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c mice
VIROLOGY
114. 175-186 (1981)
Isolation
and Characterization of a New Mouse Tumor Virus from BALB /c Mice
W. DROHAN,* Y. A. TERAMOTO,* D. MEDINA,? ‘Laboratuq
Mammary
J. SCHLOM**’
AND
of CeUular and Mokcular Bidogy, Nation& Cancer Institute, National Institutes of Health, Beth&a, Malylnnd 21l%l5, and fBaylm College of Medicine, Houston, Texas 77080
Received February 22, 1981; accepted May 29, 1981 A novel mouse mammary tumor virus (MMTV) has been isolated from mice of a subline of the BALB/cCrl Med mouse strain designated BALB/cV. Whereas breeding females of the parent BALB/cCrl Med colony have a mammary tumor incidence of l%, 47% of the breeding females of the BALB/cV subline develop mammary tumors before 10 months of age. Foster nursing experiments demonstrated this virus, termed MMTV (BALB/c), was transmitted only by milk. The novel MMTV isolate was shown to be immunologically related to, but distinct, from the MMTV variants of C3H, GR, and RI11 mice by a series of competition radioimmunoassays for the MMTV 28,000-dalton major core protein (p28), and the 52,000 (gp52)- and 36,OOGdalton (gp36) major envelope glycoproteins. Monoclonal antibodies directed against MMTV gp36 were also used to clearly distinguish MMTV(BALB/ c) from MMTV(CBH), MMTV(RIII), MMTV(GR), MMTV(C3HfC57BL). and MMTV(A). MMTV-specific proviral DNA content of mammary adenocarcinomas arising in the BALB/cV subline was examined with restriction endonucleases and the Southern blot technique, and compared to the MMTV proviral DNA content of BALB/cAnDe mammary tumors. The virus arising from these latter tumors has been termed MMTV(0). Analysis of EcoRI digests of high-molecular-weight DNA from both types of mammary tumors demonstrated additional MMTV-related proviral sequences when compared to the DNA of normal BALB/c tissues. The patterns generated with the restriction endonuclease Sac1 distinguished the additional MMTV-specific proviral information in the mammary tumors of the BALB/cV mice from the proviral information in tissues containing the GR, C3H, or RI11 MMTVs, as well as from the proviral information in the BALB/cAnDe mammary tumors. These immunological and molecular studies thus define MMTV(BALB/c) as a novel MMTV variant.
of its spontaneous mammary adenocarcinomas. Although BALB/c mice are highly susceptible to infection by the oncogenic milk-borne mouse mammary tumor viruses (MMTVs) of the high mammary tumor incidence strains, most colonies of BALB/c mice are believed to be free of infectious virus (Moore et al., 1974, 1979; Moore, 1975; Medina, 1976). BALB/c mice, however, contain endogenous proviral information related to the MMTVs of other mouse strains (Michalides et al., 1978; Cohen et al., 1979a, b; Groner and Hynes, 1980; Morris et al, 1980). This information is expressed at a low level in normal BALB/c mammary tissues, and mammary tumors, but has not been etiologically
INTRODUCTION
Although BALB/c mice have been characterized as a low-incidence mammary tumor strain (Andervont, 1945; Bentvelzen, 1972a; Nandi and McGrath, 1973), the tumor incidence reported in different colonies has varied from 1% to as high as 30% (Andervont and Dunn, 1948; Bentvelzen, 1972b; Peters et aZ., 1972; Nandi and McGrath, 1973; Moore, 1975). Significantly, the BALB/c strain differs from the high mammary tumor incidence strains, C3H, GR, and RIII, in the late appearance ‘To whom dressed.
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175
0042~6822/81/130175-12$02.00/O Copyright All rights
0 1981 by Academic Press. Inc. of reproduction in any form reserved.
DROHAN
176
linked to mammary tumorigenesis (McGrath et al., 1978; Michalides et al., 1978; Dusing-Swartz et al., 1979; Pauley et al., 1979). Occasionally, an infectious virus (sometimes referred to as MMTV(0)) has been recovered from BALB/c mice, which has been associated with a high mammary tumor incidence (Hageman et al., 1972; Fieldsteel et aZ., 1975). In this study we have examined MMTVrelated proviral information in a substrain of BALB/cCrl Med mice in which the incidence of spontaneous adenocarcinomas is almost 50% in breeding females at 10 months of age. We also compared by restriction endonuclease analyses, the proviral information of BALB/cCrl Med mammary tumors, of BALB/cAnDe mammary tumors induced by MMTV(O), and of normal BALB/c tissues. By using both molecular and immunological techniques, evidence is presented that the BALB/c virus represents a novel MMTV variant indigenous to BALB/c. The BALB/c subline has been designated BALB/cV to emphasize the presence of an infectious virus, and to distinguish it from other BALB/c sublines which express an infectious virus occasionally (MMTV[O] in BALB/cAnDe) and continuously (BALB/cNIV). MATERIALS
AND METHODS
Tissues. The BALB/cV tumors used in these experiments arose in lo-month-old breeding females and were transplanted subcutaneously into virgin BALB/cCrl Med mice. Frozen mammary tumors induced by the MMTV(0) variant found in BALB/ cAnDe were obtained from Dr. P. Bentvelzen, The Radiobiological Institute TNO, Rijswijk, The Netherlands. Normal BALB/ cV female mice were used as controls as well as BALB/cCR mice obtained from the Charles River Breeding Laboratories, Wilmington, Massachusetts. Nucleic acid studies were performed on three different pools of a transplanted BALB/cV mammary tumor. Normal GR/N mice were obtained from the National Institutes of Health through the National Cancer Institute Office of Resources and Logistics. Feline cells were infected with the RI11 and C3H variants of MMTV as previously
ET AL.
described (Howard et al., 1977). A D2 mammary tumor which has arisen from the D2 hyperplastic outgrowth line, established by Medina (1976) from a hormonally stimulated BALB/c mouse, was also examined. Viruses. MMTV(C3H) was obtained from supernatant fluids of the C3H mammary tumor cell line Mm5mt/c1 (Owens and Hackett, 1972). MMTV(RII1) was obtained from the milk of multiparous RI11 mice. MMTV(BALBicfC3H) was obtained from supernatant fluids of primary cultures of BALB/cfC3H mammary tumors. MMTV(BALB/c) was obtained from supernatant fluids of primary cultures (Howard et ah, 1977) of serial transplanted mammary tumors from BALB/cV mice. MMTV was concentrated and purified from supernatant fluids of mammary tumor cultures by ammonium sulfate precipitation and a series of discontinuous and linear sucrose density gradients as previously described (Drohan et al., 1977). MMTV(RIII) was purified from mouse milk following treatment of the milk with EDTA (Teramoto et aZ.,1977). Tumor extracts. Mammary tumors were finely minced and homogenized for 2 min on ice in 0.01 M Tris, pH 7.2, containing 0.0002 M CaClz. A volume of buffer equal to at least 10 times the tissue weight resulted in optimal homogenization. The homogenate was further disrupted by nitrogen cavitation using a cell disruption bomb (Parr Institute) at 1000 lb/in? for 5-15 min. The homogenate was centrifuged at 1000 g for 5 min and the protein in the supernatant was quantitated by the method of Lowry et al. (1951). Radioimmunoassay. The techniques of iodination and competition radioimmunoassays for purified MMTV gp52, gp36, and ~28 have been previously described (Teramoto et al., 1977; Teramoto and Schlom, 1978; Teramoto and Schlom, 1979). Briefly, competitors were added to limiting dilutions of the appropriate antisera and incubated at 37” for 1 hr. Radioactive control antigen was then added and the incubation continued for another hour at 37”. Following the addition of appropriately diluted anti-immunoglobulin, the in-
MOUSEMAMMARYTUMORVIRUSCHARACTERIZATION
177
60-70 S RNA produced identical patterns when hybridized to restricted murine DNA and analyzed by the technique of Southern (1975). In most cases, the [32P]MMTVcDNA probe did not appear to contain detectable amounts of ribosomal or other normal cellular RNAs as indicated by the absence of hybridization to normal feline tissue which does not contain MMTV sequences (Fig. 2, lane H). Furthermore, a [32P]cDNA probe synthesized from 28 S and 18 S murine ribosomal RNA, was hybridized to an EcoRI digest of high-molecular-weight murine DNA, and revealed no bands in positions corresponding to the MMTV-related DNA fragments presented in Fig. 2. Filters were soaked in 3X SSC at 65” for 30 min and then prehybridized in 3~ SSC, 0.1% SDS, 100 pg/ml denatured and sonicated canine DNA, and Denhardt buffer (0.2% each of bovine serum albumin, Fi~011,and polyvinylpyrrolidine) for 4 hr at 65”. [=P]MMTV cDNA was added at 4 X lo6 cpm/ml and hybridized to the filter for 15 hr. Filters were then washed with Gel electrophoresis, DNA transfer, and 3X SSC, 0.1% SDS, and Denhardt’s soluhybridization. After restriction endonu- tion at 5-min intervals until the wash clease digestion, DNAs were adjusted to buffer contained only 100 cpm 32P/ml. The 20% glycerol and electrophoresed in 0.8% filters were then washed with 0.3~ SSC, agarose (BRL) containing 0.02 M sodium 0.1% SDS at 15-min intervals until the acetate, 0.002 M EDTA, 0.018 M Tris base, wash buffer contained only 100 cpm 32P/ and 0.028 M Tris-HCl (pH 8.05). Bromo- ml. Filters were air dried and were autophenol blue was added as tracking dye and radiographed 75 hr with Kodak X-OMAT gels were electrophoresed at 35 V until the R film at -70”. dye migrated 13 to 16 cm. Ethidium broExtraction of high-molecular-weight mide (0.005 mg/ml) staining was used to DNA. Tissues were homogenized in DNA visualize the X marker DNAs after elec- extraction buffer (0.02 M Tris-HCl, pH 8.5, trophoresis. DNA was transferred to ni- 0.1 M NaCl, and 0.001 M EDTA) using a trocellulose filter paper by the method of Waring blender. Samples were incubated Southern (1975). Gels were treated with at 37’ for 120 min with 0.5% SDS, 1.0% denaturing solution (1.5 M NaCl, 0.5 M mercaptoethanol, and 1.0 mg/ml prediNaOH) for 20 min, neutralized (3.0 M gested Pronase. DNA was extracted by NaCl, 0.5 M Tris-HCl, pH 7.5) for 40 min, shaking the aqueous phase with l/2 vol of and treated with the 6X SSC buffer (SSC, phenol and l/2 vol of chloroform for 5 min, 0.15 M NaCl, 0.015 M sodium citrate) for centrifugation at 3400g for 15 min, and 10 min. The DNAs were transferred from removal of the aqueous phase. Extractions the gel to the nitrocellulose paper with 6X were repeated until the interphase was SSC for 15 hr. The filters were dried in a clear. DNA was precipitated in 2 vol ethyl alcohol. High-molecular-weight DNA was vacuum oven at 80” for 4 hr. Viral-specific DNA sequences were de- spooled out with a glass rod and was dissolved in 0.1X SSC. tected by hybridization of the DNA filters Competition hybridization. Hybridizato 32P-labeled DNA complementary to MMTV(C3H) 70 S RNA (MMTV cDNA). tion conditions are as described above for liquid hybridization. Briefly, unlabeled [32P]MMTV cDNA and ‘%I-MMTV(C3H)
cubation was continued for 1 hr at 37” and at 4” overnight. Following centrifugation at 10,OOOgfor 1.5 min, the supernatants were aspirated, and the pellets assayed for radioactivity. Liquid hyti’dixation. The technique for liquid hybridization has been previously described (Drohan and Schlom, 1979). Restriction endonuclease digestion of DNA. EcoRI restriction endonuclease was obtained from Boehringer-Mannheim (Indianapolis, Ind.). Sac1 was purchased from Biotec, Madison, Wisconsin. Conditions for Sac1 digestion were as provided by the manufacturer. EcoRI digestions were performed in 0.1 M Tris-HCl (pH 7.5), 0.05 M NaCl, 0.005 M MgClz, and 100 pg/ml bovine serum albumin (BSA). EcoRI restrictions at 5-10 units of enzyme per microgram DNA were incubated 4 hr at 37”. Ten micrograms of DNA was loaded per lane. EcoRI-restricted X bacteriophage DNA (New England Biolabs., Beverly, Mass.) was used to provide marker bands for molecular-weight determinations.
