Distinct transformation phenotypes induced by polyoma virus and simian virus 40 in rat fibroblasts and their control by an early viral gene function.

JOURNAL OF VIROLOGY, Feb. 1980,p.697-707

0022-538X/80/02-0697/11$02.00/0 Vol. 33, No. 2

Distinct

Transfonration

Phenotypes Induced by Polyoma

Virus

and Simian Virus 40 in Rat Fibroblasts and Their

Control

by an Early

Viral Gene Function

BERNARD PERBAL* AND MINOO RASSOULZADEGAN Centre de Biochimie, Universitede Nice,06034 Nice, France

Several transformed cell lines established from Fisher rat cells (FR 3T3)

infected withwild-type polyoma virus orsimian virus 40 or early

temperature-sensitivemutants(polyomatsa and simian virus 40tsA30)werestudied for their

transformation phenotypes.The distinct patterns which were obtained for

poly-oma and simian virus 40 transformants led to the conclusion that these two

virusesexpressdifferenttransformingabilitiesin ratcells. Theresults obtained

withtemperature-sensitive mutant-derived transformantsindicate that all of the

transformation characteristicsstudied sofarmay beunder thecontrolof a viral

function in polyoma tsa-transformed cells.

Transformed cell lines have been established recently inour laboratory from normal diploid Fisher rat cells (FR 3T3) infected either with

wild-type (WT) polyoma virus or simian virus 40 (SV40) orwith theirearly

temperature-sen-sitive (ts) mutants (polyoma tsa and SV40 tsA30). Inboth cases, transformation by thets

viruses led to two distinctclasses of cell lines: "N" transformants, which reverted to the

nor-mal phenotype athigh temperatures, and "A" transformants, which still behaved as

trans-formed cells at the restrictive temperature (8,

15).Inpolyomatsaand SV40tsA N

transform-ants,theexpression ofatleastpartof the

trans-formedphenotype thusappears tobe underthe

control of a function involving the "large T" viralpolypeptidewhich isaffected by mutations of the A/a group of both polyoma virus and

SV40 (6,14, 16).

Except fortheirtemperaturesensitivity,tsA

and N cells appearedtobevery similartoWT transformantsintheirgrowthpropertiesatlow

temperatures.However,variations incellgrowth characteristics and in theratesof

2-deoxyglucose

uptake

andof

plasminogen

activator

(PA)

pro-ductionwereobserved within these classes. This variabilityin theexpression ofthe transformed phenotypemight

correspond

totheearlier

con-cept of a stepwise progression toward the ulti-matetumoralstate (18). The

phenotypic

varia-bility was most apparent among SV40

trans-formants, as

expected

onthe basis of the pre-vious work of Risser and Pollack

(12).

Since mostof the

polyoma

transformantsexhibited a

fully transformed

phenotype

for all these char-acters,one mightask whethera

significant

dif-ferenceexists between the

transforming

abilities of the twoviruses in thissystem.

Experimental resultspresented in this report

ledus totheconclusion that there is anactual difference between thespectrumoftransformed phenotypes induced by polyoma virus and SV40 in FR 3T3cells. They also indicate thateventhe

"maximal" transformation phenotypes maybe

under the control ofaviralfunctioninpolyoma

tsa transformants. Evidence is also presented that in suchcellsagenetic drift towarda heat-stable transformedphenotype is observed incell culture, which may be due to the selection of

spontaneous cellular mutationsepistatic to the viral transforming function(s).

MATERIALS AND METHODS

Cellculture. Unless otherwise specified,cellswere grown in Dulbecco-modified Eagle medium (GIBCO Laboratories) supplemented with 10% newbom calf serum (GIBCO Laboratories). They were routinely propagated at 33°C by seeding every 3rd day at a density of 5 x 104 cells per 6-cm petri plate, one transfer thus being equivalent to three to five cell generations.

The isolation of transformed cell lines has been described previously (8, 15). After three successive isolationsstartingfromisolatedcolonies,stockswere constituted afteraminimal number of transfers and kept inliquid nitrogen for further studies.

Theability of cellstogrow withoutattachmentto asolidsubstratewasassayedinbasalEaglemedium

(GIBCO Laboratories)supplementedwith 10%serum

and agaroseaspreviouslydescribed(9).

Assays for PAproduction. (i) Preparationof substrates. Purified human plasminogen wasfrom theCentre NationaldeTransfusionSanguine, Paris,

France.Thebatchusedthroughouttheseexperiments

containedonlytwopolypeptidespecieswithapparent

molecular weights ofbetween 94,000 and 96,000 as

revealedby sodiumdodecylsulfate-polyacrylamidegel

electrophoresis. Plasminogen waskept desiccated at 697

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698 PERBAL AND RASSOULZADEGAN -20°C and dissolved before use in 0.05 M borate buffer, pH 9.0, containing 0.1 M NaCl and 1 mM EDTA.

