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Establishment of a C3Hf Mammary Tumor Cell Line Expressing Endogenous Mouse Mammary Tumor Virus: Antigenic and Genetic Relationships of This Virus with Highly Oncogenic Mouse Mammary Tumor Viruses

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JOURNAL OF VIROLOGY, Dec. 1979,p.852-859

0022-538X/79/12-0852/08$02.00/0 Vol.32, No.3

Establishment

of a

C3Hf Mammary

Tumor Cell Line

Expressing Endogenous

Mouse

Mammary

Tumor

Virus:

Antigenic and

Genetic

Relationships

of This

Virus with

Highly

Oncogenic Mouse

Mammary

Tumor

Viruses

LARRY 0. ARTHUR,* GERALD G. LOVINGER,AND GERALD SCHOCHETMAN

BiologicalCarcinogenesisProgram,Frederick Cancer ResearchCenter, Frederick, Maryland21701

Received forpublication 16 July1979

Asingle-cellclone ofC3Hfmammarytumorcells(clone14)wasdevelopedinto

a continuous cell line expressing high levels of endogenous mouse mammary

tumor virus (MMTV) with less than 0.1% murine leukemia virus expression.

Comparison of the C3Hf MMTV protein profile on sodium dodecyl

sulfate-polyacrylamide gel electrophoresis with that of C3H MMTVrevealed thatthe

protein content of thetwo viruses was quite similar. However, oligonucleotide

fingerprints obtained of MMTV 70S RNA revealed thatapproximately 20% of

thelargeoligonucleotides examinedwere

unique

toeach virus. The

oligonucleo-tidefingerprint indicated that

although

thevirusesweresimilar, they differedin

theirgeneticcontent.Thedifferencesinthetwovirusesextendedto

immunolog-ical differencesinthemajorenvelope glycoprotein,gp52.C3HfMMTVcompeted

only partially in a homologous

radioimmunoassay

for gp52 of C3H MMTV, whereas C3H MMTV gave

complete

competition,

indicating

that gp52 ofC3H

MMTV contained

type-specific

determinants not present on gp52 of C3Hf

MMTV.ComparisonofC3Hf MMTV withhighly oncogenic

C3H,

GR,andRIII

MMTVs ina

homologous

C3HMMTVgp52 assay gavetwo

patterns

ofreactivity:

complete competition by GR and C3H MMTV andincomplete competition by

C3HfandRIII MMTV.

Absorption

ofanti-C3H MMTVserum by either C3Hf

MMTV or RIII MMTV removed all antibodies against both viruses but not

against GRandC3H MMTVs. These results indicate that C3H and GR MMTVs

are more

closely

relatedtoeach other thantoRIIIandC3HfMMTVs.

Certain inbred strains of

mice,

suchas

C3H/

HeN, contain milk-transmitted (exogenous)

mousemammarytumorvirus

(MMTV)

and

ge-netically transmitted

(endogenous)

MMTV.

Foster nursing the C3H

suckling

on mice of

strains of low mammary tumor incidence

re-moves the milk-transmitted virus and reduces

the mammary tumor incidence from greater

than 95% to

approximately

40% (13).

Reintro-duction of the exogenous MMTV into young

mice restores the high mammary tumor

inci-dence(1). Thetumorsinducedbythe exogenous

MMTVoccurearly (average8months),whereas

mammary tumors which arise in foster-nursed

C3H (termedC3Hf) miceoccurlate(14months

of age). DNA-proviral sequences of the

exoge-nous virus are foundonly inmammarytumors

induced by this virus, whereas endogenous

MMTV DNA-proviral sequences can be found in all tissues, including late-arising mammary

tumors (8, 16, 21). Even though the complete

endogenousMMTVcanbe recoveredfrom

late-arisingC3Hf mammary tumors, it is still unclear

whether theendogenous MMTV is the

etiolog-icalagentfortumorformation in the C3H mouse

orwhethervirus expression was a result of tumor

development. Studies to ascertain the

tumori-genic properties of C3Hf MMTV have been

hampered by the lack ofa reproducible source ofhighly purifiedvirus in whichthe

concentra-tionorinfectious doseof MMTVcouldbe

con-trolled. Mouse milk has been usedforyears as

a source of exogenous MMTV; however, milk

was not afeasiblesourceofendogenous MMTV

duetothelow concentration of virus in the milk

of thefoster-nursed animals. Exogenous MMTV

from mammarytumorcells in culture haslargely

replaced milk-derived MMTV as a source of

virus. Tissueculture-derived exogenous MMTV

is highly oncogenic (4) and can be obtained

reproducibly inlargequantities free of

contam-inating mouse milk proteins. Since production

of MMTV from tissue culture has proven

suc-cessful for the exogenous C3H MMTV, we

es-tablished cell lines from late-arisingtumors of

C3Hf MMTVtoprovideasourceof the

endog-enousMMTV.

In thisreport we describe the establishment

852

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C3Hf CELL LINE EXPRESSING MMTV 853

of a C3Hf cell line producing high levels of

MMTV. Theprotein and nucleic acid

composi-tion of this C3Hf-MMTV was similar to theC3H

MMTV, although the two viruses were

antigen-ically andgenetically distinguishable. Based on

aradioimmunoassay (RIA)forthemajor

enve-lopeglycoprotein, gp52, the C3Hf tissue

culture-derived MMTVwasindistinguishable fromthe

MMTV expressed in C3Hf mammary tumors

andmilk,indicatingthatit was theendogenous

virus. Comparison of gp52 antigenic

determi-nants of C3Hf MMTV with determinants on

gp52 of highly oncogenic C3H, GR, and RIII

MMTVs suggested that GR MMTVwas more

closely related toC3HMMTVthan toC3Hf and

RIII MMTVs.

MATERIALS AND METHODS

Cells and virus. MMTVwasobtained from

mam-mary tumorcelllines and milk. The viruswaspurified

from the culture fluid as previously described (12).

C3H-derived MMTV was from the Mm5mt/c, line

established by Owens and Hackett (18), and

C3Hf-derived MMTVwasobtainedfrom clone(Cl) 14ofa

C3H/HeNfmammarytumorcellline describedbelow.

GR MMTVwasfromaGR/Nmammarytumorcell

linepreviouslyestablished inourlaboratory,andRIII

viruswasobtainedfrom RIIImousemilk. The C3Hf

mammary tumor cell line was established from an

adenocarcinoma ofa16-month-oldmultiparousC3H/

HeNf NIHSwiss female. Thetumor wasasceptically

removed,sliced into smallpieces (1 to5mm3),and

incubatedat40Covernightinatubecontaining

Dul-becco-modifiedEaglemediumplus 10% fetal calf

se-rum,insulin(10

,g/ml),

tylocine (60ytg/ml), penicillin

(100U/ml), and streptomycin (100

jug/ml).

The

me-dium was replenished, and the tumor pieces were

agitated by gentle pipetting. After the large tumor

pieces settledtothe bottomof thetube,themedium

containingdislodgedcells and smalltumorfragments

wastransferred to a 75-cm2tissue culture flask and

incubatedat370Cin 5%C02. Cells which grewwere

predominantly epithelialin appearance withfew

fibro-blastic-like cells. Fibroblastic cells detach from the

tissuecultureflask in the presence oftrypsin-EDTA

morerapidlythanepithelialcells andweretherefore

removedbyincubating trypsin-EDTA onthe

mono-layer until 50% of the cells were detached. The

de-tached cells were discarded and fresh medium was

added.After4to6weeks of thisselective

trypsiniza-tion, islands ofepithelial cellswerethepredominant

cell type withnoappreciable growthoffibroblasts.At

confluency,thecellsweretransferredby trypsinization

andsubsequentlysubculturedat7-dayintervalsat a

1:4split ratio.

Cells from C3Hf mammarytumor cell lines were

foundtobe free ofmycoplasma bybioassay (R. Del

Guidice,Frederick CancerResearchCenter).

Karyo-typic examinationaswellasanalysisof the

glucose-6-phosphate isomeraseisozymesconfirmed the cellsto

be ofmouse origin (C. S. Stulberg, Child Research

Center ofMichigan, Detroit;and W.A.

Nelson-Rees,

University of California, Berkeley). The cells were

neartetraploidwitha meanchromosomecountof78.

Cells were tumorigenic when inoculated into C3H/

HeN,C3H/HeNf, and nude mice. The C3Hf cells did

not grow when inoculated into GR/N and BALB/c

mice (Arthur andMassey,unpublished data).

Cellcloning. Cells were removed from flasks with

trypsin-EDTA anddilutedto 10cells per ml of

com-plete Dulbecco-modified Eagle medium, and 100 pl

was placed in each of96 wells ofa Costar 96-well

cloning dish. These dishes were incubatedat370Cin

5%C02. At 7 dayspostplanting,the wellswere

exam-ined microscopically and scored for single colonies.

Wells were refedat7-dayintervals,andwhen the cells

were 60 to80%confluent, theyweretransferredto a

30-cm2 flask and subsequentlyto a 75-cm2 flask. At

this stage cells wereincubated incompleted

Dulbecco-modified Eagle medium plus

10'

Mdexamethasone,

and MMTV in the spentmedium was monitored for

reversetranscriptase activity and MMTV gp52.

Clon-ingefficiency ranged between 10 and 20%.

Polyacrylamide gel electrophoresis.

Electro-phoreticseparation of MMTVpolypeptideswas

per-formedbyprocedurespreviously described (2).

Pro-teinwasdeterminedby the procedure ofLowryetal.

(14) withbovine serum albumin as a standard.

CompetitiveRIAs. The major envelope

glycopro-tein of MMTV (gp52) was purified by a combination

oflectin chromatography (lens culinaris coupled to

agarose polyacrylic hydrizide) andmolecular sieving

(Sephacryl S-200) as previously described (3). The

purified gp52wasiodinated with

"1I

and used as the

radiolabeled antigen in a competition RIA as

previ-ouslydescribed(3).Antisera used in the assays were

prepared in rabbits by multiple intradermal inocula-tions of purified C3H or C3Hf MMTV in complete Freund adjuvant followed by inoculation of each

MMTV in incomplete Freund adjuvant at 3-week

intervals. Animals were bled before each inoculation, andsera werestoredat-70°C until used. Antiserum

prepared againstC3H-derived MMTV was absorbed

withC3H,C3Hf,GR, or RIII MMTV by incubating 2

mgof virus with 10 ml of serum at a 1:100 dilution for

2hat370Candovernightat40C.Immune complexes

wereremoved bycentifugationat100,000 x g for 2 h.

RNA-directed DNA polymerase assay.

RNA-directed DNApolymeraseassays wereperformed by

determining [3H]dGTP incorporation by

detergent-disrupted MMTV as previously described with the

synthetictemplate, oligo(dG) -poly(rC) (12).

Isolation of MMTV 70S RNA. Approximately 6 mg ofsucrose gradient-purified MMTV was pelleted

andresuspended in2ml of TNE buffer (0.5 M

Tris-hydrochloride [pH 7.8], 0.1 M NaCL and 0.001 M

EDTA). Protein was digested by addition of proteinase

Kat afinal concentration of 50,g/mlandincubation

for 10 min at room temperature. Sodium dodecyl

sulfatewasadded to afinalconcentration of 1%, and

incubation wascontinued for 15min. The 70S RNA

waspurified by centrifugation in a 15 to 30% sucrose

gradientfor3hat40,000 rpm with a BeckmanSW41

rotor.Fractions were monitored by UV absorption at

260 and 280 nmtolocate the 70S RNA, which was

thenpooled and precipitated by addition of 0.1 volume

of2M sodiumacetate (pH 5.0) and 2.5 volumes of

cold ethanol.

Separation of oligonucleotides produced by

digestion ofMMTV RNA with ribonucleaseTL.

VOL. 32,1979

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854 ARTHUR, LOVINGER, AND SCHOCHETMAN

MMTV 70S RNAwasdigested, and the 5'-phosphate

residueswereremoved by incubating1to5gtgof RNA

with2Uof RNase T1 and0.5U of alkaline

phospha-tasein0.1MTris-hydrochloride, pH 7.6,at370C for

45 min. Thedigestion mixture (20

pl)

wastwice

ex-tracted with 100

pl

ofphenol-chloroform (1:1), and the

aqueousphasewassubsequently extracted twice with

diethyl ether. The 5' ends of theoligonucleotideswere

then labeled with 3P as described by Maxam and

Gilbert (15). The 32P-labeled oligonucleotides were

precipitated with ethanol and separated by two-di-mensional gel electrophoresis by the procedure of

DeWachter and Fiers (10). Thefirst dimension was

runina10%acrylamide gelatpH3.4and 6Murea,

andthe second dimensionwasrunin21.8%acrylamide

atpH 8.0.Radiolabeledoligonucleotideswerelocated

byautoradiography with Kodak X-OMAT X-rayfilm.

RESULTS

MMTV expression in C3Hf cell lines.

Mammary tumors were established in culture,

and cells with epithelial morphology were

se-lected as described in Materials and Methods.

MMTVexpressionwasdeterminedby analyzing

virus pellets fromspent mediumforreverse

tran-scriptaseactivityandMMTVantigen.

Approx-imately 30 ml ofDulbecco-modifiedEagle

me-diumplus

10-'

Mdexamethasonewasaddedto

culturesat80%confluency (approximately5 x

106 celLs) and incubated for 48 hat370C.

Me-diumwasremoved andclarified, andthe virus

waspelleted by centrifugationat100,000xgfor

1h.Theviruspelletswereresuspended in TNE

buffer, and thisvirussuspensionwasmonitored

forreverse transcriptase and MMTV gp52.All

valueswerenormalizedto mlof virus

suspen-sion to allow comparison of antigen and

enzy-maticactivity.Lowlevels of MMTV(13 pmolof

dGTPincorporationpermlperhand129ngof

gp52 per ml) were detected by both assaysin

viruspelletsfromC3Hfcells(Table 1).AllC3Hf

primary cultures which were established had

low MMTV expression> Treatment with

dexa-methasone, progesterone, insulin,

hydrocorti-sone, and combinations of hormones did not

increase virus yields. However, approximately

10%ofcultures establishedbysingle-cell cloning

of C3Hf cells released high levels of MMTV

aftertreatment with dexamethasone at10-5M.

Theresults of thereversetranscriptase and gp52

assays on virus pellets prepared from these

clonesaregiveninTable 1. C3Hf Cl 14yielded

virus in comparable levels to the

Mm5mt/c,

mammarytumor cellline. Inaddition, MMTV

of Cl 14 incorporated [3H]dGTP greater than

nine times more efficiently in the presence of

Mg2+thanMn2+.Reversetranscriptasewith

op-timalactivityinthepresence of

Mg2e

is

indica-tive of MMTV rather than murine leukemia

virus (MuLV). Virus pellets from 12 separate

experimentswere examined for MuLV p30 by

TABLE 1. Reversetranscriptase andMMTVgp52

invirus pelletsfrom C3Hfmammarytumorcells

andcloiewa

Reversetranscriptaseb

MMTV Celldesignation

Meg/a

gp52c(ng/

pmolincor- Mn2+ra- MI)

porated

tioe

C3H/HeNfline1 13 0.6 129

C3H/HeNfCl1 225 5.9 4292

C3H/HeNfC12 113 4.3 383

C3H/HeNfC13 13 1.3 53

C3H/HeNfC14 146 3.6 441

C3H/HeNfCl5

120 2.3 474

C3H/HeNfC16 16 0.9 1,506

C3H/HeNfC17 10 0.5 182

C3H/HeNfC18 151 5.0 378

C3H/HeNfCl9

126 5.5 408

C3H/HeNfCl10 110 1.6 154

C3H/HeNfC111 109 1.2 626

C3H/HeNfC112 15 0.4 219

C3H/HeNfCl13 436 7.0 8,051

C3H/HeNfC114 1,442 9.2 38,550

C3H/HeNf Cl15 118 6.6 440

Mm5mt/c,cells 2,171 9.6 58,623

a

Virus

pelletsprepared by centrifugationofspent

medium from eachcell linetreated with10-5M

dex-amethasone for 48 hwere resuspendedat lOOx and

testedfor reverse transcriptase activityand MMTV

gp52antigen. All values are normalizedto 1ml of virus

suspension.

bResultsareexpressed aspicomolesof[3H]dGTP

incorporated usingthesynthetic template,

oligo(dG).

poly(rC)permilliliterofvirussuspensionper hour.

'MMTV gp52 isexpressed asnanograms of gp52

permilliliterof virussuspensionandwasdetermined

with theC3HMMTVgp52competitionRIA.

dA low

Mge/Mn2'

ratio atbackground

RNA-di-rected DNA polymerase levels is not indicative of

MuLVcontamination.

competition RIAs andimmunodiffusionassays,

and less than 0.1% MuLVwasdetected inanyof

the pellets. MuLV expression in high-passage

cultures has not been ascertained (data not

shown).

Polypeptide composition of exogenous

and

endogenous

MMTVs. The protein

com-positions of

C3H

and C3Hf MMTVwere

ana-lyzed by sodiumdodecyl sulfate-polyacrylamide

gel

electrophoresis. Figure 1shows aCoomassie

brilliant blue-stained

gelofsucrose

gradient-pu-rifiedC3H- and C3Hf-MMTV. gp52, gp36, p27,

p14,

andplO were present in both viruses.

Ex-cept for the higher relative concentrations of

pp2O

and

p16

in the exogenous MMTV, the

proteinprofiles ofthe two viruses are

essentially

identical.MMTV

pp2O

is aphosphoprotein(20),

and

p16

has beenshownto be a cleavageproduct

ofp27(22).

C3H

and C3Hf

MMTV

gp52's

possess

group- and type-specific antigenic

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VOL. 32,1979

minants. With agp52 RIA, werecently found

that tumors from C3H and C3Hf mice were

immunologically distinct (3). To determine

whether the gp52 antigenic polymorphism

ex-tends to MMTV from C3H and C3Hf

cells,

viruses from both cell lines were examined in

competition immunoassays forMMTV gp52. In ahomologousassayemploying limiting dilutions ofanti-C3H MMTVserumand

1251-labeled

gp52

purified

fromC3H MMTV, both C3Hfand

C3H

MMTV competed with parallel displacement

curves(Fig.2A).MMTV derivedfromC3Hcells

gave

complete

competition, whereas

C3Hf-de-rived MMTV competedto amaximumof 90%.

The lack ofcomplete competitionsuggests that gp52ofC3Hf MMTV lacked antigenic

determi-nants found on gp52 ofC3HMMTV. The

incom-plete competition by C3HfMMTVwas not due to insufficient concentration of antigen in the

C3Hf MMTV preparation. This was

demon-stratedin aheterologousgp52 RIAwith antise-rumprepared to C3HfMMTV and

125I-labeled

gp52fromC3H MMTV where bothviruses gave

complete competition with identical

displace-ment curves (Fig. 2B). To amplify the

type-specific differences, theC3HMMTV antiserum wasabsorbed with C3HfMMTV toremove the group-specific antibodies. Thisabsorbed

antise-rum was then used in a gp52 RIA with gp52

from C3HMMTV as the radiolabeled antigen.

As shown in Fig. 2C, MMTV from

C3H cells

C3Hf CELL LINE EXPRESSING MMTV

855

C3Hf C3H Protein

MMTV MMTV Standards

iilEIIF - Bovine Serum Albumir

4h1_ - Ovalbumin

-CarbonicAnhydrase

-Myoglobin

_IiMI6 -CytochromeC

FIG. 1. Sodiumdodecylsulfate-polyacrylamidegel

electrophoresis ofsucrosegradient-purifiedMMTVs

from C3HandC3Hfmammarytumorcell lines. The

gelwasfixedand thenstained with Coomassie

bril-liantblue.

100

90- 80- 70-a 60

Xc 40

20 10

.

c

244.99 819.5 39 78 156 3126251250250050 0 2.44.9 9.819.5 39 78156 312625125025005000 2.449 9.819539 78 156 31262512502500500

ngProtein ng Protein ngProtein

FIG. 2. Immunological relatedness ofthe major envelope glycoprotein ofMMTV. Detergent-disrupted

MMTVsweretestedatserialtwofold dilutionsforthe abilitytocompetewith

125I-labeled

gp52 from C3H

MMTVfor binding limiting amounts

of

antiserum. The results are normalized for 100% binding in the

absence ofcompeting antigen. Competitions were C3HfMMTV (0) and C3H MMTV (0). Competition

immunoassays included:(A) homologousimmunoassayin whichlimitingamountsof anti-C3HMMTVserum

wasusedtoprecipitate 125I-labeledgp52; (B) heterologousimmunoassay in whichanti-C3HfMMTVserum

wasusedtoprecipitate125I-labeledgp52;(C) homologous absorptionimmunoassay in which anti-C3HMMTV

antiserumwhich had beenextensivelyabsorbed with

C3Hf

MMTVwasusedtoprecipitate

251I-labeled gp52.

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856 ARTHUR, LOVINGER, AND SCHOCHETMAN

competed togreater than 85%, whereas no

com-petition was detected with C3Hf-derived

MMTV. This clearly demonstrates that

type-specific antigen determinants on gp52 of C3H

MMTV were notpresent inC3HfMMTV.

Analysisof 70SRNAsof endogenous and

exogenous MMTVs. To determine whether

immunological differences were a result of

dif-ferences in the genetic information of the two

viruses and not the result ofposttranslational

modifications of the gp52, oligonucleotide

fin-gerprint analysis of the genomes of the two

viruses wasperformed.The70S RNA which was

purified from each virus wassubjected to

ribo-nuclease

T,

digestionand theresulting

oligonu-cleotides were labeled with32P and then

sepa-rated by two-dimensional electrophoresis.

Au-toradiograms of thelargeradiolabeled

oligonu-cleotidesaregiveninFig.3. Theautoradiograms

were superimposed to allow identification of

common and unique

oligonucleotides,

and a

[image:5.504.264.453.246.407.2]

composite of the autoradiograms is shown in

Fig. 4.Of100uniqueoligonucleotides examined,

77were commontobothviruses, indicatingthat

thegenomes aresimilar.However,the 70S RNA

ofC3Hf MMTV contained 23

oligonucleotides

which were not present in C3H MMTV, and

C3H MMTV contained 18 oligonucleotides not

present inC3HfMMTV.

Antigenic relationships of C3Hf MMTV

gp52 to gp52 of mouse mammary tumors

andmousemilk.It has been

previously

shown

that C3Hf mammary tumors and C3Hf milk

contain MMTV (19, 26). If the C3Hf MMTV

expressed bytheC3Hfcell line is the same virus asexpressedinC3Hftumorsandmilk,itshould

possess thesamegroup and typeantigenic

spec-ificities observedbetweengp52ofC3HfandC3H MMTV.

Therefore,

tumor extracts and milk

from various strains of mice were analyzed in the homologous C3H MMTV gp52 assay. As

shown in Table 2, C3H MMTV, mammary

tu-mors, and milkgive 100% competition, whereas

C3HfMMTV, mammary tumors, and milk

com-pete only 90%. When the anti-C3H MMTV

se-rum that was extensively absorbed with C3Hf

mammary tumor extract was used in the gp52

RIA, C3H MMTV and mammary tumors still

competed extensively. However, C3HfMMTV

and mammary tumors gave no competition.

These results are consistent with the C3Hf

MMTV fromC3Hf Cl14celllinebeingthe same

virus as thatexpressed in C3Hfmammary

tu-mors andmilk. As would be expected, the low

mammary tumor mouse strains, BALB/c and

8a°0

ooC-)0

o) e2

oe

-co

(0000 o

1~ *

O)

C * *0e

C?

c w

a

i

4

-J0

C4)

0

c0

10%ACRYLAMIDE

pH 3.4,UREA

FIG. 4. Composite of two-dimensional

electropho-reticfingerprint of 70S RNA from C3Hf MMTV and

C3H MMTV. Oligonucleotides were generated by

RNaseT, digestion. Those common to both viruses

(0), those unique to C3H MMTV (O), and those

uniquetoC3Hf MMTV(0)areindicated.

FIG. 3. Two-dimensional electrophoretic fingerprint of digested 70S RNA from C3Hf (A) and C3H (B)

MMTV.Oligonucleotidesweregenerated by RNaseT1digestion of70S RNAs purified from C3H MMTV and C3HfMMTV.

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C3Hf CELL LINE EXPRESSING MMTV 857

NIH Swiss, contained no detectable MMTV

gp52 in their milk. Also, no gp52 was found in

a dimethylbenzanthracene-induced BALB/c

TABLE 2. Competition by samples from mice of

various strains inaC3HMMTVgp52 RIA

Maximum % competi-tionin RIA employing C3H MMTV antiserum

Competing antigen Absorbed

Unab- with C3Hf sorbed tumor

ex-tract

Virus

C3H 100 >73a

C3Hf 90 0

Manmmary tumors

C3H 100 >93a

C3Hf 90 0

RIII 92 0

BALB/c(DMBAb-induced) 0 0

Milk

C3H 100 NT

C3Hf 88 NT

BALB/c 0 NT

SWISS 0 NT

aCompetitionwas not

carried

to completion. The

competition curves were similar to those shown in Fig.

