Cytoplasmic T antigens of mouse and human cells transformed by a simian virus 40 tsA mutant.

JOURNALOF VIROLOGY, June 1980, p.782-788 Vol.34,No. 3 0022-538X/80/06-0782/07$02.00/0

Cytoplasmic

T

Antigens of

Mouse and Human Cells

Transformed

by a

Simian

Virus 40 tsA Mutant

D.R. DUBBS,* H.OTSUKA, AND S.KIT

Divisionof Biochemical Virology, Baylor College of Medicine, Houston, Texas77030

Simian virus 40 T antigens accumulate in the cytoplasm of simian virus 40

tsA207 transformants ofprimary mouse kidney or human retinoblastoma cells

grownat40°C in 10%serum.

Usingserafromhamstersbearing largesimian

virus 40 (SV40) tumors, SV40tumor (T)

anti-gens can be demonstrated by

immunofluores-cence in the nuclei of cellslytically infected or

transformedby SV40(13, 14).AlthoughSV40 T

antigensareassumedtobesynthesizedon

poly-ribosomes in thecytoplasm, followedby

migra-tion to the nucleus, SV40 T antigens are not

usually visible by immunofluorescence in the cytoplasm of SV40-transformed cells. Even in

mouse,Syrian hamster, Chinese hamster, rabbit,

andratcells transformedby SV40 tsAmutants

(whicharedefective inanearly SV40 function) abnormallocalization of Tantigenshasnotbeen reported (2, 10, 11, 17). This paper describes a temperature-dependent SV40tsAtransformant, mKSA207 clone6 (Cl6), which under

appropri-ategrowth conditions accumulates SV40 T

an-tigens in the cytoplasm. The purposes of this

study were to: (i) define the growth conditions

under which T antigens accumulate in the

cy-toplasmofmKSA207 Cl6cells and otherSV40

tsA207 transformants; (ii) determine the effect

of the growth phenotype on localization of T

antigens, using serum-requiring and

tempera-ture-resistant (tr) variants derived from

mKSA207 Cl 6; and (iii) determine whether

in-duction ofcytoplasmicTantigens is an inherent

property of SV40 tsA207 in transforming and

lyticinfections.

The cell lines used in this study are

summa-rized inTable1.Wild-typemKS [mKS(wt)] and

ERSV40(wt) cells were grown at 37°C;

mKSA207 Cl 2, 3, 6, 9, and 10, S6/R1/R'5,and

ERA207 cells were grown at 33.5°C; and

mKSA207-CH, mKSA207-S4, and mKSA207-S6

cellsweregrown at40°C. Allcellswere grown in

Eagle minimal essential medium (Auto-Pow,

Flow Laboratories, Rockville, Md.)

supple-mented witheither 10% calf serum or 10% fetal

calfserum.SV40 T-antigen determinations were

carriedoutwithcellswhich were grown on glass

coverslips, fixed at-20°C in absolute ethanol, and placed for 15 min on dry ice. T antigens

were demonstrated by indirect

immunofluores-cence,usingascitic fluid from hamstersbearing

SV40 tumors and goat anti-hamster gamma globulinconjugatedwithfluorescein

isothiocya-nate (Antibodies, Inc., Davis, Calif.). The

ham-ster ascitic fluid was adsorbed with calfserum

and human [HeLa(BU25)] and mouse

[LM(TK-)] cells beforeuse.The hamster ascitic

fluid used in this study immunoprecipitated bothlargeTandsmalltpolypeptides.Theterms

"cytoplasmic T antigen" and "intranuclear T antigen"areusedtodefinespecific

immunoflu-orescentmaterial locatedin the

cytoplasm

and

nucleus, respectively. Cells were observed and

photographed with a Leitz Ortholux 2

micro-scope equipped with incident UV illumination.

Theresultsareshown inFig. 1,2, and3andare

summarized in Table2.

ThemKS(wt) cells,like many otherSV40(wt)

transformants of rodent cells, are temperature

independentforgrowthinlow(0.5%)serumand

are anchorage

independent

at both 33.5 and 40°C (5, 12).These

cells,

like most

SV40-trans-formedcells,expressedtheintranuclearT

anti-gen at both 37 and

40°C

in either 0.5 or 10%

serum(Table2;Fig. 1A, B,andC).No cytoplas-mic T antigen was observed in mKS(wt) cells

under anygrowthcondition studied.

