considerable controversy exists regarding the association of xenobiotics with breast cancer. Cell-ECM Extracellular Matrix) interactions are critical to cellular morphology, and alterations in cell morphology alter cell unction and gene transcription. Cells are linked to the ECM via integrin-containing focal adhesions. Other types of structures that link cells to each other or to the ECM include adherens junctions and desmosomes. Because correct issue architecture and cell morphology are critical for cellular homeostasis, we have examined whether xenobiotics disrupt cell-ECM interactions in a series of breast epithelial cell lines that represent a model of human proliferative breast disease (PBD), a risk factor for breast cancer. Preliminary results show that cells cultured on the ECM Matrigel adopt a three-dimensional morphology reminiscent of breast architecture in vivo and display differential sensitivity to Fas-mediated apoptosis. Organochlorines disrupt the levels and association of adhesion molecules associated with cell-ECM and cell-cell communication. Treatment of cells with p,p'-DDT results in increased delta-catenin levels relative to controls, whereas Kepone decreases alpha-catenin expression 2-fold and disrupts desmosomes relative to controls. The hypothesis of this research is that xenobiotics alter the levels, localization and association of cell adhesion molecules critical to the formation of focal adhesions, desmosomes and adherens junctions, which ultimately affect intracellular signaling pathways (e.g. PI 3-kinaseIAkt kinase), gene expression, cell cycle regulation, proliferation and apoptosis. We will employ organochlorine (p,p'-DDT, o,p'-DDT and Kepone) and non-organochlorine xenobiotics (DMBA, TPA, PhIP) in this proposal. Thus, the specific aims of this research, employing breast epithelial cells cultured on Matrigel, are: 1) To determine xenobiotic effects on the distribution and association of cell adhesion molecules critical to normal cell-ECM interactions and focal adhesions, adherens junctions or desmosomes. 2) To examine xenobiotic effects on PI 3-kinase/Akt signaling. 3) To determine xenobiotic effects on the cell cycle and cell cycle regulatory components (pRB, E2F, p53, cyclins and cyclin dependent kinases (cdk)). 4) To examine whether xenobiotics alter Fas-mediated apoptosis. 5) To assess whether xenobiotics at concentrations relevant to tissue levels in vivo, alter global gene expression using microarray analysis. The results of this proposal will provide novel information regarding chemical-mediated effects in breast epithelial cells and seminal data on fundamental cellular changes in human proliferative breast disease.
