Modulation of cyclin gene expression by adenovirus E1A in a cell line with E1A-dependent conditional proliferation.

Copyright C) 1994, American Society for Microbiology

Modulation of

Cyclin Gene

Expression

by Adenovirus ElA in

a

Cell Line with ElA-Dependent Conditional Proliferation

DIMITRY SPITKOVSKY,' PHILIPP STEINER,2 JIRI LUKAS,3 EMMA LEES,4 MICHELEPAGANO,S

ALMUT

SCHULZE,'

SILVIAJOSWIG,1 DIDIER PICARD,6MASSIMOTOMMASINO,7

MARTIN EILERS,2ANDPIDDERJANSEN-DURRl*

Angewandte Tumorvirologie, DeutschesKrebsforschungszentrum,1 and Zentrumfur MolekulareBiologie,2D-69120 Heidelberg, Germany; Divisionfor CancerBiology, Danish Cancer Society Research Center, DK-2100Copenhagen,

Denmark3;Massachusetts GeneralHospital Cancer Center Boston, Massachusetts

021294;

Mitotix, Cambridge, Massachusetts021395;DepartmentdeBiologieCellulaire, UniversitedeGeneve, CH-1211 Geneve 4,

Switzerland6; and Tumor VirusGroup, ImperialCancer ResearchFund, Cambridge CB21QP,

UnitedKingdom7

Received 1October 1993/Accepted 27 December 1993

Toinvestigate howadenovirus ElA controls cell proliferation,wehave fusedElAto the hormone-binding domain of the human estrogen receptor (ER) and introduced the EIA-ER chimeric gene together with an

activatedrasgeneintoprimaryratembryofibroblasts. Cell linesderived from thistransfection proliferate in

an estrogen-dependent manner. Estrogen-dependent activation of ElA-ER led to a rapidinduction of both

cyclin Eand cyclinAgeneexpression. Incontrast,levels ofcyclin Dl were stronglyreduced by activation of ElA-ER. Similar changes in cyclin gene expression were observed when primary human fibroblasts were

infected with wild-type adenovirus and when adenovirus ElA was stably expressed in NIH 3T3 cells. Our

findings suggest that activation ofcyclinA and E,but not Dl, gene expression byElAprecedesandmaybe

responsible for ElA-dependent cell proliferation. In contrast, we found that quantitative disruption of

complexes between the E2F transcription factor and the retinoblastoma protein is not required for

ElA-dependent S-phaseentry.

Recent evidence suggests a correlation between the devel-opment of certain cancers and infection with different DNA tumorviruses (for a review, see reference 42). One character-istic property of these viruses is their ability to stimulate proliferation in quiescent cells; to carryoutthisfunction, they have evolved specific genes, such as the E7 gene of human papillomaviruses (35 and references therein), the large T

antigen of simian virus 40 (reviewed in reference 9), and the ElA gene of adenovirus (see below). The deregulation of cell cycle control achieved by such oncogenes may contributetothe immortalization and transformation of mammalian cells by theseviruses.

The mechanism(s) by which these genes override cell cycle control is largely unknown. Adenovirus ElA protein may interfere with growth control by physically interacting with several cell cycle control proteins (39), including the retino-blastoma protein (pRB), p107, cyclin A, cyclin E, and cdk2

(reviewed in reference 7). The interaction between EIA and pRB (38) has attracted particular attention, as pRB has been shownto negatively regulate cell cycle progression during the G1 phase (10). One intracellular target for pRB is the E2F transcription factor (4, 5). E2F is involved in the control of several genes required for cell proliferation (reviewed in reference 23). In untransformed mammalian cells, E2F is complexedtopRB(2), and the activity of E2F is repressed by pRB in transient cotransfection experiments (11, 12, 41). Binding of ElA to pRB can disrupt the association of pRB

with E2Fin vitro (2). Disruption of pRB-E2F complexes by

*Corresponding author. Mailing address: Angewandte Tumorvi-rologie, Abteilung 620, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 242, D-69120 Heidelberg, Germany. Phone: 49-6221-424628. Fax: 49-6221-424902.

ElAmight, therefore, contribute to the stimulation of DNA

synthesis bythe viral oncoprotein.

Progression through the mammalian cell cycle is thought to

be regulated by a set of related protein kinases, termed

cyclin-dependent kinases(cdkgenefamily [20, 36]),and their regulatorysubunits,termedcyclins (reviewedin reference32). Several classes of cyclins which differ in the timing of their

expression in the cell cycle have been identified (for reviews,

seereferences6, 22, 26, and34).Evidence thatcyclins regulate

cell cycle progression isprovidedby the observations (i) that injection of antibodies to several cyclins blocks progression through the cell cycle (1, 29), (ii) that overexpression of any

oneof thecyclingenesA,Dl,orE can overcomethe cellcycle blockimposed by theRBgene(13), and (iii) that overexpres-sion ofcyclinE(25)aswellascyclinDl(33)canaccelerate the

G,

phase in mammalian fibroblasts and alter their growth factorrequirements.

Since there isample evidence that adenovirus ElA can act as atranscriptionfactor(forarecentreview, see reference 24), it ispossible that cell cycle entry may be correlated to specific changesofcyclin gene expressionmediatedby EIA. However, suchchangesare notdocumented; inparticular, it is not clear fromprevious data which cyclin genes, if any, may be targets forElA.Toaddressthis question, primary rat fibroblasts were transfected with the EJ ras oncogene and a second vector encoding a chimeric protein (ElA-ER), in which the ElA protein was rendered conditionally active by fusing it to the hormone-binding domain of the human estrogen receptor

(ER). Wefound that in cells expressing the chimeric protein ElA-ER (IREE-1 cells), proliferation is dependent on the addition of the appropriate steroid hormone. Expression of both thecyclinAandcyclinEgenesis induced byElAinthis system,while expression of cyclin Dl is downregulated.