ThemKSA207 Cl6cellsaretemperature

de-pendentforexpression ofsome growth proper-ties(6, 12). At33.5°C, growthofmKSA207Cl6

cells resembled that ofmKS(wt), and the cells

contained only the intranuclear T antigen

whether grown in 10 or 0.5% serum (Table 2;

Fig.1D). However, mKSA207 Cl6cells failedto

sustaingrowthin 0.5%serum at40°C and,when

incubated for 3 or 4 days, lost intranuclear T

antigens (Fig. IF). About 70 to 80% of cells

fluoresced only faintlyornot atallby

immuno-fluorescence,andabout 70% of Tantigens were

lostby complement fixation (6). The addition of

10% serum to cultures ofmKSA207 Cl 6 cells

that had been partially depletedofTantigens

by incubationat40°C in lowserum for several

days stimulated the cellstoenterthe Sphase of

the growth cycle. Within24 hafter addition of

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TABLE 1. Origin and growth properties ofSV40-transformed cells

Cell line Source Growth properties Reference

mKS(wt) BALB/cmousekidneycells Anchorageindependent, tr 5

transformed

by

SV40(wt) for

growth

inlowserum

mKSA207Cl6 Independent isolates of ts forgrowth in either low 6 mouse kidneycellstrans- serum or methylcellulose formed by SV40 tsA207 suspension culture

mKSA207Cl9 ts forgrowth in low serum Otsuka et al.

(unpub-mKSA207Cl10 ts forgrowth in low serum lishedexperiments)

mKSA207Cl2 tr forgrowth in low serum

mKSA207Cl3 tr forgrowth in low serum

mKSA207-CH IsolatedfrommKSA207Cl6 tr forgrowth in low serum 12

mKSA207-S4 ormethyl cellulose

sus-mKSA207-S6 pension culture

S6/R1/R'5 IsolatedfrommKSA207-S6 Serumrequirement for Otsuka et al. (unpub-growth; anchoragede- lishedexperiments) pendent

ERSV40(wt) Human retinoblastomacells' Not done transformedbySV40(wt)

ERA207 Human retinoblastomacells Not done

transformed by SV40 tsA207 whichhad been rescued from mKSA207Cl

6

aHuman retinoblastoma

cells

wereobtained from V. Riccardi, Baylor College of Medicine, Houston, Tex. Thesecells containone no. 13chromosome with an interstitial deletion.

10%serum at40°C, 95% of thecells

incorporated

[3H]thymidineinto DNA(6).Within2or3

days

afteradditionof 10%serumat

40°C,

T

antigens

accumulatedinthecytoplasm ofmany

cells,

yet

mostofthenuclei remained

relatively

T

antigen

negative (Fig. 1E). If mKSA207 Cl 6

cells,

ac-tively growing at

33.5°C

in 10% serum, were

shifted to

40°C

in 10% serum, the cells also accumulated

cytoplasmic

T

antigen,

but inthis

case they continuedto

display

the intranuclear Tantigen

(data

not

shown).

These results sug-gestthat:

(i)

preexisting

nuclear T

antigen

is lost under conditions which

preclude

cell

growth

(i.e.,inlowserum at40°C);

(ii)

addition of 10%

serum stimulates the cellsto grow,

resulting

in synthesis of new T

antigens;

and

(iii)

newly

synthesized

T

antigens

accumulatein the

cyto-plasmofmKSA207 Cl6cellsandarenot

effec-tively

transported

tothe nucleus.

Theresults withmKSA207Cl6

cells,

indicat-ing that synthesis of T

antigens,

localization of

T antigens,and loss of T

antigens

depended

on

culture conditions,

suggested

that the

growth

properties of thecells

might

play

an

important

role. Variant cell lines withaltered

growth

phe-notypeshave beenderived frommKSA207Cl 6

(12). It was,

therefore,

ofinterestto

study

these

variantcells to assess the impact of the growth

phenotype on localization of T antigens.

Three variant lines, mKSA207-CH,

mKSA207-S4, and mKSA207-S6, are

tempera-ture resistant (tr) for growth, resembling

mKS(wt). The trvariants are anchorage

inde-pendent and growwellin either 0.5 or 10% serum

at either 33.5 or 40°C (12). A serum-requiring

variant line, S6/R1/R'5, was derived from

mKSA207-S6 bytwostagesof suicide selection

based on (i) the inability to grow in methyl

cellulosesuspension cultureat40°C and (ii) the

inability to grow in methylcellulosesuspension

at 33.5°C (unpublished experiments). This cell line, in addition to being anchorage dependent

atboth33.5and40°C, also failed to grow in low

serum ateither temperature.

