TRAIL is a key target in S-phase slowing-dependent apoptosis induced by interferon-b in cervical carcinoma cells Serena Vannucchi 1,4 , Maria V Chiantore 1,4 , Gianna Fiorucci 1,2 , Zulema A Percario 3 , Stefano Leone 3 , Elisabetta Affabris 3 and Giovanna Romeo* ,1,2 1 Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanita`, Rome, Italy; 2 Institute of Molecular Biology and Pathology, National Research Council, Rome, Italy; 3 Department of Biology, University of ‘Roma Tre’, Rome, Italy Interferon (IFN)-b inducesS-phaseslowingandapoptosis in human papilloma virus (HPV)-positive cervical carci- nomacelllineME-180.Here,weshowthatapoptosisisa consequence of the S-phase lengthening imposed by IFN- b, demonstrating the functional correlation between S- phasealterationandapoptosisinduction.InME-180cells, where p53 function is inhibited by HPV E6 oncoprotein, IFN-b effects on cell cycle and apoptosis occur indepen- dently of p53. The apoptosis due to IFN-b is mediated by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a manner dependent on the S-phase deregulation. IFN-b appears to increase TRAIL expres- sion both directly at the mRNA level and indirectly by augmenting surface protein levels as a consequence of the induced S-phase cell accumulation. Moreover, the altera- tion of the S-phase due to IFN-b promotes TRAIL- dependent apoptosis by potentiating cell sensitivity to TRAIL,possiblythroughinductionofaproapoptoticNF- jB activity and TRAIL-R2 receptor expression. Interest- ingly, IFN-b-induced TRAIL-dependent apoptotic events strongly differ in the requirement of caspase activity. These results show that IFN-b may induce an apoptotic response by deregulating cell cycle. Understanding the linkage between these mechanisms appears to be of primary importance in the search for new IFN-based therapeutic strategies to circumvent cancer disease or improve clinical outcome. Oncogene (2005) 24, 2536–2546. doi:10.1038/sj.onc.1208403 Published online 7 February 2005 Keywords: TRAIL; IFN-b; apoptosis; cell cycle; SCC Introduction There is accumulating evidence that cell cycle deregula- tion may prevent or induce an apoptotic response depending upon the cellular context (Pucci et al., 2000). In mammalian cells, apoptosis can be initiated through the engagement of the death receptors, and/or the release of cytochrome c from the mitochondria. The death receptor pathway is initiated by the tumor necrosisfactor(TNF)familyofcytokines,suchasTNFa, Fas-ligand and TNF-related apoptosis-inducing ligand (TRAIL)/APO-2L, all of which can act as extracellular activators of apoptosis upon binding to their respective receptors (Armitage, 1994). These receptors recruit adapter proteins such as FADD, which in turn bind caspase-8 molecules, resulting in caspase-8 activation. Caspase-8 directly activates caspase-3 or acts by cleaving Bid, a member of Bcl-2 family, which targets mitochondria for cytochrome c release (Scaffidi et al., 1998; Stennicke et al., 1998; Gross et al., 1999). TRAIL induces apoptosis in various tumor cells without affecting normal cells; therefore, it is a promising candidate for cancer therapy (Fiorucci et al., 2005). At least four receptors bind to TRAIL with similar affinity. Both TRAIL-R1/DR4 and TRAIL-R2/ DR5 recruit caspase-8 or -10 via FADD and activate the caspase cascade, resulting in apoptotic cell death. In contrast, TRAIL-R3 and TRAIL-R4 seem to work as decoy receptors (Baetu and Hiscott, 2002). TRAIL-R3 lacks a cytoplasmic domain and exists as a glycophos- pholipid-anchored protein on the cell surface; TRAIL- R4 carries a cytoplasmic-truncated death domain that cannot transmit a death signal. The physiological functions of these multiple TRAIL-Rs remain obscure. Binding of TRAIL to TRAIL-R1 and TRAIL-R2 leads also to the activation of NF-kB by a TRAF2-NIK- IKKa/b-dependent signaling and c-Jun N-terminal kinase (Hu et al., 1999; Muhlenbeck et al., 2000). NF- kB can also be activated through TRAIL-R4. The role of NF-kB is poorly characterized and appears to be dependent on the cellular type. The possible induction of NF-kB-dependent antiapoptotic activities by TRAIL, supposed to underlie the resistance of normal cells to TRAIL, is consistent with the observation that repres- sion of NF-kB function by some chemotherapeutic agents (Ghosh et al., 2003) or interferon (IFN)-b (Shigeno et al., 2003) can enhance TRAIL-mediated cancer cell death. In contrast, it has been shown that TRAIL-dependent NF-kB activation triggers apoptosis in T cells and epithelial cell lines by inducing proapop- totic genes (Baetu et al., 2001; Shetty et al., 2002). Received 21 July 2004; revised 4 November 2004; accepted 22 November 2004; published online 7 February 2005 *Correspondence: G Romeo, c/o Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanita`, Viale Regina Elena, 299, Rome 00161, Italy; E-mail: romeo@iss.it 4 Both authors contributed equally to the manuscript Oncogene (2005) 24, 2536–2546 & 2005 Nature Publishing Group All rights reserved 0950-9232/05 $30.00 www.nature.com/onc