LETTERS NATURE CELL BIOLOGY VOLUME 7 | NUMBER 9 | SEPTEMBER 2005 909 Clusterin inhibits apoptosis by interacting with activated Bax Honglai Zhang 1 , Jin Koo Kim 1 , Chris A. Edwards 2 , Zhaohui Xu 3 , Russell Taichman 4 and Cun-Yu Wang 1,5 Clusterin is an enigmatic glycoprotein that is overexpressed in several human cancers such as prostate and breast cancers, and squamous cell carcinoma 1,2 . Because the suppression of clusterin expression renders human cancer cells sensitive to chemotherapeutic drug-mediated apoptosis, it is currently an antisense target in clinical trials for prostate cancer. However, the molecular mechanisms by which clusterin inhibits apoptosis in human cancer cells are unknown. Here we report that intracellular clusterin inhibits apoptosis by interfering with Bax activation in mitochondria. Intriguingly, in contrast to other inhibitors of Bax, clusterin specifically interacts with conformation-altered Bax in response to chemotherapeutic drugs. This interaction impedes Bax oligomerization, which leads to the release of cytochrome c from mitochondria and caspase activation. Moreover, we also find that clusterin inhibits oncogenic c-Myc-mediated apoptosis by interacting with conformation-altered Bax. Clusterin promotes c-Myc- mediated transformation in vitro and tumour progression in vivo. Taken together, our results suggest that the elevated level of clusterin in human cancers may promote oncogenic transformation and tumour progression by interfering with Bax pro-apoptotic activities. Clusterin, also known as apolipoprotein J or testosterone-repressed prostate message 2 (TRPM-2) among others, is a glycoprotein that has previously been reported to have diverse functions, including lipid trans- portation, complement inhibition and regulation of apoptosis 1,2 . The cytoplasmic form of clusterin is a protein with a relative molecular mass of ∼60,000 (M r 60K) whereas the secreted clusterin is a heterodimer con- sisting of α and β chains. Accumulating evidence suggests that clusterin is an anti-apoptotic protein associated with both cancer therapy resist- ance and tumour development 3–7 . To explore the potential anti-apoptotic function of clusterin in cancer cells, we stably expressed clusterin in the human fibrosarcoma cell line HT1080 using retrovirus-mediated infec- tion. As shown in Fig. 1a, clusterin-expressing HT1080 cells (HT1080/ Clu) and control cells (HT1080/V) were generated. A band with 1 Laboratory of Molecular Signaling and Apoptosis, Department of Biological and Materials Sciences, 2 Microscopy and Image-Analysis Core, Department of Cell and Developmental Biology, 3 Life Science Institute and 4 Department of Periodontics, Prevention and Geriatrics, School of Dentistry, University of Michigan, Ann Arbor, MI 48109-1078, USA. 5 Correspondence should be addressed to C.-Y.W (e-mail: cunywang@umich.edu) Published online: 21 August 2005; DOI: 10.1038/ncb1291 M r ∼60K (c-Clu), corresponding to the full-length intracellular clusterin, and a band with M r ∼40K (s-Clu) of the secreted subunit of clusterin, were detected by western blot analysis. To determine whether overex- pression of clusterin promoted chemoresistance, both HT1080/Clu and HT1080/V cells were treated with the chemotherapeutic drug etoposide. As shown in Fig. 1b, approximately 67% of HT1080/V cells, but only 27% of HT1080/Clu cells, were killed following etoposide treatment. Clusterin also provided protection against apoptosis induced by the topoisomerase I inhibitor camptothecin (Fig. 1c). Consistently, overex- pression of clusterin in HT1080/Clu cells inhibited DNA fragmentation and histone release induced by etoposide (Fig. 1d). Moreover, mitochon- drial release of cytochrome c and caspase-9 activation were significantly suppressed by clusterin (Fig. 1e, f). Because clusterin is also secretory, it was possible that secreted clusterin might inhibit apoptosis in HT1080 cells. We found that conditioned media from HT1080/Clu cells did not suppress etopo- side-mediated apoptosis in HT1080/V cells and other cells (see Supplementary Information, Fig. S1a). The secreted clusterin expressed in conditioned media had been confirmed by western blot analysis (see Supplementary Information, Fig. S1b). Moreover, we found that the addition of recombinant clusterin to the culture media could not inhibit apoptosis (see Supplementary Information, Fig. S1c–e). These results suggested that intracellular clusterin might be responsible for anti-apoptotic functions in HT1080 cells. In mitochondria-mediated apoptosis, it has been reported that BH3- only proteins induce Bax to undergo a conformational change 8–14 . This change results in the exposure of an amino-terminal epitope of Bax and its translocation to mitochondria, where it is oligomerized and subse- quently induces the release of cytochrome c and other apoptogenic pro- teins 8,15,16 . This activated form of Bax can be detected with the specific anti-Bax monoclonal antibody clone 6A7 (ref. 17). Therefore, we exam- ined whether clusterin might affect Bax conformational changes induced by etoposide. As shown in Fig. 2a, etoposide stimulated Bax conforma- tional changes in both HT1080/V and HT1080/Clu cells at a similar level. Moreover, we found that conformation-altered Bax was translocated to mitochondria because Bax immunostaining with the 6A7 antibodies was Nature Publishing Group ©2005