The research will evaluate the mechanisms by which hormones regulate the growth of kidney epithelial cells and the alterations that occur upon malignant transformation. The MDCK cell line is particularly advantageous for studies on hormonal regulation of growth. Medium K-1, a hormone-supplemented, serum-free medium, has been developed that permits MDCK cells to grow in the absence of fetal calf serum. Five supplements were identified (insulin, transferrin, T?3?, hydrocortisone, and PGE?1?) that permitted MDCK cells to grow over a long term and at an equivalent rate in serum-free and serum-supplemented media. Medium K-1 also has been used to grow primary cultures of baby mouse kidney epithelial cells. Medium K-1 will be used for the following studies. (1) The possibility that PGE?1? stimulates growth by increasing intracellular cAMP in MDCK cells will be examined. Available PGE?1?-independent variants of MDCK cells (variants that lack PGE?1? requirement for long-term growth in serum-free media) will be utilized. In addition, dibutyryl cyclic AMP-resistant variants of MDCK cells that are altered in their cyclic AMP-dependent protein kinase activity will be studied. The possibility will be examined that different forms of cyclic AMP-dependent protein kinases cause the protein phosphorylations that are responsible for the stimulatory effects of PGE?1? on growth and on bone formation, respectively. (2) The possibility that malignant transformation is accompanied by changes in requirements of hormones for growth will be evaluated, utilizing available tumorigenic transformants of MDCK cells. (3) Growth regulation by hormones will also be examined in primary cultures of kidney epithelial cells. Medium K-1 was utilized to select epithelial cells from primary kidney cultures whose growth responses to hormones resemble those of MDCK cells. Additional hormone-supplemented, serum-free media will be developed for primary cultures of kidney epithelial cells. A primary proximal tubule cell culture system has been developed that retains proximal tubule functions. Selective growth conditions are being developed for primary proximal tubule cells by examining their nutritional requirements (e.g., growth in glucose-free medium) and their drug sensitivity. The optimal growth requirements of kidney tubule cells derived from the cortex and medulla will similarly be determined in serum-free medium, so as to develop selective media for different types of kidney tubule epithelial cells. The functional properties of cortical and medullary cells will be evaluated. The growth and functional properties of EGF-responsive cells will be determined. (B)
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