The Inhibition of EGF-dependent Proliferation of Keratinocytes by Tyrphostin Tyroskne Kinase Blockers

Protein tyrosine kinase blockers of the tyrphostin family inhibited the EGF-dependent proliferation of human and guinea pig keratinocytes grown in culture and induced their growth arrest. These blockers also significantly inhibited the growth of epidermal keratinocytes, but not of dermal cells, in whole skin organ culture from both guinea pig and human origin. The antiproliferative activity of these tyrphostins correlated quantitatively with their potency as inhibitors of EGF receptor autophosphorylation and the EGFdependent protein phosphorylation of intracellular target proteins in the keratinocyte. Furthermore, no significant cell cytotoxicity or reduction in serine and threonine phosphorylation of many intracellular polypeptides were observed upon incubation of the cells with tyrphostins like AG213. The complete growth arrest induced by the tyrphostins is fully reversible and upon their removal the keratinocytes resumed their growth with the original growth rate. Because of the nontoxic nature of these compounds and their growtharresting properties, we suggest their use as agents to treat hyperproliferative conditions of human skin. p HOSPHORYLATION of proteins on tyrosine residues is a key biochemical reaction that mediates a large variety of cellular signals (14, 32), including the control of the cell cycle and cell differentiation. Many of the cellular plasma membrane receptors, such as the receptors for EGF, PDGF, and insulin, possess an integral, intracellular, tyrosine kinase moiety that is activated upon the binding of its specific ligand to the extracellular domain of the receptor (28, 32). The involvement of abnormally high protein tyrosine kinase (PTK) 1 activity with a large variety of growthrelated disease states has already been established (5). In many cases, the enhanced PTK activity ofoncogene products or the overexpression of their normal counterparts was found to be essential for their transforming activity (5, 24, 28). Hyperproliferation of cells leading to nonmalignant growth is also often associated with enhanced PTK activity, for example, the enhanced PTK activity of PDGF receptor which results from its exposure to sustained levels of PDGF seen in atherosclerosis and restenosis (24). The overexpression of PTK oncoproteins, which results in enhanced kinase activity, can also alter the developmental pattern of cell types into which they have been introduced (3, 10, 29). The involvement of PTKs in a wide range of disease states has thus led to the general concept that development of PTK blockers is a reasonable approach to combat hyperproliferative conditions which result from enhanced activity of PTKs Correspondence may be addressed to Alexander Levitzki, Department of Biological Chemistry, Hebrew University of Jerusalem, Jerusalem 91904,