The endogenous proviral mouse mammary tumor virus genes of the GR mouse are not identical and only one corresponds to the exogenous virus.

The endogenous proviral copies of mouse mammary tumor virus (MMTV) were selected from a gene library of GR mouse DNA. We obtained five different lambda. MMTV recombinant clones. Four of them correspond to the 3' Eco RI fragments of the endogenous proviruses an one comprises an intact MMTV provirus with 2 to 3 kb of flanking mouse genomic DNA. Heteroduplex formation followed by S1 digestion under stringent conditions shows that there is nucleotide sequence heterology among the cloned endogenous proviral copies. Only one endogenous proviral copy, associated with the mtv-2 locus, was found to be totally homologous to the exogenous proviral DNA.     

Proviruses of mouse mammary tumor virus in normal and neoplastic tissues from GR and C3Hf mouse strains.

We analyzed two experimental situations to assess the role of endogenous mouse mammary tumor virus (MMTV) DNA in the genesis of mammary carcinomas. (i) GR mice carry in their germ line one or more proviruses indistinguishable by limited restriction mapping from the proviruses introduced into cells by experimental infection with the highly tumorigenic virus isolated from GR mouse milk, MMTV(GR). Most tumors arising in GR mice contain one or more proviruses at various sites in tumor DNA in addition to those present endogenously. Detection of these new proviruses is possible as a consequence of the clonal or quasiclonal character of the tumors. (ii) C3H/He mice carry three units of endogenous viral DNA, none of which resembles the DNA of the commonly encountered strains of milk-borne MMTV. Nevertheless, MMTV-associated tumors arise late in life when these animals are removed from the influence of milk-borne virus; the responsible agent, MMTV(C3Hf), can also produce tumors in BALB/c mice. We found that tumors arising in both C3Hf/He mice and BALB/c mice infected with MMTV(C3Hf) were clonal or quasiclonal and contained one or more new copies of proviral DNA at various sites in the host genome. These new proviruses were readily distinguished from the proviruses of the common milk-borne virus strains and closely resembled unit II of endogenous MMTV DNA (Cohen et al., J. Virol., 32:483-496). Thus, in both experimental systems, we found evidence for new proviruses in mammary tumors, despite the preexistence of similar or identical proviruses in the germ line. The results suggest that the repositioning of MMTV proviruses may be required for the full expression of the oncogenic potential of endogenous MMTV DNA.     

Selective amplification of mouse mammary tumor virus in mammary tumors of GR mice.

DNAs extracted from the mammary tumors of GR mice were analyzed for mouse mammary tumor virus proviral sequences by the restriction enzyme-Southern blot procedure. The tumor DNAs contain more proviral copies of mouse mammary tumor virus than DNA from a nonmalignant tissue. The degree of proviral amplification is small (ca. one to five additional copies) and appears to be variable from tumor to tumor. The restriction patterns of the amplified proviral sequences suggest a clonal origin for the tumor mass. In addition, the restriction patterns observed after digestion with the enzymes BglII and SacI indicate that only one of the proviruses endogenous to GR mice is amplified. The amplified provirus found in GR mammary tumors is identical to the provirus that is missing in GR-Mtv-2- mice, a congenic line exhibiting a low mammary tumor incidence.     

Genetic mapping of endogenous mouse mammary tumor viruses: locus characterization, segregation, and chromosomal distribution.

The restriction endonuclease EcoRI has been used to study the inheritance of strain difference in endogenous mouse mammary tumor virus DNA sequences. This enzyme, which cleaves at only one site within the nondefective viral genome, generates DNA fragments containing mouse mammary tumor virus sequences which vary in size according to the locations of EcoRI restriction sites in the flanking mouse sequences, thereby defining unique integration sites of the viral genome. Recombinant inbred strains of mice have been used to study the inheritance of these DNA fragments which hybridize to mouse mammary tumor virus cDNA sequences. The results define 11 segregating units consisting of 1 or 2 fragments. These units were shown to segregate among the recombinant inbred strains, and in some instances linkage was established. Two units were shown to be linked on chromosome 1. Another unit was mapped to chromosome 7, which is presumably identical to the previously defined genetic locus Mtv- 1. One other mouse mammary tumor virus locus was tentatively assigned to chromosome 6. The results are consistent with the view that integration of mouse mammary tumor virus can take place at numerous sites within the genome, and once inserted, these proviruses appear to be relatively stable genetic entities.     