178
DROHAN
competitor RNA was added in increasing amounts to the reaction mixture which already contained 1000 cpm of [3H]MMTV(C3H) 60-70 S RNA and 100 pg of C3H mammary tumor cell line DNA (3 mg/ml). The hybridization reaction was carried out to a C,,t of 28,000 mol. sec. liters-’ and assayed for the acquisition of RNase resistance. Competition was determined by comparing the percentage of hybridization in the presence of competitor RNA with that of the control sample [MMTV 3H-labeled 60-70 S RNA hybridized to DNA from C3H mammary tumor cells (greater than 50% hybridization)], after subtracting the background hybridization of less than 5% [[3H]MMTV(C3H) 60-70 S RNA hybridized to canine DNA]. RESULTS
History of the BALB/cV Substrain Transmission of MMTV(BALB/c)
and
Two pairs of BALB/cCrl breeding mice, with litters, were received from the Cancer Research Laboratory, Berkeley, California, in 1970. The mice were subsequently brother and sister mated to establish a breeding colony. A mammary tumor arose in one female, from the original two litters, at 10 months of age. This mouse and its progeny were isolated and maintained as a separate subline, now designated as BALB/cV, to emphasize the presence of an infectious virus. Since 1970, this BALB/ c subline has been maintained at the Baylor College of Medicine, Houston, Texas. Through generations F4 to Fzo the mammary tumor incidence in breeding females was 47% (111/234) with mean age of tumor appearance of 10.2 months and a mean number of litters of 6.3. In contrast, BALB/cCrl Med mice have a 1% spontaneous tumor incidence in retired breeders by 15 months of age (Pauley et aZ.,(1979). All tumors that arose in BALB/cV mice were type B mammary adenocarcinomas. Electron microscopic examination of mammary tumors occurring in BALB/ cV at Fi, Fq, and Flz mice demonstrated the presence of budding and mature B particles characteristic of MMTV (Fig. 1). In order to determine the route of transmis-
ET AL.
sion of MMTV (BALB/c), reciprocal foster-nursing experiments were carried out between BALB/c and BALB/cV. BALB/cV female newborns were foster-nursed on lactating BALB/c mice by the drop-cage method. The reciprocal groups were also examined. The BALB/cVf and BALB/cf female mice were mated with the male BALB/c mice and checked weekly for the presence of mammary tumors. In addition, the fourth litter (designated F,) from both groups were saved, bred, and the mammary tumor incidence recorded. The data (Table 1) show that MMTV(BALB/c) is transmitted via the milk and can be eliminated by foster-nursing the infected mice on uninfected BALB/c female mice. The mammary tumor incidence dropped from 59% in the BALB/cfBALB/cV to 0% in BALB/cVfBALB/c. Female mice derived from the fourth litters of the two groups also exhibited similar tumor incidences.
Restriction Endonuclease BALB/c DNAs
Analysis
of
Restriction endonuclease analysis of DNA from normal and tumor bearing BALB/c tissue was undertaken to examine their MMTV-specific proviral DNA content. The restriction endonuclease EcoRI cleaves MMTV DNA at a single site, and thus, can be used to quantitate MMTVspecific proviruses in cellular DNA. The DNAs of normal tissues and mammary tumors of BALB/c mice were compared by EcoRI restriction analysis. As shown in Fig. 2, lane E, radioactive MMTV cDNA hybridizes to fragments of 14.5, 8.7, 7.2, 6.8, 5.8, and 3.0 kilobase pairs (kbp) in digest’s of liver DNA from nontumor-bearing BALB/cCR mice. Identical restriction profiles were obtained from the DNA of livers and other apparently normal tissues of mammary tumor-bearing BALB/cV mice. This restriction pattern is unique to BALB/c mice (Cohen et al., 1979a, b; Morris et al., 1980) and defines the endogenous MMTV provirus content for these tissues. The EcoRI restriction pattern from DNA of a transplanted mammary tumor of BALB/cV mice was found to contain two restriction fragments of 10.9 and 10.3 kbp
MOUSE
MAMMARY
TUMOR
VIRUS
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CHARACTERIZATION
FIG. 1. Electron microscopy of tumors arising in BALB/cV female mice demonstrates the presence of budding and mature B particles characteristic of MMTV. A and B focus on B particles found on the apical cell membranes and lumina of mammary tumor cells. Bars represent 0.1 km.
tissue DNA. The presence of the additional EcoRI restriction fragments in the BALB/ c mammary tumor DNAs (Fig. 2) suggested that all three mammary tumor tissues have either acquired exogenous viral information by infection or that endogenous BALB/c information has been amplified and reintegrated into high-molecular-weight host DNA. The restriction endonuclease PstI has been shown to cleave MMTV proviral DNA at multiple internal sites (Shank et al., 19’78; Cohen and Varmus, 1979; Cohen et ah, 1979a). Pst-I digests of DNAs from
(Fig. 2, lane F), in addition to the fragments observed in apparently normal tissues of the same mice. DNA from mammary tumors which developed from a transplantable BALB/cD2 hyperplastic alveolar nodule (Medina, 1976), and from mammary tumors induced by the MMTV(0) variant of BALB/c mice (Bentvelzen, 1974) also contained several addition EcoRI restriction fragments (Fig. 2, lanes G and D, respectively) which distinguished them from each other, from the BALB/cV mammary tumor DNA, and from the apparently normal BALB/cCR TABLE
1
MAMMARY TUMOR INCIDENCE IN FOSTER-NURSED BALB/cV
Group BALB/cVfBALB/c BALB/cfBALB/cV BALB/cVfBALB/cFl” BALB/cfBALB/cVF,’
No. tumors/ No. mice o/21 16/27 l/28 23/29
%T
Average No. litters/ mouse
0 59 4 79
a The F1 female mice were derived from the fourth
4.2 3.7 8.5 7.0 litters
AND BALB/c
FEMALE MICE
Average latent period of tumor formation (months)
Average age of mice dying without tumors (months)
0 10.0 14.0 9.8
17.4 15.0 13.8 13.8
of the BALB/c-fostered
mice.
130
DROHAN
ABCD
K’:t+a= E F G H -14.6,
-I).7,7.2-
-e..S-6.8’
/
9.0
FIG. 2. MMTV containing DNA fragments in EcoRIdigested BALB/c mammary tumors. Ten micrograms of DNA was electrophoresed on 0.8% agarose gels and transferred to nitrocellulose filter paper by the method of Southern (1975) and then hybridized to 4 X lo6 cpm/ml MMTV [ssP]cDNA. Lane A, Normal GR liver; lane B, MMTV(RIII)-infected feline cells; lane C, MMTV(CIH)-infected feline cells; lane D; BALB/ c(O)-induced mammary tumor; lane E, normal BALB/ cCR liver; lane F, BALB/cV mammary tumor; lane G, BALB/cD2 mammary tumor; lane H, uninfected feline cells. Fragments of X phage DNA cleaved with EcoRI served as size markers on ethidium bromidestained gels.
mammary tumors resulting from infection by MMTV(C3H), MMTV(RIII), and MMTV(GR) proviruses yield distinctive restriction fragments in addition to those corresponding to endogenous MMTV proviral DNA. These unique fragments are also in digests of MMTV-infected feline cells in culture. The unique MMTV(C3H) proviral DNA fragments are 3.9 and 0.9 kbp (Cohen and Varmus, 1979) while those of MMTV(RII1) are 3.6 and 0.9 kbp. The presence of any of these unique fragments in Pst-I-digested DNAs from BALB/c mammary tumors and their absence in DNA from normal BALB/cV or BALB/ cCr tissues would suggest that these tumors were infected with a nongerm line transmitted virus. MMTV-(O)-induced, BALB/cV, and BALB/cD2 mammary tu-
ET AL.
mors each contained a 3.9- and a 0.9-kbp Pst-I restriction fragment in addition to the internal PstI fragments of 5.1,4.8, 1.7, 1.3, and 0.8 kbp found in the DNAs from normal internal organs of normal or tumor-bearing BALB/c mice. This result is consistent with the BALB/cV and BALB/ c-D2 mammary tumors being infected with an exogenous variant of MMTV. To determine if we could demonstrate additional MMTV-specific DNA fragments unique to the BALB/cV mammary tumors we digested BALB/cV, BALB/cD2, and MMTV(O)-induced BALB/c mammary tumors with the restriction endonuclease SucI. Digestion of DNA from tissue culture cells infected with the MMTV(C3H) variant with the enzyme Sac1 reveals a unique restriction fragment of approximately 8.0 kbp. This 8.0-kbp band reflects the presence of a Sac1 restriction site in the long terminal repeat (LTR) of MMTV(C3H) proviral DNA and has been observed only in Sac1 digests of DNA from spontaneous C3H mammary tumors or DNA from feline tissue culture cells infected with MMTV(C3H). We have examined the pattern of MMTV-specific fragments in Sac1 digests of BALB/c mammary tumor and normal tissue DNA for the presence of the 8.0-kbp fragment. MMTV-specific restriction bands of 9.6, 7.4, and 5.8 kbp are observed in DNA of all normal BALB/c tissues, as well as in BALB/cV and D2 mammary tumors. However, in multiple Sac1 digests of DNA from the MMTV(O)-induced mammary tumor there was a pronounced restriction fragment at 8.0 kbp (Fig. 3, lanes D and D’). Except for tissues infected with the MMTV(C3H), the MMTV(O)-induced mammary tumor was the only tissue examined thus far which gave rise to a 8.0kbp Sac1 restriction fragment. These fmdings clearly differentiated BALB/cV mammary tumor proviral DNA from that of the MMTV(O)- and MMTV(C3H)-induced mammary tumors and suggests that the MMTV(0) virus may be related to the MMTV( C3H). The restriction endonuclease B&II was also used to examine the MMTV-specific DNA content of MMTV(O)-induced mam-
MOUSE A B
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CHARACTERIZATION
181
Liquid H&ridixation of Radioactive MMTV(CSH) RNA to BALB/cV and C3H Mammary Tumors
By restriction endonuclease analysis we have been able to distinguish MMTV(C3H) from MMTV(BALB/c). However, to precisely examine the nucleotide sequence homology shared between the two variants, the kinetics of liquid hybridization *.*experiments were examined. Using 3H-la*abeled 60-70 S MMTV(C3H) RNA as a probe, the final extent of hybridization with DNA from C3H mammary tumors was ‘79% with a C,-,tmof 150. In contrast, only 41% of this RNA hybridized with DNA from BALB/cV mammary tumor, with a C,t, of 800. The difference in the FIG. 3. MMTV containing DNA fragments in SacIextent of hybridization of the MMTV(C3H) digested BALB/c mammary tumors. Lane headings (A-H)and conditions as described in the legend to RNA suggests that the BALB/cV mamFig. 2. Lanes D’ and E’ are the same as lanes D and mary tumor DNA does not contain all E except that the hybridized filter was exposed to Xthe MMTV(C3H)-specific nucleic acid seray film for a shorter period of time to clearly resolve quences present in the C3H mammary the presence of an 8.0-kbp MMTV containing DNA tumor DNA. The stability of the hybrids fragments in the BALB/c(O)-induced mammary tuformed with the two DNAs was assesed mor (lane D’). All DNA samples were run on the to determine the extent of base pair missame gel. match. The T,,, of the hybrid between MMTV(C3H) 60-70 S RNA and DNA from mary tumors, as well as BALB/cV and a C3H mammary tumor was 81.6”, whereas hybrids formed with DNA from BALB/cV BALB/cD2 mammary tumors. In addition to endogenous MMTV-specific DNA found mammary tumors had a T,,, of 74.5”. Since in all BALB/c tissue, an intense 4.3-kbp a 1” reduction in T, has been shown to be equivalent to approximately 1.0 to 1.5% DNA fragment was found in MMTV(O)induced mammary tumors; no such frag- base mismatching (Drohan et aZ.,1977), we conclude that there is approximately a ment was present in BALB/cV or BALB/ cD2 mammary tumors. Digestion of DNA 7-10s base pair mismatch between from normal or mammary tumor tissues MMTV(C3H)RNA and BALB/cV mamcontaining the infectious GR, RIII, or C3H mary tumor DNA. In order to precisely compare the perMMTV variants with the restriction endonuclease BglII produces an equally in- centage homology shared between the tense 4.3-kbp fragment (Cohen et aZ.,19’79a; RNA genomes of the MMTV(C3H) and Fanning et al., 1980; Drohan and Schlom, MMTV(BALB/c), RNA was extracted from unpublished data). However, a 4.3-kbp virus produced from primary cultures of MMTV-containing DNA fragment, com- BALB/cV mammary tumors and composed of about 75% cellular DNA, is found pared to the RNA of MMTV(C3H) virus endogenously in all BALB/c DNA. Al- by competitive hybridization assay. Both though, using intensity differences of spe- unlabeled viral RNAs were used as comcific bands on Southern blots to suggest petitors for the hybridization between 3Hthe absense or presence of a unique virus labeled MMTV(C3H) 60-70 S RNA and is tenuous, these data add further evidence C3H mammary tumor DNA (Fig. 4). that the BALB/cV and BALB/cD2 mam- MMTV(C3H) RNA competed greater than mary tumors are not infected with either 97%, while MMTV(BALB/c) viral RNA competed only 82%. Therefore, approxithe GR, RIII, of C3H variants of MMTV.
182
DROHAN
0
0 /
1W 0 50100
500
1000
COMPETING
molecular-weight 60-70 S RNA, and a magnesium-dependent reverse transcriptase. Furthermore, the electrophoretic profile of MMTV(BALB/c) polypeptides from that of was indistinguishable MMTV(C3H). MMTV(BALB/c) thus has biochemical characteristics identical to those of other MMTVs (Teramoto et al., 1974).
7
0
: i : 1
15co
2coo
2500
VIRAL RNA IngI
FIG. 4. Competition hybridization of the MMTV(C3H) genome. One thousand counts per minute of ‘zI-labeled MMTV(C3H) 60-70 S RNA was hybridized in 100 Fg of DNA (3000 pg/ml) from the C3H mammary tumor cell line, MmBmt/q. To this reaction, increasing amounts of unlabeled viral RNAs were added. Hybridizations were conducted as described under Materials and Methods to a final C,t of 28,000 mol. sec. liter-‘. Symbols for competitor RNAs: MMTV(C3H) 60-70 S RNA (0); MMTV(BALB/ c) total viral RNA (A); AMV 60-70 S RNA (m); MuLV(C3H)lOT1/2 60-70 S RNA (0).
mately 15% of the RNA genomes of MMTV(C3H) and the BALB/c virus appear to be different. It should be noted that when 3H-labeled 60-70 S RNA from MMTV(BALB/c) was hybridized to the DNA from BALB/cV mammary tumors and to apparently normal internal organs of BALB/cV mammary tumor-bearing mice, the probe hybridized 65% (C&/z = 760) to the tumor DNA while hybridizing only 35% (C&z = 980) to apparently normal DNA from the tumor-bearing animal. These results are consistent with the presence of the two additional EcoRI bands in the BALB/cV mammary tumor DNA (see Fig. 2).
Biochemical Characterization MMTV(BALB/c)
ET AL.
of
MMTV(BALB/c) purified from supernatant fluids of primary BALB/cV mammary tumors had a buoyant density in sucrose of 1.17 g/ml, contained high-
Immunological Characterization MMTV(BALB/c)
of
Purified virus from supernatant fluids of BALB/cV cultures was tested for its ability to compete in group-specific radioimmunoassays (RIAs) for MMTV gp52, MMTV gp36, and MMTV ~28. All three of these RIAs were performed under conditions whereby MMTV group-specific antigenic determinants could be detected; all three assays have been previously shown to be highly specific for MMTV antigens (Teramoto et al., 1977; Teramoto and Schlom, 1978, 1979). The immunological reactivity of MMTV(BALB/c) virus from that of was indistinguishable MMTV(C3H) and all other MMTVs tested in all three RIAs. Almost identical quantities of virus preparations caused similar levels of inhibition in each RIA. Mammary tumors of BALB/cV mice were also immunologically analyzed. Cell-free extracts of BALB/cV mammary tumors contained MMTV gp52, gp36, and ~28 as determined by the appropriate RIAs. Approximately l-3% of the mammary tumor extract protein corresponded to antigens related to MMTV virion associated gp52, gp36, and ~28. Purified virus from BALB/cV cultures were also assayed in the MMTV type-specific gp52 and gp36 RIAs (Fig. 5). The MMTV(BALB/c) preparation did not compete as efficiently as MMTV(C3H) or MMTV(BALBicfC3H) in either the gp52 (Fig. 5A), or the gp36 (Fig. 5B) MMTV type-specific RIAs. The fact that MMTV(BALBicfC3H) could be distinguished from the MMTV(BALB/c) virus is noteworthy since both virions were obtained from BALB/c mice, thus ruling out the factor of host-strain-specific differ-
MOUSE
1
2
3
4
5
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1
2
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3
4
5
LOG COMPETITOR PROTEIN (ng)
for MMTV FIG. 5. Type-specific radioimmunoassays polypeptides. Purified virus preparations were used as competitors in the following type-specific RIAs: (A) type-specific gp52 RIA using anti-MMTV(C3H) at a dilution of 1:10,606 to bind ?-labeled MMTV(C3H) virions; (B) type-specific gp36 RIA using anti-MMTV(C3H) at a dilution of 1:10,666 to bind ‘%I-labeled MMTV(C3H) gp36. MMTV(C3H) (o), MMTV(BALB/fC3H) (m), MMTV(RII1) (0), MMTV(BALB/c) (A).
ences. In the group-specific RIAs, this same preparation of MMTV(BALB/c) competed identically to MMTV(C3H). Thus, the MMTV(BALB/c) appeared to also have some antigenic determinants that are not identical to those of MMTV(C3H). The MMTV(BALB/c) was further immunologically characterized with several monoclonal antibodies. These antibodies were synthesized by hybridoma cell cultures derived from cell fusions involving spleens from animals immunized with disrupted MMTV(C3H). A series of different monoclonal antibodies have been used in a solid-phase radioimmunoassay to identify antigenic differences among MMTVs isolated from several different mouse strains (Colcher et al, 1980). With the combined use of the appropriate monoclonal antibodies, the MMTV(BALB/c) could be readily distinguished from the MMTVs of C3H, C3Hf, GR, RIII, and A strains of mice (Table 2). Of the various MMTVs tested, MMTV(BALB/c) was the only virus which showed differential reactivity with monoclonal antibodies M2.1 and M1.l. Monoclonal antibody M2.1 had an endpoint titer of 1:12,800, while Ml.1 had an endpoint titer of 1:800 as tested in solid-
VIRUS
183
CHARACTERIZATION
phase radioimmunoassay against identical MMTV(C3H) virus preparation. Although M2.1 had approximately a 16-fold higher amount of antibody, it did not react with MMTV(BALB/c) while Ml.1 reacted positively with MMTV(BALB/c). Thus, the differential reactivity observed with M2.1 and Ml.1 with MMTV(BALB/c) could not be attributed to differences in titer between the two monoclonal antibodies. Both of these antibodies recognized typespecific antigenic determinants on MMTV gp36, in contrast to monoclonal antibody R2.1, which recognized group-specific antigenic determinants on MMTV gp36. These data are thus consistent with the results obtained in liquid competition RIAs. The BALB/cV isolate thus appears to have antigenic and nucleic acid sequences unique among all MMTVs thus far characterized and is therefore termed MMTV(BALB/c). DISCUSSION
BALB/c mice are generally considered a model tumor strain for investigating low-incidence spontaneous mammary tumors (Bentvelzen, 1972a). Except for two TABLE
2
IMMUNOLOGICAL CHARACTERIZATION OF MMTV WITH MONOCLONAL ANTIBODIES TO MMTV
(BALB/c) m36
MMTV
MCI Ab R2.1 M2.1 Ml.1
BALB/c +a +
C3H + + ++---
GR
C3Hf
RI11
A
+ +
+ -
+ -
+ -
Note. Hybridoma supernatant fluids containing monoclonal antibodies reactive with MMTV gp36 were added to microtiter wells containing266 ng each of the various disrupted MMTVs. After 1 hr at 3’7”, unbound antibodies were removed with three successive rinses in PBS and 50,666 cpm of ‘251-labeled Staphylococcus aureus protein A was added to the wells. Following 1 hr at 37”, unbound radioactivity was removed with PBS and the plates were autoradiographed overnight. a +, Counts per minute at least five times the background of 150 cpm.
