Regulation of the IRF-1 tumour modifier during the response to genotoxic stress involves an ATM-dependent signalling pathway Jessica Pamment 1 , Eleanor Ramsay 1 , Michael Kelleher 1 , David Dornan 1 and Kathryn L Ball* ,1 1 Cancer Research UK Laboratories, University of Dundee Medical School, Dundee DD1 9SY, UK The mechanism by which genotoxic stress induces IRF-1 and the signalling components upstream of this anti- oncogenic transcription factor during the response to DNA damage are not known. We demonstrate that IRF- 1 and the tumour suppressor protein p53 are coordinately up-regulated during the response to DNA damage in an ATM-dependent manner. Induction of IRF-1 protein by either ionizing radiation (IR) or etoposide occurs through a concerted mechanism involving increased IRF-1 expression/synthesis and an increase in the half-life of the IRF-1 protein. A striking defect in the induction of both IRF-1 mRNA and IRF-1 protein was observed in ATM deficient cells. Although ATM deficient cells failed to increase IRF-1 in response to genotoxic stress, the induction of IRF-1 in response to viral mimetics remained intact. Re-expression of the ATM kinase in AT cells restored the DNA damage inducibility of IRF- 1, whilst the PI-3 kinase inhibitor wortmannin inhibited IRF-1 induction by DNA damage in ATM-positive cells. The data highlight a role for the ATM kinase in orchestrating the coordinated induction and transcrip- tional cooperation of IRF-1 and p53 to regulate p21 expression. Thus, IRF-1 is controlled by two distinct signalling pathways; a JAK/STAT-signalling pathway in viral infected cells and an ATM-signalling pathway in DNA damaged cells. Oncogene (2002) 21, 7776 – 7785. doi:10.1038/sj.onc. 1205981 Keywords: IRF-1; protein turnover; ATM; p53; DNA damage Introduction Interferon regulatory factor-1 (IRF-1) is the founding member of a family of interferon (IFN) responsive transcription factors. IRF-1 was first described as a mediator of the interferon response that bound to the virus-inducible ‘enhancer-like’ elements of the IFN-b gene (Fujita et al., 1988; Miyamoto et al., 1988; Taniguchi, 1989). More recently, it has been recognized that activation of IRF-1 leads to the expression of genes involved in a number of different cellular processes. In addition to the anti-viral response, these include: regulation of the cell cycle (Stevens and Yu- Lee, 1992, 1994) and apoptosis (Kirchhoff and Hauser, 1999; Tamura et al., 1995); development of the T cell immune response (Matsuyama et al., 1993); suscept- ibility to transformation by oncogenes (Tanaka et al., 1994), and the response to genotoxic agents (Prost et al., 1998; Tanaka et al., 1996). Furthermore, deletion or point mutation of the IRF-1 gene (Eason et al., 1999; Willman et al., 1993), and exon skipping of IRF- 1 mRNA (Harada et al., 1994) have been linked to the development of human haemopoietic malignancies, such as leukaemia and myelodysplastic syndrome (Boultwood et al., 1993; Green et al., 1999; Willman et al., 1993), as well as, solid phase tumours of the gastro-intestinal tract (Ogasawara et al., 1996; Tamura et al., 1996). In conjunction with a recent study showing that IRF-1 can modulate tumour suscept- ibility in the presence of oncogenic lesions (Nozawa et al., 1999), the above research provides evidence that IRF-1 has tumour suppressor/modifier activity. The spectrum of IRF-1 responsive genes is depen- dent on a number of factors including stimulus, cell type and stage of development (Taniguchi et al., 1998). IRF-1 activation has been most extensively studied in haemopoietic cells during the response to viral attack or IFN-g treatment. Induction of IRF-1 gene expres- sion following stimulation by IFN-g is dependent on the JAK/STAT signalling pathway and genes down- stream of IRF-1 in this pathway include, IFN-b (Fujita et al., 1989a; Miyamoto et al., 1988), RNA dependent protein kinase (Beretta et al., 1996; Kirchhoff et al., 1995) and 2-5A synthetase (Coccia et al., 1999; Wang and Floyd-Smith, 1998). Although the role of IRF-1 in the response to viral attack is now well established there is less known about the role of this transcription factor in response to other environmental stresses, such as DNA damage. Mouse embryonic fibroblasts (MEFs) and hepatocytes that are deficient in IRF-1 are compromised in their ability to undergo growth arrest (Tanaka et al., 1996) and to repair damaged DNA (Prost et al., 1998), respectively. Furthermore, maximal induction of the cyclin depen- dent kinase inhibitor, p21 (WAF1/CIP1), following exposure of MEFs to ionizing radiation (IR), appears to be dependent on both p53 and IRF-1 (Tanaka et al., 1996). This has led to the suggestion that IRF-1 Received 27 May 2002; revised 16 August 2002; accepted 16 August 2002 *Correspondence: KL Ball; E-mail: k.l.ball@dundee.ac.uk Oncogene (2002) 21, 7776 – 7785 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc