it tial

AN IN VITRO STUDYOF ONCOGEN ESISOF PRIMARY CERVICAL CELLS BY HUMANPAPILLOMAVIRUS16

ANDCIGARETT E SMOKECONDENSATE

BY

XIAOL QNG YANG

Athesissubmitt ed to the School of Graduate Stu di es inpartial fulfilmentof the

re quire ments for th e degree of Ma s t e r of Science

Div isionof Basic Sciences Fac u l ty ofMe d i c i n e Memori a l uni ve r s it y ofNewf o und land

May 1995

st. John'5 Newf o u ndland

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L'AUTEURA ACCORDSUNBLlCENCE IRREVOCABLEET NONEXCLUSIVE PERMETTANTA LABffiLIOTHEQUE NATIONALE DU CANADADE REPRODUIRE,PRETER, DlSTRlBUER OUVENDRE DESCOPIESDE SA THESEDE QUELQUE MANIERE ET SOUSQUELQUE FORMEQUE CESOIT POURMETTREDES EXEMPLAlRESDE CETTETHESEA LA DISPOSITIONDES PERSONNEINTERESSEES.

L'AUTEURCONSERVELA PROPR!ETE DUDROITD'AlITEURQUIPROTEGE SATHESE.NI LA THESE NIDES EXTRAITS SUBSTANI'IELS DE CEU..E·

ClNE DOlVENr ETREIMPRIMES OU AUTREMENT REPRODUITSSANSSON AUTORlSATlON.

ABSfRACf

Previousepidem i ologicalstudie sind icated tha t cigarette smoking-fe mal e s are at inc r e a s e d risk of developingcervica l cancer.However. be c a use of the conf o und ingvariablp:s of these studies , and since other studies have given confli c t i ng resu l ts, co nvi nci ng evidence is lacking. Furthermore, the molecul ar ro l e of cofactors , such as hormones, in the multistagecarcinogenic proc essis stillunclear .

To ex ami ne the role of huma n papilloma virus (HPVI and cigarettesmokingin the carcinogenesisof ce r vi ca l cancer, I studied two HPV1 6 immor t a lized endocer vica l cell lines previously establishe d in thi s lab, HEN-16 an d HEN-16-2. Both lines were treated withciga r ette smoke conden s a t e le SC) at 15, 100, 125 ~g/nl. After 6-12mon ths ' tr e atme n t with CSC, 10¹ cells of treatedand unt r e at e d immo rt aliz e d cel ls with equal pa s s a ge s were injec tedinto female nudemi ce. Four to sixwee ks la t e r , on l y csc-t.reateccells but not csc-untre e ted immortalized ce l l s formed palpable tumors, wh i ch we r e subs e q ue ntl y used to derive tumorcell lin es, HEN-16T andHEN-

Furthe r cha racterizatfonof the immortaliz e d cel l line s and csc-t.re n e r c rme c celllines indicated that the esC-treated

tumorigenic cells al displayed distinct morphologies in monolayer and organotypic (raft) cultures; b} proliferated fa st e r in DMEM, a medium cont a i ni ng physLo IoqLc.iI calcium levels; c) showed in vitro anchorage-independency; d) containedand expressed similarlevels ofHPVl 6;e) exp ressed similarlevelsof somecellulargenes as so ciatedwith enhanced malignancy, c-myc, H-ras, p53, WAFl, GADD45, GADDI53, and fibronectin, compared with immortalized cells. However, f) HEN-16T and HEN-l6-2Texpressed higher le v e l s of B-myb and PCNA;an d g) obta inedenhancedce sis tence togrowthinhibition by tans forming growth rector-Br (TGF- ~l) and retinoic acid (RA). On the other hand, the two immortalized cell lines displayedhigher levels ofp53, B-/I1yb, PCNA and lower levels ofWa ft and fibronectin, compared withprimary endocervical cells.

In addition, two novel cDNAs, designatedPA.4and PA9, wh i c h were specifica ll y expressed at higher le vel s in immortalized cells and primary endocervical cells, respectively, were identified and cloned using dif fere ntia l di sp l a y assay". Further characterization of these two no vel cDNAs will be very helpful for us to fully understand the molecula rpathogenesisof cervicalcancer.

In summary, this st ud y p r-ovLded the first in vi t r o

evidence that c.tqerctte smoke can enhance the risk of developing cerv ical in HPV-infected pati ent s. Furth e r more, the in vitro system wi ll be very useful to inv est i gate the mechanism of multistep carcinogenesis of cervicalcancer.

iii

ACKNOWLEDGEMENT

I amindebted to my supervisors, Drs. Alan and Mary Pater, for their continuous support and encouragement in both academic:' and non-academic matLersand especially for their kindness in giving me the opportunity to study in one of the most fascinatingand promising field of research in medicine, thsmolecularbiology of cancer. I would like to presentthi s thes is to Dr.Mary M. Pater, whopassed away on November 2, 1994, in memory of her lovely smile and being a generoushost.

t would also like to thank my other committee meeeeee, Dr. La ura Gillespie and Dr. Martin Mulligan, fo r the ir expertise, invaluable comments and enthusiastic support throughout this program.

Iamgrateful to my colleagues, Dr. Qi Sun, Mingfeng Liu, Ge Jin, Dr. Yoshifumi Nakao, Dr. Masanobu Shindoh, Dr.Masatoshi Yokoyama, Dr. Umesh Kumar and Masuma Rahimtula, not only for their help in many ways, but also for thei r fr i e n ds hip. Further, I would like to thank Ge Jin for some of the work on tumorigenicity assays and tumor cell line init iationandEdward Evelly for histology.

Acknowledgement is also due to Dr. Richard D. Neuman, Dr . Ver na Skanes, the Faculty of Medicine and the School of Graduate Studies for their moral and financial suppor t throughout th i s program.

iv

Finally, my love and appreciationgo to my wi fe, Ya wei Hao, and my family.This thesis would not have beenpo s s i b l e witho uttheir love ,support andpat i e nc e .

TABLEOF CONT ENTS

Abstract•. ... .. .. .... . . .. . ... ... . ... . ... .. .. .... ....1 Ac knowledge men t s...••..••. •. •.. . . •... .. . •.. •• .•••.• ••.•iv

Table of content;s vi

Lis t Of tab l es... . .. .. ... . .... ..•. .. .. . . .... .. ... ... . xi i i

Lis t offigure s xiv

List ofabb rev ia t i o ns xvii

CHAPT ER1INTRODUCTI ON 1

1.1 MUltistage carci nog enes is 1

1.1.1 Physical factors ... ... ... . ... ....•...4

1.1.2 Chemical factors 5

1.1.3 Vi ral fact or s B

1. 1.4 Cellular fac tors 12'

1.2 Gen era l featuresof HPVs 19

1.3 Molecul aroncogenesis of primarycervica l cell s•..22 1.3.1 Aninvivo and in vi t r o model of

muLtistaqe oncogenes isofcervi c al cells .. .22 1.3.2 Role of HPVin onc og e n e s i s of ce r v i c a l

cells 27

1.3 .2. 1Invivoevidence 28

1.3 . 2.2 Invitro evide nc e.. . .. . ...••.... 29 vi

1.3.3 Chro mosome abnormalitiescausing

oncogenesisof cervicalcells 31

1.3.4 Cellula r genes mvo Ived inmult i st a ge oncogenesisofcerv i ca l ce ll s .... ...•. 32 1.3.4.1 Cell ula r genesre gul a t edby HPV•••.32 1.3.4. 2Cel l ulargenesregulating HPV 34 1.3.4.3 Cellulargenesnot interacting

with HPV 36

1.3.5 Role of co-factorsin multistage

carcinogenesisof ce rvvcaI cell s 38

1.3 .5. 1Hormones 38

1.3.5 .2 Other infectious agents 39

1.3.5.3Imm unere s pon se 40

1.3 .6 Tobacco smokeand cervical cancer 43 1.4 Obje c ti ve of this study .. ... . ... ...• •.44

CHAPTER 3 MATERIALSANO METHOOS 46

2.1 Mate r ial s 46

2.2Cellculture ... .... . . .. .. ....•... .. ... ...49

2.2.1 Mo nol a yer culture 49

2.2.2 0 r gan o t yp i c (ra ft) cult u re 50 2. 3Tumo r ige ne sis of HPV16 -immortali ze dce lls by

CSC.. .. . .. ... . . ... .. ... . . . .. ... .•.•...53

vii

2. 3 .1Tre a t me n t of HPV16-immortalizedcells wi t h esc •.. .• .• • . . • . ... .•..••. .. . . ...•. .••..•53 2.3.2 Invivotumorigenic ityassays !l4 2.3 . 3 Path olog i cal an alysis ...• . ...•54 2.4 Measureme n t of growth rateLind seturetton

dens ity of cervical cells...•. .. . •..•.... • ...•.••55 2. 5Soft agaroranchora ge inde pe n de n t growt h assays ..• 55