EIA-dependent modulation of cyclin gene expression was

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CYCLIN GENE REGULATION BY ElA 2207

firmed in control experiments in which ElA was delivered to fibroblasts by an adenovirus infection or, alternatively, by

stable transfection.

The potential to manipulate ElA activity by an external stimulus, as in IREE-1 cells, offers the unique opportunity to perform kinetic experiments on the stability of E2F-pRB complexes when challenged by ElA. We found that indeed ElAinterferes with E2F-pRB complexes inIREE-1 cells when grown in the presence of estrogen. However, complexes of E2F withpRBpersisted through several cell cycles after the addi-tion ofhormone to quiescent cells, indicating that disruption of suchcomplexes occurs with slow kinetics. Taken together, our dataindicate that ElA-dependent S-phase entry is correlated to specific modulation of cyclin gene expression, whereas disruption of complexes of E2F with pRB may not be required for the onset of DNA synthesis.

MATERIALSAND METHODS

Construction of plasmid pElAER. ER sequences from plasmid HE14 (17) were first subcloned into the BamHI site of pSP64 to introduce a SmaI site upstream of the coding sequences. ER sequences excised from this intermediate plas-mid weresubsequentlyligated to adenovirus-2ElA sequences at the SmaI site in plasmid pm975 (31). In this construct expression of a chimeric protein containing the 150 amino-terminal amino acids of ElA (containing CR1 and CR2; see Results) fused to the ER hormone-binding domain is driven by theElApromoter.

Cell lines. To establish cells expressing the ElA-ER con-struct, rat embryo fibroblasts were transfected with 5 ,Lg of pElAER and 5 ,ug of pEJras. Transfections were plated in Dulbecco modified Eagle medium (DMEM) containing

10%

fetal calf serum(FCS), to which estrogen was added at a final

concentration of 100 nM. In this experiment six foci of

transformed cells appeared, whereas in a control experiment notransformed cells appeared in the absence of estrogen. Cells from one focus oftransformationwere established and gave rise to the cell lineIREE-1. To obtain cells that constitutively expressadenovirus ElA, NIH 3T3 fibroblasts were infected by

a recombinant retrovirus (pMXSVneo-18) carrying the ElA

13S cDNA (a gift of R. Ralston). Subsequently, colonies were selected for growth in soft agar. One of the resulting clones was used toestablish the NIH 3T3/13S cell line. Control cells were

derivedbyinfecting NIH 3T3 cells with the empty vector. Cell cycle analysis. Cells were washed with phosphate-buffered saline (PBS), fixed in 70% ethanol, and stained by

propidium iodide as described previously (8).

Fluorescence-activated cell scanning(FACScan) analysis wasperformed by the cell-fit program, with a Becton-Dickinson FACScan sys-tem. Thymidine incorporation was measured as described previously (8).

Antibodies and Western immunoblotting. Antibodies to human cyclin Dl were obtained by immunizing a rabbit with

purifiedcyclinDl proteins as described previously (1).

Mono-clonal antibodies to cyclin E were produced by standard

procedures after BALB/c mice were immunized with a

gluta-thione-S-transferase-cyclin

Efusionprotein, as described pre-viously (18). Polyclonalantibodies to cyclin A (29) andcdk2 (27), respectively, were prepared as described previously.

Monoclonal antibodies recognizing the C terminus of ElA

(M73) were obtained from Dianova (Hamburg, Germany). Antibodies to the N terminus of ElA (M37) were obtained fromEd Harlow.Western blots were performed as described previously (27), with the enhanced chemiluminescence system

(Amersham, Inc.).

Northern (RNA) blotting. Total cellular RNA was extracted by the guanidinium thiocyanate-acid phenol method. Total RNA (10,ug) was electrophoresed on 1% agarose formalde-hyde gels and transferred to nylon membranes. Expression of

ElA

and the

ElA-ER

construct was analyzed with a 0.3-kb BstXI fragment prepared from an

ElA

cDNA clone. Expres-sion ofcyclin A was monitored with a 1.6-kb mouse probe (15) or a2.2-kbhuman probe, expression of cyclin E was monitored with a 1.5-kb mouse probe (15) or a 2.5-kb human (16) cDNA probe, and expression of cyclin

Dl

was monitored with the mouse cyll probe (19). Glyceraldehyde-3-phosphate dehydro-genase (GAPDH) expression was analyzed with a rat cDNA probe (15).

Band shift assays. A synthetic oligonucleotide encompass-ing the E2F-binding site of the adenovirus E2 promoter was incubated with extracts from different cell lines, as described previously (28). E2F-associated proteins were analyzed by incubation of the band shift reaction with specific antibodies on ice for 50 min prior to electrophoresis. For detection of cyclin A and cdk2 proteins, a polyclonal antiserum was used after affinity purification (28). Human and rat pRB were detected by monoclonal antibody XZ 55 (Dianova), which has been shownpreviously to recognize native rodent pRB (15).

