Doxorubicin-induced death in neuroblastoma does not involve death receptors in S-type cells and is caspase-independent in N-type cells Sally Hopkins-Donaldson 1 , Pu Yan 1 , Katia Balmas Bourloud 1 , Annick Muhlethaler 1 , Jean-Luc Bodmer 2 and Nicole Gross* ,1 1 Department of Pediatric Onco-Hematology, Centre Hospitalier Universitaire Vaudois, CH1011 Lausanne, Switzerland; 2 Institute of Biochemistry, University of Lausanne, CH-1066, Epalinges, Switzerland Death induced by doxorubicin (dox) in neuroblastoma (NB) cells was originally thought to occur via the Fas pathway, however since studies suggest that caspase-8 expression is silenced in most high stage NB tumors, it is more probable that dox-induced death occurs via a different mechanism. Caspase-8 silenced N-type invasive NB cell lines LAN-1 and IMR-32 were investigated for their sensitivity to dox, and compared to S-type noninvasive SH-EP NB cells expressing caspase-8. All cell lines had similar sensitivities to dox, independently of caspase-8 expression. Dox induced caspase-3, -7, -8 and -9 and Bid cleavage in S-type cells and death was blocked by caspase inhibitors but not by oxygen radical scavenger BHA. In contrast, dox-induced death in N- type cells was caspase-independent and was inhibited by BHA. Dox induced a drop in mitochondrial membrane permeability in all cell lines. Dox-induced death in S- type cells gave rise to apoptotic nuclei, whereas in N- type cells nuclei were non-apoptotic in morphology. Transfection of SH-EP cells with a dominant negative FADD mutant inhibited TRAIL-induced death, but had no effect on dox-induced apoptosis. These results suggest that S-type cells undergo apoptosis after dox treatment independently of death receptors, whereas N-type cells are killed by a caspase-independent mechanism. Oncogene (2002) 21, 6132 – 6137. doi:10.1038/sj.onc. 1205879 Keywords: apoptosis; caspases; doxorubicin; caspase-8; neuroblastoma Introduction Neuroblastoma (NB) is a heterogeneous pediatric cancer derived from the sympathetic nervous system. Children over 1 year of age with stage IV NB tumors respond poorly to treatment, with a 5 year survival rate of only 30% (Spix et al., 2001), whereas low stage tumors in infants frequently undergo spontaneous remission. Failure to undergo programmed cell death is a putative mechanism for cancer cell resistance to chemotherapy, while in contrast spontaneous remission is thought to be a result of increased apoptosis (Oue et al., 1996). Apoptosis signaling occurs via two different path- ways which converge on a common machinery of cell demise that is activated by caspases (Strasser et al., 2000). The death receptor pathway is activated by ligands of the tumor necrosis factor (TNF) family which induce the trimerization of their cognate receptors (Daniel et al., 2001). This aggregation triggers the recruitment of adaptor protein FADD, which in turn binds procaspase-8, inducing its activa- tion by limited proteolytic cleavage. Effector caspases-3 and -7 are subsequently processed and activated, which ultimately results in DNA fragmentation and death. The mitochondrial pathway is activated by endoge- neous stress such as DNA damage and is characterized by the loss of mitochondrial transmembrane potential and the release of cytochrome c from mitochondria (Green and Reed, 1998). Cytosolic cytochrome c triggers the assembly of the apoptosome which recruits and activates caspase-9 and in turn caspases-3 and -7. Anti-apoptotic Bcl-2 family members such as Bcl-2 inhibit cytochrome c release by modulating the ability of Bax and Bak to facilitate opening of pores in the outer mitochondrial membrane (Wei et al., 2001). Crosstalk between the two apoptosis signaling path- ways occurs in some cells when caspase-8 converts an inactive form of Bid into a pro-apoptotic form that interacts with Bax and induces cytochrome c release (Scaffidi et al., 1999). Recent studies have shown that in addition to apoptosis, death receptor and mitochondrial signaling may activate alternative mechanisms of programmed cell death which are caspase-independent (Holler et al., 2000; Leist and Jaattela, 2001). These alternative mechanisms include necrosis and caspase-independent apoptosis which although are rare events in normal cells may be the prominent mode of death in tumor cells which have defects in the two apoptosis signaling pathways. Several years ago, Fulda et al. (1997) reported that death in NB cells induced by chemotherapeutic drugs Received 10 April 2002; revised 15 July 2002; accepted 16 July 2002 *Correspondence: N Gross, Department of Pediatric Onco-Hematol- ogy, University Hospital Lausanne (CHUV), CH-1011 Lausanne, Switzerland; E-mail: ngross@chuv.hospvd.ch Oncogene (2002) 21, 6132 – 6137 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc