CELL CYCLE, SURVIVAL AND DEATH

JIA Hong-Ti ( 贾弘禔 )DEPT BIOCHEM MOL BIOL

INTRODUCTION For What Make Choice of This Topic

Meier P et al: Apoptosis in Development (Nature, 407:796-801, 2000)Yuan J & Yankner BA: Apoptosis in The Nervous System (Nature, 407:802-809, 2000)Evan GI & Vousden KH: Proliferation, Cell Cycle and Apoptosis in Cancer (Nature, 411:342-348, 2001)Nicholson DW: From Bench to Clinic with Apoptosis-based therapeutic agents (Nature, 407:810-816, 2000)

p53 as a cellular superhero exerts a tumor-suppressive role at multiple stages of carcinogenic progression (Nature, 411:342-348, 2001)

Mutation of p53 occurs at the point of transition from pre-malignant to invasive lesions, well after activation of some of oncogenes that are thought to trigger p53 response (Nature, 411:342-348, 2001)

Cell Cycle and Its Controls (from Mol Biol of The Cell)

ATM(ataxia telangiectaxia mutated), checkpoints and the cell cycle. P: phosphorylation; T: transactivation; B: inhibitory blockade; D: degradation (Nature, 407:778, 2000)

Apoptotic and survival pathways (Nature, 407:780, 2000)

STUDY STRATEGY

Two examples

Berns K et al: A Large-scale RNAI Screen in Human Cells dentifies New Components of The p53 Pathways (Nature, 428:431-437, 2004)

Katou et al: S-phase checkpoint proteins Tof1 and Mrc1 form a stable Replication-pausing complex (Nature, 424: 1078-1082, 2003

Berns K et al: A Large-scale RNAI Screen in Human Cells dentifies New Components of The p53 Pathways (Nature, 428:431-437, 2004)

(Nature, 428:431-437, 2004)

(Nature, 424: 1078-1082, 2003)

Katou et al: S-phase checkpoint proteins Tof1 and Mrc1 form a stableReplication-pausing complex (Nature, 424: 1078-1082, 2003)

FACTORS CAN INFLUENCE THE OUTCOMES OF PATHWAY EVENTSEVENTS IN THE CANCER PATHWAYEarly 1900s —— The idea that tumors arise from somatic genetic change originated

1970s —— Tumor formation is related to the action of specific genesa) Gene transfer:Gain-of-function genetic alteration/Oncogen (e. g., src); proto-oncogene (Ha-ras)b) Epidemiology/Study of inherited predisposition and Cell Hybridization: Loss-of-function genetic change

Nature, 411: 336, 2001

FATES OF THE CELLS AFTER LESION

Based on Molecular Damages:Phenotypic (Cell Organization or Structure) AlterationFunctional Alteration

Survival: Cell-cycle arrest Senescence CancerCell death: Apoptosis Mitotic catastrophe Necrosis

Radiation (UV, X-ray) Chemicals (chemicotherepeutic

Drugs)

Cell lesions:DNA, RNAs,proteins…

Cell-cycle arrest

Cell death: apoptosis,

Mitotic catastrophe,

Necrosis

SenescenceCancer

FACS

Comparing PKH2 fluorescence profiles of the wild-type, p21-/- and p53-/- HCT116 cell lines six days after exposure to doxorubin.

The inhibiton or knockout of p53 or p21 decreased but did not abolish drug- or radiation-induced senescence, indicating Partial Requirement for p53 and p21 in treatment-induced senescence.

Drug Resistance Update, 4:303, 2001

(43%)

(11%)

(29%)

Drug Resistance Update, 4: 303, 2001

MOLECULES IN THE CONTROLS OF THE CELL CYCLECYCLINs:Forming Cyclin-kinase complexs and determining the sub-cell location; the specific interactions with substrates or upstream regulatory enzymes, and the temporal activations of CDKs

Cyclin A: Promoting mature of oocytes and reaching a peak before Cyclin B-kinase complex formation. In human forming a Cyclin A-Cdc2 Co., implicating in DNA repair and transition to G2/M

Cyclin B: In yeast Cdc13 for entry into mitosis; Cig1 and Cig2 functions unclear. In human the kinase in Cyclin B-Cdc2 co. reaches a peak in the interphase

Cyclin C: In human reaching a peak of transcript in mid-G1, but the partner and function unclear

Cyclin D: Isoforms1, 2, and 3, forming a tetramer with Cdk4, PCNA and p21Cip1/Sdi1/Waf1/Pic1 that plays kinase role; with dephospho-Cdk2 playing anti-Cyclin role. D1 (pro-oncogene) is required for entry into G1

Cyclin E: Forming a kinase activity co with Cdk2, implicating G1/S transition

Cyclin F: Similar to Cyclin A

Cyclin G: Similar to Cyclin A in mammal; a target for p53

Cyclin H: In mammal

CDKs:A family of Ser/Thr protein kinase, forming a complex consisting of a catalitic subunit (CDK) and a regulatory subunit (Cyclin) and playing key roles in cell-cycle

Cdk1/Cdc2: Forming a active co with Cyclin B (Cdc13), being required for transitions of G1/S and G2/M

Cdk2: In yeast for G1/S transition control; Being active in late G1 and highest in S & G2 in human

Cdk3: Passing G1?

