Radiation Oncology/Radiobiology/Cell Cycle

Cell Cycle

Overview

• Decision to proliferate and enter cell cycle highly regulated process
• Binding of extrinsic growth factors typically necessary
• mTOR integrates input from growth factor receptors, cellular nutrient and energy levels, and redox status to stimulate new protein synthesis in preparation for cell cycle entry via S6K to regulate translational machinery and eIF4E to regulate cyclin D
• Regulation of cell cycle through complex of cyclins with cyclin-dependent kinases (cdk)
  • cdk levels constant throughout cell cycle
  • cdk activity regulated by concentration of cyclins, cdk phosphorylation, and cdk inhibition
  • Cyclin concentrations are cyclical
• Progression is unidirectional from G1 -> S -> G2 -> M
• Cell cycle checkpoints
  • Prevent or delay progression through cell cycle when critical events have not been completed
  • Provide time for DNA repair
  • 4 main checkpoints (please see DNA damage response for information about effect of RT on the checkpoints
    • G1 (stop)
    • S (slow)
    • Early G2 (stop)
    • Late G2/M (stop)

G1 Phase

• Preparation for synthesis: Is cell big enough, is environment favorable, is DNA undamaged?
• G1 checkpoint is integrated at p53. Please see DNA damage response for more information
  • If there is a G1 arrest, p53 increases levels of p21, which inhibits cyclinD-cdk4/6 complex
• If there is no arrest (low p53 levels), the cyclinD-CDK4/6 complex becomes active, and phosphorylates Rb protein
• Rb protein is normally associated with E2F, but upon phosphorylation dissociates from it
• E2F is a DNA transcription factor, which triggers entry into S phase
• Cyclin D and E2F levels are promoted by myc and by eIF4E

S Phase

• CDK2 kinase plays a major role in maintaining the pace of S-phase; it is a transcription target of E2F
• S phase checkpoint serves to insure that there is no replication through DNA damage that could result in double strand DNA break
  • S phase checkpoint is triggered by DNA damage -> ATM -> Chk2 -> CDC25A -> CDK2 phosphorylation
  • If there is an S phase arrest, CDC45 fails to load onto DNA, and DNA replication halts
• If there is no arrest, CDK2 remains dephosphorylated and CDC45 begins DNA replication
• CyclinD/CDK2 also inhibits p27^kip1, which promotes expression of Cyclin A and progression through S-phase
• CyclinA/CDK2 plays a key role in duplication of centromeres

G2 Phase

• CyclinB/CDK1 kinase is called the Maturation-Promoting Factor (MPF), and promotes entry into mitosis by phosphorylating multiple proteins. The complex also includes Cyclin A and CDC 20
• G2/M checkpoint is triggered by DNA damage -> ATR -> Chk1 -> CDC25C -> CDK1 phosphorylation
• Prior to mitosis, cyclin B is primarily located in the cytoplasm. When the cell is ready, Cyclin B is phosphorylated, which results in its accumulation in the nucleus
• At the same time, CDK1 must be dephosphorylated to trigger mitosis
• Targets of MPF complex include:
  • condensins, which enable chromatin condensation
  • microtubule-associated proteins involved in mitotic spindle formation
  • lamins, contributing to degradation of the nuclear envelope
  • histones H1 and H3
  • Golgi matrix, which leads to fragmentation
  • Dephosphorylation of Rb protein, prior to start G1 of the next cycle
• There are two distinct G2/M checkpoints (PMID 11809797)
  • A very rapid, dose-independent, ATM-dependent arrest of cells that were in G2 at the time of irradiation (and consequent failure to enter mitosis), which can be assayed using flow cytometry against phosphorylated histone H3 (a marker of mitosis)
  • A slower, dose-dependent, ATM-independent accumulation in G2 of cells which were in earlier stages of the cell cycle at the time of irradiation, which can be assayed using standard propidium iodide flow cytometry
  • Please see the cell cycle checkpoints for further information

Viral cell cycle promoters

• These block Rb, which leads to dissociation of E2F and cell entry into S phase
  • SV40 T Antigen
  • Adenovirus E1A
  • HPV E7

Cell cycle inhibitors

• Block cyclins to prevent entry into next cell cycle phase. They act primarily in G1 to stop progression into S
• Kip inhibitors:
  • p21^WAF1/CIP1 blocks cyclin D
  • p27^KIP1 blocks cyclin D & E
  • p57^KIP2 blocks cyclin E & A, and to lesser degree mitotic cyclin B
• INK inhibitors:
  • p16^Ink4a, p15^Ink4b, p18^Ink4c, p19^Ink4d
• Structurally related, they destabilize association of cyclin D with CDK4/6
• p21 and p16 increase in senescence and contribute to G1 arrest

Myc

• DNA transcription factor
• Multiple actions to promote entry of cells into S phase
  • Cyclin D transcription -> Rb phosphorylation -> E2F activity
  • SCF gene transcription -> KIP family (p21, p27, p57) inhibitor degradation
  • E2F transcription

Ras

• Activation of ras triggers two major pathways related to entry into cell cycle:
  • Ras -> Raf -> MEK -> MAPK -> transcription of cyclin D and block of p27 inhibitor
  • Ras -> PI3K -> PKB -> stabilization of cyclin D and block of p21 inhibitor