Casein (according to Hammarsten, Merck & Co., Inc.) was tritiated by a minor modification of the

proceduredescribedbyRiceand Means(10).Sodium [3H]borohydride (12.5 mCi;Commissariatal'Energie Atomique,Saclay, France)wasprogressivelyaddedto 2 mlofformaldehyde-activated casein (80 mg/mlin 0.2Mborate buffer, pH 9.0).The reactionwasstopped

byadjustingtopH2with1 NHCl;thesolutionwas

thenneutralized andextensively dialyzed against0.05 M boratebuffer, pH 9.0, containing0.1 M NaCl and1 mM EDTA.Specificradioactivities obtainedwere

be-tween0.6 and 2.5Ci/mmolofcasein,correspondingto thelabelingof 5 to 10% of thelysineresidues.

Hydrolysisof[3H]caseinwasfollowedby counting

theradioactivityof the supernatant afterprecipitation withtrichloroaceticacid(10%finalconcentration)and centrifugationfor 5minat2,000rpm.

(ii) Qualitativeassayfor extracellular PA.

As-says described in the next two sections deal with activator(s)excreted in the mediumorboundtothe extemal cell membrane or both. Theseassays were

performedwith intactcells,whereastotal PAactivity

wasmeasured incellextracts.

The firstassayis derived from the method described

byGoldberg (2).Cell cultures (usuallyasclonal

colo-niesin6-cmdishes)wereoverlaid with 4.5 ml ofamix containing0.5to1%agarose(IndubioseA37;Industrie Biologique

Frangaise),

2.5%(wt/vol)commercial

non-fat dried milk(Francelait), and 20,ugofpurified plas-minogen. For celLs growing on plastic, the culture

mediumwasremovedand thecells werewashed twice

with prewarmed phosphate-buffered saline before pouringtheagarosemix. Forthe detection of the PA

produced bycoloniesgrowing in softagar, the total volume of the overlay was reduced to 2 ml. Plates

wereexamined forclear proteolysisareasafter

over-nightincubationateither 33or400C.For thevarious

celllinestested,theextentofproteolysiswasusually

foundtobe correlated with thecellnumber and with

therateofactivatorproduction assayedquantitatively

asdescribedbelow. Thesensitivityofthe methodwas

such thatafour-toeight-cell microcolonyandpossibly

asingleproducingcellgaverise toadetectablelysis

areaafter8hof incubation.

(iii) Quantitative measurement. For assay of

extracellular PA, thecelllayerwasincubatedin the

presenceof[3H]casein andplasminogen for increasing periodsof time. Aspreviouslypointedout byRifkin andPollack(11),thekinetics of caseinolysis measured inthiswayisacomplexonesince it isinfluenced by

parameters such as the rate of secretion of PA, its stabilityinthe reactionmedium,and the interaction ofplasminwithplasminogenandactivator.

Cells were grown in 3.5-cm-diameter petri dishes

andwashedthree timeswith 5 mlof Eagle medium. Twomillilitersof thesamemedium containing25,ug ofpurified plasminogen and100 jgof [3H]casein was

addedtotheplates, whichwerethen incubatedatthe appropriatetemperature. Samples(200,uleach)were

taken at various time intervals, and the amount of hydrolyzedcaseinwasmeasured aftertrichloroacetic

acidprecipitation.Plasminogen-independent

caseino-lysiswasestimated in control assays without addition ofplasminogen.Spontaneous hydrolysisof[3H]casein

didnot exceed 0.1% under these conditions. Results were expressed as specific activities relative to the total cellprotein (counts per minute permilligram of protein). Protein concentrations wereassayedas de-scribed byeitherLowryetal. (5) orBramhall et al.

(1).

(iv) Total cell-associated PA.Cell cultures were washed threetimeswith 5 ml of 0.1 M

Tris-hydrochlo-ride buffer (pH 7.4) containing 0.01 M EDTA and lysed in1ml of thesamebuffercontaining0.5% Triton X-100(Serva)andnoEDTA.Samples (4001l each) of this extract were incubated in Tris-hydrochloride

buffer(pH7.4) in thepresenceof[3H]casein (200

jig)

andplasminogen (1 ug)for 45 minat370C. Unlabeled casein (1ml, 10 mg/ml) was addedtoeach reaction mixture, and the nonhydrolyzed casein was

precipi-tated with trichloroaceticacid. Theextentof caseino-lysis wasestimated from the 3Hradioactivity meas-ured in 0.5 ml of the supernatant after low-speed

centrifugation.

Uptakeof2-deoxyglucose.Ratesofuptakeof 2-deoxyglucoseweremeasuredasdescribedpreviously

(8). Valueswerenormalizedasratiosof the cell-asso-ciated3H radioactivityatagivenincubationtimeto thetotalamountofproteinonthesame coverslips.

Theamountofproteinwasdetermined byremoving

the cover slips from the scintillation mixture after counting, washing with methanol, and staining with Coomassie brilliant blue forproteindeterminationby

themethod ofBramhalletal.(1).

RESULTS

Transformed phenotypes among

poly-omavirus and SV40derivatives. Table 1 is

a summary of the growth characteristics with andwithout anchorage and the rates of hexose uptake and PA production for 16 independently

isolatedcell lines. Eight lines were transformed

by polyoma virus (either WT or tsa) and by SV40 (either WT or tsA30). In all cases, the

transformed cells and the normal control cells (FR 3T3) were grown at330Candtested after a

limitednumber ofpassages in culture (three to eight).