Characterization, chromosome assignment, and segregation analysis of endogenous proviral units of mouse mammary tumor virus.

In the course of analyzing sites of proviral integration in tumors induced by mouse mammary tumor virus (MMTV), we have isolated recombinant DNA clones corresponding to the 5' and 3' ends of four endogenous MMTV proviruses present in BALB/c and BR6 mice. This has permitted the structural characterization of each locus by detailed restriction mapping and the preparation of DNA probes specific for the cellular sequences flanking each provirus. These probes have been used to trace the segregation patterns of the proviruses, designated Mtv-8, Mtv-9, Mtv-17, and Mtv-21, in a panel of inbred strains of laboratory mice and to map Mtv-17 and Mtv-21 to mouse chromosomes 4 and 8, respectively. The unambiguous resolution of these four proviruses on Southern blots has greatly facilitated the analysis of other endogenous MMTV proviruses in these inbred mice.     

Integration of type B retroviral DNA in virus-induced primary murine thymic lymphomas

In previous studies we described the isolation and characterization of a highly leukemogenic virus, DMBA-LV, isolated from a transplanted, chemical carcinogen-induced thymic lymphoma. The virus is composed of a mixture of two unrelated retroviral genomes, one highly related to type B milk-borne mouse mammary tumor virus isolates and the other partially related to type C viral genomes. In the present study, primary thymic lymphomas induced by DMBA-LV in CFW/D, NIH Swiss, C3H/Bi/Ka, and C57BL/Ka mice were assessed for the presence of newly integrated type B retroviral DNA. All 46 primary thymic lymphomas examined contained one to four newly acquired murine mammary tumor virus proviruses. Based on the sizes of provirus-cell DNA junction fragments, the integration of newly acquired murine mammary tumor virus proviruses did not appear to be random.     

Mammary tumor virus proviral DNA in normal murine tissue and non-virally induced mammary tumors

The Southern DNA filter transfer technique was used to study the involvement of the endogenous mouse mammary tumor virus (MMTV) in the development of mammary tumors of nonviral etiology. The presence of extra MMTV proviruses in the genomes of these non-virally induced mammary tumors would indicate an integration of the provirus of an activated endogenous MMTV. Acquisition of MMTV proviruses did not seem to be an absolute requirement for the development of hormone or carcinogenically induced mammary tumors in strain BALB/c nor for hormone-induced mammary tumors in mouse strains 020, C57BL, and C3Hf. In some hormone-induced mammary tumors we did observe extra MMTV proviruses in submolar quantities, indicating that reintegration may occasionally occur and that only a part of the tumor cells acquired new MMTV DNA information. Hormone-dependent and -independent primary mammary tumors of the mouse strain GR, which are controlled by the Mtv-2 mammary tumor induction gene, all acquired extra MMTV proviruses. Most of these extra MMTV proviral-DNA-containing fragments appeared present in submolar quantities, suggesting that only part of the tumor cells acquired extra MMTV proviral information. These findings indicate that the initially transformed mammary gland cells of non-virally induced mammary tumors do not necessarily acquire extra MMTV proviral DNA information, in contrast to the MMTV-induced mammary tumors, in which all tumor cells contain extra MMTV DNA information.     

Mouse mammary tumor virus-induced tumorigenesis in sag transgenic mice: a laboratory model of natural selection.

Transgenic mice that expressed the superantigen protein encoded in the C3H exogenous mouse mammary tumor virus long terminal repeat deleted their V beta 14+ T cells during the shaping of their immune repertoire and showed no evidence of virus production in their mammary glands after infection by milk-borne C3H exogenous virus. However, they developed mammary gland tumors that had newly integrated copies of C3H exogenous virus, although the latency of tumor formation was much longer than in their nontransgenic littermates that retained their V beta 14+ T cells. After four generations, infectious C3H virus was completely eliminated from the transgenic mouse pedigree. These data support the hypothesis that endogenous mouse mammary tumor proviruses are retained in the genome as protection against exogenous virus infection and subsequent tumorigenesis and show that there may be natural selection against the virus in vivo.     

