The Transforming Growth Factor-beta (TGF-beta) gene family includes three highly homologous mammalian isoforms which act through a cell surface receptor to selectively inhibit the growth of normal mammary gland cells in vivo as well as suppress the proliferation of many transformed mammary epithelial cells. In other epithelial cells types embryologically regulated to mammary gland cells, such as keratinocytes, TGF-beta induces a late G1 arrest of the cell cycle, suggesting a similar response occurs in mammary cells. To understand the molecular basis by which TGF-beta inhibits the proliferation of mammary epithelial cells, the cell cycle arrest will be examined in a series of newly characterized mouse mammary cells that include immortalized normal, preneoplastic and tumorigenic phenotypes. As part of this characterization, the TGF-beta effects on cell morphology, differentiated functions and growth factor production will be tested. TGF-beta induced and suppressed cell cycle-regulated genes will be identified by Northern blot analysis and transcriptional nuclear run-on studies. These genes will be functionally tested by their ability to interfere with the TGF-beta growth arrest of mammary cells after transection of appropriate """"""""sense"""""""" or """"""""anti-sense"""""""" expression vectors. Disruption of the TGF-beta cell cycle arrest in transfected cells will be characterized in vitro and in vivo as well as tested for effects of mesenchyme-epithelial cell interactions. Initial efforts will focus on the mid and late G1-cyclins (Cyclin D and Cyclin E), E2F (which forms a complies with the retinoblastoma tumor suppressor gene and Cyclin A) and the p53 growth suppressor gene. The ability of TGF-beta to regulate the formation or disassembly of cyclin-kinase complexes will be also examined in growth inhibited mammary cells. The 5'-flanking regions of primary TGF-beta regulated genes will be clone, TGF-beta regulated elements functionally mapped and transcriptional regulatory factors defined by DNA binding assays. In a complementary approach, previously uncharacterized TGF-beta inducible genes that mediate the growth arrest will uncovered by subtractive cloning, and functionally tested by transient transfections of growing mammary cells. The temporal expression of the subtractive TGF-beta regulated genes will be analyzed in the normal mammary cells during the various proliferative and differentiated stages of the mammary gland as well as in transformed mouse mammary cells. The molecular reagents generated to the TGF-beta regulated growth suppressor genes by the proposed in vitro studies will be used to explore in vivo growth regulatory mechanisms of mammary cells and may eventually prove to be clinically useful in the predictive diagnosis of human breast cancer.
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