All of the transformants thus far tested ex-hibitedhigh saturation densities whengrown in

Dulbecco-modified Eagle medium supple-mented with 10% calf serum. With only one exception (SV-WT-N2 cells), all ofthem grew

well in agar medium. Slightly higher plating

efficiencieshave beenrecorded for clones origi-nally selected as agar colonies, as compared with thosederived from foci (8, 15).

Incontrast to this relatively static picture, all oftheother growth and biochemical

character-isticslisted in Table 1 were found to discriminate

betweenatleasttwoclasses of transformed phe-notypes.

(i) Generationtime. As previously reported

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PHENOTYPES OF POLYOMA AND SV40 TRANSFORMANTS 699

TABLE 1. Summary of the results oftransformation assays performed on various derivatives from the FR 3T3cell linea

Cellline Isola- Satura- Generation Growthin Growth Hexose uptake Membrane PA produc-Celllie tionb tionden

timed

(h) agarose inlow rate proteaseac- tion'

sityc medium' serum r

tivit3th

FR 3T3 5 25 - - Low Low

-SV-WT-Nl f 45 20 + - Medium Low

-SV-WT-N2 f 45 20 - - Medium Low

-SV-WT-A1 a 45 20 ++ - Medium Low

-SV-tsA30-Nl f 45 24 + - Medium Low

-SV-tsA30-N4 f 45 18 + - Medium NTi

-SV-tsA30-Al a 45 15 ++ - Medium Low

-SV-tsA30-A2 a 45 19 ++ - Medium NT

-SV-tsA30-An3 f 45 16 + - NT NT NT

PY-WT-N2 f 45 20 + + High High +

PY-WT-N3 f 45 NT + + High High +

PY-WT-A1 a 45 12 ++ + High High +

PY-tsa-Nl f 45 12 + + High High ++

PY-tsa-N3 f 45 12 + + Medium High ++

PY-tsa-Nll f 45 12 + + High High ++

PY-tsa-Al a 45 NT ++ + High High +

PY-tsa-A2 a 45 12 ++ + High High +

aEstablished from Fisherratembryonic

cells

(15).

ba,Colonies in agarose; f, focus formation (9).

cCell densitiesatthegrowth plateau.

Cells

x104 per square centimeter.

d Measured inDulbecco-modified Eagle mediumsupplemented with10%

newborn

calf serum (15).

'Measuredby seeding5x

104 cells

in soft agar andcountingthecolonies8dayslater(9). Growthability in agarosewasscoredasfollows: ++,morethan 75% of the cellinput gave risetocolonies; +, 25%; -, less than 0.002%.

fSymbols: -, slow growth and low saturationdensity similartothose observed in the presence ofhighserum concentrations (15); +, generation time and maximal cell density similarto those observed at high serum concentrations.

'Explanation ofterms:low, values similartothat shown for FR 3T3cells (Fig. 2); medium, values in the samerangeasthat shown forSV-WT-A1;high, values similartothat shown for PY-tsa-Nl.

hHydrolysis of[3H]casein in theabsence ofplasminogen after8hof incubation at 33°C (see text). Low, Slighthydrolysis(verysimilartothat of FR3T3); high,significantlyhigherrateofhydrolysis (can reachavalue which is10to15% that obtained in the presence of plasminogen).

'Symbols:++,positive reactionby the agaroverlayassayandrateof[3H]casein hydrolysissimilartothat obtainedforPY-tsa-N1cells(Fig.5); +,rateof[3H]caseinhydrolysisin thesamerangeasthatshown for PY-tsa-Al cells(Fig.5); -,nodetectablecaseinolysis byeither assay.

iNT, Not tested.

(15), all polyoma transformants tested except

one(PY-WT-N2) exhibited generation times of near 12 h at 33°C in Dulbecco-modified Eagle

medium supplemented with 10%calfserum, as

compared with 25h forFR3T3cellsunder the same conditions. SV40 transformnantsappeared

tobesignificantly different in this respect,as all

of themgrewwithdoublingtimesrangingfrom

15 to 24 h. This property of the established

transformants is in agreement with the

obser-vation that, after virusinfection, theappearance of visible foci or agar colonies requires 5 to 6

weeksat33°C for SV40,ascomparedwith 2to

3weeks forpolyomavirus.

(ii) Growthatlowserumconcentrations.

Severaltransformed cell lines and theFR 3T3

parentalline weregrownin mediumcontaining 2% calfserumuntiltheyreached agrowth

pla-teau. As previously reported (15), the FR 3T3

line attainedacell

density

of

106

cellsper 6-cm

plate

and then ceased to divide. On the other

hand,

as shown in

Fig.

1, SV40- and

polyoma

virus-transformedcells grew

beyond

the

conflu-ence state to reach

higher

saturation densities (4x

106

and7x

106

cellsper

plate,

respectively).

(iii)

Rateof

2-deoxyglucose uptake.

Cells

wereseededoncover

slips

atalow

density

and tested for therate of hexose

uptake during

ex-ponential growth

at

33°C.