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

The methylation and amplification of mouse mammary tumor virus (MuMTV) proviral DNA was investigated in normal, premalignant, and malignant tissues of GR/A mice. The proviral methylation pattern was examined with the restriction enzyme HhaI, which fails to cleave methylated DNA. MuMTV proviral DNA from liver, kidney, and heart was highly methylated. Proviral DNA was somewhat undermethylated in mammary gland cells from virgin and lactating mice and extensively undermethylated in cells from premalignant outgrowths, pregnancy-dependent tumors, and pregnancy-independent tumors. The restriction enzyme SacI was used to detect additional proviruses in the same cells. No additional proviral copies of MuMTV were detected in liver, kidney, or heart cells or in mammary gland cells from virgin mice. Some mammary gland cells from lactating mice appeared to contain additional copies of the endogenous, highly oncogenic GT-MTV-2 provirus. Premalignant outgrowth, pregnancy-dependent tumor, and pregnancy-independent tumor cells contained an average of two to three additional copies per cell of the GT-MTV-2 provirus. Thus, neoplasia in GR/A mice was directly associated with quantized increases in MuMTV proviral DNA undermethylation and GR-MTV-2 proviral DNA amplification. Restriction enzyme analysis suggested that premalignant outgrowths and pregnancy-dependent tumors both consisted largely of heterogenous cell populations, although some evidence of clonal dominance was detected.     

Integration of the DNA of mouse mammary tumor virus in virus-infected normal and neoplastic tissue of the mouse.

We have used restriction endonucleases which cleave the DNA of mouse mammary tumor virus (MMTV) at one site (Eco RI) and several sites (Pst I, Sac I and Bam HI) to study infection and mammary tumorigenesis in mice. Proviruses acquired during infection of BALB/c mice foster-nursed by virus-producing C3H females can be distinguished from the MMTV proviruses endogenous to uninfected BALB/c mice by the nature of the fragments generated with Pst I and Bam HI. Using this assay, we show that lactating mammary glands as well as mammary tumors from BALB/cfC3H mice have acquired MMTV DNA, and that a minimum of approximately 10% of normal glandular cells can be infected. The new proviruses appear to be linked to cellular DNA of mammary tumors and infected lactating mammary glands within a limited region (0.2 x 10(6) daltons) of the viral DNA; the location of this region, based upon mapping studies with unintegrated MMTV DNA, suggests that the orientation of these proviruses is colinear with linear DNA synthesized in infected cells and thus approximately colinear with the viral RNA. Comparisons of many mammary tumors and studies of lactating mammary glands with a high proportion of independently infected cells indicate that a large number of sites in the cellular genome can accommodate a new provirus; the acquired proviruses are rarely, if ever, found in tandem with each other or with endogenous proviruses. We cannot, however, distinguish between random integration and integration into a large number of preferred sites in the host genome. Since Eco RI and Bam HI cleavage of DNA from each mammary tumor generates a unique set of viral-specific fragments, we propose that the tumors are composed principally of cells derived from a subset of the many infected cells in a mammary gland; this proposal is supported by our finding that Eco RI digestion of DNA from several transplants of a primary tumor yields the pattern characteristic of the primary tumor.     

Organization of mouse mammary tumor virus-specific DNA endogenous to BALB/c mice.

We used restriction endonucleases to prepare physical maps of the mouse mammary tumor virus (MMTV)-specific DNA endogenous to the BALB/c mouse strain. The mapping was facilitated by the DNA transfer procedure, using complementary DNAs specific for the whole and for the 3' terminus of MMTV RNA to detect fragments containing viral sequences. The strategies used for the arrangement of fragments into physical maps included sequential digestions with two or three enzymes; preparative isolation of EcoRI fragments containing viral sequences; and comparisons of virus-specific fragments derived from the DNA of several mouse strains. Most of the MMTV-related DNA in the BALB/c genome is organized into two units (II and III) which strongly resemble proviruses acquired upon horizontal infection with milk-borne strains of MMTV and other retroviruses. These units contain approximately 6.0 x 10(6) Mr of apparently uninterrupted viral sequences, they bear redundant sequences totaling at least 700 to 800 base pairs at their termini, and the terminal redundancies include sequences derived from the 3' end of MMTV RNA. Units II and III are closely related in that they share 12 of 14 recognition sites for endonucleases, but cellular sequences flanking units II and III are dissimilar by this criterion. The remainder of the MMTV-related DNA endogenous to BALB/c mice is found in a single subgenomic unit (unit I) with a complexity of ca. 2 x 10(6) Mr; the structure of this unit has not been further defined. These results support the hypotheses that endogenous proviruses have been acquired by infection of germinal tissues with MMTV. The physical maps are also useful for identifying the MMTV genomes endogenous to BALB/c mice in studies of the natural history of mammary tumorigenesis.     

Dexamethasone increases the number of RNA polymerase II molecules transcribing integrated mouse mammary tumor virus DNA and flanking mouse sequences.

In mouse Ltk- cells that were transfected with recombinant bacteriophage DNA containing a complete proviral copy of an integrated endogenous mouse mammary tumor virus (MMTV) with its flanking cellular sequences, the newly acquired MMTV proviruses were transcribed in a glucocorticoid-responsive fashion. After hormone treatment of selected cell clones in culture we isolated the nuclei, elongated the nascent RNA chains in vitro, and determined the number of RNA polymerase II molecules on the transcribed MMTV DNA as well as on the flanking mouse DNA sequences. We found that the specific increase in the polymerase loading after hormone treatment is proportional to the increase in the amount of stable MMTV mRNA. When the DNA sequences which are responsible for hormone-receptor binding and for the increased MMTV mRNA levels were deleted, no increase in RNA polymerase II loading on MMTV DNA was observed. Nuclear RNA chains which were transcribed in response to hormone treatment were detected not only from the transfected MMTV DNA but also from the mouse DNA sequences adjacent to the 3' end of the provirus.     

Rearrangements in the long terminal repeat of extra mouse mammary tumor proviruses in T-cell leukemias of mouse strain GR result in a novel enhancer-like structure.

Male GR mice develop T-cell leukemia at low frequency late in life. These leukemia cells invariably contain large amounts of mouse mammary tumor virus (MMTV) RNA and MMTV proteins and have extra MMTV proviruses integrated in their DNA. We show here that the extra MMTV proviruses are all derived from the endogenous MMTV provirus associated with the Mtv-2 locus and that the T-cell leukemias are clonal with respect to the acquired MMTV proviruses. The extra MMTV proviruses in six transplantable T-cell leukemia lines studied had rearranged, shortened long terminal repeats (LTRs); each T-cell leukemia, however, had a different LTR rearrangement within its extra MMTV provirus. The alteration within the extra LTRs of T-cell leukemia line 42 involved deletion of 453 nucleotides and generation of a tandem repeat region consisting of regions flanking the deletion. This alteration generated a sequence similar to the adenovirus enhancer core sequence. The viral RNAs in the T-cell leukemias contained corresponding alterations in their U3 regions. These results demonstrate that expression of MMTV in T-cell leukemias of GR mice may be the consequence of the generation of a novel enhancer, which could also stimulate expression of any adjacent cellular oncogene.     

Variation in the number of copies and in the genomic organization of ecotropic murine leukemia virus proviral sequences in sublines of AKR mice.

DNAs isolated from individual mice of four AKR sublines (AKR/J, AKR/N, AKR/Cum, and AKR/Boy) were examined by hybridization of electrophoretically separated restriction enzyme fragments to a 500-base pair, 32P-labeled probe specific for env sequences of ecotropic murine leukemia virus. Variation in the number of proviral DNA copies and in their genomic organization, as reflected by the location of restriction enzyme sites in flanking cellular sequences, was observed both between and within AKR sublines. Evidence is presented for the continual acquisition of new proviruses in the four sublines studied. The ecotropic proviral DNA copies present in the four AKR sublines can be related to their genealogy; each subline contains two or three copies of proviral DNA in common with other sublines and from one to six unique ecotropic proviruses. Overall, a new copy appears about every 12 generations of inbreeding. Some of the unique proviral DNA copies contain internal alterations, as reflected by restriction enzyme maps that differ from those of prototype ecotropic proviruses.     

