Effect of actinomycin D on the expression of herpes simplex virus-common surface antigen in cells transformed by herpes simplex virus type 2.

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0022-538X/79/01-0161/09$02.00/0 No. 1

Effect of Actinomycin

D

on

the Expression

of Herpes Simplex

Virus-Common Surface

Antigen in Cells

Transformed by

Herpes Simplex

Virus Type

2

SUSUMU KIMURA,'* KATSUICHIROOKAZAKI,2 NAGAYUKIYOSHIDA2AND

YOSHINARI OHNISHI'

Department of Bacteriology, Schoolof Medicine, TokushimaUniversity,1 andDepartment ofMicrobiology,

Schoolof Pharmacy, TokushimaUniversity of ArtsandScience,2 Tokushima770,Japan

Received forpublication7April1978

Using rabbit antiserum hyperimmune toherpes simplex virus (HSV) type 1,

the expression of HSV-common surface antigen(s) was studied by indirect

im-munofluorescencetestsin cells transformedby HSVtype2and in derivedtumor

cells. The following resultswere obtained. (i) AntiserumtoHSV type 1 reacted

specifically with surface antigenpresent onthe plasma membrane of both HSV type2-infected and HSVtype2-transformed hamstercells.(ii)The expression of

this antigen was enhanced in the absence ofactive protein synthesis in

trans-formedcells, but notin tumorcells, after culture for3to5 h at37°C. (iii) This

enhancement ofexpressionwasmaintained for20h in thepresenceof actinomycin

D, but this prolonged expression required active protein synthesis. (iv) The

enhancing effect observed in thepresenceof actinomycin D continued forsome

timeafter removal of the drug, for example, for20h after5h oftreatmentwith

2 ,ug/ml of actinomycin D perml. Actinomycin D hadno detectable effect on

antigen expression in tumor cells. (v) Theprotease inhibitor antipaininhibited the actinomycin D-enhanced expression withoutcausing significantcell damage

but didnotmodify the transient enhanced expression of antigen when cellswere

seeded in theabsence ofactinomycin D. These results indicate that in transformed cells antigen expression canbe enhanced inatleasttwoways.

It is well known that mammalian cells are

transformed

oncogenically

in vitro by herpes simplex virus

(HSV)

type1

(HSV-1)

andtype 2

(HSV-2) (1, 5, 6, 13-16, 28). Viral DNA

se-quences (4, 10, 19),

HSV-specific

RNA (2), and

HSV-specific

antigen (1,5, 13-16, 28) have been demonstrated in cells transformed by HSV-2. Moreover, viral genetic information has been

concluded to be present in HSV-transformed cells fromthefollowing findings. (i) Acquisition of

HSV-specific

thymidine kinasehasbeen

dem-onstratedinthymidine kinase-negativemouseL

cells after in vitro transformation with HSV-1

(20). (ii) Complementation between

tempera-ture-sensitive (ts) mutants ofHSV-2 and

resi-dent, functional HSV genes in HSV-2-trans-formedcells has been demonstrated atthe

non-permissivetemperature (12, 17).

Recently,ithas been demonstrated that

sev-eral type-common and type-specific HSV anti-genscanbeidentifiedby

immunological analysis

of HSV-1- and HSV-2-induced glycoproteins

(30) and that type-common antigen(s) is also

detectableonthesurface ofcells transformed

by

HSV(21, 22).

In an attempt to

identify

the

HSV-specific

geneproducts

expressed

intransformedcells,we

observedanHSVtype-commonsurface

antigen

(CSA) which increased after

actinomycin

D(Act

D) treatment of cultures transformed by a ts

mutantofHSV-2. Thispaper reports studieson

theexpression of CSAintransformedandtumor

cells, the doses ofAct D which enhance

CSA

expression and the kinetics ofits

effect,

andthe effect of a proteaseinhibitor,

antipain,

onAct

D-enhancedCSA

expression.

MATERIALS AND METHODS

Cell lines. HSV-2-transformed cells and the

de-rivedtumorcellsused in thisstudywereall isolated

by the routine method fromhamster (inbredstrain

LSH)embryo fibroblast cellcultures,asreported

pre-viously by Kimura etal. (13). Transformed cell line

155-4(passages130to180)wasobtainedafter infection ofcellswith theDNA-negative,mutantB5 of HSV-2

strain 186 at the nonpermissive temperature (38°C)

and then passagedserially at37°C,because thiscell line wasnotstabletomaintenanceat38°C.Celllines

U-15, U-26, and U6V-7 (passages 131 to 135) were

obtained after infection with UV-inactivated strains

186 (U-15 andU-26) and333 (U6V-7) ofHSV-2,

re-161

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spectively.Two otherhamstertumorcelllines,

TSV-5 (passages232 to 249) andBAT-6 (passage> 100),

induced by simian virus40andbovineadenovirus type

3,werekindly suppliedby T. Kurimura(Tottori

Uni-versity,Tottori, Japan) and Y. K.Inoue(Kyoto

Uni-versity, Kyoto, Japan),respectively.Primaryhamster

embryofibroblasts(HEF)wereobtained from

13-day-old hamster embryos (inbred National Institutes of

Healthstrain). All cellsweregrownat37°CinEagle

minimum essential medium (MEM) supplemented

with 10%fetal calf serum(FCS) and 0.075% NaHCO3

for cultures in closed vessels and 0.225%NaHCO3for

cultures in open vessels.

Virus. HSV-1 (strain KOS), HSV-2 (strain 186),

and the DNA-negative ts mutant (ts B5) of HSV-2

(strain186) used in thisstudywerekindly providedby

P. A. Schaffer (SidneyFarber CancerInstitute,

Har-vard Medical School, Boston, Mass.). Virus stocks

were grown in human embryonic lung cell cultures

withEagle MEMcontaining 5%FCS,andvirus assays

wereperformedasdescribedpreviously (8, 25).Virus

stocks had the following titers inhuman embryonic

lung cell cultures: HSV-1, 6 x 107 PFU/ml at 37°C;

HSV-2, 2 x 107 PFU/ml at 37°C; ts B5, 2 x 107

PFU/mlat340C.

Antisera. Two hyperimmune rabbit antisera to

HSV-1(strainKOS) werepreparedindependently by

immunizing albino rabbits intraperitoneally with

HSV-1 in Freundcomplete adjuvant (Difco

Labora-tories,Detroit, Mich.)onceaweek for10weeks. The

animalwasbled 1 week after the finalinjection.The

neutralizingantibody titerstoHSV-1 of thetwo

anti-serawere1:1,280 and1:320,respectively,in 50%plaque

reductiontests.Anti-HSV-1serum(serumA in Table

1) possessing a higher neutralization titer was used

mainly in thisstudy. Additionalhyperimmunerabbit

antiserumtostrain HF of HSV-1waskindly provided

by Y.K.Inoue and usedas areference antiserum with

aneutralization titer of1:640.Fluorescein-conjugated

goatantiserumtorabbit 7Simmunoglobulin (FGAR)

waspurchased from HylandDivision,Travenol

Lab-oratories,Inc., CostaMesa,Calif.Nonspecific

reactiv-ity was removed from these antiserabytheabsorption

technique described below.

Absorption technique.Theabsorption technique

describedbyFlanneryetal.(9)wasused,with minor

modifications. Four milliliters of undiluted rabbit

anti-HSV-1serumwasmixed with1gof rabbit liverpowder

suspended in4ml of Trisbuffer(pH 7.4). The mixture

wassonically treated inanicebathat 10kcyclesfor

two 1-minperiods,shakenat370 for 1 hand at40C

overnight,and thencentrifugedat100,000xgfor 1 h.

The supernatant fluid was mixed with the pellet of

human embryonic lung cells (108 cells), sonically

treated, incubated at 370C for 1 h and at4°C

over-night,and thencentrifugedat100,000xgfor 1h. The

supernatantwasabsorbedathirdtimewith108intact

HEF cells for 1 h at 370C and subjected to sonic

treatment. Thesuspensionwasincubatedat37°C for

1 h and at 4°C overnight and then centrifuged at

100,000 xg for 1h.Absorbed anti-HSV-1 serum was

used todetectHSV-CSA.Fortestingthespecificityof

thisantiserum,fourdifferentcelltypes(155-4,TSV-5,

and HSV-2- and ts B5-infected HEF) were used in

absorptionexperiments.

Celllines 155-4andTSV-5wereseeded in150-mm

petridishes(FalconPlastics,Oxnard, Calif.)atinocula

ofabout1x 107cells perdish andharvested 3 h later

by gentle

trypsinization. HEF cellswereinfectedata

multiplicity of 3PFU/cellwith HSV-2at37°C orts

B5at34°C. The HEF cellsinfected with HSV-2 and

ts B5were harvested with a rubberpoliceman 10 h

and 20 h later, respectively, when the numbers of

HSV-CSA-positivecellsweremaximal. The antiserum

(0.7 ml)wasabsorbedagaintwice,with each cellpellet

containing6x107cells,bythe abovemethod,andthe

absorbedserum was testedforHSV-CSA reactivity.

FGAR serum (4 ml) was also absorbed with 1 g of

rabbit liverpowderand108HEF cells.

Detection ofHSV-CSA. Indirect

immunofluores-cence tests were used for detection of HSV-CSA.

Before experiments, the test cells were grown and

passagedatleast three times understrictly controlled

conditions: thatis, 10 ml ofcellsuspension (105cells

perml) wasinoculated intoaculture bottle(50 cm2)

andincubated at37°C for48h.After thistime, the

culturewasalmost confluent(about 90%)andthecell

number had increased four fold (i.e., 4 x 105 ml).

Conditioned cellsuspensions (105 cells perml) were

seeded into 5 ml ofmedium in 60-mm petri dishes

(Falcon Plastics) and incubated at 37°C in a CO2

(5%)incubator. Atdesignated times,cellsweretreated

with 0.25% trypsin (1:250; Difco Laboratories) for 1

min andwashed three times withbarbital buffer(pH

7.4) containingbarbital(3x

1i-'

M), sodium barbital

(1.8x 10-3M),0.85% NaCl, Mg2+ (5x 10-4 M),Ca2+

(1.5x 10-4 M),and 0.1% gelatin (GBB) (31) by

cen-trifugationat800xgfor10min.Samplesof1dropof

antiserum absorbed with various cellsornormalrabbit

serum at a final dilution of 1:4 were added to cell

suspensions (6 x 105 to 10 x 105 cells) incentrifuge

tubes. Thesuspensionswereincubatedat37°C for30

min,washed threetimes withGBB, and then treated

for30 min at 37°C with 1 drop of absorbed FGAR

serumdiluted1:8andwashedthreetimes with GBB.

