Phorbol ester-induced generation of reactive oxygen species is protein kinase C beta-dependent and required for SAPK activation

Treatment of human U-937 myeloid leukemia cells with 12 O-tetradecanoylphor bol-13-acetate (TPA) is associated with protein kinase C (PKC) beta II-medi ated activation of the stress-activated protein kinase (SAPK) pathway. The present studies demonstrate that the TPA response of U-937 cells includes t he generation of reactive oxygen species (ROS), By contrast, the TPA-resist ant U-937 cell variant (TUR), which is deficient in PKC beta II expression, failed to respond to TPA with the induction of ROS. Moreover, we show that TPA-induced ROS production is restored in TUR cells stably transfected to express PKC beta II, The results also demonstrate that TPA-induced ROS prod uction is required for activation of the MEK kinase-1 (MEKI-1)--> SAPK path way. In concert with this observation, treatment of U-937 with H2O2 as a so urce of ROS is associated with activation of the MEKK-1-->SAPK cascade. The se findings indicate that PKC beta II is required for TPA-induced ROS produ ction and that the MEKK-1-->SAPK pathway is activated by a ROS-mediated mec hanism.