The trvariants, like mKSA207 Cl6 parental

cells, accumulated Tantigensinthe cytoplasm

when growing at 40°C in 10% serum (Table 2;

Fig. 1H). Occasionally, mKSA207-S6 contained

some cytoplasmic T antigen even when grown in 10%serum at33.5°C (Fig. 1G). However, the

trvariants also

displayed bright

nuclear

fluores-cence atboth temperatures in 10% serum (Fig.

IG andH). These variants, whichgrowwell in

0.5%serum at40°C, didnotlose intranuclearT 34,1980

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784 NOTES

I

FIG. 1. Micrographsof mKS(wt), mKSA207 Cl 6, mKSA207-S6, andS6/Rl/R'5after immunofluorescent stainingforTantigens (X250). mKS(wt) cells grownfor2daysat(A)37°C in10%oserum, (B)40°C in 10% serum, and (C) 40°Cin 0.5%serum.mKSA207 Cl6cells(D) grownfor2daysat33.5°C in 10% serum, (E) grownfor2daysat33.5°C in 10%oserum, depleted of T antigens by incubationfor4days at40°C in0.5%

serum, and thenshiftedto10%serum at40°C for3days, and (F) incubatedat40°Cin0.5%serumfor3days. mKSA207-S6 cells grownfor3daysat(G)33.5°Cin10%oserum,(H) 40°C in 10% serum, and(I) 40°C in0.5% serum.S6/RJ/R'5cellsgrownfor2daysat(J) 33.5°Cin10%oserum(K),40°Cin10%oserum,and(L)40°Cin 0.5%serum.

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[image:4.504.50.439.33.299.2]

FIG. 2. Micrographs of human retinoblastoma cells transformned by SV40(wt) and SV40 tsA207 after immunofluorescent staining for T antigens (x250). ERSV40(wt) cells grown for 2 days at (A) 36°C in 10% serum,(B)40°C in10% serum, (C) 36°C in 1% serum, and (D) 40°C in 1% serum.ERA207 grown for 3 days at (E)33.5°C in 10% serum, (F) 40°C in 10% serum, (G) 33.5°C in 1% serum, and (H) 40°C in 1% serum.

1

FIG. 3. Micrographsof CV-1 cellslytically infectedand 3T6 cellsabortivelyinfected with SV40 tsA207or

SV40(wt) after immunofluorescent staining for T antigens (x250). (A) Uninfected CV-1 cells; CV-1 cells

infectedwithSV40 tsA207for48hat(B)33.5°Cand(C)40°C. (D)CV-1 cellsinfectedwithSV40(wt)for48h

at40°C.(E) Uninfected3T6cells.3T6cellsinfectedwithSV40 tsA207for48 hat(F)33.5°C and(G)40°C.(H)

3T6 cellsinfectedwithSV40(wt)for48hat40°C.

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[image:4.504.51.438.354.616.2]

[image:5.504.61.252.84.260.2]

786 NOTES

TABLE 2. Presenceof cytoplasmic (C)ornuclear (N) TantigensinSV40-transformed cells

Localization ofTantigenby IF' in cells growing in: Cell line 0.5% serumat: 10%serum at:

33.50C 400C 33.50C 400C

mKS(wt) N N N N

mKSA207Cl6 N Nib N C±N

mKSA207Cl 6-CH N N N C + N

mKSA207 C1 6-S4 N N N C+N

mKSA207 Cl 6-S6 N N N±C C+N

S6/R1/R'5 NJ NJ N±C C+N

mKSA207 Cl2 N N N C + N

mKSA207Cl3 N N N C+N

mKSA207 Cl9 N NJ N C±N

mKSA207Cl10 N NJ N C±N

ERSV40(wt) N N N N

ERA207 NJ NJ N C±N

'IF,Immunofluorescence.

b , Nuclear T antigendecreased in most cells.

antigenafter3 or 4days,asdidmKSA207Cl 6 cells, nor did they accumulate

cytoplasmic

T antigeninlowserum at400C

(Fig.

1I).

