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.     

Characterization and chromosome assignment of the human homolog of int-2, a potential proto-oncogene.

int-2 is one of two cellular genes (int-1 and int-2) currently implicated in the genesis of mammary carcinomas by mouse mammary tumor virus and may constitute a novel cellular proto-oncogene. Using low-stringency hybridization with mouse int-2 probes, we established that homologous genes exist in a variety of mammalian species, including humans, but failed to detect related sequences in other classes and phyla. Recombinant bacteriophage clones and a single cosmid encompassing the human int-2 gene were isolated and characterized by restriction enzyme mapping. A survey of nine primary human breast tumors, three breast tumor cell lines, and three normal individuals revealed no evidence for gross amplification or rearrangement of the int-2 locus. Three distinct restriction fragment length polymorphisms were observed which could prove useful in future linkage studies. By a combination of in situ hybridization of metaphase chromosomes and somatic cell genetics, the human int-2 gene was mapped to chromosome 11, band q13.     

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.     

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.     

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.     

The int-2 gene product acts as an epithelial growth factor in transgenic mice.

The induction of mammary tumors by mouse mammary tumor virus (MMTV) is thought to occur through proviral activation of one or more cellular genes. One of these, int-2, encodes a 27 kd protein which exhibits striking homology to the basic fibroblast growth factor family. To assess directly the role of the int-2 protein in cell proliferation, we have established transgenic mice which carry the int-2 gene driven by the MMTV promoter/enhancer. Expression of the int-2 gene in female transgenic mice results in pronounced mammary gland hyperplasia. Interestingly, expression of the MMTV-int-2 transgene in the prostate gland of male carriers results in a benign, but dramatic, epithelial hyperplasia similar to benign prostatic hypertrophy (BPH), a common but poorly understood disorder in human populations. Together, these results indicate that the int-2 product can act as a potent growth factor in these epithelial tissues.     

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.     

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.     

The different activation of int genes in mammary carcinomas developed in three mouse strains harboring mouse mammary tumor viruses derived from DD/Tbr.

RNA expressions of common integration site (int) genes and several oncogenes were investigated in mammary carcinomas spontaneously developed in different three strains of mice; DD/Tbr, NIH Swiss and BALB/c which harbor DD-MMTV derived from DD/Tbr mouse. Latter two strains of mice were designated NIH/Mtv+ and BALB/Mtv+, respectively. An increased expression of int-1 (wnt-1) and int-2 genes was observed in 56% (9/16) and 50% (8/16) of mammary carcinomas of DD/Tbr mice, respectively. Either int-1 or int-2 RNAs were expressed in 81% (13/16) of the carcinomas of DD/Tbr mice. IN NIH/Mtv+ mice, activation of int-1 and int-2 was observed in 41% (7/17) and 24% (4/17) of mammary carcinomas, respectively. Either int-1 or int-2 RNAs were expressed in 47% (8/17) of the carcinomas examined in this strain. In BALB/Mtv+ mice, on the other hand, either int-1 or int-2 gene were transcribed into RNAs at low frequency (33%: 3/9). These results suggest that the frequency of activation of int genes in mammary carcinomas induced by the same DD-MMTV in three strains of mice is genetically defined characteristics of these strains, and that the involvement of int-1 and int-2 genes in virus-induced mammary carcinogenesis may be influenced by genetic properties of animals. The activation of int-1 and int-2 genes did not clearly correlate with an increase in the expression of oncogenes examined; H-ras, K-ras, N-ras, myc, raf, fgr, fms, erB, mos, and src genes.     

Identification of protein products encoded by the proto-oncogene int-1.

The proto-oncogene int-1 is activated by adjacent insertions of proviral DNA in mouse mammary tumor virus-induced tumors and has transforming activity in certain mammary epithelial cell lines. The gene is normally expressed in the central nervous system of mid-gestational embryos and in the adult testis. We raised antibodies against synthetic int-1 peptides and used these to identify protein products of the gene in cells transfected or infected with retroviral vectors expressing int-1. Four protein species of 36,000, 38,000, 40,000, and 42,000 Mr were immunoprecipitated by antibodies against two different int-1 peptides and were not present in control cells. Partial degradation with V8 protease showed the four species to be structurally related to each other and to int-1 polypeptide synthesized in vitro. Treatment of the cells with tunicamycin prevented the appearance of all but the 36,000-Mr species, suggesting that the slower-migrating forms are glycosylated derivatives. The unglycosylated 36,000-Mr species migrated faster in polyacrylamide gels than the in vitro translation product of int-1 and has probably undergone cleavage of an amino-terminal signal peptide.     

The proto-oncogene int-1 encodes a secreted protein associated with the extracellular matrix.

The proto-oncogene int-1 plays an important role in mammary tumorigenesis when activated by proviral insertions of the mouse mammary tumor virus. In normal mouse tissues the gene is expressed in the embryonic neural tube, suggesting a developmental function, while in Drosophila the homolog of int-1 is the segment polarity gene wingless. In order to study the protein products of int-1 we have derived fibroblast cell lines infected with multiple copies of a retroviral vector expressing int-1 cDNA. By Western blot analysis and immunoprecipitation we have identified a 44 kd form of int-1 protein which is secreted from these cells. The 44 kd species is distinct from the major intracellular forms of int-1 protein as judged by its slower mobility in SDS-polyacrylamide gels and by its longer half-life in pulse-chase experiments. Under normal growth conditions, little or none of the 44 kd protein is detectable in the cell culture medium but instead the majority is found associated with the extracellular matrix (ECM). The protein appears to bind heparin in vitro, suggesting that it might bind glycosaminoglycans in the ECM. These data support the view that int-1 protein may play a role in cell-cell communication over short distances.     

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.     

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.