Effect of protein kinase C inhibitor (PKCI) on radiation sensitivity and c-fos transcription

Purpose: The human genetic disorder ataxia-telangiectasia (AT) is a multisy stem disease characterized by extreme radiosensitivity. Although ionizing r adiation was known to induce c-fos transcription and cellular protein kinas e C (PKC) induces the expression of this immediate response gene, little is known about how mutated AT (ATM) or PKC-mediated signal transduction pathw ay modulates the c-fos gene transcription and gene expression, Here we have studied the effect of PKC inhibitor (PKCI) on radiation sensitivity and c- fos transcription in normal and AT cells, and also studied whether PKCI eff ect on c-fos occurs in Ras-dependent pathway. Methods and Materials: Normal (LM217) and AT (AT5BIVA) cells were transfect ed with PKCI expression plasmid and integration and overexpression of PKCI was evaluated by polymerase chain reaction and northern blotting, respectiv ely. Cells were irradiated at a dose of 5 Gy/min with Cs-137 irradiator and harvested 48 h after irradiation and investigated apoptosis with TUNEL met hod. The c-fos transcription activity was studied by performing compute ass isted tomography (CAT) assay of reporter gene after transfection of c-fos C AT plasmid into LM and AT cells. Overexpression of Ras protein in transfect ed cells was shown by western blotting. Results: Our results demonstrated for the first time a role of PKCI on the radiation sensitivity and c-fos transcription in LM and AT cells. PKCI incr eased radiation induced apoptosis in LM cells (5% to 20%) but reduced apopt osis slightly in AT cells. The basal c-fos transcription activity is 70 tim es lower in AT cells than in LM cells. This c-fos transcription activity wa s repressed by overexpression of PKCI in LM cells but not in AT cells. Afte r induction of c-fos by Ras protein, overexpression of PKCI repressed c-fos transcription in LM cells but not in AT cells. Conclusions: Overexpression of PKCI increased radiation sensitivity and rep ressed c-fos transcription in LR I cells but not in AT cells, and this is r elated with Ras, These results suggest that the effect of PKCI on c-fos tra nscription activity is related with Ras dependent signal transduction pathw ays and these mechanisms are different between normal fibroblasts, LM and A TM mutated, AT cells, The data obtained by this study provided evidence for novel transcriptional difference between LM and AT cells and this may be a reason for increased radiation sensitivity of AT cells. (C) 2001 Elsevier Science Inc.