Thus,

the

trvariants

appeared

tobe somewhat less

defec-tive in ability to transport T

antigen

to the

nucleus than wasparental mKSA207Cl6.

Theserum-requiring variant S6/R1/R'5 also accumulatedcytoplasmicTantigenwhengrown at400Cin10%serum

(Fig.

1K).The

S6/R1/R'5

cells grew only to a low saturation

density

in

10% serum ateithertemperature, and manycells

lost intranuclear T antigen as the cells

ap-proached confluence(Fig. 1Jand

K). S6/R1/R'5

cells failed togrow in 0.5%serum ateither 33.5 or 40°C, and many cells lost intranuclear T antigenin 2 or 3

days (Fig.

1L).

Theseresults suggest that transportofT

an-tigens tothe nuclei wasdefective inmKSA207 Cl6cellsgrowingat40°Cin 10%serumand that

thisdefect was presentto somedegreeinall cell

lines derived from mKSA207 Cl6, irrespective

of thegrowthphenotype.In anattempt to learn

whether the altered localization of T antigens

wasdue to a tsdefect of thecell, localization of

SV40 T antigens was studied in other SV40

tsA207transformants.

Fourindependent lines of mouse kidneycells

transformed by the samestock of SV40 tsA207

that was used totransformmKSA207

Cl

6were

studied. Twoof theselines,mKSA207

Cl

9 and

Cl 10, were temperature dependent forgrowth

in lowserum. LikemKSA207Cl6 cells,Cl 9 and

Cl 10 cells lost T antigens when incubated at

40°C in low serum (Table 2). The other two

lines, mKSA207 Cl2andCl 3, resembled the tr

variants inabilityto grow at either temperature

in low serum and did not lose intranuclear T

antigen whenincubated at 40°C in 0.5% serum.

All fourtransformants accumulatedcytoplasmic

T antigen when grown at 40°C in 10% serum

(Table2).

Localization of T antigens was also examined

inSV40(wt)- andSV40tsA207-transformed

hu-man retinoblastoma cells [ERSV40(wt) and

ERA207, respectively]. For these studies, the

SV40 tsA207 used for transformation was

res-cued from mKSA207 Cl 6 cells by fusion with

CV-1 cells using UV-irradiated Sendai virus.

The rescued virus was plaque purified and

shown to be ts for both viralreplicationand viral

DNA synthesis before transformation ofhuman

retinoblastoma cells. The results inFig. 2A to D

show that T antigens were confined to the

nu-cleus in ERSV40(wt) at either 36 or 40°C in

either 10 or 1.0% serum. In contrast, when

ERA207 cells were incubated in 1.0% serum at

either 33.5 or 40°C, they tended to lose

intra-nuclear T antigen (Fig. 2G and H), and when

incubated at 40°C in 10% serum, T antigens

accumulated in thecytoplasm (Fig. 2F). These results indicated that aberrant localization of T antigens wasprobablynotdue to a tsdefectin

the cell transport system since it occurred in a

number of tsA207 transformants but did not

occur in wt transformants. Therefore, the

pos-sibility was considered that aberrant

cytoplas-micaccumulation of TantigensintsA207

trans-formantsmightbe due to theabnormal

proper-ties of the tsA gene products synthesized at

400C.

Previous studies by Tegtmeyer et al. (18)

showed that at41°CSV40 tsA mutantsinduced,

in both infectedmonkey cells and transformed

rabbit cells, the overproduction of a 100,000-daltonprotein (largeTantigen) immunoprecip-itableby hamster anti-SV40tumor sera. Immu-nofluorescent studies indicated that the nuclei of wild-type-infected cells stained more uni-formlythantsA-infected cells. Further, the cy-toplasmof monkey cellsinfected by tsA58, but not bywild-type virus, was distinctly immuno-fluorescent in most but not all infected cells. (The localization of T antigen in tsA-trans-formedcells was not reported.) Therefore,

local-ization ofT antigens wasstudied in CV-1 cells

lytically infected and 3T6 cells abortively

in-fected with SV40 tsA207. Forthese studies, virus

rescued frommKSA207Cl 6cellswasused. At

24 and 48 h after lytic infection with either

SV40(wt) orSV40tsA207 at either 33.5 or 40°C,

CV-1 cells displayed brightlyfluorescent,

T-an-tigen-positive nuclei (Fig. 3B, C and D). Very little,ifany, immunofluorescencewasobserved