2.6 pr ob e la b ellingprotocols 56

2.6 . 1 Radioa c t iveprob e sforNorther nand Sout he rnbl ot hyb ri di za ti o n.. . ... .•..••.56 2.6.2 No n-ra d i o a cti ve pr o be s for insitu

hybr idiz ation•. . . . .• •.. .• . .. . .... .. . ... . . ..57

2.7Assays formRN.~exp re s s ion 58

2.7. 1Ex tr actionof RNA and mRNA 58

2."7•2 No rth e r nbl o tassays•.•••..••...•.••..••.•60 2.7.3 Rever se transcr i ption-polymerasech a i n

rea ct i o n (RT- PCR) as s ay s..•. • .•• .... ... ...61

2.7.4 In situhybrid iza ti on assays 62

2.6Protoco l s forDNAdet e ctio n ... .•...•...••..•..•63 2.8.1 prepara tio n of hi ghmcLe cuLat-weigh tDNA••• ••64 2.8.2 South e rnblo t as says..•.. . .•. .. .. . •.. •..• •.••64 2.9Indire c t immuno fluores ce nceassa ys ••..••••.•. ..•...65 2.10Treatmentof cer vic a l ce ll s with tumor necros is

factora(TNF-p), TGF-pl, and M ...•66 viii

2.11ntr re renttaI di s p la y ass ay s... .... • .. ....•67

2.11.1RT- PCR 67

2. 11 .2 Re a mp lificatio n and confir mat ion... .. .. ..• •71

2.11 .3 Cloning 72

2.11.4 Sequencing an dhomo l o g y comp a r ison withGe nBa nt:.sequences ... ....•.... . . .73

CHAPTER 3 RESULTS . . 74

3.1 Tumorigeni c i t y of ca c-treetec cell li nes 74 3.2 Morpho l og y of primarycells , and HPV16-

immorta li zed and csc-t. rens rcrne dcellline s 76

3.2.1Mo nol ay e r cul ture 79

3.2.2 Raft cu lture B2

3.3 Growth characteristi csofprimar y cell san d immo rt a liz e dandcs c-t .ren s rcrmedcell lin e s ... ... •82 3.4 Presenceandexpr e s s i o n ofHPV1 6 DNA in

irrunor t a li zed and eSC-transformed cell s...• . .BB 3.5 Expres s i o n of cancer- r el atedcellul arge ne s 94

3.5.1 Oncogene s... .... ... .. .... . . ..• .. .•..•.•. 94 3.5.2 Tumor suppressorge ne s ... .. . . . ...• •...•94 3.5 . 3 DNA rep lic a t i o nandre pai r genes.... •...98 3.5 .4Senes c e n c e- r e l a tedfibr onectin (FN) gene ..• .106 3.6 Response of primary , inunort aliz ed and tumor

ix

ce ll s to TNF-p,TGF-p l and RA 112 3.6.1TNF-p.. .•... •.... ... ... . . . .. ... . . .112 3.6.2TGF-pl.. . .... ... ... .... ... .. ...•..120 3.6.3 RA.••.... •.•. ...• ..• •. .... ..• .•.•.••125 3.7Identification, isol a t i o n and character ization

of no v el genesdifferentiallyexpressed in

end o cerv i ca l cells 130

3.7.1Id en t if i ca t i onofdifferentiallyex presse d genesbydi f fe re n t i a l displayassays ]30 3. 7. 2 Isolation ofcDNAsandNo rt h e r nhybr-ad lza t.Lon

assays of dif feren tia llyexpres s e dmRN1\s •••• 133 3.7.3DNAsequen ceanalysisof twotsote c e o

clones , 136

CHAPTE R 4 lllSC USSIO NANDFUTUREDIRECT IONS 139 4.1 Invitro evidence of cigarettesmoke

asco-factorin cervical oncogene sis 139

4.2 Role of HPV and cell ul a r genes in

inunortali zationof primaryce r vi c a l cells 1401 01.2.1HPV... .. ... ..•... . .. ... •. ... .. 144

4.2. 2Oncogenes 145

01.2 .3 Tumor supp r es s o r genes.... ... ...• . . . ... 117 01.2. 01DNA replic at i on- and repair- relate d gene s... 150

4.2. 5aenes cenc e -x eLated genes 15 3 4.3Roleof HPVandcellula r genesinmai lignant

pr ogre s si on of immort a li z e d cells 154 4.4 Res pons e of cervical ce lls at dif f er ent stages

ofonc o ge nes i s to cytoki nesandRA••• • ••••••••••••160 4.4 . 1Cyto ki n es...•.•.•.•.• •••. •.•.•. •• •••••• ••••.160 4.4.2 RA... ... ... ... • .• .• .••.• .167 4.5 Identif icati o n of genesin vo lved inoncogenes i s

of cer vical cance r bymRNAdif f e re nt i a l display..•169

4.6 Future direc t i on s 174

REFERENCES 176

xi,

LIST OFTABLES

Table 1 Chemical carc i nog ensand assoc ia ted human

cancers _ 6

Table2 Oncogenesamp li fi e d in human cancers..•.•••. .. . ..14 Ta b l e 3 Deleti onof chromosomes and loss of

tumor suppr essor gene s inhuman cancer.. • • ••. • •••15 Table 4 Bas icfeat u res and func tion of HPV ORFs

and prot e i ns , ... ... ...•.. ....•:3 Table5 List of5' primers us ed in diffe re nt i al

displayassa ys .•... • • ... ••• •...•....•.• . . •.• •70 Table6 Tumorigenic i tyof HPV16-immortali ze d and

esC-treated immo r t a li ze d cells in nu de mice 15 Table 7 Transcription of cell ul a rgenesin primary,

immort a li ze d and eSC-tr ansformedce lls••.•.. ..•.•95

xii

[.1ST OF FIGURES

Figure 1NuIt.Lstaqe carcinoge n esi sofcan c e r...•.•. .... ..3 Figure2 nenet tc chanqos as s ocf eted with

mu ltLsteqe carcinoge nesi sofcolonca n c e r 18 Figure3 cencn r c organizationof HPV16 •••• • •• • • • • • •• ••••••21 Figure4 In vivoandinvitro mode l sonthe mu LtLs ta q e

car c ino ge ne s i s of cervical ca nc er 26 Figure 5Or ga natyp i c (ra f t ) culture syste m 52 Figure6Schemati c rep resent ation ofmRNAdif ferential

dis p layme t h o d...•. • . •.. .•..•• ••• •• •• ••.•• ••. • •69 Figure7 Histo l o g y of tumors forme d by csc-t.reeted

immor ta li zed cell lines...•..•..., .• ... ...78 Figure BMorph o l o gy of cervical cells grownin monolayer

culturewithkeratino cytegro wth med ium (KGM) and Dulbecco 'smodified Eagle 's med ium IONEMl••••81 Figure9Morp holo gy of cervical cells gro wnin

ra f t cu l t ur e•...•..•...•.• • ...• •••.•• • • ...••••.••84 Figure 10 Prolife r ati o nof cervica l cells in KGM

andDMEM..••. .... •.•. • .••.. .•....••.•. • . •• •• • • ••67 Figure11 Anch orage- i n d ep e n den t growth of

csc-t

xii i

incer v i c a l cells.. ...•. .. ... . ... .93 Fi gure13Expre ss ionofmRNAforc-myc, B-myband

a-re s incervica l cel ls . ...• ..•.•• •.. .97 Fiqure14 Expre s s i onof mHNA forp5 3, wild- typep53

ac tiv at e d - f r agmen t 1 (Wafl )anddelet ion in col on cancer (DCC ) in cer vi c alce l ls ...• ..• .100 Fi gure 15 Exp ressi on ofmRNA forprolif e rating cell

nucl e a r anti g e n {PCNAl, GAOD45and GADDI53 in cervica l cells .... .. ... .. .. . . .. .... •..••103 Figure16 Indirec t inunu nofluorescence analys i s of

peNA in cervicalcells cu lture d in

monolaye r andra ft syste m... . ...•....105 Fiqure17Express i o n ofFNmRNAincer vi c a l ce l ls •..•.•..109 Figura18 Ind irect immunof l u o r es c e n c eana l ys isof

FNin cervica lcells ... ... ..•..•••. .. l11 Figure19Ef fect ofTNF-ponprolif erati on of

cerv i cal cells... . . . ... .. . ..•.. . ...•..• .•...114 Figure20 Effect of TNF-I:>on mRNA expr e s sionof

