Large-T-antigen-p53 complex formation is not cold sensitive in a cold-sensitive transformant induced by simian virus 40 mutant tsA1499.

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0022-538X/84/030997-05$02.00/0

Large-T-Antigen-p53 Complex Formation

Is Not

Cold Sensitive in

a

Cold-Sensitive Transformant Induced by Simian Virus 40

Mutant

tsA1499

NOEL BOUCK,* JOHN FIKES,AND M. KATHLEENRUNDELL

Department ofMicrobiologylImmunology and Cancer Center, Northwestern University Medical and DentalSchools,

Chicago, Illinois 60611

Received24August 1983/Accepted9November1983

Flll rat cells transformed by simian virus 40 mutant tsA1499 are cold sensitive for the

expression

of

transformation. Yet, unlike Flll cells transformed by

tsA58, they

do not lose the

ability

to stabilize the

transformation-associated hostcell

protein

p53atthe temperatureatwhichtransformation isextinguished.

Although it has been clear for a number of years that the

maintenance of the neoplastic phenotype in a cell

trans-formedby simian virus40 (SV40) depends on the continual

functioning of the virus-encoded protein known as large T

antigen (2, 21, 30, 38, 46, 51), exactly how T antigen

performs this feat remainsobscure.

LargeTantigen isa708-amino-acidprotein which is found

tobeassociatedinhighconcentrations withvarious nuclear

fractions of the cell (45) and in low concentrations with the

plasma membrane(20).Multiple,genetically separable func-tions have been assigned to large T antigen (50), many of

which could conceivably be crucial in keeping a host cell

transformed. Some of these functions, however, can be

active in virusesthatareunable to maintain transformation

and, therefore, appear unlikely to be sufficient for the

maintenanceofmalignancy.Theseinclude the ability to bind to the SV40 origin of DNA replication (7), to initiate viral DNA synthesis (10), to regulate viral transcription (10), to conferimmortality (9), and tostimulate rRNAtranscription and DNAsynthesis in thehost cell genome (44).Some

large-T-antigen functionscanbe lostfromavirus without impair-ingitsabilitytotransformandtherefore appear unnecessary

formaintaining transformation. These functions includethe

ability to bind to the origin (49), to initiate viral DNA

synthesis (31, 48), and to help with adenovirus replication (1). Oftheknownfunctions oflarge Tantigen, only two have not been convincingly dissociatedfrom the maintenance of

transformation: an ATPase activitywhich copurifies with T

antigen (6, 15, 49) and the ability of T antigen to from a

complexwithand stabilizeahost cellprotein with a molecu-larweightof about 53,000 (p53)(24).

The most attractive current hypothesis suggesting the mechanismby whichTantigen might act to maintain

trans-formation focusesontheability of T antigen to complex with

p53. p53 is a highly conserved (23), very short-lived (36)

phosphoprotein with an associated protein kinase activity

(22) thatappears toplay a regulatory role in normal cellular growth control, as microinjected antibodies againstp53 can haltseruminductionof normal cellular DNA synthesis (33).

p53

levels are usually low in normal cells. In cells

trans-formedby SV40, virtuallyallcellularp53is complexed with aportion of T antigen (32), and the level and stability of the

p53 protein are increased manyfold (36, 37). It has been

* Correspondingauthor.

proposed that these high levels of p53 complexed with T

antigen act in a positive way to maintain the unregulated growthof theSV40-transformed cell(25).

This suggestion is enhanced by the finding that early

transformation-associated antigensoftwoother DNA virus-es,adenovirusandEpstein-Barr virus, also formcomplexes with andincrease the levels of p53 (28, 42). Inaddition, p53 levels are elevated in a wide array of variously induced

primarytumorsand tumorcelllines, especially inmice (13,

26,40, 41),butalso in humantumorcells(12, 14).However, thereare clearexceptions to these correlations ofhigh p53

levels and transformation. Some tumorcells of human (12) andmouseorigin (36) andsome primary mousetumors(40) do not showhigh p53levels. Inlymphocytes (34, 35), some cultured cells (14), and early embryos (5), high levels are seen in the absence oftransformation. As hasbeentrue of some earlier correlatesoftransformation, elevated levels of p53 could beafrequent consequence of theexpressionof the

rapid-growth phenotype ofthetransformed cell ratherthan

thefundamentalcause oftumorigenic growth deregulation.

