Cell cycle and 2556

Chalermchai Mitrpant Outline • Overview of the • Regulatory mechanisms controlling cell cycle • Progression of the cell cycle

• Checkpoint of the cell cycle M • Phases of the cell cycle C • G1/S transition G2

• (G1, S, G2 phases) initiation (M) (C) • G2/M transition S G1 • Anaphase initiation • DNA damage Checkpoint • Medications interfering cell cycle • dependent apoptosis Phases of the cell cycle Phases of the cell cycle and its DNA content

G1 (2n) Homologous chromosomes S (2n4n) (replicated) G2 (4n)

M (4n2n)

kinetochore

Cell cycle: G1-S-G2-M G1 Timing for each phases of the cell cycle

Restriction point for cell ready to M-phase: mitosis undergo Mitosis and cytokinesis

Interphase

G1: (Growth phase1)

G0: quiescent or senescent

S: DNA replication

G2: (Growth phase2) Overview of cell cycle control • Two irreversible points in cell cycle – Replication of genetic material – Separation of sister chromatids • Cell cycle regulation is controlled by Go&Stop signal – Progression & Checkpoints Regulatory mechanism controlling cell cycle -Cyclin dependent kinase (Cdk) system P Cyclin Positive Feedback

Cyclin-Cdk

Cdk Wee-1 CAK Inactive Inactive Inactive Active : Inhibitory kinase CAK: CDK-activating kinase CDC25: Phosphatase • Progression G1/S through /Cdk activity • Progression G2/M through /Cdk activity Cyclin-Cdk is controlled by ubiquitin proteasome system during G1/S and G2/M progression

M-phase M-cyclin APC E3 system

G1/S-phase G1/S- SCF E3 system Enzymatic cascade in Ubiquitin proteasome system Ub Ub Ub G1/S-Cyclin

SCF APC

M-Cyclin Ub Ub Ub Regulatory mechanism controlling cell cycle

S / M phase kinase : cdk + cyclin

Molecular cell biology Regulatory mechanism controlling cell cycle Regulation of cell cycle progression

Mitogenic stimuli

Cyclin D

Retinoblastoma P (RB)

E2F

G1/S-phase cyclin

Other transcriptional activity DNA Replication

Cyclin B The cell cycle is also controlled by inhibition

Spindle checkpoint G2 / M checkpoint • Chromosomes attached at • Replication completed metaphase plate • DNA integrity Inactive Active Active Inactive Cdk/cyclin B M APC cytokinesis (MPF) C

G2 mitosis

G1

S Cdk/G1/S cyclin MPF = Mitosis Inactive Promoting Factor Active APC = Anaphase G1 / S checkpoint • Growth factors Promoting Complex G1/S transition Extracellular signals induce cell to commit S-phase entry

Growth factor

• Platelet derived growth factor (PDGF) gives mitogenic signal through Receptor tyrosine kinase (RTK)

• made by platelets in blood clots

• binding of PDGF to cell receptors stimulates in connective tissue promoting wound healing

Extracellular signals induce cell to commit S-phase entry Growth factor signaling growth factor

nuclear pore

nuclear membrane P P cell division cell surface Cdk receptor protein kinase P cascade P chromosome P cytoplasm nucleus Role of Retinoblastoma sensitivity protein Rb Retinoblastoma sensitivity protein (Rb) in G1/S progression

CDK substrate Mitogenic

stimuli E2F transcription factor

Rb-E2F: inactive

Rb-P + E2F Retinoblastoma P G1/S protein expression protein (RB)

P E2F

G1/S-phase cyclin Cyclin A

Other transcriptional activity DNA Replication

Cyclin B Expressed Cyclin-Cdk during cell cycle progression

M-cyclin M-CDK

CDK-cyclin combinations CDK/cyclin in G1/S Wee1-like inhibitory kinase CAK: G1/S-CAK CDC25

SCF-dependent ubiquitylation of cyclin Cell cycle Checkpoint (G1/S)

Cell cycle arrest when DNA damage found p53: transcription factor Cell cycle Checkpoint protein p53 phosphorylation : CDK inhibitor

DNA repair & Apoptosis G1/S checkpoint by p53

Cell cycle Checkpoint (G1/S) How is p53 activity induced by DNA damage? DNA damage and cell cycle arrest

p53: transcription factor p53 transcribes p21. Cell cycle checkpoint p53 induces E3 MDM2 transcription.

