Cell Cycle Regulation

• Why do cells divide? (GRR)

  • Grow in size
  • Replace old and damaged cells
  • Reproduction
• Categories of cells 

  • Cells that no longer divide after they differentiate 
  • Cells that normally don't divide but can be induced to divide with proper stimulus
  • Cells with relatively high level of mitotic ability
• Checkpoint control system: signals indicate if key cellular processes have been completed correctly
• Checkpoints -> where cell cycle is controlled by STOP and GO  chemical signals at critical points
• • G1/S (restriction point): can DNA synthesis begin?
  • G2/M: has DNA synthesis been completed correctly?
  • Spindle checkpoint: are all chromosomes attached to spindle? can sister chromatids separate correctly?
• Cyclin

  • regulatory subunit
  • Constant cycle of synthesis and degradation during cell division
  • Activates cyclin dependent kinase (Cdks) and thereby help control progression from one stage of the cell cycle to the next
• Cdks(cyclin-dependent protein kinase)

  • Dependent on cyclin
  • catalytic subunit
  • Enzyme that adds negatively charged phosphate groups to other molecules (phosphorylation), which indicates that the cell is ready to pass into the next stage of the cell cycle
• G1: Cdk4/6 + Cyclin D

  • Commit the cell to DNA replication
• R point (late G1): Cdk2 + Cyclin E

  • Help promote passage thru restriction point in late G1
• S-phase promoting factor: Cdk2 + Cyclin A

  • Initiate DNA replication
• M-phase promoting factor: Cdk1 (cdc2) + Cyclin B

  • Promote the events of mitosis
• cyclin oscillates during the cycle
• Protein phosphatases that modify Cdks:

  1. Wee1 kinase: Phosphorylates inhibitory sites in Cdks; primarily involved in controlling entry into mitosis
  2. Cdc25 phosphatase: removes inhibitory phosphates from Cdks
  3. Protein phosphatase 2 (PP2A): Dephosphorylates of Cdc25 and Wee1 during anaphase and telophase
• Phosphatases decrease when cdk activity increases
• Cdk inhibitory proteins (CKIs)

  1. p27
  2. p21
  3. p53
• p27

  • Suppresses G1/S-Cdk and S-Cdk activities in G1
  • Helps cells to withdraw from cell cycle when they terminally differentiate
• p21

  • Suppresses G1/S-Cdk and S-Cdk activities following DNA damage in G1;
  • transcriptionally activated by p53
• p53

  • Promotes transcription of genes that induce cell cycle arrest or apoptosis in response to DNA damage or other cell stress
  • Regulated by Mdm2
• Ubiquitination

  • Is the addition of ubiquitin molecules residues of a protein directing the proteins for degradation via the proteasome or by lysosomes
  • Involves ubiquitin ligases
• APC/C - Anaphase promoting complex or cyclosome
• cdc20

  • APC/C-activating subunit in all cells
  • Triggers initial activation of APC/C at metaphase-to- anaphase transition, stimulated by m-cDk activity
• SECURIN

  • Binds into and inhibits the activity of separase
• SEPARASE

  • Cleave subunit of cohesin separating the sister chromatids
• cdh1

  • APC/C activating subunit that maintains APC/C activity after anaphase and throughout G1
  • Inhibited by Cdk activity
• SCF (Skp, Cullin, F-box containing complex): Catalyzes ubiquitylation of regulatory proteins involved in G1 control, including CKIs
• Three processes that determine cell size (GDS)

  1. Cell Growth
  2. Cell Division
  3. Cell Survival
• SIGNAL MOLECULES THAT REGULATE CELL GROWTH, DIVISION AND SURVIVAL

  1. Mitogen
  2. Growth Factors
  3. Survival Factors
• MITOGEN

  • Stimulates cell division
  • Triggers G1/S-Cdk activity that relieves negative controls of cell cycle
• GROWTH FACTORS

  • Stimulates cell growth (an increase in cell mass) by promoting the synthesis of proteins and other macromolecules
• SURVIVAL FACTORS

  • Promote cell survival by suppressing programmed cell death
• Mitogens that stimulate cell division

  1. Platelet Derived Growth Factor (PDGF) 
  2. Epidermal Growth Factor
  3. Erythropoietin
  4. Transforming Growth Factor-β
• PLATELET-DERIVED GROWTH FACTOR (PDGF): Stimulate cells to divide, such as fibroblasts, smooth muscle cells and neuroglial cells
• EPIDERMAL GROWTH FACTOR (EGF): Acts on epithelial and nonepithelial cells
• ERYTHROPOIETIN: Induces the proliferation of red blood cells
• TRANSFORMING GROWTH FACTOR-ẞ (TGF- ẞ): Inhibits the proliferation of several cell by blocking cell-cycle progression
• E2F

  • Promotes transcription of genes required for G1/S progression
  • Binds with Cdk and cyclin A producing the S promoting factor
• Rb (Retinoblastoma protein)

  • Tumor suppressor protein that serve as a guardian of the restriction-point gate
  • Prevents excessive cell growth by inhibiting cell cycle progression until a cell is ready to divide
• pRB-E2F pathway 

  • pRB serves as a break in cell cycle by binding to E2F
  • Inactivation (hyperphosphorylation) of pRb helps switch the progress of the cell cycle to mitosis
• Phosphorylation inactivates pRB since it cannot bind to E2F
• MTT assay: quantitative colorimetric method to determine the cell proliferation