Introduction Apoptosis is a common, conserved, endogenous cell suicide program required for proper embryonic development and maintenance of homeostasis in adult tissues (Vaux and Korsmeyer, 1999; Jacobson et al., 1997). Apoptosis is characterized by a biochemical cascade and morphological changes, including activation of caspases, translocation of phosphatidylserine from the inner to the outer layer of the plasma membrane, blebbing of cytoplasmic membranes, chromatin condensation and fragmentation into apoptotic bodies (Kerr et al., 1972; Thornberry and Lazebnik, 1998; Hengartner, 2000). Recently, a novel non-apoptotic programmed cell death (PCD) process designated paraptosis was described by Sperandio et al. (Sperandio et al., 2000). The features of paraptosis differ from those of apoptosis and involve cytoplasmic vacuolation, mitochondrial swelling and absence of caspase activation or typical nuclear changes, including pyknosis and DNA fragmentation (Sperandio et al., 2000; Wyllie and Golstein, 2001). There is increasing evidence that this alternative, non-apoptotic PCD exists in parallel with apoptosis. For instance, T9 glioma cells expressing membrane macrophage colony-stimulating factor were killed by polymorphonuclear leukocytes and macrophages with vacuolization of the mitochondria and endoplasmic reticulum (ER) resembling paraptosis (Chen et al., 2002). Similarly, the neuropeptide substance P and its receptor, neurokinin-1 receptor, mediated a non-apoptotic form of PCD resembling paraptosis in some cases (Castro-Obregon et al., 2002). Activated microglia triggered neuronal cell death with ultrastructural characteristics of marked vacuolation, slightly condensed chromatin and severely disintegrated perikarya, following blockage of the caspase cascade (Tanabe et al., 1999). Apaf-1-deficient embryonic stem (ES) cells died through a non-apoptotic mechanism associated with loss of mitochondrial membrane potential after treatment with diverse cell death stimuli (Haraguchi et al., 2000). Overexpression of extramitochondrial Omi/HtrA2 induced atypical PCD, which was neither accompanied by an increase in caspase activity nor inhibited by caspase inhibitors (Suzuki et al., 2001). In addition, ceramide induced non-apoptotic PCD with necrotic- like morphology in human glioma cells in the presence of pan-caspase inhibitors or during overexpression of Bcl-xL (Mochizuki et al., 2002). These examples support the theory that cells have other intrinsic programs for death that are distinct from apoptosis. Despite the elucidation of morphological characteristics, the precise biochemical mechanism for non-apoptotic PCD and the basic components of the death machinery are currently unknown. Tumor necrosis factor receptor (TNFR) superfamily proteins play important regulatory roles in cellular responses ranging from cell proliferation and differentiation to cell survival or 1525 Accumulating reports demonstrate that apoptosis does not explain all the forms of programmed cell death (PCD), particularly in individual development and neurodegenerative disease. Recently, a novel type of PCD, designated ‘paraptosis’, was described. Here, we show that overexpression of TAJ/TROY, a member of the tumor necrosis factor receptor superfamily, induces non- apoptotic cell death with paraptosis-like morphology in 293T cells. Transmission electron microscopy studies reveal extensive cytoplasmic vacuolation and mitochondrial swelling in some dying cells and no condensation or fragmentation of the nuclei. Characteristically, cell death triggered by TAJ/TROY was accompanied by phosphatidylserine externalization, loss of the mitochondrial transmembrane potential and independent of caspase activation. In addition, TAJ/TROY suppressed clonogenic growth of HEK293 and HeLa cells. Interestingly, overexpression of Programmed cell death 5 (PDCD5), an apoptosis-promoting protein, enhanced TAJ/TROY-induced paraptotic cell death. Moreover, cellular endogenous PDCD5 protein was significantly upregulated in response to TAJ/TROY overexpression. These results provide novel evidence that TAJ/TROY activates a death pathway distinct from apoptosis and that PDCD5 is an important regulator in both apoptotic and non-apoptotic PCD. Key words: Paraptosis, Non-apoptotic, PDCD5, TFAR19, TAJ/TROY Summary An alternative form of paraptosis-like cell death, triggered by TAJ/TROY and enhanced by PDCD5 overexpression Ying Wang 1 , Xianting Li 1 , Lu Wang 2 , Peiguo Ding 1 , Yingmei Zhang 2 , Wenling Han 2 and Dalong Ma 1,2, * 1 Laboratory of Medical Immunology, School of Basic Medical Science, Peking University, Xueyuan Road 38, Beijing 100083, China 2 Center for Human Disease Genomics, Health Science Center, Peking University, Beijing 100083, China *Author for correspondence (e-mail: madl@bjmu.edu.cn) Accepted 18 November 2003 Journal of Cell Science 117, 1525-1532 Published by The Company of Biologists 2004 doi:10.1242/jcs.00994 Research Article