Mechanisms of expression of herpes simplex virus-common surface antigens in clonal cells of a herpes simplex virus type 2-transformed line.

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JOURNAL OF VIROLOGY,May 1981,p.420-429 0022-538X/81/050420-10$02.00/0

Mechanisms of

Expression

of

Herpes

Simplex Virus-Common

Surface

Antigens in

Clonal Cells of

a

Herpes Simplex Virus

Type

2-Transformed Line

KATSUICHIROOKAZAKI,' YOSHINARIOHNISHI,' NAGAYUKI YOSHIDA,2ANDSUSUMU

KIMURA3*

Department of Bacteriology, School of Medicine, University of

Tokushima,'

andDepartmentof Microbiology, School of Pharmacy, Tokushima University ofArts and Science,2 Tokushima 770, and

DepartmentofMicrobiology, Kochi MedicalSchool, Oko, Nangoku, Kochi 781-51,3Japan

Received24September 1980/Accepted 19 January 1981

Rabbit antiserum hyperimmune to herpes simplex virus type 1 was used to

studytheexpression of herpessimplex virus type-commonsurfaceantigens(CSA)

byindirectimmunofluorescencetestsinthreerepresentativecell clonesisolated

from a herpes simplex virus type 2-transformed hamster line, 155-4. These three clones showed differentphenotypeswith respect to CSAexpression: (i) a CSA-positivetype (clone 155-4-213), in which the antigensincreased soon (5 h) after seedingat370C,but notaftertreatmentwithactinomycinD; (ii)aCSA-inducible type (clone 155-4-03), in which the antigens increased after tretment with acti-nomycin D (2,ug/ml) for20h, butnotafter seeding only;and(iii)aCSA-negative type (clone 155-4-16),in whichthe antigensdid not increase after seeding orafter actinomycin Dtreatment.CSA expressionintheCSA-positivetypewasinhibited by 2-deoxy-D-glucose, but not by puromycin, suggesting that the expression required glycosylation, but notactive proteinsynthesis. CSA expressionin this

type was insensitive to the protease inhibitors antipain and

p-nitrophenyl-p'-guanidinobenzoate. On theotherhand, actinomycinD-induced CSA expression in the CSA-inducible type wasinhibited by both 2-deoxy-D-glucose and puro-mycin, suggesting thatthe induced expression required both glycosylation and protein synthesis. CSAexpression inducedin this type wassensitive to the two protease inhibitorsatconcentrations havinglittleeffect onoverallcellular metab-olismor cell viability.These resultsindicate thatCSA expressionsin the CSA-positivetypeand theCSA-inducibletype areenhnaced bydifferent mechanisms.

Various mammalian cells have been

trans-formedoncogenicallyinvitrobyherpessimplex virustypes 1 and2 (HSV-1 and HSV-2, respec-tively) (2, 12, 13, 25, 27, 29, 44). Viral DNA

sequences (11, 15, 33), HSV-specific RNA (7),

andHSV-specific antigens(2,12,25,27,44) have beendemonstratedincells transformed by

HSV-2.Moreover, viralgenetic information has been

concluded tobe present inHSV-2-transformed

cells from reports that complementation

be-tween temperature-sensitivemutantsof HSV-2

andresident HSV genes intransformedcells has

been demonstrated at the nonpermissive

tem-perature (24, 30).

SeveralHSV type-specificand type-common

antigens havebeenidentifiedrecently in

HSV-infectedcellsby immunological analysis (16, 34,

46), and type-common antigens have also been

detectedonthesurface ofcells transformed by

HSV (4, 18, 38, 39).

In an attempt to identify the HSV-specific

geneproducts

expressed

inHSV-2-transformed hamster

cells,

wedemonstratedthatHSV

type-commonsurfaceantigens

(CSA)

canbedetected

specifically

in transformed and derived tumor

celllinesby immunofluorescencetestsand that

HSVCSAexpressioncanbeenhancedin

trans-formed cellline 155-4 inatleasttwoways: one

way is after seeding at 37°C, without active

protein synthesis, and the other way is in

re-sponseto actinomycin D (ActD) and involves

protein synthesis (26).We have shownrecently

byclonal analysisof the cellsthat the cellline

consists of at least three different phenotypes

with respect to the expression of HSV CSA,

namely, aCSA-positive type, a CSA-inducible

type, and a CSA-negativetype. This paper

de-scribes a study of the mechanisms of CSA

expressionintwodifferentcelltypes

(CSA-pos-itive type andCSA-inducibletype).

