Vol.41,No. 3 JOURNALOFVIROLOGY,Mar.1982,p.1007-1013
0022-538X/82/031007-07$02.00/0
Methylation and Amplification of Mouse
Mammary
Tumor
Virus DNA in Normal, Premalignant, and Malignant Cells of
GR/A
Mice
THOMAS G.FANNING, ARTEMIOS B. VASSOS,t ANDROBERT D.CARDIFF* Department ofPathology, School of Medicine, University of California, Davis, California 95616
Received 8 June1981/Accepted4November1981
The methylation andamplification ofmousemammarytumorvirus (MuMTV) proviral DNAwasinvestigated in normal, premalignant, and malignant tissues of GR/A mice. The proviral methylationpatternwasexamined withthe restriction enzyme HhaI, which fails to cleave methylated DNA. MuMTV proviral DNA from liver, kidney, and heartwashighlymethylated. ProviralDNAwassomewhat undermethylated in mammary gland cells from virgin and lactating mice and extensivelyundermethylated incells frompremalignant outgrowths,
pregnancy-dependent tumors, and pregnancy-independent tumors. The restriction enzyme Sacl was used todetect additional proviruses in the same cells. No additional proviral copies of MuMTV were detected in liver, kidney, or heart cells or in mammaryglandcellsfromvirgin mice. Somemammarygland cells fromlactating mice appearedtocontain additional copies of theendogenous, highly oncogenic GR-MTV-2provirus. Premalignant outgrowth, pregnancy-dependenttumor, and pregnancy-independenttumorcells containedanaverageoftwo tothree addition-alcopiesper cellof the GR-MTV-2 provirus. Thus, neoplasia in GR/A micewas directly associated with quantized increases in MuMTV proviral DNA under-methylation and GR-MTV-2 proviral DNA amplification. Restriction enzyme
analysis suggested that premalignant outgrowths and pregnancy-dependent tu-mors both consisted largely of heterogenous cell populations, although some evidence of clonal dominancewasdetected.
Mouse mammary tumor virus
(MuMTV)-in-duced mammary tumors in mice are
character-ized by increased levels of MuMTV proviral
DNA, RNA, and proteins (2, 21). Recently, it
has been shown that MuMTV(C3H)-induced
mammary tumors inBALB/c mice contain
un-dermethylated copies of MuMTV(C3H) proviral DNA (7). This finding is significant in view of
recently proposed mechanisms concerning the
regulation ofeucaryoticgenes.Inparticular, the suggestion that activegenes are
undermethylat-ed (4, 14, 24, 26), as has been found with the
globin(28, 31) and ovalbumin (16, 18)genesand
certain eucaryotic viruses (13), mayalso apply
toactive MuMTVproviruses (7).
Premalignancy in mice is thought to be a
precursorof the malignantstate(6, 10).
Prema-lignant tissues in BALB/c mice appear to be
composed of the descendents ofone or afew
cells.Inthisrespecttheymoreclosely resemble
mammary tumors than normal tissue (5). Ithas
beenproposed thatmammary tumorsarise from
transformed cells within the premalignant cell
population (10). Alternatively, certain tumors
tPresent address: UniversityofIllinoisCollege of Medi-cine, Urbana,IL 61801.
mayarisedenovo. Forexample, the
pregnancy-dependenttumorsfound insome mousestrains,
including GR/A, arise duringpregnancy or
hor-monal stimulation of the animal and regress
uponparturitionorremoval of the hormone (2,
30).
MuMTV-inducedmammarytumors
(pregnan-cy-independent tumors) contain more proviral copies of MuMTV than are found in normal, nonmalignant tissues (8, 9,,11, 15, 20, 22). We
have analyzed various normal, premalignant,
and malignant tissues ofGR/A mice to
deter-mine whether a correlation exists between
MuMTV undermethylation and amplification.
