30 August 2006
Cell Cycle
Dr. Ivana de la Serna, Ph.D.



Definition


  • Cell reproduces by performing an orderly sequence of events in which it duplicates its contents and then divides in two
  • In multicellular species, cell division is required to:
    • Produce a functioning organism
    • Replace cells that die
    • Millions of cells are manufactured each second simply to survive

Eukaryotic Cell Division


  • Contain multiple chromosomes and organelles
  • Mitochondria divide by splitting
  • Golgi and ER undergo fragmentation

Stages of the Cell Cycle


  • Interphase
    • G1 phase
    • S phase (synthesis)
    • G2 phase
  • Mitosis
  • Timing and order of phases is crucial for proper function
  • Some cells can withdraw from the cell cycle and go to G0 phase
    • Some cells can be reactivated from G0, others cannot

S Phase

  • Replication of nuclear DNA
  • Cells contain a diploid (2n) amount of DNA before S phase
  • Cells contain double (4n) amount of DNA in preparation for cell division
  • Duplicated chromosomes consist of two identical chromatids, called sister chromatids

G2 Phase

  • Metabolism of RNA, regulatory proteins and enzymes necessary for mitosis to take place
  • DNA is analyzed for possible errors, and errors are corrected before mitosis
  • During this period, cell has 2 complete diploid sets of chromosomes

Mitosis

  • Interphase
    • DNA is not condensed with intact nuclear envelop
  • Early Prophase
    • Chromosomes become more condensed
  • Late Prophase
    • Spindle apparatus is forming
  • Prometaphase
    • Spindle apparatus is visible
  • Metaphase
    • Chromosomes line up
  • Anaphase
    • Chromosomes split
  • Late Telophase
  • Telekinesis

G1 Phase

  • Cell volume is restored to normal (2n)
  • Metabolism of RNA, regulatory proteins, and enzymes necessary for DNA
  • Cells can withdraw from the cell cycle into G0 (outside) during G1 and stop dividing for long periods of time or indefinitely

Cell Cycle Research


  • Biochemical analysis of animal eggs and embryos
    • Giant fertilized xenopus eggs contain large amounts of proteins needed for cell division and undergo division rapidly
    • Inject test substances into eggs to determine their effect on cell cycle progression
    • Prepare cell extracts and reconstitute many events of the cell cycle in vitro
  • Identification of yeast cell division cycle mutants
    • Genetically analyzing cell cycle because of ease of obtaining mutants
  • Yeast cell cycle mutants are conditional mutants that produce a mutant product under one set of conditions but not another
    • Typically temperature dependent
  • At permissive temperature, cells divide normally
    • At restrictive temperatures, cells continue cell cycle until they are unable to complete
  • Length of yeast is indicative of what kind of cell cycle mutation they have
  • Mammalian cell fusion experiments

Mitosis Promoting Factor

  • Abbreviated MPF
  • Cytoplasm of cell arrested in metaphase was injected into a G2 arrested egg, causing the G2 arrested egg to enter mitosis
    • Demonstrates presence of a factor promoting initiation of mitosis
      • MPF required for chromatin condensation, nuclear envelop breakdown, fragmentation of ER and Golgi apparatus, reorganization of microtubules to form mitotic spindle
  • MPF is a complex consisting of cyclin B and cyclin-dependent kinase (M-Cdk)

Cyclin B

  • Cyclically increases and decreases during post-fertilization period
  • Cyclin B rises and falls just before cell cleavage

Cdc25 and Wee 1

  • Cdc25 deficit results with elongated cells
    • Increased G2 phase
  • Wee1 deficits result in small cells
    • Decreased G2 phase

S-CDKs

  • S + G1 cell results in entry to S phase
    • Resulting cells will have half the amount of DNA (n)
  • S + G2 cell stays in G2
    • G2 cell already has twice the normal amount of DNA (4n)
  • G1 + G2 cell stays in G2 cell
  • Some kind of diffusible cell cycle progression factors in S phase promotes the cell cycle progression in G1 phase, but is blocked in G2 phase

Regulation of Cell Cycle


  • Cell cycle has different stages which different processes sequence
    • Processes are timed and have a specific unidirectional sequence
    • Different cycles possible
    • Sensors to detect completion of each stage
  • Checkpoints are points in cell cycle at which cell can be arrested if previous events have not been completed
    • e.g. Progression through G1 and G2 is delayed if DNA is damaged to allow time for repair
    • Checkpoints can allow control system to be regulated by extracellular signals
    • Operate by negative signals – negative signals allows progression through cell cycle even if previous phase is not complete

Cyclin Dependent Kinases

  • Abbreviated Cdks
  • Activity rises and falls through cycle
  • Lead to cyclic changes in phosphorylation of intracellular proteins that initiate or regulate events in cell cycle
  • Cdks dependent on cyclins for activity
  • Amount of Cdks remain the same throughout cell cycle but activity is cylical
  • Activity can be suppressed by inhibitory phosphorylation and by inhibitory proteins
    • Phosphorylation of inhibitory sites by Wee 1 inhibit Cdk activity
    • Dephosphorylation by Cdc25 increases Cdk activity
  • Cdk inhibitor proteins (CkIs) bind to cyclin-Cdk complex and change active site conformation

Cyclins

  • Bind to Cdks to cyclically regulate their activity
  • Cyclins undergo a cycle of synthesis and degradation with each cycle
  • Activity is terminated by degradation
  • Classes of cyclins:
    • G1/S-cyclins
    • S-Cyclins
    • M-Cyclins
    • G1-Cyclins

Regulation by Proteolysis

  • Cyclines are destroyed by dependent mechanism that marks protein for destruction in proteasomes
  • Ubiquitin ligases catalyze ubitquitin-transfer reactions
    • Covalent attachment of small protein ubiquintin to lysine residues
      • Targets protein for degradation by proteasome

Regulation by Transcription

  • Cyclin levels are controlled by transcription and cyclin synthesis level

S-Phase Regulation


  • S-phase cyclin-Cdk Complexes (S-Cdks) initiate DNA replication once per cycle
  • Origin Recognition Complex (ORC) binds to replication origins and serves as landing pads for regulatory proteins
  • Cdc6 is present at low levels during cell cycle and increases transiently during early G1
    • Binds to ORC and Mcm proteins to form pre-replicative complex
  • Activation of S-Cdk (Cdk2 and S-cyclin) in late G1 initiatives DNA replication and causes Cdc6 to dissociate
    • Cdc6 dissociation prevents ORC from starting replication again, blocking re-replication of DNA

G2-Phase Regulation


  • Damaged DNA sends out signals to proteins that phosphorylated and inactivate Cdc25
  • Blocks dephosphorylation and activation of M-Cdk, blocking mitosis
    • When DNA is repaired, signals turn off and mitosis resumes
  • Lack of growth factors also results in inhibition of cyclin-cdk activity thrugh association with CKIs

M-Cdk Regulation

  • Activate after S-phase and accumulates
  • Phosphorylation by Cdk-activating kinase (CAK) is important for later activation
  • In G2, M-Cdk is phosphorylated and inactivated by Wee 1
  • M-Cdk is dephosphorylated and activated by Cdc25 right before mitosis
    • Active M-Cdk phosphorylates and activates Cdc25 and inactivates Wee 1 in a positive feedback loop when ready for mitosis
      • Allows a all-or-none start of mitosis

Spindle Attachment Regulation


  • Ensures all chromosomes are properly attached
  • Sensor mechanism detects state of kinetochore
    • Unattached kinetochore sends out negative signal blocking Cdc-20 APC activation and chromatid separation
  • Non-disjunction can occur if this checkpoint fails
  • Chromosome defects are one of the causes of cancer

Chromosome Separation Regulation


  • M-Cdk also induces assembly of mitotic spindle and ensures replicated chromosomes attach to spindle
  • APC triggers sister chromatid separation
  • APC is activated by Cdc20
    • Proteolysis of securing by APC activates protease called separase
      • Cleaves the cohesion complex which falls away from chromosomes, allowing separation

Mitosis Termination Regulation


  • M-Cdk must be inactivated for mitosis to end
  • Occurs by ubiquintin-mediated proteolysis of M-cyclins by Cdc20-APC and by dephosphorylation of Cdk1
  • Proteolysis of cyclin B ensures the unidirectionality of M --> G1 transition

