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 and 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. More recent, Int6 has been found to be a highly conserved gene through evolution varying little in sequence homology from fission yeast to humans. Int6 protein functions are pleiotropic and its intracellular location varied. To determine whether truncated Int6 (Int6sh) has a biological function in cellular transformation, we transfected Int6sh into the human mammary epithelial cell line (MCF10A) and the mouse mammary epithelial cell line (HC11) and tested for acquisition of anchorage-independent growth. Both epithelial lines were transformed to anchorage-independent growth following transfection with Int6sh. Transfected 3T3 fibroblasts were also transformed to anchorage-independent growth by mutant Int6. Stably transfected Int6sh/MCF10A cultures were established and transplanted into Nu/Nu mice either directly into the #4 mammary fat pad or introduced into the intrascapular fat in combination with Matrigel.Similar experiments were done with Int6sh/HC11 cells.In both experiments Int6sh-transfected epithellial cells showed an increased capacity to produce growth in vivo compared to control cells. 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. From 11 Int6sh-positive founder lines only two showed expression of WAP-Int6sh in their mammary glands. Transplantation of WAPInt6sh mammary gland indicates that the presence of the transgene modifies ductal branching morphology resulting in atypical outgrowths. Serial passage of these transplants to determine their prwemalignant nature is in progress. 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. We have now carried several clonal outgrowths through multiple transplant generations. Growth senescence occurs in the 4th generation in some of the implants. During this process, the capacity to produce secretory lobules is lost independent of the ability to produce a complete ductal branching morphology. In addition premalignant and fully malignant progeny appeared stochastically within individual aging outgrowths. Our transplantation studies indicate that 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. To test the consequence of stem cell aging on tumor susceptibility, we infected WAP-TGF-b1 transgenic mammary glands which show premature aging of their mammary stem cells with MMTV at different points during mammary development to evaluate the preventive strategy of prematurely aging stem cells to reduce the risk of breast cancer.Our results indicate a strong prophylactic effect of early stem cell aging against mammary tumorigenesis induced by mouse mammary tumor virus (MMTV).

Agency
National Institute of Health (NIH)
Institute
Division of Basic Sciences - NCI (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC010021-06
Application #
6559097
Study Section
(LTIB)
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2001
Total Cost
Indirect Cost
Name
Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Booth, Brian W; Jhappan, Chamelli; Merlino, Glenn et al. (2007) TGFbeta1 and TGFalpha contrarily affect alveolar survival and tumorigenesis in mouse mammary epithelium. Int J Cancer 120:493-9
Booth, Brian W; Boulanger, Corinne A; Smith, Gilbert H (2007) Alveolar progenitor cells develop in mouse mammary glands independent of pregnancy and lactation. J Cell Physiol 212:729-36
Boulanger, Corinne A; Mack, David L; Booth, Brian W et al. (2007) Interaction with the mammary microenvironment redirects spermatogenic cell fate in vivo. Proc Natl Acad Sci U S A 104:3871-6
Mack, David L; Boulanger, Corinne A; Callahan, Robert et al. (2007) Expression of truncated Int6/eIF3e in mammary alveolar epithelium leads to persistent hyperplasia and tumorigenesis. Breast Cancer Res 9:R42
Clarke, Robert B; Smith, Gilbert H (2005) Stem cells and tissue homeostasis in mammary glands. J Mammary Gland Biol Neoplasia 10:1-3
Boulanger, Corinne A; Wagner, Kay-Uwe; Smith, Gilbert H (2005) Parity-induced mouse mammary epithelial cells are pluripotent, self-renewing and sensitive to TGF-beta1 expression. Oncogene 24:552-60
Lowther, William; Wiley, Korah; Smith, Gilbert H et al. (2005) A new common integration site, Int7, for the mouse mammary tumor virus in mouse mammary tumors identifies a gene whose product has furin-like and thrombospondin-like sequences. J Virol 79:10093-6
Smith, Gilbert H (2005) Label-retaining epithelial cells in mouse mammary gland divide asymmetrically and retain their template DNA strands. Development 132:681-7
Triplett, Aleata A; Sakamoto, Kazuhito; Matulka, Laurice A et al. (2005) Expression of the whey acidic protein (Wap) is necessary for adequate nourishment of the offspring but not functional differentiation of mammary epithelial cells. Genesis 43:1-11
Wagner, Kay-Uwe; Smith, Gilbert H (2005) Pregnancy and stem cell behavior. J Mammary Gland Biol Neoplasia 10:25-36

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