Inall cases, kinetics

oftransportwereobserved toremain linearfor

atleastthefirst30minof incubation.

Results obtained with six

representative

cell

linesareshownin

Fig.

2.Foreverycell

line,

the

rate of

2-deoxyglucose

uptake

wasincreased as

compared

with thatofnormalFR 3T3cells. Two classes oftransformants

could, however,

be

dis-tinguished:

afirst group,

exemplified by

the

PY-tsa-Al and

-Nil

lines,

was characterized

by

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700 PERBAL AND RASSOULZADEGAN

3~~eS ,~.m'".,.3

FIG. 1. Typical saturation density ofFR3T3, SV-tsA30-Al,andPY-tsa-Al cell lines.Cells(5x104/60-mm petri dish)wereseeded andgrownfor 15daysin the presenceof2%ocalfserum. Thegrowth mediumwas changedeveryotherdayatthebeginninganddailyat the endoftheexperiment. Monolayerswerestained with Giemsa(Rhone-Poulenc, Lyon,France).

°

15 v

E/

C\j =5

0

v15 30 45 60

Time of incubation (min)

FIG. 2. Kineticsof 2-deoxyglucose uptakein

nor-mal andtransformedFR 3T3 cells. Cellswereseeded on coverslips, grown at33°C, and assayed for 2-deoxyglucoseaspreviously reported (8).Lines repre-sent the average ofseveral independent

measure-ments. Symbols: A, PY-tsa-Al; V, PY-tsa-Nll; *,

SV-tsA30-Nl;x,SV-tsA30-Al;0,PY-tsa-N3;O, FR

3T3 normalcells.

rate of uptake at least five times higher than that of the normal cells, whereas in cell lines

such as PY-tsa-N3, tsA30-N1, and

SV-tsA3O-Al, these valueswereonly increased bya factor of1.5 to 2.

As shown in Table 1, all of the SV40-trans-formed lines tested exhibited a relatively low

rateofuptake, whereassevenof eight polyoma transformants behaved like the PY-tsa-A1 and

TABLE 2. External proteases and PA in FR 3T3 cells and variousrepresentative transformants

Hydrolysis of[3H]caseinin the mediumby intactcellsa Cellline

Without With plas-plasminogen minogen

FR 3T3 1,390 4,500

SV-tsA30-N1 2,050 3,100

SV-tsA30-A1 610 3,700

SV-WT-A1 1,500 3,700

SV-WT-N1 2,100 3,200

PY-tsa-Nl 2,200 20,500

PY-tsa-A1 1,900 7,000

PY-WT-N2 1,250 6,700

Therateofhydrolysisof

[3H]casein

wasmeasured asindicated in thetextin cells grownat33°C;values represent the ratios ofacid-soluble 3H radioactivity

after6hof incubationat330Ctothe total cellprotein.

-Nil

lines

(PY-tsa-N3

being theonly exception

observedamong this set of polyoma

virus-trans-formedlines).

(iv)

Surface proteases and external PA.

Hydrolysisof

[3H]casein

(activityreleased in the

mediumorbound to the

external cell

membrane orboth[see above]) was measured either in the presence of plasminogen (assaying both PA and nonspecific protease activities) or in its absence

(protease activity). The plasminogen-independ-entactivity was slightly increased in most

trans-formantsascompared with that of FR 3T3 cells. Theplasminogen-dependent activity was, how-ever, again clearly different in the two sets of transformants; whereas

all

SV40

transformants

exhibitedlow levels of activity, as did the origi-nal normal

cells,

significantly higher values were

observed for polyoma

virus-transformed

lines

(Tables1and 2). In addition, whencolonies were tested by the qualitative casein overlay assay, none of the SV40 transformants induced a de-tectable caseinolysis, whereas

all

of the polyoma

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PHENOTYPES OF POLYOMA AND SV40 TRANSFORMANTS 701

transformants were clearly positive (data not

shown).

Withonly fewexceptions, the 15independent

transformants tested so far may therefore be

distributedamong twoclasses of transformation phenotypes.Afirstgroupof lines ("low

transfor-mation levels") wascharacterized by relatively long generationtimes,poor growth atlowserum

concentrations, amoderate increase in 2-deoxy-glucose uptake, and no detectable increase in PA production. A second group ("maximal transformation") wasclearly different from the normal phenotype for all of these characters. The fact that the spectrum oftransfornation phenotypes inducedbypolyoma virus in FR 3T3

rat fibroblasts differed

significantly

from the widely distributedrangeof transformation levels induced

by

SV40 (12) will be further docu-mentedbelow.

