PROLONGED C-JUN EXPRESSION IN IRRADIATED ATAXIA TELANGIECTASIA FIBROBLASTS

Purpose: Ataxia telangiectasia (AT) is an autosomal recessive disorder associated with radiation sensitivity and anincreased incidence of leukemia, lymphoma, and some solid tumors. After exposure to ionizbtg radiation, cells from patients with AT demonstrate an attenuated G,-phase checkpoint. Because c-jun is known to reguiate, in part, the exit from G, and the onset of DNA replication, we analyzed c@n transcription in irradiated .4T fibroblasts. Methods and Materials: ATSBI fibroblasts were irradiated and RNA was extracted and assayed for c-jun expression by Northern blot analysis. Transcriptional regulation of c-&n was evaluated by use of the 5’ untrzmslated region of the jun promoter linked to the chloramphenicol acetyl transferase (CAT) reporter gene. Deletion mutants of the RSRF, SP-1, AP-1, and CCAAT domains within the jun promoter linked to the CAT reporter were transfected into AT531 cells. Transfectants were irradiated, and CAT expression was qua&Bed. After x-irradiation, nuclear protein binding to CCAAT was evaluated by an electrophoretic mobility shii assay. Results: X-ray-mediated c-&n expression was sustained in ATSBI cells as compared to only transient expression -irradiated normal diploid fibroblasts. Mutation of either the AP-1 or CCAAT domains within the c-jun promoter reduced transcription by 50% and combined deletion of both AP-1 and CCAAT cis-aetingelementsentirely eliminated radiation-mediated transcriptional activation. Electrophoretic mobility gel &ii assay of t&e nuclear proteins isolated from irradiated AT fibroblasts demonstrated their increased binding to the CCAAT sequence at 30 mln after irradiation. Competition for nuclear protein binding to the CCAAT sequence with excess cold CCAAT demonstrated that protein binding to this sequence was specific. These findings were distinct from induction by phorbol esthers in that the RSRF c&-acting element and DNA segments upstream of -’ 132 base pairs do participate in e.j,n induction by phorbol esthers but not by radiation. Conclusions: Radiation-mediated transcriptional regulation of c-jun is prolonged in AT fibroblasts and is regulated in combinatorial control by the AP-1 and CCAAT domains, and transcriptional regulation is distinct from that induced by phorbol esthers. Copyright 0 1996 Elsevier Science Inc. Radiation, Ataxia telangiectasia, c&n, CCAAT box.