Washed suspensions were mounted in 50% glycerin

solution,and 103 cells persample were observed with

a reflected light fluorescence microscope (Olympus

OpticalCo.Ltd., Tokyo, Japan). The number of viable

cellswasdeterminedby trypan blue exclusion.

To test the effects ofvarious conditions on the

expression of HSV-CSA in 155-4 cells (see Table2),

cellsdetachedby pipettingweresuspendedin1ml of

phosphate-buffered saline withoutCa2" and Mg2' and

treated with 1 ml oftrypsin (0.5%) or 1 ml of

phos-phate-buffered saline without Ca2' andMg2"for 1 and

3 min, respectively. Trypsinization was stopped by

addition of15ml ofEagle MEM containing 10% FCS

andbycentrifugation. Some cell suspensions withor

without trypsinization wereimmediately assayed for

indirectimmunofluorescence. Other suspensions (1 x

105to25 x 105 cells per sample) wereincubated with

5ml ofEagle MEM containing 10% FCS, in 60-mm

dishes asmonolayers orintubes (15 by 150mm) as

suspensions, for3h at4or37°C and then assayed for

indirectimmunofluorescence.

Inhibitors. Act D and puromycin-dihydrochloride

(PM) were obtained from Boehringer Mannheim,

Mannheim,West Germany, and Nutritional

Biochem-icalsCo., Cleveland, Ohio, respectively. The protease

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SURFACE ANTIGEN IN HSV-2-TRANSFORMED CELLS 163

inhibitor antipain [(1-carboxy-2-phenylethyl)

car-bamoyl-L-arginyl-L-valyl-argininal] (26) was kindly

providedby T. Uchida (Osaka University, Osaka,

Ja-pan). Ten times-concentrated stock solutions were

prepared, passed through 450-nm membrane filters

(Millipore Corp., Bedford,Mass.) and storedat-20°C.

Routinely, cells were treated with Act D (0.5 to 4

,ug/ml)whentheywereseeded into60-mmdishesand

then incubated for the desired time at 37°C. For

further incubation withoutAct D,cells werewashed

twice with Tris bufferandreincubatedinfresh Eagle

MEM containing10%FCSat37°C.Antipain (0.5 mM)

wasalsoaddedto ActD-treated and untreated cells

at the time ofseeding, and the cells wereincubated

with antipain for30 h, even when the medium was

changed.

Incorporation of[3H]leucine.A5-,uCiportion of

L-[4, 5-3H]leucine (40 to 60 Ci/mmol; New England

Nuclear, Boston, Mass.) was added to 60-mm petri

dishes for10h. Thecells werethenwashedoncewith

cold(4°C) Trisbuffer, harvested, and centrifuged at

800xgfor10min. Pelletssuspended in1ml ofcold

Tris bufferweresonicallytreated inanice bathat10

kcycles for20s,treated with1mlof20%trichloroacetic

acid for 20min at 0°C, and then boiled for 15 min.

The suspensionswereappliedtoWhatman GF/Cfilter

disks, usinganaspirator. The diskswerewashed three

timeswith 5%trichloroacetic acid andoncewith

ethyl-alcohol, dried, and placed in scintillation vials. Ten

milliliters ofscintillationfluidwasaddedtoeach vial,

andradioactivitywasmeasured inaliquid scintillation

counter(Packardmodel 3385). RESULTS

Specificity of anti-HSV-1 serum. To test

the specificity of the reaction of anti-HSV-1

serum with HSV-CSA on HSV-2-transformed

cells, three HSV-2-infected, five HSV-2-trans-formed, and two other DNA virus-induced

tu-morcellsanduninfected HEFcellsweretested

for reactivitytothree anti-HSV-i seraabsorbed

withHEF cells (Table 1). HSV-2at370C andts

B5atthe permissivetemperature (340C) caused

anincrease in the number ofCSA-positive cells

with timeafter infection,tomaximaof 10 h(58% positive cells) and 20 h (48% positive cells),

respectively,after infection; however,atthe

non-permissivetemperature (38°C),tsB5induceda

maximum of only 13% positivecellsat10hafter infection, and the number of positivecellsthen

rapidly decreased. A positive reaction (ranging from4 to36%CSA-positive cells) wasobserved

in thefive HSV-2-transformed cell lines, regard-less of the strain ofHSV-1 used for the

antise-rumpreparation; themostpositive reactionwas

found in ts B5-transformed (155-4) cells (23to

36%CSA-positive cells).No specific reactionwas

observed in tumorigenic transformed cells

in-ducedwith simian virus 40orbovineadenovirus

type 3 or in control HEF cells ('0.7%

CSA-positive cells). In the experiments carried out

below, antiserum to strain KOS of HSV-1

(se-rum A in Table 1), which has ahigher titer of

neutralizing antibody, wasused to detect HSV-CSA. Both HSV-2 at 370C and ts B5 at 340C

(Fig. 1A) induced predominantly ringlike

fluo-rescence, withafew dotted and arc-shaped

re-gions of fluorescenceonthe surface of HEFcells.

