c mice with a reported high mammary tumor incidence have acquired new mammary tumor virus DNA sequences

VIROLOGY 100, 101-109 (1980)

Mammary Tumors from BALB/c Mice with a Reported High Mammary Tumor Incidence Have Acquired New Mammary Tumor Virus DNA Sequences V I N C E N T L. MORRIS, 1 J A N E T E. VLASSCHAERT, CYNTHIA L. BEARD, MARK F. MILAZZO, AND WAYNE C. B R A D B U R Y Department of Microbiology and Immunology, University of Western Ontario, London, Ontario N6A 5C1 Canada Accepted August 26, 1979 Various laboratories have reported differences in the mammary tumor incidence caused by the endogenous mouse mammary tumor virus (MMTV) in BALB/c mice. In order to resolve these differences, we have compared the MMTV specific nucleic acids extracted from BALB/c normal organs and tumor tissue obtained from laboratories reporting either a high or low BALB/c mammary tumor incidence. Hybridization kinetics and restriction endonuclease analysis indicate that mammary tumor tissue from laboratories reporting a high mammary tumor incidence contains integrated MMTV-specific DNA that is not found in normal organs from these mice and is therefore not in the germ line. Furthermore we cannot detect these acquired MMTV DNA sequences in a BALB/c mammary tumor from a laboratory reporting a low mammary tumor incidence. INTRODUCTION

The BALB/c mouse strain is potentially very valuable in mouse mammary tumor virus (MMTV) research, for even though it normally contains no exogenous MMTV, it remains extremely susceptible to infection with exogenous MMTV (Nandi and McGrath, 1973; Bentvelzen, 1974). However, the usefulness of BALB/c mice has been limited by the controversy surrounding the endogenous MMTV in this strain. Some laboratories report that BALB/c mice are associated with a low (less than 1%) mammary tumor incidence and that no viral antigens are produced by lactating mammary glands (Andervont, 1945: Medina and DeOme, 1968; Nowinski et al., 1967). However, other laboratories report that the endogenous MMTV in BALB/c mice causes a high (7-30%) mammary tumor incidence in aging BALB/c mice (Bentvelzen, 1970, 1972; Hageman et al., 1972; Peters et al., 1972). In addition, if MMTV is isolated from these mammary tumors, it is reported

to be highly virulent and to cause a high incidence of mammary tumors when inj ected into newborn BALB/c mice (Bentvelzen, 1972; Hageman et al., 1972). In order to resolve this difference, we have obtained normal organ and mammary tumor tissue from laboratories reporting either a high or low mammary tumor incidence. The nucleic acids extracted from these tissues have been examined by conventional hybridization kinetics and restriction endonuclease analysis. Our results indicate that mammary tumors from certain BALB/c colonies reporting a high mammary tumor incidence contain integrated MMTV DNA sequences that are not encoded in the germ line of these mice. Furthermore, these MMTV DNA sequences are indistinguishable by restriction endonuclease analysis with P s t l from sequences found in mammary tumor DNA extracted from high mammary tumor incidence mouse strains (i.e., C3H, GR). MATERIALS AND METHODS

Sources of virus, tissue, and animals. Mammary tumor bearing and normal

1 Author to whom requests for reprints should be addressed. 101

0042-6822/80/010101-09502.00/0 Copyright © 1980by AcademicPress, Inc. All rightsof reproductionin any formreserved.

lO2

MORRIS ET AL.

BALB/c mice were provided by Dr. P. Bentvelzen (Radiobiology Institute, Rijswijk, The Netherlands): mammary tumors from BALB/cCR mice were supplied by Dr. R. L. Peters (Microbiological Associates, Inc., Walkersville, Md.). BALB/c mice with mammary tumors were also obtained from Dr. P. Blair (University of California, Berkeley). MMTV (C3H) derived from the Mm5mt/cl mammary carcinoma cell line (Fine et al., 1974) was provided by the Frederick Cancer Research Center (Bethesda, Md.). R N A and D N A extraction. Tissues were homogenized with a Virtis blender for 2 min in 0.05 M Tris-HC1, pH 7.4, 0.01 M EDTA, 1% SDS. The homogenates were extracted three times with redistilled phenol:chloroform:isoamyl alcohol (50:48:2) and precipitated with ethanol. The RNA was then separated from the bulk of the DNA by a modification of the procedure of Smith et al. (1974). We resuspended the RNA in 0.2 M phosphate buffer, pH 6.8, 8.0 M urea, 1% SDS, applied it to an hydroxylapatite (Bio-Rad) column at 40 °, and eluted it with the same buffer (under these conditions the DNA remained bound to the column). The RNA was dialyzed three times against 100 vol of 0.02 M TrisHC1, pH 7.2, 0.003 M EDTA, 0.5% SDS. After dialysis the RNA was treated with DNase as previously described (Varmus et al., 1973), extracted with phenol: chloroform (50:50), and ethanol precipitated. Identical hybridization results were obtained when RNA was extracted with buffers at pH 9.2 using the procedures of Varmus et al. (1973) (unpublished observations of authors). DNA for hybridization kinetic analysis was extracted as previously described (Morris et al., 1977). DNA was prepared for restriction enzyme analysis as described by Cohen et al. (1979). Hybridization reagents and conditions. The ~4C-labeled mouse unique sequence DNA was prepared from BALB/c 3T3 cells as described by Morris et al. (1977). Labeled MMTV cDNA was synthesized in a reaction catalyzed by AMV DNA polymerase (supplied by J. Beard and the Office of Program Resources and Logistics, National Cancer Institute) using oligomers of

calf thymus DNA as primers (Goulian, 1968). The cDNA was synthesized with MMTV RNA isolated from virions purified from the Mm5mt/cl cell line using reaction conditions described by Shank et al. (1978). For kinetic analysis the cDNA was labeled with [3H]dCTP (Amersham, 25.5 Ci/mM). The MMTV [3H]cDNA (1.1 × 107 cpm/ t~g) was isolated as previously described (Morris et al., 1979). For restriction endonuclease analysis the cDNA was labeled with [32P]dCTP (Amersham, 350 Ci/mM); the MMTV [32P]cDNA (2-4 × 108 cpm/ ttg) was isolated using the procedures of Cohen et al. (1979). Restriction endonuclease analysis of DNA. High molecular weight DNA was completely digested with EcoRI (Miles, Elkhart, Ind. or Bohringer-Mannheim, St. Laurent, P.Q.) in 0.1M Tris, pH 7.5, 0.05M NaC1, 0.01 M MgC12 at 37° for 16-18 hr, Pst-1 (Boehringer-Mannheim) in 0.02 M Tris, pH 7.5, 0.01 M MgCl~, 0.05 M (NH4)2S04, 100 t~g/ml gelatin at 30° for 16-18 hr or B a m H I (Boehringer-Mannheim) in 0.1 M Tris, pH 7.5, 0.01 M MgC12 at 37° for 16-18 hr. Completeness of reactions was monitored using h bacteriophage DNA (Miles, Elkhart, Ind.). In general a fivefold excess of enzyme was used in each digestion and reaction volumes were approximately 0.1 ml. The fragments were separated by electrophoresis in 0.7% agarose gels, transferred to nitrocellulose strips, and annealed with MMTV [32p]_ cDNA as previously described (Southern, 1975; Shank et al., 1978; Cohen et al., 1979). Approximately 8500 cpm of MMTV [32P]cDNA was added per square centimeter of nitrocellulose paper. The molecular weights of the mouse DNA restriction endonuclease fragments were determined using EcoRI fragments of h bacteriophage DNA (Miles, Elkhart, Ind.) orEcoRI fragments of ~ plac bacteriophage DNA supplied by Dr. G. Mackie (University of Western Ontario, London, Ontario). RESULTS Determination of the Quantity of MMTVSpecific R N A in BALB/c Tissues We have compared the amount of mouse mammary tumor virus (MMTV)-specific

ENDOGENOUS MMTV IN BALB/c MICE

RNA in BALB/c tissues obtained from laboratories which have reported differences in the mammary tumor incidence in aging, retired BALB/c breeders (Medina and DeOme, 1968; Bentvelzen et al., 1970). Unlabeled R N A is extracted from normal organs or mammary tumor tissue and is then annealed to tritium-labeled single-stranded DNA that is complementary to MMTV 70 S RNA (MMTV [3H]cDNA). At a Crt of 6.6 × 104 we find only 18-28% annealing between MMTV [3H]cDNA and normal organ RNA from either BALB/c (HT) 2 or BALB/c (LT) a mice (Fig. 1). Even though this amount of annealing is too low to determine a precise Crtli2, we can say that there is less than one genome equivalent of MMTV R N A per cell (Table 1). RNA from a BALB/c (LT) mammary tumor anneals with MMTV [aH]cDNA with a Crt,2 of 1.9 x 104 (Fig. 1, Table 1). This C~tlj2 also corresponds to less than one genome equivalent of MMTV R N A per cell (Table 1). These results are similar to those previously reported for primary BALB/c tumors (Dudley et al., 1978; Michalides et al., 1978). However, RNA from all of the BALB/c (HT) primary mammary tumors that were examined, anneal with MMTV [3H]cDNA at a Crtll2 of 7.5-15 which indicates that there are approximately 1200 to 2500-fold more MMTVspecific RNA sequences in BALB/c (HT) mammary tumor cells than in BALB/c (LT) mammary tumors (Fig. 1, Table 1). Levels of MMTV-specific RNA similar to that observed with BALB/c (HT) mammary tumors are seen in mammary tumors of high mammary tumor incidence strains (i.e., Rl11, C3H, GR; Varmus et al., 1973) and in BALB/c mice infected with exogenous MMTV isolated from C3H mice (McGrath et al., 1978).