420

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MATERIALS AND METHODS Cell lines. Transformed cell line 155-4 (passages 165 to 185) was obtained byaroutine methodafter theinfection of hamster (strainLSH) embryo fibro-blast (HEF) cellswith the DNA-negative tempera-ture-sensitive mutant (tsB5) of HSV-2 strain 186 at the nonpermissive temperature (38°C), as reported previously by Kimura et al. (25). The cell line was passagedseriallyat37°Cbecause it was not stable at 38°C. Clones from cell line 155-4 were isolated as follows. 155-4cells (approximately 30 cells per dish), which hadbeen subcultured for 180 passages in vitro, wereseeded into60-mmpetri dishes (Falcon Plastics, Oxnard, Calif.) and incubated in Eagle minimum es-sentialmedium (MEM) containing 20% fetal calf

se-rum (FCS) at 37°C in a CO2 (5%) incubator. At 10

days afterseeding, when there were4 to8cell colonies per dish, a total of30colonies were collected from several dishes, using a steel cylinder and

typsiniza-tion. Cellcloneswere passaged in Eagle MEM con-taining 10% FCSat37°C. Allclones were used between passages 9 and 24. All cells used in this study were grown in Eagle MEM supplemented with 10% FCS and 0.075%NaHCO3 for cultures in closed vessels and 0.225% NaHCO3 for cultures in open vessels.

Antisera. Rabbit antiserumhyperimmuneto HSV-1 (strain KOS) was prepared as reported previously (26). Theneutralizingantibody titer to HSV-1 of this anti-HSV-1 serum was 1:1,280 in 50% plaque reduction tests.Fluorescein-conjugatedgoatantiserum to rabbit

7Simmunoglobulin (FGAR)waspurchased from

Hy-landLaboratories, Inc., Costa Mesa,Calif.Nonspecific reactivity was removed from the two antisera by the absorption technique described previously (26). In brief,4ml ofundiluted rabbit anti-HSV-1 serum was absorbed with 1 gof rabbitliver powder, 108human embryoniclung cells,and10'HEFcells. FGAR (4 ml) wasalso absorbed with1gofrabbit liver powder and 108 HEFcells. The absorbed anti-HSV-1 serum used

in thisstudyreacted specificallywithHSV CSAon

HSV-2-transformedandHSV-2-infectedcells,butnot

withtumorigenictransformedcellsinduced with sim-ian virus 40 or bovine adenovirus type 3 orcontrol HEFcells(26).

Detection of HSV CSA The procedure of the indirectimmunofluorescence testfor thedetection of HSV CSA has been describedindetailbyKimuraet al. (26). The cells weregrown andpassagedat least three timesbeforetests.Cells (5x

105)

weretreated withAct D(0.5to 4ug/ml)atthe time ofseeding with 5 ml of medium into60-mm petri dishes. Inhibitors werealsoaddedtoActD-treatedcellsatthe time of seeding.Atthe indicatedtime, cellsweredispersedby treatmentwith 0.25% trypsin for 1 min and washed threetimes with barbital buffer(pH 7.4)containing3

X10-3 Mbarbital, 1.8x10-3Msodiumbarbital,0.85% NaCl,5x 10-4MMg2+, 1.5x 10-4MCa2,and0.1% gelatin(GBB).Samplesof1dropofabsorbed antise-rum at a final dilution of 1:4 were added to cell suspensions (6x105to 10x105cells).After30minof incubationat37°C,cellswerewashedthreetimeswith GBB and then incubated with 1 drop of absorbed FGARdiluted1:8for30minat37°C.Finally,the cells werewashed threetimes withGBB and mountedina 50%oglycerol solution, and 103cells persamplewere

observed with a reflected-light fluorescence micro-scope(Olympus OpticalCo.Ltd., Tokyo,Japan). The number of viable cellswasdeterminedby trypanblue exclusion.

For testsontheeffects ofvarious conditions on the

expressionofHSV CSAinclone 155-4-213 (see Table

1), cells detached by pipetting were suspended in 1 ml ofphosphate-buffered saline withoutCa2" and

Mg2e

[PBS(-)]and treatedwith1ml of trypsin(0.5%) or 1 ml of PBS(-) for 1 to 3 min. Trypsinization was

stopped by the addition of 15 ml of Eagle MEM

containing 10% FCS and centrifugation. Some cell

suspensions with or without trypsinization were

im-mediately assayed for indirect immunofluorescence.

Other suspensions(1x105 to25 x105cells per sample) were incubated with 5 ml of Eagle MEM containing 10%FCS in 60-mm dishes as monolayers or in tubes (15 by 150mm) assuspensions for5h at 4 or37°C andthen assayed for indirectimmunofluorescence.

Inhibitors. Act D andpuromycindihydrochloride (PM) were obtained from Boehringer Mannheim,

Mannheim, West Germany, and Marker Chemicals Ltd., Jerusalem, Israel, respectively. The protease in-hibitor antipain

[(l-carboxy-2-phenyl-ethyl)carbam-oyl-L-arginyl-L-valyl-argininal] (42) was kindly pro-videdby T. Uchida, Osaka University, Osaka,Japan,

andp-nitrophenyl-p'-guanidinobenzoate

dihydrochlo-ride(NPGB)wasobtained from ICNPharmaceuticals, Life SciencesGroup Inc., Cleveland, Ohio. 2-Deoxy-D-glucose was purchased from Sigma ChemicalCo., St. Louis, Mo. Stock solutions concentrated 10 times wereprepared, passedthrough 450-nm membrane fil-ters (Millipore Corp.,Bedford, Mass.),andstored at -200C.

Incorporations of[3Hlthymidine, [3H]uridine,

and[3H]leucine.The radioactiveprecursors

[methyl-3H]thymidine (40to60Ci/mmol),[5-3H]uridine (>25 Ci/mmol), and L-[4,5-3H]leucine (40to60Ci/mmol) wereobtainedfrom NewEngland NuclearCorp., Bos-ton, Mass. For the examination of DNA and RNA syntheses, cultureswerelabeled with either [3H]thy-midine (0.5,Ci/ml) or[3H]uridine (0.1 XCi/ml) con-taining 0.1 ,ug of thymidine per mlor0.1

,Ag

of uridine per ml, respectively, for 8 h afterseeding. For the examination ofproteinsynthesis, cultureswerelabeled with[3H]leucine (1.6t,Ci/ml) for20h afterseeding. The cellswerethen washedoncewith cold(4°C) Tris buffer, harvested, andcentrifuged at 800 xg for 10 min.Pelletssuspendedin1ml ofcold Tris bufferwere sonicated inanice bathat10 kc for 20sand treated with1ml of 20% trichloroacetic acid for20minat0°C. The[3H]leucine-labeledsampleswerethenboiled for 15 min. The suspensionswere applied to Whatman GF/C filterdisks,usinganaspirator. The diskswere washed three times withcold 5% trichloroacetic acid and once with ethyl alcohol, dried, and placed in scintillation vials. A 10-mlamountofscintillation fluid was then added to each vial, andradioactivity was measured inaliquidscintillation spectrometer(model

3385;Packard InstrumentCo., Inc.,Rockville,Md.).

RESULTS

Expressionof HSV CSA in variousclones

of transformed line 155-4. We isolated three

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422 OKAZAKI ET AL.

representative clones, 155-4-213, 155-4-03, and

155-4-16, from cell line 155-4 transforned by

HSV-2 (tsB5 of strain186). These clones showed

different phenotypes with respect to the

expres-sion ofHSV CSA (Fig. 1). In clone 155-4-213

(CSA-positive type), CSA expression was

en-hanced (about 40% positive) soon (5 h) after

seeding at 37°C; thereafter, the expression de-creased with an increase in the cell number, returningto theoriginallevel (about 12% posi-tive) 48hafterseeding.Act D(2

,ug/ml)

didnot

affect this CSA expression. In clone 155-4-03 (CSA-inducible type), CSA expressionwas low

(11 to 15%positive) for30hafterseeding.

How-ever, theexpression oftheseantigensgradually

increased after treatment with Act D(2

I.g/ml),

reachinga maximum (35 to 40% positive) 20h

after treatment. Inclone155-4-16(CSA-negative

type),CSA expression didnotincrease within 30

hafter seeding or after Act D treatment. The number of viable cells decreased to a similar

extentin these three cell clonesupon treatment

with Act D.

Conditions

affecting expression

of HSV CSA in the

CSA-positive

type. The condi-tions inducing CSA expression in clone 155-4-213wereexamined byvarying thetimeof tryp-sinization, the size of the cell inoculum, the

incubationconditions, andthemedia(Table1).

No increase inCSA was observed in 155-4-213

cells thatwere immediately assayed forthe de-tection of CSA without trypsinization or after trypsinization for 1 or 3min or that were

incu-bated at 4°C for 5 h after trypsinization for 1

min. A slight increase in CSA was observed

when: (i) cells were incubated at 37°C for 5 h without trypsinization; (ii) smaller inocula

(105

'd

155-4 155-4-21

*~40

0 20 U)

o 20

0.

;W°O IS15-4 155-4-21

EX)

9

0loft f

c 5

cells) or larger inocula (2.5 x

106

cells) were

used; (iii) cells were incubated at 37°C for5 h

aftertrypsinization for3min;and(iv) cellswere

incubated with PBS(-), MEM without serum,

or MEMcontaining a low concentration (5%) of

serum. The increase in CSA in 155-4-213 cells

wasmarked only when the cells were trypsinized

for 1min and incubated at an appropriate

inoc-ulum (5 x 105 cells per dish ortube) for 5 h at

37°C with MEM containing 10 or 20% serum. Anincrease inCSAwasobservedwithcell sus-pensions andeven morewithmonolayers.Thus,

incubation of cells at37°C after trypsinization

was necessaryfor an increase in CSA in clone

155-4-213, andtrypsinization itself was not

suf-ficient.