Although the intensityofstaining of 155-4cells

TABLE 1. DetectionofHSV-CSA inHSV-2-infectedand-transfornedhamster cellsby indirect

immunofluorescencea

No.ofceH

~~~~~~~CSA-positive

cells

Cell type No. ofcell Virus used(strain)

SerumA Serum B Serum C

HSV-2 infectedHEF,at37oCc HSV-2(186) 58.0

tsB5 infectedHEF,at

34oC'

HSV-2(tsB5of strain186) 48.0

tsB5infectedHEF,at380Cd HSV-2(tsB5ofstrain186) 13.0

U6V-7 135 HSV-2(333) 10.4 4.2 8.4

U-15 Te 86 HSV-2(186) 8.0 7.9 8.1

U-26 T 126 HSV-2(186) 5.2 5.5 5.9

155-4 138 HSV-2(tsB5 ofstrain 186) 36.1 23.4 35.0

155-4T 114 HSV-2(tsB5 of strain 186) 7.0 9.0 7.0

TSV-5 246 SV40 0.5 0.7 0.6

BAT-6 >100 BAD-3f 0.3 0.6 0.6

HEF 5 None 0.4 0.5 0.5

a

All cells

exceptinfected

cells

wereharvested3to5 h afterseedingat37°C and stained withanti-HSV-1

serun absorbed with HEFcells.

bAtotal of

103 cells

werecounted.SeraAandBwererabbit antiserapreparedindependently againststrain

KOS of HSV-1 andserumCwasrabbitantiserumtostrain HFofHSV-1.Neutralizingantibodytitersto

HSV-1of seraA,B, and Cwere1:1,280,1:320,and1:640,respectively,in50%plaquereductiontests.

cHEF

cells

infectedwith HSV-2(3PFU/cell)wereharvested10hafterinfection.

d HEF

cells

infected withtsB5(3PFU/cell) at34and

380C

wereharvested20and 10h,

respectively,

after

infection when thenumbers ofpositivecellsweremaximal.

'T representscultured

cells

derived fromtumorspassedoncein vivo.

fBAD-3,Bovine adenovirustype3.

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FIG. 1. SurfacefluorescenceobservedinHSV-2-infectedandtsB5-transformed (155-4)cellsaftertreatment

with anti-HSV-I serum absorbed with various cells in indirect immunofluorescence tests. (A) HEFcell

infectedwithtsB5(20hafterinfectionat

34QC)

stainedwith anti-HSV-IserumabsorbedwithHEFcells. (E

155-4cells3 hafterseedingwithanti-HSV-Iserumabsorbedwith HEF cells. (C)155-4cells 3hafterseeding stained with anti-HSV-I serum absorbed with TSV-5cells. (D) 155-4 cells 3 h afterseedingstained with normalrabbitserum orwithanti-HSV-I serumabsorbed withHSV-2-infected, tsB5-infected,or155-4cells. was slightly less than that of the two types of

HSV-infectedcells,distinctring-shapedand dot-ted regions offluorescence were observed

pre-dominantly, with a few arc-shaped regions of fluorescence onthe surface oftransformedcells

(Fig. 1B).

To confirm thespecificityof thereaction, anti-HSV-1serum wasabsorbed with TSV-5(simian

virus 40-induced tumor cells), HSV-2-infected,

ts B5-infected, or the transformed 155-4 cells

andthen tested forHSV-specificreactivity with

155-4 cells. Reactivity with 155-4 cells was

re-tained after absorption with TSV-5 cells (Fig. 1C); however, afterabsorption with HSV-2- or

ts B5-infected or 155-4 cells, the reactivity of this antiserumwasreducedtothe level of that of normal rabbitserum (Fig. 1D). These results indicate that the surfacefluorescence observed with transformed 155-4 cells is HSVspecific.

Expression ofHSV-CSA in transformed

and tumor cells. Preliminary experiments

showed that 155-4cellsdoubled every 25 hand that when5 x 105cellswereinoculated into

60-mm dishes at 37°C, CSA-positive cells were

more numerous 3 to 5 haftertrypsinization and

seeding (30to 40% positive) than after 15 to48

h (8 to 20%positive). To testwhether trypsini-zation itself induces activation of CSA, it was

carried out atvarying times, with varying sizes of cellinoculum, and under varying incubation conditions (Table2). No activation of CSA was observed in 155-4 cells that weresimply trypsin-ized for 1 or 3 min or after addition of fresh medium. PartialactivationofCSA wasobserved when: (i) cells were not trypsinized; (ii) cells

were trypsinized for 1 min and then incubated at4°C for 3 h; (iii) large inocula (2.5x 106cells) or small inocula (105 cells) were used; and (iv) cells were incubated for 3 h at 4 or 37°C after trypsinization for 3 min. Activation of CSA in 155-4cells was markedonly when the cellswere

trypsinized for 1 min andincubated at an appro-priate inoculum (5 x 105 cells per dish ortube) for 3 h at 37°C. Activation was observed with cell suspensions and even moremarkedly with monolayers. Thus, trypsinization was necessary

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SURFACE ANTIGEN IN

TABLE 2. Various conditionsaffecting theexpression of HSV-CSAin155-4 cells

a

.. Cellinoculum/ Incubation conditions CSA-positive

Trypsinization

sample(x

105)

Stateb Time (h) Temp(°C) cells

(%)c

None 5 Noincubation 12.2

5 Suspension 3 4 13.1

5 Monolayer 3 37 14.4

Medium

changed

1min 5 Noincubation 6.2

25 Noincubation 9.6

3mm 5 Noincubation 5.0

aTrypsinization wasperformedasdescribed in the text.

b

Cells

wereinoculated into dishes(monolayer)orintubes (suspension) with gentleshaking. No incubation,

Cell suspensionswereimmediately assayed for indirect immunofluorescence.

cAtotal of103

cells

werecounted.

dTheculture medium was replaced by fresh medium2days after seeding.

butnotsufficient for activation.

Next, the relationship between CSA expres-sion and cell growth was examined in trans-formed(155-4) andtumor (155-4T) cellsduring

incubation at37°C (Fig. 2). In 155-4 cells a peak of CSA-positive cells (36% positive) was ob-served 3 to 5 h after seeding, and then the

number ofpositivecellsdecreasedwithincrease

in cellnumber.Only 15%ofthecellswere

posi-tive 25 h after seeding, when the cell number hadalmostdoubled, andonly 8%wereobserved 48 h after seeding, when the cell number had

increased fourfold (data not shown). On the otherhand, although thedoublingtime of 155-4Tcellswassimilartothatof155-4cells(about

25h), they didnotshowapeak of

CSA-positive

cells within 25 h after seeding, although a low but constant level of5 to8%positive cells was

observed

during

this

period.

Effect of Act D on expression of HSV-CSA in transformed and tumor cells. Pre-liminary experiments with 155-4 cells showed

thata

high

percentageof

CSA-positive

cells was

induced by treatmentwith 1 to 2

jig

ofAct D per ml for 15 h. To confirmthiseffect,155-4and

155-4T cellsweretreated withActD (2,ig/ml)

for 25 h from the time of cell

seeding,

and at

intervalsCSA-positivecells and viable cellswere

counted(Fig. 2).Inuntreated155-4cell

cultures,

the percentage of

CSA-positive

cells decreased from 3 hafterseeding;but thepercentage in Act D-treated cell cultures remained

high

for20h.

50

if-ae

CD

._e

-W

v

E

m

c

0 10 20 0 10

Hours after inoculation

FIG. 2. Relationship between expressionof HSV-CSA and cell growth in transformed (155-4) and tumor (155-4T) cells in thepresenceand absenceof Act D (2tLg/ml). Symbols: 0, absence ofAct D; *,

presenceofAct D.

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166

Thus, the initial increase ofCSA-positive cells afterseedingwasmaintainedbytreatmentwith Act D. Fluorescence staining in Act D-treated cellcultureswasalso more intense than that in untreated cell cultures (not shown). The viable cells in ActD-treatedcellcultures decreasedto

four-tenths of the cell inoculum (5 x 105 cells perdish) 10h afterseeding. On the otherhand,

treatment with Act D didnotaffectCSA

expres-sion in 155-4T cells for 25 h after seeding,

al-though theproportion of viable cells decreased to the same extent in 155-4 cells treated with ActD.

Effective time oftreatment and dose of actinomycinD. Totry to find out the time at which Act D treatmentaffectedCSAexpression,

155-4cells were treatedwithAct D(2

[ig/ml)

for

3 to 15 hand then incubated infresh medium without Act D foratotal of 30 h. As shown in

Fig.3, 155-4cell culturestreated with -Act Dfor

5, 10,and15h retainedabout40%CSA-positive cells foratleast20hafter removal ofthe ActD,

but incell cultures treated with Act D for3 h and in control cultures the numbers of CSA-positive cells decreased with cell growth; the number ofpositivecells decreased morerapidly

in control cultures than in those treated with Act Dfor3h.

2

E

c

0 10 20 30 Hours ater removal d drug

FIG. 3. Effect oftimeofAct Dtreatment on

expres-sionofHSV-CSA and cell growth in 155-4 cells. After treatmentwith Act D(2

tog/ml)

for various times (3 to 15 h), cells were washed (O h in this figure) and reincubated infreshmedium without Act D at 37°C.

(A) Expression of CSA; (B) cell growth. Symbols:0,

control cells in medium changed3 h after seeding;

0, 3-h treatment with ActD; A, 5-h treatment with ActD;A, 10-h treatment with Act D; O, 15-h treat-mentwith Act D.

Next, to examine the effect of the concentra-tion of Act D onCSA expression in 155-4cells, cellsweretreatedwith various concentrations of Act D(0.5 to 4

tog/ml)

for 10 h(Fig. 4). A linear

relationship wasobserved between the

concen-tration of Act D (0.5 to 2 ,ig/ml) and

CSA-positive cells. Althoughthe viable cell number wasthesameincells treated with 0.5 to4

tog

of Act D perml,addition of 2,ug/mlresulted in the greatestCSA expression;ahigher dose(4

,tg/ml)

inhibited itsexpression.

EffectofpuromycinonHSV-CSA expres-sion in transformed cells. To determine

whether new protein synthesis is required for CSAexpressionin 155-4

cells,

PMwasaddedto

cultures,andCSA-positivecells, viable cells, and theincorporation of[3H]leucineintocells were

then scored.Theinitialincrease ofCSA-positive cells observed in untreated 155-4 cells was in-hibited only partially by 70

jig

of PM per ml (Fig. 5A), a level which almost completely

in-hibited theincorporation of[3H]leucine into vi-able cells for20h (Fig.

5G).

TheresponseofAct

D-affected, CSA-positive cells was greatly

in-hibited by the same concentration of PM. In

controls, the number ofviable cellswassimilar

inculturestreated with Act Dalone, PMalone, or ActDandPM (Fig. 5B).

Toconfirm theinhibitoryeffectof PM on Act

D-affected CSA expression, various concentra-tions (1 to 50,ug/ml) of PM wereadded to 155-4 cells treated with Act D for 10 h (Table 3). The Act D effect was inhibited partially by 10 and 20

jig

of PM per mland completely by 50

,tg/ml,

but itwas notaffectedwith theaddition of 1

,Ag

of PM per ml. Similar results were

obtained withexperimentson theeffectof PM on incorporation of [3H]leucine into the cells.

Theseresultsindicate that newproteinsynthesis

8

2

6

4

2 (

0 0.5 1 2 4

Concentrationodactinomycin D

lpg/mlI)

FIG. 4. Effect of concentration of Act D on HSV-CSAexpression in 155-4 cells. Act D (0.5 to 4 ,ug/ml)-treated and un,ug/ml)-treated cells were incubated for 10 h

at37°C. Points in this figure represent the average

valueoftwo separate tests. Symbols:0,cellnumber;

0,CSA-positivecells.

W

-,

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SURFACE ANTIGEN IN HSV-2-TRANSFORMED CELLS 167

20 / °

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> 20

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ur

O

CD

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-!E

E

:3

1-z

un

1-E

0

10

5

0

30

20

10

0

0 5 10 15

[image:7.501.75.221.59.378.2]

Hours after seeding

FIG. 5. Effect ofPMonHSV-CSA expression,cell

growth, and incorporation of['Hileucine in 155-4

cells treated with andwithout Act D.ActD(2 ug/ml)-treated and unug/ml)-treated(control) cellswereincubated with and without PM(70,ug/ml)at37°C for20h. (A)

Expression of CSA; (B)cellgrowth; (C) incorporation

of ['H]leucineinto105viablecells.Symbols:0,

con-trol cells withoutPM; 0, control cells with PM; A,

Act D-treated cells without PM; A, Act D-treated

cells with PM.

is required for Act D-affected CSA expression, but itishardly required for the initialincrease

of CSA in untreated cells.

Effect of antipain on HSV-CSA

expres-sionintransformed cells.Anapproachtothe studyof themechanism(s) ofCSA expressionis

provided by the protease inhibitor antipain,

which prevents A phage induction in

Esche-richiacolibyUV irradiationataconcentration

of 0.5 mM (18).NeithergrowthnorCSA

expres-sion in 155-4 cells was affected by addition of

antipain (0.5 mM) (Fig. 6). However, theboost

in CSAexpressionin thepresenceof Act Dwas

abrogated by0.5 mMantipain (0.05mMhadno

effect and isnotshown);theviablecellnumber

in Act D-treated cultures wasnotdecreasedby

this concentration (0.5 mM) ofantipain. When

antipain (0.5 mM) was added to the cellsafter

removal of Act D, CSA-positive cells also de-creased to 16.3 and 13.2% by 10 and 20 h, re-spectively, after addition of antipain.

DISCUSSION

The specificity of the reaction between anti-HSV-1 serum and HSV-CSA present on the

plasmamembrane of cells transformed by HSV-2 was demonstrated in the present study. The evidence was as follows: (i) HSV-2-transformed cellsand the derivedtumorcellsgave apositive

reactionwithHSV-1antiserum, but otherDNA virus-induced tumor cells did not; and (ii) the

reactivity ofthis antiserum in ts B5-transformed

cells (155-4) was significantly decreased after

absorption withHEFcells infectedwith HSV-2 (wild type or ts B5) or with homologous 155-4

cellsbut not with normal HEF cells or simian virus 40-induced tumor hamster cells. These

findings show thatthe antiserum reactedwith a geneproduct(s), detected asCSA, that was

ex-pressedspecificallyinHSV-2-transformedcells;

however, it isnotknownwhether the CSA

ex-pressed in 155-4 cells is identical to CSA ob-served in theinfected cells.

The present study also demonstrated that

CSA expression was enhanced in transformed

155-4cells3to 5hafter cell seedingat37°C,but not inthe derivedtumor(155-4T)cells,and that

the increase of CSA in 155-4 cells was not an immediate consequenceof trypsinizationbut

re-quired incubation ofthe cells at

37°C

for 3 h. Theexpression ofCSAis not temperature

sen-sitiveinthesecellsat37°C,sinceapeakof CSA-positive cells

(34%

positive) was also found in 155-4cells 10hafter seedingat

34°C,

andonly

a low level (1 to 7%) of the positive cells was

detected in 155-4 T cells at

34°C

as well as at

37°C throughout70h afterseeding (manuscript

in preparation). Furthermore, the finding that

CSA was not enhanced in tumor cells could indicate that either CSA expressionismore

re-pressed in tumor cells or the viral gene(s)

re-sponsible forCSA expression is reduced in the tumor cells. The latter

possibility

is consistent with thereport that the sequence

complexity

of

HSV-2DNA intumorcellsis lower than that in theoriginal HSV-2-transformedcells

(10).

It iswellknownthatAct DbindstoDNA and inhibits DNA, RNA, andprotein syntheses (3,

23). Thisdrugisalso

tumorigenic (27)

and

