Cell Cycle Regulation and Checkpoints

Chi-Wu Chiang, Ph.D. IMM, NCKU

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From cell division to a full grown body

-----Accompanied with cell division, cell growth and cell death

cell mass
Cell mass=cell number+cell size
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Cell division, cell growth, and cell death are independently regulated but linked
Intrinsic programs Regulated cell cycle progression Programmed cell death Extracellular signal molecules Mitogens Growth factors Survival factors Apoptosis-inducing factors Proliferation inhibitors
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Introduction to cell cycle and cell cycle checkpoints

Cell cycle
A cell reproduces by performing an orderly sequence of events in which it duplicates its contents and then divides in two

The phases of cell cycle

Embryonic cell cycle and somatic cell cycle

Somatic cell cycle

30 min

6h
Early embryonic cell cycle

M G2 S G1

12h

M S

6h
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Yeast provides a good genetic system for study on cell cycle

Mostly proliferating in a haploid state Easy for genetic manipulation Cdc genes and cdc mutants ----cell-division-cycle genes

Cdc mutants selected by temperature sensitive mutations

A mutant that cannot complete the cell cycle, cannot be propagated Temperature-sensitive mutants Functioning in permissive condition (low temp.) and nonfunctioning in restrictive condition (high temp.)

Xenopus oocyte: a giant cell for study cell cycle biochemically

1 mm in diameter Carrying 100,000 times more cytoplasm than an average cell in the human body

After fertilization occurs, the first division takes about 90 minutes, and the next 11 divisions occur at 30-mim intervals, producing about 4096 cells within 7 hours Each cycle is divided into S and M phase without detectable G1 or G2 phases 10

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Studying the cell cycle in a cell-free system

Observation of repeated nuclei decondense and DNA replication and mitosis In vitro

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Measurements of cell cycle progression

Propidium iodide (PI) staining

EMBO 2003, 22:5459

BrdU staining
(bromodeoxyuridine)

PI staining
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Oncogene 2001, 20:4507

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The cell-cycle control system

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The control of cell cycle

Operates like a timer that triggers the events in a set sequence The system of switches is binary (ON/OFF) and launches events in a complete, irreversible fashion The control system is independent of the events it controls The system is highly adaptable and can be modified to suit specific cell types
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The Cyclin-Cdk complexes: the major components of the cell cycle control system

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Cyclically activated protein kinases control cell cycle progression

Cyclin-dependent kinases (Cdks)The go system (or the engine ) Expression is constant through the cell cycle Kinase s activity oscillates in the cell cycle Cyclical changes in Cdk activity are controlled by an array of enzymes and other proteins Among these, cyclins are the major molecules
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The cyclins
Four classes of cyclins: 1. G1-cyclinshelp to promote passage through Start or the restriction point in late G1 2. G1/S-cyclinsbind Cdks at the end of G1 and commit the cell to DNA replication 3. S-cyclinsbinds Cdks during S phase and are required for the initiation of DNA replication 4. M-cyclinspromote the events of mitosis
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The major Cyclin-CDK complexes

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Regulation of cell cycle

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Regulation of the cyclin-Cdk complex

Post-translational modification Transcriptional regulation Cyclical proteolysis Inhibitors of Cdks

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Activation of the cyclin-Cdk complex

Partial activation of Cdks by cyclin association Full activation by Cdk-activating kinase (CAK) phosphorylation Study on crystal structure of CDK2 revealed:

Active site hindered by inhibitory T-loop

Exposure of Tloop by cyclin binding

Phosphorylation by CAK causes conformation change

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Regulation of the cyclin-Cdk complex :post translational modification

Inhibition of Cdks by inhibitory phosphorylation Human Cdk1 and also Cdc2 in yeast: Inhibition: Phosphorylation by Wee1 kinase at Tyr15 Cdc25 phosphatase dephosphorylates the p-Tyr15 and activates the Cdk

PP2A
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The inhibition of a cyclin-Cdk complex by Cdk inhibitor proteins (CKI) CKI (Cdk inhibitor proteins):
(during G1) The INK family, P15, p16, p18, and p19 target to CDK4, 6 (during all phases) The CIP/KIP family, p21, p27, and p57 target to most CDKs Binding of CKI renders Cdk inactive by conformation change and rearranged structure of active site

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Cyclical proteolysis regulates cyclin-Cdk activity

Regulated by SCF complex in G1 and S phase SCF complex(Ubiquitin ligase) Targets:G1/S cyclins, some CKIs (such as p27Kip)

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Cyclical proteolysis regulates cyclin-Cdk activity

