Research Article Antisense Suppression of the Chloride Intracellular Channel Family Induces Apoptosis, Enhances Tumor Necrosis Factor a-Induced Apoptosis, and Inhibits Tumor Growth Kwang S. Suh, Michihiro Mutoh, Michael Gerdes, John M. Crutchley, Tomoko Mutoh, Lindsay E. Edwards, Rebecca A. Dumont, Pooja Sodha, Christina Cheng, Adam Glick, and Stuart H. Yuspa Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, Maryland Abstract CLICs are also found in a soluble form in the cytoplasm (2–5). mtCLIC/CLIC4 is a p53 and tumor necrosis factor a (TNFa) Crystallographic analysis of the structure of soluble CLIC1 regulated intracellular chloride channel protein that local- indicates homology to the glutathione transferase family of izes to cytoplasm and organelles and induces apoptosis proteins. It is hypothesized that soluble CLICs may become when overexpressed in several cell types of mouse and activated as anion channels or channel regulators when ‘‘auto- humanorigin.CLIC4iselevatedduringTNFa-induced inserted’’ into intracellular membranes (6). apoptosis in human osteosarcoma cell lines. In contrast, Among the CLIC family proteins, the biological functions of inhibition of NFKB results in an increase in TNFa-mediated CLIC4 have been most thoroughly studied. CLIC4 is expressed in apoptosis with a decrease in CLIC4 protein levels. Cell lines many cell types. In skin keratinocytes, CLIC4 was first localized to expressing an inducible CLIC4-antisense construct that also mitochondria and cytoplasm and later was localized specifically reduces the expression of several other chloride intracellular to the inner mitochondrial membrane by immunogold electron channel (CLIC) family proteins were established in the microscopy (7, 8). Other reports have localized CLIC4 in the trans- human osteosarcoma lines SaOS and U2OS cells and Golgi network in pancreatic cells, endoplasmic reticulum in rat a malignant derivative of the mouse squamous papilloma hippocampal HT-4 cells, and large dense core vesicles in line SP1. Reduction of CLIC family proteins by antisense neurosecretory cells (9–11). CLIC4 has also been associated with expression caused apoptosis in these cells. Moreover, CLIC4- the actin cytoskeleton in membrane ruffles and lamellipodia. À antisense induction increased TNFa-mediated apoptosis Electrophysiologic analysis suggests that CLIC4 has Cl selective in both the SaOS and U2OS derivative cell lines without channel activity (4, 10, 12, 13). CLIC4 is highly conserved in altering TNFa-induced NFKB activity. Reducing CLIC proteins different species with nearly 95% identity in amino acid sequence in tumor grafts of SP1 cells expressing a tetracycline-regulated indicating an important functional role in cellular physiology (8). CLIC4-antisense substantially inhibited tumor growth and CLIC4 associates with dynamin I, actin, tubulin, and 14:3:3 isoforms induced tumor apoptosis. Administration of TNFa i.p. mod- in neuronal cells, suggesting it may also play a role in cell signaling estly enhanced the antitumor effect of CLIC reduction (14). This is consistent with the recently reported induction of in vivo. These results suggest that CLIC proteins could serve CLIC4 in transforming growth factor-h and serum-activated as drug targets for cancer therapy, and reduction of CLIC human breast fibroblasts, where it was associated with trans- proteins could enhance the activity of other anticancer differentiation to myofibroblasts (15). drugs. (Cancer Res 2005; 65(2): 562-71) CLIC4 is a direct response gene for p53 transactivation, and the up-regulation of CLIC4 is strongly associated with p53-mediated Introduction apoptosis (7). CLIC4 overexpression induces apoptosis character- ized by changes in the intrinsic mitochondrial apoptotic pathway Chloride intracellular channel (CLIC) family of proteins (p64, CLIC1-5, and parchorin) is frequently localized to intracellular such as loss of mitochondrial membrane potential, cytochrome c organelles in multiple cell types. The putative chloride ion gating release, and caspase activation (7). CLIC4 also translocates to the activity of some members of this family suggests that CLIC nucleus in cells induced to undergo apoptosis by a variety of proteins function to regulate organellar volume, ionic homeosta- stress inducers (16), and nuclear-targeted CLIC4 is strongly sis, and pH (1). CLIC1 to CLIC5 are similar in size and highly proapoptotic even when the mitochondrial death pathway is inhibited by genetic deletion of Apaf1 (16). Exposure to tumor homologous, whereas p64 and parchorin have distinct NH2- terminal domains but share strong sequence similarity to the necrosis factor a (TNFa) also increases CLIC4 transcripts and other family