2C and 5C.NT, Not tested.

bDMBA, Dimethylbenzanthracene.

mammary tumor. Surprisingly, mammary

tu-morextracts ofthe high mammary tumor

inci-denceRIIImicecompetedtothesame extent as

C3Hf MMTV.

Antigenic relatednessof gp52 of MMTVs from C3H, C3Hf, GR, and RM mice. The

RIA employing

"2I-labeled

gp52 from C3H

MMTV and its homologous antiserum, which

distinguishes C3H and C3Hf MMTVs, allowed

an analysis of the antigenic relatedness of MMTV from different mice.

Detergent-dis-rupted MMTVs from C3H,C3Hf, GR, and RIII

mammary tumor cell lines and milk were

ex-amined in thehomologous gp52RLA (Fig. 5A). The differences in the extent of competition

weresufficiently pronouncedtodistinguish C3H and GR MMTV from C3Hf and RIII MMTVs. Although the slopes ofthe competition curves were similar, GR and C3H MMTVsgave

com-plete competition, whereas C3Hf and RIII

vi-rusescompetedto amaximum of90to92%. This

demonstrates that all determinants on C3H MMTV gp52 detected

by

the assay are also

present on GR-NMMTVgp52. Incontrast, all

four MMTVs competed completely ina heter-ologous RIA with

"251-labeled

gp52 from C3H

MMTV and limiting dilutions of anti-C3Hf

MMTVantiserum(Fig.5B). This demonstrated that gp52 determinants shared

by

C3Hf and C3H MMTVwerealsocommon toRIII and GR

MMTV.

100r

i

90-0 80 70 a

60 cb

Ee

c

e!

\1

I&t

I, \

XI -d A

2 449 9 8 19 5 39 78 156 312625 1200 2500 5000

ngProtein

100 90

a0

70 > 60

.50

c e 0

30 20 10

ngProtein

a a a a

I * .

a a.

\O @~~~~

c

24499.819.539 78156 312 6251200 25005000 ngProtein

FIG. 5. Immunological relatedness of the major MMTV envelope glycoproteins. Detergent-disrupted

MMTVsweretestedat serialtwofolddilutionsfortheabilitytocompetewith 215I-labeledgp52 fromC3H

MMTVfor binding limiting amounts ofantiserum. The results are normalizedfor 100% binding in the

absenceof competing antigen. CompetitorswereC3HfMMTV (0),C3H MMTV(0),GR MMTV(O),andRIII

MMTV(-). Competitionimmunoassaysincluded: (A) homologousimmunoassayinwhichlimitingamounts

ofanti-C3H MMTVserum wereusedtoprecipitate 1251I-labeled gp52; (B) heterologous immunoassayin which

anti-C3HfMMTVserum wasusedtoprecipitateIMI-labeledgp52; (C) homologous absorptionimmunoassay

in which anti-C3H MMTVserumwhichwasextensivelyabsorbedwithC3HfMMTVwasusedtoprecipitate

125I-labeledgp52.

In 50 Co eL 40

30 20 10

VOL. 32,1979

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[image:6.504.53.450.131.580.2] [image:6.504.59.447.384.580.2]
(7)

858 ARTHUR, LOVINGER, AND SCHOCHETMAN Theincomplete competition by RIIIMMTV

inthe

homologous

assay

(Fig. 5A)

indicated that

the gp52's of C3Hf and RIII MMTVs lacked antigenic determinantspresentonthe

gp52's

of C3H and GR MMTVs. If the

missing

determi-nants were identical, then absorption of C3H

MMTV antiserum with C3Hf MMTV would

alsoremove all the antibodiesto RIII MMTV

gp52. As shown in Fig. 5C, RIII and C3Hf

MMTVsgave no

competition

inthe RIA with

"2'I-labeled

gp52 from C3H MMTV and C3H

MMTVantiserum absorbed with C3Hf MMTV.

However, C3H MMTV and GR MMTV

com-peted greater than 80%. Similar results were

obtained with C3H MMTV antiserum

exten-sively absorbed with RIII MMTV (data not

shown). Absorption of the antiserum with GR andC3H MMTV removed all

precipitating

an-tibodies for

'25I-labeled

gp52. This demonstrates

that

gp52's

from C3H and GR MMTVs contain

unique

antigenic

determinants

lacking

on

gp52's

fromboth C3Hf and RIII MMTVs.

DISCUSSION

Theresultspresented here describe the estab-lishment of the first C3Hfmammarytumorcell line producing

high

levels of

endogenous

MMTV.

Comparison

of thisMMTVwithC3H MMTVrevealed that thetwoviruseswere

sim-ilar

morphologically

(datanotshown) and both

required

Mg2e

for

optimum

reverse

transcriptase

activity. The

polypeptide

profile,

asdetermined by sodium

dodecyl

sulfate-polyacrylamide gel

electrophoresis,

was

essentially

identical forthe

twoviruses.

