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0022-538X/94/$04.00+0

Copyright© 1994,American SocietyforMicrobiology

Reversible Repression of Papillomavirus Oncogene Expression

in Cervical Carcinoma Cells: Consequences for the

Phenotype

and E6-p53 and E7-pRB Interactions

MAGNUS VON KNEBEL DOEBERITZ,* CLAUDIA

RITYMMULLER,

FRANKAENGENEYNDT,

PIDDERJANSEN-DURR, ANDDIMITRYSPITKOVSKYt FSP 6 AngewandteTumorvirologie, Deutsches

Krebsforschungszentnum,

D-69120

Heidelberg, Germany

Received 20 October 1993/Accepted 19 January 1994

The transforming genes E6 and E7 of high-riskhuman papillomaviruses are consistently expressed in papillomavirus-associated neoplasms of the anogenital tract. In papillomavirus type 18-associated SW 756 cervical carcinomacells, transcription of the viral E6-E7 genes is blocked by dexamethasone. Herein we show thatdexamethasone-mediated repression of the E6-E7 genes results in loss of the neoplasticphenotype of SW 756 cells. Withdrawal of dexamethasone restores E6-E7 expression and neoplastic growth. Moreover, reconstitution of E6-E7 gene expression by adexamethasone-inducible expression vector renders the neoplastic

phenotype resistant to dexamethasone. These results clearly indicate that the continuous expression of the viral E6-E7oncogenesis requiredtomaintaintheneoplasticgrowth properties of SW 756cervicalcancer cells. Theviral E6 protein destabilizes the p53 tumor suppressor gene product in vitro. Since low levels of p53 have beenobservedinpapillomavirus-transformed keratinocyte cell lines,it wasspeculatedthatdegradationof p53 by E6 contributes to papillomavirus-associated growth deregulation. Consistent with this hypothesis, we detectedasignificant increaseinp53 levelsupondexamethasone-inducedrepression of papillomavirus E6-E7 oncogeneexpression.Nop53 increasewasobservedindexamethasone-treatedcells in which the viral oncogene expression was restored. The viral E7 protein has been shown to complexwith the retinoblastoma tumor suppressorgene product (pRB).In some cells,this interaction has been shown to release the transcription factorE2Ffrom its complex withpRB,and it has been hypothesized that E7-induced,increasedlevelsof free E2F contribute to the transforming potential of the viral oncogenes. In gel shift experiments, we detected relatively stable complexes of pRB and E2F in all SW 756-derived cells, independent ofthe level of E7 expression. This suggests that E7-mediated releaseofE2Ffrom its complexwithpRB mightnotberequired to maintain theneoplastic phenotype ofhuman papillomavirus-associated cancer cells, althougha possibly relevantpartial E7-mediated release of E2F frompRB cannot beexcluded.

Human papillomaviruses (HPVs) induce benign and dys-plastic lesions on either cornified or mucosal epithelia (re-viewed in reference56).Manyepidemiological and experimen-talstudies have shown thatsomegenotypes (mostimportantly HPVtype16[HPV-16]andHPV-18),whicharereferredto as high-riskpapillomaviruses, contribute to themalignant trans-formation of infectedcells,particularlyintheanogenital tract (reviewed inreference 57). Transfection ofhigh-risk

papillo-mavirus DNA into primary human keratinocytes cultured in vitro results in immortalization of therespective cells(12, 31, 42).Thisimmortalizing activityismediatedbytwoviralgenes, designatedE6and E7(26,37, 47).

Incervical carcinoma cells, fragments of the high-risk pap-illomavirusgenomesincludingthe E6 andE7 genes areusually integratedinto the host cellchromosomes(10, 13, 49,

50).

The

integrated E6 and E7genes aretranscribedinto polycistronic fusion transcripts, which include the E6 and the E7 open reading frames (ORFs) and in addition cellular sequences derived from the3'-flankingchromosomalregion(2, 28, 48, 52, 53, 66).Thesecellular sequencesdonotencode

proteins

(48)

but contribute to the stability of the

polycistronic

fusion

*Corresponding author. Mailingaddress: FSP 6Angewandte

Tu-morvirologie, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld242,D-69120Heidelberg,Germany.Phone:49-6221-424663.Fax: 49-6221-424852.

tOnleavefrom: Russian CancerResearchCenter, Moscow,Russia.

transcripts (34). The E6 ORF is represented at the mRNA level in either two differentially spliced forms, referred to as E6* andE6**,or asfull-length E6 ORF.SplicedE7transcripts have not been identified so far. The persistent continuous

transcription of the high-risk HPV E6-E7 genes in cervical carcinomacells,either inprimarytumors orin celllines which werecultured formany yearsinvitro,suggeststhatexpression

ofone orbothpapillomavirusgenes arerequiredtoinitiate the

neoplastictransformation and also tomaintain themalignant phenotype offullytransformed cervical carcinoma cells. This

hypothesis has been supported by experiments in which the

expressionof E6-E7 antisenseRNAin cervical carcinoma cells ledtoefficientinhibition of cellgrowthin vitro(54,61)and loss of tumorigenicity in nude mice

(62).

Hence, biochemical functions mediatedbyeither theE6ortheE7proteinorboth appear to be critical for the

neoplastic

growth of

HPV-associated anogenitalcarcinoma cells.

Analysisof theE6 geneproductofhigh-riskHPVsrevealed an approximately 150-amino-acid basic zinc-binding

protein

which contains four repeats ofa

CysXXCys

motif. E6 binds

efficiently to double-stranded DNA and

might

function as a transcriptional regulator

(1,

5,

18,

19).

In

addition,

the E6 protein of the high-risk HPV genotypes

complexes

with the

p53tumorsuppressor

protein

(64)

andstimulates its

degrada-tion (7, 27, 46). Transfection of HPV-16 E6

expression

plas-mids into HeLa cells results in further reduction of the low level ofendogenouswild-type

(wt)

p53 activity

(22).

Elimina-2811

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0 0 (0 (0

LO L)

Cl Cl

I

- HPV 18 E6/ E7

FIG. 1. Morphology ofthe SW 756cervicalcarcinoma cellsbefore(leftpicture)and afterdexamethasone(dex.)treatment(rightpicture)for 2weeks.Northern blotanalysisof the HPV-18 E6-E7 mRNAdemonstratedthe absence ofdetectableHPV-18 E6-E7 mRNAin dexamethasone-treatedSW756cells(58).

tion of functional, active wt p53 by E6 has therefore been proposed to contribute to the transforming activities of the E6-E7 genes in cervical carcinogenesis (24). In line with this hypothesis, inactivating p53 mutations were frequently ob-served in therareHPV-negativecervical and analcarcinomas,

whereasmostof the HPV-associatedcancersretain thewtp53 genes (8, 9, 45). With immunoblot or immunoprecipitation

assays, the wtp53 protein is only hardly detectable in HPV-18-associated HeLacervicalcancercells, despitethe presence of translatablep53mRNA(35). However, endogenouswtp53 was easily detectable in HeLa cells after stabilization with a mutant mouse p53 (36). Taken together, these data suggest that wt p53 might be continuously destabilized by E6 in HPV-associated HeLa cervical carcinoma cells.