HPV16andc-myr:..•••.•. ...•••.•....••.•• ••••..•116 Figure21Detect i on ofJE ge neincer v icalcel l s...•.. 119 Fi gure 22 Effectof TGr- 1:>on prolife r ati o n of

cervica l cells ...•...••..••... 122 Figure 23Efhlc t of TGr-p onmRNAexpr e s sion of

xi v

HPV16, c-myc and piS.•. . . •.. . . .•. •.• ••• . •••.•.124 Figure24Effect of AAon prolife r ation of

cervi ca l cells 127

Figure2SEffectof AA onmRNAexpress i onof

HPV16 andc- myc...•••.•...••.. .• •....••••.. ••••129 Figure26 Ident ifi cat i o nofPA4 and PA9 by

mRNAdiff e ren tial d.Lspl.a y ...•. •. .,.,••...•••••.13 2 Figure27Expressi onof PA4emd PA9mRNAsin

ce rv i ca l cells 135

Figu r e28Nucleotideseqoences ofPA4ana

PA9 cDNAs•••..•..•••• ••••••••••.•,••• .••• ••• •,.1 3 8

LIST OFABBREV IATIONS

AIDS AcquiredInmu uoderLc -Iency syndr-ome API Activator pr o t e in 1

ATL Adult Tcell Leukemt.a

BCIP 4-b r o mo-5 ~chl o ro -3-i ndo ly Iphoe phut.e BL Burkit t'slymphoma

bp base pa i r s

BRL Bethe s da Research La bo ra torie s BSA Bovi n eseru m albumi n

°c ^{degre e}ce^{ts Iuc}

CAT Ch l o ramphenico lacetyl transf e rase cd k cyc Ld n dependen Lkinase eDNA CompLenerrta r y DNA

CIN Cervicalio t r aepithe li a l neopl asia Cipl Cdk-interactingprotein 1

CMV Cytomeg alovirus

CPM Cou n ts per minute CSC ci ga re t t e smokeco ndens e te De C Del et i onincoIon cancer DEPe Diet hy lpyr ocarlJonate

DMEM DulJ:ecco 'smodi f iedEagle 's medium DNA Deoxyr i bo n u cleic aci d

DNa s eI peoxyr IbcnuLease I dNTP Deoxynucl eo t ide tri pho sph ate D'!'T Dithiothrei to l

EBV Epste i n- Bar r virus res Fetal calfsurum FITC Fluores ce inisoth i o c yn at e FN Fibronect i n

EGF Epiderma l growthfa ct o r xvi

GR GRE K8M KGM HBV HCC HEN HIV HPV HSC HTLV INF IL Kb kDa LCR mRNA MSEE NCR NBT NF-IL6 HMU

NPC ORF

Gluc o cor t i c o i dreceptor Gluc o cor t i c oid respons e eleme nt Kerat i noc yte~Ja~icmed ium Kera tinocy t egrow t hmed iu m Hep a tit i s Bvtru c Hepatoc e llul a r ce rci nome Human end oc ervic aI ce l ls Humanirrununodefic Ienc y vi rus Humanpapi Ll omavLrus Herpes si mple x virus Huma n'r-ce ri teukemtavirus Int erfe ron

rnterJeu kin Kil o b ase Kll odalto n Long control r eqLon Messenge r RNl\.

Me th a ne s u lfontc acid ethyles t er Non c odingregioh

Ni tr oblue tel r a z o lium Nuclea r factor forin t erleuki n6 Nttrosomcthy l u r ea

Nasophar yngea1carcin o ma Ope n rea ding frame

xviL

PBS Phosph a t e buffe r edsali ne PCNA Proliferat i nqcoII nuclea r anti g e n PCR Polymer a s echaillre a c tion RA Ret i no i cilci d

RACE Rapi dampLifioatlonofcDNAends Rb RetInubLastorna(June

RNA Ribonu cl eica;hl RNase Rib o n u c1~~a s e Rpm Revolu t ionspe rminutes RT Reverse transc ri pt a s e

Sl d l Senesc entcell-derivedinhibito r I

sec Sq u amo u seelj~ilr ci nornil SV40 Simi a n virus 40

TEMED Tet r a- met hyl e t hyle n e d i amine TGF Trans f o r mi ny qrowth fac t o r TNF Tumor necrosi s fac to r

TPA 12-0-tetrad ecanoyLpborbo 1-13-ace taL l"

URR Upst r e amregul a t o r region W Ult r av i ole t

Waf! Wi ld- t ypep~J3 »cr tva t ed-rrecne nt 1

xvi ii

CHAPTER 1 INTRODUCTION

1.1 Multistagecarcinogenesis

In 1954 ,Armitage and Dol lstudi e dth eage and incidenc e of 17 common types of cance rs and co n c luded th a t ca rc i nogen esis wa sat least a six or seven stage proc e s s (Ar mita ge and Doll, 19 54). Th is lead to the con c e pt of mul t istage carcf n c ce n e sIs of ca nce rs . since the n, this concep t has been pr ove n clear ly and impro v edsignifi cantl yby epi d e miologi cal and mol ecula r gen etic studies. As shown in Figu r e I, mult ist a gecarcino g e nes is canbe divide din t o four steps : In1tia t l on , promot i o n , mal ignant prog ress i on an d metas tas i s . However,insome case s, no tall of th e s est e p s can beident if ied cl e a rly. The initiationste p isbeli eved to be caus e d usually by genot oxlc agents , especially phy s ical fact ors, such as ult r aviole t (UV) ligh t and irradi a t i on, chemicalca rcino g ens and viru s es, whi ch ca use geneticchang e s in a sing l e cel l . After initi a tio n , the ce ll sti llmay ha v e the normal phenotype. However, when the cellbe come s exposed to some tumor promoting reagents and experience s fur ther genet ic al ter at ions, such as mutat ion, acti vat i on or inactivati on or on cogenesand tus or suppressor genes, a si ngle cellacquiressome heritable form of growth advantage and

Fig ure1 Multi stagecarcinogenesi s of cance r. The sYJIbols repres e nt: 0,nor.al ce lls; 0, nor.a l cell s withqenetic changes; . , premalignantcells; ., maliqnan t cel l s.See thetexttor details.

0 0 0 000°0

Normal[tlls 000°0

I

Normalcells OO,P O 0°0°0

I

Prtmalignant~~II, 00.°0 0°0°0

I

ElpaRs ionor 00 0 premalignant cells

0

·.·0

I

g.~,~

0·0· 0

I

mlligna nlcells

-"". .' ..

11 \

Furthergeeet te changes

Mela, lalic«II,

expands to form a clonal malignant tumor. Finally,add itional changes allow the outgrowth of clones with metasta tic potential to othe r tissues. Her e, I will discuss how ind i vidu a l factorsfunction inthe mUltistage carcinogenes is of human cancer.

1.1.1 Physical fa ctors

UVlightandioni z i ng radiation are recognizedas the two mos t impo r t ant phys i c alcausative factors inthedevelopment of cancer (Adam and Cox,1991).

UVlightha s wavelengthsbetween 200 run and 400nmand is composed of fN-A (200-280 nm) , tN-B 1280-320 nm) and uv-c (3 2 0-400 rim}, each ha vinq differenteffe c tson the e ktnand eye. Generally, the shorter the wavelength, th e more destructivethe radiati on. Thus, UV-Aha s the mostdamaging effects on human cells. Sincealmost allth e UV-A is absorbed by the oxygen and ozone of the atmosphere , it is a minor fa c t or . However, a considerableamountof UV-B penetratesthe atmosphere, especially in the Arctic an d Antarctic regions wh e r e a severe seasona l depletion 0f ozone layer is observed. Sincethe spectrumofUV-B is veryclo s e to the absorptio n spectrum ofDNA, itcan be easily absorbedby DNT\molecules and cause damage to DNA. The damaged DNAis normally repairedbythe DNA repairsysteminthe cells. However,when

the re pa i r syste m is also impai r e d, UV-B may causefurther ge netic changes an d, fina ll y, induce cance r. UV light is believed tobeoneof the majorca uses ofmalignan t melano ma an dot her forms of skinca n cers(S t ern andLa n g, 1988) .

Ionizing radiationcons i sts ofel ec tromagn e tic radi ation, suc hasX- andy-rays, and subatomi cpart i cles, suchas alpha pa r t i cl e s, elect r on s, ne u t r o ns, and pro t on s.Epidemiologica l st u d i e s indicatethat ionizingradiationisbelieve dto be one of thecausative factorsfor the developmentof leukemiaand skin, bone, breast, lung, thyroid , st o ma ch, colon, blad der, esophagus, and liv e r ca nc e r (BEI R IV, 1988). rurtbermore, ce llul a r andmolecu la r bi ological stud ies have conf irmed tha t ioniz i n g radiation can be inv o l ve d in the initiati o n and promotio n pr oc essof multi s t a g e carc i n o gen e s is of cancer by causingce llinac ti v a ti on, chromos omedamage an dmutat ion of gen es , such as ras, directlyor by in t e rac t i ng withwate r to for m hydroxyl radicals that can da ma g e DNA(Ada m and Cox , 19 91 ).

1.1.2 Chemical factors

Epidemiologica l and ani ma ltoxicological stud i e s ha ve led tothe general accept a nce thatchemical carc i nog e ns areone of

Table 1. Chemica l carcinogens as soc i a t e d with human cancers.

