Host genetic background effect on the frequency of mouse mammary tumor virus-induced rearrangements of the int-1 and int-2 loci in mouse mammary tumors.

The frequency with which int-1 and int-2 are rearranged in mouse mammary tumors by mouse mammary tumor virus (MMTV)-induced insertional mutagenesis is a consequence of the host genetic background. In 75% of C3H mammary tumors, int-1 is rearranged by MMTV insertion, whereas only 30% of BALB/cfC3H tumors contain a virus-induced rearrangement of int-1. This difference is significant (P less than 0.005) and could not be accounted for by the potentially additive effect of the genetically transmitted Mtv-1-encoded virus in C3H mice. Similarly, MMTV-induced rearrangement of the int-2 gene in mammary tumors of the R111 mouse strain (59%) occurred at a significantly (P less than 0.025) higher frequency than in BALB/cfR111 (25%) mammary tumors. Moreover, in BALB/cfR111 mammary tumors, there is evidence that rearrangement of int-1 and int-2 does not occur independently (P less than 0.025). These results suggest that the long history of inbreeding for high tumor incidence of C3H and R111 mouse strains has selected for the fixation of host mutations which either complement the action of the particular int gene or affect the sensitivity of specific subpopulations of mammary epithelium to infection by particular strains of MMTV.     

Incomplete proviral genome of mouse mammary tumour virus is present on chromosome Y of NIH Swiss and some genetically related mouse strains.

An incomplete proviral genome of endogenous mammary tumour virus (MMTV) was found in DNA of several strains of mice. This MMTV-related sequence was assigned to the Y chromosome since it was clearly observed in male mice only. This MMTV provirus contained a sequence related to LTR (long terminal repeat), but not to gag-pol and env genes. NFS, NIH Swiss/S, STS/A, and DD/Tbr mice have this sequence but BALB/cHeA, SHN, SLN, C57BL/6NJcl, C3H/HeNJcl and CBA/JJcl mice are negative. In the strains containing this sequence, a DNA test for the sequence makes it possible to easily distinguish the DNAs of male or female mice.     

Genetic marker patterns and endogenous mammary tumor virus genes in inbred mouse strains of Japan

In order to establish the genetic relatedness of the inbred mouse strains kept in Nara, genetic marker patterns were determined in conjunction with a study on endogenous mammary tumor viral genes in these strains. Isoenzyme patterns combined with patterns of other genetic markers, show that the unrelatedness between various inbred strains of the dd stock is as high or even higher as between strains of known different origin and geneology. Based on endogenous viral gene patterns the dd stock derived mice can be subdivided into three group, DDD, DDN, DDO, KF and DD/Tbr. The DD/Tbr and its foster-nursed substrain (DD/Tbrf) have the lowest number of endogenous viral genes, i.e. two, while the other strains carry 4-6 such genes. The SLN and SHN strains, derived from a Swiss stock, have a similar pattern of viral genes different that of all other strains studied, also strains of Swiss origin from other sources, such as the NFS and the GR.     

Horizontal transmission of the mouse mammary tumor virus in cage mates of the same and opposite sex of low and high mammary cancer strain mice

Horizontal transmission of mouse mammary tumor virus (MTV) was investigated in cage mates of the same and opposite sex of low (BALB/c) and high mammary cancer strains (DD/Tbr, SHN and GR). By MTVp27 and MTVgp52 radioimmunoassay, MTV antigen expression was found in the salivary glands, mammary glands and secondary male genital organs of the MTV-free BALB/c strain. Infectivity and oncogenicity were also found in DDf or BALB/c mice by inoculating extracts of salivary gland and/or seminal vesicle in high mammary cancer strains. It is suggested that the primary source of the infectious agent in cases of caged animals of the same sex is saliva, while the primary source in cases of caged animals of the opposite sex is the seminal fluid, although additional infection through saliva cannot be ruled out in the latter case.     

