D) ATM
    Answer: C) Inactive CDC20
35. Which protein is negatively regulated at the spindle checkpoint?
    A) p53
    B) CDC20
    C) p21
    D) CDK2
    Answer: B) CDC20
36. How is the spindle checkpoint deactivated?
    A) DNA damage repair
    B) E2F activation
    C) Redistribution of motor proteins
    D) p53 degradation
    Answer: C) Redistribution of motor proteins
37. What attaches sister chromatids to opposite poles?
    A) Cytoskeleton
    B) Microtubules
    C) Kinetochores
    D) Ribosomes
    Answer: B) Microtubules
38. What complex mediates chromosome separation in anaphase?
    A) CDK4
    B) p21
    C) APC
    D) TGF-β
    Answer: C) APC
39. What does APC tag for degradation?
    A) Cyclin D
    B) CDK4
    C) Securin
    D) ATM
    Answer: C) Securin
40. Straggler chromosomes are detected by which checkpoint?
    A) G1
    B) G2
    C) Spindle
    D) S-phase
    Answer: C) Spindle

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🧬 Miscellaneous:

41. What type of feedback is G1 checkpoint arrest?
    A) Negative
    B) Neutral
    C) Positive
    D) Feedforward
    Answer: C) Positive
42. Which protein acts as the “master brake” of the cell cycle?
    A) p53
    B) E2F
    C) pRb
    D) CDC20
    Answer: C) pRb
43. Which checkpoint is directly involved in tumor suppression?
    A) G0 checkpoint
    B) G1 checkpoint
    C) S checkpoint
    D) Cytokinesis checkpoint
    Answer: B) G1 checkpoint
44. What condition may lead a cell to enter G0?
    A) Spindle misalignment
    B) Proper mitosis
    C) Lack of growth stimuli
    D) Apoptosis
    Answer: C) Lack of growth stimuli
45. Which cells can re-enter the cycle from G0 upon stimulation?
    A) Neurons
    B) Skeletal muscle
    C) Lymphocytes
    D) RBCs
    Answer: C) Lymphocytes
46. What triggers re-entry of stable G0 cells?
    A) UV exposure
    B) Cytokines or stimuli
    C) Chromosome duplication
    D) Cyclin A
    Answer: B) Cytokines or stimuli
47. Which protein is phosphorylated to initiate S-phase entry?
    A) E2F
    B) pRb
    C) p21
    D) CDK2
    Answer: B) pRb
48. CDK2 plays a role in transition between which phases?
    A) G2 to M
    B) S to G2
    C) G2 to S
    D) G1 to S
    Answer: D) G1 to S
49. What molecule activates p27?
    A) TGF-β
    B) CDK1
    C) Cyclin D
    D) APC
    Answer: A) TGF-β
50. Which of the following is NOT checked by any checkpoint?
    A) Chromosome attachment
    B) DNA damage
    C) Organelle duplication
    D) Growth factors
    Answer: C) Organelle duplication