Ourfindingsdemonstrate thatsuchacorrelation
exists. This,together with the data of others (7),
suggeststhatin certainmammarycellsMuMTV
DNA is amplified and that the amplified DNA
escapes the host methylatingprocess. Alterna-tively,undermethylation ofMuMTV DNAmay
allow amplification of endogenous proviral DNA.
MATEIUALS AND METHODS
GR/A mice were obtained from the Cancer Research Laboratory, Berkeley,Calif.Pregnancy-dependent
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1008 FANNING, VASSOS, AND CARDIFF
RESULTS
MethylationofMuMTV proviralDNA in
nor-mal,premalignant,andmalignantceils.Weused therestriction enzyme HhaItodistinguish
meth-ylated
fromunmethylatedMuMTV DNA. HhaIcleavesatthesequenceG-C-G-C,butnot atthe
sequence G-meC-G-C (3). We digested the
DNAs from GR/A mouse liver, kidney, and
heart and from the mammary glands of virgin
and lactating mice with HhaI and visualized
the MuMTV proviral restriction fragments by
theblot-transfer method of Southern (27). Most
of the proviral DNA detected in the liver,
kidney, and heart samples remained high
molecular weight (Fig. 1, lanes 2-4). This
indi-cates that the proviral DNA in these samples
washighly methylated. Mammary gland DNAs
1 2 3 4 5 6 7 I from virgin and lactating mice exhibited a
promi-1. HhaIrestrictionpattersof MuMTV pro- nent
MuMTV-specific fragment having
amolec-INA in nonmalignant GR/A and GR-Mtv-2- ular
weight
ofapproximately
6 x106
(Fig.
1,tissues. Approximately10Lgof each DNA was lanes 5 and 6). In addition, mammary gland
d with HhaI and analyzed by the Southern DNA fromlactatingmice exhibitedanumberof
procedure and autoradiography. Lane 1, preg- faint lower-molecular-weight fragments, many
independent tumor DNA; lane 2, liver DNA; of which appeared identical to those found in
kidney DNA; lane 4, heart DNA; lane 5, GR/A tumor DNA (Fig. 1, compare lanes 1 and mammary gland DNA from a virgin mouse; lane 6,
mammarygland DNA from a lactating mouse; lane 7, mammary gland DNA from a virgin GR-Mtv-2-mouse; and lane 8, mammary gland DNA from a lactating GR-Mtv-2- mouse. The 6 x 106-dalton MuMTV proviral fragment discussed in the text is indicated by the arrow.
mors(plaques)(30) wereobtained from 18- to 20-day-old pregnantGR/Amice. These lesions can bereadily visualizedin a late pregnant animal due totheir high vascularity and general lack of alveolarity and secre-tion. Hyperplastic alveolar nodules (1, 10) were ob-tainedfrom tumor-bearing, nonlactating, nonpregnant GR/Afemales. They were serially transplanted into gland-freefatpads (1) to obtain the tissues referredto in the text as premalignant outgrowths. GR-Mtv-2-mice were kindly provided by J.Hilgers,The Nether-landsCancer Institute, Amsterdam.
Tissue DNAs (11) were digested with restriction enzymes(NewEngland BioLabs, Beverly, Mass.)and electrophoresed into agarose gels,and the restriction fragmentsweretransferred to nitrocellulose filters(11 12, 27). Phagelambda DNAwasincluded in alldigests tomonitor theextentofcleavage. The nitrocellulose-bound MuMTVrestrictionfragments werehybridized with [32P]cDNAmadeusing calf thymusprimers and 18 to28S MuMTV RNA(cDNA,p)or, inoneinstance (seeFig.6), with nick-translated(25) cloned MuMTV
DNA (an equimolar mixture of MuMTV PstI 0.9 x
106-, 1.1 x 106-,and 2.5 x 106-daltonfragments [17])
kindly provided by J. Majors and H. Varmus, Univer-sityofCalifornia,San Francisco. After hybridization, thefilterswere washed andautoradiographed accord-ing to published procedures (8).
cDNAeP
and radiola-beled cloned MuMTV DNA gave nearly identicalpatterns (see Fig. 6) indicating the specificity of the
cDNA,,pprepared inthis laboratory.