184
DROHAN ET AL.
instances (Bentvelzen, 19’72a;Fieldsteel et aZ.,1975), there is usually no transmission of exogenous milk-borne virus in this strain, nor is there production of infectious virus by mammary tumors. Although Cohen et al. (1979a) have mapped three endogenous MMTV proviral loci in BALB/c mice, the expression of MMTVrelated viral RNA has been detected in normal tissues and mammary tumors only at very low levels (one to six copies of viral RNA per cell) and usually in the absence of detectable translational products (McGrath et al, 19’78;Dusing-Swartz et al., 1979; Pauley et al, 1979). In this report, we have described the isolation and characterization of virions from spontaneously arising mammary tumors of a subline of BALB/cCrl mice. Three features of this subline are of interest: (a) the incidence of spontaneous mammary adenocarcinomas in the BALB/ cV strain was abnormally high (about 47% at 10 months in breeding females) as compared to other BALB/c strains, (b) mammary tumors contained type B particles characteristic of MMTV, and (c) this MMTV variant was transmitted by milk and not through the female germ cells. This latter observation is in accord with previous observations on the transmission of other MMTV variants (MMTV-S, MMTV-P, MMTV-L) when introduced into the BALB/c strain (Benvelzen and Daams, 1969). Two possible explanations for the expression of MMTV in BALB/cV mice were considered. First, we have identified a subline of BALB/c mice which spontaneously expressed endogenous MMTV information, or second, that the BALB/cV subline contains an infectious MMTV varient. In order to distinguish between these alternatives we characterized virus and mammary tumor tissues of the BALB/cV strain of mice biochemically, immunologically and by restriction endonuclease analysis. Our results indicated the following: (a) virions produced from primary explants of BALB/cV mammary tumors are immunologically distinguishable from all other MMTV variants, (b) [=P]-cDNA probe synthesized from the MMTV(BALB/
c) 60-70 S RNA hybridized to a greater extent with BALB/cV mammary tumor tissue than to BALB/cV normal liver tissue, suggesting the presence of additional viral-specific sequences, perhaps as the results of infection, in BALB/cV mammary tumor, (c) restriction endonuclease analysis of DNA from BALB/cV tissue revealed additional MMTV-specific DNA fragments in BALB/cV mammary tumors not found in DNA from normal BALB/cV livers. The patterns of restriction fragments resulting from digestion of BALB/ cV mammary tumor DNA suggests that the tumor tissue has acquired viral DNA sequences which can be distinguished from those of any endogenous or infectious MMTV variant thus far reported. The virus we have described in this work is distinguishable from all known endogenous and exgenous MMTV variants, and consequently it’s presence in BALB/cV mice can not be explained simply by an infection of BALB/cV mice with one of the laboratory strains of MMTV. It is possible, however, that this may represent a rare instance in the BALB/c population in which an endogenous MMTV is expressed in its infectious form and, subsequent to its escape from germ-line replication, the viral genome became altered by mutation or recombination. ACKNOWLEDGMENTS We wish to thank Mses. D. Rath, S. Reed-Chambers, F. Nicholson, F. Kittrell, J. Thomas, F. Miller, and Mr. J. Taubenberger for their technical assistance. We thank Drs. D. Howard and D. Colcher for their help in some experiments and Dr. R. Callahan for helpful advice. This was supported in part by National Cancer Institute Contracts NOl-CP-43223 and NOl-CP-01018. REFERENCES ANDERVONT,H. B. (1945). Fate of the C3H milk influence in mice of strains of C and C5’7 black. J. Nat. Cancer Inst. 5.383-390. ANDERVONT,H. B., and DUNN, T. B. (1948). Efforts to detect a mammary-tumor agent in strain C mice. J. Nat. Cancer Inst. 3.235-243. BENTVELZEN,P. (1972a). The biology of the mouse mammary tumor virus. ht. Rev. Exp. Path1 11, 259497.
MOUSE MAMMARY TUMOR VIRUS CHARACTERIZATION BENTVELZEN,P. (1972b). Hereditary infections with mammary tumor viruses in mice. In “RNA Viruses and Host Genomes in Oncogenesis” (P. Emmelot and P. Bentvelzen, eds.), pp. 309-337. North American Elsevier, New York. BENTVELZEN, P. (1974). Host-virus interactions in murine mammary tumorigenesis. B&him. Biophys. Acta 355, 236-251. BENTVELZEN,P., and DAAMS, J. H. (1969). Herititary infections with mammary tumor viruses in mice. J. Nat. Cancer Inst. 93,1025-1035. COHEN, J. C. (1980). Methylation of milk-borne and genetically transmitted mouse mammary tumor virus proviral DNA. CeU 19.653-662. COHEN, J. C., MAJORS, J. E., and VARMUS, H. E. (1979a). Organization of mouse mammary tumor virus-specific DNA endogenous to BALB/c mice. J. Virol. 32,483496. COHEN,J. C., SHANK, P. R., MORRIS,V. L., CARDIFF, R., and VARMUS,H. E. (1979b). Integration of the DNA of mouse mammary tumor virus-infected normal and neoplastic tissue of the mouse. Cell 16, 333-345. COHEN,J. C., and VARMUS,H. E. (1979). Endogenous mammary tumor virus DNA varies among wild mice and segregates during inbreeding. Nature (London) 278,418423. COLCHER,D., HORAN HAND, P., TERAMOTO,Y. A., WUNDERLICH, D., and SCHLOM,J. (1981). Monoclonal antibodies define diversity of mammary tumor viral gene products in virions and mammary tumors of the genus Mus. Cancer Res. 41, 14511459. DROHAN, W., KETTMANN, R., COLCHER, D., and SCHLOM,J. (1977). Isolation of the mouse mammary tumor virus sequences not transmitted as germinal provirus in the C3H and RI11 mouse strains. J. ViroL 21, 986-995. DROHAN, W., and SCHLOM,J. (1979). Diversity of mammary tummor viral genes within the genus Mus, the species Mus musculus and the strain C3H. J. Viral. 31, 53-62. DUSING-SWARTZ,S., MEDINA, D., BUTEL, J. S., and SOCHER,S. H. (1979). Mouse mammary tumor virus genome expression in chemical carcinogen-induced mammary tumors in low- and high-incidence mouse strains. Proc. Nat. Acad. Sci. USA 76, 5360-5364. FANNING, T. G., PUMA, J. P., and CARDIFF, R. D. (1980). Selective amplification of mouse mammary tumor virus in mammary tumors of GR mice. J. Viral. 36, 109-114. FIELDSTEEL, A. H., DAWSON,P. J., KURAHARA, C., and BROOKS,R. E. (1975). Unusually high incidence of spontaneous mammary carcinomata in a colony of BALB/c mice. Brit. J. Cancer, 741-771. GAUTSCH,J. W., LERNER,R., HOWARD,D., TERAMOTO, Y. A., and SCHLOM,J. (1978). Strain-specific markers for the major structural proteins of highly on-
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cogenic murine mammary tumor viruses by tryptic peptide analyses. J. ViroL 21.688-699. GRONER,B., and HYNES, N. E. (1980). Number and location of mouse mammary tumor virus proviral DNA in mouse DNA of normal tissue and of mammary tumors. J. ViroL 33,1013-1025. HAGEMAN, P., CALAFAT, J., and DAAMS, J. H. (1972). The mouse mammary tumor viruses. In “RNA Viruses and Host Genome in Carcogenesis” (P. Emmelot and P. Bentvelzen, eds.), pp. 283-300. NorthHolland, Amsterdam. HOWARD, D. K., COLCHER,D., TERAMOTO, Y. A., YOUNG, J. M., and SCHLOM,J. (1977). Characterization of mouse mammary tumor viruses propagated in heterologous cells. Cancer Res. 37,26962704. LOWRY,0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. C/rem. 193,265275. MCGRATH, C. M., MARINEAU, E. J., and VOYLES, B. A. (1978). Changes in MuMTV DNA and RNA levels in Balb/c mammary epithelial cells during malignant transformation by exogenous MuMTV and by hormones. Virology 87.339-353. MEDINA, D. (1976). Preneoplastic lesions in murine mammary cancer. Cancer Res. 36.2589-2595. MICHALIDES,R., VANDEEMTER,L., NUSSE,R., ROPCKE, G., and BOOT, L. (1978). Involvement of mouse mammary tumor virus in spontaneous and hormone-induced mammary tumors in low-mammary-tumor mouse strains. J. Viral. 27,551-559. MOORE,D. H. (1975). Mammary tumor virus In “Cancer: A Comprehensive Treatise” (F. F. Reeker, ed.), pp. 131-167. Plenum, New York. MOORE,D. H., CHARNEY,J., and HOLBEN,J. A. (1974). Titrations of various mouse mammary tumor viruses in different mouse strains. J. Nat. Cancer Inst. 52,1757-1761. MOORE, D. H. SARKAR, N. H., HOLEXN, J. A., and SHEFFIELD,J. B. (1979). Idiopathic mammary tumors in BALB/c mice. Int. J. Cancer 23.713-717. MORRIS, V. L., VLASSCHAERT,J. E., BEARD, C. L., MILAZZO, M., and BRADBURG,W. C. (1980). Mammary tumors from BALB/c mice with a reported high mammary tumor incidence have acquired new mammary tumor virus DNA sequences. Virology 100.101-109. NANDI, S, and MCGRATH, C. M. (1973). Mammary neoplasia in mice. Advan. Cancer Res. 17.353-414. OWENS,R. B., and HACKETT, A. J. (1972). Tissue culture studies of mouse mammary tumor cells and associated viruses. J. Nat. Cancer Inst. 49, 13211332. PAULEY, R. J., MEDINA, D., and SOCHER,S. (1979). Murine mammary tumor virus expression during mammary tumorigenesis in BALB/c mice. J. ViroL 29,483-493.
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PETERS,R. L., RABSTEIN,0. S., SPAHN, G. J., MADISON,R. M., and HUEBNER, R. J. (1972). Incidence of spontaneous neoplasms in breeding and retired breeder BALB/c mice throughout the natural life span. Znt. J. Cancer 10,273-279. 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. SOUTHERN,E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. BioL 38.503-517. TERAMOTO,Y. A., KUFE, D., and SCHLOM,J. (1977). Type-specific antigenic determinations on the ma-
jor external glycoprotein of high- and low-oncogenie murine mammary tumor viruses. J. ViroL 24, 525-533. TERAMOTO,Y. A., PUENTES,M. J., YOUNG, L. J. T., and CARDIFF, R. D. (1974). Structure of the mouse mammary tumor virus: Polypeptides and glycoproteins. .Z. ViroL 13,411-418. TERAMOTO,Y. A., and SCHLOM,J. (1978). Radioimmunoassays demonstrating type-specific and groupspecific antigenie reactivities for the major internal structural protein of murine mammary tumor viruses. Cancer Res. 38.1990-1995. TERAMOTO,Y. A., and SCHLOM,J. (1979). Radioimmunoassays for the 36,996 dalton glycoprotein of murine mammary tumor viruses demonstrate type, group, and interspecies determinants. J. Viral. 31, 334-340.
