Vitamin A is an essential nutrient for the maintenance of the differentiated state of adult epithelia. In the breast, Vitamin A acts both as an antiproliferative signal and as a signal for the expression of differentiated attributes. Defects that interfere with the ability of cells to respond to Vitamin A may lead to enhanced growth and decreased differentiation (hallmarks of cancer), and thus may represent potential mechanisms of carcinogenesis/cancer progression. Cellular response to Vitamin A, or retinol, is mediated by a family of nuclear receptors for retinoic acid, i.e., retinol must be oxidized to retinoic acid (RA) or other RAR ligands in order to evoke a response. The cellular retinol binding protein I (CRBPI), by virtue of its ability to bind retinol and present it to the appropriate metabolizing enzymes (thus preventing inappropriate oxidation), has been postulated to play an important rode in the synthesis of RA from retinol. Once formed, RA binds to one of three nuclear retinoic acid receptors (RARalpha, Beta, gamma), which are ligand-dependent transcription factors and are activated by bound RA; RARs then regulate the transcription of specific target genes. Preliminary data indicate that the Vitamin A response pathway is multiply defective in breast cancer cells. Two specific defects have been targeted for study based on the high frequency with which they occur. These are the underexpression, relative to normal breast epithelial cells, of CRBPI and RARbeta. We will address 3 open questions (1) The mechanism of RAR beta inactivation; (2) The mechanism of CRBPI inactivation; and (3) The effect of ectopically expressed CRBPI. To address (1), we will use MDA-MB-231 subclones that differ in their endogenous expression of RARbeta, and pursue our finding that the defect in subclone A4 (non-expressor) is in cis. Our first step will be to sequence the A4 RARbeta promoter/RARE region. Strategies for pursing alternative mechanisms besides beta promoter mutation are outlined. In aim (2), we will pursue the hypothesis, based on Southern blot data, that methylation of the CRBPI gene accounts for its inactivation in breast cancer. Finally, in aim (3), we will study whether T47D and MCF-7 clones expressing CRBPI (already on hand) differ in their sensitivity to retinol with regards to growth, apoptosis, and target gene regulation. These and similar MDA-MB-231 clones (also on hand) will also be analyzed with regards to retinol metabolism in collaboration with Dr. Joseph Napoli.