Infection of fibroblasts with adenovirus. Primary human fibroblasts

(MS107,

provided by E.-M. deVilliers) (28) that were obtained from the oral cavity of a healthy human were infected at about 50% confluency by adenovirus 5 or the

ElA-deficient mutant

d1312,

as described previously (14). For infection, cells were placed in serum-free medium and incu-bated for 1 h with virus at 10 PFU per cell. After 1 h the medium wasreplaced by DMEM supplemented by 10% FCS. For detection of ElA in infected fibroblasts, immunofluo-rescence was performed on coverslips with infected MS 107 cells. Coverslips were rinsed twice in PBS, and cells were fixed for 6 minatroomtemperature in 3.7% formaldehyde in PBS. Coverslips were dipped in ice-cold acetone for 20 s and then washed in 70% ethanol and PBS. The monoclonal anti-ElA antibody M73, diluted 1:20 in PBS containing 5,ug of bovine serum albumin, was applied for 60

min

at 37°C. After three PBSwashes fluorescein-conjugated goat anti-mouse immuno-globulin G was applied. After further incubation for 45 minat 37°C, cells were washed three times with PBS and then

counterstained with DAPI (4',6-diamidino-2-phenylindole) (1

,ug/ml)

inPBS for 15minat37°C. Cells were washed twice with PBS,mounted on glass slides with Mowiol (Calbiochem), and examined with a Leitzfluorescence microscope.

RESULTS

Characterization of a cell line expressing

EIA-ER

fusion proteins. Primary rat embryo fibroblasts were transfected with a vector encoding the EJ ras oncogene together with an expression vector encoding a chimeric protein, in which ElAis fused inframe to asteroidreceptor hormone-binding domain. Initial experiments using full-length ElA fused to the

hor-mone-binding domain of the glucocorticoid receptor (31) revealedtransformingactivity even in the absence of hormone (data notshown). Since this result may reflect inappropriate spacing between the hormone-bindingdomain and the active domain of ElA (reviewed inreference 30), we constructed a vector coding for a fusionprotein

(ElA-ER)

(Fig. 1A)inwhich theN-terminal 150 amino acids of adenovirus type 2ElAare linked to thehormone-bindingdomain of the human ER. Both conserved regions 1 and 2 of ElA (CR1 and CR2 [21]) are retained in this construct. Two weeks after transfection, foci wereobserved on plates in which cultures were grown in the

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A EIAcDI

A(codonI

Ad EIA-5'region

B

-EIA ER

-EIA

200.

150 E o 150

5-E00

2 3 4

time (days)

5 6

time (days)

90.

80.

70.

60.

0 50.

40.

30.

20.

10.

() 4 12 16 24 48 72 96 120

time(hrs)

FIG. 1. Characterization ofIREE-1 cells. (A) Structure ofpElAER.pElAERcontains theEIApromoter-enhancer driving expressionofthe

ElA-ERfusion protein, comprising amino acids 1 to 150 of Ad2 EIAfused to amino acids 282 to 595 of the human ER.Furthermore, this

pSP64-derivedvectorcontains the simianvirus 40 BamHI-BclIfragment (nucleotides2533 to2770) comprisingthe simian virus 40poly(A)site.

(B) Expression of the EIA-ERgeneinIREE-1 cells. RNA from 293 cellsandIREE-1cellswasisolated andhybridizedtoaprobederivedfrom

EIAsequences.EIA expressionin293 cellsis indicatedbytheappearanceofa0.8-kbmRNA,whereas inIREE-1 cellsanmRNA withalength

ofabout 1.6 kbwasdetected.(C) Growth propertiesofIREE-1cells.(Upper panel)IREE-1cellswereseeded induplicatein DMEM(10% FCS), containing either100 nM estrogenor noestrogen. Cellswerecountedevery24 h.(Lower panel)Cellswerekeptin hormone-free mediumfor1

week and restimulated by the addition of 100 nMestrogen.At the indicatedtime, samplesof the cellswereanalyzed byFACScan. Theproportion of cells inG,phase isshown.(D) Growth propertiesof control cells. Ratembryofibroblasts(REF)and RatIA cellswereseeded induplicate in DMEM(10% FCS), containing either 100 nMestrogen(+ oes)or noestrogen(- oes).Cellswerecountedevery24 h.

presence ofestrogen but not on plates from which estrogen

was omitted. Cell lines were established from individual foci

and grown in DMEM supplemented with 10% FCS in the

presenceof 00nMestrogen.One of these celllines,IREE-1,

wasused forsubsequent experiments. To monitor expression of the EIA-ERgeneinIREE-1 cells, RNAwasprepared from

IREE-1 cells and hybridizedtoanElA-derived labelledprobe.

The presenceofanmRNAwith alength of 1.6 kb in IREE-1

cells demonstrates expression of the transfected gene (Fig.

IB). Using amonoclonal antibody to the N-terminal part of ElA (M37), we detected a protein of the expected size (75 kDa) inextracts from IREE-1 butnot from control cells (see below) (Fig. 2B).