Chemicals

Alcohol

Aflato xin Alkylating agents Arsenic& 1 ts compounds

Beta l quid Ch lorambucil Cyclophosph a mide DiethystiIb oe st r ol Estrogens , steroidal Melphalan

Nickel &its compounds Nitrites

Progestin Tobaccosmoke

Vinyl chloride

Cancers/s1 tes

Oral cavit y, pha r ynx, larynx, esophagus, li ve r

Liver Leukemia

Skin, lung, li ve r angiosarcoma periungular

Oral cavity LeUkemia Bladder , le uke mi a

Cervix/vagina , breast, testis Endometr ium , breast Leukemia Na s a l sinus, lung Colon

Cervi x

Lung,blad der, or al cavity, lar ynx, phar yn x, esop hagus , pancreas, renal pelv i s Angiosa rcoma, lung

"Adaptedpa r ti a ll y fromIARC, 19 87.

the mos t impo rt a n t causativefactors forth e dev e lopment of human ca ncer.Table1 lists some of the chemicalca r ci nogen s that can cause human ca ncers .

From the Table 1 we notice that a specific chemica l causesonly certaintypes of tumors. Forexamp l e , arsenic and its compounds can induce skin, lung and live r cancer;however, chlorambucilonlycauses le u kemi a . The mechanism for this ca rcinogen-specificis unknown.

One of the most convincing pieces of evidence that imp li c a t e chemical carcinogensin multistage carcinogenesis ca me from stud ies on the for mat i o n of mouse skin turno r s (Be renb l um and Shubik, 1949; Balmain and Brown, 1966). In the s e experiments, a singlechemical carcinogen, such as MNNG or NMU, wa s first appliedto the skin of mouse. Thisresulted in the initiation of an unkno wnnumbe r of cells whichwould persist for a very long time without showing any apparent cha ngesif they were leftwithout further treatment . However, if a second class of chemicalagent, a tumor promoter, suchas te tradecanoyl phorbol l3- a c e t ate (TPA) , was applied at the same time or even a ye a r later, benign tumors called papillomas appeared. A small portion of these papillomas de ve l ope din t o fully malignanttumors with or withoutfurther applicationsof tumor'promoters. Further molecular biological studies indicatedtha t thetumors were derived from the clonal

expansion of cells bearing the .ras proto-oncogenewhich had been activated by mutation after tre at me nt with the first chemical carcinogen (Ba l mi n and Brown, 1988). Therefore, chemical carcinogens are believed to be involved in the initiation and promotion of cancers by activating proto- oncogenes or LnectIvat Lnq tumor suppressor genes.

1.1.3 Viral factors

Viruses have been implicated in the etiology of up to 20'1.

of all cancers in the world (Vousden and Farrel, 1994). Thus, studying the relationshipbetween viruses and human cancers has become one of the most extensively studied areasin cancer research.

Until now, only five viruses, including three DNA viruses, Epstein-Barrvirus (EBV), hepatitis B virus (HBV), human papillomavirus (HPV) and two RNAviruses, humanT-cell le u kemi a virus I (HTLV I) and human immunodeficiency virus (HIV) are well known to be the factors associated with several human cancers.

EBV is a member of human herp e s v i r u s family, which inf ectsabout90% of the worldpopulation (Keitf and Liebowiz , 1990). Previous studies indicate that EBVma y be the causative agent of two human neoplasms , Burkitt's lymphoma (BL) and nasoph aryngealcarcinoma (NPC) (Keiff and Liebowiz, 1990).The

epidemiological association of EBVwith BL wa s further support ed by the ability of EBV to immortali ze human lymp hocy tes in cu lture (Hammerschmidt and Sugden, 1989). Howeve r , until now, the molecularmechanism for EBV in the etiology ofBL and NPCis still not clearly de fi ne ddue to the la r g e 180 kbp size of the EBV genome and the la c k of cell eurture systems for virus propagation (Vousden and Farrel, 1994).

HPVs are a large groupof epitheliotropic papovaviruses whichinf e c t awidevariety of cutaneousand mucosal epithelia includi ng thosein theskin, mouth, cervix , vulva , larynxand respiratorytract (Howley, 1991).Of the 70typ e s of HPVsthat have been ide nt i f i e d so far, mos t are rel ated to benign geni tal or skinwarts. Onl ya fe w of them, especia llyHPV16 andla, areinvolvedin the developmentofhuman cancers,such as cervica l ,anal and la r yngeal carcinomas (de Villiers,1994). The associationofHPVs with cerv i calcanc er , the secondmost commonfemale ma l i gna nc y 1n the world, has bee n extensively studied (for rec e ntre v i ews seezur Haus e n, 199 4; zur Hause n and deVilliers , 1994a,b). Thiswill be dis cu s s ed in more detail insection1.2and 1.3.

Hep a t it i s Bvirus (HBV) is a 3.2 kb par tia ll y double strandedandpa r tia lly singlestranded ci r c ularDNA vi rus. It

is a member of the hepatitis viruses and infects at least 300 million people in the world (Be a s l e y and Hwang, 1991). Epidemiolog ical studies indicate that HBV is a major risk factor for thedevelopment of hepatocellular carcinoma (HCCl (Be a s l e y et a1.,1981). Despite the close association between HBV rncrcence and hepatocellular carcinoma, the exact mechanisms by which HBV contributes to malignant progression remain to be elucidated. It has been reported that HBV can integrate into the human DNA genome and persist for life.This integration is thought to allow HBV to escape the immune response and initiate the subsequent development of HCC (Be a s l e y et a L,,1981 ) . In addition, integration of HBV near some genes, such as c-myc, cyclin A or retinoic acid receptor pmay cause the deregulation of the genes, which is related to the multistagecarcinogenesis of Hec (Nagaya et a1., 19 B1 ).

Furthermore, HBV X protein can interact with the p53 tumor suppressor gene and inactivate thetr a ns activating function of the gene (Feitelson et a1., 1993). All of the above observationsindi c a t e thatHBV may initiate the carcinogenesis of hepatocellularcarcinoma by interacting with some cellular gene products.

HTLV-1is a retrovirus which infects from O.025~, (inthe U.S.A .) to 30% (in Japan), of the huma n population of

10

differentcountries during th€dr life time tc enn and Chen, 1990 ). Epidemiolog i cal studi e s link thisre tro vi r us toa type of cancer calledadult T cel! leukemia rATL).Sinc e only 2\of the peoplewhoarein f e c t e d by HTLV-I developATL and HTLV-I ca nonl yimmor t alize periph e ralbloodlymptJocytes. HTLV-Ihas been only re g a r de d as the in itiating agent in the carcinogenesi s of ATL (Ho l! sberg and Hatle r , 1993). Oth e r cha nge s such as chromoson.al rearra ngements or somatic mut a t i on s are necessary for the full transfor matio n of prima ry lymp hocy t e s (Holl s b e r and Hafle r , 1993 ).

HIV is anothe r retrov a rus and is linked to acq ui red immunodeficiency synd ro me (AIDSI (Ga llo and Mo ntagnier. 1988 ). The hallmark of AIDS is tbe gradual lo s s of T4 ce ll lympho c yt e s and the sub sequent development of immunodeficien t st a te . The absenc e of T cells leads to the ex p a n s ionofpolyclonalBcellpopul ation sand. finally, the formationof lymphomas.Molecular genetics stud i e s have sho wn thatrearra ngement and ectfvat.tcn of the c-m}'C loc us isoneof th e major pathomechanisms in the development of lymp homas (Kno wles et aL, 1988) . Benign hyperpla stic lymph nodes obtaine d from patients infect e d byAIDScontained an intact c- myc gene locus and oligoclonal IgH, whereas freque nt mono clona l c-my cgene rearrange ment and ol1goclona l 19Hgene rearr angement have be e n observed in lympho id neop l a s ms

11

(Knowles et a L, , 1988), suggesting that infectionof HIV is only an initiating agent in the multistep process in AIDS patients and that the alteration of other ce llulargenes such as c-mycmay also contribute to this process. In addition, a higher incidence of several types of tumors, such as rectal, cervical, oral, testicularcancers, has also been described irlAIDS patients, suggesting the possiblerole of this virus in the oncogenesis of these cancers dueto immunosuppression (E-r a u , 1994).

In summary, all these DNAvi r us e s and RNA virusescan be regarded to be necessary but not sufficient agents in the multistep carcinogenesis of cancers. Other changes cr.i ticalin tumor progression and metastasis.

1. 1.4 Callularfa c to r s

Molecular genetic ucudt ea indicate that development of cancer is largely a consequence of abnormal expression function of specific cellulargenes (Solomonet a L,, 1991). Specific cellular genes are either activated or inactivatedin the process of mUltistage carcinogenesis. Genes Who s e activation or abnormal function of the gene productcan induce neoplastic transformation of nonmalignant cells are called oncogenes. On the contrary, genes for which inactivationof the gene product can induce neoplastic transformation of

12

beni g nceiieare called tumo r suppress or gen es .

Until now, at le ast 50 di ffer en t oncoge n e s have be e n ide n ti fied that induce chan ges neces sary for ma l i gnar.t cell gro wth.Table2 lis t s onc oge ne s amplifi e d insomeofthe most commontumors.

A certai n kind of canc er is usuall y caused by dereg ulati on of several of the onco genes in this table, sugges ti ngthat multiplegeneticalterationsare required for invo l ve d inthe mUltistagecarcinogenesisof thecancer.

Whil e oncogenes pr e vt o usLywere regarded as the maj or cellul a r fac to r in human cancers, withthe dev el opment of mol ecular genet ics within the past 10 years , an in c r e as i ng number of reportsha ve shownaltera t i ons in tumor suppre ssor gene s I So l omonet al ., 19911. 'rbus, it is now believed that mutation and deletion of ch romosomes for tumo r sup pre s s or gene s plays acentral ro le inthe genes i s andprogres si on of humancancers (Weinb e r g , 1989 ; Lev in,199 31 .Table 3 shows the correl at ionbet weenhuman cancers anddele tion of chromos omes or tumor suppres s or ge n es.

Comparing iable 2 with rebae 3, one basically can perce ivethatbothonc o gene s andtumor suppressorgenesmay be invo lve d in a specific type of cancer. For example, in colon cance r,boththe activation of erca t, L-myc, N-myc , H-ra s and

13

Table 2: Oncoqenes amplified in hurnan cancers.

Cancer Oncogenes

Bladder H- r a s , K-ras , int-2

Brain erbBl, sis

Breast erbB2, n-c-ee, c-myc, int-2

Cervical c- myc, H-ras, erbB2

Col o r e c t a l a-rae, x-a-ee, c-myb, c-myc Ga s t ric erbBl, hst, c-myc, c- myc, N-ras, yes Lung erbB1, c-myc ,r.-myc ,N-myc,n-za s ,

x-eas

Melanoma H-ras

Neuroblastoma c-myc , N-myc

Ovaria n erbB2, K-ras

Pancreas x-r-as ,c-myc

Prostate c-myc

Stomach c-ave, int-2, hst

Testicular c-myc

... Cole and KeIeker , 1981; Cooper , 1990; Daviesant Vousden, 1993; Mitraet al.. 199<1.

14

Table 3. Delet ion of' chr omosomes and los s of'human tumor suppr e s sorglaReSinhumancanceee-

Tumor/site Chromosome Loss of tumor

deletion suppressor ge ne

Bladder 13ql4 Rb

17p13 p53

Breast 5q2l APe

13ql 4 Rb

l7pl3 p53

17 BRCAl

l8ql1 Dee

cer vLx 3p, llq,17 q

Colon 5q2l APe

17p13 p53

l8ql1 DCe

Liver 4, l6q

Lung 3p25 VHL

5q21 APe

13ql4 Rb

l7pl3 p53

Melanoma 9p2l MTSl

Ip,6q, 9q ?

Ne ur o b l a s t oma lp, 14 q

Neurofibromatosis l7q l1 Nfl

22ql2 Nrz

Osteosarcoma 13q14 Rb

17p13 p53

Prostate IIp, 16q

Stomach lq, 12q

Wilms llp13 "Tl

llp15 H19

*We inber g, 1989;Solomon etaL,, 1991; Levin, 19 93 ; Yokoya et al., 19 9 3 .

15

K-rasoncogenesandtheinactiv atio nofAPe,Rb, p5J andVHl, tumor suppr essor genes wer e found. However, howare these oncogenes an d tumor suppressor gen es invo lve d in the oncoge n e s i s of a certain cancer? Is their functionaltered s IrnuLt anaousLy or subsequently during the pr oc e s s of carcinogene sis? Studies on the multistep carctno cenests of colon ca nc e r have shed some lighton this que s t i on s.

Figure 2 shows the gene t i c changes occurring during evolution of a typical colon carcinoma (Fe aro n and Volgelstein , 1990).Wh il e the tumor suppressor ge ne , APC, inactivated by somati c mutations at the earliest st a ge s of colo ncarcinogenesis, H-r a s activationand DCC losswere only found in most of the late adenomas. Inactiva tionof p53is detectedonly in the fu ll carcinoma (Figure 2). This model clear ly illustratesthat alterationsof oncogenes and tumor suppressor genes may

carcinogenes is . However , there

at differe nt stages of also some examp l e s of activationof certain oncogenes and ina c tivat i onof certain tumo r supp resso r genes occurring at th e sa me stage of carcinogenesis (Mc Don a l d and Ford, 1991). Thu s , coope ratio n betwe en oncogenes, such as H-r a s and c-myc, or be t ween oncoqenesandtumor suppressor genes, suchas inactivationof Rb and activation of c-myc , may al s o be necessary for the multistepcarcinogenesisof cancer.

16

Figure AlteratioDs ass ociated. with mUltistage carcinogenesis of col on cancer. Act ivation of speci fic oncogenesand inactivation of specific tumor suppressorgen e s arest at i s t i c a lly associatedwith4.of the 6st eps in tumor progre s s i on (Fea ronsand volgelstein, 1990 ).

17

!t--_.

'i

I'

1. 2 . Gene r al featuresof HPVs

Human papil lomaviruses are small , naked, ico s a h edr a l double- s trandedcirc ularDNAof about8,000bps. TheY bel ong to the sub-familypapillovirinae (Howl e y, 1991).

Un til now, approx ima tely 70 typ e s of HPVs have been id e n t ifie d based on a comparisonof the DNA sequencesof E6, E7, and L1 open reading frames (ORFs ). Ov e r 30 types are associatedwith anogenital lesion s (de Villiers,1994).These HPVsca n be furthe r cl a s si fi e d as eitherhigh risk, such as HPV 16, 18, 31, 33, 39 and52,or low risk, such as HE'V6 and 11, basedonthe relative tende nc yof the lesions toprogress in to genita l carcinomas (de Villiers, 1989). Generally, high ris k BPVs are frequently fo und in high gra d e cerv i c a l in t r a e p ithe li a l neoplasia (CI N) , which has a highertendency fo r malignant conversion . In con tras t , low risk HE'Vs commonly found in benigngenital condylomas and nasal and laryngeal papillomas, which have a lower tendency for malignantconv ers i on (Shahand Howl ey , 1990 ).

The 8,000bp genome of HPVs is general lyorganizedinto thre e distinctregions:earlyregi on (E), lateregio n(Lj , and long control region (Le R) es shown in Figure 3. The early regionis composedof 6ORFs , designatedE1, E2, E4, E5, E6, E7, wh Lcl, are involved in viral DNA replication and transcriptionand cellular transformationand maturation. The

19

Pigure3.Ge n? aUc organizati onof HPV1 6 (Ada pte d frOlll Munget et a1.. 1992 ) . All papillomavirusescontain a double-stranded circular DNA genome of approximately 8 kb. The major transcriptional promoter for HPV16 is des ignated P97.

Transcr iptionoccursonly in a cl ockwisemanner . Nucleotide positions are indicated in the inner circle. The early ORFs deduced from the DNA sequence are designated 51to57, and the late ORFs are designated Ll and L2. The viral long control region CLCR) contains DNA elements that regulate HPV transcriptionand replication by interacting with viral and cellular transcriptionfact orsandreplication factors. Short vertical lines in the LCR represent thelo c atio n s of the E2 bi ndi n g sites. ACCN.GGT.Note thatove rlap between some of the early ORFs and late ORFs is observedin HPV16 and otherHPVs.

20

LeR

6000

HPV-16

21

El

late region is composed of two ORFs, des i gn a ted Ll and J~2,

which encode theviral capsidproteins. The LCR, al s o called the noncoding region (NCR) or upstream regulator y region (ORR), isfound between the3' end of the late region and5' end of the early region and provides binding si tes for sev eral cellular and viral transcription and replication factors (Howley, 1991; Hoppe-Sey lerand Butt, 1994 ).

The basic features andfunct i ons of each ORF and their proteins are listed in Table 4, and they wi ll be further discussed insucceedingsections.

1.3 . Molecular oncogenesis of primarycervicalce l l s 1.3 . 1 An in vivoandinvi t ro modelof multistage oncoqsnesill of cervicalcells

As stated above, the development of cancer is a muLtistage process, in wh i ch mu lt i p l e genetic changes arc required for the full tr a n s f o r ma t i on of nor mal cells. This mul t i s t a g e nature of canceralso clearlycan beobserved in the oncogenesis of human cervica l cellsbothin vivc andin vitro. Figure <Iill u s t r ate s a mode l of the in vivoand in vitrooncogenesisof cervica lcanc er. In this model, high risk

22

Table 4 Basicfeaturesandfunctions ofHPV ORFs andproteins•

QRF Protein5! ze

Feate es ^Functions

(' 0. )

E1 68-7Z 1.Consel'Ved hil1y wellamongHPVs 1.IncreaseimmortaliZiltlooc;ap;llc:ltyofHf'Vgen~

Z.Bindsto minimalreplication Of'igin 2.Requi'"ed togetherwith£2forvlr. 1replication 3.lnt erKt,wlthE2protein

4.Usuallytan bedtletedInHPV-eontaining

,."Inomo>

EZ 48 1.Cont;tin,DNAbIndlngaI'Iduansa etlvatitlg 1. Transactiwtlll orrepressHPVIIlllP'nsiort demol.

