Evidence from in vivo and in vitro studies has shown that functional differentiation (i.e., synthesis and secretion of milk) of mouse mammary epithelial cells (MMEC) in response to lactogenic hormones is profoundly influenced by that extracellular matrix (ECM). Recent experiments in our laboratory indicate that the keratin composition of normal MMEC can also be modulated by the ECM, whereas preneoplastic and neoplastic MMEC may be insensitive to this stimulus. The purpose of the proposed research is to test the hypothesis that the ability of MMEC to perform mammary-specific differentiated function is coordinately expressed with a particular set of keratins and that both of these activities are subject to control by the ECM. MMEC cultured on different types of ECM will be used in experiments to address the following questions: 1) Is the set of keratins expressed by normal MMEC in vivo a requisite for obtaining synthesis and/or secretion of the full complement of milk components? 2) Do any culture systems which support certain aspects of differentiated function allow the cells to retain their exact in vivo pattern of keratins? 3) If coordinate expression of functional ability and a particular set of keratins exists, what is the role of keratin filaments in this activity? 4) If the distribution and arrangement of keratin filaments in MMEC is disrupted, what is the effect on synthesis and secretion of milk components? 5) Are mammary preneoplastic and/or tumor cells constrained to an aberrant pattern of keratin expression and unable to respond to modulation by the ECM? 6) Can the latter cells induced to produce/secrete milk components under in vivo or culture conditions that are permissive for normal MMEC? Synthesis and secretion of milk components and expression of keratins will be analyzed by biochemical assays, immunocytochemistry, and electron microscopy. Experiments on functions of keratin filaments will involve the use of acrylamide to perturb the arrangement and distribution of the filaments. Results of these studies should provide new information of ECM control of differentiated function in mammary cells, modulation of keratin expression by the ECM, the role of keratin filaments in cells performing an epithelial-specific function, and defects in differentiation associated with malignant progression of mammary cells.
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