,...I
0
-S.i....
...
*::
-...
.t.o1 2 3 4 5 6 7
FIG. 2. HhaIrestriction patternsofMuMTV
pro-viral DNA inpremalignant and malignant GR/Amouse
tissues. Analyseswereperformedasdescribedin the
legend to Fig. 1. Lane 1,pregnancy-independent
tu-mor DNA; lane 2, liver DNA; lane 3, premalignant
outgrowth DNA (sample 1); lane4,premalignant out-growth DNA (sample 2); lane 5, pregnancy-dependent
tumorDNA(sample1);lane6, pregnancy-dependent tumorDNA (sample 2); and lane7, HhaI restriction
pattern of GR-MTV-2-infected rat cell DNA. The
arrow indicates the6 x 106-dalton MuMTV proviral
fragment discussed in thetext.
FIG. viral D mouset
digeste4 blotting nancy-i lane 3,
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[image:2.491.48.239.71.263.2] [image:2.491.254.443.343.565.2]MuMTV DNA METHYLATION AND AMPLIFICATION 1009
genetically inherited MuMTV proviruses (15,
22). Do the MuMTVrestriction fragments
gener-ated
by
HhaIcleavage originate
from one orfrom more than one of these proviruses? The
congenic
line GR-Mtv-2- ismissing
MuMTV proviral DNA (19) and does not develop earlymammary tumors (29).
Specifically,
the linelacks
only
oneof theendogenous
proviruses
(11,15), which we have designated the GR-MTV-2
provirus (11).HhaI digestion of mammary gland
DNAs from virgin and lactating
GR-Mtv-2-* mice demonstrated the presence of several
MuMTVrestrictionfragments, includingthe 6x
106-dalton
fragment(Fig. 1, lanes 7 and 8). Thus,^uj4ese'.w_* theHhaI-generatedrestriction fragments seen in
Fig. 1 and 2 cannot be attributed solely to the GR-MTV-2 provirus. Many of the fragments, however, are probably derived from GR-MTV-2
sinceHhaI digestion ofGR-MTV-2-infected rat
cell DNA (12) shows apatternsimilar to thatof
GR/A tumor DNA(Fig. 2, lane 7).
Amplification of MuMTV proviral DNA in
12 3 4 5 6 normal, premalignant, and maligant cells. We
have previously shown that MuMTV proviral
riu. 3. Saci restrictionpatterns of MuMTV
provi-ral DNA innonmalignant GR/A mousetissues. Ap-proximately 10 Fjg ofeach DNA was digested with SaclandanalyzedbytheSouthernblotting procedure andautoradiography. Lane1;pregnancy-independent tumorDNA; lane 2, liver DNA; lane 3,kidneyDNA; lane 4, heart DNA; lane 5, mammary gland DNAfrom
avirginmouse;and lane 6, mammary gland DNA from alactatingmouse.The 4.0x106-and 1.6x106-dalton fragments derivedfrom the GR-MTV-2 provirus are
indicated by thearrows. 4-04O__4
6). Most of the lower-molecular-weight
frag-mentsaroseby cleavageatsites withina
provi-ral genome(T. G. Fanning,R.D.Cardiff,andJ.
P. Puma, manuscript in preparation). Thus, of
the fivenormal tissuesanalyzed, onlymammary
gland cells contained proviral DNA that was
undermethylatedatseveral sites withina provi-ral genome.