Resultsreportedsofar also indicate that the

E

4-

I-0

C)

0_X

r

transformation levelsobservedin various trans-formantsare notrelated in any simple way to

the selection procedureor to the A and Ntypes

among tsa- andtsA30-transformedcells. It was

of interesttodeterminewhether in thetsNlines

which expressed the maximal transformation levels (polyoma tsa-Nl and -Nil), all of the

characters assayedare temperaturedependent. Temperaturedependence of the saturation

den-sity of these lines,aswellastheir growthrates

and ability to grow in low serum and in agar

medium, has beenreported previously (15). Temperature sensitivity of hexose

up-take

andof PA

production

inpolyoma tsa-N transformants. (i) 2-Deoxyglucose up-take. Cellsweregrownateither33or

410C,

and kinetics of

[3H]2-deoxyglucose

uptake were

measured at both temperatures. As shown in Fig. 3, the initialrates of hexose uptake were

decreasedby about 50% in tsa-Npolyoma

trans-pytsN3

D

FR3T3

15 30 45 60 90

TIME OF INCUbATION (min)

FIG. 3. Thermosensitivityof 2-deoxyglucoseuptakeintypeN

transformed

celllines. Therate

of

deoxyglu-coseuptakewasmeasuredaspreviouslydescribed(8)ateither33°C (open symbols)or41°C(closedsymbols)

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702 PERBAL AND RASSOULZADEGAN

formants grown at

41°C

relative to a parallel culture grown at

33°C,

whereasa 1.5- to2-fold increase was observed for FR 3T3

cells,

tsa-A

transformants,

and WT

polyoma

transformants.

A similar observation was

reported

previously forSV40

tsA30-transformed

cells(8).

Asshown inFig. 4, whenPY-tsa-N1cells were

shifted from 33 to

410C,

their rate of uptake dropped quickly and within 4 h after the

tem-perature shiftwasdown tothe level measured under thesameconditions in culturesgrown at 410C (Fig. 3A,30min).

(ii) PA. As shown inFig. 5,

production

of PA

asmeasured

by

the

plasminogen-dependent

hy-drolysis of

[3H]casein

wasfoundtobe

tempera-turesensitiveinPY-tsa-N1cells.Thesame

con-clusion was reached

independently (data

not

shown) from

qualitative

overlay

assays on iso-lated colonies. Since these assays

only

reflect the presence of PA on the outside of the cells (membraneandmedium), similarmeasurements

20

pyLs Ai

n 15_

AiJ

_.

/

_

10

D

0

o pyLsNi

w

CsJ

5-0 2 4 6 8

TIME (hours)

FIG. 4. Temperature-shift experiments with PY-tsa-Nl andPY-tsa-Al cell lines. Cells were seeded onplastic cover slips(Thermanox; Lux Corp) ata density of 5x 104cells percover slip (9 by 35 mm). They were grown inDulbecco-modified Eagle me-dium (GIBCO Laboratories) buffered with 50 mM HEPES

(N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid; Merck & Co., Inc.)for 70 h at33°C. Cover slips were then shifted to the restrictive tem-perature (41IC) forincreasing periods of time. The rate of2-deoxyglucose uptake was measured by in-cubating the cover slips for 30 min under the condi-tions described previously (8).

were

performed

on cell extracts to determine whether a transport

mechanism,

rather than

synthesis by itself, might

be the

temperature-sensitive

rate-limiting step.

Triton X-100 extracts were prepared as

de-scribed abovefrom cellsgrownatboth

temper-atures andshifted from 41 to

330C

for various

periods

oftime. PA activities in such extracts werefoundtobeabout 10-fold higher thanthe

activitiesmeasured

previously

in theintactcells

(Table 3),

whichmayreflecteitherthepresence

of

large

amountsofinternalPA or anactivation process in the presence of Triton X-100. The

specific activity

of extracts from tsa-N1 cells

grownat

410C

was20-to30-foldlowerthan that ofsimilar extractspreparedfrom cellsgrownat

lowtemperatures.Upon temperatureshiftsfrom

41 to

330C,

the

activity

was

fully

restored within

a short period oftime (less than 2.5 h). It is

therefore

likely

that the temperature-sensitive viralfunctionacts at acontrollevelinthe intra-cellular

synthesis

of PA rather than on a

trans-port mechanism.

Preliminary

resultsfrom simi-lar experiments performed in the presence of

cycloheximide suggest that the appearance of PA activity upon a temperature shift-down is

dependentonactiveproteinsynthesis andmight therefore representaninduction of denovo

syn-thesis of PA.

(iii) Uncoupling

oftemperature

depend-ence oftransformation

characteristics

in tsa-Ntransformants after high numbersof

passages in culture. A fully thermosensitive phenotype wasalso obtainedwith PY-tsa-N11

cells when these cells were tested soon after

their isolation. However, when cells at

higher

passage numbers were examined, they were

foundtostillbetemperaturedependent for their

rate of2-deoxyglucose uptake, but to produce

high

levelsofPAirrespective of the temperature (datanotshown). Itthereforeseems likelythat during successive generations in culture, var-iants expressing this transformation character undera different control may be selected. To furtherdocumentthisescape fromthe

temper-atureviral control, wegrew early passage PY-tsa-Nl cells at330C forlarge numbersof gen-erationsandtested thematvarious

timnes

forthe

amount of PAreleasedin themediumateither

33 or 410C. As shownin Table 4, the

41/330C

ratios increased progressively to a point (130 generations)atwhichthetemperatureeffectwas barelysignificant. Further studies arerequired

to determine whether clones which have lost

theirtemperatusensitive behavior contain

re-vertant viralgenomes.This does notseem

likely,

however, in view of the observation reported above that hexose uptakeandcloning efficiency in agar remain thesame as in theoriginal