members in the COOH terminus (CLIC module; protein and causes CLIC4 to translocate to the nucleus ref. 2). Common to all members is a hydrophobic region in the independent of p53 (8, 16). CLIC module consistent with a transmembrane domain, although TNFa induces apoptosis in some cell types and is in clinical trials for the treatment of certain cancers (17). The interaction of TNFa with its receptor can activate a death pathway through caspase-8 and caspase-3 leading to a cytochrome c–independent Note: K.S. Suh and M. Mutoh contributed equally to this work. apoptotic response (18). However, TNFa can simultaneously Requests for reprints: Stuart H. Yuspa, Cell Carcinogenesis and Tumor induce an antiapoptotic response through its activation of the Promotion, Room 3B25, MSC 4255, 37 Convent Drive, Bethesda, MD 20892-4255. K Phone: 301-496-2162; Fax: 301-496-8709; E-mail: [email protected]. downstream transcription factor NF B and subsequent induction I2005 American Association for Cancer Research. of inhibitors of apoptosis and other NFKB response genes to Cancer Res 2005; 65: (2). January 15, 2005 562 www.aacrjournals.org Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. CLIC4 Antisense Induces Apoptosis and Inhibits Tumor Growth blunt the apoptotic response (19). In experimental settings, this proapoptotic response. We considered this an important under- can be overcome by inhibiting the TNFa-mediated nuclear taking because CLIC4 could be a collateral target in biological translocation of NFKB using the mutant form of the NFKB approaches to cancer therapy with TNFa. cytoplasmic binding partner IKB (20, 21). The mutant IKB (IKBsr) cannot be phosphorylated and degraded and thus does not dissociate from NFKB to allow nuclear translocation and Materials and Methods DNA binding. Whereas this has been an effective tool to Cell Culture. Tet-On U2OS cell lines were purchased from Clontech understand the antiapoptotic activity of NFKB, this antiapoptotic (Palo Alto, CA). Both Tet-On U2OS, p53 Tet-On SaOS cell line (26), and their pathway could compromise the clinical effectiveness of TNFa as derivatives were maintained in DMEM/10% fetal bovine serum. SP1 an antitumor agent (22). keratinocytes and its derivatives were maintained as described previously The death receptor pathway together with inhibition of NFKB (7). Recombinant human and murine TNFa were obtained from is considered the major route through which TNFa induces Calbiochem (San Diego, CA). apoptosis in experimental settings, but other pathways, such as Immunoblot Analysis. Cells were lysed into 100 AL M-Per (Pierce, p53 and mitogen-activated protein kinases, have also been Rockford, IL), and 30 Ag of proteins were resolved by SDS-PAGE and implicated in TNFa-mediated apoptosis (23). These pathways transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, K may contribute to cell killing by TNFa independently of NF B MA). Antibodies against the COOH terminus of CLIC4 (8) were used at a (24, 25). Because expression and nuclear translocation of 1:10,000 dilution. The following antibodies were also used: rabbit polyclonal proapoptotic CLIC4 are induced by TNFa, we embarked on a anti-NFKB, Bax, Bcl-2, and anti-IKBa antibodies were from Santa Cruz study to determine where CLIC4 might fit into the TNFa Biotechnology (Santa Cruz, CA); anti-h-actin mouse monoclonal antibody Figure 1. IKBsr or CLIC4-antisense expression does not inhibit TNFa-induced nuclear translocation of CLIC4. p53 Tet-On SaOS cells (A, B, and D) and Tet-On U2Os cells (A and C) were pretreated with (A) null adenovirus (N), (B) Tet-Off adenovirus (T) to induce antisense expression, (C) IKBsr adenovirus (I), or in (D) combination of IKBsr and Tet-Off adenoviruses (I+T) with 10 MOI for 17 hours, exposed to 25 ng/mL TNFa for 30 minutes (ÀTNF or +TNF), and fixed in 2% paraformaldehyde. NFKB, IKB, and CLIC4 were examined by immunofluorescence using confocal microscopy. Insets in A-D, confocal images correspond to the antibody designated in parenthesis. CLIC4 immunostaining for B and D: overexposed confocal image in order to detect CLIC4 staining when CLIC4 antisense is expressed. www.aacrjournals.org 563 Cancer Res 2005; 65: (2). January 15, 2005 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2005 American Association for Cancer Research. Cancer Research Figure 2. IKBsr enhances TNFa-mediated apoptosis and reduces CLIC4 protein independent of CLIC4 promoter activity. p53 Tet-On SaOS cells (A, C, and E) and Tet-On U2OS cells (B, D, and F) were untreated or pretreated with 50 MOI of null Ad or IKBsr adenovirus for 24 hours prior to 25 ng/mL TNFa. Annexin V fluorescence-activated cell sorting analysis was done 24 hours after TNFa (A and