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Methylation and amplification of mouse mammary tumor virus DNA in normal, premalignant, and malignant cells of GR/A mice.

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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. HhaI

cleavesatthesequenceG-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

a

molec-INA in nonmalignant GR/A and GR-Mtv-2- ular

weight

of

approximately

6 x

106

(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 identical

patterns (see Fig. 6) indicating the specificity of the

cDNA,,pprepared inthis laboratory.

,...I

0

-S.i....

...

*::

-...

.t.o

1 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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MuMTV DNA METHYLATION AND AMPLIFICATION 1009

genetically inherited MuMTV proviruses (15,

22). Do the MuMTVrestriction fragments

gener-ated

by

HhaI

cleavage originate

from one or

from more than one of these proviruses? The

congenic

line GR-Mtv-2- is

missing

MuMTV proviral DNA (19) and does not develop early

mammary tumors (29).

Specifically,

the line

lacks

only

oneof the

endogenous

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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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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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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Figure

FIG. HhaIdigeste4blottingviralmousenancy-ilane pro- D tlactatingmammarymammarymammarymouse;MuMTVindicated1
Fig. 1GR-MTV-2however, and 2 cannot be attributed solely to the provirus. Many of the fragments, are probably derived from GR-MTV-2
FIG. 5.mammarydaltonshownmorDNA. Densitometertracingsof Saclpatterns in Fig. 3 and 4. (A) liver DNA, (B) lactating gland DNA, (C) pregnancy-dependent tu- DNA, and (D) pregnancy-independent tumor Arrows indicate the 1.6 x 106- and 4.0 x 106- GR-MTV-2 proviral fragments.

References

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