The experiments reported here test whether the expres-sion ofthe transformed phenotype always correlates with thestabilization ofp53inanSV40-transformed cell line. The

results indicate that transformation can be lost without

significantreduction in the p53-T-antigen complexorinp53

stability andthat thep53 stabilizingfunction of Tantigenis notsufficientto maintainthe transformed state.

Amatched setof cell lines (38) wereusedwhich wereall selected and recloned in agar after virus infection of one parental rat fibroblast line, Flll (for culture methods, see reference 38). The lines were derived from infection with different T-antigen mutants and varied in the effect of temperature on their ability to express transformation. Transformationhas beenmeasuredasthe abilitytoclone in

softagar, anassay whichhas been showntobea strict and

appropriate in vitro correlate of neoplasia in

SV40-trans-formedratcells (50).

Table 1 illustrates the phenotypes of the cell lines as measured inafractionfrom each of the cultures used for the

pulse-chaseexperiments reportedbelow. Normal Flll does notclone in agar, whereas itswild-type-transformed

deriva-tive, F(wt)40,clonesefficientlywhether grown and testedat the high (39.3°C) or the low (31.5°C) temperature. Flll transformedby mutant tsA58 expressestransformation

effi-ciently onlyatthelow temperature,asis characteristic of the

majority of cells from various species transformed by this 997

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998 NOTES

TABLE 1. Effect of temperature on expression of transformation

Cellline Temp Agar plating Relative agar plating Phenotype

efficiency(%) efficiency(%)

Flll 31.5 <0.02 <0.04 Normal

39.3 <0.02 <0.03 Normal

F(wt)40 31.5 39 48 Transformed

39.3 43 59 Transformed

F(tsA58)A 31.5 67 56 Transformed

39.3 0.1 0.29 Normal

F(tsA1499)1 31.5 0.36 0.42 Normal

39.3 29 21 Transformed

Fisher rat fibroblasts of cell line Flll and cloned derivatives transformed by wt 45-54, by tsA58 or by tsA1499 were grown at the temperatureindicated for3days and testedatthat

temperatuire

for theabilitytocloneonplasticand toclone in0.3% softagar (38). Relative agarplatingefficiency = (agarplatingefficiency/plastic plating efficiency) x 100.

mutant(2, 21, 30, 38).Flll, transformed bymutanttsA1499

(39), expresses transformation efficiently only at the high

temperature. This cold-sensitive phenotype occurs in the

majority ofclonestransformed by the unusual mutant (38),

which contains adeletion at the carboxy-terminal end ofT

antigen, resulting in ashortenedprotein (dlT).

Thestability ofp53 and ofthep53-T-antigen complexwas tested inpulse-chaseexperiments under conditions inwhich transformationwaseitheron oroff(Fig. 1).Brotherdishes of

cells growing at high or lowtemperature were pulsed with

[35S]methionine

and chased for variousperiods oftime. An identical amount ofextractfromeach dishwas immunopre-cipitated withmonoclonalantibodiestoeither

p53

orlargeT

antigen, and amountsofp53 and Tantigen were visualized on fluorograms of sodium dodecyl sulfate gels. The

wild-typetransformantbehaved asexpected (32). LargeTantigen

was stable, the

mnajority

of itwas not complexed with p53, and, as reported previously (3, 4), it appeared to complex with p53 slowly as the amount of

pulse-labeled

T antigen precipitated by

anti-p53

increased with time,

especially

at high temperature. p53 was also stable in the

wild-type

transformant and ran on thesegels as a doublet with label appearing first in the

slower-moving

band and then

equili-brating betweenthetwo. Thisapparent increasein

mobility

ofp53has beenseen

by

othersaftershort

pulses

(3)and may be due todifferences in

phosphorylation

(3) or to

modifica-tions in primary structure (29, 47). In these rat cells, the

faster-moving

band labels more

heavily

with

32p

(data not shown), butbothbandscancomplex withT

antigen,

asboth

areprecipitated by

anti-T-antigen

antibodies.

The tsA58-transformed ratcellsbehave in the same man-ner as mouse cellstransformed

by

this same mutant(4, 17, 27)

(Fig. 1).