As shown in Fig. IC (upper panel), IREE-1 cells grow

exponentially in thepresenceofserumandestrogen;therewas

nosign of growth arrestunder these conditions foratleast 12 months(datanotshown). In the absence ofestrogen,the cells do not grow but remain quiescent for several weeks. To analyze the kinetics of ElA-dependent S-phase entry,IREE-1 cellgrowthwasarrested by withdrawal ofestrogenfor 1 week and then restimulated by the addition ofestrogen. At several times after the estrogen addition, samples were taken and

analyzed for their distribution in different phases of the cell cycle by FACScan. The results of this experimentareshown in

Fig. IC (lower panel). Itappearsthat IREE-1 cell growthwas

arrested in

GI

in the absence of estrogen, and cells entered S-phase after readdition of hormone, reaching a peak after

about 48 h. Withdrawal ofestrogenafter 48 h ledto rearrestof cell growth into G1. Estrogen-dependent S-phase entry of IREE-1 cellswasconfirmed inaparallel experiment measuring

[3H]thymidine incorporation (data not shown). To

demon-stratethatestrogen-dependent proliferation of IREE-1 cells is mediated by the EIA-ER chimeric gene, we established

growth curves for nontransfected primary rat embryo fibro-blasts and foran establishedratfibroblast cell line, RatlA, in theabsence andpresenceofestrogen.For bothcell types,the proliferation ratewasnot significantly altered by the addition of 100 nM estrogen tothe medium (Fig. 1D). The results of theseexperiments indicate that estrogen-dependent prolifera-tion of IREE-1 cells reflects their dependence on an EtA

function forgrowth, rather than activation of the endogenous ERby the steroid. This conclusion is supported byourfinding

thatrat fibroblasts do not express detectable levels of the rat

ER(7a).

Activation ofcyclin A and cyclin Egeneexpression by ElA.

We determined whether activation of cell proliferation in IREE-1 cells was accompanied by changes in cyclin gene

expression. RNAwas prepared from quiescent IREE-1 cells

and from IREE-1 cells at different times after readdition of estrogenandanalyzed by Northern blotting.

Estrogen-depen-Io +oestroen

-oestrogen

tt

oestrogen

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CYCLIN GENE REGULATION BY ElA 2209

dl312 Ad5

A

anti ETA

DAPI

)

cn

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Or-mxco a:: cm 22 2 4m

B

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dent proliferation was accompanied by a rapid induction of

cyclinA and cyclin E mRNA levels (Fig. 3A). In additional experiments,wedeterminedthatmaximalinduction of cyclinE

mRNAlevelswasreachedat8 hafter theaddition ofestrogen, whileby 4 h half-maximal activation was observed (Fig. 3A)

(datanotshown).Toanalyzewhetherthisinduction is

revers-ible,RNAwasalso preparedfromgrowing IREE-1cells and from cells shifted to estrogen-free medium for 4 h. mRNA levels ofbothcyclinsAand Ewerefoundtobemuchhigherin cells grown in the presence of estrogen than in the cells

withdrawn from estrogen(Fig. 3B). Higherlevels ofcyclinA mRNAwerereflected in similarchangesof theproteinlevels

(Fig. 3B). Rat cyclin E was not detectable by antibodies to humancyclinEavailable tous.

Controlexperiments demonstratedthattheadditionof 100 nMestrogendidnotinducesignificant changesinthe level of

cyclin Ainratembryo fibroblasts and RatlAcells (Fig. 3C).

Similarly, the level of cyclin E mRNA was not affected by

FIG. 2. ElA expression in different cell lines. (A) Expression of ElA in adenovirus-infected fibroblasts. Thepresenceof ElA in cells infected by AdSord1312wasanalyzed by indirect immunofluorescence with the M73 antibodyandcounterstaining with DAPI. As shown here,

a large proportion ofAd5-infected cells expressed ElA at 12 hpi (Table 1). (B)Comparison of ElA protein levels in different cell lines. Extractswereprepared from growing IREE-1 cells, NIH 3T3/13S cells,

293 cells,Ad5-infected MS 107 cells, and control cells (3T3, RatlA,

andREF),asindicated. Eachextract(20,ug)wasseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The EIA and ElA-ERproteinsweredetected by Western blotting with the anti-ElA

antibody M37.

estrogeninRatlA cells(Fig. 3D).Takentogether,thesedata indicate that activation of cyclin Aand cyclin Egene

expres-sion, observed in IREE-1 cells after theadditionofestrogen, reflects activation of the ElA-ER fusion protein and is not

[image:4.612.72.566.71.511.2]

mediated by activation ofthe ratER.

TABLE 1. ElA-positive nucleiwerecountedat differenttimes of adenovirusinfection,asoutlined in thelegendtoFig. 2A

Time(hpi) ElA-positive_nuclei()

0.0~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

O

...

6...0

12... 85±5

16... 80±6

24... 84±3

31...3 aResultsareaverages ± standarddeviations fortwoindependentinfection

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Toconfirm thatadenovirus EIAcanupregulate expression

of cyclins E and A, we infected primary human fibroblasts

(isolate MS 107) with adenovirus type 5 (Ad5) and the ElA-deficientmutantd1312.Immunofluorescence showed that expressionof EIAcouldfirst be detectedat 12 hpostinfection

(hpi) in cells infectedwith Ad5,atwhich time about85% of the nucleiwerebrightly stained(Fig. 2A); d1312-infected cells did

notshow anysignal. During theAd5infection, nuclearstaining disappeared at about 30 hpi (Table 1), consistent with the

previous observation that EIA proteins have a short half-life andnegatively regulate theirownsynthesis(3). Control

exper-imentswerealsoperformedwith 293cells,expressingEIAand

A

z

cb. ._

.o-

=: ct

-*

CZ*_

&

ef~C

12

0 3 24 72 76

time(hrs)

ElB,and in NIH3T3/13Scells. NIH3T3/13Scclls, derived by infection with a recombinant retrovirus (see Materials and Methods), express the 289-amino-acid (13S) ElA protein.

Expression of EIA in the cell lines used in this study was

demonstratedbyWesternblotting (Fig. 2B).