Wenext examined MuMTVproviral
methyl-ation in premalignant outgrowth,
pregnancy-dependent tumor, and pregnancy-independent
tumorcells. We observed extensive
undermeth-ylationof MuMTVproviralDNAin thesecells
(Fig. 2). With several minor exceptions, the
restrictionpatternsfor eachcell typewere
iden-tical. Eachcell type exhibited a predominant 6x
106-daltonfragment and numerous
lower-molec-ular-weight fragments. Thus, we conclude that
bothpremalignancyandmalignancyin theGR/A
mouseareassociated with extensive
undermeth-ylation of MuMTV proviral DNA. MuMTV
pro-viral DNA has been previously shown to be
undermethylated in pregnancy-independent
tu-morcells of the GR/A mouse(7).
GR/A mice harbor five to six endogenous,
1 6-* -4
1
2
3
4
5
6
FIG. 4. Saclrestriction patterns of MuMTV provi-ral DNA inpremalignant and malignant GR/A mouse tissues. Analyses were performed as described in the legendtoFig. 3. Lane 1, pregnancy-independent tu-morDNA; lane 2, liver DNA; lane 3, premalignant outgrowth DNA (sample 1); lane 4, premaliant out-growth DNA (sample 2); lane 5, pregnancy-dependent tumorDNA(sample 1); and lane 6, pregnancy-depen-denttumorDNA(sample 2). Arrows indicate the 4.0x
106- and 1.6 x 106-daltonGR-MTV-2 proviral
frag-ments. 40- i
VOL.41,1982
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[image:3.491.47.239.66.314.2] [image:3.491.252.444.340.574.2]1010 FANNING, VASSOS, AND CARDIFF
A
16 4'0
4.
B
FIG. 5. Densitometer tracings of Sacl patterns shown in Fig. 3 and 4. (A) liver DNA, (B) lactating mammary gland DNA, (C) pregnancy-dependent tu-mor DNA, and (D) pregnancy-independent tumor DNA.Arrows indicate the 1.6 x 106-and 4.0 x 106-dalton GR-MTV-2 proviral fragments.
amplification in GR/A mammary tumors
in-volves increased numbers of the GR-MTV-2
provirus (11). These additional proviruses are
readily detected by comparing tumorand liver
DNAs digested with certain restriction
en-zymes. Forexample, Sacl cleaves the amplified
GR-MTV-2 provirus three times, giving riseto
two internal fragments. Since these two
frag-ments are releasedfromevery GR-MTV-2
pro-virus, a simplecomparisonof SacI-cleaved liver
DNA (which hasonly the endogenous
comple-mentofproviralDNA [22])with asecond
sam-ple ofSacI-cleaved DNA immediately allowsa
determinationof the number of GR-MTV-2
pro-viruses in thesecond sample.
SacIcleaves the GR-MTV-2 provirus intotwo
proviral fragments of 4.0 x 106 and 1.6 x 106
daltons (11, 12). Using the criteria outlined
above,wedetected noamplificationofthe
GR-MTV-2provirus inliver, kidney,orheartcells,
orinmammaryglandcells fromvirginmice
(Fig.
3). Inpremalignantoutgrowths andin pregnan-cy-dependent tumors we were able to detect amplificationsimilarto thatfound in
pregnancy-independent tumors (Fig. 4). Integrated
densi-tometer tracings, as reported previously (11),
confirmed that the premalignant outgrowths,
dependent tumors, and
pregnancy-independenttumors eachcontained an average
oftwotothree additionalcopies of GR-MTV-2
proviral DNA per cell (Fig. 5). Although not
apparent fromFig. 3, densitometer
tracings
sug-gested that mammary glandcells fromlactating
mice also exhibited a slight increase in
GR-MTV-2 copy number (Fig. 5B).
Heterogeneity of premalignantoutgrowths and
pregnancy-dependent tumors. MuMTV-related tumorigenesis in the mouse is usually accompa-nied by a pronounced homogeneity of the
pre-malignant or pre-malignant tissue. The MuMTV
restriction patterns observed in BALB/cfC3H premalignant outgrowths, BALB/cfC3H tumors,
GR/A tumors, andC3Hf tumors have been
inter-preted as indicating that these tissues are more homogenous (clonal dominant) than MuMTV-infected normal mammary tissue (5, 8, 9, 11, 15).