Genetics of mouse mammary tumor virus-induced mammary tumors: linkage of tumor induction to the gag gene

Retroviruses are believed to induce tumors by acting as insertional mutagens that activate expression of cellular protooncogenes. Indeed, almost 90% of mouse mammary tumor virus (MMTV)-induced mammary tumors in C3H/He mice show upregulation of Int protooncogenes. We have analyzed three different MMTV variants [MMTV(C3H), MMTV(HeJ), and a genetically engineered MMTV hybrid provirus (HP)] for tumorigenicity in mice from two distinct genetic backgrounds. All three viruses were tumor causing in BALB/cJ mice. However, only MMTV(C3H), but not MMTV(HeJ) or HP, induced mammary tumors in C3H/He mice. All of the viruses were infectious on either background and up-regulated expression of Int genes in tumors they induced. Like HP, MMTV(HeJ) was found to be a genetic recombinant between endogenous Mtv1 provirus and exogenous MMTV(C3H). Sequence comparison of MMTV variants linked the tumorigenicity of MMTV(C3H) to the gag region of the retrovirus.     

Specific activation of the cellular Harvey-ras oncogene in dimethylbenzanthracene-induced mouse mammary tumors.

Genomic DNAs from dimethylbenzanthracene-induced BALB/c mouse mammary tumors arising from the transplantable hyperplastic outgrowth (HPO) line designated DI/UCD transformed NIH 3T3 cells upon transfection. Transforming activity was attributed to the presence of activated Harvey ras-1 oncogenes containing an A----T transversion at the middle adenosine nucleotide in codon 61. DNAs from untreated DI/UCD HPO cells and radiation-induced and spontaneous mammary tumors from the DI/UCD HPO line failed to transform NIH 3T3 cells. The results indicated that the mutation activation of Harvey ras-1 oncogenes was specific to dimethylbenzanthracene treatment in the mouse mammary tumor system.     

A new common integration region (int-3) for mouse mammary tumor virus on mouse chromosome 17.

Mus musculus subsp. musculus (Czech II) mammary tumor DNA frequently contains an integrated proviral genome of the mouse mammary tumor virus (MMTV) within a specific 0.5-kilobase-pair region of the cellular genome (designated int-3). Viral integration at this site results in activation of expression of an adjacent cellular gene. We mapped int-3 to mouse chromosome 17 by analysis of PstI-restricted cellular DNAs from mouse-hamster somatic cell hybrids. Restriction analysis of cellular DNA from (C3H/OuJ X Czech II) X Czech II backcross mice established the gene order T-H-2-int-3. These results demonstrated that the int-3 locus is distinct from two other common integration regions for mouse mammary tumor virus (designated int-1 and int-2) in mammary tumor DNA and suggest that several cellular genes may be at risk for virally induced activation during mammary tumor development.     

Mammary tumorigenesis in feral mice: identification of a new int locus in mouse mammary tumor virus (Czech II)-induced mammary tumors.

A population of Mus musculus subsp. musculus (Czech II), recently isolated from the wild, lack endogenous mouse mammary tumor virus (MMTV) proviral genomes. Some of these mice carry an infectious MMTV [designated MMTV (Czech II)] that is transmitted in the milk and is associated with mammary tumor development. This virus is distinct from laboratory strains of MMTV present in inbred mice. An MMTV (Czech II) genome was found within a 0.5-kilobase region of the cellular genome in five of 16 Czech II mammary tumors. MMTV insertion at this site activates expression of a 2.4-kilobase species of RNA from a previously silent cellular gene. This region of the cellular genome was designated int-3 since it is unrelated to the int-1 and int-2 loci. The int-3 locus does not appear to correspond to other proto-oncogenes but is well conserved among mammalian species.     

The nucleotide sequence of the human int-1 mammary oncogene; evolutionary conservation of coding and non-coding sequences.

The mouse mammary tumor virus can induce mammary tumors in mice by proviral activation of an evolutionarily conserved cellular oncogene called int-1. Here we present the nucleotide sequence of the human homologue of int-1, and compare it with the mouse gene. Like the mouse gene, the human homologue contains a reading frame of 370 amino acids, with only four substitutions. The amino acid changes are all in the hydrophobic leader domain of the int-1 encoded protein, and do not significantly alter its hydropathic index. The conservation between the mouse and the human int-1 genes is not restricted to exons; extensive parts of the introns are also homologous. Thus, int-1 ranks among the most conserved genes known, a property shared with other oncogenes.     

Oncogene expression in mammary epithelial cells.