G1 Phase Regulation

  • Restriction point: Prevents entering S phase if conditions are unfavorable
    • Growth factors required 2-3 hours prior to initiation of S phase
  • Damaged DNA can delay S phase
  • M-Cdk destruction by Cdc20-APC leads to inactivation of Cdc20-APC
    • Allows M-cyclin accumation
  • Accumulation of cyclins inhibited by:
    • Hct-APC activity
    • Decrease transcription
    • Increased CKI synthesis

Rb Protein

  • Rb binds to E2F transcription factors and blocks transcription of S phase genes
    • S phase genes normally regulated by E2F transcription factors
  • Stimulated by growth factors resulting in accumulation of active G1-Cdk (cdk4/6 and cyclin D)
  • Phosphorylation of Rb by G1-Cdk leads to inactivation and release of E2F and activation of S phase gene expression

E2F Transcription Factors

  • Increases transcription of its own gene
  • Dependent on transcription of cyclin A and E leading to increased G1/S-Cdk and S-Cdk activites
    • This turns on Rb phosphorylation, promoting E2F release
  • Increase G1/S-Cdk and S-Cdk activates enhances phosphorylation of Hct and CKIs, leading to their inactivation or destruction

CKI

  • Cdk inhibitors
  • Regulate G1/S transition
  • Inactivate cdk-cyclin complexes and prevent phosphorylation of Rb
  • Two families
    • INK4 family – cdk4, 6, p16, p15, p18, 19
    • KIP family – inhibits all G1 and S phase cdk complexes
      • Includes p21, p27, and p57

DNA Damage Regulation


  • DNA damage activates p53 protein
  • p53 transcriptionally regulates several genes including p21, which binds G1/S-Cdk and S-Cdk to block entry into S phase
  • In undamaged cells, p53 is kept at low levels by interactions with Mdm2 protein that acts as a ubiquitin ligase to target p53 for destruction by proteosomes
  • DNA damage activates protein kinases that phosphorylate p53, reducing interaction with Mdm2.
    • p53 accumulates to high levels, activating p21 which inhibtits G1/S-Cdk and S-Cdk
    • Can arrest cells in G1 or apoptosis in response to DNA damage

Regulatory Proteins to Know

  • Protein kinases and phosphatases
  • CKIs
  • Gene Regulatory Proteins
    • E2F
    • p53

Rb and p53 Tumorigenesis

  • Rb and p53 – associated with tumor suppressing activities
    • p53 protein is lost or mutated in over 50% of all human cancers
      • Cause Li-Fraumeni syndrome, rare from of inherited cancer; affected individuals display cancers in variety of sites
    • Rb gene is located on human chromosome 13
      • Mutation on Rb may be inherited, and a second copy may be lost by somatic mutation (two-hit hypothesis), resulting in predisposition to cancer development
      • Acts in tumor suppression by blocking cell cycle progression, promoting apoptosis, and DNA repair upon damage to prevent proliferation of abnormal cells
  • Cyclin D – mutants associated with cancer
    • Cyclin D1 – esophageal, breast, and gastic cancers
    • Cyclin D2 – colorectal cancer
  • CDK4 – sarcomas and gliomas
  • CDKI – such as p16 mutations, associated with head and neck, pancreatic, and non-small cell lung carinomas

Chemotherapy

  • Alkaloids
    • Block M phase, prevent chromosome spindle formation
    • Derived from plants
    • Treat Wilm’s tumor, lung, breast and testicular cancer
    • Vincistine and Vinblastine
  • Antitumor antibiotics
    • Block S phase, bind DNA
    • Intravenous administration
    • Treat testicular cancer, leukemia
    • Doxorubicin and Mitomycin-C
  • Antimetabolites
    • Block S phase, block cell growth
    • Mimicking nucleotides during DNA synthesis
    • Administered orally or intravenously
    • Treat gastric, breast, and ovarian cancers
    • 6-mercaptopurine and 5-fluorouracil
  • Cdk Inhibitors
    • Block progression of cell cycle by inhibiting Cdks
    • Ongoing clinical trials testing flavopiridol, roscovitine, and other small molecules