114. 175-186 (1981)
Isolation
and Characterization of a New Mouse Tumor Virus from BALB /c Mice
W. DROHAN,* Y. A. TERAMOTO,* D. MEDINA,? ‘Laboratuq
Mammary
J. SCHLOM**’
AND
of CeUular and Mokcular Bidogy, Nation& Cancer Institute, National Institutes of Health, Beth&a, Malylnnd 21l%l5, and fBaylm College of Medicine, Houston, Texas 77080
Received February 22, 1981; accepted May 29, 1981 A novel mouse mammary tumor virus (MMTV) has been isolated from mice of a subline of the BALB/cCrl Med mouse strain designated BALB/cV. Whereas breeding females of the parent BALB/cCrl Med colony have a mammary tumor incidence of l%, 47% of the breeding females of the BALB/cV subline develop mammary tumors before 10 months of age. Foster nursing experiments demonstrated this virus, termed MMTV (BALB/c), was transmitted only by milk. The novel MMTV isolate was shown to be immunologically related to, but distinct, from the MMTV variants of C3H, GR, and RI11 mice by a series of competition radioimmunoassays for the MMTV 28,000-dalton major core protein (p28), and the 52,000 (gp52)- and 36,OOGdalton (gp36) major envelope glycoproteins. Monoclonal antibodies directed against MMTV gp36 were also used to clearly distinguish MMTV(BALB/ c) from MMTV(CBH), MMTV(RIII), MMTV(GR), MMTV(C3HfC57BL). and MMTV(A). MMTV-specific proviral DNA content of mammary adenocarcinomas arising in the BALB/cV subline was examined with restriction endonucleases and the Southern blot technique, and compared to the MMTV proviral DNA content of BALB/cAnDe mammary tumors. The virus arising from these latter tumors has been termed MMTV(0). Analysis of EcoRI digests of high-molecular-weight DNA from both types of mammary tumors demonstrated additional MMTV-related proviral sequences when compared to the DNA of normal BALB/c tissues. The patterns generated with the restriction endonuclease Sac1 distinguished the additional MMTV-specific proviral information in the mammary tumors of the BALB/cV mice from the proviral information in tissues containing the GR, C3H, or RI11 MMTVs, as well as from the proviral information in the BALB/cAnDe mammary tumors. These immunological and molecular studies thus define MMTV(BALB/c) as a novel MMTV variant.
of its spontaneous mammary adenocarcinomas. Although BALB/c mice are highly susceptible to infection by the oncogenic milk-borne mouse mammary tumor viruses (MMTVs) of the high mammary tumor incidence strains, most colonies of BALB/c mice are believed to be free of infectious virus (Moore et al., 1974, 1979; Moore, 1975; Medina, 1976). BALB/c mice, however, contain endogenous proviral information related to the MMTVs of other mouse strains (Michalides et al., 1978; Cohen et al., 1979a, b; Groner and Hynes, 1980; Morris et al, 1980). This information is expressed at a low level in normal BALB/c mammary tissues, and mammary tumors, but has not been etiologically
INTRODUCTION
Although BALB/c mice have been characterized as a low-incidence mammary tumor strain (Andervont, 1945; Bentvelzen, 1972a; Nandi and McGrath, 1973), the tumor incidence reported in different colonies has varied from 1% to as high as 30% (Andervont and Dunn, 1948; Bentvelzen, 1972b; Peters et aZ., 1972; Nandi and McGrath, 1973; Moore, 1975). Significantly, the BALB/c strain differs from the high mammary tumor incidence strains, C3H, GR, and RIII, in the late appearance ‘To whom dressed.
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be ad-
175
0042~6822/81/130175-12$02.00/O Copyright All rights
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DROHAN
176
linked to mammary tumorigenesis (McGrath et al., 1978; Michalides et al., 1978; Dusing-Swartz et al., 1979; Pauley et al., 1979). Occasionally, an infectious virus (sometimes referred to as MMTV(0)) has been recovered from BALB/c mice, which has been associated with a high mammary tumor incidence (Hageman et al., 1972; Fieldsteel et aZ., 1975). In this study we have examined MMTVrelated proviral information in a substrain of BALB/cCrl Med mice in which the incidence of spontaneous adenocarcinomas is almost 50% in breeding females at 10 months of age. We also compared by restriction endonuclease analyses, the proviral information of BALB/cCrl Med mammary tumors, of BALB/cAnDe mammary tumors induced by MMTV(O), and of normal BALB/c tissues. By using both molecular and immunological techniques, evidence is presented that the BALB/c virus represents a novel MMTV variant indigenous to BALB/c. The BALB/c subline has been designated BALB/cV to emphasize the presence of an infectious virus, and to distinguish it from other BALB/c sublines which express an infectious virus occasionally (MMTV[O] in BALB/cAnDe) and continuously (BALB/cNIV). MATERIALS
AND METHODS
Tissues. The BALB/cV tumors used in these experiments arose in lo-month-old breeding females and were transplanted subcutaneously into virgin BALB/cCrl Med mice. Frozen mammary tumors induced by the MMTV(0) variant found in BALB/ cAnDe were obtained from Dr. P. Bentvelzen, The Radiobiological Institute TNO, Rijswijk, The Netherlands. Normal BALB/ cV female mice were used as controls as well as BALB/cCR mice obtained from the Charles River Breeding Laboratories, Wilmington, Massachusetts. Nucleic acid studies were performed on three different pools of a transplanted BALB/cV mammary tumor. Normal GR/N mice were obtained from the National Institutes of Health through the National Cancer Institute Office of Resources and Logistics. Feline cells were infected with the RI11 and C3H variants of MMTV as previously
ET AL.
described (Howard et al., 1977). A D2 mammary tumor which has arisen from the D2 hyperplastic outgrowth line, established by Medina (1976) from a hormonally stimulated BALB/c mouse, was also examined. Viruses. MMTV(C3H) was obtained from supernatant fluids of the C3H mammary tumor cell line Mm5mt/c1 (Owens and Hackett, 1972). MMTV(RII1) was obtained from the milk of multiparous RI11 mice. MMTV(BALBicfC3H) was obtained from supernatant fluids of primary cultures of BALB/cfC3H mammary tumors. MMTV(BALB/c) was obtained from supernatant fluids of primary cultures (Howard et ah, 1977) of serial transplanted mammary tumors from BALB/cV mice. MMTV was concentrated and purified from supernatant fluids of mammary tumor cultures by ammonium sulfate precipitation and a series of discontinuous and linear sucrose density gradients as previously described (Drohan et al., 1977). MMTV(RIII) was purified from mouse milk following treatment of the milk with EDTA (Teramoto et aZ.,1977). Tumor extracts. Mammary tumors were finely minced and homogenized for 2 min on ice in 0.01 M Tris, pH 7.2, containing 0.0002 M CaClz. A volume of buffer equal to at least 10 times the tissue weight resulted in optimal homogenization. The homogenate was further disrupted by nitrogen cavitation using a cell disruption bomb (Parr Institute) at 1000 lb/in? for 5-15 min. The homogenate was centrifuged at 1000 g for 5 min and the protein in the supernatant was quantitated by the method of Lowry et al. (1951). Radioimmunoassay. The techniques of iodination and competition radioimmunoassays for purified MMTV gp52, gp36, and ~28 have been previously described (Teramoto et al., 1977; Teramoto and Schlom, 1978; Teramoto and Schlom, 1979). Briefly, competitors were added to limiting dilutions of the appropriate antisera and incubated at 37” for 1 hr. Radioactive control antigen was then added and the incubation continued for another hour at 37”. Following the addition of appropriately diluted anti-immunoglobulin, the in-
MOUSEMAMMARYTUMORVIRUSCHARACTERIZATION
177
60-70 S RNA produced identical patterns when hybridized to restricted murine DNA and analyzed by the technique of Southern (1975). In most cases, the [32P]MMTVcDNA probe did not appear to contain detectable amounts of ribosomal or other normal cellular RNAs as indicated by the absence of hybridization to normal feline tissue which does not contain MMTV sequences (Fig. 2, lane H). Furthermore, a [32P]cDNA probe synthesized from 28 S and 18 S murine ribosomal RNA, was hybridized to an EcoRI digest of high-molecular-weight murine DNA, and revealed no bands in positions corresponding to the MMTV-related DNA fragments presented in Fig. 2. Filters were soaked in 3X SSC at 65” for 30 min and then prehybridized in 3~ SSC, 0.1% SDS, 100 pg/ml denatured and sonicated canine DNA, and Denhardt buffer (0.2% each of bovine serum albumin, Fi~011,and polyvinylpyrrolidine) for 4 hr at 65”. [=P]MMTV cDNA was added at 4 X lo6 cpm/ml and hybridized to the filter for 15 hr. Filters were then washed with Gel electrophoresis, DNA transfer, and 3X SSC, 0.1% SDS, and Denhardt’s soluhybridization. After restriction endonu- tion at 5-min intervals until the wash clease digestion, DNAs were adjusted to buffer contained only 100 cpm 32P/ml. The 20% glycerol and electrophoresed in 0.8% filters were then washed with 0.3~ SSC, agarose (BRL) containing 0.02 M sodium 0.1% SDS at 15-min intervals until the acetate, 0.002 M EDTA, 0.018 M Tris base, wash buffer contained only 100 cpm 32P/ and 0.028 M Tris-HCl (pH 8.05). Bromo- ml. Filters were air dried and were autophenol blue was added as tracking dye and radiographed 75 hr with Kodak X-OMAT gels were electrophoresed at 35 V until the R film at -70”. dye migrated 13 to 16 cm. Ethidium broExtraction of high-molecular-weight mide (0.005 mg/ml) staining was used to DNA. Tissues were homogenized in DNA visualize the X marker DNAs after elec- extraction buffer (0.02 M Tris-HCl, pH 8.5, trophoresis. DNA was transferred to ni- 0.1 M NaCl, and 0.001 M EDTA) using a trocellulose filter paper by the method of Waring blender. Samples were incubated Southern (1975). Gels were treated with at 37’ for 120 min with 0.5% SDS, 1.0% denaturing solution (1.5 M NaCl, 0.5 M mercaptoethanol, and 1.0 mg/ml prediNaOH) for 20 min, neutralized (3.0 M gested Pronase. DNA was extracted by NaCl, 0.5 M Tris-HCl, pH 7.5) for 40 min, shaking the aqueous phase with l/2 vol of and treated with the 6X SSC buffer (SSC, phenol and l/2 vol of chloroform for 5 min, 0.15 M NaCl, 0.015 M sodium citrate) for centrifugation at 3400g for 15 min, and 10 min. The DNAs were transferred from removal of the aqueous phase. Extractions the gel to the nitrocellulose paper with 6X were repeated until the interphase was SSC for 15 hr. The filters were dried in a clear. DNA was precipitated in 2 vol ethyl alcohol. High-molecular-weight DNA was vacuum oven at 80” for 4 hr. Viral-specific DNA sequences were de- spooled out with a glass rod and was dissolved in 0.1X SSC. tected by hybridization of the DNA filters Competition hybridization. Hybridizato 32P-labeled DNA complementary to MMTV(C3H) 70 S RNA (MMTV cDNA). tion conditions are as described above for liquid hybridization. Briefly, unlabeled [32P]MMTV cDNA and ‘%I-MMTV(C3H)
cubation was continued for 1 hr at 37” and at 4” overnight. Following centrifugation at 10,OOOgfor 1.5 min, the supernatants were aspirated, and the pellets assayed for radioactivity. Liquid hyti’dixation. The technique for liquid hybridization has been previously described (Drohan and Schlom, 1979). Restriction endonuclease digestion of DNA. EcoRI restriction endonuclease was obtained from Boehringer-Mannheim (Indianapolis, Ind.). Sac1 was purchased from Biotec, Madison, Wisconsin. Conditions for Sac1 digestion were as provided by the manufacturer. EcoRI digestions were performed in 0.1 M Tris-HCl (pH 7.5), 0.05 M NaCl, 0.005 M MgClz, and 100 pg/ml bovine serum albumin (BSA). EcoRI restrictions at 5-10 units of enzyme per microgram DNA were incubated 4 hr at 37”. Ten micrograms of DNA was loaded per lane. EcoRI-restricted X bacteriophage DNA (New England Biolabs., Beverly, Mass.) was used to provide marker bands for molecular-weight determinations.
