Inhibition of protein kinase C activity promotes heat-induced apoptosis in RIF-1 but not in TR-4 cells

Abstract

Purpose: Inhibition of protein kinase C (PKC) activity has been demonstrated to reduce thermotolerance (TT), presumably by decreasing heat shock protein (HSP) production. Therefore, the interest was in evaluating this relationship further in two isogenic murine tumour cell lines: RIF-1 and its thermoresistant TR-4 selectant. Materials and methods: TT was induced in RIF-1 and TR-4 cells (45 C for 15min, then 37 C for 6h) with or without Ro31-8220, a specific inhibitor of PKC. PKC activity was assayed by determining the catalytic transfer of ATP to a specific substrate peptide. Survival was determined using the clonogenic assay. Apoptosis was quantitated by counting the number of cells demonstrating apoptosis after staining with acridine-orange/ ethidium bromide. Production of the inducible form of HSP70 was assessed using Western blot. Results: At 2 mum Ro31-8220, >80% of PKC activity was inhibited in both cell lines, which was associated with no cytotoxicity at 37 C. Basal HSP70 level was ~10-fold higher in the TR-4 compared with the RIF-1 cells. Upon TT induction, HSP70 level increased significantly in both cell lines, which was suppressed in the presence of Ro31-8220, but the relative amount of HSP70 remained high in the TR-4 cells. At 24h, heat-induced apoptosis increased from 4 to 38% in RIF-1 cells in the presence of Ro31-8220, which was associated with a 26% reduction in clonogenic survival after thermotolerant heating. In contrast, 1% of TR-4 cells demonstrated apoptosis even with the highest dose of Ro31-8220, and no effect on survival was observed. Conclusion: Inhibition of PKC activity reduces HSP70 induction, which in turn is associated with promotion of heat-induced apoptosis in RIF-1 cells. However, the survival signals in the TR-4 cells are so strong, that even 80% inhibition of PKC activity has minimal impact on heat-induced apoptosis and survival in this thermoresistant cell line.