Tyrphostins are low-molecular-weight synthetic inhibitors of protein tyrosine kinase, which block cell proliferation. Since platelet-derived growth factor (PDGF) is thought to figure prominently in disorders of vascular smooth muscle cells (VSMC), such as atherosclerosis, hypertension, and restenosis, we examined whether tyrphostins would inhibit PDGF-induced mitogenesis in VSMC. In this communication, we demonstrate that tyrphostins with the benzenemalononitrile nucleus inhibited PDGF-dependent growth of VSMC as well as PDGF-dependent DNA synthesis in these cells, with the concentrations for 50% inhibition ranging from 0.04 to 9 microM. Up to 30-fold higher tyrphostin concentrations were required to inhibit serum-stimulated DNA synthesis of VSMC. The effect of the tyrphostins is reversible, since on their removal a normal proliferative response to PDGF was resumed. Tyrphostins also inhibited PDGF-receptor autophosphorylation and PDGF-induced phosphorylation of intracellular substrates, including the phosphorylation of phospholipase C-gamma, with a potency ratio similar to their antimitogenic activity. The expression of c-fos mRNA, a mitogenic nuclear signal, was also reduced in PDGF-stimulated VSMC treated with tyrphostins at concentrations which inhibit PDGF-induced mitogenesis. It is concluded that tyrphostins are potent reversible inhibitors of PDGF-induced mitogenesis which act by inhibiting the tyrosine kinase activity of the PDGF receptor and the subsequent signaling cascade. Tyrphostins may be useful in the study and treatment of VSMC proliferation disorders.

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SUMMARY The activity of cAMP-dependent and cAMP-independent protein kinases, a class of enzymes involved in the regulation of cell proliferation was measured in rat colonic epithelium. Sequential cell populations harvested by a stepwise scraping technique from colonic crypt regions were identified by histology and incorporation of [3HJ-thymidine into DNA. cAMP-independent phosphorylation of casein, in the presence of [y-32PJATP, was markedly suppressed by quercetin, a bioflavonoid known to inhibit G-type casein kinase, protein kinase-C and tyrosine protein kinase. Conversely, the cyclic nucleotide regulatable form requiring histone as substrate was responsive to the action of the heat stable protein kinase inhibitor. The protein kinase species were characterised and partially purified by DEAE-cellulose chromatography. The activity ofcAMP-dependent protein kinase in colonic cytosols (pmol 32P/min/mg protein, means (SE)) increased from 129.4 (15.9) in superficial ceUl populations to 238-5 (31.4) in lower crypt cell fractions (p<0 01). Colonic cAMPindependent protein kinase activity increased from 87-3 (15-6) in surface cell preparations to 17841 (300) in lower crypt cell populations (p<002). A comparable activity gradient was observed in membrane fractions. The activity gradient persisted when the results were expressed as a function of celular DNA. These findings indicate that protein kinases display a defined topological segregation along the colonic crypt regions and that during migration to the lumen colonic cells attenuate enzyme signals supposedly related to tissue growth.The colonic epithelium provides a unique model of cells at different stages of differentiation aligned in an orderly pattem along the crypt-lumen axis.'2 Differentiation of colonic cells is accompanied by congruent morphological and biochemical changes.'2 Synthesis of nucleic acids is restricted to mitotically active young cells in the crypt regions: during the migration toward the lumen, the maturing cells acquire the typical absorptive and secretory functions and limited life expectancy.'2 Senescent colonocytes are shed into the bowel lumen and replaced by cells migrating from the proliferative zones. The sizes of the proliferative and functional compartments along the crypt surface axis are maintained within precise boundaries by multiple homoeostatic mechanisms.' 2

Primary embryonic fibroblasts isolated from beige (Chediak-Higashi) mice develop pathognomonic giant granules in vitro. Inclusion in the culture medium of carbamylcholine (carbachol) or phorbol myristate acetate (PMA) results in the generation of morphologically normally granules. Chediak-Higashi fibroblasts are highly susceptible to shape changes induced by colchicine. This abnormal property is also corrected by carbachol and PMA.