The rationale behind using restriction
en-zymes to ascertain homogeneity has been
dis-cussed in detailelsewhere (8). Briefly, enzymes
(notinhibited by DNA methylation) are chosen
which cleave the MuMTV proviral DNA once or twice near the middle of the genome. Since
MuMTV integrates at many sites in the mouse
genome (8) the appearance of restriction
frag-ments, in addition to those found in normal
tissue DNAs, indicates that the sample is more homogenous than the randomly infected cell population.
Wecleaved the DNAs from two premalignant
outgrowths and two pregnancy-dependent
tu-A
B
. .
*.
.tZ:di.it
..h
4
1 2 344 2 3 4
FIG. 6. XbaIrestrictionpatternsofMuMTV provi-ral DNA in GR/A pregnancy-dependent tumors and premalignant outgrowths. Approximately 10 ,ug of each DNA was digested with XbaI andanalyzedby theSouthernblotting procedure andautoradiography. (A) Lane 1,pregnancy-dependenttumorDNA(sample 1); lane 2,pregnancy-dependenttumorDNA(sample 2); lane 3, premalignant outgrowthDNA (sample 1); and lane 4,premalignant outgrowth DNA (sample 2). Theprobe used todetect the MuMTVproviral
frag-ments wascDNAr,p. (B) Lanes1-4, thesametissue DNAs usedin part(A). Theprobe usedtodetect the MuMTV proviral fragments was radiolabeled cloned MuMTVproviral DNA. The arrowhead between slots 3and4identifies themostprominent additional provi-ralDNAfragment detected in these samples with the clonedMuMTVprobe.
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[image:4.491.55.243.77.269.2] [image:4.491.260.446.357.518.2]MuMTV DNA METHYLATION AND AMPLIFICATION 1011
mors with the enzymes XbaI, KpnI, and
BamHI. The results of the XbaI digests are shown in Fig. 6. The restriction patterns of each
DNA, usingcDNArCpas a probe, were identical
(Fig. 6) and were identical to those obtained
after XbaI cleavage ofGR/A liver DNA (not
shown). A similar result wasobtained with the
KpnI and BamHI digests. Further, we have
analyzed three additional premalignant
out-growths and sevenpregnancy-dependent tumors
with a variety of enzymes. The patterns were
identical to GR/A liver DNA when cDNArep was used to detect MuMTV proviral DNA frag-ments.
However, we observed faint additional
MuMTV fragments in these samples when
cloned 32P-labeled MuMTV DNA was used as
probe (Fig. 6B). We estimated, on the basis of autoradiographic intensity, that the cells con-taining these detectable MuMTV proviruses
made up no more than 15 to 20o of the tissue
mass. The use of cloned 32P-labeled MuMTV
DNA gave a similar result with the KpnI and
BamHIdigests. Thus, although the premalignant outgrowths and pregnancy-dependent tumors containedanaverageof two to three additional
GR-MTV-2 proviruses per cell (Fig. 4), they
probably consist of many different cell popula-tions, each containing MuMTV proviruses
inte-gratedinto unique sites in thecell genome.
DISCUSSION
We have demonstrated that the DNAs
ob-tainedfromnonmammary GR/Amouse tissues
contain only the endogenous number of
MuMTV proviruses and that these proviruses
arehighlymethylated. In contrast, both amplifi-cation and undermethylation of MuMTV pro-viruses were readily detected in premalignant
andmalignant mammary tissue DNAs.
Ourexperiments were designed to investigate
three phenomena that occur during MuMTV-induced tumorigenesis in mice: the increase in
MuMTV proviral copy number per cell, the
apparent undermethylation of these additional
proviruses, and the tendency for only a subset of infected cells to proliferate, giving rise to the
bulkofthe tumor mass.