At low temperature, where transformation is

expressed, the cells are

indistinguishable

from

wild-type

transformants, except for the

slightly

increased

lability

of largeTantigen.Athightemperature, where thetransformed phenotypeisextinguished,Tantigenisquitelabile, andp53

is extremely unstable. Little

p53-T-antigen

complex is formed in these cellsathightemperature,for,althoughboth

p53 and T antigen are clearly present, precipitation of one

protein withthemonoclonalantibodiesspecificfor the other was seen onlyin veryoverexposed gels.

RatcellstransformedbytsA1499arecold sensitiveforthe

expression of transformation to approximately the same

extent that tsA58 transformants areheat sensitive (Table 1). Yet, the amountofp53-T-antigen complexand the stability ofp53 do not appear to change withgrowth temperature, although under the same conditions, a dramatic change is observed in F(tsA58)A (Fig. 1A and B). In F(tsA1499)1 at low temperature, as well as at high temperature, p53 is

clearly complexed with T antigen and is reasonably stable.

Despite the fact that these cells are as isogenic as we can

make them,there are severalconsistentdifferencesbetween thisline and the wild-type and tsA58transformants. (i) The

slower-migrating component ofp53 remainsheavily labeled and does not equilibrate with the faster component in the tsA1499 line. (ii) A larger proportion oftotal T antigen is

complexed with p53 in the tsA1499 transformant. This is

due,in part, to thefactthat thereisless total Tantigen inthis

line than in the other lines. (iii) p53 is less stable in the tsA1499transformants,morenoticeably soafteraveryshort pulse (Fig. 1A)than after alonger pulse (Fig. 1B).However, although these differences may limitsomewhat direct com-parisonsamongdifferentlines,noneof theunique character-istics ofthe tsA1499 line appeartobecausallyrelatedtothe

expression oftransformation,astheyallpersistatbothhigh

and low temperatures, regardless of whether transformation is expressed or extinguished. For example, densitometry tracings ofthegeldepicted in Fig.1B, withanexposurethat

brings the relevant bands well within the experimentally

determined linear range ofgel

sensitivity,

show that28%of

theinitial p53 remained in the tsA1499transformantaftera 22-hchaseat31.5°C and48%remainedafterthe same chase at 39.3°C. In the tsAS8 transformant, 24% of initial p53 remainedafter 22 hat31.5°C, whereas nonewasdetectable at 39.3°C. Thus, in the tsA1499 lineexpressing the normal

phenotypeatlow temperature, p53was asstableasitwasin the tsA58-transformed line, whichwas fully transformed at this temperature.

Todetermine whether some difference could be detected

by sedimentationin thep53-T-antigen complex whichcould account for the shift in transformed phenotype in

F(tsA1499)1, extracts from the three cell lines growing at high and low temperatures were run on 5 to 20% sucrose

gradients, and individual fractions across thegradient

pre-cipitated withanti-T-antigenantibodieswere run onsodium

dodecyl sulfategels (Fig. 2).

Inthewild-type transformant, sedimentation profilesforT J.VIROL.

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A

F II

F(wt)40

p p

1

2

3 r-

____-

-F(ts A58)A

p

1

2

3

- -_

31*50

_ _-_

T-

_- -_

-m

F(tsA

1499)l

F(tsA58)A

31.50 39*30 39 30

dl

T-p

53=

p 1 2 p 1 2 p 1 2

FIG. 1. (A) Stability of p53. Cells described in Table 1werelabeled inquadruplicate with [35S]methionine for 50 minat31.50Corfor20min

at39.30C andeither extractedatonce(lanep)orchasedin media containingafivefoldexcessof cold methionine for1h(lane1), 4h (lane 2),or

24 h(lane 3). Extracts made in buffer containing 0.15MNaCl, 1%sodium deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecylsulfate, 10 mMphosphate buffer (pH 7.4), and 1% Trasylolwereimmunoprecipitated with monoclonal antibodiestop53,PAb 122 (18)(uppertworowsof

panels),orwithmonoclonal antibodiestolarge T antigen, PAb 419 (11, 19) (lowertworowsof panels). Monoclonal antibodieswerepurified

bya50%ammoniumsulfatecutfrom culturesupernatantsof hybridomasgrownin staphylococcus-adsorbedfetal calfserum. Immunoprecipi-tates were run onOrtec 20% gels and fluorographed asdescribed previously (38). Control precipitations ofeach extractwith ammonium sulfatecutsof media showednobands.Sequential immunoprecipitations showed that antibody concentrations weresufficienttoprecipitate allbuttrace amountsof recognized proteins. (B) Stability of p53 afteralong pulse.Methodswere asdescribed for(A),exceptthat cellswere

labeledfor 1 hat39.3°C and for 2 h and 15min at31.50C. Chase timeswere6h(lane 1)or22h (lane 2).

antigenandp53didnotvarywithtemperature. NoTantigen

was seen at the top of the gradient, where monomers and

dimers would be expected in these standardgradients (32). There was a peak of T-antigen oligomers just below the

middleof thegradient, and heavy p53-T-antigen complexes could be seen infractions nearthe bottom.