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PHENOTYPES OF POLYOMA AND SV40 TRANSFORMANTS

pytsa Ni pytsa Ai

C3

Cl.. o 7v E5 A

C 0

2 4 60 2 4 6

Tm.c of incubation (houlrs)

FIG. 5. PAsynthesis by normal andtransformed FR 3T3 cells incubated at either 33 or41°C.Normal cells, polyoma transformants, andSV40transformants were grown at33°Cuntil theyreached a density of about 2

x 104 cells per 35-mm plastic petri dish. Some plates were left at33°C, whereas others were shifted to the restrictivetemperature (40 to410°C) for 24 to 48 h. Cellswere washed three times with 5 ml of prewarmed Dulbecco-modified Eagle medium without calf serum; 2 ml of medium containing 25 piofplasminogen and 100 pgof[3H]casein was added to the plates. At the indicated times, the amount of [ H]casein hydrolyzed was measured in a 2(X-ylsample. The proteinconcentration was measured after incubation of the cells in the presence of 0.2 N NaOH (1 ml). It was checked each time that the plasmin generated during the incubation

did not affect the cell morphology. Symbols: open,33°C;closed,41°C.

TABLE 3. Temperaturedependence of PA activity inextractsfrom PY-tsa-NI cells

~~con~tio~ Hydrolysisof Overlay Culture conditions

[3H]caseina

assayb

33°C,2days 420,000 ++

41C,2days 15,000

41and33°C,0 h 17,000

41and 33°C,2.5h 370,000 +

41and33°C,4.5h 496,000 ++

41and33°C,7h 300,000 ++

aTriton X-100extracts werepreparedasdescribed

in the text. Theactivity is expressed as [3H]casein

hydrolyzed (counts per minute) permilligramof pro-tein in theextract.

bCellsweregrownat

330C

and incubatedfor48h at41°C. Plateswerethen shiftedto33°Cforincreasing periodsof time andincubated backtothe restrictive temperature to perform the qualitative assayas de-scribed inthelegendto Fig. 1.See Table 4, footnote b, forexplanationofsymbols.

Ni andtsa-Nllcelllines

(data

notshown). Frequencyof

maximally

transformed

de-rivatives after mass infection of FR 3T3 cells with either

polyoma

virus or SV40. Among the set of 16

independent

transformed lines testedatthis

point

(Table 1),

most

polyoma

transformants exhibited maximal

transforma-TABLE 4. Loss of temperature sensitivity of PA production inPY-tsa-NIcellsupon continuous

growth in cell culture

[3H]caseinhydrol-

[3H]2-ysis PA

deoxyglu-No. of Temp over- cose

up-gener- (OC) Activity 41°C/ lay as- take

ations inex- 330C Sayb (41Cra tractsa ratio

33iCora

5 33 300,000 0.03 ++ 0.5-0.6

5 41 10,000

10 33 260,000 0.10 ++ 0.5-0.6

10 41 28,000

80 33 223,000 0.45 ++ NT

80 41 102,000 +

130 33 212,000 0.66 ++ 0.5-0.6

130 41 140,000 +

aTotal PA measuredin Triton X-100 extracts

ex-pressedascountsperminutepermilligram ofprotein.

bPerformedas in Fig. 1. Symbols: ++, clear large

halos after 8 h ofincubation; +, need to wait 24 to 48 h to seeclearhalos; -, no halos within 36 h of incu-bation.

CMeanratio established from severalindependent measures(30-niinuptake). NT,Not tested.

tionlevels, whereas allof the

SV40-transformed

lines,although showinghighsaturationdensities and the ability to grow in agar medium,

ex-hibited relatively low levels of hexose uptake

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704 PERBAL AND RASSOULZADEGAN andnodetectable PAproduction. To determine

whether this observation reflectedanactual

dif-ferencebetween thetransforming abilitiesofthe twoviruses, rather thanarandomphenomenon

due to the relatively small number of clones analyzed, weused the qualitativeoverlayassay for PA production (see above) as a means for

establishing the proportionsofmaximally

trans-formed derivatives in a statistically significant way.

Parallelcultures of FR 3T3cells wereinfected

witheither WT polyoma virus orWT SV40 at

identical multiplicities (250 PFU/cell), and transformants were selected at 33°C either as

fociovergrowing attached monolayersoras

col-onies in agarose medium, under the conditions

previously used forestablishing the transformed cell linesdescribed above(8, 15). After the plates

were scored for the number of transformants,

both the attached cultures and the agarplates

were overlaid with the casein-plasminogen-aga-rosemixture, and the number of PAproducers wasdetermined after either 12-hor2-day

incu-bationsat33°C. The numberof haloswas never

observedtochange with the incubation time. As shown in Table 5, the frequencies of PA-producing clones obtainedamongthe

transform-ants were quite different after SV40 and poly-oma virus infections. The lowest values were

observed among transformants selected as foci

afterSV40infection; in thiscase, thefrequency

wasalso dependentontheselectionprocedure, ashigher proportions of PA producerswere

ob-served among the colonies grown in agar

me-dium.Highfrequencieswereobservedwith pol-yomavirus among both foci and agarcolonies.