MuMTV proviral amplification during
mam-marytumorigenesis is well established (8, 9, 11,
15, 20, 22). Originally, the GR/A mouse was
thought to represent a special case in which amplification did not occur (2, 20, 22). Recent findings, however, have shown that additional
MuMTV proviral copies are present in GR/A
mammary tumors, albeit in lownumbers (9, 11,
15). The additional MuMTVproviruses found in
GR/Amammary tumors appear to originate from
the endogenous, highly oncogenic GR-MTV-2
provirus (11). Our resultsdemonstrate that
GR-MTV-2 amplification can be detected in many
GR/A tissues thatare known (2, 21)toexpress
MuMTV (Fig. 3 and 4). This includes
pregnan-cy-dependent tumors which regress during the
postpregnancy period. The apparent slight
de-gree of amplification seen in the mammary gland
tissue of lactating mice (Fig. 5) may reflect a
situationin whichonly a small percentage (5 to
20%) of the mammarygland cells contain
addi-tional GR-MTV-2 proviruses. Some of these
additional proviruses may be actingastemplates
for viral RNA transcription, genome length copies of which are packaged and released from
thetissue as the milk-borne virion.
Proposals linking the transcriptional activity
of certain DNA sequences with their degree of methylation have been put forward (4, 14, 24,
26). Active cellular genes (16, 18, 28, 31) and
retroviruses (13) appear to be less methylated
than inactive genes. Indeed, the active
MuMTV(C3H) provirusfound in the mammary
tumorsofBALB/cfC3H miceis
undermethylat-ed(7). In the GR/A mouse, the mammary gland
cells of lactating mice, premalignant outgrowth
cells, and pregnancy-dependent and
-indepen-dent tumor cells contain actively transcribed
MuMTV proviral sequences (2, 21). These cells
also contain undermethylatedMuMTVproviral
DNA (Fig. 1 and 2). Thus, our results are
consistent with the concept that
undermethylat-ed proviral DNA is transcriptionally active in
these tissues.
We have previously shown that the only
MuMTV provirus amplified in detectable
amountsinGR/Amammary tumorswasthe
GR-MTV-2 provirus (11). This provirus, however,
does not appear to be the only MuMTVprovirus
undermethylated in GR/A mammary gland
tis-sue.Mammaryglandtissueof GR-Mtv-2-mice,
lacking the GR-MTV-2 provirus (30), also
con-tains some undermethylated MuMTV proviral
DNA(Fig.1).Althoughlackingthehighly onco-genic GR-MTV-2 provirus, MuMTV antigens
canbefound in the milk oflactating
GR-Mtv-2-females (23). GR-Mtv-2-mice, however, donot
produce infectious MuMTV virions (30).
Our results are consistent with thefollowing
model: duringlactation the GR-MTV-2provirus
is amplified/undermethylated to a limited
de-gree in some mammary cells. The amplified/
undermethylated GR-MTV-2provirus (and
pos-sibly other undermethylated MuMTV
provi-ruses) is transcribed intoMuMTVmRNAs, but
only genomic-sized copies of GR-MTV-2 are
packaged and released into the milk (12). The
event(s) whichtriggerstheemergence of
prema-lignant cells involves additional amplification,
undermethylation, and transcription of
GR-MTV-2 proviruses. The proviral amplification, undermethylation, and gene expression found in
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premalignant and malignant GR/A tissues, and
the absence of all three in nonmammarytissues,
suggestthat all three phenomena are interrelated
and areconsistent with the model.
Finally, our results (Fig. 6) suggest that
pre-malignant outgrowths andpregnancy-dependent
tumors ofGR/A mice consist largely of mixed
cellpopulations. These cell populations are not
completely random, however, since we
ob-served faint additional MuMTV proviral DNA
fragments in the DNAs of these tissues. This
implies that certain cells enjoyedaweak
selec-tive advantage during thegrowth of the tissue.
