Prof. R. V. Skibbens

November 22, 2010

BIOS 10: BioScience in the 21st Century

Cell Cycle, Cell Division and Cancer (Part 2)

Directionality - clocks go in only one direction

G1 doesn’t have replication-inducing activityS but not G2 can not induce G1 cell to replicate DNAS cells has feedback controls that ensure replication completionS cells has feedback controls that ensure replication completion

Molecular mechanismF ‘ i i ’For ‘gaining’

and then ‘losing’ an Activity . . .

Master Regulator of the Cell Cycle:Cyclin-dependent Kinase (CDK)

Cell cycle progressionCell cycle progressionrequires CDK activity Controls

timing of divisonsequence of divisionq

Respond to cues

Partners in crime:Cyclin binds/activates kinase subunit

Different catalyticCDK kinase proteinsCyclin-CDK complexes:

budding vertebrate

CDK kinase proteins

G1 Cdc28 CDK4, CDK6

CLN3 Cyclin D1-3y

G1/S Cdc28 CDK2

CLN1 2 Cyclin E. . . And

CLN1,2 Cyclin E

S Cdc28 CDK2

CLB5 6 C li A

Different cyclins

CLB5,6 Cyclin A

M Cdc28 CDK1

CLB1-4 Cyclin B

Cyclin-dependent Kinase (CDK) Microtubule re-organization(MAPs)

Condense chromosomes(Histone 1)

Nuclear envelope breakdown(Lamins)

y p ( )

Cell cycle progressionrequires CDK activity

(MAPs)

requires CDK activity

iff liDifferent cyclins -

different substrates

Unidirectionality -Unidirectionality -

cyclin expressiondand

Transcription of DNA replication factors

(Polymerase, RFCs)

Initiate DNA replication (ORC)

CDK regulation C li d d iCyclin degradation

Mitotic cyclin degradation = Anaphase Promoting ComplexAnaphase Promoting Complex

or APC

G1 and S-phase cyclindegradation

SCF (Skp1-Cullin-F boxcomplex)

Low CDK G1 llG1 cells can enter Quiescence

(G0)( 0)

GDifferentiated cells

orG0or

Cells that controlthe ‘clock’ via

t lexternal cues

Understanding cancerUnderstanding canceroften involves under-

standing how cells Reenterh ll l i h bthe cell cycle in the absenceof cues!

G1 (and S) cyclin-CDK typically activated by external cues: Mitogens!!

Releasing the brakesthat keep cells in G1

g

MITOGENSPDGF

Platelet-derived growth

EGFfactor

epidermal growth factor

TGF-

epidermal growth factor

Transforming growth factor

G1 cyclin-CDK activated by external cues: Mitogens!!

Releasing the brakesthat keep cells in G1 Mitogen receptor

i llactivates small GTPase ->

Ras pathwayRas pathway

MAP kinase

PDGF

MAP kinase activates TFsEGF

TGF-

G1 cyclin-CDK activated by external cues: Mitogens!!

Releasing the brakesthat keep cells in G1 Mitogen receptor

i llactivates small GTPase ->

Ras pathwayRas pathwayPDGF

MAP kinase

TF i d MYC

EGFMAP kinase activates TFs

TFs induce MYC expressionTGF-

T d !! Th t t fTada!! The target ofMitogen activation . . .

Actions of MYC:

1. Express G1 cyclins

GG0

Actions of MYC (plus Max binding partner):1. Express G1 cyclins - G1-CDK will phosph Rb p y p p

Mitogen-induced

?

E2F transcription factorE2F transcription factor

E2F - transcription factor . . .

Rb (retinoblastoma protein) binds E2F –> E2F OFF(inhibits cell cycle progression until external signal)

E2F - regulating a S-phase transcription factor . . .

Rb (retinoblastoma protein) binds E2F –> E2F OFF(inhibits cell cycle progression until external signal)

G1-CDK phosphorylates Rbturn inhibitor off -> E2F ON

Actions of MYC (Mitogen induced!):1. Express G1 cyclins - G1-CDK will phosph Rb p y p p

Cancer pathways:

Oncogene – a stuck accelerator in a Driver’s Ed Car . . .(2 of everything)(2 of everything)

Causes:mutation that falsely signals mitogeny g gdosage – too much

Overactive Accelerators - dosage effectsGeneral mechanisms of gene expansiong p

General mechanisms of gene expansion

Expansion of genes that regulate cell cycle progression:

Myc proto-oncogene amplificationhomogenously staining regions (HSR)