Analysis of BALB/c Tissues for Newly Acquired M M T V DNA Sequences Since there is a 1200- to 2500-fold difference in the MMTV-specific RNA levels 2 BALB/c mice from laboratories (Dr. Bentvelzen and Dr. Peters) reporting a high (7-30%) mammary tumor incidence. 3 BALB/c mice from a laboratory (Dr. Blair) reporting a low (less than 1%) mammary tumor incidence at any age.

103

80-

c 40-

•

10'

•

•

102 103 Crt (mol-$ec/L)

•

o

10'

o

10'

FIG. 1. Quantification of MMTV-specific RNA in BALB/c tissues. MMTV-Specific [aH]cDNA (700 cpm/ sample) was annealed for 72 hr to various dilutions of RNA extracted from the following tissues: (Q) BALB/c (HT) mammary tumor; (O) BALB/c (LT) mammary tumor; (A) BALB/c (HT) pooled normal organs; (A) BALB/c (LT) pooled normal organs.

present in BALB/c (HT) and BALB/c (LT) mammary tumors, we have looked for differences in the MMTV-specific DNA sequences present in mice from these BALB/c colonies. Restriction endonuclease analysis using Pstl is very useful for this purpose, since it can distinguish acquired MMTV DNA sequences (i.e., milk-borne MMTV from C3H or GR mice) from endogenous MMTV DNA sequences in certain strains of mice (i.e., BALB/c) (Cohen et al., 1979). Even if the proviruses are positioned at many sites throughout the cellular genome, Pstl digestion of milk-borne C3H or GR MMTV proviral DNA produces fragments of 2.5 and 0.6 × 106 daltons that are not present in endogenous BALB/c MMTV DNA (Fig. 2; Cohen et al., 1979; Dr. J. C. Cohen, personal communication). BALB/c (LT) mammary tumor DNA lacks the 2.5 and 0.6 × 106-dalton Pstl fragments and therefore these tumor cells only contain endogenous MMTV DNA integrated into their genomes (Fig. 3, lane 2; Table 1). However, when DNA from all three primary BALB/c (HT) mammary tumors, obtained from Dr. P. Bentvelzen (Radiobiology Institute, Rijswijk, The Netherlands), are digested with Pstl, the MMTV-specific band at 2.5 x 106 dalton is seen. Pstl digests of BALB/c (HT) mammary tumor DNA from animal 2 also yield the 0.6 × 106-dalton band (Fig. 3, lane 1; Table 1). The 0.6 x 106-dalton band is not distinct for mammary tumor DNA from BALB/c (HT) animals I and 3 (Fig. 3, lanes 3, 4; Table 1); however, because of the

104

MORRIS ET AL. TABLE 1 SUMMARYOF MMTV-SPECZFICRNA AND DNA IN BALB/c TISSUES Endonuclease analysis MMTV-Specific RNA

Mouse strain

Tissue

C,t,2

BALB/c (LT) BALB/c (LT) Animal 1 Animal 2 Animal 3 BALB/c (HT) BALB/c (HT) Animal 1g Animal 2g

Pooled normal organs

_e

Animal 3g Animal 4~

Mammary tumor 1.9 × 104 Mammary tumor N.T. Mammary tumor N.T. Pooled normal organs e Mammary tumor Normal organs Mammary tumor Mammary tumor Mammary tumor

10 N.T. 7.5 15 N.T.

Presence or Relative MMTV- MMTV DNA absence of Genome specific DNA level copy number acquired equivalent determined by per diploid MMTV DNA per cella kinetic analysisb celF sequence ~ <1

1.0

5

N.T/

0.3-0.4 N.T. N.T. <1

N.T. 1.0 N.T. 1.1

N.T. N.T. .5 5

N.T. N.T. Absent N.T.

650 N.T. 866 433h N.T.

1.5 N.T. 1.8 1.5 N.T.

6 5 10-11 8 N.T.