GR-MTV-2 isanendogenousproviruspresent in
every GR/Amouse cell. Thus, many cells may
simultaneously emerge from the normal cell
populationaspremalignant.This is in contrast to
the homogenous nature of
MuMTV(C3H)-in-fectedBALB/cpremalignanttissue(5).The
pre-malignantto malignanttransition in GR/Amice
mustentail the emergence ofaselectpopulation
ofcells, sinceGR/Amammarytumorsare more
homogenous than premalignant tissues (9, 11, 15).
ACKNOWLEDGMENTS
Wethank L. J. T.Young, J. P. Puma, D. W.Morris,and V. Pathekfor helpanddiscussionand J.Majorsand H. Varmus for thegiftof cloned MuMTV DNA.
This work wassupported byPublic HealthServicecontract NO1-CP-61013from the Virus CancerProgram, Division of CancerCauseandPrevention,National CancerInstitute,and by Public Health Service grant 5RO1-CA21454 from the National CancerInstitute.
LITERATURECITED
1. Ashley, R. L., R. D.Cardiff,D.J.Mitchell,L.J.Faulkin, andJ.K. Land.1980.Developmentandcharacterization of mouse hyperplastic mammaryoutgrowth lines from BALB/cfC3Hhyperplasticalveolar nodules.CancerRes. 40:4232-4242.
2. Bentvelzen,P.,andJ. Hilgers. 1980. Themurine mammary tumorvirus,p.311-355.InG. Klein(ed.),Viraloncology.
RavenPress,NewYork.
3. Bird,A.P.,andE.M.Southern.1978. Use ofrestriction enzymestostudyeukaryoticDNAmethylation:the meth-ylation patternin ribosomal DNAfromXenopuslaevis. J. Mol. Biol.118:27-47.
4. Brown,D. D. 1981.Geneexpressionineukaryotes.
Sci-ence211:667-674.
5. Cardiff, R. D., T. G.Fanning,D. W.Morris,R. L.Ashley, and L.J. Faulkin. 1981. Restriction endonuclease studies ofhyperplastic outgrowthlines from BALB/cfC3H hyper-plasticmammarynodules.CancerRes.41:3024-3029. 6. Cardiff,R. D., S.R.Wellings, and L.J. Faulkin. 1977.
Biologyof breastpreneoplasia.Cancer(Phila.) 39:2734-2746.
7. Cohen, J.C. 1980.Methylationof milk-borne and geneti-callytransmitted mousemammarytumorvirusproviral
DNA. Cell19:653-662.
8. Cohen,J.C.,P. R.Shank,V. L.Morris,R.Cardiff,and H. E. Varmus. 1979. Integration ofthe DNAofmouse
mammarytumorvirusin virus-infected normal and neo-plastictissues of themouse.Cell 16:333-345.
9. Cohen, J. C., and H. E. Varmus. 1980. Proviruses of
mouse mammary tumorvirus in normal and neoplastic
tissues from GR and C3Hf mouse strains. J. Virol. 35:298-308.
10. DeOme, K. B., L. J.Faulkdn,H. A. Bern, and P. B.Blair. 1959. Development of mammary tumors from hyperplas-tic alveolar nodules transplanted into gland-free mamma-ry fatpads of female C3H mice. Cancer Res. 19:515-520. 11. Fanning, T. G., J. P. Puma, and R. D. Cardiff. 1980. Selectiveamplification of mouse mammary tumor virus in mammary tumorsof GR mice. J. Virol. 36:109-114. 12. Fanning, T. G., J. P. Puma, and R. D.Cardiff. 1980.
Identification and partialcharacterizationof an endoge-nousformof mouse mammary tumor virus thatis tran-scribed into the virion associated RNA genome.Nucleic Acids Res. 8:5715-5723.
13. Guntaka, R. V., P. Y. Rao, S. A.Mitalais,and R. Katz. 1970. Modification of avian sarcomaproviral DNA se-quences in nonpermissive XCcellsbut not inpermissive chickencells.J.Virol.34:569-572.