Z.Formso;kners 2.ReqJiredtogether,.,.;th E1for vinlr~ic.tlon 3.htlffilctswithE lprotein

~ ^{4.Usuallydeleted}In HPV·eontalnlngCU"d nornas

E4 16-10 1.Significantdivergence amongHPVs 1.Vlfal maturation 2.MostabundantproteinInHPV-lnteetedcells2.Collaps e otker atincytoslreleton 3."'t_Uwlth eyt~er.tlns

4.U:p'essedinditferentii ting keriiltinoc:ytes

1.u....lydel.l~orlIsruptedIn~~ 1.Weak onc;~is C¥l Cff$

2:.EnhancedEGF-medatedmitogenicSll1lal transduction

E6 18·19 1.Produces unspticed £6and£6*andE6", 1.Required together with E7forIrnmorta~zatlon Int ernallyspliced rnRNA and transformation

2.In_blndinllprot eio 2.Tr_ ti v.at ion

3.hterxts withcellulatproteinsuch as pS3

24

Figure 4. In vivo and. in vil:ro model s of the multistage oncogenesis of cervicalcancer.Each ste p willbede s c r ibed in de t a il inthe te xt.

25

INV /VO INVfTRO

Metastasis

1 " - ''''''''''''''' 1

feetlon""thIofIV_____

I

I

7

I

I

rpes

. ^,

7

IAAd"",g~""'1

I

I

Adlitklnal modfica lionof hostce lklargenesarid

geneexpression

m'l

Increased InsublityofdwO'T\O$Oml int egrationof vir alDNA Modificationof host

t

("my"

"T

Otherfact ors(hormon n,he sl.11'virus.lnYnunologlc stat us, smolle,etc)

I

I

4 -nfectkrlo fHPV(hlgh ...-HostiTmunesuppres .

I

I

1

I

I

1

I

I

I

I

26

HPVs are regardedas the initiatingagentfor th e deve l opme n t ofce r vica l cancer.The pre s e n c e ofHPV is responsible for eIN I low grade lesionsinvivo andan ext e n de d life span in cell cultu re (in vi t ro) . Furt hermodi f i ca tio n s ofce llu la r genes result in CIN II and III hi gh grade le s i o n s in vivo or immortalizationin vitro. Howeve r, thes e eventsare notenoug h totr ans f o r m the normalcervical cells into inva s i ve tumors. Other factors, such as hormones and smoking are requiredto ma ke furtherchanges in other cellular genes to transform some of the hi gh grade neoplasias or immortali7.ed cells to form invasive tumors. I willdiscussthisprocessin detai lin the next section.

1.3.2 Role of HPV in oncogenesis of cervical cells Cervical cancer is the secondmos t commoncancer am'lnq womenin the world (Howley , 1988). It was recognized as a sexuallytr ansmi tt ed disease mor e than acenturyago an d since thennumerousinfectious agents havebeen suggestedto playa causative role in cervical cancer Ifor revie w se e zurHausen andde Villiers, 1994 ).In 1976, zurHausen firs tpr op os e d a hypothe sis th at links human papillomaviruses to cerv ical carcinoma. Over the subsequ ent 20 years, a la r ge body of epidemiological and molecu lar biologi cal evidenc e ha s accumula t e d to firmly sup port a role for HPVs in the

27

development of cervical cancer(fo r reviews see Howley , 19 88:

zur Hausen, 19 91,19 9 4; zur Hausen and deVi lliers, 1994a,b;

Garland et a1., 1992; Daviesand Vousden,19 :J3:Lowyet a1., 1994; Ponten et a1., 1995). Here, I brieflydiscuss the in vi vo and in vitro evidence that suppo rtthe role of HPVin the etiologyof cervical cancer.

1.3.2.1 Invivoev i de nce

Thein vivo data linking HPV withcarcinomaof the cervix are as follows:

1) HPVDNA,especiallyhigh risk HPVs such as HPV16 and HPV18 , can be detected in over 90%of cervical car cinoma biopsies (zu r Hausen,1991).

2) HPVDNAof the same types can be detectedbo th in primary cervical cancer and metastati clesions (Lowy et a1., 1994).

3) In morpholo gicallybenign lesi ons, th e viral genomeof HPV is usually maintained in an episomal stat e while in malignantlesions an d high grade intraepithelial ne opla s i ait is often int e gr a ted into the hos t cell chro mosomal DNA.

Integra t io ngenerally occurswit hintheE1 or E2 ORFs, le aving the ORFs of the transformingonc og ene s,E6 and E7, intact and active ly tr anscribed in HPV-positlve cance r cells (Cull en, 1991). Only a small perce nt a ge of ma li gn a nt tumors ha r bor

28

episoma l DNAlCu llen, 19911 .

4) Theintervalbetween primary inf e ction ofHPVandth e appearanceof cerv i c a l can c e r is usually severaldecades. In addition, only a small percentage of wome nwith clinically appa r e nt HPVinfec tion will eventually develop cervical carcinoma, suggesting thatHPVisne c es s arybut not sUfficient for malignantprogression l zur Hausen, 19941Ponten et al., 1995).

1.3 .2.2 Indt r oevide nce

Most of th e evidenc e for the role of HPV in the oncogenesis of cerv i c al cancer comes from the in vit ro st ud i e s, wh i chare sumrnar1t.e dinthe following :

1) Transfecti onwiththehigh risk HPVs, suchas HPV1 6 and HPV1S, or the i r transfo rmi ng qen es , E6 and E7, can ini t ia t e immortalization of ectocervical and endocer vi cal ce ll s (Pirisiet a1., 1987 ; Woodworthet a1., 1988; Ts ut s urn i et aL., 19921. The £6 and £7 ORFs of HPV16 and HPVl a are suf fi ci e nt to immortalize ce rvi c al cel ls in vitro IHawley - Nelson et a1., 1989; Kaur et al., 198 9, Tsutsumi et al . , 1992). The E7 geneof HPV16 andHPV18,whe npla cedunde r the con trol of a st ro ngpro mot e r,ca n immo rta lize huma n ce rvi c a l epitheli al ce ll s withlow effici enc y (Halber t et al., 1991 ) ; the E6 ge neof HPValoneca n not immortalizehuman cer vi c al

29

cells, but canenh anc e the effic iencyof illllllortalizationby the 81 gene (Ha lb er t et al., 1991) or immortalize human mammaryepi theliaI cells (Ba n d et al., 1990) .Other genes of HPV, such as El, E2, Eol, E5, can no t immortalize cervical cells (pi ri si et aI., 1987). However , disruption of Elor 82 can enhance the immortalization capac ityof the HPV genome (Durst et al .,19 8 1;Mungeret al., 1989; Sang et al., 1992). 2)The E6an dE7 oncoproteinsof HPVs areresponsible for the immortalizationof genitalcells byHPVs , sincecontinued expression of theE6 and E7 isnec e ssar y for the infinite proliferat ion of cervica l carcinoma cel ls (Munger et a L .• 1989a; Mansur and Androply, 1993) .

3)The immo r t a li ze dce l l susua l l y contai n int e g r a t edHPV DNA, expressvir al transcripts, are defective in their abili ty to termina llydifferentiate and resemble moderate or high gradeeIN (Woodworthet aL,, 1990; sun et al,, 1992).

4) The E7prot e i n from high riskHPVs can bin d to the tumor suppressor gene, Rb(Dyson et al., 198 9 ) , an d the E6 protein can bind to p53 and acceler ate it s deg radat ion (Wer ness et aI., 1989; Munger et al., 1989b;Scheffneret al., 1990 ) . Inactivationof thetumorsuppressor genes, Rb andp5 3 , by HPV is nec es s ar y bu t not sufficient for the immo r t a li za ti o n and tumori genicityof pr i ma r y cervicalcellsin vitro (Chen et al ,, 1993; Jewers et al . , 19 92). P5 3 is usua l lywil d-type in

30

HPV-positivecervica lcarcinoma celllinesandmu tat ed in HPV- ne g a t iv e ce rvical ca rci nomacell lines (Pa r ket a1., 19941 .

51Altho ugh invitroE6-E7-immortalizedcervical cells nonmalignant in nudemi c e (Ha wl e y-Nelsonet al. , 1989, Watanab1eet al ., 198 9), additionaltrans tec tion wit h thev- usoncogene or wi t hHe rp es Simplexvirus canco n ve r t themto tumorigeni c cells (Durs t at aL; , 1990; DiPaol oet aI., 1987, 199 0, 1994). Furthermore, co-t ranstection of HPV 16 and 18 withanoncogene, suc h as c-mycor H-ras, cantrans form rodent cellsandprima r y humanfi brob l a s t s and keratinocytesinvitro IDi Pa o10 et aI. , 1989; Durst et aL, , 1990; PeI , 19931, suggesting HFVs may cooperate with oncogenes in the onc oge ne s i s of canc er .