MDM2 ubiquitylates p53. How is p53 activity induced by DNA damage? DNA damage and cell cycle arrest

DNA damage DNA kinase

p53 phosphorylation p21: CDK inhibitor Phosphoinositide Checkpoint kinase 2 DNA repair 3 kinase Apoptosis

p53 mutation in >50% of cancers

S phase initiation Regulation of S phase initiation (a) Control of S-phase by regulated proteolysis of P27: (i) P27 is a Cdk inhibitor (CKI) specific for the G1/S-phase Cdk-Cyclin complex Mitogenic stimuli (ii) This proteolysis is initiated by Cyclin D the Cyclin D which phosphorylates P27 making it a target for ubiquitin RB

Conjugating enzyme E2F

G1/S-phase cyclin Cyclin A p P27 Other Tx Activity DNA Replication

Cyclin B Regulation of S phase initiation (b) Regulation of pre replication complexes: Cdks simultaneously 1. Activate initiation of replication ATP ADP 2. Prevent re-initiation at origin sites by phosphorylation &

disassociation of McM’s from the P ORCs (origin recognition complex) which are permanently associated with replication origins. G1/S Cyclin Cdk

Minichromosome maintenance (McM) (DNA helicase) G2/M transition Regulatory mechanism controlling cell cycle Cyclin-Cyclin dependent kinase (Cdk) system

P Cyclin

Cyclin-Cdk Cdc25

Cdk Wee-1 CAK inactive inactive inactive active

G2 Mitosis Initiation of Anaphase (Spindle assembly checkpoint) Model for Induction of Anaphase by Regulation of Complexes

• “Anaphase inhibitor” () is involved in the stabilization of a multicomplex of called • Cohesins tie together sister chromatids at the centromeric attachments and other sites along the chromatid pairs and maintain this association despite the pulling of the kinetochore attached spindle fibers Regulation of Sister Chromatid Pairing by the Cohesin Complexes

• Sister chromatid pairing is mediated by the cohesin complex in which Smc1/Smc3 together with Scc1/Scc3 form a bridge between the two chromatids • Scc1 is the critical protein that serves as a “linker” between Cdc20 cohesin complexes on the two sister chromatids • APC/Cdc20 mediates release of the anaphase inhibitor (securin) by activating securin degradation resulting in activation of and cleavage of Scc1 linkages on the cohesin complexes

Regulation of Mitotic Exit

• Regulated degradation of mitotic cyclins is a result of a corresponding activation of APC activity via phosphorylation by mitotic cdk and Cdh-1 binding • This is an example of two protein complexes which regulate each other in concert with cell cycle regulation

Cdh-1

cyclin B degrades Summary of the Dual Effect of APC on Mitosis

P + Cdh-1

[Securin] + Cdc20

Active Separase breaks Cohesin links Summary of the Dual Effect of APC on Mitosis DNA damage checkpoint throughout the cell cycle Signal transduction of checkpoint responses DNA damage checkpoint throughout the cell cycle Sensor

Mediator

Transducer

Effector Cell cycle arrest in response to DNA damage DNA damage induces cell cycle arrest DNA repair and apoptosis Summary of cell cycle arrest Medications interfering cell cycle Topoisomearse II inhibitor Spindle assembly inhibitor Bleomycin induces dsDNA cleavage Bleomycin activates cell cycle arrest DNA repair and apoptosis Apoptosis P53 dependent apoptosis

• Transcriptional activation; proapoptotic genes (Puma, Noxa and Bax) • Transcriptional independent process

• Antagonise Bcl-XL and Bcl-2 complex through Bax and Bak oligomerization • Disruption of mitochondrial structure; complex formation with cyclophilin D Why is it important

Between G1 and S phase, cell wish to assure whether cell is ready for cell division

This checkpoint

S is stand for synthesis

Mitosis G2 Prophase Anaphase mm pp

m p + m p Metaphase Telophase G1 Mitosis

Cell cycle and apoptosis Cyclin-Cdk is controlled by ubiquitin proteasome system during G1/S progression

Cyclin-dependent kinase (CDK): key molecule

Cyclin: activator

Wee1: inhibitory kinase CAK: CDK-activating kinase CDC25: phosphatase Mitosis (M-phase) controlled APC-dependent ubiquitylation of by CDK and cyclin cyclin Role of SCF to regulate G1/S progression Cyclin-Cdk is controlled by ubiquitin proteasome system during G1/S progression

M-phase M-cyclin APC E3 system

G1/S-phase G1/S-cyclins SCF E3 system