POTENTIATION OF ETOPOSIDE-INDUCED APOPTOSIS BY STAUROSPORINE IN HUMANTUMOR-CELLS IS ASSOCIATED WITH EVENTS DOWNSTREAM OF DNA-PROTEIN COMPLEX-FORMATION

Protein kinase inhibitors have demonstrated potential for use in the t herapy of human cancers, in particular leukemia. Staurosporine, a prot ein kinase inhibitor with broad specificity, enhances the cytotoxic ef fects of various antitumor agents with different modes of action. The topoisomerase II inhibitor, etoposide, has shown clinical activity aga inst a wide range of tumor types. Purpose: The purpose of this study w as to assess the effects of staurosporine on etoposide-induced cell de ath processes in a human tumor of epithelial origin. Methods: Modulati on of etoposide-induced apoptosis by staurosporine in HeLa cells was a ssessed by cell morphology, extraction of low molecular weight DNA, qu antitation of DNA-protein complexes, and measurements of rates of DNA synthesis. The effects on cellular genes implicated in apoptosis were determined by Northern and Western blotting, along with assays of cycl in-dependent kinase activities. Results: Staurosporine exhibited a two - to three-fold potentiation of apoptosis caused by etoposide in HeLa cells when applied concurrently, or immediately following etoposide re moval, but did not alter the quantity of DNA-protein complexes produce d by etoposide. Etoposide-induced apoptosis, and its potentiation by s taurosporine, were associated with reduced c-myc expression, and a mod erate increase in p21(WAF1/CIP1) mRNA and protein levels. Inhibitors o f cyclic AMP-dependent protein kinase and protein kinase C, which exhi bit greater specificity than staurosporine, were without effect on apo ptosis caused by etoposide, whereas use of the tyrosine phosphatase in hibitor, vanadate, resulted in its abrogation. The potentiation of eto poside-induced apoptosis by staurosporine was associated with a signif icant increase in cyclin A-dependent kinase activity. In addition, eto poside caused substantial inhibition of DNA synthesis. Conclusion: The se results indicate that staurosporine potentiates apoptosis through e vents which occur downstream of DNA damage, and implicate unscheduled activation of cyclin A-dependent kinase during inhibition of DNA synth esis as a possible cause.