The E7 gene of the high-risk HPV genotypes encodes a 98-amino-acid zinc-binding phosphoprotein which contains twoCysXXCys motifs(5).The E7proteinisphosphorylated by

casein kinase II at two adjacent serine residues (4, 16). In contrast to the E6 protein, E7 is sufficient to transform established rodent fibroblast cell lines (30, 41, 63). Recent studies have shown that the E7proteinof thehigh-riskHPVs can complex with various host cell proteins, including the retinoblastomatumorsuppressorprotein(pRB)(11, 14, 15, 38,

55). High-affinity binding of E7 to pRB appears to be one major determinant of its transforming functions in rodent cells

(21, 44). Binding of E7 to pRB in vitro has been shown to

interferewith thecomplex formation of the pRB protein with the transcriptionfactor E2F(6).Dissociation of the pRB-E2F

complex has also been observed in HPV-16-immortalized

keratinocytes and HPV-18-positive HeLa cervical carcinoma cells(6, 40). Hence, it has been hypothesized that the disrup-tion of the pRB-E2F complex by the E7 protein and the resulting release of increased amounts of the transcription factor E2Fis acritical eventfor the growth-deregulating and transforming properties of the viral E7 oncoprotein (40). However, mutant E7 proteins which have lost the capacity to

complexwith pRB do not lose the immortalizing activity in primary human keratinocytes (29).

Hereinwedescribeatissue culture model consisting of a set of cell clones derived from the HPV-18-positive cervical car-cinoma cell line SW 756(17),in which the transcription of the

viral genes can be blocked by the glucocorticoid hormone

dexamethasone (58).Viral geneexpression could be restored eitherbyremoval of dexamethasoneorbyreconstitution of the HPV-18E6-E7expression byanexogenousexpressionvector. The characterization of the growth properties of these cells indicated that theneoplasticphenotype isclearlylinkedtothe continuous expression of the viral E6-E7genes. The steady-state level ofp53 is increased in cells in which the HPV gene

expressionhas beenblocked butnotin those in which the HPV gene expression was reconstituted either by dexamethasone withdrawal orbydexamethasone-dependent activation of the HPV-18 E6-E7 expression plasmid. Since the p53 mRNA levels were affected neither by viral gene expression nor by dexamethasone, these data strongly suggest that continuous HPV geneexpression in SW 756 cervical cancercellsindeed reduces thesteady-state level ofp53.

Band shift experiments suggest that expression of the E7 protein in SW 756 cells mightnot result in the disruptionof mostof thepRB-E2Fcomplexes, althoughapartialrelease of E2Ffrom pRB in theE7-expressing cellscannotbe excluded. This observation indicates that the complete release of the

transcriptionfactor E2F from itscomplex with the pRBtumor suppressorprotein may notbe necessary forthe

papillomavi-rus-mediatedgrowth regulation in SW 756 cervical carcinoma cells. Whetherapartial E7-mediated release ofE2FfrompRB contributestothegrowth control by the viral oncogene prod-uctshas tobeclarified by further experiments.

MATERIALS AND METHODS

Cell lines. The SW 756 cell line was derived from a squamous cell carcinoma of the cervix (17). Cellswere con-tinuouslycultured in Dulbecco's modified Eagle medium sup-plemented with 10% fetal calf serum. To obtain subclones harboring the dexamethasone-inducible HPV-18 E6-E7 ex-pression plasmid (pM7) (Fig. 2), SW756 cells were transfected with thepM7 and pSV2neo plasmids in a molar ratio of 20:1 by standardprocedures.Atotal of200geneticin(G418)-resistant clones were isolated and raised to cell lines as described previously (61). Five of them were selected for further analysis. Whenindicated, cellswere grown in culture medium supple-mented with 1 ,uM dexamethasone.

Cell growth assays. (i)

[3H]thymidine

incorporation. The

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proliferationrateof cells culturedineitherthepresenceorthe absence of dexamethasone (1 ,uM)wasanalyzedaspreviously described (61). Each value was determined 24-fold, and the

averagevalue and the standard deviationwerecalculated with

the Sigma Plot 5.0 statistical softwareprogram(Jandel

Scien-tific, Erkrath, Federal Republic ofGermany).

(ii) Plating efficiency. Cells

(103)

were seeded in

10-cm-diameter dishes and cultured for 2 weeks in culture medium either supplemented or not supplemented with

dexametha-sone(1 ,uM). Cellswerethenfixed in 3% formalin and stained in 0.2% crystal violetin the dishes.

(iii) Growthinsemisolid medium. Cells(103)wereseeded in

3 ml of 0.25% low-melting-point agarose (SeaKem; FMC)

dissolved in culture medium either withorwithout dexameth-asone (1 ,uM). This suspensionwaslayeredona0.5%agarose

medium basallayerin6-cm-diameter dishesand incubated for 4weeks in 5% CO2at37°C. Eachtestwasperformedatleast in triplicate. After 8 weeks, the numbers ofgrowing colonies

were determinedand the standard deviationswere calculated. Northern (RNA)blotanalysis. Toanalyzetheexpressionof p53 or E6-E7 mRNA within the isolated clones, cytoplasmic RNAof cellscultured in thepresenceorabsence of dexameth-asone was isolated as previously described (23) and blotted

onto nylon membranes after electrophoresis ina 1% agarose

gel. Filterswere hybridized with random-primed 32P-labelled HPV-18 E6-E7 cDNA (48) or wt p53 cDNAprobes (kindly provided by R. Klein).

PCR amplification and thermocycle sequencing of the p53 cDNA. Heat-denaturedcytoplasmicRNA(5 ,ug)of the

respec-tive cell lines was reverse transcribed into single-stranded cDNA in a reaction mixture of 40 ng of random hexanucleo-tides (Boehringer GmbH, Mannheim, Federal Republic of Germany), 0.4 mM deoxynucleoside triphosphates (dNTPs), 200 U ofMoloneymurine leukemia virusreversetranscriptase

(GIBCO BRL)inanappropriatebuffersystem(GIBCO BRL)

at 37°Cfor 1 h(32). Foramplification of thep53 cDNAs,2 jil

of the reversetranscription reactionproduct was subjectedto PCR in a total volume of 100 ,ul of 200 ,uM dNTPs, 1.5 mM

Mg2Cl, 250 nM the respective primers, and 1 U of Taq polymerase dissolved in the buffer system supplied by the

manufacturer of the Taq polymerase (GIBCO BRL). After heat denaturation at 93°C for 5 min, amplification was

per-formed in 35 cycles of primer annealing at 57°C for 1 min,

polymerization at 72°C for 3 min, and heat denaturation at

93°Cfor 40s. The reactionwascompleted by afinal polymer-ization stepat72°Cfor 8min.Theamplification productswere

purifiedwith the Geneclean IIkit (Bio 101) as recommended by the supplier. The p53 cDNA including exon 1 to 11 was

amplifiedwith four overlapping primer systems, which were

selected with the helpof the HUSAR software program. The

p53cDNAsequencesusedasprimerswerelocated for the first

pairatnucleotides(nt)83to 102(TAGAGCCAC CGT CCA

GGGAG)and atnt543 to 525 (CGGAAACCG TAG CTG

CCCT). The secondprimer pairwaslocated at nt 173 to 190

(CTT CCCTGGATT GGCAGC) and nt 900 to 881 (CAG

TCA GAG CCAACC TCAGG).The thirdprimer pairused

waslocatedat nt464to483(GCCCCT GCA CCA GCC CCC

TC)andnt900to881(CAGTCA GAGCCA ACC TCAGG). The fourthprimer pairwaslocatedat nt 856 to 876(TAGTGT

GGT GGTGCC CTATGA)and nt 1430 to1413(GAGGCT

GTC AGT GGG GAA). These primers were also used to

initiate the asymmetric PCR sequencing reaction. Since the

primer located at nt 173 to 190 did not permit in all cases

undoubtful reading ofthesequence ofexons4 to7,the third

primer pairwasused foramplificationandsequencingofthese

exonsequencesof thep53 mRNA.

All p53

amplimeres

were

sequenced

with the CircumVent

Thermal

Cycle

Dideoxy

DNA

Sequencing

Kit

(New England

Biolabs)

bythe methodof the

supplier.