In Ad5-infected, but not d1312-infected MS 107 cells, we

observed a strong increase in cyclin E mRNA (Fig.4A) and protein (Fig. 4B) at 18 hpi,demonstrating that cyclinE gene

expression is induced in an ElA-dependent fashion. High

levels ofcyclinE werealsoobservedin293 cells(Fig. 4A).As

already suggested by the rapid induction ofcyclin E mRNA upon activation ofElA-ER in IREE-1 cells, these data

indi-cate that EIA acts as a potent inducer of cyclin E gene

expression,independentof theproliferativestatusof thetarget

cells.

Surprisingly, nosignificant changesincyclinA gene

expres-sion were observed after infection of human fibroblasts with either wild-type Ad5 ord1312 (Fig. 4A and B). However, in

contrast to IREE-1 cellskept inthe absence ofestrogen, MS

107 fibroblasts were actively proliferating when ElA was

introduced into these cells by adenovirus infection. Thus,

activation ofcyclinA gene expression byEIA maybe detect-able inquiescent cellsonly.Totestthishypothesis,weanalyzed

expression ofcyclin A in control NIH 3T3 cells and in NIH

3T3/13S cells, both in the absence (0.5% FCS) and in the

presence (10% FCS)of externalgrowthfactors.Incellsgrown

in the presence of 10% FCS, nodifference in cyclin Alevels could be detected, regardless of ElA expression. However, cyclin A expression was downregulated in control cells in

response to serum deprivation, whereas cells that expressed

EIAfailed to downregulatecyclinA (Fig.4C). Weconclude that EIA-dependent upregulation of cyclin A reflects the

B

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c -n

*

7i

-cYclin DA

-* 0., cyliD

_0 -cyclinE

3T3

IREE-i

Q .-

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> u0

ta 2

rA o +

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C

c --

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15 wL

oestrogen I-

I

+

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-

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I

_ - -cyclin A D

oestrogen

I

- |+|

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A - cyclin E -cyclin Dl

0

_ -cyclinDl

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FIG. 3. Estrogen-dependent changes of cyclin gene expression in IREE-1 cells. (A) Changes ofcyclin mRNAs during estrogen stimulation of IREE-1 cellsby Northern blot.IREE-1 cell growth was arrested by hormone-free medium and restimulated by the addition of estrogen. After 72 hthe medium was replaced by hormone-free medium, and the cells were kept for 4 h in the absence of estrogen. At the time indicated RNA was prepared and hybridized to probes derived from cyclin A, cyclin

Dl,

and cyclin E, as indicated (also see panel B). mRNA levels were quantitated by densitometric scanning. (B) Changes of cyclin gene expression induced by estrogen are reversible. (Left panel) RNA was prepared fromIREE-I cellsgrowinginthe presence of estrogen (+ oes) or from cells shifted to estrogen-free medium for 4 h (-oes). Total cellular RNA was hybridized

toprobes derived from cyclin A, cyclin DI, cyclin E, and GAPDH, as indicated. (Right panel) Extracts were prepared from IREE-1 cells growing inthe presence of estrogen (+ oes) or from cells shifted to estrogen-free medium for 24 h (-oes).Extracts were also prepared from growing NIH 3T3-cells(10% FCS) or serum-starved NIH 3T3 cells (0.5% FCS). Control lanes contained either cyclin A, in vitro translated from a bacterial expression vector (29), or extracts fromRTI12 cells, derived from a human tumor expressing cyclinDI (18a).CyclinDIappears as a double band in Westernblots performed with extracts from rodent cells, whereas only one band is visible in extracts of human cells (Fig.4B).(C) Expression ofcyclinAandcyclin

Dl

isnot changed by estrogen in rat embryo fibroblasts (REF) andRatlAcells.Rat embryo fibroblasts andRatIAcells were grown inthe absence of oestrogen (-) or treated with 100 nM estrogen for 48 h (+). Levels of cyclinAand cyclinDI were analyzed by Western blotting.(D) Expression of cyclinEis notchanged by estrogen inRatlAcells.RatlA cells were grown as described for panel C; expression of cyclin Ewasanalyzedby Northern blotting.

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CYCLIN GENE REGULATION BY ElA 2211

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FIG. 4. ElA-dependent changes of cyclin gene expression in adenovirus-infected human fibroblasts. (A) ElA-dependent changes of cyclin mRNAlevels in infected MS 107 cells.MS107 cells were infected byAdS or d1312 or mock infected. RNA was prepared at 18 hpi and probed with cyclin A-, Dl-, and E- and ElA-specific probes, as indicated. For a control, RNA was also prepared from 293 cells and included as a reference. (B) ElA-dependent changes of cyclin protein levels in infected MS 107 cells. Extracts were prepared from MS 107 cells infected as described for panelA.Therelative amount of cyclin A, Dl, and E was determined by Western blotting. For a control, extracts were also prepared from HaCaT cells, either growing in the presence of 10% FCS or serum starved (0.5% FCS). (C) Modulation of cyclin A and cyclinDl gene expression byserum

starvationinNIH3T3/13S cells and control cells. Extracts were prepared from NIH3T3/13Scells that were either grown in the presence of 10% FCS or kept in the presence of0.5% FCS for 48 h, as indicated. Extracts were also prepared from control NIH 3T3 cells that had been treated inthe same way. Each extract (20pLg)was analyzed byWestern blotting.

proliferativestatusof the targetcells and may, therefore, be an indirect consequence of ElA-induced cell proliferation.

Repression of cyclinDl gene expression by ElA. In contrast

tocyclins A and E, activation of ElA-ER in quiescent IREE-1 cells led to agradual decrease of cyclinDl mRNA(Fig. 3A) andprotein (datanotshown; also see below). Full repression ofcyclin DlmRNArequired exposure to estrogen for at least 72 h(Fig. 3A). Subsequent removal of estrogen for 4 h led to

arapid increaseincyclinDl mRNA and protein (Fig. 3B). In other rodent fibroblasts, e.g., NIH 3T3, cyclin Dl is barely

detectable in serum-starved cells but highly expressed in

growing cells (Fig. 3B) (40). Furthermore, the addition of estrogen did not affect the level ofcyclin Dl in rat embryo fibroblasts andRatlAcells(Fig.3C), indicating that repression of cyclin Dl, as observed in IREE-1 cells, depends on the presence of the ElA-ER protein. These data suggest that

expression ofElAcandownregulate expression of the cyclin Dl gene.

This conclusion was confirmed by the observation that

infection of MS 107 fibroblasts by Ad5 led to a substantial reduction ofcyclin DlmRNA(Fig. 4A)andprotein(Fig. 4B).

Infection withd1312didnotaffectcyclinDl expressionin MS 107 cells, suggesting that repression requires an active ElA protein. To investigatethe possibilitythat expression ofElA might change the cell cycle profile of an infected culture,

FACScan analyses were performed with Ad5-infected and

mock-infectedMS107cells. From theseexperiments itappears thatat16hpiaslight increasein the

G1

fraction resulted from adenovirus infection (52% in

Gl)

compared with mock-in-fected cells (45% in

Gl)

(data

not

shown).

This observation rulesoutthe formalpossibilitythat thechangesincyclinEand Dl levels whichwe observedaredueto amajoralteration in the cellcycle profileof cellsat16hpi duringanAdS infection.

Cyclin Dl expressionwas notdetectable in293cells,further

supporting the

hypothesis

thatthis gene may be

repressed by

an adenovirusearlygene(Fig.

4A).

In NIH3T3 controlcells,

as well as in established human keratinocytes (HaCaT) (Fig.

4B), cyclin Dl is highly expressed when the cells are grown in medium containing 10% FCS but is reduced upon serum starvation.Incontrast tothisobservation, cyclin Dl expression wasstrongly downregulated in proliferating NIH 3T3/13S cells, compared with proliferating control NIH 3T3 cells (Fig. 4C), indicating that ElA-expressing cells fail to upregulate cyclin Dl expression upon cell proliferation. Taken together, our data suggestthat expression of the cyclin Dl gene is repressed

byElA.

ElA-dependent changes ofE2Fmultiprotein complexes. We nextaddressed thequestion of whether complexes of E2F and pRB would be sensitive to ElA-dependent progression of IREE-1cells toS phase. First, we monitored the status ofE2F in rapidly growing IREE cells (with estrogen) and after the removal of estrogen for2 days. Complexes betweenE2Fand pRB were not detected in extracts from cells which were

growingin the presence of estrogen and, hence, containedan

active ElA protein. The removal of estrogen led to the appearance ofa newcomplex which contains the RBprotein (Fig. 5A). The protein composition of the other complexes

seeninFig.5Aremains obscure. However, the results shown in Fig.5Arule out the association of either cyclinAorcdk2with E2Fin any of thecomplexes observed. The antibodiestocyclin

A and cdk2, whichwere used in this experiment, have been shown previously to recognize the relevant antigen in E2F bandshiftexperiments using extractsfrom rat cells (15) (Fig. 6). These results demonstrate that activation of ElA-ER by

estrogen can indeed disrupt E2F-pRB complexes. Similar changesintheE2Fcompositionwereobservedduring adeno-virus infection of MS 107 fibroblasts(Fig. 6),inagreement with

current hypotheses (23). In a control experiment, we found that ElA-ERprotein iscoprecipitatedwhenpRB is immuno-precipitated from IREE-1 cells (data not shown),

indicating

association of bothproteins.

Second, we asked whether dissociation of E2F-pRB

com-plexes would bean earlyeventduring restimulation of

prolif-eration in cells in which growth was arrested by hormone

VOL.68,1994

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[image:6.612.114.520.73.240.2]

IREE-I+ oestrogen IREE-1-oestrogen

x<

<

+-+ WC+- + L + + + + +

3c v~ S EvE >=S

+ +++; + + +

qVf04,<S_rO_t t

LA

B +oestrogen [hrs] . 4 24 48 72 control

Rbantibody | + . + + + + +

E2FRb[

wEM

]E2F Rb

FIG. 5. E2F complexesinIREE-1cells.(A)E2F-RBcomplexeswereinducedby estrogenwithdrawal fromgrowing IREE-1cells.IREE-1cells

weregrowninthepresence ofestrogen.Half ofthe culturewaskeptinthe absence ofestrogenfor 48h,while control cellswerefurther grown

inthepresenceofestrogen.Extractswerepreparedfrom the cells and used for band shiftexperiments. Multiprotein complexesformedonthe

labelled DNAfragmentaredesignated"E2F-complexes";thecompositionof suchcomplexeswasanalyzed bythe addition ofwild-type (WT)or mutant(mut) competitor oligonucleotides (oligo)and antibodiestopRB, p107, cyclin A, cdk2,EIA,cdc2,andp13suc,asindicated (seeMaterials andMethods). (B) Disruption ofE2F-pRB complexes byaddition of estrogentoquiescent IREE-1cells occurredslowly. IREE-1cellgrowthwas

arrestedbyhormone-free mediumand restimulatedbythe addition of estrogen. At the timesindicated, samplesweretaken. Extracts of thesecells

wereanalyzedinband shiftexperimentsin thepresenceorabsence ofpRBantibodies,asindicated.In the controllanes,extractsofIREE-1cells

growninthepresenceofestrogenforseveral weekswereanalyzed.