However,

thetwo viruses could be

distinguished

onthe basis of the

antigenic

prop-erties of their

major

glycoprotein,

gp52. Al-though

group-specific antigenic

determinants comprised themajor

reactivity

of the gp52 mol-ecules, the results of the gp52 RIAs

clearly

dem-onstrated that C3H MMTVgp52 contained

an-tigenic determinants notfound on the gp52 of C3Hf MMTV. Theseantigenic type-specific dif-ferences are not

unexpected

inview of the ob-served differencesinthe

oligonucleotide

finger-printsof thetwoviruses

reflecting

differences in

theirgeneticcontent.

Immunological type specificity of MMTV

from mammary tumors and milk has been

re-ported by Blair (6, 7) and Teramoto et al. (25)

by usingimmunodiffusionand intact virus

RIAs,

respectively. We have demonstratedtype

speci-ficityof gp52 for C3H and C3HfMMTVs and

furtherthat thepattern ofreactivity extendsto

C3HfmammarytumorsandC3Hfmilk (3; Table

2). This indicated that tissue culture-derived

C3Hf MMTV was the same MMTV as that

expressedinthemilk andmammary tumors of C3Hf mice.

Previous studies of gp52 performed in this laboratoryrevealed that C3H and GR MMTVs

were more

closely

relatedtoeach other thanto

RIII MMTV (23). This was based on results derived from (i) type-specific RIAs for C3H

MMTV gp52, (ii)

tryptic peptide

maps of cell

surface-labeledgp52of MMTVsgrownina com-mon cell, and (iii) type-specific cytotoxic anti-bodies inseraof C3Hmammarytumor-bearing mice which were directed

against

cell surface

MMTVgp52. Interestingly, results of the gp52

RIApresented here revealed that C3Hf MMTV

was moreclosely relatedtoRIIIMMTVthan to

C3H and GR MMTVs. Since removal of the

group-specific antibodiestogp52by absorption with either C3HforRIII MMTV removed activ-ity against bothMMTVs, butnotagainst gp52's of C3H and GR MMTVs, it can be concluded that the same type-specific antigenic

determi-nants were missing on thegp52's of C3Hfand

RIII MMTVs. This pattern of relatedness, which groups C3H and GR MMTVs together and distinguishes them from C3Hf and RIII MMTVs, wasalso observed with MMTV

neu-tralizing antibodies fromseraof C3Hmammary

tumor-bearing mice (Massey et al., submitted

forpublication). It is therefore possiblethat the

type-specificgp52determinantsmeasured in the

RIA are the same determinants as those

de-tected by the

neutralizing

antibodies of natural

sera.

When the degree of oncogenicity of the

MMTVs is compared in C3Hf and BALB/c

strains ofmice, both C3H and GR MMTVsare

highly oncogenic, whereas C3Hf and RIII

MMTVare

considerably

less oncogenic (5, 17,

24,26). Thisagreeswith theirgrouping basedon

type-specific

antigenic determinants of gp52.

Studies of murine leukemia virus indicate that

thegeneration of highlyoncogenic viruses

cor-relates with a change in the major envelope

glycoprotein,

gp7O.

It has been reported

that

thesechangesareduetogenetic recombination between

ecotropic

MuLV and endogenous

MuLVsequences coding for

gp7O

determinants

whichmay confer thymotropic properties to the

recombinant virus (9, 11). Further studies

will

berequired to determine whether the acquisi-tionoftype-specific differencesongp52's of C3H

andGRMMTVs alsoarosebyrecombinational

eventsand whether thesedeterminantsare

in-volvedinenhancing infectivity andsubsequently

oncogenicity of the MMTVs for mammary

glands of various strains ofmice.

ACKNOWLEDGMENTS

Wegratefully acknowledge the assistanceof Charles Ben-tonforlarge-scale production and purification of individual MMTVs.WealsoexpressourappreciationtoRichardBauer

andSharon Bladenfor excellent technical assistance.

J. VIROL.

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(8)

C3Hf CELL LINE EXPRESSING MMTV 859

This work wassupported by Public Health Service contract N01-CO-75380 with the National Cancer Institute.

LITERATURE CIED

1. Andervont,H.B.,M. B.Shimkin,and W. R.Bryan.

1942.Techniquesuitable forquantitativestudiesonthe mammarytumorinciter of mice. J. Natl. Cancer Inst. 3:309-318.

2. Arthur, IOO.,R. F.Bauer,L S.Orme,and D. L Fine. 1978.Coexistence of the mouse mammarytumorvirus (MMTV)majorglycoproteinandnatural antibodiesto

MMTV in sera ofmammary tumor-bearingmice. Vi-rology 87:266-275.

3. Arthur, L 0., and D. L Fine. 1979. Immunological

characterization of mouse mammarytumorvirusp1O

and its presence in mammarytumorsandseraof

tumor-bearing mice. J.Virol.30:148-156.

4. Arthur, L 0.,D.L Fine, and P. Bentvelzen. 1978. Oncogenicity ofmurine mammary tumor virus

pro-ducedin tissue culture. J. Natl. Cancer Inst. 60:461-464.

5.Bentvelzen,P., J.Brinkhof,andJ. J. Haaijman. 1978.

Genetic controlofendogenousmurinemammary tumor virusesreinvestigated.Eur. J. Cancer 14:1137-1146. 6. Blair,P. B. 1970.Immunologyofthe mouse mammary

tumorvirus: comparison of the antigenicity of mam-marytumor virus obtained from several strains of mice. Cancer Res.30:625-631.

7. Blair, P. B. 1971. Strain specificity in mouse mammary tumorvirus virionantigens.CancerRes. 31:1473-1477. 8.Cohen,J.C.,P. R.Shank,V.LMorris,R.Cardiff,

and H. E.Varmus.1979.Integration of the DNA of mousemammarytumorviusinvirus-infectednormal andneoplastictissue of themouse.Cell 16:333-345. 9.Devare, S.G., U. R. Rapp, G. J. Todaro, and J.R.

Stephenson.1978.Acquisition of oncogenicity by

en-dogenousmousetypeC viruses: effects ofvariationin envandgaggenes. J.Virol.28:457-465.

10. DeWachter,R., and W.Piers. 1972.Preparative

two-dimensional polyacrylamide gelelectrophoresis of3P2 labeledRNA. Anal. Biochem.49:184-197.

11. Elder, J. H., J. W. Gautsch, F. C. Jensen, R. A. Lerner, J. W.Hartley,and W. P. Rowe. 1977. Bio-chemical evidence that MCFmurineleukemiaviruses

areenvelope (env) gene recombinants. Proc. Natl. Acad. Sci. U.S.A. 74:4676-4680.

12. Fine, D. L, L. 0. Arthur, J. K. Plowman, E. A.

Hillman, andF.Klein. 1974. In vitro system for pro-duction of mousemammarytumorvirus.Appl.

Micro-biol. 28:1040-1046.

13. Heston,W.E.,M. K.Deringer,T. B.Dunn, and W. D.

Levillain.1949.Factors in thedevelopmentof sponta-neous mammary gland tumors in agent-free strain C3Hb mice. J. Natl. Cancer Inst. 10:1139-1151. 14. Lowry,0. H., N. J.Rosebrough,A. LFarr,and R. J.

Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275.

15.Maxam,A.M.,and W.Gilbert.1977.Anewmethod for

sequencingDNA. Proc.Natl. Acad. Sci. U.S.A. 74:560-564.

16.Michalides,R.,G.Vlahakis,and J.Schlom.1976.A biochemicalapproach to thestudy of the transmission of mousemammary tumor viruses in mouse strains RIII and C3H. Int. J. Cancer 18:105-115.

17. Moore,D. H.,J. A. Holben,and J. Charney. 1976. Biological characterization of some mouse mammary tumorviruses. J. Natl. Cancer Inst. 57:889-896. 18. Owens,R.B.,and A. J.Hackett.1972.Tissue culture

studies of mouse mammary tumor cellsand associated viruses.J.Natl. Cancer Inst.49:1321-1332.

19. Pitelka,D.R.,K. B.DeOme,and H. A.Bern. 1960.

Virus-likeparticles in precanceroushyperplastic

mam-mary tissuesof C3H and C3Hf mice. J. Natl. Cancer Inst. 25:753-777.

20.Sarkar,N.H.,E. S.Whittington,J.Racevskis,and

S.L Marcus. 1978. Phosphoproteinsofthemurine

mammarytumorvirus.Virology91:407422. 21. Schlom, J.,D.Colcher,W.Drohan,R.Ketterman,R.

Michalides, G.Vlahakis,and J.Young. 1977. Dif-ferencesin mouse mammary tumor viruses. Cancer 39: 2727-2733.

22.Schochetman, G.,LArthur,D.Fine,and R.Masmy.

1978.Mousemammarycell surface antigens (CSA). I. Mouse mammary tumor virus (MMTV) and murine

leukemia virus(MuLV)surface-associatedantigens,p. 115-141. In R. Ruddon (ed.), Biological markers of

neoplasia: basic andapplied aspects.Elsevier

North-Holland, Inc.,New York.

23. Schochetman, G.,L.0.Arthur, C.W.Long,and R. J.

Masey. 1979. Micewithspontaneous mammary tu-morsdeveloptype-specific neutralizing andcytotoxic antibodies againstthe mousemammarytumorvirus

envelope proteingp52. J.Virol. 32:131-139.

24. Squartini,F., andM. Bistocchi. 1977. Bioactivity of

C3H andRHI mammarytumorviruses invirginfemale Balb/c mice: brief communication. J. Natl. Cancer Inst.

58:1845-1847.

25. Teramoto,Y.A.,D.Kufe,and J.Schlom.1977.

Type-specificantigenic determinants on themajorexternal

glycoproteinofhigh-andlow-oncogenic murine mam-marytumorviruses. J.Virol.24:525-533.

26. vanNie,R.,and A. A. Verstraeten.1975. Studies of

genetic ransmissionofmammarytumorvirusbyC3Hf mice. Int. J. Cancer 16:922-931.

VOL. 32,1979

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Figure

FIG. 2.MMTVMMTVsabsencewasimmunoassayswasantiserum Immunological relatedness of the major envelope glycoprotein of MMTV
Fig. 4.the Of 100 unique oligonucleotides examined,77 were common to both viruses, indicating that genomes are similar
TABLE 2. Competition by samples from mice ofvarious strains in a C3HMMTVgp52 RIA

References

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