178
DROHAN
competitor RNA was added in increasing amounts to the reaction mixture which already contained 1000 cpm of [3H]MMTV(C3H) 60-70 S RNA and 100 pg of C3H mammary tumor cell line DNA (3 mg/ml). The hybridization reaction was carried out to a C,,t of 28,000 mol. sec. liters-’ and assayed for the acquisition of RNase resistance. Competition was determined by comparing the percentage of hybridization in the presence of competitor RNA with that of the control sample [MMTV 3H-labeled 60-70 S RNA hybridized to DNA from C3H mammary tumor cells (greater than 50% hybridization)], after subtracting the background hybridization of less than 5% [[3H]MMTV(C3H) 60-70 S RNA hybridized to canine DNA]. RESULTS
History of the BALB/cV Substrain Transmission of MMTV(BALB/c)
and
Two pairs of BALB/cCrl breeding mice, with litters, were received from the Cancer Research Laboratory, Berkeley, California, in 1970. The mice were subsequently brother and sister mated to establish a breeding colony. A mammary tumor arose in one female, from the original two litters, at 10 months of age. This mouse and its progeny were isolated and maintained as a separate subline, now designated as BALB/cV, to emphasize the presence of an infectious virus. Since 1970, this BALB/ c subline has been maintained at the Baylor College of Medicine, Houston, Texas. Through generations F4 to Fzo the mammary tumor incidence in breeding females was 47% (111/234) with mean age of tumor appearance of 10.2 months and a mean number of litters of 6.3. In contrast, BALB/cCrl Med mice have a 1% spontaneous tumor incidence in retired breeders by 15 months of age (Pauley et aZ.,(1979). All tumors that arose in BALB/cV mice were type B mammary adenocarcinomas. Electron microscopic examination of mammary tumors occurring in BALB/ cV at Fi, Fq, and Flz mice demonstrated the presence of budding and mature B particles characteristic of MMTV (Fig. 1). In order to determine the route of transmis-
ET AL.
sion of MMTV (BALB/c), reciprocal foster-nursing experiments were carried out between BALB/c and BALB/cV. BALB/cV female newborns were foster-nursed on lactating BALB/c mice by the drop-cage method. The reciprocal groups were also examined. The BALB/cVf and BALB/cf female mice were mated with the male BALB/c mice and checked weekly for the presence of mammary tumors. In addition, the fourth litter (designated F,) from both groups were saved, bred, and the mammary tumor incidence recorded. The data (Table 1) show that MMTV(BALB/c) is transmitted via the milk and can be eliminated by foster-nursing the infected mice on uninfected BALB/c female mice. The mammary tumor incidence dropped from 59% in the BALB/cfBALB/cV to 0% in BALB/cVfBALB/c. Female mice derived from the fourth litters of the two groups also exhibited similar tumor incidences.
Restriction Endonuclease BALB/c DNAs
Analysis
of
Restriction endonuclease analysis of DNA from normal and tumor bearing BALB/c tissue was undertaken to examine their MMTV-specific proviral DNA content. The restriction endonuclease EcoRI cleaves MMTV DNA at a single site, and thus, can be used to quantitate MMTVspecific proviruses in cellular DNA. The DNAs of normal tissues and mammary tumors of BALB/c mice were compared by EcoRI restriction analysis. As shown in Fig. 2, lane E, radioactive MMTV cDNA hybridizes to fragments of 14.5, 8.7, 7.2, 6.8, 5.8, and 3.0 kilobase pairs (kbp) in digest’s of liver DNA from nontumor-bearing BALB/cCR mice. Identical restriction profiles were obtained from the DNA of livers and other apparently normal tissues of mammary tumor-bearing BALB/cV mice. This restriction pattern is unique to BALB/c mice (Cohen et al., 1979a, b; Morris et al., 1980) and defines the endogenous MMTV provirus content for these tissues. The EcoRI restriction pattern from DNA of a transplanted mammary tumor of BALB/cV mice was found to contain two restriction fragments of 10.9 and 10.3 kbp
MOUSE
MAMMARY
TUMOR
VIRUS
179
CHARACTERIZATION
FIG. 1. Electron microscopy of tumors arising in BALB/cV female mice demonstrates the presence of budding and mature B particles characteristic of MMTV. A and B focus on B particles found on the apical cell membranes and lumina of mammary tumor cells. Bars represent 0.1 km.
tissue DNA. The presence of the additional EcoRI restriction fragments in the BALB/ c mammary tumor DNAs (Fig. 2) suggested that all three mammary tumor tissues have either acquired exogenous viral information by infection or that endogenous BALB/c information has been amplified and reintegrated into high-molecular-weight host DNA. The restriction endonuclease PstI has been shown to cleave MMTV proviral DNA at multiple internal sites (Shank et al., 19’78; Cohen and Varmus, 1979; Cohen et ah, 1979a). Pst-I digests of DNAs from
(Fig. 2, lane F), in addition to the fragments observed in apparently normal tissues of the same mice. DNA from mammary tumors which developed from a transplantable BALB/cD2 hyperplastic alveolar nodule (Medina, 1976), and from mammary tumors induced by the MMTV(0) variant of BALB/c mice (Bentvelzen, 1974) also contained several addition EcoRI restriction fragments (Fig. 2, lanes G and D, respectively) which distinguished them from each other, from the BALB/cV mammary tumor DNA, and from the apparently normal BALB/cCR TABLE
1
MAMMARY TUMOR INCIDENCE IN FOSTER-NURSED BALB/cV
Group BALB/cVfBALB/c BALB/cfBALB/cV BALB/cVfBALB/cFl” BALB/cfBALB/cVF,’
No. tumors/ No. mice o/21 16/27 l/28 23/29
%T
Average No. litters/ mouse
0 59 4 79
a The F1 female mice were derived from the fourth
4.2 3.7 8.5 7.0 litters
AND BALB/c
FEMALE MICE
Average latent period of tumor formation (months)
Average age of mice dying without tumors (months)
0 10.0 14.0 9.8
17.4 15.0 13.8 13.8
of the BALB/c-fostered
mice.
130
DROHAN
ABCD
K’:t+a= E F G H -14.6,
-I).7,7.2-
-e..S-6.8’
/
9.0
FIG. 2. MMTV containing DNA fragments in EcoRIdigested BALB/c mammary tumors. Ten micrograms of DNA was electrophoresed on 0.8% agarose gels and transferred to nitrocellulose filter paper by the method of Southern (1975) and then hybridized to 4 X lo6 cpm/ml MMTV [ssP]cDNA. Lane A, Normal GR liver; lane B, MMTV(RIII)-infected feline cells; lane C, MMTV(CIH)-infected feline cells; lane D; BALB/ c(O)-induced mammary tumor; lane E, normal BALB/ cCR liver; lane F, BALB/cV mammary tumor; lane G, BALB/cD2 mammary tumor; lane H, uninfected feline cells. Fragments of X phage DNA cleaved with EcoRI served as size markers on ethidium bromidestained gels.
mammary tumors resulting from infection by MMTV(C3H), MMTV(RIII), and MMTV(GR) proviruses yield distinctive restriction fragments in addition to those corresponding to endogenous MMTV proviral DNA. These unique fragments are also in digests of MMTV-infected feline cells in culture. The unique MMTV(C3H) proviral DNA fragments are 3.9 and 0.9 kbp (Cohen and Varmus, 1979) while those of MMTV(RII1) are 3.6 and 0.9 kbp. The presence of any of these unique fragments in Pst-I-digested DNAs from BALB/c mammary tumors and their absence in DNA from normal BALB/cV or BALB/ cCr tissues would suggest that these tumors were infected with a nongerm line transmitted virus. MMTV-(O)-induced, BALB/cV, and BALB/cD2 mammary tu-
ET AL.