The reactionproducts were

separated

in 6%

urea-polyacrylamide gels

which were

subsequently

dried and

exposed

to

X-ray

films

(X-Omat;

Kodak).

Preparation ofprotein extracts. Extracts were

prepared

as

previously

described

(40). Briefly,

cells grown in either the absenceorthe presenceof dexamethasonewerewashed twice with ice-cold

phosphate-buffered

saline, harvested,

and

resus-pended

in 1.5 volumesof

lysis

buffer

(20

mM

N-2-hydroxyeth-ylpiperazine-N'-2-ethanesulfonic

acid

[HEPES; pH 7.9],

0.4M sodium

chloride,

25%

glycerol,

1 mM

EDTA,

2.5 mM

dithio-threitol,

1 mM

phenylmethylsulfonyl fluoride).

Cellswere

kept

within this buffer for 20 minon ice and

subsequently

frozen and thawed. The cell

lysate

wasthen

vigorously

vortexed and

centrifuged

for 10 minat

13,000

rpmin an

Eppendorf

centri-fuge.

After the

protein

concentration of the supernatantwas

determined,

itwasstored in

liquid nitrogen.

Western blot

analysis.

Western blots

(immunoblots)

were

performed

as

previously

described

(40). Equal

amounts of

protein

extracts were

electrophoretically separated

on 10%

denaturing

polyacrylamide gels

and blottedonto

Poly

Screen membranes

(DuPont NEN).

After

being

stainedwithPonceau redtocontrol for

protein

transfer anda

subsequent

destaining

in

H20,

filters were blocked in 5% skim milk dissolved in

PBS-0.1%

Tween 20 and then incubated with either an

anti-HPV-18 E7 rabbit antiserum diluted 1:500

(51)

or

anti-p53

monoclonal

antibody

1801

(Dianova, Hamburg,

Federal

Republic

of

Germany)

diluted

1:1,000.

Bound antibodieswere

detected with the enhanced chemiluminescence detection sys-tem

(Amersham

Inc.)

asrecommended

by

the

supplier.

Band shift assays. Two

complementary

synthetic

oligonu-cleotides,

including

the distal E2F

binding

site of the adeno-virusE2promoter

(40),

wereannealed and5' end labelled

by

T4

polynucleotide

kinase with

[_-32P]ATP.

The DNA was

purified by gel

electrophoresis

anddilutedto 104

cpm/,ul.

Band

shiftassayswere

performed

as

previously

described

(40).

The

pRB-E2F

complex

wasdetected

by

inducing

retarded

mobility

(supershift)

with the monoclonal

antibody

C36

(65),

which

specifically

binds to

pRB

(40). E2F-cyclin

A

complexes

were

detected

by

inhibition of the

E2F-cyclin

A

complex

formation with an

affinity-purified

anti-cyclin

A antiserum. An

affinity-purified

antiserum directed

against

the mammalian cdc2-associated factorsuc13servedas a

negative

control. The band

shift reaction mixturewasincubated with these antiseraonice for 50 min

prior

to

electrophoresis

as

previously

described

(40).

RESULTS

Establishment of SW 756 cervical carcinoma cells with or

withoutHPV-18 gene

expression.

Dexamethasonetreatmentof the

HPV-18-positive

cervical carcinoma cell line SW 756 leads

to selective down

regulation

of the HPV-18 E6-E7

transcrip-tion

(58)

and alsoto

significant changes

of thecell

morphology

(Fig.

1).

An

analysis

of the

underlying

transcriptional

mecha-nisms revealed that down

regulation

of the E6-E7 mRNA upon dexamethasone treatment is mediated in cis

by

cellular

flanking

sequences at the

integration

site in this

particular

HPV-associatedcervical carcinoma cell line

(58).

If

dexameth-asone is withdrawn from the culture

medium,

the HPV-18

E6-E7

transcription

is reinitiated and the

morphology

of the cellsrevertsbackto the

phenotype

ofuntreated SW 756 cells

(data

not

shown). Therefore,

wetested whether down

regula-tion of the viral oncogenes or

HPV-independent

effects of

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SW 756 dex. HPV 18

t1r.ITV-LTR EG E7 Cell.Seq.

HPV 18

E6-E7

FIG. 2. pM7expressionvector.The 7-23 cDNA clone (48)which encloses thefull-lengthE6ORF, the E7ORF, andapolyadenylation signal withinthecellularsequenceswasisolated from the SW 756cells. It wascloned3'tothecentralHaeIIIfragment of themousemammary tumorviruslong terminalrepeat(MMTV-LTR)inapUC8vector.

dexamethasone areresponsible for the

phenotypic

alteration ofdexamethasone-treated SW 756 cells. Thus,weconstructed a glucocorticoid-inducible HPV-18 E6-E7 expression vector (pM7) (Fig.2).HPV-18 E6-E7 sequenceswerederived froma complete cDNAclonewhich had been isolated from SW 756 cells (48) and includes thefull-length E6ORF,the E7 ORF,

and

3'-flanking

cellular sequences. Wetransfected thisplasmid

into SW

756

cells together with a selectable marker gene

(pSV2neo) and selected about 200 geneticin-resistant cell clones. Northern blots with RNApreparedfrom dexametha-sone-treated cells were hybridized with an HPV-18 E6-E7 probe. Most of the isolated clones expressedsimilar levels of the E6-E7 mRNA upondexamethasone treatment compared

with untreated controls. However, few clones expressed re-ducedor noE6-E7transcriptsupondexamethasonetreatment. Arepresentative set ofthese cloneswas further investigated

(Fig. 3). In dexamethasone-treated clones Pl andP5, similar

levels ofE6-E7 transcriptswere expressed comparedwith in untreated controls. Inclones P2andP3,the E6-E7 expression level was only partially reconstituted upon dexamethasone treatment. The clone P4 expressed no detectable E6-E7 mRNAupon dexamethasone treatment, although it hasbeen

selectedby thesameprocedure. Therefore,this clone servedas a negative control.

Hybridization

of the same RNAs with a

radiolabelled

mouse mammary tumor virus long terminal repeat fragment revealed that the HPV-18 E6-E7 transcripts detected in dexamethasone-treated cellswere initiatedwithin themouse mammarytumorviruslong terminal repeat of the exogenous pM7 plasmid. Hence, expression of the E6-E7

genesin the dexamethasone-treated cellscannotbesimply due toloss of the glucocorticoid responsiveness of the cells (data not

shown).

This conclusion is further underlined by the

dexamethasone-induced

effects

on glucocorticoid-regulated

cellular factors, asfor example, the epidermal growth factor receptororthemajor histocompatibility complex class I anti-gens (59, 60),whichwereall modulated by dexamethasone in theanalyzed cell clones.

Dexamethasone-mediated repression of the E6-E7 mRNA inSW 756 andP4 cells leads to a severereduction of the E7 protein (Fig. 4), whereas reconstitution of the E6-E7 mRNA results in E7protein levels comparable to those in parental cells, as indicated for example for clone P5 in Fig. 4. If dexamethasone is withdrawn from the medium of the SW 756 orP4cells,theexpression of the viral genes is reinitiated(Fig. 4). TheE7protein reaches its original levels about 48 h after removal ofdexamethasone.

The growth pattern of these clones in the presence of

P1 P2 P3 P4 P5

dex. dex. dex. dex. dex

28 S

18 S

[image:4.612.327.560.81.313.2]

5S

FIG. 3. Northernblotanalysisof the E6-E7 mRNA in the SW 756 cells andpM7-transfectedsubclones. About 10 ,ugofcytoplasmicRNA wassubjectedtoNorthern blotanalysis.Theethidium bromide-stained gel is shown to demonstrate that identical amountsof RNA were

loaded. Thefilterwashybridized withanE6-E7-specific,32P-labelled probe. dex., dexamethasone.

dexamethasone isclearly linkedtothe level of HPV-18 E6-E7 geneexpression(Fig. 5).Theplating efficiencyof theparental

SW 756 cells and the P4cellswas significantlyreduced when the cells were seeded in

culture

medium supplemented with dexamethasonecomparedwith that of cells seeded in medium without dexamethasone. The plating efficiency and cellular morphology of P1 and P5 cellswere notaffectedby dexameth-asone.Theplatingefficiencyof clones P2and P3 is reduced but still detectable upon dexamethasone treatment.