Analysis of tissue-specific methylation patterns of mouse mammary tumor virus DNA by two-dimensional Southern blotting.

We used a two-dimensional Southern blotting procedure to analyze the tissue-specific methylation patterns of the five endogenous mouse mammary tumor viruses in the GR/A mouse strain. Our findings suggest that in certain tissues (brain, kidney, and liver) all proviruses are extensively methylated. In other tissues (spleen, placenta, and testes) all proviruses are hypomethylated to some degree. In these tissues individual proviruses display both quantitative and qualitative differences in methylation. We interpret the general patterns of tissue-specific hypomethylation in terms of a "hitch-hiker" model: mouse mammary tumor virus proviral methylation patterns reflect the tissue-specific activity of neighboring sequences. The observation that certain sites on particular proviruses are differentially methylated in a tissue-specific fashion may reflect tissue-specific differences in the makeup or conformation, or both, of proviral-containing chromatin.     

Tumorigenesis by mouse mammary tumor virus: Evidence for a common region for provirus integration in mammary tumors

We have prepared specific probes for unique-sequence cellular DNA adjacent to each of the newly integrated proviruses in tumors induced by mouse mammary tumor virus (MMTV). The use of such probes to screen a large number of independent mammary tumors in the BR6 strain of mouse has indicated that in at least 17 out of the 40 tumors examined so far, an MMTV provirus has integrated into a common chromosomal domain. A 10 kb Eco RI fragment of single copy DNA from this region has been isolated and partially characterized by restriction enzyme mapping. Of the proviruses located within this fragment in different tumors, all but one are complete, in the same orientation, and clustered within about 3 kb of cellular DNA. These findings are consistent with an insertional mutagenesis model for tumorigenesis by MMTV, in which the integration of a provirus in a particular region of cellular DNA may activate a neighboring oncogene. The region we describe here appears to be different from that reported for mammary tumors in the C3H strain of mouse.     

Hormonal regulation of mouse mammary tumor growth

In view of reports that human breast cancer cells secrete growth factors that can replace estradiol in sustaining tumor growth [1], we have investigated whether hormone independent (HI) GR mouse mammary tumors can sustain growth of estrogen-depleted hormone dependent (HD) tumors. HD GR mammary tumor TSl 106 was grafted subcutaneously in the right flank of estrone plus progesterone treated castrated (020 X GR)F1 mice. After 2 weeks the estrone treatment was stopped and the mice received 50, 100 or 150 mg HI GR mammary tumor TSl 104 in the left flank. However, the regression of the HD tumor due to estrone depletion was not prevented or retarded by the HI grafts. In other experiments we investigated integrations of mouse mammary tumor virus (MMTV) proviral DNA in the DNA of GR mammary tumors. We could demonstrate the presence of two cell populations in tumor TSl 96, both HD but differing in MMTV DNA integration events. Our data indicate that exogenous integrations of MMTV proviruses can take place in mouse mammary tumor DNA without loss of hormone dependency of the tumors. Like in GR/Mtv-2+ mice, mammary tumor transplants differing in MMTV proviral integrations are also observed in 020/Mtv-2+ mice.     

Absence of mouse mammary tumor virus proviral amplification in chemically induced lymphomas of RF/J mice.

RF/J mice are susceptible to the induction of thymic lymphomas by the carcinogens 3-methylcholanthrene and N-methyl-N-nitrosourea. Given the association of mouse mammary tumor virus (MMTV) with certain thymomas, we examined genomic DNA from chemically induced lymphomas of RF/J mice for new MMTV proviruses. Of 13 tissue culture lines derived from 3-methylcholanthrene-induced tumors, 5 had acquired new proviruses. MMTV amplification coincided with the appearance of viral mRNAs and proteins. However, no primary tumors or animal-passaged tumors contained new proviruses. These observations indicate that MMTV does not have a role in the tumor induction process, although it may become activated and amplified in tissue culture lines derived from tumors.