These frequencies were not found to be de-pendent on themultiplicity of infection (0.1 to

250 PFU/cell), an observation reinforcing the conclusion that there is an actual difference

between the transforming abilities of these two viruses which cannotbe accounted for by acci-dental variations in the titers of the virus stocks. Table 5 also shows the results of an

experi-mentinwhichanonproducingestablished SV40

transformant (SV-tsA30-A2) was infected with either polyoma virus or SV40. "Supertrans-formed" PA producers could be observed, again

atagreaterfrequency with polyoma virus than

with SV40. These clones also grew faster than SV-tsA30-A2 cells, since they appeared as dis-tinct foci on the layers of transformed cells grown onplastic platesat33°C.

Withpolyoma virus, thediameter of the

cas-einolysis area wasnotrelatedtothesize ofthe colony, whereas the halo sizeswerealwaysmore

uniformly distributed and correlated with the diameter of the colony for SV40 transformants (Fig. 6). This observation suggests thatPA pro-duction insomecases,butnotinothers,maybe

correlated with the growth rate of the cells in suspension.

DISCUSSION

A number of independently isolated rat cell

lines transformed by SV40 and polyoma virus

werestudied for their transformation phenotype

incell culture.These lineswereestablished un-deralimited number ofdefinedconditions (cell

growth,selection procedure,useof either WTor early tsmutants,etc.) andtherefore offered an

approach for the identification of possible cor-relations between transformation characteris-tics. Their phenotype wassystematically com-pared with theparental diploid established FR 3T3 lineatearlypassagesincell culture.

Theresults obtained ledustothe operational distinction between a maximally transformed

[image:8.514.69.465.516.646.2]

state and several intermediate phenotypes as

TABLE 5. Frequency of PA-producingtransformants after polyoma virus and SV40 infections of normal rat cells(FR3T3) and nonproducing SV40-transformed cells

Transfor- No of clones No. of

casei-Cell line Virus Selection' mation fre- tse oyi ao

quencyb tested nolysis halos

FR 3T3 None aorf 0

FR 3T3 SV40 a 35 528 90 17

FR3T3 Polyoma a 15 800 765 96

FR 3T3 SV40 f 20 403 12 3

FR 3T3 Polyoma f 20 300 204 68

SV-tsA30-A2 None a 200 0 0

SV-tsA30-A2 SV40 a NT NT NT

SV-tsA30-A2 Polyoma a 152 124 82

SV-tsA30-A2 SV40 f 700 119 17

SV-tsA30-A2 Polyoma f 900 333 37

aa,

Colonies in

agarose

medium;

f,focus formation (15).

bNumber oftransformants per104infected

cells.

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PHENOTYPES OF POLYOMA AND

SV40

TRANSFORMANTS

705

I

FIG. 6. PA-induced caseinolysis by WT SV40 transformants(A)andby WTpolyoma transformants (B)growninagarmedium.

previously reported for polyoma virus-infected

hamstercells(18) and for SV40-infectedmouse

cells (12). Thus, all polyoma virus-transformed

ratcellson oneside exhibiteda

well-character-ized transformed phenotype for any property which was assayed (growth characteristics on

solid substrate withhighor lowserum

concen-trations, abilitytogrowwithoutanchorage,rate

of hexose uptake, and production of PA), whereasall of the SV40 transformants studied expressed intermediatelevels of transformation.

Although all transformantsshowedanincreased rate ofdeoxyglucose uptake, noneof theSV40

transformants produceddetectable amounts of

PA. A similar dissociationof PA excretion from

othertransformation characteristicshasalready been reported in some Rous sarcoma

virus-transformed cells (19). Thelatter cells showed

levels of PA similar to that of the uninfected

cells, although theyexhibitedseveralproperties of transformed cells, including increased sugar transport, altered cellularmorphology, high

ef-ficiencyofgrowthinagar,andhighincidenceof

wing webtumors (20). Thesecretion of PA

in-hibitors bytransformed cellshas also been re-ported in severalcases(4, 13). Ourpreliminary results obtained with SV40-transformed cells

havenotrevealed the detectionofany PA

inhib-itor. The lack of PA secretion by SV40

trans-formants, togetherwith the intermediate

trans-formationphenotypeobserved forgrowthin2%

serum,growthinagar,and rate ofdeoxyglucose

uptake, may reflectdifferences in the transform-ing abilities of SV40 and polyoma virus. Al-though conflicting results have been reported regarding the correlation between tumorigenic-ityandPAproduction (3, 4, 7, 13),the

compar-isonbetween the different phenotypes observed

in culture and the ability to induce tumors in animals has been undertaken. Preliminary

re-sults indicate that the polyoma transformants

induce tumor formation in various animals (young Fisher rats,nudemice, and chicken

cho-rioallantoid membrane), whereas the SV40 transformantsaremuchless tumorigenic under

the same conditions (M. Rassoulzadegan, F. Birg, G. Meyer,andF. Cuzin,unpublisheddata).

