Involvement of Reactive Oxygen Intermediates in the Induction of c-jun Gene Transcription by Ionizing Radiation

Previous work has demonstrated that the cellular response to ionizing radiation includes transcriptional activation of the c-jun gene. The signaling events responsible for this response, however, remain unclear. The present studies have examined the effects of ionizing radiation on c-jun expression in a variant of HL-60 cells, designated HL-525, which is deficient in protein kinase C (PKC)-mediated signal transduction. The results demonstrate that these cells expf ess low levels of PKCα and PKCβ transcripts and exhibit an attenuated induction of c-jun expression following treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA). In contrast, HL-525 cells respond to ionizing radiation with an increase in c-jun mRNA which is more pronounced than that in wildtype HL-60 cells. These cells similarly respond to ionizing radiation with increased expression of the jun-B, jun-D, c-fos, and fos-B genes. Nuclear run-on assays demonstrate that X-ray-induced c-jun expression in HL-525 cells is regulated by increases in the rate of c-jun gene transcription. Moreover, mRNA stability studies in irradiated HL-525 cells demonstrate that the half-life of c-jun transcripts is prolonged compared to that in wildtype cells. Studies with TV-acetyl-L-cysteine (NAC), an antioxidant, suggest that X-ray-induced transcriptional activation of the c-jun gene is mediated at least in part through the formation of reactive oxygen intermediates (ROIs). In this context, H₂O₂ also induced c-jun expression in HL-525 cells, and this effect was inhibited by NAC. We further demonstrate that the induction of c-jun expression by X-rays, as well as H₂0₂, is inhibited (1) by prolonged exposure to TPA or bryostatin and (2) by H, 7, a nonspecific inhibitor of PKC-like protein kinases, but not HA, 1004, a more selective inhibitor of cyclic nucleotide-dependent protein kinase activity. Taken together, these results indicate that ionizing radiation induces c-jun gene transcription through the formation of ROIs and that a protein kinase, perhaps a PKC isoform distinct from PKCα and PKCβ, is also involved in this signaling pathway.