Anchorage-independent growth of these cells in semisolid

medium is also linked to theexpressionlevelof the viral genes. Theparental SW 756 cells and the clone P4, which both do not express theE6-E7 mRNAin the presence of dexamethasone

(Fig.3and4), donotgrowin semisolid mediumsupplemented with the hormone, even if they were cultured for several months(Fig. 6).Incontrast,clones P1 and P5 did not show any difference of theirgrowth propertiesin soft agar, either withor without dexamethasone. The cloning efficiency of clones P2 and P3 was reduced butstill detectable upon hormone treat-ment compared with that ofuntreated controls. In addition, the proliferation rate of the cells directly correlated to the expression level of the HPV-18 E6-E7 genes (Fig. 7). Cells withactiveE6-E7expression (SW 756,P1,P2, P3, P4, P5, P1 dex., and P5 dex. [Fig. 3]) growsignificantly faster than cells with reduced expression of the E6-E7 genes (P2 dex. and P3 dex. [Fig. 3]) or cells in which the E6-E7 mRNA is not detectableby Northern blot analysis (SW 756 dex. and P4 dex.

[Fig. 3]).

Steady-state level of wt p53 in SW 756 cells expressing different levelsoftheHPV-18 E6-E7 genes.The E6 protein of thehigh-risk HPVs has been shownto induce the premature degradation of thewtp53 tumorsuppressor gene in vitro(7, 27, 46, 64). A reduced half-life of the p53 protein has been observed in HPV-immortalized keratinocytes compared with

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P4

SW 756

P5

abode

acde

abcde

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HPV

18

E7

FIG. 4. Westernblotanalysis of the HPV-18 E7 protein expression of SW 756, P4, and P5 cells cultured either in the presence (lanes a) or in theabsence (lanes e)of dexamethasone (1 F.M)for 2 weeks. At 4 h (lanes b), 24 h (lanes c), and 48 h (lanes d) prior to protein extraction, dexamethasonewaswashed outandcells were subsequently cultured in dexamethasone-deficient medium.Westernblots wereincubated with a polyclonal rabbit anti-HPV-18 E7 antiserum. Bound antibodies were visualized by the enhanced chemiluminescence detection system (Amersham).

E6 negative controls, suggesting that E6 might also in living cells support the degradation of the p53 gene product (25). Direct thermocycle sequencing of the complete cDNA ofthe

p53 gene expressed in the SW 756 cells, and the respective subclones described here, revealed that all SW 756 clones expressthep53wtgene(datanotshown).Toanalyzewhether

modification of the HPV-18 E6-E7expression level results in

concomitant changesof the steady-state levelofwtp53in SW 756 cells, we performed a Western blot analysis of protein extractsof theparental SW 756 cells andofthe clones P4 and

PS, grown either in the absence or in the presence of dexa-methasone. As shown in Fig. 8a, dexamethasone-mediated inhibition of HPV-18 E6-E7 gene expression resulted in a

significantincrease of thep53steady-state level in SW 756 and P4cells.Whendexamethasonewaswithdrawn from the culture medium, both cell lines again started to express the viral oncogenes and subsequently expressed reduced levels of the

p53

protein. No increase of the

p53

steady-state level was observed in dexamethasone-treated PS cells compared with

that in the untreated controls. This is consistent with the

control dex.

SW 756

P1

P2

P3

P4

SW

756

SW

756

dex. (P4).

E6-E7neg.

SW 756 dex.

(P5)

E6-E7 pos.

FIG. 5. Growthcharacteristicsof SW 756cellswithorwithout HPV-18 E6-E7 geneexpression.Cells

(103)

wereculturedinmedium either with

orwithoutdexamethasone(dex.) (1 ,uM).After 2weeks,cellswerefixedand stainedasdescribedinMaterialsandMethods.

Single

colonies of stained cellswerephotographed.neg.,negative;pos.,positive.

PS

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111

control

m

dexamethasone

(1 ,uM)

control dex.(11JM)

SW

756

P4

P5

[image:6.612.70.552.86.308.2]

SW 756 P1 P2 P3 P4 PS

FIG. 6. Anchorage-independent growth of SW 756 cells withorwithout HPV-18E6-E7geneexpression. Cells (103)wereseededin softagar

and cultured for 4 weeks. The numbers of outgrown colonies weredetermined in triplicate experiments, and the standarddeviations were

calculated.dex., dexamethasone.

finding that dexamethasonetreatmentof the clone P5 doesnot result in reducedexpression of the viraloncogenes.

QuantitativeNorthern blot analysisofp53 mRNAlevels in therespectiveclones revealed similar levels of thep53 mRNA, independent of E6-E7 expressionlevelorhormonetreatment (Fig. 8b). This suggeststhat thetranscriptional control of the p53gene isnotaffected in these cells.Therefore, alteredp53 levels observed herearemostlikelyinducedby posttranscrip-tionalregulatory pathways.This observationstronglysupports the hypothesis that the HPV E6 gene product continuously stimulates thedegradationofwtp53in HPV-linkedanogenital

cancercells.

liicontr

20000 dexar

18000

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-

14000-E

12000-C.) 8000

6000-

4000-2000

-Gel shift analysis of E2F-protein complexes in SW 756-derived cells with inducible HPV-18 E7expression.Toanalyze whether growth regulation by the papillomavirus genes

re-quires theE7-mediated release of the E2Ftranscriptionfactor from itscomplexwithpRB, thestateofthe pRB-E2F complex

wasanalyzed ingel retardation assays (Fig. 9). Extractswere

preparedfromP4andP5 cells,growneither in thepresence or

in the absence of dexamethasone, and incubated with a

32P-labelled oligonucleotide encompassing the E2F

recogni-tion sequenceof theadenovirus E2promoter (40).The band shift pattern obtained with extracts from P4 cells, in which expressionof E7wasrepressed by dexamethasone, revealeda

ol

rethasone

(1

IAM)

SW 756 P1 P2

P3

P4

P5

FIG. 7. Proliferationrateof SW 756 cellseither withorwithoutHPV-18 E6-E7geneexpression. Cellswereseeded in 96-well tissue culture

plates and incubated in the absence or presence of dexamethasone for 2 weeks. [3H]thymidine (0.2 jiCi)was added to each well, and the

incorporatedhigh-molecular-weight radioactivitywasdetermined 48 h later.Foreach cell line either withorwithoutdexamethasone treatment, 24valuesweredeterminedand the standard deviationswerecalculated.

U) 0

L-o 120

Ca

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100

a

80

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a

P4

SW 756

P5

abcde acde abcde

;I;I;£Al"i

E7

p53

b

0

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10 10 0 0

a.

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p53

FIG. 8. (a)Westernblot analysisofwtp53in SW 756 cellswithor

without HPV-18 E6-E7 gene expression. Cells were cultured in the

presence of dexamethasone (1 jiM) for2weeks (lanes a).

Dexameth-asone wasthenwithdrawnandextractswerepreparedafter4h (lanes

b), 24 h (lanes c),or48h(lanes d). Cells whichwerenottreatedwith

dexamethasone (lanes e) served as controls. Protein extracts were

subjectedtoWesternblot analysis, and thep53 proteinwasvisualized

by the monoclonal antibody 1801 (AB 2; Dianova). The rate of HPV-18 E6-E7 gene expression was determined by incubating the

same filters with apolyclonal rabbit anti-HPV-18 E7 antiserum (51).