Mouse strains which develop tumors at a high incidence with characteristics very similar to human cancers have been derived over the last 8 years. The tumors are caused by defined genetic alterations in the mouse genome. Three areas of research have contributed to the derivation of these mouse strains: (1) Molecular analysis of human tumors has shown that distinct oncogenes and tumor suppressor genes are consistently involved in a high percentage of primary tumors. (2) Regulatory enhancer-promoter sequences have been identified which direct gene expression to specific target cells, preferentially mammary epithelial cells. (3) The introduction of recombinant DNA molecules into fertilized mouse eggs by microinjection and integration of the injected DNA into the genome of injected cells has given rise to mutant mouse strains with unique and defined genetic alterations. Studies with different promoter-oncogene combinations introduced into transgenic mouse strains have led to the following general conclusions: (1) Oncogenes expressed in mammary gland cells predispose transgenic mice to mammary tumors. (2) The oncogenic potential of individual oncogenes in mammary epithelial cells differs. (3) Oncogene expression initially often causes a preneoplastic state affecting growth and differentiation parameters of cells. (4) The expression of different oncogenes synergizes to reduce tumor latency. Synergism can also be observed with physiological growth signals like estrogen or growth hormone. The oncogenes with a role in mammary carcinomas which have been investigated in transgenic mice will be described here. The phenotypic consequences of oncogene expression and the implications for the multistep carcinogenesis model will be discussed.     

Expression of the int-1 and int-2 loci in endogenous mouse mammary tumor virus-induced mammary tumorigenesis in the C3Hf mouse.

The int-1 locus appears to be involved in over 80% of C3H exogenous mouse mammary tumor virus (MMTV)-induced mouse mammary tumors, and the int-2 locus appears to be involved in approximately 10% of these tumors. Analysis of 46 C3Hf mammary tumors resulting from endogenous, rather than exogenous, MMTV infection revealed that only 41% expressed int-1 RNA, while 2% expressed int-2 RNA. Our results suggest that in addition to the int-1 and int-2 loci, other loci may be involved in endogenous-MMTV-induced mammary tumors of the C3Hf mouse.     

Mammary tumor induction loci in GR and DBAf mice contain one provirus of the mouse mammary tumor virus

The mammary tumor induction genes Mtv-1 in mouse strain DBAf and Mtv-2 in strain GR control the complete expression of the endogenous mouse mammary tumor virus (MMTV). We have used a combination of genetic, biochemical and molecular biological methods to identify and correlate specific copies of the endogenous MMTV proviral genes with the biological properties of the tumor induction genes Mtv-1 and Mtv-2. These Mtv induction genes contain specific MMTV proviral information, as was concluded from restriction enzyme analysis and molecular hybridization of DNAs of congenic mouse strains and of progenitors of backcross populations. The congenic strains differed from the parental strains GR and 020 only in the Mtv-2 gene, one lacking the Mtv-2 gene (GR/Mtv-2-) and one having obtained this gene (020/Mtv-2+). The gain or loss coincided with two Eco RI cellular DNA fragments containing MMTV DNA information. Since Eco RI cuts the exogenous proviral variant of MMTV DNA once, we assume that these two cellular DNA fragments contain one MMTV provirus. The same cellular DNA fragments containing MMTV DNA information segregated together with MMTV expression in the offspring population of the backcross. In a similar backcross analysis of the induction gene Mtv-1 it was also demonstrated that the Mtv-1 gene comprises one MMTV provirus. These data indicate that Mtv induction genes contain specific but different MMTV proviral genes and that nly a limited number of the MMTV proviruses present in the cellular DNA is associated with the control of proviral expression.     

Mammary tumorigenesis in feral Mus cervicolor popaeus.

A pedigreed breeding population of feral Mus cervicolor popaeus with a high incidence of mammary tumors, arising between 6 and 14 months of age, is described. These mice were chronically infected with a type B retrovirus which is distantly related to the mouse mammary tumor virus (MMTV) of inbred strains of Mus musculus. MMTV-induced mammary tumors in inbred mice frequently (80%) contained an insertion of the viral genome into the int-1 or int-2 loci of the tumor cellular genome. These two cellular genetic loci were also altered by viral insertion in 11 of 20 M. cervicolor popaeus mammary tumor cellular DNAs tested. Results of our study of mammary tumorigenesis in feral mice demonstrate that viral-induced rearrangement and activation of the int loci are not limited to the genetic background of inbred mice selected for highly infectious MMTV and a high incidence of mammary tumors.     

