The CZECHII mouse colony represents a unique experimental model for identifying insertional mutations caused by mouse mammary tumor virus (MMTV) because they lack endogenous germline MMTV proviral sequences which are present in all other laboratory strains of mice. CZECHII mice and their MMTV-induced hyperplasias are utilized to identify early-acting genes and, subsequently, to determine which MMTV-induced mutations might be involved with progression from hyperplasia to tumor to metastasis. By taking advantage of the clonal dominant nature of the preneoplasias, their tumors and metastases, we hope to identify genes commonly affected by MMTV during these mutagenic events, and differentiation of normal mammary tissue in vivo.Using this experimental model, a new transforming INT gene (INT6) was identified in a premalignant CZECH mouse mammary outgrowth.Int6, was originally discovered as an integration site for MMTV in the somatic DNA of a clonally-derived premalignant mammary outgrowth line (CZZ-1) and in the tumors and metastases arising from this line. The MMTV insertion occurred within an intron of Int6 resulting in the transcription of a truncated Int6 mRNA from one allele. The other allele was found to be unaffected, suggesting that the MMTV-induced mutation was dominant. It was discovered that the DNA sequence of Int6 is identical to the gene encoding eukaryotic initiation factor 3 (eIF3) protein p48. The eIF3 p48 subunit is thought to negatively regulate initiation of translation by the eIF3 complex. This discovery raises the possibility that MMTV- interrupted Int6 is a dominant negative mutation of eIF3 p48 regulatory function. To determine whether truncated Int6 has a biological function in cellular transformation, we transfected truncated Int6 into the human mammary epithelial cell line (MCF10A) and tested for acquisition of anchorage-independent growth. Truncated Int6 expression was driven from the eukaryotic elongation factor-1a (EF-1a) gene promoter in the transfected MCF10A cells. Stably transfected Int6sh/MCF10A cultures were established and seven independent anchorage independent Int6sh/MCF10A clones were picked from soft agar assays. Each of these clones showed high steady state levels of the truncated Int6 transcript when analyzed by RT-PCR and Northern blot. All 7 anchorage-independent Int6/MCF10A clones were transplanted into Nu/Nu mice either directly into the #4 mammary fat pad or introduced into the intrascapular fat in combination with Matrigel. Subsequent to the observation of the transforming ability of truncated Int6 in MCF10A cells, we ligated Int6sh to the whey-acidic-protein (WAP) promoter and generated mice with truncated Int6 expression targeted to the mammary gland. The consequences associated with the expression of Int6sh in the mammary epithelium are under study. In my studies on the identification, characterization and analyses of mammary epithelium-specific stem cells, several important breakthroughs have been made. We have demonstrated the existence of clonogenic progenitor cells among the mammary epithelium capable of producing lobular growth but not ductal branching morphogenesis and, conversely, progenitors capable of producing ductal morphogenesis but not lobulo-genesis. These cells are present in small numbers among the mammary population and appear to be produced by a third clonogenic cell which is capable of producing an entire functional mammary gland and replicating itself in the process. Utilizing genetically-marked mammary epithelial cells we have conclusively demonstrated that an entire functional mammary gland can regenerated in vivo from the progeny of a single mammary tissue-specific stem cell. We further proved that this clonally-derived gland contains the three types of clonogenic epithelial cells described above. Our estimates indicate that these multipotent mammary cells can divide symmetrically up to 44 doublings before becoming proliferatively senescent. Our hypothesis is that these multipotent epithelial cells represent the immediate targets for oncogenic transformation in the mammary gland. Continuing studies of serially-transplanted clonally derived normal Czech mouse mammary outgrowths have revealed that growth senescence of lobular versus ductal-limited progenitors is developed independently during serial transplantation suggesting that primary mammary stem cells produce these multipotent surrogates by completely different mechanisms which are subject to aging at different rates. To test the consequence of this disparity in aging, we are subjecting WAP-TGF-b1 transgenic mammary glands which show premature aging of their mammary stem cells to infection by MMTV at different points during mammary development to evaluate the preventive strategy of prematurely aging stem cells to reduce the risk of breast cancer.
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