Conditions inducing expression of HSV CSA in theCSA-inducible type. The effectof

the concentrationofAct D onthe expression of

CSAinclone155-4-03 wasexaminedby treating the cells with various concentrations of Act D

(0.5 to 4

tig/ml)

for 20 h(Fig. 2). A high

corre-lationwas observed between the concentration

of Act D (0.5 to 2 ,ig/ml) and the number of

CSA-positive cells. Although the viable cell numbers were almost the same in cultures

treated with 0.5 to 4

,tg

of Act D per ml, the

additionof 2

,ig

of Act Dresultedinthe greatest CSA expression, whereas a higher dose (4 ,ig/ ml) slightly inhibited its expression. Therefore,

in theexperiments describedbelow,2,ug of Act

D per ml was used for the induction of CSA

expressionin 155-4-03 cells.

For thedeterminationof theeffective time of

additionof Act D on the induction ofCSA, 155-4-03cellsweretreated with Act D (2,Lg/ml) at various times (0, 3, 5, and 10 h) after seeding

0 10 20 30 0 10 20 30 0 10 20 30 0

Hours

after

seeding

FIG. 1. Relationshipbetween the expression of HSV CSA and cell growth in HSV-2-transformed cell line 155-4andthree clonesfrom the line at 37°C in the presence and absence of Act D (2,ug/ml). Symbols: 0, absenceofActD;0,presenceof Act D.

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TABLE 1. Various conditionsaffecting the expression of HSV CSA in clone 155-4-213

Cell inoculumper Incubation conditions SA-positive

Exptno.andtreatment sample(X105) Sa l (h) Temp(°C) cells (%)b

1. Trypsinizationc

None 5 Noincubation 9.5

5 Monolayer 5 37 16.7

1min 5 Noincubation 6.8

5 Suspension 5 4 10.8

5 Suspension 5 37 32.4

3min 5 Noincubation 7.0

2. Medium andserumd

PBS(-) without serum 5 Monolayer 5 37 15.4

MEM without serum 5 Monolayer 5 37 20.8

MEM with 5%serum 5 Monolayer 5 37 24.8

MEM with 10%serum 5 Monolayer 5 37 38.2

MEM with20% serum 5 Monolayer 5 37 34.0

a

Celis

wereinoculated into dishes (monolayer) or tubes (suspension) with gentle shaking. No incubation,

Cellsuspensionswereimmediately assayed for indirectimmunofluorescence.

'Atotal of103

cells

werecounted.

Trypsinizationwasperformedasdescribed in the text.

d

Cells

weretreatedwith trypsin (0.25%) for1min andsuspendedin5mlofPBS(-) or Eagle MEM containing various concentrations of FCS.

3o o

E

.~0

o o5 2 4 Cancmnitonof oinonycinD

[image:4.500.59.450.88.313.2]

(Mg/mi)

FIG. 2. Effect ofthe concentration ofAct D on

HSV CSAexpression in clone 155-4-03. Act D(0.5to

4pg/ml)-treatedanduntreated cellswereincubated for20hat37°C. Symbols: 0, CSA-positive cells;0,

cell number.

(Fig. 3). When ActD wasaddedtothe cellsat

thetime ofseeding,HSVCSAincreasedgreatly by20 h afterseeding.The addition of Act Dat

3 h after seeding induced CSA afteralong lag

(about 17h). NoincreaseinCSAwasobserved

uponthe addition of thedrugat5or 10h after

seeding. Thesefindings indicate that the

addi-tion of Act D within 3 h after seeding was

necessaryforthe induction ofCSAin clone

155-4-03.

Forthe determination of the effective period

of treatment with Act D, 155-4-03 cells were

treated with Act D (2 ag/rnl) for3, 5, and 10 h

fromthe time of seeding and then incubated in

freshmedium without Act D foratotal of 30h.

In cells treated with Act D for 5 and 10 h,CSA

expression wasinduced and a high percentage

(35to40%) ofCSA-positivecellspersistedfor 20

h after the removal of Act D. However,in cells

treated with Act D for 3h, thenumber of

CSA-positive cells did not increase greatly (data not shown).

Effect of PM on the expression ofHSV CSA in the CSA-positivetypeand CSA-in-ducible type.To determine whether new

pro-tein synthesis is required for CSAexpression in

clones 155-4-213 and 155-4-03, we added PM to

cultures with or without Act D (2 ,ug/ml) and

determined the numbers and percentages of

CSA-positive cells and viable cells and the in-corporation of [3H]leucine into the cells. As

shown in Fig.4A,CSAexpression inclone

155-4-213 with or without Act D was notsuppressed

by PM (70,ug/ml),whereas theincorporationof

[3H]leucine into viable cells was almost com-pletely inhibited by this concentration of PM (Fig. 4G).

On the other hand, the induction of CSAby

Act D in clone 155-4-03 was suppressedto the

level of that in untreated cultures by 70 tg of

PM per ml (Fig. 5A), a concentration which

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40

- 30

2

i

20

4A 10

0

15

12

9

6

1

3

0

10 15 20

[image:5.500.58.252.61.339.2]

Hours ftersuing

FIG. 3. Effect ofthe timeofadditionofActDafter

cellseedingontheexpression ofHSVCSAinclone 155-4-03. At the indicated times (0, 3, 5, and 10h) after seeding,cellsweretreated with ActD(2 pg/ml) and reincubatedat37°C. (A) Expression of CSA; (B)

cellgrowth. Symbols:0,control(untreated) cells;0,

additionofAct Dat0 hafter seeding; A,additionof Act Dat3 hafter seeding;A,additionofAct Dat5

h after seeding; O, addition ofAct D at 10h after seeding.

greatly inhibited the incorporation of

['H]leu-cine into the cells (Fig. 5G). CSA expressionin

this clone in the absence of Act D was not

suppressed by the same concentration of PM.

Thenumbers of viable cells in clones 155-4-213

and 155-4-03 were similar in cultures treated

with Act Dalone, PM alone, and Act D plus PM

(Fig. 4B and 5B). These results indicate that

new protein synthesis is required for Act

D-inducedCSA expression inclone155-4-03,but is

notrequired forCSA expression in clone

155-4-213afterseeding.

Effect of 2-deoxy-D-glucose on the

ex-pression of HSV CSA in the CSA-positive

type and CSA-inducible type. We next

ex-amined whether glycosylation is necessaryfor

CSA expression in clones155-4-213and155-4-03

by treating them with various concentrations (1,

5, 10, and 20 mM) of2-deoxy-D-glucose, which

inhibits glycosylation of viral glycoproteins (9,

10,22). CSA expression in clone 155-4-213after

seedingwasinhibited inadose-dependent

man-nerby2-deoxy-D-glucose (Fig. 6A).The number

of viable cells in this clone also decreasedslightly

upon treatmentwith thedrug.

CSA inductionbyAct D in clone 155-4-03was

inhibited greatly bythe addition of

2-deoxy-D-glucose (more than 5 mM), although CSA

expression in the clone without Act D was not

affected by 2-deoxy-D-glucose (Fig. 6B). CSA

inductionbyAct D in 155-4-03 cells was more

sensitive to 2-deoxy-D-glucose than was CSA

expression in 155-4-213 cells afterseeding. The

number of viable cells in clone 155-4-03 de-creasedgreatlyathigherconcentrations(10and 20mM) of2-deoxy-D-glucose in the absence of

ActD,but not in the presence of Act D. These

findings suggest thatglycosylationisnecessary forCSAexpressionboth in 155-4-213 cells after

e 30

i

20

I^ 10

u

E^

=

-C0

_ _

u

C

0 5 10 15 20

Hoursafterseeding

FIG. 4. Effect ofPMonHSVCSAexpression,cell

growth, and incorporation of [3H]leucine in clone 155-4-213 incubated with andwithout Act D. Act D (2pg/ml)-treatedand untreated(control)cellswere

incubatedwith andwithout PM(70ug/ml) at37°C for20 h.(A) Expression of CSA; (B)cellgrowth; (C)

incorporation of [3H]leucine into 105 viable cells. Symbols:0,control cellswithoutPM; *, control cells

withPM; A,ActD-treated cellswithoutPM;A,Act

D-treated cellswith PM.

C

P-E

.5

U)1

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40

:-,

-.Z

'a

=

In

30

20

10

0

8

4

0

16

8

0

0 5 10 15 20

Hours after seeding

FIG. 5. Effect ofPMonHSV CSAexpression,cell growth, and incorporation of[3Hileucine in clone 155-4-03 incubated with andwithout Act D. Act D(2

Pg/ml)-treatedanduntreated(control)cellswere

in-cubated with andwithout PM(701tg/ml)at37°Cfor 20 h. (A) Expression of CSA; (B) cellgrowth; (C) incorporation of[3Hileucine into 105 viable cells. Symbols:0,control cells withoutPM; *,control cells

withPM; A,ActD-treatedcells withoutPM;A,Act

D-treated cells with PM.

seeding and in 155-4-03 cells after treatment

withAct D.