mors each contained a 3.9- and a 0.9-kbp Pst-I restriction fragment in addition to the internal PstI fragments of 5.1,4.8, 1.7, 1.3, and 0.8 kbp found in the DNAs from normal internal organs of normal or tumor-bearing BALB/c mice. This result is consistent with the BALB/cV and BALB/ c-D2 mammary tumors being infected with an exogenous variant of MMTV. To determine if we could demonstrate additional MMTV-specific DNA fragments unique to the BALB/cV mammary tumors we digested BALB/cV, BALB/cD2, and MMTV(O)-induced BALB/c mammary tumors with the restriction endonuclease SucI. Digestion of DNA from tissue culture cells infected with the MMTV(C3H) variant with the enzyme Sac1 reveals a unique restriction fragment of approximately 8.0 kbp. This 8.0-kbp band reflects the presence of a Sac1 restriction site in the long terminal repeat (LTR) of MMTV(C3H) proviral DNA and has been observed only in Sac1 digests of DNA from spontaneous C3H mammary tumors or DNA from feline tissue culture cells infected with MMTV(C3H). We have examined the pattern of MMTV-specific fragments in Sac1 digests of BALB/c mammary tumor and normal tissue DNA for the presence of the 8.0-kbp fragment. MMTV-specific restriction bands of 9.6, 7.4, and 5.8 kbp are observed in DNA of all normal BALB/c tissues, as well as in BALB/cV and D2 mammary tumors. However, in multiple Sac1 digests of DNA from the MMTV(O)-induced mammary tumor there was a pronounced restriction fragment at 8.0 kbp (Fig. 3, lanes D and D’). Except for tissues infected with the MMTV(C3H), the MMTV(O)-induced mammary tumor was the only tissue examined thus far which gave rise to a 8.0kbp Sac1 restriction fragment. These fmdings clearly differentiated BALB/cV mammary tumor proviral DNA from that of the MMTV(O)- and MMTV(C3H)-induced mammary tumors and suggests that the MMTV(0) virus may be related to the MMTV( C3H). The restriction endonuclease B&II was also used to examine the MMTV-specific DNA content of MMTV(O)-induced mam-
MOUSE A B
C
MAMMARY
TUMOR
D E F (3 H D’ E’
VIRUS
CHARACTERIZATION
181
Liquid H&ridixation of Radioactive MMTV(CSH) RNA to BALB/cV and C3H Mammary Tumors
By restriction endonuclease analysis we have been able to distinguish MMTV(C3H) from MMTV(BALB/c). However, to precisely examine the nucleotide sequence homology shared between the two variants, the kinetics of liquid hybridization *.*experiments were examined. Using 3H-la*abeled 60-70 S MMTV(C3H) RNA as a probe, the final extent of hybridization with DNA from C3H mammary tumors was ‘79% with a C,-,tmof 150. In contrast, only 41% of this RNA hybridized with DNA from BALB/cV mammary tumor, with a C,t, of 800. The difference in the FIG. 3. MMTV containing DNA fragments in SacIextent of hybridization of the MMTV(C3H) digested BALB/c mammary tumors. Lane headings (A-H)and conditions as described in the legend to RNA suggests that the BALB/cV mamFig. 2. Lanes D’ and E’ are the same as lanes D and mary tumor DNA does not contain all E except that the hybridized filter was exposed to Xthe MMTV(C3H)-specific nucleic acid seray film for a shorter period of time to clearly resolve quences present in the C3H mammary the presence of an 8.0-kbp MMTV containing DNA tumor DNA. The stability of the hybrids fragments in the BALB/c(O)-induced mammary tuformed with the two DNAs was assesed mor (lane D’). All DNA samples were run on the to determine the extent of base pair missame gel. match. The T,,, of the hybrid between MMTV(C3H) 60-70 S RNA and DNA from mary tumors, as well as BALB/cV and a C3H mammary tumor was 81.6”, whereas hybrids formed with DNA from BALB/cV BALB/cD2 mammary tumors. In addition to endogenous MMTV-specific DNA found mammary tumors had a T,,, of 74.5”. Since in all BALB/c tissue, an intense 4.3-kbp a 1” reduction in T, has been shown to be equivalent to approximately 1.0 to 1.5% DNA fragment was found in MMTV(O)induced mammary tumors; no such frag- base mismatching (Drohan et aZ.,1977), we conclude that there is approximately a ment was present in BALB/cV or BALB/ cD2 mammary tumors. Digestion of DNA 7-10s base pair mismatch between from normal or mammary tumor tissues MMTV(C3H)RNA and BALB/cV mamcontaining the infectious GR, RIII, or C3H mary tumor DNA. In order to precisely compare the perMMTV variants with the restriction endonuclease BglII produces an equally in- centage homology shared between the tense 4.3-kbp fragment (Cohen et aZ.,19’79a; RNA genomes of the MMTV(C3H) and Fanning et al., 1980; Drohan and Schlom, MMTV(BALB/c), RNA was extracted from unpublished data). However, a 4.3-kbp virus produced from primary cultures of MMTV-containing DNA fragment, com- BALB/cV mammary tumors and composed of about 75% cellular DNA, is found pared to the RNA of MMTV(C3H) virus endogenously in all BALB/c DNA. Al- by competitive hybridization assay. Both though, using intensity differences of spe- unlabeled viral RNAs were used as comcific bands on Southern blots to suggest petitors for the hybridization between 3Hthe absense or presence of a unique virus labeled MMTV(C3H) 60-70 S RNA and is tenuous, these data add further evidence C3H mammary tumor DNA (Fig. 4). that the BALB/cV and BALB/cD2 mam- MMTV(C3H) RNA competed greater than mary tumors are not infected with either 97%, while MMTV(BALB/c) viral RNA competed only 82%. Therefore, approxithe GR, RIII, of C3H variants of MMTV.
182
DROHAN
0
0 /
1W 0 50100
500
1000
COMPETING
molecular-weight 60-70 S RNA, and a magnesium-dependent reverse transcriptase. Furthermore, the electrophoretic profile of MMTV(BALB/c) polypeptides from that of was indistinguishable MMTV(C3H). MMTV(BALB/c) thus has biochemical characteristics identical to those of other MMTVs (Teramoto et al., 1974).
7
0
: i : 1
15co
2coo
2500
VIRAL RNA IngI
FIG. 4. Competition hybridization of the MMTV(C3H) genome. One thousand counts per minute of ‘zI-labeled MMTV(C3H) 60-70 S RNA was hybridized in 100 Fg of DNA (3000 pg/ml) from the C3H mammary tumor cell line, MmBmt/q. To this reaction, increasing amounts of unlabeled viral RNAs were added. Hybridizations were conducted as described under Materials and Methods to a final C,t of 28,000 mol. sec. liter-‘. Symbols for competitor RNAs: MMTV(C3H) 60-70 S RNA (0); MMTV(BALB/ c) total viral RNA (A); AMV 60-70 S RNA (m); MuLV(C3H)lOT1/2 60-70 S RNA (0).
mately 15% of the RNA genomes of MMTV(C3H) and the BALB/c virus appear to be different. It should be noted that when 3H-labeled 60-70 S RNA from MMTV(BALB/c) was hybridized to the DNA from BALB/cV mammary tumors and to apparently normal internal organs of BALB/cV mammary tumor-bearing mice, the probe hybridized 65% (C&/z = 760) to the tumor DNA while hybridizing only 35% (C&z = 980) to apparently normal DNA from the tumor-bearing animal. These results are consistent with the presence of the two additional EcoRI bands in the BALB/cV mammary tumor DNA (see Fig. 2).
Biochemical Characterization MMTV(BALB/c)
ET AL.
of
MMTV(BALB/c) purified from supernatant fluids of primary BALB/cV mammary tumors had a buoyant density in sucrose of 1.17 g/ml, contained high-
Immunological Characterization MMTV(BALB/c)
of
Purified virus from supernatant fluids of BALB/cV cultures was tested for its ability to compete in group-specific radioimmunoassays (RIAs) for MMTV gp52, MMTV gp36, and MMTV ~28. All three of these RIAs were performed under conditions whereby MMTV group-specific antigenic determinants could be detected; all three assays have been previously shown to be highly specific for MMTV antigens (Teramoto et al., 1977; Teramoto and Schlom, 1978, 1979). The immunological reactivity of MMTV(BALB/c) virus from that of was indistinguishable MMTV(C3H) and all other MMTVs tested in all three RIAs. Almost identical quantities of virus preparations caused similar levels of inhibition in each RIA. Mammary tumors of BALB/cV mice were also immunologically analyzed. Cell-free extracts of BALB/cV mammary tumors contained MMTV gp52, gp36, and ~28 as determined by the appropriate RIAs. Approximately l-3% of the mammary tumor extract protein corresponded to antigens related to MMTV virion associated gp52, gp36, and ~28. Purified virus from BALB/cV cultures were also assayed in the MMTV type-specific gp52 and gp36 RIAs (Fig. 5). The MMTV(BALB/c) preparation did not compete as efficiently as MMTV(C3H) or MMTV(BALBicfC3H) in either the gp52 (Fig. 5A), or the gp36 (Fig. 5B) MMTV type-specific RIAs. The fact that MMTV(BALBicfC3H) could be distinguished from the MMTV(BALB/c) virus is noteworthy since both virions were obtained from BALB/c mice, thus ruling out the factor of host-strain-specific differ-
MOUSE
1
2
3
4
5
MAMMARY
1
2
TUMOR
3
4
5
LOG COMPETITOR PROTEIN (ng)
for MMTV FIG. 5. Type-specific radioimmunoassays polypeptides. Purified virus preparations were used as competitors in the following type-specific RIAs: (A) type-specific gp52 RIA using anti-MMTV(C3H) at a dilution of 1:10,606 to bind ?-labeled MMTV(C3H) virions; (B) type-specific gp36 RIA using anti-MMTV(C3H) at a dilution of 1:10,666 to bind ‘%I-labeled MMTV(C3H) gp36. MMTV(C3H) (o), MMTV(BALB/fC3H) (m), MMTV(RII1) (0), MMTV(BALB/c) (A).
ences. In the group-specific RIAs, this same preparation of MMTV(BALB/c) competed identically to MMTV(C3H). Thus, the MMTV(BALB/c) appeared to also have some antigenic determinants that are not identical to those of MMTV(C3H). The MMTV(BALB/c) was further immunologically characterized with several monoclonal antibodies. These antibodies were synthesized by hybridoma cell cultures derived from cell fusions involving spleens from animals immunized with disrupted MMTV(C3H). A series of different monoclonal antibodies have been used in a solid-phase radioimmunoassay to identify antigenic differences among MMTVs isolated from several different mouse strains (Colcher et al, 1980). With the combined use of the appropriate monoclonal antibodies, the MMTV(BALB/c) could be readily distinguished from the MMTVs of C3H, C3Hf, GR, RIII, and A strains of mice (Table 2). Of the various MMTVs tested, MMTV(BALB/c) was the only virus which showed differential reactivity with monoclonal antibodies M2.1 and M1.l. Monoclonal antibody M2.1 had an endpoint titer of 1:12,800, while Ml.1 had an endpoint titer of 1:800 as tested in solid-
VIRUS
183
CHARACTERIZATION
phase radioimmunoassay against identical MMTV(C3H) virus preparation. Although M2.1 had approximately a 16-fold higher amount of antibody, it did not react with MMTV(BALB/c) while Ml.1 reacted positively with MMTV(BALB/c). Thus, the differential reactivity observed with M2.1 and Ml.1 with MMTV(BALB/c) could not be attributed to differences in titer between the two monoclonal antibodies. Both of these antibodies recognized typespecific antigenic determinants on MMTV gp36, in contrast to monoclonal antibody R2.1, which recognized group-specific antigenic determinants on MMTV gp36. These data are thus consistent with the results obtained in liquid competition RIAs. The BALB/cV isolate thus appears to have antigenic and nucleic acid sequences unique among all MMTVs thus far characterized and is therefore termed MMTV(BALB/c). DISCUSSION
BALB/c mice are generally considered a model tumor strain for investigating low-incidence spontaneous mammary tumors (Bentvelzen, 1972a). Except for two TABLE
2
IMMUNOLOGICAL CHARACTERIZATION OF MMTV WITH MONOCLONAL ANTIBODIES TO MMTV
(BALB/c) m36
MMTV
MCI Ab R2.1 M2.1 Ml.1
BALB/c +a +
C3H + + ++---
GR
C3Hf
RI11
A
+ +
+ -
+ -
+ -
Note. Hybridoma supernatant fluids containing monoclonal antibodies reactive with MMTV gp36 were added to microtiter wells containing266 ng each of the various disrupted MMTVs. After 1 hr at 3’7”, unbound antibodies were removed with three successive rinses in PBS and 50,666 cpm of ‘251-labeled Staphylococcus aureus protein A was added to the wells. Following 1 hr at 37”, unbound radioactivity was removed with PBS and the plates were autoradiographed overnight. a +, Counts per minute at least five times the background of 150 cpm.