(b) Northern blot analysis of the p53 mRNA steady-state level.

Cytoplasmic RNA (10,ug) isolated from cells cultured for 2 weeks

either in thepresence orin the absence ofdexamethasone(dex.)were

electrophoresed in a 1% agarose gel and blotted onto GeneScreen

Plus nylon membranes(NEN DuPont). Thefilterwashybridized with

a32P-labelledwtp53cDNAprobe (kindlyprovided by R. Klein).

complex pattern of retarded bands. The four more slowly

migrating complexes involve binding of E2F, since these complexes are selectively reduced by competition with

unla-belledwt E2Foligonucleotide but notwith amutated version

of this fragment which has been shown previously to be

deficient for E2F

binding

(40). To detect cellular proteins

complexed

to E2F in this extract, specific antibodies were addedtothe band shift reaction. IfE2F-associated proteinsare

recognized by

theantibodies,the retardedband will befurther

shifted

by

the bound antibody (supershift). Alternatively, if

binding

ofthe

antibody

interfereswithformationof

multipro-teincomplexes, such complexes are selectively eliminated, as wasshownpreviouslyforcyclinA-E2Fcomplexes fromseveral human celllines(40).In extractsfromdexamethasone-treated

P4

cells,

one complex is specifically supershifted by a

pRB-specific antibody.

Furthermore, the complex with the lowest

mobility

is eliminatedby additionof a polyclonal antibodyto

cyclin

A. These results indicate that in the absence of E7

proteins, E2F is complexed to pRB and cyclin A in

dexa-methasone-treated P4 cells. Similar results were previously obtained for primary human fibroblasts (40). To analyze the

influence of E7 expression on E2F complexes, extracts were alsoprepared from untreated P4 cells and either dexametha-sone-treated cells or untreated P5 cells, which all express similar high levels of the HPV-18 E7 oncoprotein (Fig. 4).

These extractsyielded very similar band shift patterns com-paredwiththat ofdexamethasone-treatedP4cells, which lack the E7protein. Inparticular, complexesofE2F with cyclin A andpRBwere observed also in this case, although these cells express high levels of theHPV-18 E7 protein. Densitometric

analysisof the autoradiogram shown in Fig. 9suggested that the relative ratio ofDNA-protein complexes which contain

only free E2F are about 40% reduced in E7-deficient cells

(dexamethasone-treated P4 cells)compared with that in cells which express the E7 protein (P4, P5, and P5 dex.). However, inrepeated experiments, this reduction rate variedfrom noor only slight reduction to about 40%, as shown in Fig. 9. Persistence of E2F-pRB complexes and E2F-cyclin A com-plexes, independentofHPV-18 E7 expression, in allanalyzed

clones suggests that E7 does not induce acomplete release of E2F from its complex with pRB and cyclin A in the SW 756-derived cervical carcinoma cells. However, a partial E7-mediated release of E2F from pRB cannot be excluded on the basis of the gel retardation experiments.

DISCUSSION

Humananogenital carcinomas can arise afterlong-standing

persistent infections by high-risk types of the HPVs. The consistentexpression of the E6-E7 genes suggests that molec-ular functions exerted by gene products encoded by one of these genesorbothplay a key role notonly in the initiation but also in the maintenance of the neoplasticphenotype of HPV-associated carcinoma cells. In vitro studies indicated that both genes have transforming properties for avariety of cultured cells.However, transformation of different host cells is possibly due to different molecular mechanisms. The E7 gene, for example, is sufficient for the malignant transformation of established rodent cells (30, 41,63) but notofprimaryrodent orhumancells, yet it is expressed inextremely high levels (20, 26, 37). For transformation of rodent cells, the pRB-binding domain of E7 appears to be important, whereas for transfor-mation of primary human keratinocytes this domain may be dispensable (29, 44). The E6 gene is sufficient toimmortalize primary human mammaryepithelialcells(3),but it doesnotby itself transform primary cervical epithelial cells. This also raises the possibility that molecular mechanisms involved in immortalization of keratinocytes act differently from those which are required to maintain the neoplastic

phenotype

of cervical cancer cells.

Herein we present a cell culture system in which the

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P4(E7-poS.) P4dex.(E7-neg.) P5(E7-pos.)

E 4j1

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L.

wLC\ \l- \j

b

.w

M

P5 dex.(E7-pos.)

6 8 x

t- 0

;_- Z Cl. c

K a 1-= co u

E E 0

0 0 Y C. 8.

u Y u- ;:-cv c c

t ui m m

.f--E2F-cyclinA-complex

-E2F-RB-complex

; -freeE2F

i i

FIG. 9. Gel shiftanalysis ofE2F-multiprotein complexes.Extractswereprepared fromgrowingP4and P5 cellswhichwerecultured either in the absence or in the presence of dexamethasone (dex.; 1 ,uM) for 2 weeks. Extracted protein (8 pLg) was incubated with labelled E2F oligonucleotide and analyzedin thebandshiftassay. Specificityofcomplexeswas determinedbyaddition ofa20-foldexcessofwt or mutant oligonucleotide, asindicated. freeE2F, complexes offree E2F bound to the oligonucleotide. Complexes containing cyclinA and pRBwere

identifiedby addition ofspecificantibodies. Theirpositionsinthegelare indicated.

expressionof thepapillomavirus oncogenesE6 and E7canbe

blocked bydexamethasone in the cervical carcinoma cell line SW 756. Dexamethasone-mediatedrepressionof the viralgene

expressionresulted in loss of theneoplastic phenotypeof these cervical carcinoma cells. If the expression of viral genes is restored either bywithdrawal of dexamethasone or bystable introduction ofavectorpermittingtheexpressionof the viral

genes in dexamethasone-treated SW 756 cells, the neoplastic growthpropertiesarerestored. Thephenotypeofthe SW 756

subclones P1 andP5,described in thisreport,is consistent with the statement that growth properties of these cervical

carci-nomacells are indeed modulated bythe viraloncogenes and are not related to dexamethasone treatment. This clearly indicates that the continuous expression of the viral E6-E7

genesisessential for theneoplastic growthofHPV-associated

cervical carcinoma cells, even when they were cultured for

decades intissueculture. Thisresult furthersupportsprevious observations indicating that inhibition of HPV oncogene

ex-pression by antisense RNAresults in severe reduction of the

growth and loss of tumorigenicity of cervical carcinoma cells (54, 61, 62).

Recentbiochemical studiesrevealed that both transforming viral gene products can interact with proteins involved in

cellulargrowth regulation (38). The E6 protein stimulates the degradation of the cellular p53 tumor suppressor protein in

vitro (7, 27, 46). In HPV-16-immortalized human keratino-cytes,onlysmallamountsofwtp53withareduced half-lifecan

bedetected(25). Hence, it hasbeenproposedthatfunctional inactivationofwtp53 by the viralE6proteincontributestothe transformation of cervical keratinocytes (24). However, the

role ofcontinuousdepletionofp53 for the maintenance of the transformedstateof cervicalcancercells hasnotbeen assessed sofar. Theextremelylow level of thep53proteinin HPV-18-associated HeLa cervical carcinoma cells (36) favors the hypothesis that p53 might be continuously degraded by E6 even after many years of tissue culture passage to permit continuousgrowth of thecancercells. The datareportedhere demonstrate a direct inverse correlation between the level of the p53 tumor suppressor protein and the expression of the HPV E6-E7 genes in cells derived from a human cervical carcinoma.Weobservedasignificantincrease ofwtp53 levels inSW 756 cervical carcinoma cells in which viral gene expres-sion has been blocked by dexamethasone. Withdrawal of dexamethasone results in reexpression of the viral oncogenes and reduced levels ofp53. In cells in which viral gene expres-sion isnotblockedby dexamethasone (clone P5), no increase ofp53 is detectable, indicating that increased p53 levels are not an inherent effect of dexamethasone treatment. The specific detection of theHPV-18 E6protein in cervical carcinoma cell linesbyimmunoblotting or immunoprecipitation experiments is still hampered by technical problems, since the available antibodies cross-react with a similar-size protein which is ubiquitously expressed in human keratinocytes (43). However, the direct correlation between E6-E7 mRNA levels and re-duced levels of the p53 protein observed in the experiments reported here indicates that p53 is down regulated by HPV gene expression. Since the levels of p53 mRNA were not

changedinthe cellsinvestigatedhere,weconclude thatp53 is regulated at a posttranscriptional level, most likely by E6-mediated destabilization.

u

3 cn m

I

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Recently, it has been shown that E6 disrupts the p53-mediated cellular response to DNA damage in cells in which the HPV-16 E6 gene has been artificially introduced (33). These data suggest that the E6-stimulated degradation of p53 favors the accumulation of mutations within the host cell genome and thereby renders E6-expressing cells at a higher risk for further genetic damage. Thus, the continuous degra-dation of wt p53 by E6 may not contribute only to the early steps ofcarcinogenesis. Our data support the hypothesis that decreased levels of p53 in fully transformed cervical cancer cells might represent an important factor for continuous tumor progression driving the E6-expressing cells in more malignant

phenotypes (67).

The growth properties of the cells described here were clearlycorrelated with the level of HPV gene expression and thus also with reduced levels of p53. It is therefore attractive to speculate that reduced growth of the dexamethasone-treated SW 756 or P4 cells is due to increased levels of endogenous p53. However, this question requires further investigation.

The E7 gene of the high-risk HPVs represents the major transforming function in transformation experiments of either rodent or human primary cells (20, 30, 41, 63). In recent studies, it has been shown that the E7 protein can complex with various cellular proteins including the retinoblastoma tumor suppressor protein (pRB) (11, 15, 38, 55). Mutational analysis of the E7 protein indicated that binding to the pRB protein is critical for its transforming functions in rodent cells (21, 44). Binding of E7 to pRB has been reported to disrupt the complex of pRB with the transcription factor E2F. In fact, a complete release of the E2F transcription factor from its binding to pRB has been observed in HPV-immortalized

keratinocytesand in the HPV-associated cell line HeLa (40). E2F regulates the expression of several growth-promoting genes, andrelease of E2F from its complex with pRB leads to

transcriptionalactivation of these genes (reviewed in reference 39). Hence, it has been proposed that E7-mediated release of increased amounts of E2F from its complex with pRB may represent animportant molecular aspect of the transforming functions of this protein in anogenital carcinogenesis. How-ever,recentstudies suggested that binding of E7 to pRB is not essential for the immortalization of primary human keratino-cytes(29). Analysis of the pRB-E2F transcription factor com-plex in SW 756 cervical carcinoma cells by gel retardation assays revealed that pRB-E2F complexes are relatively stable in SW 756-derived cells, independent of the level ofE7. The relative intensity ofDNA-protein complexes containing only free E2F wasslightly reduced in SW 756-derived cells, which do notexpress the HPV-18 E7 protein (P4 dex.). However, in contrast to HPV-18 E7-expressing HeLa cells or HPV-16-immortalized primary human keratinocytes, a significant

pro-portion of E2F was still complexed with pRB in HPV-18 E7-positive, SW 756-derived cells. These results suggest that the complete E7-mediated disruption of the pRB-E2F com-plex may not be critical for the neoplastic phenotype of

HPV-18-associated SW 756 cervical cancer cells. Whether a partialE7-mediated release of E2F from its complex with pRB eventuallycontributes to the growth control activity of the viral oncogeneproducts in the SW 756-derived cells requires fur-therinvestigation.

Using specific mutants of the E6 and E7 genes, the experi-mental system described here will be useful to delineate

domainswithin the E6 and E7 proteins which are involved in growth regulation of HPV-associated cervical cancer cells. Suchexperiments might help to define at the molecular level thefunctions which are essential for the continuousgrowthof

cervical carcinoma cells and therefore could provide a molec-ular basis for targeted therapeutic interventions.

ACKNOWLEDGMENTS

We thank H. zur Hausen for stimulating discussions and critical review of the manuscript. Critical reading of the manuscript by Martin Scheffner is gratefully acknowledged. Oligonucleotides used in this study were kindly provided by H. J. Delius.

This work was in part supported byaBMFT grant of the Verbund klinisch-biomedizinische Forschung to M.V.K.D. and a grant of the EC program Human capital and mobility to P.J.-D.

REFERENCES

1. Androphy, E. J., N. L. Hubbert, J. T. Schiller, and D. R. Lowy. 1987. Identification of the HPV-16 E6 protein from transformed mouse cells and human cervical carcinoma cell lines. EMBO J. 6:989-992.

2. Baker, C. C., W. C. Phelps, V. Lindgren, M. J. Braun, M. A. Gonda, and P. M. Howley. 1987. Structural and transcriptional analysis of human papillomavirus type 16 sequences in cervical carcinoma cell lines. J. Virol. 61:962-971.

3. Band, V., J. A. De-Caprio, L. Delmolino, V. Kulesa, and R. Sager. 1991. Loss of p53 protein in human papillomavirus type 16 E6-immortalized human mammary epithelial cells. J. Virol. 65: 6671-6676.

4. Barbosa, M. S., C. Edmonds, C. Fisher, J. T. Schiller, D.R.Lowy, andK.H. Vousden. 1990. The region of the HPV E7 oncoprotein homologous to adenovirusElAand SV40 large T antigen contains separate domains for rb binding and casein kinase ii phosphory-lation. EMBO J. 9:153-160.

5. Barbosa, M. S., D. R. Lowy, and J. T. Schiller. 1989. Papilloma-virus polypeptides E6 and E7 are zinc-binding proteins. J. Virol. 63:1404-1407.

6. Chellappan, S., V. B. Kraus, B. Kroger, K.Munger,P. M. Howley, W. C. Phelps, and J. R. Nevins. 1992. Adenovirus ElA, simian virus 40 tumor antigen, and human papillomavirus E7 protein share the capacity to disrupt the interaction between transcription factor E2F and the retinoblastoma gene product. Proc. Natl. Acad. Sci. USA 89:4549-4553.

7. Crook, T., J. A. Tidy, and K. H. Vousden. 1991. Degradation of p53 can be targeted by hpv E6 sequences distinct from those required for p53 binding and trans-activation. Cell 67:547-556.

8. Crook, T., D. Wrede, J. Tidy, J. Scholefield, L. Crawford, and K. H. Vousden. 1991. Status of c-myc, p53 and retinoblastoma genes in human papillomavirus positive and negative squamous cell carci-nomas of the anus. Oncogene 6:1251-1257.

9. Crook, T., D. Wrede, J. A. Tidy, W. P. Mason, D. J. Evans, and K. H. Vousden. 1992. Clonal p53 mutation in primary cervical cancer: association with human-papillomavirus-negative tumours. Lancet 339:1070-1073.

10. Cullen, A. P., R. Reid, M. Campion, and A. T. Lirincz. 1991. Analysis of the physical state of different human papillomavirus DNAs in intraepithelial and invasive cervical neoplasm. J. Virol. 65:606-612.

11. Davies, R., R. Hicks, T. Crook, J. Morris, and K. Vousden. 1993. Human papillomavirus type 16 E7 associates with a histone Hi kinase and with p107 through sequences necessary for transfor-mation. J. Virol. 67:2521-2528.