mu-tagenic (7, 11) in animals. The present

study

demonstratedthatincreased CSA

expression

in transformed cells after cell

seeding

could be maintained bytreatmentwithAct

D,

and that new protein

synthesis

was

required

for CSA

expression affected by Act D but not for the initial increase of CSA

expression

in the absence

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TABLE 3. Effectof PMonHSV-CSA in 155-4 cells treated with Act D

Incorporationof

Treatment (10 h) CSA-positivecells(%)b Viablecells/plate(x 104) [BH]leucine (cpm/10' viablecells)

Untreated 23.5 50.0 8,038

Act D 42.4 13.5 6,779

PM (50,ug/ml) 16.7 5.9 761

Act D+PM(1,ug/nl) 43.9(1.04)c 8.0(0.59)d 4,786(0.71)e

ActD+PM(10yg/ml) 10.7(0.25) 6.8(0.50) 1,179(0.18)

Act D+PM(20,g/ml) 10.1 (0.24) 6.4(0.47) 983(0.15)

Act D+PM

(50,ug/ml)

2.0(0.05) 5.6(0.41) 478(0.07)

a 155-4

cells

weretreatedsimultaneouslywith Act D (2

iog/ml)

and various concentrations of PM and then

incubated for10hat37°C.

b Atotal of

103 cells

werecounted.

'Values in parentheses represent the ratios ofCSA-positivecells in cultures treated with ActDand PMto

those incultures treated with Act Donly.

dValues in parentheses represent the ratios of cell numbers in cultures treated withAct Dand PMtothose

incultures treated with ActDonly.

eValues inparentheses represent the ratios ofincorporationof

[3H]leucine

into

cells

treated with Act D and

PMtothat intocells treated withActDonly.

50

30

20

10

0 B

12

8

C4

u~~~~

0

0 5 10 15 20 25

Hours

FIG. 6. Effect of antipainonHSV-CSAexpression in 155-4 cells. Act D(2,ug/ml)-treatedanduntreated (control) cells incubated for 5 h at 37°C with or

withoutantipain (0.5 mM) werewashed(0h in this

figure) and reincubated in fresh medium with or

without antipain (0.5 mM). (A)Expression of CSA; (B) cell growth. Symbols: 0, control cells without

antipain; 0, control cellswith antipain; A,Act

D-treated cells withoutantipain;A,ActD-treated cells

withantipain.

of Act D.Thiseffectof Act DonCSA expression

continued for 20 h after removal of the drug,

once cellshad been treated with2

,ug/ml

for5h. However,Act Ddid notaffect CSAexpression inthederivedtumorcells. Perhaps inan

analo-gous way, Epstein-Barr virus-associated

mem-braneantigenin atested Burkitt

lymphoma

cell

lineshowednoresponse to anymetabolic inhib-itor(includingActD)or toanychangeinculture conditions (32).

Theproteaseinhibitorantipain isarelatively nontoxic (29),

low-molecular-weight

compound,

which inhibitstrypsin- andpapain-likeproteases in particular (26). It is very interesting that

antipain inhibitedActD-enhancedCSA expres-sionwithoutcausingsignificant cell damage but didnotinhibit theinitial increase ofCSA

expres-sionin theabsence ofAct D.Roberts and Rob-erts (24) have shown thatAprophage inE. coli is induced by proteolytic cleavage of the A re-pressor. Recently, Meynetal. (18) have shown thatantipain inhibitsAphage induction (oneof the SOS functions) in E. coli by blocking

pro-teolytic inactivation of therepressor.Therefore,

itisconceivablethat theputative repressor(s) of the CSA

function(s)

is inactivated by a

pro-tease(s) inducedoractivatedbyActD, leading

to the appearance of CSA. Another possible explanation ofthe effect ofAct D would be an

inhibition ofa repressor at the transcriptional level. Of course, these possibilities are highly

speculativeatpresent.

In any case, our findings indicate that two kinds of CSA expression occur in transformed

155-4

cells.

One type is expressed soon after

seeding, without the need for further protein

synthesis, and is insensitive to antipain. The other isexpressed in response to Act D, requires

protein synthesis, and is sensitive to antipain. Protease(s), therefore, seems to play an impor-tant role in the enhanced level of expression.

Sinceit seems that cellline 155-4 respondsin a

heterogeneousway with respect toCSA

expres-168

KIMURA ET AL.

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SURFACE ANTIGEN IN HSV-2-TRANSFORMED 169

sion, clonal analysis of cells from the line isnow

inprogress.

ACKNOWLEDGMENTS

WearegratefultoDavid Schlessinger for critical reading of themanuscript.

LITEIATURE CITED

1. Boyd, A. L., and T. W. Orme.1975.Transformationof

mousecellsafter infection withultraviolet

irradiation-inactivatedherpessimplexvirustype2.Int. J. Cancer 16:526-538.

2. Collard, W., H. Thorton, and M. Green. 1973. Cells transformed by human herpesvirustype 2 transcribe virus-specific RNA sequences shared by herpesvirus types1and2.Nature(London)New Biol. 243:264-266. 3.Cooper,H. L., andR. Braverman. 1977. The mecha-nismby which actinomycinDinhibitsproteinsynthesis inanimal cells. Nature(London)269:527-529. 4. Davis,D.B., and D. T.Kingsbury,1976.Quantitation

