Breast cancer represents the most common malignant neoplasm occurring in women from developed countries world-wide. In the United States, breast cancer accounts for over 40,000 deaths per year, and numerous studies have documented a steadily increasing incidence of the disease over the last 30-40 years. Delineating the basic growth control mechanisms of mammary epithelial cells is crucial to nearly every aspect of breast cancer research; and endocrine, paracrine, and autocrine mechanisms of growth control all appear to be important to the development and progression of breast neoplasia. The intent of this proposal is to increase our understanding of the growth control mechanisms essential in mediating the proliferation/growth inhibition of mammary epithelial cells and to identify novel factors involved in this process. This project will focus on characterizing and cloning a heparin-binding growth factor derived from normal human mammary epithelial cells (HMEC), and on developing new ways of identifying and cloning growth factors from HMEC.
The specific aims of the project are two-fold: l) to purify, fully characterize, and clone an apparently novel heparin-binding stimulatory factor derived from conditioned media supporting HMEC cells, and 2) to implement an expression cloning strategy for identifying and cloning novel epithelial cell growth mediators. To achieve these aims, we have developed a bioassay that quantitates the growth effects of HMEC conditioned media on MCF-7 cells. This assay is capable of identifying both stimulatory and inhibitory agents, is suitable for screening a large number of conditioned medias, and measures actual cell proliferation as opposed to increased DNA synthesis. In preliminary experiments, we have demonstrated that the conditioned media from HMEC cells markedly stimulates the growth of MCF-7 cells. This activity is heat insensitive, has a molecular weight of greater than 10 kD, is blocked by neutralizing antibody to the EGF receptor, is inhibited by the heparin analog pentosan polysulfate, and is not inhibited by neutralizing antibody specific for EGF or TGFalpha. As these findings suggest a novel stimulatory activity, the cloning strategy in goal 2 will be implemented. In addition, parallel efforts will be undertaken to purify sufficient quantities of the identified stimulatory activity via heparin-sepharose affinity chromatograph to obtain partial amino acid sequence data. For goal 2, an expression cloning strategy which relies on the MCF-7 cell proliferation bioassay and a COS cell transient expression system has been developed. This strategy allows for the cloning of growth mediating substances without the requirement of developing specific antibody or obtaining partial amino acid sequence data, and specific conditions for the MCF-7 cell bioassay have been developed which foster identification of both stimulatory and inhibitory activities. With this strategy, cDNA expression libraries from HMEC cells will be probed for growth stimulatory/inhibitory activity.
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