Cell Division and PathogensPathobiology 552

Denise Gallowaydgallowa@fhcrc.org

• The Cell Cycle

• Growth arrest vs. apoptosis

• Telomeres and telomerase

• Human papillomaviruses and the cell cycle

• Immortalization and transformation

S – phaseReplication of DNA

M- phaseSegregation ofchromosomes

G1-phaseGap 1

G2-phaseGap 2

M G1

SG2

CYC DCDK 4

CYC ECDK 2

CYC ACDK 2CYC B

CDC 2CYC ACDC 2

CDKinase

Transcription

Degradation(PEST, D-box)

CKI

T161 - P

CAK(CDK activating kinase)

Binding of cyclin to CDK

Binding of inhibitor

P - T14 Y15

Wee1/Myt-1 kinase CDC25

phosphatase

Cks

Cyclin

CKI

Control of G1/S Transition

E2F

Rb/p107/p130

CYC DCDK4p16INK4A

(p15, p18, p19)

CYC ECDK2p21CIP1

(p27, p57)

p53 ARF

HDAC

Ac Ac

AcAc

Ac Ac

AcAc

HDAC HAT

X

MG1

SG2

p53 p53

DNA Damage

ATM/p53 Signaling Pathway

ATM

Chk2

p53

p21

CDK2

CDC2

E

B

ATM/p53 Signaling Pathway

G1 arrest

G2 arrest

G1 G2S M

S/M

SpindleG2/M G1/S

Re-rep.

S

G2/M

p53

p21

Growth arrestApoptosis

ARF

MDM2

Oncogenes,

Growth factors

kinases

DNA damage

Hypoxia

Redox

Pathogen infection stimulates apoptosis

Linear chromosomes pose 2 problems for cells:

1. End-replication problem

2. Distinguishing chromosome ends from ds DNA breaks

Why do cells need telomeres and telomerase?Why do cells need telomeres and telomerase?

The end-replication problemThe end-replication problem

DNA replication

5’

3’

leading strand

lagging strand

RNA primer removal and Okazaki fragment ligation

5’

3’

Telomeres are protein/DNA protective Telomeres are protein/DNA protective structures at the ends of chromosomesstructures at the ends of chromosomes

~ 10 Kb human~ 40 Kb mouse

3 ´

TTAGGG TTAGGGAATCCC AATCCC

telomerestelomeres

Q-FISH to detect TTAGGG (PNA probes)

G-strand overhang

TRF1

TRF1

TRF2

TRF2

“closed” telomere

“open” telomere

t-loop

Telomere-binding proteinsTelomere-binding proteins

Rad50/Mre11/Nbs1

telomerasetankyrase

PARP5Ku

DNA-PKcsKu

pot-1

Ku

DNA-PKcs

tin2

Griffith 1999

Telomere structure caps chromosome endsTelomere structure caps chromosome ends

zoom in on telomere

TRF2

hPOT1

nucleosomes

G strand

3’

5’

C strand

T-loop

TRF1

adapted from Neidle and Parkinson (2003) Current Opinion in Structural Biology

3’

Telomeres and telomeraseTelomeres and telomerase

CCCAATCCCAATCCC5’GGGTTAGGGTTAGGGTTAGGGTTAGGGTT3’

10-15 Kb ds telomeric DNA

~200 ntss 3’ overhang

AAUCCCAA

hTRhTERThTERT

Telomerase

Regulation of telomerase expressionRegulation of telomerase expression

Primarily by regulating expression of the hTERT catalytic subunit

AAUCCCAA

hTERThTERT

The hTERT and hTR components are sufficient for in vitro activity.Kilian 1997, Meyerson 1997, Nakamura 1997

Expression of hTERT subunit is sufficient to induce telomerase activity inmany different cell types.

Bodnar 1998, Counter 1998, Vaziri and Benchimol 1998

While most somatic cells do not express hTERT, stem cells, germ cells, and tumor cells DO express hTERT.

Kim 1994, Sharma 1995, Chiu 1996, Kolquist 1998

Regulation of hTERT expressionRegulation of hTERT expression

CpG Island

No correlation of DNA methylation with transcriptional activity of the promoter

TSA treatment to inhibit histone deacetylases induces telomerase activity in some cell types

Cong 2000, Takakura 2001, Hou 2002

Chromatin modifications may play a key role in activity

Ac Ac

Ac Ac

Ac Ac

Ac Ac

Ac Ac

Ac Ac

Ac Ac

Ac Ac

SV40 Large T antigen Rb, p53

Small Tpp2A

Ad E1A Rb

E1B p53

HPV E6 p53, hTert

E7 Rb

DNA Tumor Virus Oncoproteins

The human papillomavirusThe human papillomavirus

LCR E6E7

L1

L2E5

E4

E2

E1HPV~8000 bp

Viral oncogenes

Viral capsidproteins

Transcriptional control and viral DNA replication

Expression of viral genes in the stratified epitheliumExpression of viral genes in the stratified epithelium

Basement membrane

Basal

Supra-basal

Squamous

HPV infects basal cells

Early gene expression and viral DNA replication

Late gene expression and capsid formation

Virus release

Infected epithelium

Normal epithelium

RBp107, p130

RBp107, p130

E7

RBp107, p130

S4ATPase c-jun,

junB, junD,c-fos

Mi-2 p21,p27

TBP M2PK

Acidα-gluc

E7E2F

Ub.

E2F

Activates transcriptionRepresses transcription

Activates telomerasein epithelial cells

(E3 Ub. ligase)

E6

p53 E6-AP

Ub Ub

bax

E6 hDLG

MCM7

ERC55 pax.

IRF3

GAP

bakPKN

CBP/p300

tyk2

Mupp1

MAGI

hScribble

Rb

Targets of the E6 and E7 oncoproteinsTargets of the E6 and E7 oncoproteins

S phase genesE2F

E7

S phase genesE2F

p53

E6

E6-AP

E6

AAUCCCAA

induces

telomeraseactivity

Regulation of the G1/S transition

E2F

Rb/p107/p130HDAC

PcG

CYC DCDK4

p16INK4A (p15, p18, p19)

CYC ECDK2

p21CIP1 (p27, p57)

p53 ARFMDM2 ?

E7E7E7

E6E6E6E6

E E X

Myc

Ma

x

TERT

Sin3A

NF

X1

-91

HDAC

Working Model for regulation of the hTERT Promoter in Normal HFKs

E E XM

ycM

ax

TERTMyc

Ma

x Ac Ac

AcAcAc

AcAc

Ac

Working Model for E6 Activation of hTERT

E6APE6 NFX1-91

?

NFX1-123

?HAT HAT

?

+ E6 + E7

HPV-16 E6 and E7 can efficiently immortalizeHPV-16 E6 and E7 can efficiently immortalizeepithelial cells epithelial cells

Days in culture

Po

pu

lati

on

do

ub

lin

gs

Senescence

+ E6 or E7

Induction of telomerase and loss of Rb/p16 pathwayInduction of telomerase and loss of Rb/p16 pathwayare required for immortalization of epithelial cellsare required for immortalization of epithelial cells

Days in culture

Po

pu

lati

on

do

ub

lin

gs

+ Telomerase AND Loss of Rb/p16

Senescence

+ Telomerase OR Loss of Rb/p16

HPV E7Inactivation of Rb/p16/cyclin D

HPV E6Inactivation of p53/p21/ATMBlock apoptosisActivation of telomerase

Activation of genes for invasion, metastasis, angiogenesis