Protein kinase C inhibition by UCN-01 induces apoptosis in human glioma cells in a time-dependent fashion.
Recent studies in our laboratory have shown that UCN-01 (7-hydroxystaurosporine), which is a derivative of the non-selective protein kinase inhibitor staurosporine that exhibits relative selectivity for protein kinase C (PKC), is a potent inhibitor of glioma growth in in vitro and in vivo models. This agent exhibits both cytotoxic and cytostatic effects, depending on the time period of drug exposure. In the present study, we examined whether UCN-01-induced cytotoxicity correlated with the induction of apoptosis, and characterized further the time course of this process as a prelude to application of UCN-01 in clinical trials. We first demonstrated that the cytotoxic effects of UCN-01 were associated with the induction of morphological features of apoptosis. Secondly, we identified electrophoretic features of apoptosis semiquantitatively at a series of time points using field inversion gel electrophoresis. These studies showed a peak in the induction of high-molecular-weight DNA fragmentation after 3-6 days of drug treatment. Thirdly, we measured the percentage of cells undergoing apoptosis at various time points using a terminal transferase-catalyzed in situ end-labeling technique, which confirmed a time- and concentration-dependent increase in apoptotic cell numbers. This correlated with a progressive decrease in the percentage of cells that were viable as assessed by trypan blue exclusion. Cell killing peaked within 2-4 days after beginning UCN-01 treatment, but continued at a lower level in the ensuing days. Taken together, these studies demonstrated that extended periods of exposure to UCN-01 are needed for optimal manifestation of cytotoxic effects against glioma cells, a factor that must be taken into consideration in the design of future clinical trials with this agent for malignant gliomas.
Bredel
M
M
Department of Neurosurgery, University of Pittsburgh School of Medicine, Pennsylvania, USA.
Pollack
I F
IF
Freund
J M
JM
Rusnak
J
J
Lazo
J S
JS
D000470:Alkaloids / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis; D001932:Brain Neoplasms / Q000201:enzymology / Q000473:pathology*; D004396:Coloring Agents; D004587:Electrophoresis, Agar Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005910:Glioma / Q000201:enzymology / Q000473:pathology*; D006801:Humans; D020287:In Situ Nick-End Labeling; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D019311:Staurosporine / Q000031:analogs & derivatives; D013997:Time Factors; D014343:Trypan Blue; D014407:Tumor Cells, Cultured
D000470:Alkaloids; D000970:Antineoplastic Agents; D004396:Coloring Agents; D004791:Enzyme Inhibitors; C054852:7-hydroxystaurosporine; D011493:Protein Kinase C; D019311:Staurosporine; D014343:Trypan Blue