Cdk4: Forming co with Cyclin D1

Cdk5,6 and 7:

E2F:A transcriptional factor binding to E2F binding site in target genes; inactived by binding with pRb and activated by releasing from E2F-pRb co and inducing cell proliferation

CKIs : Cdk inhibitorsp21, p27, p57 function in S-G2-M transitionp21, p57 in G2-M transitionp15, 16, 21, 27, 18, 19, 57 in G1P27, p57 in G0-G1-S transition

pp pp

S期基因的转录

G1期cdk

非磷酸化的RB

活性被抑制的E2F 有活性的E2F

G1/S期Cyclin ES期

Cyclin A

正反馈调节

磷酸化的RB

pp pp

S期基因的转录

G1期cdk

非磷酸化的RB

活性被抑制的E2F 有活性的E2F

G1/S期Cyclin ES期

Cyclin A

正反馈调节

磷酸化的RB

Checkpoint controls

The Checkpoint Regulatory Mechanism Has An Important Role in Maintaining The Integrity of The Genome

Nature, 408: 433, 2000Genes Dev, 15: 2809, 2001Curr Opin Cell Biol, 14:237, 2002

G1/S PHASE CHECKPOINT PATHWAY

Chromosome Instability and Immunodefiency Syndrome (ICF Syndrome) Caused by Mutations in A DNA Methyltransferase Gene (DNMT3B Gene)

The recessive autosomal disorder known as ICF syndrome (for immunodeficiency, centromere instability and facial anormalies; Mendelian Inheritance in Man No 242860) is characterized by variable reductions in serum immunoglobulin levels which cause most ICF patients to succumb to infectious diseases before adulthood. The cytogenetic abnormalities in lymphocytes are … multiradiate chromosomes (1q, 2 x 16q, 16p, 2 x 1q, 2 x 1p) (chr. 1,16,and 9 unmethylation in iuxtacentromeric regions). GL Xu et al found that the mutations in both alleles of a gene encoding DNA methyltransferase 3B.

Nature, 402:187, 1999

Nature, 411: 366, 2001

G1-PHASE CHECKPOINT PATHWAYATM-CHK2-CDC25A-CDK2

axis forms a rapid response system;

ATR-CHK1-CDC25A-CDK2;

CDC25A-CDK4;

ATM-CHK2/ATR-CHK1-P53-p21 pathway

(Oncogene, 22:5834, 2003)

Oncogene, 22:5834, 2003

S-PHASE CHECKPOINT PATHWAY

ATM-CHK2-CDC25A-CDC45 axis forms a rapid response system

BRCA1 as a ATM target

Mre11-NBS1-RAD50 complex is required for radioresistant DNA synthesis (RDS)

MDC1, a newly discovered BRCT-repeat protein as a mediator to recruit repair proteins (Nature 421:952; 961 2003)

SMC (Genes Dev 16:560,2002)

E2F

S-phase Checkpoint Proteins TOF1 and MRC1 Form A Stable Replication-pausing Complex (Nature, 424:1078,2003)

This is particularly important in S-phase of the cell-cycle,when genomic DNA is most susceptible to variuous enviromental hazards. When chemical agents damage DNA, activation of checkpoint signalling pathways results in a temporary In cessation of DNA replication. A replication-pausing complex is believed to be created at the arrested forks to activate further checkpoint cascades, leading to repair of the damage DNA.

Thus, checkpoint factors are thought to act not only to arrest replication but also to maintain a stable replication complex at replication forks. Recently, Katou et al demonstrated that the checkpoint regulatory proteins Tof1 and Mrc1 interact directly with the DNA replication machinery in S cerevisiae. When hydroxyurea blocks chromosomal replication, this assembly forms a stable pausing sttructure that serves to anchor subsequent DNA repair events.

G2/M-PHASE CHECKPOINT PATHWAY

Oncogene, 22:5834, 2003

A key effector of G2 checkpoint is CDC2 (CDK1) :

ATM-CHK2- CDC25C-CDC2 axis

ATR-CHK1- CDC25C-CDC2 axis

ATR-CHK1- CDC25A-CDC2 axis

Weel-CDC2 inhibition

PLK1 (-) and PLK3 (+) (polo-like kinase family) play a crucial roles in initiation and exit from mitosis

P21-PCNA-CDC2-Cyclin B complex excludes CDC25C(-)

Radiation Res., 159:426, 2003

Caffeine,as a nonspecific inhibitor of ATM and ATR, abolishes mutil-checkpoint responses (including radiation-induced G2 phase checkpoint) and sensitizes cells to radiation-induced killing.

Initiation of a G2/M Checkpoint after Ultraviolet Radiation Requires p38 Kinase(Nature, 411: 102, 2001)

ROLE OF p21 IN TRANSIENT AND PERMANENT CELL CYCLE ARREST AND MAINTENANCE OF GENOME STABILITY

Cancer Lett., 179: 1-14, 2002

P21 INDUCTION APPEARS TRANSIENT RATHER THAN SUSTAINED IN ALL PHYSIOLOGICAL SITUATION, INCLUDING NOT ONLY DAMAGE-INDUCED TEMPORARY GROWTH ARREST (QUIESCENCE) BUT ALSO PERMANENT ARREST ASSOCIATED WITH SENESCENCE

Drug Resis Update, 4:303, 2001

TREATMENT-INDUCED SENESCENCE AND MITOTIC CATASTROPHE IN TUMOR CELLS IF NOT APOPTOSIS

Drug Resistance Update, 4: 303, 2001

Cells with multiple micronuclei (MN) and apoptotic cells(A)

Drug Resistance Update, 4: 303, 2001

Oncogene, 22:5834, 2003

Nature Reviews/Neurosci., 4:1, 2003

Apoptosis Pathways

Figure 5A

Late anaphasee Late telophase

DNA damage triggers Chk2-dependent centrosome inactivation, which induces defects in spindle assembly and chromosome segregation and mitotic catastrophe(Cell, 113, 87-99 , 2003)

H3(Ser10) Phosphorylation

late-prophase

anaphase

metaphase

telophase

Thank you!