1.3.3 Ch romosome abno rmalitiesas cause or cervical canceee cyt og e ne t icaMl ysis of ce r v i c a l tumors ha s shown tha t chro mos ome s 1, 3, 11, and 17 are of t e n abnormal l:e y s si er, 1999),and that tra nsfo rmat ionis as sociatedwith non-random abnormalitiesof chromosomes I, 11, 19, and 20 (Di Pa olo et aI., 1993). In addition,aneuplo i dy is ottenobs e rved in HPV- immor t aliz edcells (Smith and Peri ra-Smi th, 1990). Recent studie s in di c a t e that E1, but no t E6, 1s respon sible fo r the chromosomal instabilitiesin HPV-po s i t ive cells (Has h idaand

31

raeumctc, 1991). Thus, ch ro mosomal loss, duplication, or rearrange m ent mi ght lead to inactivation of some tumor suppressor genes such as p53,DeCor act ivatio nof ce ll ula r oncogenes such c-syc, which suggests a role of abno rmalitiesin the mechanism of oncogenes is of cervical cancers (referred to Section1.1.4 ).

1.3.4 Ce11ular genes involvedin multistagQoncogenesi!lof cervical cells

1.3 . 4.1 Cellulargen e s ragulat ed byHW e

Aswe knowfromTable 4, the HPVE6 and E7 products are zinc bi nd ing proteins, implying that they may modul a t e cellularge ne expressionby binding to theirpromoter reglons. In addi tion, th e E6 andE7 proteinsalso contai n peot ein- proteinbi n di ngand transact lvating domai n s, whi ch maybe also ve ryimportant r oz thetransformi ng act i v it y of HPVs.

The amino te rmi n al half of the E7 protein contains regionsof similarity with the tr ansforming prote i n sof the DNA tumo r vi r us e s , adenovirusEla and SV40large T antigen, implying that these protein s may ac tby a common me chan i sm (Phe lpset a1., 1986).Asexpected, prev i o us invitrostudies demonstratedthat all the three pr ot ein s ca n rormacomp l e x wi t h a tumorsuppressorgen e pr oduc t,Rb, which is a nega t i v e

32

regulatorof ce ll grow th (Munger et aL,, 1992). Since pRb inducesG1growth inh i b i tio n by bindingand inactivating the tre n s c - s oc icn factor, E2F, bind i n g of E7 to the hyp op hos pho r yla t e d form of Rb resultsin the rel e a s e of E2F frompRb. Thispermits the ac ti va tion byE2F of oncogenessuch as c-myc, c-fos,or B-myb, and consequentlythepro gressionof th e cell into S phaseof thece llcycle (ScheUneret aL,, 199 4). In additi on, E7has been showntointe r act wi thother cellular pr o t e i ns , such as pl07, p130, hi s ton e H1 kinase, p33"IOl,cyclinA and B-myb ITornma sino et a L,, 1993; Ar royo et al., 1993; Dav i e s et a1., 19~3; Lam et a L., 199 4). The activation, such as forB-myb, or inactivation, suchas for Rb, ofthese proteinsby the E7protein mustplay an important role in the oncoge nesis of cervicalcells.

Unli ke E7,E6 doesnot display sequence similaritywith thetransto r mi ng proteins of othe r DNA tumor viruses.However, a cl e a r functional simila rity is found in that HPV E6, adenovirus E1b and SV40 la r ge T antigen all targetanother tumor suppressor gene protein, p53 (Munge r et a L,, 1992). Interactionof the HPV E6 proteinwith p53 through ace llu la r protein, E6-AP, results in thera piddegradation of the p53 pr otei n (Scheffner et a1., 1990; HUiBregtse et al., 1991), abro gationof the transact!vatingac t!vi tyof p53 (Lec h ne r et aL,, 1992) and loss of the p53-me d i a t e d cellular re s po n s e to

33

DNA damage (Kessiset al., 1993; Hickman etaL., 1994), which are re s po n s i b l e for the subsequent accumulation of ge netic changes aseoctated with cervical tumorigenesis. Furthermo re , E6 has recently been shown to interact with at least seven other cellular proteins, designated pp212, pp 18 2 , plOO,pSI, p15, p53 and p33 (Keen et a L,, 1994). The role of the interaction of E6 with these proteins needs to be fur t he r characterized.

In addition, cellular genes that are up- or down- regulated by p53, suchas Rb and pl01, may be alsoimportant in the etiology of cervical cancer. However, the roles of these gene products in the oncogenesisof cervical cancer have not been well characterized.

1.3.4.2 Cel l ular gonos regul atingHPV express i on From the abov ediscussion, it can be concluded that the expressionof HPV plays a central role inthe oncogenesis of HPV-associated cervical carcinoma . Therefore, it is of particular interest to understand the re g u l a t o r y mech a n i s m resultingin the activation of HPVoncogene exp r e s s i on in this carcLnoqenesie process.

The 500-1000 bp LCR of IIPVs is known to fun c tio n transcriptional re gu l a t o r y element in almost all the HPV- containing cells. It is the major determinant of the

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differentialimmo r t a li z a t i o n activities of some HPVssuch as HPV16 and HPVla tacne nceuket aI., 1991). Previous studies indicatedthat, in addition to th e binding of E1and E2 to the LCR, cellular proteins ar e the main fac tors that participate in the transcriptional control of HPV oncogene expression (zur Hausen, 1994). A numbe r of cellular transcriptionfactors have been identified thatbind tothe LCR reg ions ofHPVl 6 (API, Nfl, oct-f , TEF- 1, Spl, and GR) and asvia (API, Nfl, Oct-I, KRF-l, Spl, GR, YY1) (For reviewsee Hoppe-Seyler and Butz, 1994) . The binding of these transcri ptionfactorscan eitheractivate (e.g . API , NFl, and GR) or repress (e.g. Oct-l and YY1), the transcrip tion of HPVs. Inadd ition,other proteins.such as nuclear factor for in te r l e u ki n 6 expression (NF- IL6) and TGF- 13 I, have also been shown to suppressHPV expression (Bran etOIL, 1990,1992; Kyo et a L,, 1993).The regulation of HPVby these factors will be discussed furtherin section1.3. I.

Severalobservations have indicatedthepo s s ib l e roleof deregul ationof the HPV LCRinthe carcinogenesis of primar y cervical cells. For examp l e , Ma y et OIL (1994) found that deletions or mutations in the HPVl6 promoter regionresulted in the loss of a YY1bind i ng site in some cervical tumors containing ep i s omal HPV DNA. Chloramph e nicol acetyl tr ans f e r a s e (CAT) assayson the promote r activityof HPV16 in

35

cervicalcancer-derivedHT3, Si Haand CaSH cells showedthat the CAT activityincreased5- to 6-foldunder the control of thispartially mutated or deleted LCR.Since YY1was shownto serve asa negative regulator of HPV expression,deletions or mutations within HPV promotermay alter the regulationof £6 and E7 expression, allowingescape from therepressionby YYl.

Loss of someparts ofchr omo s ome 11 havebeen reported in several cervical carcinomas (Teys sler, 1989). In addition, it ha s been shown by several researchers that transfer of chromosome 11into cervica lcarcinoma cell linescan suppress th e i r tumorigenicity (Se x on et a L, , 1986), ind icatingthat a suppresso r gene may exist on chromosome11. The inactivation of this gene might causece r v i c a l cance r.The tumor suppressor gene was proposed to act through the inhibition of HPV expression by bindingthe LCR(Sc he gge t and van derxc o r c e a , 1994)•

1.3.4 .3 CellulargenGSnotinteracting withUP\'

From the above discussio n, it seemsthatHPV has become themos t importantfactor in the etiology of cervical cancer.

However, the long latency between HPV infection and tumor emergence (zurHa us en , 1991), non-malignant characteristicsof HPV-immorta li zedcells (Hawley-Nelson etal ., 1989), and the existence of cell lines derived fro mcervical carc i nomas

36

(C33A, H3T) or cervical carcinoma specimens devoid of HPV (Park. et al., 1994), all suggest that HPV is not sufficient for the full transformation of primary cervical cells and that deregulationof cellular genes other than HPV may be also involved in the multistage carcinogenesis of cervical cells.

One cellular gene that is not related to HPV is a tumor suppressor gene DeC. The DCC gene product is a ce ll surface protein which may be responsible for cell-cell communication (Gao et al., 1993). The inaction of DeC by mutations or deltionshas been reported to be responsible for several human cancers inclUdingcolon, breast and lung cancer (Gao et al., 1993;Thompson et a1., 1993). Cytogenetic analysis of the HPV- IS-immortalizedkeratinocytecells exposed to nitrosometh1urea (NM U) detected a deletion of part of the long arm of chromosome IS, which harbors DeC. Further analysis indicated that the expression of DeC in NMU-transformed cells was greatly decreased (Klingelhuz et a L,, 1993), indicating that ina c t i va tio n of DeC expression may be important in the NMU- induced carcinogenesis. However, the role of Dee in the oncogenesis of primary cervical cell has not been clearly identified.