of theviralDNA present incells transformedby UV-irradiated herpes simplexvirus.J. Virol. 17:788-793. 5. Duff, R., and F. Rapp. 1971. Properties of hamster

embryo fibroblasts transformedin vitroafterexposure

toultraviolet-irradiatedherpes simplexvirustype2.J. Virol.8:469-477.

6. Duff, R.,and F.Rapp.1973.Oncogenictransformation of hamsterembryocellsafterexposure toinactivated herpes simplex virustype 1.J. Virol. 12:209-217. 7. Epstein, S., E. Arnold,J. Andrea,W.Bass,and Y.

Bishop.1972.Detectionofchemicalmutagensbythe dominant lethal assay in the mouse. Toxicol. Appl. Pharmacol. 23:288-325.

8. Esparza, J.,D. J.M.Purifoy,P.A.Schaffer,and M.

Benyesh-Melnick. 1974. Isolation, complementation andpreliminary phenotypiccharacterizationof temper-ature-sensitivemutantsofherpessimplexvirustype2. Virology57:554-565.

9. Flannery,V.L.,R. J.Courtney,andP. A. Schaffer.

1977.Expressionofanearly,nonstructural antigenof herpes simplex virus incells transformed in vitro by herpes simplexvirus. J. Virol.21:284-291.

10.Frenkel, N.,H.Locker,B.Cox,B.Roizman,and F.

Rapp.1976.Herpes simplexvirus DNAintransformed

cells:sequencecomplexityin fivehamstercelllinesand

onederived hamstertumor.J.Virol. 18:885-893. 11.Haruta, M. 1968.Teratogenic effects ofactinomycin-D

onddOmouseembryos.Acta Pathol.Jpn.18:267-286.

12. Kimura, S.,J.Esparza,M.Benyesh-Melnick,and P.

A. Schaffer. 1974. Enhancedreplicationof tempera-ture-sensitive mutantsofherpes simplexvirustype2

(HSV-2) at the nonpermissive temperature in cells

transformedbyHSV-2.Intervirology3:162-169. 13. Kimura, S.,V.L.Flannery,B.Levy,and P. A.

Schaf-fer. 1975.Oncogenictransformationofprimaryhamster

cells by herpes simplexvirustype2 (HSV-2) andan

HSV-2temperature-sensitivemutant.Int. J. Cancer15:

786-798.

14. Kucera,L.S.,and J. P. Gusdon.1976.Transformation of humanembryonicfibroblastsbyphotodynamically inactivatedherpes simplexvirustype2 atsupraoptimal

temperature.J. Gen.Virol. 30:257-261.

15. Kutinova L., V. Vonka, and J. Broucek. 1973.

In-creasedoncogenicitv and synthesis of herpesvirus

anti-gensinhamstercellsexposedtoherpes simplextype2

virus. J.Natl.Cancer Inst. 50:759-766.

16. Macnab,J. C. M. 1974. Transformationofrat embryo

cells bytemperaturesensitivemutantsof herpes

sim-plex virus.J. Gen. Virol. 24:143-153.

17. Macnab, J. C. M., andM. C. Timbury.1976. Comple-mentationofts mutantsbyaherpes simplexvirus

ts-transformed cell line. Nature(London) 262:233-235. 18. Meyn, M. S., T. Rossman,and W. Troll.1977. A

pro-tease inhibitor blocks SOS functions in Escherichia coli:antipain preventsArepressorinactivation,

ultra-violetmutagenesis, andfilamentous growth.Proc. Natl.

Acad. Sci.U.S.A.74:1152-1156.

19.Minson, A. C., M. E. Thouless, R. P. Eglin, and G. Darby.1976.The detection of virus DNAsequencesin

aherpestype2transformed hamster cell line(333-8-9). Int.J. Cancer17:493-500.

20. Munyon, W., E. Kraiselburd,D. Davis, and J. Mann. 1971.Transfer ofthymidine kinasetothymidine kinase-less L cells by infection with ultraviolet-irradiated herpessimplex virus. J. Virol.7:813-820.

21. Rapp,F.,and R. Duff.1972.Invitrocelltransformation byherpesviruses. Fed. Proc.31:1660-1668.

22. Reed, C. L., G.H.Cohen, and F.Rapp. 1975. Detection ofa virus-specific antigen on the surface of herpes simplex virus-transformed cells. J. Virol. 15:668-670. 23. Reich, E., A. Cerami, and D.Ward. 1967. Actinomycin,

p.714-725. InD.Gottlieb andP.Shaw (ed.), Antibiot-ics, vol.1.Springer-Verlag,NewYork.

24. Roberts, J. W., and C. W. Roberts. 1975.Proteolytic cleavage of bacteriophage Lamdarepressor in

induc-tion. Proc.Natl. Acad. Sci. U.S.A.72:147-151. 25. Schaffer,P.A., G. M. Aron, N. Biswal, and M.

Ben-yesh-Melnick.1973.Temperature-senstivemutantsof herpes simplex virustype1:isolation, complementation andpartial characterization. Virology 52:57-71. 26. Suda, H.,T.Aoyagi,M. Hamada, T. Takeuchi, and

H.Umezawa. 1972.Antipain,a newproteaseinhibitor isolated from actinomycetes. J. Antibiot. 25:263-266. 27. Svoboda, D., J. Reddy, and C.Harris. 1970. Invasive

tumorsinduced inratswithactinomycinD.Cancer Res. 30:2271-2279.

28. Takahashi, M.,and K. Yamanishi. 1974. Transforma-tion of hamsterembryo and humanembryo cellsby temperaturesensitivemutantsofherpes simplexvirus

type2.Virology61:306-311.

29. Umezawa,H. 1972.Enzymeinhibitors of microbial ori-gin,p.29-32.UniversityofTokyoPress, Tokyo. 30. Vestergaad,B.F.,and P. C.Grauballe.1977.Crossed

immunoelectrophoreticidentification ofpartially puri-fied typecommonandtypespecific herpes simplexvirus

glycoprotein antigens.Proc. Soc.Exp.Biol. Med. 156: 349-353.

31. Williams,C.A.,and M. W. Chase(ed.).1968.Appendix II.Buffer.Methods Immunol. Immunochem. 2:388. 32. Yata, J.,G.Klein,J.Hewetson,andL.Gergely.1970.

Effect ofmetabolic inhibitorsonmembrane

immunoflu-orescencereactivityof established Burkittlymphoma celllines.Int. J. Cancer 5:394-403.