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

Purpose: Inhibition of protein kinase C (PKC) activity has been demonstrate d to reduce thermotolerance (TT), presumably by decreasing heat shock prote in (HSP) production. Therefore, the interest was in evaluating this relatio nship further in two isogenic murine tumour cell lines: RIF-1 and its therm oresistant TR-4 selectant. Materials and methods: TT was induced in RIF-1 and TR-4 cells (45 degrees C for 15 min, then 37 degrees C for 6 h) with or without Ro31-8220, a specif ic inhibitor of PKC. PKC activity was assayed by determining the catalytic transfer of ATP to a specific substrate peptide. Survival was determined us ing the clonogenic assay. Apoptosis was quantitated by counting the number of cells demonstrating apoptosis after staining with acridine-orange/ ethid ium bromide. Production of the inducible form of HSP70 was assessed using W estern blot. Results: At 2 mu M Ro31-8220, > 80% of PKC activity was inhibited in both c ell lines, which was associated with no cytotoxicity at 37 degrees C. Basal HSP70 level was similar to 10-fold higher in the TR-4 compared with the RI F-1 cells. Upon TT induction, HSP70 level increased significantly in both c ell lines, which was suppressed in the presence of Ro31-8220, but the relat ive amount of HSP70 remained high in the TR-4 cells. At 24 h, heat-induced apoptosis increased from 4 to 38% in RIF-1 cells in the presence of Ro31-82 20, which was associated with a 26% reduction in clonogenic survival after thermotolerant heating. In contrast, <1% of TR-4 cells demonstrated apoptos is even with the highest dose of Ro31-8220, and no effect on survival was o bserved. Conclusion: Inhibition of PKC activity reduces HSP70 induction, which in tu rn is associated with promotion of heat-induced apoptosis in RIF-1 cells. H owever, 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 an d survival in this thermoresistant cell line.