Regulated by APC complex in M phase APC: anaphase promoting complex APC complex (Ubiquitin ligase) Target: M cyclin

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The G1, G1-S, and S phase

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The G1 phase is a state of stable Cdk inactivity

To reach a stable G1 for growth, Cdk reactivation is prevented
Decline in M-cyclin and in Cdc20-APC Decrease in M-cyclin transcription Increase in Cdh1-APC* activity which targets to M-cyclins Sic1, a Cdk inhibitor ( CKI), inactivates M-Cdk *Cdh1 is a close relative of Cdc20 *Cdh1-APC targets to M-cyclins to inactivate M-Cdk after mitosis
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Initiation of S phase in budding yeast

Accumulation of G1-cyclin that is resistant to Sic-1 and Hct-1-APC G1-Cdk stimulates G1/S-cyclin synthesis G1/S-Cdk stimulates S-cyclin synthesis, and increase in S-Cdk activity G1/S-Cdk phosphorylates Sic1 and Hct-1 and blocks their activity
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Initiation of S phase in animal cells

In G1, cells are in a state of Hct-1 activation, accumulation of a CKI, and inhibition of cyclin gene expression (M-cyclin) In late G1, activation of G1-Cdk reverses the inhibitory state of G1 G1-Cdk and G1/S-Cdk phosphorylate Rb, an inhibitor of cell cycle progression, and results in release of E2F

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Mitogens stimulate activation of Ras and a MAP kinase cascade to trigger cell division

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Mechanisms of Myc-promoted cell cycle entry

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Control of the initiation for DNA replication

(formed by Orc proteins)

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Figure 17-23 Molecular Biology of the Cell ( Garland Science 2008)

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The control for DNA replication

Regulation of pre-replicative complex (pre-RC) by S-CDK
ORC: Origin recognition complex Binds to the replication origin through the cell cycle Cdc6, Cdt1 associate with ORC at early G1 help to recruit Mcm proteins, a DNA helicase

S-Cdk causes: Start of DNA replication, by ORC phosphorylation, MCM activation, and replication firing

Control only one replication, by Cdc6 degradation and by Mcm export from nucleus
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The M phase
:Mitosis and cytokinesis

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Regulation of entry into mitosis

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Entry into mitosis

M-cyclin accumulates by reduced degradation before M phase in embryonic cell cycles M-cyclin increases in transcription in most cell types Inactivated M-cyclin-Cdk complex accumulates due to inhibitory phosphorylation Cdc25 phosphatase dephosphorylates and activates M-Cdk Polo kinase and active M-Cdk activate Cdc25 Active M-Cdk inactivates Wee1

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Multiple roles of M-Cdk in mitosis

Induce the assembly of mitotic spindle Ensure replicated chromosomes attach to the mitotic spindle Chromosome condensation Nuclear envelope breakdown Reorganization of the Golgi apparatus and endoplasmic reticulum
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Mitosis

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Mitosis, continued

Exit

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Sister chromatid separation is triggered by proteolysis

Separase is required for sister chromatids separation Securin, can inactivate separase; degradation is promoted by APC APC: anaphase-promoting complex, a highly regulated ubiquitin ligase; promote the destruction of several mitotic regulatory proteins Cdc20 activates APC
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The spindle-attachment checkpoint

Ensure that all chromosomes are properly attached to the spindle before sister-chromatid separation occurs A sensor mechanism monitors the state of the kinetochore, the specialized region of the chromosome that attaches to microtubules of the spindle Improper attachment of kinetochore to the spindle sends out a negative signal to the cell-cycle control system, blocking Cdc20-APC activation and sister chromatid separation Several proteins, including Mad2, are recruited to unattached kinetochores. Mad2 binding results in Inhibition of Cdc20-APC and blocking securin destruction
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Exit from mitosis and start of G1

Complex changes at the end of mitosis The mitotic spindle must be dissembled Chromosomes decondensed The nuclear envelope reformed Inactivation of M-Cdk is required for exit from mitosis
Cdc20-APC complex mediated ubiquitin-dependent proteolysis of M-cyclin

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Exit from the M phase and entry of G1 phase in somatic cells

Decline in M-cyclin and in Cdc20-APC Decrease in M-cyclin transcription Increase in Cdh1-APC* activity which targets to M-cyclins Sic1, a Cdk inhibitor ( CKI), inactivates M-Cdk *Cdh1 is a close relative of Cdc20 *Cdh1-APC targets to M-cyclins to inactivate M-Cdk after mitosis
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DNA damage checkpoints and p53 activation

Normally, P53 is targeted for ubiquitin-dependent degradation by Mdm2, a ubiquitin ligase p53 is phosphorylated by DNA damage-activated kinase and is released from binding to Mdm2

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An overview of the cell-cycle control system

Accelerators (cyclin-CDK) and brakes (checkpoints)

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From cell dividing to senescence

-gal-positive Flat morphology Abnormal nuclear morphology

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Cell senescence observed by Hayflick

In 1965, Leonard Hayflick first suggested that there was a finite limit to the cultivation period of diploid cell strains and this was attributable to intrinsic factors which are expressed as senescence at the cellular level . Hayflick proposed that normal cells cannot divide indefinitely because they are programmed for a set proliferative lifespan.

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Cellular senescence
:A state of irreversible growth arrest
Can be triggered by multiple mechanisms such as: telomere shortening, DNA damage, and INK4a/ARF derepression

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Cell 130, 223-233, 2007

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Telomere

Molecular Cell Biology, 6th edition

Nature Reviews Cancer 1:203, 2001

GGGTTA repeats (near thousands) in human s telomere

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Telomere shortening and senescence

Human diploid fibroblasts (HDFs) undergo 60-80 doublings in culture, after which they cease division and develop a senescent phenotype Telomerase expression in HDFs is sufficient to bypass the cell senescence and crisis

50 Nature Reviews Cancer 1:203, 2001

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The telomere problem of a linear DNA replication

The must be 5 to 3 polymerization, the lagging stand, and the telomere

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Maintaining telomere length immortalizes cells

Exogenous expression of telomerase reverse transcriptase (TERT) at any stage in the replicative period allows immortalization of these cells

52 Nature Reviews Cancer 1:203, 2001

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Telomere hypothesis
Most adult cells have limiting amounts of telomerase that are not sufficient for preventing telomere loss, resulting in the shortening of telomeres with ages Telomerase-deficient mice with short telomeres show reduced function of various stem cell compartments including those in the bone marrow and skin Telomerase-deficient mice show a decrease in both the median and maximum lifespan, and these effects become more pronounced with each subsequent generation Terc transgenic mice show increased mortality due to cancer, but show evidence improved tissue regeneration as well as a slight increase in maximum life span

53 Nature 2007, 448:767-774

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Telomere stabilization: an important step in tumor development

Most human cells don t express telomerase, whereas most human tumors express telomerase Those tumors that don t express telomerase maintain the telomere by an mechanism of alternative lengthening of telomeres (ALT) Expression of dominant-negative telomerase reverse transcriptase (TERT) results in apoptosis of tumor cells

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Age-dependent increase in the number of senescent cells in differentiated tissues

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Genomic instability in aging cells

DNA damage and mutations accumulate with age in mammals Cytogenetically visible lesions such as translocations, insertions, dicentrics, and acentric fragments accumulate in aging mammalian cells

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The role of DNA repair in aging

DNA damage
DNA repair Defect in DNA repair Genomic instability Recovery and regenerate

? Aging

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Genomic instability increased not only in aging cells but also in most cancer cells

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Excessive oncogenic induction can result in cell-cycle arrest or apoptosis

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OIS: Oncogene-induced senescence

ARF-p53 pathway p16INK4a-RB pathway Implication: in response to the activation of mitogenic oncogenes, checkpoint-mediated failsafe mechanisms such as apoptosis or cellular senescence may be recruited to terminate a pre-malignant condition

Trends cell biol 11, s27-s31, 2001 60 Nature Rev Cancer 3, 286-295, 2003

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Pathways to senescence

Nature Reviews Cancer 1:203, 2001 61

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Assignments for next week s class

Cdk2 suppresses cellular senescence induced by the c-myc oncogene, Nat Cell Biol. 2010 Jan;12(1):54-9 Please introduce some key background information regarding this paper, including the motivation or hypotheses for pursuing this study, cdk2, myc, relationship between cdk2 and myc, oncogene-induced senescence (such as Ras), etc. What has been found in this paper is different from the finding in previous studies regarding myc activity in cell proliferation and apoptosis? Why authors think myc-induced senescence in the absence of cdk2 is not due to blocking apoptosis? And myc-induced cell proliferation is independent of cdk2? Why authors conclude that Arf-p53-p21 and p16-pRb pathways were integrally required for myc-induced senescence in cdk2-/- cells?

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Assignments for next week s class, continued,

Did authors conclude that cdk2 regulates myc-induced senescence through controlling myc-induced genotoxic stresses? Why? Why oxidative stress may contribute to myc-induced senescence? What is the role of cdk2 in this? Since CDK2 is dispensable in many cancer cell lines, why authors still think inhibition of CDK2 can be a therapeutic consideration for treatment of cancer?

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