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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.