Treatment of human HL-60 leukemic cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with activation of protein kinase C (PKC) and induction of monocytic differentiation. An HL-60 variant cell line, termed HL-525, derived from long-term exposure to TPA (Homma et al, Proc Natl Acad Sci USA 83: 7316, 1986) is resistant to TPA-induced differentiation and displays decreased PKC beta expression compared with the HL-60 parent line. However, this variant exhibits features of granulocytic differentiation, including nitroblue tetrazolium reduction, when exposed to all-trans retinoic acid (ATRA). Whereas treatment of HL-525 cells with ATRA or TPA alone had no effect on features of monocytic differentiation, these agents in combination resulted in cellular adhesion, nonspecific esterase staining, and induction of the c-fms (monocyte growth factor receptor) gene. In order to measure PKC expression associated with the reversal of TPA resistance by ATRA, we exposed HL-525 cells to ATRA and analyzed PKC-mRNA and protein levels. Exposure of HL-525 cells to ATRA for 3 days resulted in induction of PKC beta transcripts, whereas there was little change in PKC alpha mRNA levels. ATRA treatment was also associated with an increase in PKC activity and an induction of cytosolic PKC beta protein levels. These findings are consistent with the hypothesis that ATRA reverses TPA resistance in HL-525 cells by enhancing the expression of PKC.

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 express low levels of PKC alpha and PKC beta 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 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-jun transcripts is prolonged compared to that in wild-type cells. Studies with N-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.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumor necrosis factor (TNF) production following X-irradiation has been implicated in the biological response to ionizing radiation. Protein kinase C (PKC) is suggested to participate in TNF transcriptional induction and X-ray-mediated gene expression. We therefore studied radiation-mediated TNF expression in HL-60 cells with diminished PKC activity produced by either pretreatment with protein kinase inhibitors or prolonged 12-O-tetradecanoylphorbol-13-acetate treatment. Both treatments resulted in attenuation of radiation-mediated TNF induction. Consistent with these results, we found no detectable induction of TNF expression following X-irradiation in the HL-60 variant deficient in PKC-mediated signal transduction. The rapid activation of PKC following gamma-irradiation was established using an in vitro assay measuring phosphorylation of a PKC specific substrate. A 4.5-fold increase in PKC activity occurred 15 to 30 s following irradiation, which declined to baseline at 60 s. Two-dimensional gel electrophoresis of phosphoproteins extracted from irradiated cells demonstrated in vivo phosphorylation of the PKC specific substrate Mr 80,000 protein at 45 s following X-irradiation. These findings indicate that signal transduction via the PKC pathway is required for the induction of TNF gene expression by ionizing radiation.