Effects of protease inhibitors on the

expression of HSV CSAintheCSA-positive

type and CSA-inducible type. Our previous

study (26) demonstrated that theprotease

inhib-itorantipain (0.5 mM) inhibited Act D-enhanced

CSA expression in the parent cell line 155-4

withoutcausing significantcelldamagebut that

it didnotinhibitanincreaseinCSA expression

in the absence of Act D in thesame cellline. In

this study, the effects of antipain and another

proteaseinhibitor, NPGB,onCSA expressionin

clones 155-4-213 and 155-4-03 were examined.

As shown in Fig. 7, CSA expression and the

number ofviablecellsin clone 155-4-213 in the

presence and absence of Act D were hardly

affected by antipain (0.5 or 1.0 mM).

Further-more, CSA expression in this clone 5 h after

seedingwas not affected by NPGB, even at a high concentration(0.1mM;seeFig. 9A).

On the other hand, Act D-induced CSA

expression in clone 155-4-03 was inhibited in a dose-dependentmannerby antipain(Fig. 8) and NPGB (Fig. 9B) without significant cell damage. Higher doses of antipain (0.5 and 1.0mM) and NPGB (0.1 mM) suppressed theinduced expres-sion to the level of the control culture (not treated with Act D), but these high doses of antipain and NPGB didnotinhibit CSA expres-sion in this clone in the absence ofAct D. To determine whether the doses of antipain and NPGB used in this study affect DNA,RNA, or proteinsynthesis in155-4-03cells,wemeasured the effects of thesecompounds onthe incorpo-rations of [3H]thymidine,

[3H]uridine,

and

[3H]leucine

intomacromolecules in thecellsin

the presence and

abs9nce

of Act D (Table 2).

Higher doses of antipain (0.5 and1.0 mM) and NPGB (0.05 and 0.1 mM) did not inhibit the incorporation of any precursors into 155-4-03 cellswithorwithoutActDtreatment, indicating that theseproteaseinhibitors,at concentrations

40

130

120

uo

40

o

(0

20

I0

0

o1 5 10 20

Conbniof of2-doxy-D-gkucose

(mm)

I

IE

[image:6.500.60.233.60.371.2]

i

FIG. 6. Effect of2-deoxy- D-glucoseon HSV CSA

expression inclones 155-4-213 and 155-4-03.(A)

155-4-213cellswereincubated with andwithout 2-deoxy-D-glucose (1to20mM)at37°C for5h.Symbols:0,

CSA-positive cells;0, cellnumber. (B)Act D(2pg/

ml)-treated and untreated (control) 155-4-03 cells

wereincubatedwith and without2-deoxy- D-glucose (1 to 20mM) at 37°C for20 h. Symbols: 0,

CSA-positivecells in controlcultures;0, ceUnumberin

control cultures; A, CSA-positive cells in Act

D-treated cultures; A, cell number in Act D-treated

cultures.

. A.155-4-213

.S

- 4

''^ v ^ *~~~~~~~21

I.. a 2

B.155-4-03 0

4

r ,-- .

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426 OKAZAKI ET AL.

40-S30

.~20

10

0

B

_6

04

4

0 5 10 15 20

[image:7.500.78.230.60.322.2]

Hoursafterseeding

FIG. 7. EffectofantipainonHSV CSAexpression

and cellgrowth in clone 155-4-213 incubated with and without Act D. Act D (2

pg/ml)-treated

and untreated (control) cells were incubated with and withoutantipain at37'Cfor20h. (A) Expressionof

CSA; (B) cellgrowth.Symbols: 0,control cells with-outantipain; *, control cells withantipain (1 mM); A, Act D-treated cells without antipain; U, Act D-treated cells withantipain (0.5 mM); A,ActD-treated cellswithantipain (1mM).

that suppress Act D-induced CSA

expression,

didnotaffect overall cellular metabolism.

DISCUSSION

We have recentlyfound that the anti-HSV-1

serumused in this study reacts with both early

and late HSV CSA induced in HSV-2-infected

HEFcellsand that the HSVCSA detected with

thisantiserum reaches a maximum before virus

infectivityreaches amaximum in infectedcells (S. Kimura, K. Okazaki, and N. Yoshida,

sub-mitted forpublication). Our previous study (26)

alsodemonstrated that the reactivity of the

an-tiserum in HSV-2-transformed cell line 155-4

wassignificantlydecreased after absorption with HEFcellsinfected with HSV-2 or with

homol-ogous 155-4cells,but not with uninfected HEF

cellsorsimian virus 40-induced tumor cells.

