A retrovirus vector expressing the putative mammary oncogene int-1 causes partial transformation of a mammary epithelial cell line

In mammary tumors induced by the mouse mammary tumor virus (MMTV), the int-1 gene is frequently activated by adjacent proviral insertions and is thereby strongly implicated in tumorigenesis. To seek a direct biological effect of int-1 that would validate its proposed role as an oncogene, we constructed a retrovirus vector containing the gene and examined its effects on tissue culture cells. Expression of int-1 in a mammary epithelial cell line caused striking morphological changes, unrestricted growth at high cell density, and focus formation on a monolayer, although the cells were not tumorigenic in vivo. This partial transformation induced by int-1 was not observed in cells infected by an otherwise identical virus bearing a frameshift mutation in the gene. These findings strongly support the hypothesis that int-1 plays a functional role in MMTV-induced mammary tumorigenesis.     

Mouse mammary tumor gene int-3: a member of the notch gene family transforms mammary epithelial cells.

Expression of a 2.3-kb RNA species is induced in mammary tumors as a consequence of insertional mutagenesis at the int-3 locus by the mouse mammary tumor virus. The nucleotide sequence and biological activity of this mammary tumor-specific int-3 RNA species were determined. It contains an open reading frame which encodes a 57-kDa protein. The translated protein possesses six nearly contiguous 32-amino-acid repeats which are related to a similar motif in the Saccharomyces cerevisiae cdc-10-encoded cell cycle protein. In addition, the int-3 cdc-10 repeats are bounded by the PEST amino acid sequence motif which is commonly found in proteins having a rapid turnover and may represent sites for phosphorylation. The int-3 cdc-10 repeat sequences are 50% identical to a portion of the intracellular domain of the neurogenic Drosophila notch gene product. Activation of expression of a recombinant int-3 genomic DNA fragment encoding the 2.3-kb RNA species in HC11 mouse mammary epithelial cells in vitro induces anchorage-independent growth in soft agar.     

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.     

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.     

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 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.     

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.     

Structure and nucleotide sequence of the putative mammary oncogene int-1; proviral insertions leave the protein-encoding domain intact

Many mammary tumors induced by mouse mammary tumor virus (MMTV) contain a provirus in the same region of the host-cell genome, leading to expression of a putative cellular oncogene called int-1. Here we present the structure and nucleotide sequence of int-1. We have established several proviral insertion sites exactly by nuclease S1 analysis or by molecular cloning and DNA sequencing. The protein-encoding domain of int-1 is distributed over four exons. At the 5' end of the gene two overlapping exons were detected, one of which is preceded by a TATA box. The deduced int-1-encoded protein has 370 amino acids, with a preponderance of hydrophobic residues at the NH2 terminus. Proviruses are found at both sides of the gene, usually oriented away from the gene. Downstream integrations occur frequently in the long 3' untranslated region of the last exon. One upstream provirus is inserted in the 5' untranslated region and, unlike the other upstream insertions, in the same orientation as the int-1 gene. Proviral integrations always leave the protein-encoding domain intact, providing further evidence that the int-1 protein contributes an essential step in mammary tumorigenesis.     

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.     

Nonuniform expression of a mouse mammary tumor virus-driven int-2/Fgf-3 transgene in pregnancy-responsive breast tumors.

We have developed transgenic mice in which expression of the mouse int-2/Fgf-3 gene is regulated by a single long terminal repeat from mouse mammary tumor virus. Such mice contain and transmit a replica of the activated int-2/Fgf-3 allele present in a spontaneous mammary tumor from a BR6 mouse. Although free of infectious mouse mammary tumor virus and with a different genetic background, the transgenic mice develop pregnancy-responsive mammary epithelial proliferations that are similar to the early stages of tumorigenesis in the BR6 strain. Histological examination revealed that most of these tumors showed pronounced tubular and acinar structures, features usually associated with morphological differentiation. In some cases, the tumors were locally invasive, causing disruption of the dermis which manifested itself as local hair loss. In situ hybridization showed that patterns of transgene expression in the abnormal glands were markedly nonuniform. In contrast, mouse mammary tumor virus-induced neoplasms showed more uniform expression of int-2/Fgf-3, as did the urogenital epithelial proliferations that occur among males of this transgenic line. These data suggest that mammary tumors in virally infected animals may depend primarily on autocrine stimulation by the int-2/Fgf-3 gene product, whereas both autocrine and paracrine mechanisms may contribute to tumors and hyperplasias found in transgenic animals.     

Mouse mammary tumor virus integration regions int-1 and int-2 map on different mouse chromosomes.

Two regions of mouse DNA which constitute common provirus integration sites in tumors induced by mouse mammary tumor virus have been identified and designated int-1 and int-2. By examining a series of hamster-mouse somatic cell hybrids, we mapped the int-2 locus to mouse chromosome 7 and confirmed the previous assignment of int-1 to chromosome 15. This constitutes proof that int-1 and int-2 are discrete genetic loci. It is therefore possible that proviral activation of two distinct cellular genes may result in the same neoplastic disease.     

A putative int domain for mouse mammary tumor virus on mouse chromosome 7 is a 5'' extension of int-2.

We extended the physical map of the mouse int-2 locus by demonstrating that the site of insertion for mouse mammary tumor virus DNA in plaque-type mammary tumors of GR mice is directly linked to int-2. An additional example of proviral integration is described in which a provirus in a presumed enhancer-insertion mode 15 kilobases upstream of the int-2 promoters is capable of activating expression of the gene at levels typical of other virally induced mammary tumors.