Cell+Cycle


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

toc

=**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