Insertion mutation of the int-1 and int-2 loci by mouse mammary tumor virus in premalignant and malignant neoplasms from the GR mouse strain

Mouse mammary tumor virus (MMTV)-induced mammary adenocarcinomas can develop from several different premalignant precursors common in GR mice. Insertion mutagenesis of the mammary protooncogenes int-1 and int-2 was studied in this multistep system by analyzing samples from various stages of neoplastic development for novel int-1 and int-2 restriction fragments generated by MMTV provirus integration. int-1 and int-2 insertion mutations were observed in both premalignant lesions and malignant tumors. Some of the tumors with insertion mutations were experimentally derived from insertion mutation-free premalignant precursors. Each class of neoplasm examined had a characteristic frequency of int-1 and int-2 insertion mutations; however, no correspondence was observed between neoplasm morphology and mutation of either gene. These results indicate that insertion mutation of the int-1 and int-2 loci by MMTV provirus can be involved in the earliest identifiable stages of neoplastic development as well as during progression of premalignant lesions to tumors. Insertion mutation of int-1 and int-2 is therefore not stage specific in this system.     

Generation of a tumorigenic milk-borne mouse mammary tumor virus by recombination between endogenous and exogenous viruses.

Two novel exogenous mouse mammary tumor viruses (MMTV), BALB2 and BALB14, that encode superantigens (Sags) with Vbeta2+ and Vbeta14+ specificities, respectively, were found in the BALB/cT mouse strain. BALB/cT females were crossed with AKR/J males to generate F1 females. Foster nursing of BALB/cT mice on (BALB/cT x AKR/J)F1 mothers resulted in the generation of a new mouse strain, BALB/cLA, that had acquired a new exogenous MMTV (hereafter called LA) with a Vbeta6+/Vbeta8.1+-T-cell-specific Sag. Sequence analysis of the long terminal repeats of the BALB2, BALB14, and LA viruses indicated that LA virus resulted from recombination between BALB14 and the endogenous Mtv-7 provirus. Mtv-7 is expressed only in lymphoid tissues but not the mammary glands of Mtv-7-containing mouse strains such as AKR. In contrast, LA virus was highly expressed in the mammary gland, although it had the sag-specific region from Mtv-7. The LA virus, as well as different recombinant viruses expressed in the mammary glands of (BALB/cT x AKR/J)F1 mice, acquired a specific DNA sequence from BALB14 virus that is required for the mammary-gland-specific expression of MMTV. Since the Sag encoded by LA virus strongly stimulated cognate T cells in vivo, selection for recombinant virus with the Mtv-7 sag most likely occurred because the increased T-cell proliferation resulted in greater lymphoid and mammary gland cell infection. As a result of the higher virus titer, 80% of BALB/cLA females developed mammary gland tumors, although the incidence was only 40% in BALB/cT mice.     

Novel common integration sites targeted by mouse mammary tumor virus insertion in mammary tumors have oncogenic activity

Non-acute transforming retroviruses like mouse mammary tumor virus (MMTV) cause cancer, at least in part, through integration near cellular genes involved in growth control, thereby de-regulating their expression. It is well-established that MMTV commonly integrates near and activates expression of members of the Wnt and Fgf pathways in mammary tumors. However, there are a significant number of tumors for which the proviral integration sites have not been identified. Here, we used high through-put screening to identify common integration sites (CISs) in MMTV-induced tumors from C3H/HeN and BALB/c mice. As expected, members of both the Wnt and Fgf families were identified in this screen. In addition, a number of novel CISs were found, including Tcf7l2, Antxr1/Tem8, and Arhgap18. We show here that expression of these three putative oncogenes in normal murine mammary gland cells altered their growth kinetics and caused their morphological transformation when grown in three dimensional cultures. Additionally, expression of Tcf7l2 and Antxr1/Tem8 sensitized cells to exogenous WNT ligand. As Tcf7l2, Antxr1/Tem8, and Arhgap18 have been associated with human breast and other cancers, these data demonstrate that MMTV-induced insertional mutation remains an important means for identifying genes involved in breast cancer.