12. Durst, M., R T. Dzarlieva-Petrusevska, P. Boukamp, N. E. Fusenig, and L. Gissmann. 1987. Molecular and cytogenetic analysis of immortalized human primary keratinocytes obtained after transfection with human papillomavirus type 16 DNA. Oncogene 1:251-256.

13. Durst, M., A. Kleinheinz, M. Hotz, and L. Gissman. 1985. The physical state of human papillomavirus type 16 DNA in benign and malignant genital tumours. J. Gen. Virol. 66:1515-1522. 14. Dyson, N., P. Guida, K.Munger,and E. Harlow. 1992.

Homolo-gous sequences in adenovirusEIAand human papillomavirus E7 proteins mediate interaction with the same set of cellular proteins. J.Virol.66:6893-6902.

15. Dyson, N., P. M. Howley, K.Munger,and E. Harlow. 1989. The human papillomavirus-16 E7 oncoprotein is able to bind to the

on November 9, 2019 by guest

http://jvi.asm.org/

(10)

retinoblastoma geneproduct. Science 243:934-937.

16. Firzlaff,J. M.,D. A. Galloway,R. N. Eisenman, andB. Luscher. 1989. The E7 protein of human papillomavirus type 16 is phos-phorylated bycasein kinase II. New Biol. 1:44-53.

17. Freedman, R. S., J. M. Bowen, A. Leibovitz, S. Pathak, M. J. Siciliano, H.S.Gallager, and B.C. Giovanella. 1982. Character-ization ofacell line (SW 756)derivedfrom a humansquamous cellcarcinomaof the uterine cervix. In Vitro 18:719-726. 18. Grossman,S.R.,and L. A.Laimins. 1989. E6 proteinof human

papillomavirus type 18 binds zinc. Oncogene4:1089-1093. 19. Grossman,S.R.,R.Mora,andL. A.Laimins. 1989. Intracellular

localization and DNA-binding propertiesof human

papillomavi-rus type 18 E6 protein expressed with a baculovirus vector. J. Virol.63:366-374.

20. Halbert, C.L.,G. W.Demers,andD. A.Galloway.1991.The E7 geneof human papillomavirustype 16is sufficient for immortal-ization of humanepithelialcells. J. Virol. 65:473-478.

21. Heck, D. V., C. L. Yee, P. M. Howley, and K. Munger. 1992. Efficiency ofbinding the retinoblastoma proteincorrelates with the transforming capacity of the E7oncoproteins of the human papillomaviruses.Proc.Natl. Acad. Sci. USA 89:4442-4446. 22. Hoppe-Seyler, F.,and K. Butz. 1993. Repression ofendogenous

p53transactivation function in HeLacervical carcinoma cellsby humanpapillomavirustype 16E6,humanmdm-2,andmutantp53. J.Virol. 67:3111-3117.

23. Hoppe-Seyler, F., K. Butz, C. Rittmuller, and M. von Knebel Doeberitz. 1991. A rapid microscale procedure forthe

simulta-neous preparation ofcytoplasmic RNA, nuclear DNA binding proteinsandenzymaticallyactive luciferaseextracts.Nucleic Acids Res. 19:5080.

24. Howley,P.M.,M.Scheffner,J. Huibregtse,and K.Munger.1991. Oncoproteins encoded by the cancer-associated human papillo-maviruses target the products of the retinoblastoma and p53

tumorsuppressor genes. ColdSpringHarborSymp. Quant. Biol. 56:149-155.

25. Hubbert, N.L., S. A. Sedman,andJ.T. Schiller. 1992. Human papillomavirustype 16 E6increases thedegradationrateofp53in humankeratinocytes. J.Virol. 66:6237-6241.

26. Hudson, J. B.,M. A.Bedell,D.J. McCance,and L. A. Laimins. 1990. Immortalization and altereddifferentiationof human kera-tinocytesinvitrobythe E6 and E7 openreadingframes of human papillomavirustype18. J. Virol. 64:519-526.

27. Huibregtse, J. M., M. Scheffner, and P. M. Howley. 1991. A cellularproteinmediates association ofp53withthe E6 oncopro-teinof humanpapillomavirus types 16or 18.EMBO J. 10:4129-4135.

28. Inagaki,Y.,Y.Tsunokawa,N.Takebe,H.Nawa,S.Nakanishi,M.

Terada,andT.Sugimura. 1988.Nucleotide sequences of cDNAs for human papillomavirus type 18 transcripts in HeLa cells. J. Virol.62:1640-1646.

29. Jewers,R. J., P. Hildebrandt, J. W. Ludlow, B.Kell, and D.J. McCance. 1992. Regions of human papillomavirus type 16 E7 oncoprotein requiredforimmortalizationof humankeratinocytes. J.Virol. 66:1329-1335.

30. Kanda, T.,A.Furuno,and K.Yoshiike. 1988. Human papilloma-virus type 16 openreadingframe E7 encodesatransforming gene forrat3Y1 cells.J.Virol. 62:610-613.

31. Kaur, P.,andJ.K.McDougall. 1988. Characterization of primary human keratinocytestransformedbyhuman papillomavirus type 18. J.Virol.62:1917-1924.

32. Kawasaki, E. 1990. Amplification of RNA, p. 21-27. In M. A. Innis, D. H. Gelfand, J. J. Sminsky, and T. White (ed.), PCR protocols:aguidetomethods andapplications.AcademicPress, Inc.,NewYork.

33. Kessis, T. D., R.J. Slebos,W. G. Nelson, M. B. Kastan, B. S. Plunkett, S.Han,A. T.Lorincz, L. Hedrick, and K. R. Cho. 1993. Human papillomavirus 16 E6 expression disrupts the p53-medi-ated cellular response to DNA damage. Proc. Natl. Acad. Sci. USA90:3988-3992.

34. Le,J.-Y.,and V.Defendi. 1988. Aviral-cellularjunction fragment fromahumanpapillomavirustype16-positivetumor iscompetent intransformationof NIH 3T3cells. J. Virol.62:4420-4426. 35. Matlashewski, G., L. Banks, D. Pim, and L. Crawford. 1986.

Analysis of humanp53 proteins and mRNA levels in normal and transformed cells. Eur. J. Biochem. 154:665-672.

36. May, E., J.R.Jenkins,and P.May.1991.Endogenous HeLap53 proteinsareeasily detected in HeLa cells transfected withmouse

deletionmutantp53gene. Oncogene6:1363-1365.

37. Munger, K.,W.C.Phelps,V.Bubb,P. M.Howley,and R.Schlegel. 1989.The E6 and E7 genes of the humanpapillomavirus type 16 togetherarenecessaryand sufficient for transformation ofprimary human keratinocytes. J. Virol. 63:4417-4421.

38. Monger, K., M. Scheffner, J.M.Huibregtse, andP. M.Howley. 1992. Interactions of HPV E6 and E7oncoproteinswithtumour

suppressor geneproducts.Cancer Surv. 12:197-217.

39. Nevins, J. R. 1992. A link between the Rb tumor suppressor protein and viraloncoproteins. Science258:424-429.

40. Pagano, M., M.Durst, S.Joswig,G.Draetta,and P.Jansen-Durr. 1992. Binding of the human E2F transcription factor to the retinoblastoma protein but not tocyclin a is abolished in HPV-16-immortalized cells.Oncogene 7:1681-1686.

41. Phelps, W. C., C. L. Yee, K. Monger, and P. M. Howley.1988. The human papillomavirus type 16 E7 gene encodes transactivation andtransformation functions similar to those of adenovirus ElA. Cell53:539-547.

