BIOCHEMICAL EVENTS IN PHORBOL ESTER EFFECTS ON NORMAL AND TUMOR CELLS

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Studies have continued on the unique phosphoprotein, pp17, which undergoes rapid phosphorylation in HL60 promyelocytic leukemia cells in response to treatment with phorbol ester (TPA). By tryptic peptide analysis of specific proteins recovered from 2-dimensional electrophoretic gels, the nonphosphorylated precursor (p17) of pp17 has been identified. A novel and inexpensive method for measurement of radioactivity in single proteins directly on 2-dimensional gels was devised in this laboratory by R. Braverman. Using this technique, the cellular content of p17 and the rate and extent of its phosphorylation to pp17 after TPA treatment was quantitated. p17 was found to be one of the major cytosolic proteins in HL60 cells (0.5% of total cytosolic protein). About 50% of preexisting p17 is phosphorylated within 15 minutes of addition of TPA to HL60. These findings indicate that phosphorylation of p17 is one of the most rapid, and quantitatively significant biochemical responses to TPA treatment. Since this occurs in the context of abrupt cessation of cell growth and onset of monocytoid differentiation, the possible role of p17 and pp17 in these processes is being explored. The only other phenomenon known to occur in this time frame in this system is the rapid and transitory activation of the c-fos cellular oncogene. The possibility of a functional interrelationship between these two phenomena is being explored. The possible role of p17 and its phosphorylation to pp17 in cell growth regulation in other cell types is also being explored. Preliminary results indicate increased phosphorylation of p17 in lymphoid cells which exhibit reduced cell growth rate in response to serum deprivation.