withdrawal. Cell growth was arrested by keeping the cells in hormone-free medium for 1 week. Estrogen was added, and thecellswere harvested at several times after theaddition of thehormone. Thestatusof E2Fwasanalyzed bythebandshift

technique. Asexpected, E2F-pRB complexesweredetectedin cells in the absence ofestrogen. Surprisingly, suchcomplexes persisted for atleast 3 days after the hormone addition(Fig. 5B), althoughatthat time thecells hadgone throughthe first cellcycles(Fig. 1).Weconcludethatquantitativedisruptionof

E2F-pRB complexes byElA isnotrequiredfor ElA-mediated entry intoS phase.

DISCUSSION

We have analyzed the effects of adenovirus ElA on the

expression ofcyclingenes and thecomposition of E2F

multi-protein complexes in two experimental systems. First, we

constructed a cell line expressing EJ ras together with a

chimeric gene (ElA-ER) encompassing the N-terminal 150 codons of the ElA cDNA fused in frame to the hormone-binding domain of the human ER. Cells that express this

chimericgeneproliferate inahormone-dependentmanner.As

discussed in Results, appropriate controls were included to

ruleouteffects ofestrogenthatarenotrelatedtothepresence

of the ElA-ER protein. Second,we developed aprotocol for

quantitative infectionofgrowing primary human fibroblastsby

adenovirus 5,expressingElA. Conclusionsderived from such

experimentswerecontrolled by additional experiments

involv-ingthe ElA-deficient virusd1312aswell astwo different cell

lines, expressing either ElA and E1B (293 cells) or only the

13Sform of ElA(NIH3T3/13S cells).Fromtheseexperiments

we make the following conclusions.