184
DROHAN ET AL.
instances (Bentvelzen, 19’72a;Fieldsteel et aZ.,1975), there is usually no transmission of exogenous milk-borne virus in this strain, nor is there production of infectious virus by mammary tumors. Although Cohen et al. (1979a) have mapped three endogenous MMTV proviral loci in BALB/c mice, the expression of MMTVrelated viral RNA has been detected in normal tissues and mammary tumors only at very low levels (one to six copies of viral RNA per cell) and usually in the absence of detectable translational products (McGrath et al, 19’78;Dusing-Swartz et al., 1979; Pauley et al, 1979). In this report, we have described the isolation and characterization of virions from spontaneously arising mammary tumors of a subline of BALB/cCrl mice. Three features of this subline are of interest: (a) the incidence of spontaneous mammary adenocarcinomas in the BALB/ cV strain was abnormally high (about 47% at 10 months in breeding females) as compared to other BALB/c strains, (b) mammary tumors contained type B particles characteristic of MMTV, and (c) this MMTV variant was transmitted by milk and not through the female germ cells. This latter observation is in accord with previous observations on the transmission of other MMTV variants (MMTV-S, MMTV-P, MMTV-L) when introduced into the BALB/c strain (Benvelzen and Daams, 1969). Two possible explanations for the expression of MMTV in BALB/cV mice were considered. First, we have identified a subline of BALB/c mice which spontaneously expressed endogenous MMTV information, or second, that the BALB/cV subline contains an infectious MMTV varient. In order to distinguish between these alternatives we characterized virus and mammary tumor tissues of the BALB/cV strain of mice biochemically, immunologically and by restriction endonuclease analysis. Our results indicated the following: (a) virions produced from primary explants of BALB/cV mammary tumors are immunologically distinguishable from all other MMTV variants, (b) [=P]-cDNA probe synthesized from the MMTV(BALB/
c) 60-70 S RNA hybridized to a greater extent with BALB/cV mammary tumor tissue than to BALB/cV normal liver tissue, suggesting the presence of additional viral-specific sequences, perhaps as the results of infection, in BALB/cV mammary tumor, (c) restriction endonuclease analysis of DNA from BALB/cV tissue revealed additional MMTV-specific DNA fragments in BALB/cV mammary tumors not found in DNA from normal BALB/cV livers. The patterns of restriction fragments resulting from digestion of BALB/ cV mammary tumor DNA suggests that the tumor tissue has acquired viral DNA sequences which can be distinguished from those of any endogenous or infectious MMTV variant thus far reported. The virus we have described in this work is distinguishable from all known endogenous and exgenous MMTV variants, and consequently it’s presence in BALB/cV mice can not be explained simply by an infection of BALB/cV mice with one of the laboratory strains of MMTV. It is possible, however, that this may represent a rare instance in the BALB/c population in which an endogenous MMTV is expressed in its infectious form and, subsequent to its escape from germ-line replication, the viral genome became altered by mutation or recombination. ACKNOWLEDGMENTS We wish to thank Mses. D. Rath, S. Reed-Chambers, F. Nicholson, F. Kittrell, J. Thomas, F. Miller, and Mr. J. Taubenberger for their technical assistance. We thank Drs. D. Howard and D. Colcher for their help in some experiments and Dr. R. Callahan for helpful advice. This was supported in part by National Cancer Institute Contracts NOl-CP-43223 and NOl-CP-01018. REFERENCES ANDERVONT,H. B. (1945). Fate of the C3H milk influence in mice of strains of C and C5’7 black. J. Nat. Cancer Inst. 5.383-390. ANDERVONT,H. B., and DUNN, T. B. (1948). Efforts to detect a mammary-tumor agent in strain C mice. J. Nat. Cancer Inst. 3.235-243. BENTVELZEN,P. (1972a). The biology of the mouse mammary tumor virus. ht. Rev. Exp. Path1 11, 259497.
MOUSE MAMMARY TUMOR VIRUS CHARACTERIZATION BENTVELZEN,P. (1972b). Hereditary infections with mammary tumor viruses in mice. In “RNA Viruses and Host Genomes in Oncogenesis” (P. Emmelot and P. Bentvelzen, eds.), pp. 309-337. North American Elsevier, New York. BENTVELZEN, P. (1974). Host-virus interactions in murine mammary tumorigenesis. B&him. Biophys. Acta 355, 236-251. BENTVELZEN,P., and DAAMS, J. H. (1969). Herititary infections with mammary tumor viruses in mice. J. Nat. Cancer Inst. 93,1025-1035. COHEN, J. C. (1980). Methylation of milk-borne and genetically transmitted mouse mammary tumor virus proviral DNA. CeU 19.653-662. COHEN, J. C., MAJORS, J. E., and VARMUS, H. E. (1979a). Organization of mouse mammary tumor virus-specific DNA endogenous to BALB/c mice. J. Virol. 32,483496. COHEN,J. C., SHANK, P. R., MORRIS,V. L., CARDIFF, R., and VARMUS,H. E. (1979b). Integration of the DNA of mouse mammary tumor virus-infected normal and neoplastic tissue of the mouse. Cell 16, 333-345. COHEN,J. C., and VARMUS,H. E. (1979). Endogenous mammary tumor virus DNA varies among wild mice and segregates during inbreeding. Nature (London) 278,418423. COLCHER,D., HORAN HAND, P., TERAMOTO,Y. A., WUNDERLICH, D., and SCHLOM,J. (1981). Monoclonal antibodies define diversity of mammary tumor viral gene products in virions and mammary tumors of the genus Mus. Cancer Res. 41, 14511459. DROHAN, W., KETTMANN, R., COLCHER, D., and SCHLOM,J. (1977). Isolation of the mouse mammary tumor virus sequences not transmitted as germinal provirus in the C3H and RI11 mouse strains. J. ViroL 21, 986-995. DROHAN, W., and SCHLOM,J. (1979). Diversity of mammary tummor viral genes within the genus Mus, the species Mus musculus and the strain C3H. J. Viral. 31, 53-62. DUSING-SWARTZ,S., MEDINA, D., BUTEL, J. S., and SOCHER,S. H. (1979). Mouse mammary tumor virus genome expression in chemical carcinogen-induced mammary tumors in low- and high-incidence mouse strains. Proc. Nat. Acad. Sci. USA 76, 5360-5364. FANNING, T. G., PUMA, J. P., and CARDIFF, R. D. (1980). Selective amplification of mouse mammary tumor virus in mammary tumors of GR mice. J. Viral. 36, 109-114. FIELDSTEEL, A. H., DAWSON,P. J., KURAHARA, C., and BROOKS,R. E. (1975). Unusually high incidence of spontaneous mammary carcinomata in a colony of BALB/c mice. Brit. J. Cancer, 741-771. GAUTSCH,J. W., LERNER,R., HOWARD,D., TERAMOTO, Y. A., and SCHLOM,J. (1978). Strain-specific markers for the major structural proteins of highly on-
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cogenic murine mammary tumor viruses by tryptic peptide analyses. J. ViroL 21.688-699. GRONER,B., and HYNES, N. E. (1980). Number and location of mouse mammary tumor virus proviral DNA in mouse DNA of normal tissue and of mammary tumors. J. ViroL 33,1013-1025. HAGEMAN, P., CALAFAT, J., and DAAMS, J. H. (1972). The mouse mammary tumor viruses. In “RNA Viruses and Host Genome in Carcogenesis” (P. Emmelot and P. Bentvelzen, eds.), pp. 283-300. NorthHolland, Amsterdam. HOWARD, D. K., COLCHER,D., TERAMOTO, Y. A., YOUNG, J. M., and SCHLOM,J. (1977). Characterization of mouse mammary tumor viruses propagated in heterologous cells. Cancer Res. 37,26962704. LOWRY,0. H., ROSEBROUGH, N. J., FARR, A. L., and RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. C/rem. 193,265275. MCGRATH, C. M., MARINEAU, E. J., and VOYLES, B. A. (1978). Changes in MuMTV DNA and RNA levels in Balb/c mammary epithelial cells during malignant transformation by exogenous MuMTV and by hormones. Virology 87.339-353. MEDINA, D. (1976). Preneoplastic lesions in murine mammary cancer. Cancer Res. 36.2589-2595. MICHALIDES,R., VANDEEMTER,L., NUSSE,R., ROPCKE, G., and BOOT, L. (1978). Involvement of mouse mammary tumor virus in spontaneous and hormone-induced mammary tumors in low-mammary-tumor mouse strains. J. Viral. 27,551-559. MOORE,D. H. (1975). Mammary tumor virus In “Cancer: A Comprehensive Treatise” (F. F. Reeker, ed.), pp. 131-167. Plenum, New York. MOORE,D. H., CHARNEY,J., and HOLBEN,J. A. (1974). Titrations of various mouse mammary tumor viruses in different mouse strains. J. Nat. Cancer Inst. 52,1757-1761. MOORE, D. H. SARKAR, N. H., HOLEXN, J. A., and SHEFFIELD,J. B. (1979). Idiopathic mammary tumors in BALB/c mice. Int. J. Cancer 23.713-717. MORRIS, V. L., VLASSCHAERT,J. E., BEARD, C. L., MILAZZO, M., and BRADBURG,W. C. (1980). Mammary tumors from BALB/c mice with a reported high mammary tumor incidence have acquired new mammary tumor virus DNA sequences. Virology 100.101-109. NANDI, S, and MCGRATH, C. M. (1973). Mammary neoplasia in mice. Advan. Cancer Res. 17.353-414. OWENS,R. B., and HACKETT, A. J. (1972). Tissue culture studies of mouse mammary tumor cells and associated viruses. J. Nat. Cancer Inst. 49, 13211332. PAULEY, R. J., MEDINA, D., and SOCHER,S. (1979). Murine mammary tumor virus expression during mammary tumorigenesis in BALB/c mice. J. ViroL 29,483-493.
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PETERS,R. L., RABSTEIN,0. S., SPAHN, G. J., MADISON,R. M., and HUEBNER, R. J. (1972). Incidence of spontaneous neoplasms in breeding and retired breeder BALB/c mice throughout the natural life span. Znt. J. Cancer 10,273-279. 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. SOUTHERN,E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. BioL 38.503-517. TERAMOTO,Y. A., KUFE, D., and SCHLOM,J. (1977). Type-specific antigenic determinations on the ma-
jor external glycoprotein of high- and low-oncogenie murine mammary tumor viruses. J. ViroL 24, 525-533. TERAMOTO,Y. A., PUENTES,M. J., YOUNG, L. J. T., and CARDIFF, R. D. (1974). Structure of the mouse mammary tumor virus: Polypeptides and glycoproteins. .Z. ViroL 13,411-418. TERAMOTO,Y. A., and SCHLOM,J. (1978). Radioimmunoassays demonstrating type-specific and groupspecific antigenie reactivities for the major internal structural protein of murine mammary tumor viruses. Cancer Res. 38.1990-1995. TERAMOTO,Y. A., and SCHLOM,J. (1979). Radioimmunoassays for the 36,996 dalton glycoprotein of murine mammary tumor viruses demonstrate type, group, and interspecies determinants. J. Viral. 31, 334-340.