Expansion of genes that regulate cell cycle progression:

Examples ofMyc proto-oncogene amplification

homogenously staining regions (HSR)homogenously staining regions (HSR)Double minute chromosomes

Myc expansion is downstream of mitogen . . .h t b t it i i l i l tiwhat about mitogenic signal mis-regulation

U l t d t i t SUnregulated entry into S

Overactive Accelerators – loss of controlreceptor active without mitogenreceptor active without mitogen

PDGF receptorpNo longer needs

PDGF forActivationActivation

Overactive Accelerators – loss of controlreceptor active without mitogen - Dosage

HER2 receptor - transmembrane tyrosine kinase(EGFR-like ) at high levels no longer

ll l l

receptor active without mitogen Dosage

( ) g gNeeds ligand for activation small molecule

tyrosine kinaseinhibitors ->

Her2EGF-likeHer-2 OE accounts

20-30% of all

gefitinib

dimerizationNameschange

HER2

Estrogen

E-receptor

breast cancers

changeERE

E receptor

Herceptin: Ab thatbl k H 2 di i ti

Tamoxifenblocks Her2 dimerization

Cancer pathways:

Oncogene – a stuck accelerator

Causes:mutation that falsely mimics mitogeny gdosage – too much no longer dependent on mitogen

Cancer pathways:

Rb, p53, viruses and BRCA1

Tumor Suppressors – Brakesneeded to slow car down! – Driver ED car!!!!!!!!

Loss of heterozygosity (LOH)mutation need not be the same!!mutation need not be the same!!

Without Rb E2F free to promote S-phaseWithout Rb, E2F free to promote S phase

Bypasses G1-CDK activation via Mitogens

X

p53 - gatekeeper of the cell53 t t d i 1/2 f ll

53 hi hl bl

p53 mutated in ~1/2 of all cancers . .

p53 highly unstable

Associates with Mdm2 X Made for destructionUb-ligase co-factor

p53 - gatekeeper of the cell

DNA d i d d

ATM/ATR

CHK1DNA damage-inducedp53 phosphorylation

CHK1

p53 - Mdm2 release-> stabilizationby p53 kinase

ATM = ataxia telangiectasia mutatedATM = ataxia telangiectasia mutatedATR = ATM related

i i h di i ipatients with predisposition to cancerand extremely UV sensitive

p53 - gatekeeper of the cell

Cells replicationDamaged DNA

Xp53 is transcription factor

induces p21 expression

X

p21 IS a CKI!! CDK-Inhibitor

BRAKES!!!

p21 IS a CKI!! CDK Inhibitor

BRAKES!!!cell locked in G1

Viral mechanisms of cancer progression

papilloma virus genome stably maintained in epithelial cells

Viral mechanisms of cancer progression

papilloma virus genome stably maintained in epithelial cells

Viral E7 upregulated- binds and inactivates

RbRb

Viral E6 upregulatedp g- drives p53

proteoloysis

Breast Cancer Gene 1 - BRCA1 (tumor suppressor –cell cycle brakes)1 1863cell cycle brakes) 1 1863

5 98 1650 1859Ring finger

BARD1BRCT

p300 CPBMRN d b p53 pRB

BACH1/FANCJCtIP

Mre11,Rad50,Nbs1BRCC complex =

BRCA1/2, BARD1

E3 Ub ligase

CtIPH2AX

C iChromatin remodeling

,Rad51, etc . . .

E3 Ub-ligase Co-activatorChromatin remodelingDNA damage signal

Modified from Boulton 2007

BRCA1 (Wt)BRCA1 slows growth

G1788V*Vector

*G1788V present inpresent in

human brca1patients

Humphrey et al., 1997; Coyne et al., 2004K1601E

DNAreplication

Chromosometisegregation

1. Cancer cells typically aneuploid – inappropriate chromosome number

2 Cancer unregulated cell growth

chromosome segregation - microtubules

2. Cancer – unregulated cell growth

Tumor Suppressor Genes Oncogenes

3. Cancer cells often exhibit heightened migration

Oncogenes

Cell motility - actin

Metastasis – cell migration and ignoring cues

leaving home without a forwarding address

Scratch test

Wildtype cells Tumor cells

Scratch test

TimeTime

Tumor cellsShow Increased

mobility

Zhu et al 2004

mobility

Cell motility – how do cells move and where?

Limiting cell movementReducing chromosome segregation Infidelityg g g y

Adhering to cell cycle cues: accelerators and brakes