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. Theoligonucleo-tidefingerprint indicated that
although
thevirusesweresimilar, they differedintheirgeneticcontent.Thedifferencesinthetwovirusesextendedto
immunolog-ical differencesinthemajorenvelope glycoprotein,gp52.C3HfMMTVcompeted
only partially in a homologous
radioimmunoassay
for gp52 of C3H MMTV, whereas C3H MMTV gavecomplete
competition,indicating
that gp52 ofC3HMMTV contained
type-specific
determinants not present on gp52 of C3HfMMTV.ComparisonofC3Hf MMTV withhighly oncogenic
C3H,
GR,andRIIIMMTVs ina
homologous
C3HMMTVgp52 assay gavetwopatterns
ofreactivity:complete competition by GR and C3H MMTV andincomplete competition by
C3HfandRIII MMTV.
Absorption
ofanti-C3H MMTVserum by either C3HfMMTV 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,
suchasC3H/
HeN, contain milk-transmitted (exogenous)
mousemammarytumorvirus
(MMTV)
andge-netically transmitted
(endogenous)
MMTV.Foster nursing the C3H
suckling
on mice ofstrains 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).
Theme-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 theradiolabeled 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)
wastwiceex-tracted with 100
pl
ofphenol-chloroform (1:1), and theaqueousphasewassubsequently 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-'
Mdexamethasonewasaddedtoculturesat80%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
isindica-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 474C3H/HeNfC16 16 0.9 1,506
C3H/HeNfC17 10 0.5 182
C3H/HeNfC18 151 5.0 378
C3H/HeNfCl9
126 5.5 408C3H/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 centrifugationofspentmedium 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 atbackgroundRNA-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 proteincom-positions of
C3H
and C3Hf MMTVwereana-lyzed by sodiumdodecyl sulfate-polyacrylamide
gel
electrophoresis. Figure 1shows aCoomassiebrilliant blue-stained
gelofsucrosegradient-pu-rifiedC3H- and C3Hf-MMTV. gp52, gp36, p27,
p14,
andplO were present in both viruses.Ex-cept for the higher relative concentrations of
pp2O
andp16
in the exogenous MMTV, theproteinprofiles ofthe two viruses are
essentially
identical.MMTV
pp2O
is aphosphoprotein(20),and
p16
has beenshownto be a cleavageproductofp27(22).
C3H
and C3HfMMTV
gp52's
possessgroup- and type-specific antigenic
deter-J. VIROL.
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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
gp52purified
fromC3H MMTV, both C3HfandC3H
MMTV competed with parallel displacement
curves(Fig.2A).MMTV derivedfromC3Hcells
gave
complete
competition, whereasC3Hf-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 C3HMMTVfor binding limiting amounts
of
antiserum. The results are normalized for 100% binding in theabsence 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
MMTVwasusedtoprecipitate251I-labeled gp52.
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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 theresultingoligonu-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
shownthat 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 milkfrom 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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[image:5.504.63.453.476.627.2]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. Thecompetition 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 C3HMMTV 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 alsopresent on GR-NMMTVgp52. Incontrast, all
four MMTVs competed completely ina heter-ologous RIA with
"251-labeled
gp52 from C3HMMTV and limiting dilutions of anti-C3Hf
MMTVantiserum(Fig.5B). This demonstrated that gp52 determinants shared
by
C3Hf and C3H MMTVwerealsocommon toRIII and GRMMTV.
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 030 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
ip
on November 10, 2019 by guest
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[image:6.504.53.450.131.580.2] [image:6.504.59.447.384.580.2]858 ARTHUR, LOVINGER, AND SCHOCHETMAN Theincomplete competition by RIIIMMTV
inthe
homologous
assay(Fig. 5A)
indicated thatthe gp52's of C3Hf and RIII MMTVs lacked antigenic determinantspresentonthe
gp52's
of C3H and GR MMTVs. If themissing
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 C3HMMTVantiserum 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 demonstratesthat
gp52's
from C3H and GR MMTVs containunique
antigenic
determinantslacking
ongp52's
fromboth C3Hf and RIII MMTVs.
DISCUSSION
Theresultspresented here describe the estab-lishment of the first C3Hfmammarytumorcell line producing
high
levels ofendogenous
MMTV.
Comparison
of thisMMTVwithC3H MMTVrevealed that thetwovirusesweresim-ilar
morphologically
(datanotshown) and bothrequired
Mg2e
foroptimum
reversetranscriptase
activity. The
polypeptide
profile,
asdetermined by sodiumdodecyl
sulfate-polyacrylamide gel
electrophoresis,
wasessentially
identical forthetwoviruses.
However,
thetwo viruses could bedistinguished
onthe basis of theantigenic
prop-erties of their
major
glycoprotein,
gp52. Al-thoughgroup-specific antigenic
determinants comprised themajorreactivity
of the gp52 mol-ecules, the results of the gp52 RIAsclearly
dem-onstrated that C3H MMTVgp52 containedan-tigenic determinants notfound on the gp52 of C3Hf MMTV. Theseantigenic type-specific dif-ferences are not
unexpected
inview of the ob-served differencesintheoligonucleotide
finger-printsof thetwoviruses
reflecting
differences intheirgeneticcontent.
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 thantoRIII MMTV (23). This was based on results derived from (i) type-specific RIAs for C3H
MMTV gp52, (ii)
tryptic peptide
maps of cellsurface-labeledgp52of MMTVsgrownina com-mon cell, and (iii) type-specific cytotoxic anti-bodies inseraof C3Hmammarytumor-bearing mice which were directed
against
cell surfaceMMTVgp52. 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 naturalsera.
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 reportedthat
thesechangesareduetogenetic recombination between
ecotropic
MuLV and endogenousMuLVsequences coding for
gp7O
determinantswhichmay 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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C3Hf CELL LINE EXPRESSING MMTV 859
This work wassupported by Public Health Service contract N01-CO-75380 with the National Cancer Institute.
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VOL. 32,1979