The mouse mammary tumor virus (MMTV) is a potent somatic mutagen, integrating within the host genome and disrupting normal gene expression. In the Czech II feral strain of mice the virus is transmitted through the milk and induces pregnancy independent adenocarcinomas in 20% of infected animals. Identifying the sites of MMTV integration provides a powerful means to isolate genes and characterize potential pathways involved with the development of cancer. To this end we have employed a PCR based approach using MMTV as a tag to help identify the flanking genomic sequences. The genomic DNA from each tumor was first digested with either EcoR1 or BamH1, both known to cut the viral genome. This created fragments containing both viral sequences and the flanking genomic sequences with either EcoR1 or BamH1 compatible ends. These linear fragments were then self-ligated creating a circle around which primers, complementary to the viral sequence, can be used to amplify the intervening host genomic sequence. The amplified product can be subsequently cloned and sequenced. We are currently identifying these sites of integration in tumors collected from Czech II mice.We have found that expression of the Notch4/Int3 locus is activated in mouse tumors as a consequence of insertional mutagenesis by MMTV. We have established two Notch4/Int3 transgenic mice strains that express the intracellular domain of Notch4/Int3 protein. One is driven by MMTV-LTR promotor and the other is by mammary gland specific whey acidic protein (WAP) promotor. In both, highly malignant tumors developed in the mammary gland.In virgin female MMTV-LTR-Notch4/Int3 mice, normal duct differentiation was remarkably retarded. In WAP-Notch4/Int3 females, ductal differentiation was normal but alveolar development was inhibited. These findings suggest that the activated Notch4/Int3 gene can mediate tumorigenesis in different fashions depending on when it is expressed during mammary gland development. To identify the genes involved in Notch4/Int3-mediated tumorigenesis, we have examined differential gene expression by Atlas Mouse cDNA Expression Array (Clontech). The results were confirmed by conventional Northern blot analysis. WAP-Notch4/Int3 tumors could be categorized into three types based on the milk protein genes that were expressed. MMTV-LTR-Notch4/Int3 mammary tumors expressed no milk protein genes. In addition, we have found that two genes involved in stem cell proliferation and self-renewal were highly expressed in Notch4/Int3 mammary tumors. Based on our results, it is suggested that activated Notch4/Int3 gene mediates tumorigenesis of mammary gland by affecting a proliferation of stem cells at different stage of mammary gland development as defined by the MMTV-LTR- and WAP- promoters.