Inthe tsA58 transformant grownat lowtemperature, p53 sedimented as in the wild-type transformant, although T

antigen banded more broadly withmore label in the

mono-mer-dimer region. At high temperature, as reported

previ-ously in other tsA58-transformed cells (4, 17), T antigen sedimented largely in the monomer-dimer region, and p53

was notdetectableoverthe background.

InthetsA1499 transformant, Tantigensedimentedovera

broaderrangethan in the other cells tested, butp53wasseen

in the same fractionsas in the parallel wild-type gradients.

There was nostriking difference in patternbetween cellsat

different temperatures where the transformed phenotype clearly varied. When thesegradients wereprecipitatedwith

anti-p53insteadof anti-T-antigenserum,similarresultswere

obtainedfor the distribution of the p53-T-antigencomplex, and no p53 was detectable sedimenting outside of the complex region (datanot shown).

In both the tsA58- and tsA1499-transformed cells, the maintenance of transformation appears to depend on T

antigen,as transformation is sensitiveto temperature shifts

dl

T-p

53=

F(tsA 1499)1

p

1

2 3

p

53=-anti-p53

39.30 p 53=

_

-

-p

53=-31

.50

anti-T

B F I

Il

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1000 NOTES

tsA1499 transformants expressing the normal phenotype is functional. It is possible that the p53-T-antigen complex is

50 functionally

cold sensitive without

displaying

metabolic

la-31@5

bility.

Alternatively, the p53-T-antigen complex could be

perfectly functionalatlow temperature, butits activity isnot involved in themaintenance of transformation. Although the latterexplanation is most appealing,anassayfor the activity

39.30

ofp53 orp53-Tantigen or both will probably be needed to distinguish rigorously between these alternatives.

FtsA1499)1

dT- * 31.50

p53-dl T- 3930

p53-F(tsA58)A

T- *

-31.5

p53-

T-39.30

p53-FIG. 2. T-antigen-p53 complexes. Cells described in Table 1

werelabeled with32pfor120minateither 31.5or39.3°C,with twice

as much label atthe lowertemperature. Tomaintain temperature

controls, all media were prewarmed, and plates were left undis-turbed inincubatorsfor the pulse period. Extracts prepared in Tris-bufferedsaline (pH 7.4) containing 0.5% Nonidet P-40were

centri-fuged in parallel through 5 to20% linear sucrosegradients by the

procedure of McCormick and Harlow(32). Fractions were

collect-ed,immunoprecipitated with monoclonal anti-T antigen, andrun on

gelsasinFig. 1,exceptwithout fluorography. Sedimentation is from righttoleft. dl Tmarks the shortenedTantigen produced bymutant

tsA1499.

only in the mutant transformants and not in the wild-type transformants (Table 1) (38). Yet, only in the tsA58

trans-formant is the loss of transformation accompanied by a

dramatic decrease in p53 stability and loss of the p53-T-antigen complex. In the tsA1499 transformant, p53 is quite stable whether transformation is being expressed or not.

These results indicate that p53 stabilization alone is not sufficient to maintain the transformed state. The possibility that p53 stabilization, in additionto being insufficient, may

sometimesalsobeunnecessarytomaintaintransformation is raisedbyobservations that (i) in SV40, twoplasmid-cloned deletedgenomes are ableto transform, although their

trun-catedlargeT antigens failto stabilizep53 (8, 43); and (ii) in adenovirus, fully transformed cells can be derived from

transfections with DNA fragments unable to make a

full-sized p58, the adenovirus tumorantigen known tocomplex with and stabilize p53 (16, 41a).

The data presented in this paper do not address the question of whether the persistent p53-T-antigen complex in

Wewould liketothank Ed Harlow for the hybridoma producing PAb 419, John Carinofor help with early experiments, and Mary Headand Karen Mayerfor excellent technical assistance.

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