Present Absent Present Present Present

Determined as previously described (Varmus et al., 1973). b Value of the MMTV DNA copy number per diploid cell in BALB/c (LT) pooled normal organs was set at 1.0 and other values were determined relative to it. c Value determined from EcoRI digest of DNA extracted from mouse tissues (see text and Materials and Methods). Determined by endonuclease analysis using Pstl (see text and Materials and Methods). e Too little annealing to determine value exactly. f Not tested. o Supplied by Dr. P. Bentvelzen (see Materials and Methods). h Determined with MMTV cDNA prepared using the endogenous DNA polymerase associated with detergent-activated virions (isolated from RIII mouse milk) as previously described (Morris et al., 1977). The MMTV was supplied by Dr. D. Moore (Hahnemann Medical College, Philadelphia, Pa.). Supplied by Dr. R. L. Peters (see Materials and Methods). d i f f e r e n c e in m o l e c u l a r w e i g h t , t h e b a n d a t 0.6 x 106 d a l t o n is g e n e r a l l y m u c h f a i n t e r t h a n t h e one a t 2.5 x 106 d a l t o n s . I n a d d i tion, t h e 2.5 a n d 0.6 x 10S-dalton P s t l fragments were obtained with DNA extracted from a primary tumor supplied b y a s e p a r a t e l a b o r a t o r y (Dr. R. L. P e t e r s , Microbiological A s s o c i a t e s , Inc., W a l k e r s ville, M d . ) also r e p o r t i n g a n e l e v a t e d m a m m a r y t u m o r i n c i d e n c e ( F i g . 3, lane 6; P e t e r s et al., 1972). T h e s e r e s u l t s i n d i c a t e t h a t mammary tumor tissue from BALB/c (HT) mice c o n t a i n n e w l y a c q u i r e d M M T V p r o v i r a l D N A t h a t is n o t p r e s e n t in m a m m a r y t u m o r t i s s u e f r o m B A L B / c (LT) mice. This r e s u l t w a s c o n f i r m e d u s i n g t h e r e s t r i c tion e n d o n u c l e a s e BamHI; an M M T V specific 0.7 x 10 e BamHI f r a g m e n t is u n i q u e to a c q u i r e d M M T V p r o v i r u s e s a n d is n o t p r e s e n t in B A L B / c e n d o g e n o u s D N A

( F i g . 2; C o h e n et al., 1979). B A L B / c ( H T ) m a m m a r y t u m o r D N A ( a n i m a l 2) dig e s t e d w i t h BamHI h a s t h e 0.7 x l 0 sd a l t o n f r a g m e n t while t h e B A L B / c (LT) m a m m a r y t u m o r D N A d o e s n o t ( F i g . 4). In addition, we found that normal organ DNA from a BALB/c mouse with a primary m a m m a r y t u m o r lacks t h e 2.5 a n d 0.6 x 10S-dalton Pstl f r a g m e n t s ( F i g . 3, lane 5; T a b l e 1). T h e s e r e s u l t s e s t a b l i s h t h a t t h e a c q u i r e d M M T V p r o v i r a l D N A is n o t in t h e g e r m line of t h e B A L B / c ( H T ) mice a n d d i f f e r s f r o m t h e e n d o g e n o u s BALB/c MMTV.

Measurement of MMTV-Specific D N A Sequences in BALB/c Tissue I n o r d e r to f u r t h e r c o m p a r e t h e M M T V specific D N A s e q u e n c e s in B A L B / c (LT)

ENDOGENOUS MMTV IN BALB/c MICE

and BALB/c (HT) normal organ and mammary tumor tissue, we examined the kinetics of annealing of MMTV pH]cDNA and DNA from BALB/c tissue. Using this technique we find that DNA extracted from BALB/c (HT) and BALB/c (LT) normal organs and BALB/c (LT) mammary tumors have similar levels of MMTVspecific DNA per cell (Table 1). This result agrees with previous reports (McGrath et al., 1978; Cohen et al., 1979). However, several mammary tumors from BALB/c (HT) animals appear to have small but reproducible increases in MMTVspecific DNA levels compared with what is found in the BALB/c (LT) mammary tumor (Table 1, Fig. 5). However, since these differences are at the level of resolution of hybridization kinetic analysis, we have confirmed and expanded these results using the restriction endonuclease EcoRI.

Quantification of MMTV-Specific Sequences Using EcoRI

DNA

In addition to kinetic analysis, the restriction endonuclease EcoRI is useful in quantifying integrated MMTV proviral DNA sequences. EcoR1 cleaves unintegrated MMTV DNA from C3H or GR and the endogenous MMTV in BALB/c once (Fig. 2; Shank et al., 1978; Cohen et al., 1979; Dr. J. C. Cohen, personal communiVIRAL RNA . . . . . . .

