SHORT REPORTS Regulation of apoptosis by fau revealed by functional expression cloning and antisense expression Mirna Mourtada-Maarabouni* ,1 , Lucy Kirkham 1 , Farzin Farzaneh 2 and Gwyn T Williams* ,1 1 School of Life Sciences, Keele University, Keele, ST5 5BG, UK; 2 Molecular Medicine, Rayne Institute, GKT School of Medicine, King’s Denmark Hill Campus, 123 Coldharbour Lane, London SE21 7AN, UK Functional expression cloning is a powerful strategy for identifying critical steps in biological pathways indepen- dently of prior assumptions. It is particularly suitable for the identification of molecules crucial to the control of apoptosis. Our screen for sequences suppressing T-cell apoptosis isolated a sequence antisense to fau (Finkel– Biskis–Reilly murine sarcoma virus (FBR-MuSV)-asso- ciated ubiquitously expressed gene). The fox gene in FBR murine osteosarcoma virus is also antisense to fau and several reports have indicated that fau displays tumour suppressor and oncogenic properties in different contexts. Our observations indicate that the fau antisense sequence suppresses expression of endogenous fau mRNA and produces resistance to apoptosis induced both by the glucocorticoid analogue dexamethasone’ by ultraviolet radiation, and by the anticancer drug cisplatin. In all cases, colony-forming ability is protected, indicating that fau affects the critical events prior to commitment to cell death. Overexpression of fau in the sense orientation induces cell death, which is inhibited both by Bcl-2 and by inhibition of caspases, in line with its proposed role in apoptosis. Oncogene (2004) 23, 9419–9426. doi:10.1038/sj.onc.1208048 Published online 15 November 2004 Keywords: fau; functional expression cloning; Finkel– Biskis–Reilly osteosarcoma virus; apoptosis Apoptosis is a tightly regulated physiological process, which is crucial to the control of many biological functions. It plays a central role in the physiology of the immune response, cellular differentiation, malignant trans- formation and in the development of degenerative diseases (Green and Evan, 2002; Rathmell and Thompson, 2002). Active cell death by apoptosis is also crucial for cytotoxic therapy of various cancers (Williams, 1991; Ferreira et al., 2002; Rathmell and Thompson, 2002). It has therefore become clear that improved understanding of the mechan- ism of apoptosis at the molecular level should lead to the identification of appropriate targets for drug therapy. Despite the significant progress of the last decade, precise knowledge of the molecular pathways involved in apop- tosis remains incomplete and potentially many critical genes remain to be identified. Recently, appreciation of the molecular complexity of the processes of apoptosis control and drug sensitivity has highlighted the advantages of functional strategies for identifying those molecules that act at controlling, that is, rate-limiting, steps. Our research programme aims to identify novel genes involved in apoptosis using cDNA library functional expression cloning. One invaluable advantage of this approach is that it uses selection strategies that result in the identification of genes based entirely on their function, so that no prior knowledge of the gene sequence is assumed or required (Whitehead et al., 1995; Zannettino et al., 1996). The identification of many molecules involved in lymphocyte function (Starr et al., 1997), lymphomas and other cancers (Li et al., 1997; Lund et al., 2002; Mikkers et al., 2002; Suzuki et al., 2002) has highlighted the success of this approach. Notable successes have also been achieved by using this approach in identifying novel apoptosis controlling genes such as LUCA15/RBM5 novel tumour suppressor gene (Sutherland et al., 2000), protein phosphatase 4 (Mourtada-Maarabouni et al., 2003) and vATPase subunit E (Anderson and Williams, 2003). In this study, we report the isolation of fau (Finkel–Biskis–Reilly murine sarcoma virus (FBR-MuSV)-associated ubiqui- tously expressed gene) by functional expression cloning and its identification as an apoptosis regulatory gene. Our functional expression system comprised a retro- viral cDNA library generated from the murine factor- dependent FDCP-1 cell line as the source of apoptosis regulators (Rayner and Gonda, 1994), the W7.2 mouse thymoma cells (Danielsen et al., 1983) used as screening hosts and dexamethasone followed by g-radiation as the apoptotic stimuli. The host for retroviral infections W7.2c was a subclone of the W7.2 mouse thymoma cell line with a low frequency of spontaneous apoptosis (Mourtada-Maarabouni et al., 2003). W7.2 cells have been used in many apoptosis studies, particularly for the study of dexamethasone-induced apoptosis. W7.2 cells infected with the FDCP1 retroviral cDNA library (Mourtada-Maarabouni et al., 2003) were cultured for 6 days with 20 nM dexamethasone, followed by 500 cGy g-irradiation to induce apoptosis. These two stimuli were used consecutively to increase the Received 3 March 2004; revised 20 July 2004; accepted 20 July 2004; published online 15 November 2004 *Correspondence: GT Williams; E-mail: g.t.williams@keele.ac.uk or M Mourtada-Maarabouni; E-mail: bia19@biol.keele.ac.uk Oncogene (2004) 23, 9419–9426 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $30.00 www.nature.com/onc