inthecytoplasm. Similarly, 3T6cellsabortively

infected with SV40(wt) or SV40 tsA207

ex-hibited only intranuclear T antigen (Fig. 3F, G

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NOTES 787

and H).In noinstanceweregranularaggregates characteristic of cytoplasmic T antigen of SV40 tsA207 transformants observed in infected cells. Whether or not SV40 tsA207 transformants

lose nuclear T antigen, synthesize new T

anti-gens, oraccumulate T antigens in the cytoplasm

apparently dependsonwhether or not the cells are proliferating. Hence, the localization of T antigens depends on the growth phenotype of thecellsaswellasonculture conditions. These conclusions are supported by thefollowing ob-servations. (i) Intranuclear T antigen was lost

fromSV40tsA207transformantswhich failed to

sustain growth in low serum at

400C

(i.e., mKSA207 C16,Cl9,andCl10,S6/R1/R'5,and

ERA207) (Table2). Tantigen was also lost, but

more slowly, from two transformants (S6/R1/ R'5 and ERA207) which grew poorly in low serum at

330C.

Apparently, preexisting intra-nuclearTantigenwasthermallyinactivated and synthesis ofnewantigenswascell cycle depend-ent (1, 19). Wild-type transformants [mKS(wt) andERSV40], tr variants (mKSA207-CH, -S4, and-S6), andSV40 tsA207 transformants which

grew at 40°C in low serum all continued to

display intranuclear T antigen (Table 2). (ii) Addition of 10%serum to mKSA207 Cl 6 cells

which hadlost nuclearTantigenstimulated cell

proliferation and synthesis ofTantigens. (iii)T antigens accumulated in the cytoplasm ofall SV40 tsA207 transformants whenactively

grow-ing at

400C

in 10% serum, suggesting thatthe

transportof newly synthesizedT antigens to the

nucleus might be defective in these cells. It shouldbepointedout,however, that considera-ble Tantigen, butnotall,wastransportedtothe nucleus in tr variants growing at 40°C in 10%

serum. Whetheror notthe

improved

abilityto

transport Tantigentothenucleusat

400C

isa

contributing factorinthetemperature-resistant growth phenotype of thesecells isnotknown.

At the present time, it is not known why

aberrant accumulation ofT

antigens

ismanifest only in tsA207 transformants when grown at

400C

in 10% serum and not in SV40 tsA207-infected

monkey

or mousecells.Nordoweknow whether the antigens which accumulate in the cytoplasm aresmallt

antigen, large

T

antigen,

truncated T

antigens,

or acombination of these (16).

Cell

fractionation

procedures

with wild-typepolyoma virus-infectedNIH-3T3cells sug-gested that

large

T

antigen

was

predominantly

nuclear, whereas smallt

antigen

was found in

the

cytosol

fraction

(15).

Perhaps

at

400C

in

SV40 tsA207

transformants,

little tis overpro-ducedandaccumulatesinthe

cytoplasm.

Other possibilitiesare that

aggregation

of T

antigens,

complexingof T

antigens

withcellular

proteins

ormembranes, orcontrol oftransportof T an-tigens to the nuclei may be different in SV40 tsA207 transformants than in lytically infected cells.

Two other systems have been described in

whichSV40Tantigenoccursinthe cytoplasm.

Adefective SV40(PARA)mutant wasdescribed byButel and co-workers (3,4, 7) in which the SV40Tantigen occurred in the perinuclear re-gionin bothlytically infected and transformed cells.Lewis et al. (9) have described a nondefec-tive SV40-adenovirus hybrid, Ad2+ND1, which containsonlyasegmentof the earlySV40region inserted intotheadenovirustype 2 genome. This segment of SV40DNAcodes forthe U antigen

which canbe detected in the perinuclear region

of cellsafter lyticinfection (9).Unlikethe defec-tive SV40(PARA) mutant, however, hamster cells transformedby

Ad2+ND,

failed to express anySV40antigens (8).

Thisinvestigation was aided by National Science Founda-tion grantPCM-7818901 and by Public Health Service grants 1-K6-Al-2352-17 and CA-06656-17 from the National Institute ofAllergy and Infectious Diseases and the National Cancer Institute, respectively.

We thank Judith Rotbein for technical assistance, Marion Hazen for photographic assistance, and Vincent Riccardi for the humanretinoblastoma cells.

LITERATURE CITED

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