-----

PROVIRAL DNA EcoR1

I CELL DNA

DIGESTION

? PROVIRAL DNA B~m- H1 DIGESTION

PROVIRAL DNA

MW

1

MW

2

3

4

MW

5

6

FIG. 3. Examination of BALB/c tissues for acquired MMTV DNA sequences. DNA was extracted from various tissues, digested with P s t l , electrophoresed in a 0.7% agarose gel, and annealed with MMTV cDNA after transfer to nitrocellulose filter sheets (see Materials and Methods). (1) BALB/c (HT) mammary tumor 2 DNA (5 tLg). (2) BALB/c (LT) mammary tumor DNA (5 t~g). (3) BALB/c (HT) mammary tumor 3 DNA (5 t~g). (4) BALB/c (HT) mammary tumor 1 DNA (5 t~g). (5) BALB/c (HT) normal organ DNA (5 tLg) from an animal with a mammary tumor (animal 2). (6) BALB/c (HT) mammary tumor 4 DNA (5 tLg). The spread of the bands is greater in some lanes than in others due to differences in the times of electrophoresis and to small differences in the conditions of electrophoresis; however, in each electrophoresis run the molecular weights of the DNA fragments were determined using EcoRI fragments of X bacteriophage DNA. In addition, BALB/c normal organ DNA digested with P s t l was included in each gel run as a reference DNA.

(A.)

Pstl DIGESTION

I

105

l

CELL DNA

) CELL DNA

FIG. 2. Diagram of Pst l, BamHI, and EcoRI restriction endonuclease cleavage pattern for MMTV (C3H) and MMTV (GR), arrows indicate restriction sites of proviral or cellular DNA. The molecular weights of the viral fragments are in units of 10" daltons. These diagrams are from Shank et al. (1978) and Cohen et al. (1979).

cation). Each copy of MMTV proviral DNA that is integrated into the BALB/c genome should therefore yield two fragments upon digestion with EcoRI. The size of the fragments is determined by the distance between the EcoRI site in the provirus and the nearest EcoRI sites in the surrounding cellular DNA sequences. Therefore, the use of EcoRI in combination with agarose gel electrophoresis and the Southern blotting procedure provides a very accurate way of detecting even small differences in integrated viral DNA copies present in tissues from various sources (Cohen et al., 1979). However, this procedure will only detect a MMTV DNA sequence that is integrated into the host cell DNA in a unique manner with respect to the surrounding host cell EcoRI

106

MORRIS ET AL.

1

2

3

FIG. 4. Digestion of BALB/c DNA with BamHI. DNA was extracted, digested with BamHI, electro° phoresed in a 0.7% agarose gel, and annealed ~ith MMTV cDNA after transfer to nitrocellulose filter sheets (see Materials and Methods). (1) BALB/c (HT) mammary tumor 2 DNA (5 tLg). (2) BALB/c (LT) mammary tumor DNA (5 t~g). (3) 13~P]pGM4 plasmid DNA digested with BamHI (supplied by Dr. G. Mackie, University of Western Ontario) which produces fragments of 4.7 and 2.9 × 106 daltons (Dr. G. Mackie, personal communication, University of Western Ontario). The arrow refers to the 0.7 × 10~-dalton fragment present in the BALB/c (HT) mammary tumor DNA but absent in BALB/c (LT) mammary tumor DNA (see text). The molecular weights of the DNA fragments are determined using EcoRI fragments of h bacteriophage DNA and the BamHI fragments of pGM4 DNA.

visualized as five bands (two of which form a doublet) by autoradiography after hybridization with [3~P]cDNA (Fig. 6. lane 2). Identical results were obtained with BALB/c (HT) pooled normal organ DNA (Table 1). This result suggests that BALB/c normal organs have two MMTV-specific DNA copies plus a subgenomic fragment in a haploid genome (Table 1) and agrees with what has been previously found for BALB/c liver DNA (Cohen et al., 1979; Drs. J. C. Cohen and H. E. Varmus, personal communication). This number represents a minimum copy number because it is possible that fragments of homogenous size are not resolved; however, the MMTV-specific DNA copy numbers obtained with this technique are in good agreement with those obtained by hybridization kinetic analysis (Morris et al., 1977; McGrath et al., 1978; Cohen et al., 1979). The pattern of MMTV-specific E c o R I fragments generated from BALB/c (LT) tumor tissue DNA is indistinguishable from that observed for BALB/c (LT) and BALB/c (HT) pooled normal organ DNA (Fig. 6, lanes 2 and 6; Table 1). The 1.0 × 107dalton band is faint in the BALB/c (LT) O

~ 4o

lo2

ld

lo"

lo°

C o t (mol-sec_./t.)

sites. Thus newly acquired MMTV sequences will only be detected in mammary tumors that are clonal or semiclonal (Cohen et al., 1979). We employed this technique to look for differences in MMTV specific DNA copy numbers present in BALB/c (HT) and BALB/c (LT) normal organ and mammary tumor tissue. DNA extracted from pooled normal organs of BALB/c (LT) mice yields five MMTVspecific EcoRI fragments of approximately 4-10 × 106 daltons. These fragments are

FIG. 5. Kinetic analysis of MMTV-speeifie DNA in BALB/c mammary tumor tissues. MMTV-Speeifie [3HIeDNA (500 epngsample) was annealed for various times with DNA (3.5 mg/ml) extracted from a (O) BALB/e (HT) mammary tumor or a (&) BALB/e (LT) mammary tumor. Mouse unique sequence [;4CIDNA (750 cpm/ sample) was also included in each reaction to serve as an internal standard and to allow quantification of the MMTV specific sequences (Morris et al., 1977). The unique sequence DNA annealed with a Cd,2 of 1.1 x liP for BALB/c (I-IT) tumor DNA and a Cdt/2 of 1.0 x liP for BALB/c (LT) tumor DNA (data net

sho~).

ENDOGENOUS MMTV IN BALB/c MICE

tumor DNA pattern because it is occasionally difficult to transfer high molecular weight DNA from the agarose gel to the nitrocellulose paper. When BALB/c (HT) mammary tumor DNA is similarly analyzed, we observe the same five bands as are present with normal organ DNA; in addition one to six extra bands are observed which are not present with BALB/c normal organ DNA; these extra bands correspond to an increase in as many as three integrated MMTV DNA copies per haploid genome (Fig. 6, lanes 1, 3, and 5; Table 1). Similar increases in MMTV DNA copies have been observed when BALB/c mice are infected with MMTV (C3H) (Morris et al., 1977; McGrath et al., 1978; Cohen et al., 1979). When normal organ DNA from a BALB/c (HT) mouse with a mammary tumor is completely digested with EcoRI, the resulting MMTVspecific fragments are identical to those observed with BALB/c (LT) normal organ DNA (Fig. 6, lanes 2 and 4). This result is consistent with the P s t l data that BALB/c (HT) mammary tumor DNA has acquired MMTV DNA sequences that are not in the germ line of the BALB/c (HT) mouse. DISCUSSION

Hageman et al. (1972) have claimed to isolate the endogenous mouse mammary tumor virus from BALB/c mice, which is reported to be transmitted through the gametes (Bentvelzen, 1972, 1974). This virus does not express itself in young BALB/c mice, but MMTV antigens and particles are produced in only aging BALB/c mice (Bentvelzen, 1972; Hageman et al., 1972). This virus is reported to be associated with a 7-30% mammary tumor incidence (Bentvelzen et al., 1972; Peters et al., 1972). However, a paradox is reported to exist with this virus because even though it is not normally expressed in young BALB/c mice, if it is injected into newborn BALB/c mice, it is highly virulent and virions are easily observed (Bentvelzen, 1972; Hageman et al., 1972). In addition, the level of the MMTV-specific R N A in mammary tumors resulting from injection of new born BALB/c mice .with ~his virus is approx~

107 MW

1

2

3

4

5

6

FIG. 6. Comparison of EcoRI digestions of BALBic normal organ and mammary tumor DNA. DNA was extracted from various tissues, digested with EcoRI, electrophoresed in a 0.7% agarose gel, and annealed with MMTV cDNA after transfer to nitrocellulose filter sheets (see Materials and Methods). (1) BALB/c (HT) mammary tumor 1 DNA (5/~g). (2) BALB/c (LT) pooled normal organ DNA (10/~g). (3) BALB/c (HT) mammary tumor 3 DNA (5/zg). (4) BALB/c (HT) normal organ DNA from animal with mammary tumor 2 (5/~g). (5) BALB/c (HT) mammary tumor 2 DNA (5 /zg). (6) BALB/c (LT) mammary tumor DNA (5/zg). The arrows indicate the molecular weights of the endogenous MMTV DNA sequences found in BALB/c normal organ DNA. Bands at positions not designated with arrows are found in BALB/c mammary tumor DNA but not in BALB/c normal DNA. The spread of endogenous MMTV DNA EcoRI bands is greater in some lanes than in others due to differences in the times of electrophoresis and to small differences in the conditions of electrophoresis; however, in each electrophoresis run the molecular weights of the DNA fragments were determined using EcoRI fragments of k or h plac bacteriophage DNA. In addition, BALB/c normal organ DNA digested with EcoRI was included in each electrophoresis run as a reference DNA.