14. Holliday, R., and J. E. Pugh. 1975. DNA modification mechanisms and gene activity during development. Sci-ence187:226-232.
15. Hynes, N. E., B. Groner, H.Diggelmana,R. VanNie, and R.Mlchalides. 1980.Genomiclocationofmouse mamma-ry tumorvirus proviralDNAinnormal mousetissue and inmammary tumors.Cold Spring Harbor Symp. Quant. Biol.54:1161-1168.
16. Kuo,M.T., J. L. Mandel, and P. Chambon. 1979. DNA methylation: correlation with DNase I sensitivity of chicken ovalbumin andconalbumin chromatin. Nucleic AcidsRes.7:2105-2113.
17. Majors, J. E., and H. E. Varmus. 1981. Nucleotide sequences athost-proviral junctions formouse mammary tumorvirus.Nature (London) 289:253-258.
18.Mandel, J. L., and P.Chambon.1979. DNAmethylation: organspecific variationsin themethylationpattern within and aroundovalbumin and otherchick genes. Nucleic AcidsRes.7:2081-2103.
19. Mlchaldes,R., L. Van Deemter, R. Nusse, and R. Van Nie. 1978.Identificationof the Mtv-2 generesponsiblefor the earlyappearance of mammary tumorsintheGR mouseby nucleic acid hybridization.Proc. Natl. Acad.Sci. U.S.A. 75:2368-2372.
20. Mlchalides, R., G. Vlabhakis, andJ. Schlom. 1976. A biochemical approachtothestudy of thetransmission of mouse MTV in mouse strains RII and C3H. Int. J. Cancer18:105-115.
21. Moore, D. H., C. A. Long, A. B. Vaidya, J. B.Sheffield,A. S.Dion, and E. Y. Lasargues. 1979. Mammarytumor virus. Adv. Cancer Res. 29:347-418.
22. Morris, V. L., E.Mederlos,G. M. Rigold, J. M. Bishop, and H. E. Varmus.1977. Comparisonof mouse mammary tumorvirus-specificDNAininbred,wild andasianmice, and in tumors andnormalorgansfrom inbred mice. J. Mol. Biol.114:73-91.
23. Nusse, R., J. de Moes, J.Hilkens,and R. VanNIe.1980. Localization ofageneforexpression ofmousemammary tumor virusantigens in the GR/Mtv-2- mouse strain. J. Exp. Med.152:712-719.
24. Olson, C. B. 1979. 5-methylcytosine, 5-azacytidine and development: a synthesis. Speculations Sci. Technol. 2:365-373.
25. Rlgby,R. W., M.Dekmann,C.Rhodes,and P.Berg. 1977. Labelling deoxyribonucleic acid to high specific activityin vitroby nick translation with DNApolymerase I.J. Mol.Biol. 113:237-251.
26. RIgg,A. D.1975. Xinactivation,differentiation and DNA methylation. Cytogenet.Cell Genet.14:9-25.
27. Southern, E. M. 1975. Detection ofspecific sequences among DNAfragments separated by gel electrophoresis. J.Mol. Biol. 38:503-517.
28. van der Poeg,L.H.T., and R. A. Flavell. 1980. DNA methylationin the human-y,B-globin locus inerythroid andnonerythroidtissues.Cell 19:947-958.
29. VanNie, R., and J. de Moes. 1977. Development ofa congenic line of the GR mouse strain without early
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VOL.41, 1982 MuMTV DNA METHYLATION AND AMPLIFICATION 1013
mammarytumors.Int. J.Cancer20:588-594. 31. WaalwUk,C., and R. A.Flavell.1978. DNAmethylation 30. Van Nie,R., and A. Dux. 1971.Biologicalandmorphologi- at aCCGGsequence in the large intron of the rabbit P-cal characteristics ofmammary tumors in GR mice. J. globingene:tissuespecific variations. Nucleic Acids Res. Natl.Cancer Inst. 46:885-897. 5:4631-4641.
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