42. Pirisi, L., S. Yasumoto, M. Feller, J. Doniger, and J. A. DiPaolo. 1987.Transformation of human fibroblasts and keratinocytes with human papillomavirus type 16 DNA. J. Virol. 61:1061-1066. 43. Roggenbuck, B., P.M.Larsen,S. J. Fey, D. Bartsch, L.Gissmann,

and E.Schwarz. 1991. Humanpapillomavirus type 18 E6*, E6, and E7proteinsynthesis in cell-free translation systems and compari-son of E6 and E7 in vitro translation products to proteins immunoprecipitated from human epithelial cells. J. Virol. 65: 5068-5072.

44. Sang,B.C., and M. S. Barbosa. 1992.Single amino acid substitu-tions in "low-risk" humanpapillomavirus (HPV) type 6 E7protein enhancefeatures characteristic of the "high-risk" HPV E7 onco-proteins.Proc.Natl. Acad. Sci. USA 89:8063-8067.

45. Scheffner,M., K. Monger, J. C. Byrne, and P. M. Howley. 1991. The state of the p53 and retinoblastoma genes in human cervical carcinoma cell lines.Proc.Natl. Acad. Sci. USA88:5523-5527. 46. Scheffner, M., B. A. Werness, J. M. Huibregtse, A. J. Levine, and

P. M. Howley. 1990. The E6 oncoprotein encoded by human papillomavirustypes 16 and 18promotes the degradation of p53. Cell 63:1129-1136.

47. Schlegel, R., W. C. Phelps, Y. L. Zhang, and M. Barbosa. 1988. Quantitative keratinocyte assay detects two biological activities of humanpapillomavirus DNA and identifies viral types associated with cervical carcinoma. EMBO J. 7:3181-3187.

48. Schneider-Gaedicke, A., and E. Schwarz. 1986. Different human cervical carcinoma cell lines show similar transcription patterns of human papillomavirus type 18 early genes. EMBO J. 5:2285-2292. 49. Schneider-Maunoury, S., 0. Croissant, and G. Orth. 1987. Inte-gration of human papillomavirus type 16 DNA sequences: a possibleearly event in the progression of genital tumors. J. Virol. 61:3295-3298.

50. Schwarz, E., U. K. Freese, L. Gissmann, W. Mayer, B. Roggen-buck, A. Stremlau, and H. zur Hausen. 1985. Structure and

transcription of human papillomavirus sequences in cervical car-cinoma cells.Nature (London)314:111-114.

51. Seedorf, K., T. Oltersdorf, G. Kraemmer, and W. Roewekamp. 1987. Identification of early proteins of the human papilloma viruses type 16 (HPV 16) and type 18 (HPV 18) in cervical carcinoma cells. EMBO J. 6:139-144.

52. Shirasawa, H., Y. Tomita, A. Fuse, T. Yamamoto, H. Tanzawa, S. Sekiya, H. Takamizawa, and B. Simizu. 1989. Structure and expressionof an integrated human papillomavirus type 16 genome amplifiedin a cervical carcinomacell line. J. Gen. Virol. 70:1913-1919.

53. Smotkin, D., and F.0.Wettstein. 1986. Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancer-derived cell line and identification of the E7 protein. Proc. Natl. Acad. Sci. USA83:4680-4684.

54. Steele, C., P. G. Sacks, K. Adler-Storthz, and E. J. Shillitoe. 1992. Effecton cancercells of plasmids that express antisense RNA of human papillomavirus type 18. Cancer Res.52:4706-4711.

on November 9, 2019 by guest

http://jvi.asm.org/

(11)

55. Tommasino, M., J. P.Adamczewski, F. Carlotti, C. F. Barth, R. Manetti, F. Contorni, F. Cavalieri, T. Hunt, and L. Crawford. 1993. HPV 16 E7 protein associates with the protein kinase p33CDK2 and cyclin A. Oncogene 8:195-202.

56. von Knebel Doeberitz, M. 1992. Papillomaviruses in human

dis-ease.I.Pathogenesis and epidemiology of human papillomavirus

infections. Eur. J. Med. 1:415-423.

57. vonKnebel Doeberitz, M. 1992. Papillomaviruses in human

dis-ease. II. Molecular biology and immunology of papillomavirus

infections andcarcinogenesis. Eur.J.Med. 1:485-491.

58. vonKnebel Doeberitz, M., T.Bauknecht, D. Bartsch, and H.zur

Hausen.1991. Influence ofchromosomal integrationon

glucocor-ticoid-regulated transcription ofgrowth-stimulating

papillomavi-rusgenesE6 and E7 incervical carcinoma cells. Proc. Natl. Acad. Sci.USA 88:1411-1415.

59. vonKnebelDoeberitz, M., L. Gissmann, and H.zurHausen.1990. Growthregulating functions of human papillomavirus early

pro-teins in cervical cancer cells acting dominant over enhanced

epidermal growth factor receptor expression. Cancer Res. 50: 3730-3736.

60. von Knebel Doeberitz, M., S. Koch, H. Drzonek, and H. zur

Hausen.1990. Glucocorticoid hormones reduce the expression of

majorhistocompatibility class I antigensonhumanepithelial cells.

Eur. J. Immunol. 20:35-40.

61. von Knebel Doeberitz, M., T. Oltersdorf, E. Schwarz, and L. Gissmann. 1988.Correlation of modified human papilloma virus early gene expression with altered growth properties in C4-1 cervical carcinoma cells. Cancer Res. 48:3780-3786.

62. vonKnebelDoeberitz, M., C. Rittmuller, H.zurHausen,andM. Durst.1992.Inhibition of tumorigenicity of cervicalcancercells in nude mice by HPVE6-E7 antisense RNA. Int. J. Cancer 51:831-834.(Letter.)

63. Vousden, K. H., J. Doniger, J. A. DiPaolo, andD. R.Lowy. 1988. The E7 open reading frame of human papillomavirus type 16 encodesatransforminggene.OncogeneRes.3:167-175. 64. Werness,B.A., A. J. Levine, andP. M.Howley.1990.Association

ofhumanpapillomavirus types 16 and 18 E6 proteinswithp53.

Science 248:76-79.

65. Whyte, P. K., J. Buckovich, S. Korowitz,M.Friend, R. Raybuck,R. Weinberg,and E.Harlow. 1988.Associationbetweenanoncogene

and anantioncogene: the adenovirus ElAproteins bind to the retinoblastomageneproduct. Nature (London) 334:124-129.

66. Yee,C., I. Krishnan-Hewlett,C.C.Baker,R.Schlegel,and P.M. Howley. 1985. Presence and expression of humanpapillomavirus

sequences in humancervical carcinoma cell lines. Am. J. Pathol. 119:361-366.

67. zurHausen,H. 1991. Humanpapillomavirusesin thepathogenesis ofanogenitalcancer.Virology 184:9-13.

on November 9, 2019 by guest

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Figure

FIG.1.2treated weeks. Morphology of the SW 756 cervical carcinoma cells before (left picture) and after dexamethasone (dex.) treatment (right picture) for Northern blot analysis of the HPV-18 E6-E7 mRNA demonstrated the absence of detectable HPV-18 E6-E7 mRNA in dexamethasone- SW 756 cells (58).
FIG. 2.enclosessignalIttumor was pM7 expression vector. The 7-23 cDNA clone (48) which the full-length E6 ORF, the E7 ORF, and a polyadenylation within the cellular sequences was isolated from the SW 756 cells
FIG. 4.polyclonalthedexamethasone Western blot analysis of the HPV-18 E7 protein expression of SW 756, P4, and P5 cells cultured either in the presence (lanes a) or in absence (lanes e) of dexamethasone (1 F.M) for 2 weeks
FIG. 7.plates24incorporated values Proliferation rate of SW 756 cells either with or without HPV-18 E6-E7 gene expression
+3

References

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