The principle goal of this project is to assess the growth modulatory effects of a protein isolated from rat liver that causes reversible inhibition of the proliferation of rat liver epithelial (RLE) cells in culture. A highly potent preparation of this inhibitor protein has been obtained using a new purification procedure involving DEAE-cellulose and gel filtration chromatography followed by high resolution chromatofocusing and hydrophobic interaction FPLC. Normal RLE cells were markedly sensitive to the antiproliferative effects of this inhibitor (ID-50 200 pg/ml), whereas aflatoxin-transformed RLE cells exhibited low sensitivity (ID-50 1.5 ng/ml). Rat hepatoma cells UVM 7777 and human hepatoma cells Hep-G2 were resistant to the cytostatic effects of the inhibitor; however, human breast carcinoma cells (MCF-7) and rat hepatoma cells (Reuber) were affected at relatively higher concentrations (ID- 50 1.0 ng/ml). On the contrary, proliferation of normal rat kidney fibroblasts (NRK) and human foreskin fibroblasts was stimulated in response to this inhibitor. Measurement of tyrosine kinase activity in RLE cells treated with this liver- derived growth inhibitor using a novel non-denaturing gel electrophoretic assay, indicated a reduction in cytoplasmic tyrosine kinase which was accompanied by an increase in membrane-associated kinase activity. The identify of these kinases and their role in the growth regulation is currently under investigation. Experiments examining the cell cycle specificity of this inhibitor using microcinematography technique are also underway.