Sincepreviousfindings suggested thatcellline

155-4 consisted of several different cell types

with respect to CSA expression (26), 30 clones

wereisolated from this line and tested forCSA

expression. These isolated clones were classified

into three different phenotypes withregard to

CSAexpression,namely,aCSA-positivetype,a

CSA-inducible

type, and a CSA-negative type (Kimuraetal., submittedforpublication). CSA expression by the CSA-positive type occurred soonafterseedingat37°C.Oftheclonesisolated, 20% belonged tothis type,and clone 155-4-213 wasusedas arepresentative. CSA expressionin thisrepresentative clonerequired incubationof thecellsfor5hat37°Caftertrypsinization and

was not affected by Act D treatment. CSA

expression in the

CSA-inducible

type occurred

after treatment of thecellswith Act Dfor 20h,

but notafterseeding. Oftheclonesisolated,

33%

belonged to this type, and clone 155-4-03 was

used as a representative. Act D-induced CSA expressionin the

representative

clonewas

great-estwhen2

jig

ofAct D permlwasaddedtothe

cellsatthetimeofseeding.In theCSA-negative

type,CSAexpressiondidnotincrease after

seed-ingorafter Act Dtreatment.Of theclones,47%

belonged to this type, and clone 155-4-16 was

usedas arepresentative.

40

Be 30

@ 20

LPa- 10

0

12

I?

0

E

.-8

4

Hoursater seeding

FIG. 8. Effectofantipain on HSVCSAexpression and cellgrowthin clone 155-4-03 incubated with and without Act D.Act D(2

lg/ml)-treated

and untreated (control)cells were incubated with and without anti-painat37°C for20h.(A)ExpressionofCSA; (B) cell growth. Symbols: 0, control cells without antipain;

0, control cells with antipain (1 mM); A, Act D-treated cells withoutantipain;x,ActD-treatedcells withantipain (0.01 mM);*, ActD-treatedcells with antipain (0.1 mM); O,ActD-treated cells with anti-pain (0.5 mM); A,ActD-treated cells with antipain

(1 mM).

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This study also demonstrated that CSA expressions inthe CSA-positivetype(clone

155-4-213) and theCSA-inducibletype (clone

155-4-A.

155-4-213

I-L

-...-...

B. 155-4-03

W___ ____ __--_

~

-,_

o

0.01 0.5 0.1

Concentration of NPGB(mM)

FIG. 9. Effect ofNPGBonHSV CSAexpression in

clones155-4-213and 155-4-03. (A)NPGB(0.01 to0.1 mM)-treated and untreated 155-4-213 cells were

in-cubatedat 37°C for5h. Symbols: 0, CSA-positive cells;0,cellnumber.(B)Act D(2

pg/ml)-treated

and

untreated(control)155-4-03 cellswereincubated with

and without NPGB(0.01to0.1mM)at37°C for20h. Symbols: 0, CSA-positive cells in controlcultures;

0, cellnumber in controlcultures; A, CSA-positive

cells in Act D-treatedcultures;A,cellnumber in Act D-treated cultures.

03) were enhanced by different mechanisms.

CSAexpressionin clone155-4-213 wasinhibited

by2-deoxy-D-glucose,but not byPM, suggesting

that theexpression required glycosylation, but

not activeprotein synthesis. This expression in

the clonewasinsensitiveto theprotease

inhibi-tors antipain and NPGB. On the other hand,

ActD-inducedCSAexpression inclone 155-4-03

was inhibited by both 2-deoxy-D-glucose and

PM, suggesting that the induced expression

re-quired bothglycosylationandprotein synthesis. Thisinducedexpression in the clone was

sensi-tive to antipain and NPGB at concentrations

that had littleeffectonoverall cellular

metabo-lism (DNA, RNA,and proteinsyntheses)orcell viability.

It iswell known that Act D binds to native

DNA and inhibits RNA, DNA, and protein

syntheses (8,40). Thisdrug alsoinduces muta-tions (14) and chromosome abnormalities (32,

36) in eucaryotic cells and has tumorigenicity

(43) andteratogenicity (20) inanimals. Itisnot clear how Act D induces CSA expression in clone 155-4-03. We recently found that CSA expression inclone 155-4-03 (about 40% ofthe cells) is also induced by treatment with the anthracycline antibiotics adriamycin (0.25 ,ug/ ml) and daunomycin (0.25 ,ug/ml), which are

known to bind to DNA (1, 3, 37) and inhibit

both RNA and DNAsyntheses (19,23), asdoes Act D; however, no CSA was induced in this clonebytreatmentwith

5-iododeoxyuridine,

cy-tosine

arabinoside,

or

mitomycin

C

(D. Kako,

Y.

Ohnishi,

and S.

