Potentiation of etoposide-induced apoptosis by staurosporine in human tumor cells is associated with events downstream of DNA-protein complex formation.
Protein kinase inhibitors have demonstrated potential for use in the therapy of human cancers, in particular leukemia. Staurosporine, a protein kinase inhibitor with broad specificity, enhances the cytotoxic effects of various antitumor agents with different modes of action. The topoisomerase II inhibitor, etoposide, has shown clinical activity against a wide range of tumor types.


OBJECTIVE
The purpose of this study was to assess the effects of staurosporine on etoposide-induced cell death processes in a human tumor of epithelial origin.


METHODS
Modulation of etoposide-induced apoptosis by staurosporine in HeLa cells was assessed by cell morphology, extraction of low molecular weight DNA, quantitation 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 cyclin-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 removal, but did not alter the quantity of DNA-protein complexes produced by etoposide. Etoposide-induced apoptosis, and its potentiation by staurosporine, were associated with reduced c-myc expression, and a moderate increase in p21WAF1/CIP1 mRNA and protein levels. Inhibitors of cyclic AMP-dependent protein kinase and protein kinase C, which exhibit greater specificity than staurosporine, were without effect on apoptosis caused by etoposide, whereas use of the tyrosine phosphatase inhibitor, vanadate, resulted in its abrogation. The potentiation of etoposide-induced apoptosis by staurosprine was associated with a significant increase in cyclin A-dependent kinase activity. In addition, etoposide caused substantial inhibition of DNA synthesis.


CONCLUSIONS
These results indicate that staurosporine potentiates apoptosis through events which occur downstream of DNA damage, and implicate unscheduled activation of cyclin A-dependent kinase during inhibition of DNA synthesis as a possible cause.
Lock
R B
RB
Henry Vogt Cancer Research Institute, J. Graham Brown Cancer Center, Department of Medicine, University of Louisville, KY 40292, USA.
Thompson
B S
BS
Sullivan
D M
DM
Stribinskiene
L
L
D017209:Apoptosis / Q000187:drug effects*; D001943:Breast Neoplasms; D016203:CDC2 Protein Kinase / Q000378:metabolism; D042846:CDC2-CDC28 Kinases; D002460:Cell Line; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004261:DNA Replication / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004357:Drug Synergism; D005047:Etoposide / Q000633:toxicity*; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009363:Neoplasm Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D009363:Neoplasm Proteins; D005047:Etoposide; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D019311:Staurosporine