Revised Manuscript Received June 9, 1992 abstract: 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 kinaseC (PKC)-mediated signal transduction. The results demonstrate that these cells express low levels of PKCa and PKC/3 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 thanthat in wild-type 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-juntranscripts is prolonged compared to that in wild-type 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 inpart 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 H2O2, is inhibited (1) byprolonged exposure to TPA or bryostatin and (2) by H7, a nonspecific inhibitor of PKC-like protein kinases, but not HA1004, 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 PKCa and PKC/3, is also involved in this signaling pathway.

The response of eukaryotic cells toionizing radiation includes activation of DNA repair mechanisms, cell cycle arrest, mutagenesis, and lethality. However, the events responsible for the control of these responses remain unclear. Ionizing radiation has been postulated to induce such effects either by direct interaction with DNA or through the formation of reactive oxygen intermediates (ROIs) which damage DNA (Hall, 1988). Recent studies have further suggested a role for the activation of immediate-early genes in the response to ionizing radiation. For example, exposure of cells toX-rays is associated with activation of the c-jun/c-fos and EGR-1 gene families which code for transcription factors (Sherman et al., 1990a; Hallahan et al., 1991a). Other studies have demonstrated that ionizing radiation induces expression and DNA binding activity of the nuclear factor kB (NF-kB)(Brach et al., 1991). The activation of transcriptionfactors may represent a critical control point in transducing early nuclear signals to longer term changes in gene expression that reflect the response to X-ray-induced damage. Indeed, other studies have demonstrated that irradiation of cells is associated with