The frequency ofPA-producing transformants

was measured after polyoma virus and SV40 infectionof FR 3T3 cells. Theresults obtained clearly showthat these two virusesinduce

dif-ferent patterns of transfornation in rat cells.

After polyomavirusinfection,96% of the trans-formantsselectedinagarosewere PAproducers, whereas only17% of the coloniesobtained under

thesameconditionswithSV40wereproducers.

Itseemsthat agarose selection leads to ahigher proportion ofPA producersamong both SV40

and polyoma transformants as compared with the selectionoffoci growingonplastic (Table 5).

It isthen possiblethat growth in agarsuspension requiresa"moretransfornedstate" than is re-quired forgrowth as foci on plastic. The super-infection of a nonproducer SV40 tsA30

trans-fornant with WTSV40 and WT polyoma virus shows that the existence of a viral genome

(SV40) in thetransformed cellsdoes not prevent

the expression ofanother since supertransfor-mationby WTpolyoma virus resultsin a much

higherfrequency of PA-positive colonies (38%)

than is observed when the cells are

retrans-formed with WT SV40 under the same

condi-tions(17%).

It therefore appears that different levels of

transfornation areinduced by SV40and poly-omavirus.Whether thesedifferencesarerelated

inmolecularterms, to the organizationofviral sequencesincells,or totheexpression of partic-ularviralproteins remainstobeestablished. It has been reported previously that ratcellsare

nonpermissive for SV40 replication, whereas

they aresemipermissive forpolyoma virus

rep-lication (reviewedinreference 17). Itwould be

of interest to determine whether the transfor-mation levelswhich are reported in this study

arerelatedtodifferentlevelsofcellular permis-sivity.Inany case, the distinct nature of thetwo

viruses appears in ourhands to be one of the criticaldeterminants of the

variability

observed among thetransformedphenotypes.

Twoclasses oftransformants have been

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PERBAL

TABLE 6. Summaryofthethermosensitivetransformationcharacteristics in PY-tsa-NI cells

Saturation Rate of[3H]casein

hy-Growth densityin Genera- Platingeffi- Rateof 2- drolysis' Production

Cells_Ct(eOmcptm _containingmeim _timbetion _agaroseciency_me-

mn

deoxyglu-ofP

_{Without (overlay}

)10% calf se- (h) diumC minogen plasmino-

assay)f

rumi gen

PY-tsa-Nl 33 >10 12 14 14,000 20,500 2,200 +

41 1.5 22 <0.01 5,500 3,400 2,700

FR 3T3 33 5 25 <0.002 2,000 4,500 1,800

41 5.1 24 <0.002 2,500 4,800 1,800

a

Cells

per 6-cmplatex 10'.

bFromSeif and Cuzin(15).

cExpressedasthe percentage of the

cell

input (5x

104

cells)growinginagarose medium. d Measuredas

previously

reported (8).

'Expressedas countsperminute per6hof incubation per

milligram

ofprotein.

fSymbols: +,clear halos after8h ofincubation;-,nodetectablehydrolysisafter 36 h of incubation.

tained with either SV40orpolyomavirus:

trans-formed celllines oftypeN were found toregain the normal phenotype at high temperatures,

whereastypeAcell linesstillexpresstheir

trans-formation characteristics at the nonpermissive

temperature (8, 15). The different phenotypes described with the present set ofcell lines did

not permit a differentiation between A and N

transformants (polyoma tsa and SV40

tsA30).

However, recent studies led to the conclusion

that theproductionof PAmaybesusceptibleto

the growth conditions applied toinfected cells

during the selection of transformed cells (B. Perbal,manuscriptinpreparation).

Allof thetsNcell lineswerefoundtoexpress their transformationphenotypeat33°Cbut not

at 40to41°C. As shown in Table 6 for

PY-tsa-Ni cells,the rates of bothdeoxyglucose uptake and PA production reverted with generation time, saturation

density,

and

plating

efficiency

inagarandonplastictothe valuesobtainedfor

FR 3T3 cells. Thus, the

expression

ofa whole

setofvarious transformationcharacteristics ap-pears to be dependent upon the

expression

of

the viral functions defined

by

tsa

(and tsA30)

mutations.Temperature-shift

experiments

show that the transition from one phenotype tothe othertakesplacewithin a shortperiod oftime. The drifts of PA production and cloning effi-ciencyonplastic,which werereportedabove for

polyomatsN cellsgrown for severalgenerations,

suggest that their control is unlikely to be a

simple oneandprobably involves bothcellular

and viral factors as previously reported with Rous sarcomavirus-transformed cells(20).

ACKNOWLEDGMENTS

We thankF.Cuzin for helpfulcriticism of this work and F. Birg and G. Fareed forthe critical reading of themanuscript. The technicalassistance of C. Bonifacino and L.Carbone is

gratefully acknowledged. We also thank M. Ducasse, L. Fer-aud, and R. Rahmani fortheir kind help in the study of the PY-tsa-Nl cell line and to Francelait S. A. for the generous gift of nonfat driedmilk.

This work wasmade possible by grants C. R. L. 78.1.083.1 and AT79-114from the Institut National de laSant6 et de la Recherche Medicale.

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