PROLONGED C-JUN EXPRESSION IN IRRADIATED ATAXIA-TELANGIECTASIA FIBROBLASTS

Purpose: Ataxia telangiectasia (AT) is an autosomal recessive disorder associated with radiation sensitivity and an increased incidence of l eukemia, lymphoma, and some solid tumors. After exposure to ionizing r adiation, cells from patients with AT demonstrate an attenuated G(1)-p hase checkpoint. Because c-jun is known to regulate, in part, the exit from G(1) and the onset of DNA replication, we analyzed c-jun transcr iption in irradiated AT fibroblasts. Methods and Materials: AT5BI fibr oblasts were irradiated and RNA was extracted and assayed for c-jun ex pression by Northern blot analysis. Transcriptional regulation of c-ju n was evaluated by use of the 5' untranslated region of the jun promot er linked to the chloramphenicol acetyl transferase (CAT) reporter gen e. Deletion mutants of the RSRF, SP-1, AP-1, and CCAAT domains within the jun promoter linked to the CAT reporter were transfected into AT5B I tells. Transfectants were irradiated, and CAT expression was quantif ied. After x-irradiation, nuclear protein binding to CCAAT was evaluat ed by an electrophoretic mobility shift assay. Results: X-ray-mediated c-jun expression was sustained in AT5BI cells as compared to only tra nsient expression in irradiated normal diploid fibroblasts. Mutation o f either the AP-1 or CCAAT domains within the c-jun promoter reduced t ranscription by 508 and combined deletion of both AP-1 and CCAAT cis-a cting elements entirely eliminated radiation-mediated transcriptional activation. Electrophoretic mobility gel shift assay of the nuclear pr oteins isolated from irradiated AT fibroblasts demonstrated their incr eased binding to the CCAAT sequence at 30 min after irradiation. Compe tition for nuclear protein binding to the CCAAT sequence with excess c old CCAAT demonstrated that protein binding to this sequence was speci fic These findings were distinct from induction by phorbol esthers in that the RSRF cis-acting element and DNA segments upstream of -132 bas e pairs do participate in c-jun induction by phorbol esthers but not b y radiation. Conclusions: Radiation-mediated transcriptional regulatio n of c-jun is prolonged in AT fibroblasts and is regulated in combinat orial control by the AP-1 and CCAAT domains, and transcriptional regul ation is distinct from that induced by phorbol esthers. Copyright (C) 1996 Elsevier Science Inc.