mately 10,000-fold higher than that found in spontaneous BALB/c mammary tumors (Michalides et al., 1978). However, other laboratories have reported a low (less than 1%) mammary tumor incidence and no production of viral antigens by mammary glands in BALB/c mice regardless of age (Nowinski et al., 1967; Medina and DeOme, 1968). We have therefore compared tissues obtained from laboratories which differ in their reported BALB/c mammary tumor incidence. Our results indicate substantial differences in MMTV nucleic acids present in mammary tumors from laboratories reporting low and high BALB/c mammary tumor

108

ENDOGENOUS MMTV IN BALB/c MICE

incidences. We find that BALB/c mammary tumor~ from laboratories reporting a low mammary tumor incidence have low levels of MMTV-specific RNA (less than one copy per cell) and no observed change in the MMTV-specific DNA sequences compared with BALB/c normal organs. However, when mammary tumors from a laboratory reporting a high mammary tumor incidence in BALB/c mice are examined, very different results are observed; we find a 1200to 2500-fold increase in MMTV-specific RNA compared to MMTV-specific RNA in BALB/c normal organs. In addition E c o R I digestion of BALB/c (HT) mammary tumor DNA reveals an increase of as many as three MMTV-specific DNA copies per haploid genome compared to MMTVspecific DNA sequences in BALB/c normal organs. In addition, the extra DNA sequences do not appear to be transmitted via the gametes since they are not present in normal organs from a mammary tumorbearing animal. The P s t l and B a m H I data provide strong evidence that the mammary tumors from BALB/c mice reported to have an increase in mammary tumor incidence in old age, have acquired MMTV DNA sequences not present in the germ line of these mice; furthermore, these MMTV DNA sequences are indistinguishable by restriction endonuclease analysis with P s t l from sequences found in mammary tumor DNA extracted from high mammary tumor incidence mouse strains (i.e. C3H, GR). This result is also consistent with the close immunological relationship of the virus isolated from BALB/c (HT) mice and MMTV-S (Hageman et al., 1972; Daams et al., 1973). There are two possible sources of these newly acquired MMTV DNA sequences. These newly acquired sequences may result from accidental infection of the highly susceptible BALB/c mouse with a strain of MMTV associated with a high mammary tumor incidence. The tumors may arise in old age because the animals either are infected relatively late in life or because they are infected with a relatively low level of virus. However, once this virus is recovered from these mammary tumors it behaves like the MMTV present in the milk of C3H or GR; it is highly virulent and produces a high tumor incidence when injected into newborn BALB/

c mice. Two of the possible sources of infection of the BALB/c mice could be through the milk (due to a mix up of animals, etc.) or by aerosols (Blair and Lane, 1974). A second possible explanation for the observed data would be the amplification, modification, and integration of MMTV DNA sequences in BALB/c (HT) mammary tumors but not in BALB/c (LT) tumors. Therefore our data indicate that BALB/c mice that differ in their mammary tumor incidence also differ with respect to the MMTV-specific sequences they contain. It is this difference which presumably accounts for the increase in mammary tumor incidence in aging BALB/c mice in some laboratories. ACKNOWLEDGMENTS We thank Drs. P. Blair, P. Bentvelzen, R. L. Peters, J. Beard, D. Moore, and G. Mackie for supplying the materials mentioned in the text. MMTV (C3H) and AMV DNA polymerase were graciously supplied by the Office of Program Resources and Logistics, National Cancer Institute. We thank Drs. H. E. Varmus and J. M. Bishop for helpful discussions and for assistance in obtaining some of the materials used in this study, and to Drs. H. E. Varmus, J. M. Bishop, and J. C. Cohen for reading this manuscript. We are grateful to Drs. J. C. Cohen and S. Hughes for sharing unpublished procedures and results with us. This work was supported by Grant CA 21311 from the U. S. Public Health Service National Cancer Institute awarded to V.M.W.B. is a postdoctoral fellow in the laboratory of Dr. Samuel Dales and is supported by a grant from the U. S. Public Health Service awarded to Dr. S. Dales. REFERENCES ANDERVONT, H. B. (1945). Fate of the C3H milk influence in mice of strains C and C57 black. J. Nat. Cancer Inst. 5, 383-390. BENTVELZEN, P. (1972). Hereditary infections with mammary tumor viruses in mice. In "RNA Viruses and Host Genome in Oncogenesis" (P. Emmelot and P. Bentvelzen, eds.), pp. 309-337. American Elsevier, New York. BENTVELZEN, P. (1974). Host-virus interactions in murine mammary carcinogenesis. Biochim. Biophys. Acta 335, 236-259. BENTVELZEN, P., DAAMS, J. H., HAGEMAN, P., and CALAFAT, J. (1970). Genetic transmission of viruses that incite mammary tumor in mice. Proc. Nat. Acad. Sci. USA 67, 377-384. BLAIR, P., and LANE, M. A. (1974). Immunologic evidence for horizontal transmission of MTV. J. Immunol. 113, 1446-1449.