By checking 22 protooncogenes for amplification in 50 primary, untreated squamous cell carcinomas of uterine cervix, Mitra et a L, (1994) observed that amplification of erbB2

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(HER2!n eul wa s fo und in 14% of the cases. In addition, 2 tumo r s wither bB2 amplificationsho wed add iti o na l restriction fragments, suggestingthatthe amplificationoferbB2 may play an impo r t a n t role intumorigenes is.

In addition, deregulationof several other gene products, such as the proliferation cell nuclear antigen (pe NAl, fibronectin, in t e g ri n and theTGF-a. receptor, in immortalized cellshas also been reported (Ll et al. , 1992; Hodivala et aI., 19 9 4;Ka r a ki t s os et a L,, 1994;Noda et a1.,1994; Shin at al. , 1994).Studying the regulatio n of the s e cel l u l arge ne s , eapec.ia Ll yinHpV-ne ga tive cells,wil l be very helpful fo r us to fully understand the multistage oncogenesis ofcer vi c a l

1.3.5 Role of co-factors in multistage carcinogenesis of cervicalcellsin vi voandin vitro

Several co-factors have been ide n t i f ied by epidemiologicaland molecular biologicalapproaches. 1.3.5. 1.Hormon es

Previ o u s epidemiologica l stud i es ind icate d that chronic of ore I contraceptive pills whlch contain steroid ho r mone s is an important riskfact or for the developmentof cer v ica l cancers IHllde s he i rn et al. , 1990). In experimental

38

st ud ies, theste ro i d ho rmones,dexamethasone andproge s teron e , havebeen shown tobe essential fo r the expressionof HPV16 in primarycervicalcells IMitt a l et a1.,1993 ) .Most impor ta ntly, thes e ho rmon es ha vealsobe e nsho wnto enhance the expressi on of HPV16 fromthr e eGREs of the enhancer andtoenha nce the immortali zatio n and trans fo rma ti oneff i cienciesof highri s k HPVs (Pat eret al., 19BB; Crooket al. , 19BB: Pater etaL,, 1990; Pat er et aL,, 1994; Mi ttal et a L, , 19 93 ) . Thus, coop e r a tionof HPVand hormones may playsan importan t rol ein theonc o ge nes isof primar y cerv i c a l cells.

1.3.5.2 Other infectious age n t s

Va r ious sexually transmit t e d age nts, including herpe s simpl e x virus (HSV), cytome ga l o vir us (CMV) and HIVhave been re garde d as the co-facto r s in ce rvical cancer (Herri n gton , 1995 ). Recent experimenta l studi e s ind icate tha t HSV 6 not onl y infected genital epith e lialcells, but alsoupregul ated the expres si on of HPV(DiPaol oet aI, 1994), and tr an sformed HPV- inunor talizedhuman genital cellsinto tumors (DiPaoloet aI, 1990 ), sugges tin gthat HSVhas a potential roleinthe tumorigene sis of cervica l cell s . Several repo r ts also sho w that squamous Intraepithelial le s i ons of the cervix progressedmorerapidlyintocervicalcancersin HIV-infected women(Ma i man et a1., 1990 ) , suggesting tha t HIV may act

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co-factorand promote the malignant progressionof cervica l IesIo ne, Furtherinvi ere studies alsoindicatethatthe HIV-l regulatorypro tei n, tat, incombi na tionwith the E2 pr otein of HPV16, cantr a ns a ct i vate the HPV 16 P97promoter in ce r v i c a l ca r c i no ma cells. tatca n al s oreverse E2- me d i a t e d repression of the HPV16 promoter (Verno net al., 1993).

1.3 .5.3 Immune respo nse

Inimmunoc ompe t e nt indi vi d u a l s the cell-mediatedinunune response influences the persiste nce or regression of HPV inf e c tio ns. When HPVs infect the cells, the host usually infiltrates the lesions with in fl a mma t o ry cells, includ ing macrophages, cytotoxic T-l ympho c yt e s , and natu ral killer cells . Thesecells can secretea varietyof regulators known as cytok ines andlyrnpho ki n e s , including TN'Fa andp, TGF-p, interleukin (ILl 1, 2, 3, 4, 5, 6, 7and B and inte r f e r on (I N'F) ex,pandy, whichcan repress the growthof HPV-infe c ted cells and HPV expression. Therefore, the in fil t r at i o n of macrophages,T-lymphocy tes, andnat u r a l killer cells is often obs e r v e d to be fo llowed by regres s ion of HPV infe c t ion (Fierlbe ck etaL, , 19891. However, a disorder of the immune response should leadtope r s i ste n c eor progressionof vira l Infect.L o n , Therefore , depres sionof immune response has been

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regarded as a co-factor for the oncogenesisof cervical cells.

Se v e r al in vivo and in vitro experiments have been ca r r i e d outtounderstandth erol e of the immune re s po ns e in the carc in ogenes is of cervical cancer.

Se ve ra l in vivo st udies demonstrated an as s ct at Lon between impa i red immune functi on and the development of cervical can cers. Firstly, pa tie nt s withhistologicallyproven inva siv e squamous ce l l carcinoma of the ce r vix ha ve becn obs e rve d to haveinunune defec tscharacterizedby a decrease in Tcel l numbersandCD4+ helpercells, and impairedfuncti on of Lang e r ha n s's cells, compared with healthy controls (Ca s tello etal., 198 6).Secondly, womenwhohavebe en immun osuppresscd and have HPVin f ection have bee nobs e r ved to develop in vas ive cervica l ca nc e r rapidly (Si l l ma n et al., 198 4).Thi rdly, the inci de nc e ofinva s i ve cervical cancer increaseswith advancing age, ind i c a t i ngtha t immunese nes c e nc e , whichinc r e a s e s with age may con t ri bute to this ca nc er (Ma nd e lbl at t, 1993 ). Finall y, women who are infec tedby both HIV and HPVhave a markedly increased incidence of cervical cancer.Womenwith con c ur rentHPVandHI Vinfect i o nare 42time s more likelyto haveeIN than womenwithout either virus (Fe i ngol d et a l,, 1990 ).

The following in vitr o e>:periments studied mainly the basic me c ha n i s m underlying the immune response to vira l

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infection usingmolecular biological approaches. Firstly, it has beenshownthat TNF-a: , TGF-[J, IL- l and IFN-ycan re p re s s HPV expression at the transcriptional level in HPV- immortalized cells (Wood wo r t h et aL., 1992 ; Braun et al. , 1990; Kyo et al., 1994). However, in some tumor cell lines, inse nsitivity to TNF-a and TGF- ~l has also been observed (Braun et al., 1990, 1992; Rosl et a L,, 1994),indicating that escape from th e immuneresponse may be a mechanismfor the tumorigenesis of cervical cells. Secondly, cocu1tivation experiments with activated macrophages re ve a l e d that nonmalig nant fi b r ob l a s t-He l a hybrid cells res pon d to macrophages bysuppressingHPV gene expression. Howeve r, this re s p ons e was not detectable in malignant fib robl ast-Hela cells, indicating that the crucial step in malignant conversion involved a disruption of animmune responsepathway that normally suppresses HPV transcription in immortalized cells(Roslet al., 1994). Thirdly, reduced expr essionof class MHCclass I genes in HPV16-positive cervical carcinomacell lines was observed (Cronune et ei, 1993), indicating that insufficient presentation of antigenic domai ns of viral oncop rot eins due to diminished expression of MHC class I molecules may be a cause for malignant prog r e s s i o nin vivc,

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1.3.6 Tobac co smokeandcervi cal ca ncer

Ep i d e mi o l ogi c al investigations and animal studies over the past several decadeshave clear ly de lin e a t e d that tobacco, particularlycigarette smoking, is one of the major cause of humancan cers including lung, oral cavity, pharynx, larynx, es opha g us , urinary bladde r, renal pelvis andpanc reas (IARe, 1986). Th e mos t imp or t a n t of these is lung cancer.

Experimenta l studies have shown that toba cco smoke contains more than 6000 chemicals which includetoxicagents, such as nicotine, ammo n i a, formic acid and carbon mon ox ide;

carci no gens, such as benzene, benzo[a)pyrene, N-nitroso compounds, forma l de hyd e , 2-n i t r o p r opa ne and vinyl chloride ; and twnor promoter agents, suchas pheno l. Therole of some of these carcinogens in the carcin ogenes is have been clearly demon st r at e d (for re v i e w see Hoffmannandwynd e r, 1986).

An as s o ci at i onbe t we e n cigarettesmoking and cervica l ca nc e r was first notedin196£ (Na guibet al., 1966) .In 1977, a speci fic hyp o t hesis regarding th e causit ive role of

ciga ret te smoking in cervi cal proposed

(Winkelstein, 1977). Since then, inc r easi ng epidemio logical invest igations hav e been carri ed out. The ev i de n c e that supp orts the hyp othe si s is asfoll ows: 1) The relat i v e risk for thedevel o pment of invasive cervical can c e r ishi g he r in cigare t t e-smok e rs tha n innon-s moker s.Wit h an increa s e inthe

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