Kimura, manuscript

in prepa-ration).Furthermore,wefound that in this clone

themaximallevel ofCSA

expression

(about

40%

of the

cells)

could be inducedby the simultane-ousadditionof lower doses ofAct D

(0.5 ug/ml)

TABLE 2. Effectsof antipainandNPGBonincorporationsof[3H]thymnidine, [3H]uridine,and[3H]leucine into clone 155-4-03 in the presenceandabsenceof ActD

Incorporation (per 105 cells)of:

Group Treatment

[3H]thymidine

[3H]uridine [3H]leucine

(cpmx10-3) (cpmx10-3) (cpmx10-4)

A Untreated 1.45 3.55 4.90

Antipain(1.0mM) 1.86(1.28)a 4.55(1.28) 5.72(1.17)

NPGB(0.1mM) 1.45(1.00) 3.38(0.95) 6.03(1.23)

B Act D(2,g/ml) 1.40 0.23 2.67

Act Dplus antipain (0.5 mM) 1.48(1.06)b 0.25(1.09) 2.84(1.06) Act Dplus antipain (1.0 mM) 1.36(0.97) 0.28(1.22) 2.73(1.02)

Act Dplus NPGB (0.05mM) 1.58(1.13) 0.23(1.00) 3.34(1.25)

Act Dplus NPGB(0.1mM) 1.51 (1.08) 0.28(1.22) 2.60(0.97) aValues withinparentheses ingroup A represent the ratio ofcounts per minute incorporated into cells

treated withantipainorNPGBtothatincorporatedinto untreated cells.

bValues withinparentheses in group B represent the ratioofcounts per minuteincorporated into cells

treated withAct Dandantipainorwith Act Dand NPGBtothatincorporatedintoceUlstreated with Act D only.

40

. 20

- 10

0 0

040

& 30

cn

() 20

90

0

_6%fr

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and

adriamycin

(0.1

,ug/ml),

each of which alone inducedonly low levels of

expression (each

about 20% of the

cells),

and that the simultaneous addition of themosteffective doses of Act D

(2

,ug/ml)

and

adriamycin

(0.25

Ag/ml)

did not

cause CSA induction in morethan 40% of the

cells.Thus, these doses of the

drugs

donotshow additive or

synergic

effects

(Kako

et

al.,

manu-script in

preparation).

These

findings

suggest that CSA induction in clone 155-4-03is dueto a

common

effect(s)

of Act D and

anthracycline

derivatives andnot to aside action ofa contam-inant(s) in Act D.

Antipain, a protease inhibitor of microbial origin, isa

relatively

nontoxic(45), low-molecu-lar-weight compound, which inhibits trypsin-and papain-like proteases in

particular

(42).

NPGBis oneof several activesite-specific

inhib-itorsof serineproteases, such astrypsin,

throm-bin, plasminogen activator, and

plasmin

(5, 6, 17). Asdescribedabove, thesetwo protease in-hibitors reducedActD-inducedCSAexpression inclone 155-4-03 (CSA-inducible type), indicat-ing that protease(s) plays an

important

role in CSA inductionbyActD. It has beenshownthat A prophage in Escherichia coli is induced by proteolyticcleavage of the Arepressor (41). An-tipain inhibits A phage induction (one of the SOSfunctions) inE.coliby

blocking proteolytic

inactivation ofthe repressor (31). Therefore, it is conceivable that aputative repressor(s) ofa

CSA function(s) isinactivated

by

aprotease(s) induced or activatedin the cellsrespondingto ActD,leadingtotheappearance ofCSA. Simi-larly, it wasreported

recently

that protease in-hibitors,including

antipain,

canblock the chem-ical (28) or physical (35) induction of an

en-dogenous

xenotropic

typeCvirus from Kirsten

sarcomavirus-transformedmouse

celLs,

suggest-ingarole of

proteolysis

invirusinduction. On the otherhand, theincreased expression ofCSA in clone 155-4-213 (CSA-positive type) afterseeding maybe due toa translocation or modificationof apreexisting antigen(s) (possibly protein). Furthermore, CSA expression in this

clone was not affectedby treatment with Act D,

PM, or the two protease inhibitors. Although

the mechanism of this phenomenon is not

known, it might be explained by supposing that

arepressor(s) reponsible for CSA expression is

notproduced or is produced in aninactiveform

inthecellsorthat arepressor-binding site(s) of

DNA is altered so that it is insensitive to the

repressor present in the cells. In a

repressor-controlledsystem, such as the A phage and Lac

system of E. coli, the presence ofconstitutive

regulator and operator mutations has been es-tablishedpreviously (21).

Furtherexperiments are in progress to

deter-mine whether the antigens expressed by the

CSA-positivetype are identical to those induced intheCSA-inducibletype.

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