ENDOGENOUS MMTV IN BALB/c MICE

COHEN, J. C., SHANK, P. R., MORRIS, V. L., CARDIFF, R., and VARMUS, H. E. (1979). Integration" of the DNA of mouse mammary tumor virus in virus-infected normal and neoplastic tissue of the mouse. Cell 16, 333-345.

DAAMS, J. H., HAGEMAN, P., CALAFAT, J., and BENTVELZEN, P. (1973). Antigenic structure of murine mammary tumor viruses. Eur. J. Cancer 9, 567-572. DUDLEY, J. P., BUTEL, J. S., SOCHER, S. H., and ROSEN, J. M. (1978). Detection of mouse mammary tumor virus RNA in BALB/c tumor cell lines of nonviral etiologies. J. Virol. 28, 743-752.

FINE, D. L., PLOWMAN, J. R., KELLEY, S. P., ARTHUR, L. 0., and HILLMAN, E. A. (1974). Enhanced production of mouse mammary tumor virus in dexamethasone treated 5-iodoxyuridinestimulated mammary tumor cell culture. J. Nat. Cancer Inst. 52, 1881-1886. GOULIAN, M. (1968). Incorporation of oligodeoxynucleotides into DNA. Proc. Nat. Acad. Sci. USA 61, 284-291. HAGEMAN,P., CALAFAT,J., and DAAMS,J. H. (1972). The mouse mammary tumor viruses. I n "RNA viruses and Host Genome in Oncogenesis" (P. Emmelot and P. Bentvelzen, eds.), pp. 283-300. American Elsevier, New York. MCGRATH, C. M., MARINEAU, E. J., and VOYLES, B. A. (1978). Changes in MuMMTVDNA and RNA levels of BALB/c mammary epithelial cells during malignant transformation by exogenous MuMTV and by hormones. Virology 87, 339-353. MEDINA, D., and DEOME, K. B. (1968). Influence of mammary tumor virus on the tumor-producing capabilities of nodule outgrowth free of mammary tumor virus. J. Nat. Cancer Inst. 40, 1303-1308. MICHALIDES, R., VAN DEEMTER, L., NUSSE, R., ROPCKE, G. and BOOT, L. (1978). Involvement of mouse m a m m a r y tumor virus in spontaneous and hormone-induced m a m m a r y tumors in lowmammary-tumor mouse strains.J. Virol. 27, 551559. MORRIS, V. L., KOZAK, C., COHEN, J. C., SHANK,

109

P. R., JOLICOEUR, P., RUDDLE, F., and VARMUS, H. E. (1979). Endogenous mouse mammary tumor virus DNA is distributed among multiple mouse chromosomes. Virology 92, 46-55. MORRIS, V. L., MEDEIROS, E., RINGOLD, G. M., BISHOP, J. M., and VARMUS, H. E. (1977). Comparison of mouse mammary tumor virusspecific DNA in inbred, wild and Asian mice, and in tumors and normal organs from inbred mice. J. Mol. Biol. 114, 73-92. NANDI, S., and MCGRATH, C. M. (1973). Mammary neoplasia in mice. Advan. Cancer Res. 17, 353414. NOWINSKI, R. C., OLD, L. J., MOORE, D. H., GEERING, G., and BOYSE, E. A. (1967). A soluble antigen of the mammary tumor virus. Virology 31, 1-14. PETERS, R. L., RABSTEIN, L. S., SPAHN, G. J., MADISON, R. M., and HUEBNER, a. J. (1972). Incidence of spontaneous neoplasms in breeding and retired breeder BALB/c Cr mice throughout the natural life span. Int. J. Cancer 10, 273282.

SHANK, P. R., COHEN, J. C., VARMUS, H. E., YAMAMOTO, K. R., and RINGOLD, G. M. (1978). Mapping of linear and circular forms of mouse mammary tumor virus DNA with restriction endonucleases: Evidence for a large specific deletion occurring at high frequency during circularization. Proc. Nat. Acad. Sci. USA 75, 2112-2116.

SMITH, M. J., HOUGH, B. R., CHAMBERLIN,M. E., and DAVIDSON, E. H. (1974). Repetitive and nonrepetitive sequences in sea urchin heterogeneous nuclear RNA. J. Mol. Biol. 85, 103-126. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98, 503-517.

VARMUS, H. n., QUINTRELL, N., MEDEIROS, E., BISHOP,J. M., NOWINSKI,R. C., and SARKAR,N. H. (1973). Transcription of mouse mammary tumor virus genes in tissues from high and low tumor incidence mouse strains. J. Mol. Biol. 79, 663679.