(i) expression of the cyclin E gene is rapidly induced in

adenovirus-infected growing fibroblasts but not in cells

in-Ad 5 infection dl312 infection

I

Mock

0

<U0

~~~~~00

0

0 .w0

-E2FcyclinA JE2F Rb

I*Mh'

I!!fI

N

J

FIG. 6. ElA-dependent changesinE2Fmultiprotein complexes in

adenovirus-infected MS 107cells. Extractswere prepared from

AdS-infected cells, d1312-infected cells, ormock-infected cells at 16 hpi.

E2Fcomplexeswereanalyzedasdescribed inthe legendtoFig. 5.mut

oligo, mutantoligonucleotide; WToligo,wild-type oligonucleotide. A

I I

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CYCLIN GENE REGULATION BY EIA 2213

fected with the ElA-deficient mutant d1312. High levels of cyclinEgene expressionwerealsofoundin 293cells.Similarly,

the addition of estrogen to quiescent IREE-1 cells led to a strongincrease ofcyclinE mRNA,detectable onlyafew hours

after the additionof the hormone.Takentogether,these data suggest that the cyclin E gene is rapidly turned on in an

EIA-dependent pathway, consistent with the assumption that

only veryfew intermediary events, ifany, are requiredbefore activationof the cyclin Egene. Furthermore, activation of this

genedoesnot merely reflect ElA-mediatedproliferation,since

it is observed also in growing cells.

(ii) Like cyclin E, cyclin A expression was also substantially increased upon activation of the EIA-ER protein in IREE-1 cells. However, incontrast totheresults obtained withcyclinE, expression of the cyclin A gene was not changed by EIA in growing fibroblasts infectedwith adenovirus. Since cyclin A is already expressed to a considerable extent in such cells, we

reasonedthat induction ofcyclin A by ETA maybe restricted

to quiescent cells. Consistent with this hypothesis, we found

that in NIH 3T3/13S cells the cyclin Alevel is similar to that found in control cells when cells are grown in 10% FCS.

However, unlike incontrol cells,cyclinAgeneexpressionwas not reduced by serum starvation in NIH 3T3/13S cells,

indi-cating that EIA can mimick serum-dependent cell cycle acti-vation.Weconclude that theElA-dependent increaseincyclin

Agene expression, observedselectivelyinquiescent cells,may representaconsequence ofcellcyclemodulationbyEIA(21),

rather than direct activation of the cyclin A gene by the viral

oncoprotein.

(iii) Incontrast totheresultsobtained withcyclinsAand E,

adenovirus infection of human fibroblasts led to a severe

reduction ofcyclin DI levels inthecells. Thiseffectapparently

depends on EIA expression, since it is also observed in 293

cells and NIH3T3/13Scells,whereasd1312-infected fibroblasts

expressnormal levels ofcyclinDI.

Cyclin

DI

expression

isalso

drastically reduced upon activation of the EIA-ER

protein

in IREE-1 cells. Takentogether,ourdatashow that

expression

of cyclin DI is downregulated by ETA. This

activity

of ETA overridesthenormal regulationofcyclin DI by

growth

factors

(40) (Fig. 3C and 4B and C) and may reflect ETA's

ability

to specifically repress transcription from several genes

(3).

(iv) ComplexesbetweenE2F andpRB were

disrupted

at 16

hpi in the adenovirus infection (Fig. 6) aswell as in 293 cells (data notshown) (18). Disruption ofE2F-pRB

complexes

was

also observed in IREE-1 cells grown in the presence of

estrogen (Fig.

5A).

These data confirm that also in IREE-1

cells, complexesbetweenpRB andE2Fare

disrupted by

ETA.

However, disruption ofcomplexes between E2F and

pRB

in

these cells occurs too

slowly

to account for the

rapid

ETA-dependentS-phaseentryobserved in

estrogen-treated

IREE-1

cells. Consistent with this

interpretation, Wang

et al.

(37)

had

previously demonstrated that ETA's

pRB-binding

domain is

notrequiredforthestimulation ofDNA

synthesis

in

quiescent

rat fibroblasts. As proposed by these

investigators,

the inter-action ofETAwith pRBmayprevent immortalizedcellsfrom

reentering the Go phase. The

experiments

reported

here

extend these findings and raise the

question

whether the

disruption by EIA of

pRB-E2F

complexes

plays

any role in

progression to S phase.

Although

at present the conclusions relyonexperimentswith a

single

cell line

expressing

ETA-ER

chimericproteins,thedata

clearly

argue

against

amodel which invokes disruption of E2F-pRB

complexes

as a

key

step for

ElA-mediatedS-phaseentry,atleast in the cellscharacterized

in this study. The absence of any detectable

changes

in the

level ofE2F-pRB

complexes

in

estrogen-treated

cells forupto

72h,equivalent to at leasttwo

population

doublings

(Fig.

1),

is most

easily

reconciled with the notion that the

stability

of such

complexes

isnotrelatedtothe

position

ofthecells in the

cycle.

This view is further

supported by

a recent report

by

Schwarzetal.

(33a), demonstrating

thatE2Fremainsboundto

hypophosphorylated

pRB

throughout

S

phase

in

synchronized

human

cells,

irrespective

ofthe overall

phosphorylation

level of

pRB.

Similarly,

the

pRB

species

found in

complexes

with E2F in

growing

IREE-1 cells may

correspond

to residual

hypophosphorylated

pRB.This

assumption

is

supported by

our

finding

that theaddition ofestrogen to IREE-1cells doesnot

result in

significant changes

of

pRB

phosphorylation,

a consid-erable

degree

of

pRB

phosphorylation being

detected also in the absence of estrogen

(data

not

shown).

We conclude

that,

by

hormone

withdrawal,

IREE-1 cell

growth

is arrested in a

stage at which

pRB

is

already

partially

phosphorylated.

This

finding

is

unexpected

but may

actually

be correlated to the

remarkably

high

level of

cyclin

DI in

resting

IREE-1

cells,

as

cyclin

DI is known to contribute

significantly

to

pRB

phos-phorylation

(iSa).

The data

reported

here are consistent with the

hypothesis

thatadenovirus

ETA

mediates its

mitogenic

effect

by

activating

the

expression

of the genesfor

cyclin

E and

cyclin

A,

both of whichwereshowntocontribute tocell

cycle

progression.

The alternative

possibility,

that EIA-mediated

proliferation

would

involve the known

capability

ofanotherG1

cyclin,

cyclin

DI,

to

facilitate

progression through

G1

(1, 33),

isnot

compatible

with

our

findings.

On the contrary,

ElA-dependent S-phase

entry coincides even with

repression

of

cyclin

Di,

a property that

ETA

shares with another

transforming

oncogene,

c-Myc

(15).

While the

significance

of this

repression

is not

clear,

recent

data indicate that continuous

expression

of

cyclin

Dl at the

GI-S

boundary

prevents the onset of DNA

synthesis

(25a),

indicating

that

cyclin

DI may block

progression

through

the

cell

cycle

at the

G,-S

boundary.

Whereas suchfunction

might

provide

a clue to the

as-yet-unexplained

repression

of

cyclin

DI

by

transforming

oncogenes,further

experiments

are

clearly

required

to address this

question.

Our data

provide

a new

example

for the use of the

inacti-vation domain of steroid receptors to block the function ofa

heterologous

protein (reviewed

inreference

30).

The

ETA-ER

fusion

protein

describedinthisreport contains the functions of the

oncoprotein

necessaryforimmortalization of rodent

fibro-blasts,

including

the

capability

to modulate

expression

of the

three

cyclin

genes

analyzed

in this

study.

These

findings

may

provide

a clue to understand the interference of

ETA

with cellular

growth

control, by

identifying

known cell

cycle

regu-latory

genesas

potential

mediators ofElA's

oncogenic

activity.

The

experimental

system

presented

here should enableus to

revealsomedetails of the molecular mechanism

underlying

the observed

biological

effects.

ACKNOWLEDGMENTS

P.J.-D. and D.S. thankH. zurHausenforcontinuoussupport

and

members of the laboratory for critically

reading

the

manuscript.

We thank Claude Kedingerfor

providing

adenovirus5 anddl312 stocks. We thank Birgit Martin for

assembly

of the

manuscript

and Ulrike Ackermann for photography. We are grateful to Steve Reed for

providingrabbitpolyclonalantiserato

cyclin

E. WethankH.Weidauer (HNO-Klinik, Universitat

Heidelberg)

for

early

passage of MS 107 fibroblasts. D.S. and P.S. made similarcontributionstothis work and should be consideredequalfirst authors.

P.S.wassupported bygrantsfrom the RocheResearch Foundation and the Freiwillige Akademische Gesellschaft, Basel. Work in the

laboratory of M.E. was

supported by

the Bundesministerium fur

Forschung und

Technologie (grant

BCT

0381-5).

Collaboration

be-tweenM.P., M.E. andP.J.-D.was

supported

by

aresearchgrantfrom

the Human Frontiers Science

Program

Organisation.

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