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[Cancer Research 65, 562-571, January 15, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Antisense Suppression of the Chloride Intracellular Channel Family Induces Apoptosis, Enhances Tumor Necrosis Factor {alpha}-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

Requests for reprints: Stuart H. Yuspa, Cell Carcinogenesis and Tumor Promotion, Room 3B25, MSC 4255, 37 Convent Drive, Bethesda, MD 20892-4255. Phone: 301-496-2162; Fax: 301-496-8709; E-mail: yuspas{at}mail.nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
mtCLIC/CLIC4 is a p53 and tumor necrosis factor {alpha} (TNF{alpha}) regulated intracellular chloride channel protein that localizes to cytoplasm and organelles and induces apoptosis when overexpressed in several cell types of mouse and human origin. CLIC4 is elevated during TNF{alpha}-induced apoptosis in human osteosarcoma cell lines. In contrast, inhibition of NF{kappa}B results in an increase in TNF{alpha}-mediated apoptosis with a decrease in CLIC4 protein levels. Cell lines expressing an inducible CLIC4-antisense construct that also reduces the expression of several other chloride intracellular channel (CLIC) family proteins were established in the human osteosarcoma lines SaOS and U2OS cells and a malignant derivative of the mouse squamous papilloma line SP1. Reduction of CLIC family proteins by antisense expression caused apoptosis in these cells. Moreover, CLIC4-antisense induction increased TNF{alpha}-mediated apoptosis in both the SaOS and U2OS derivative cell lines without altering TNF{alpha}-induced NF{kappa}B activity. Reducing CLIC proteins in tumor grafts of SP1 cells expressing a tetracycline-regulated CLIC4-antisense substantially inhibited tumor growth and induced tumor apoptosis. Administration of TNF{alpha} i.p. modestly enhanced the antitumor effect of CLIC reduction in vivo. These results suggest that CLIC proteins could serve as drug targets for cancer therapy, and reduction of CLIC proteins could enhance the activity of other anticancer drugs.

Key Words: CLIC • TNF{alpha} • NF{kappa}B • apoptosis • tumor therapy • chloride channel


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chloride intracellular channel (CLIC) family of proteins (p64, CLIC1-5, and parchorin) is frequently localized to intracellular organelles in multiple cell types. The putative chloride ion gating activity of some members of this family suggests that CLIC proteins function to regulate organellar volume, ionic homeostasis, and pH (1). CLIC1 to CLIC5 are similar in size and highly homologous, whereas p64 and parchorin have distinct NH2-terminal domains but share strong sequence similarity to the other family members in the COOH terminus (CLIC module; ref. 2). Common to all members is a hydrophobic region in the CLIC module consistent with a transmembrane domain, although CLICs are also found in a soluble form in the cytoplasm (2–5). Crystallographic analysis of the structure of soluble CLIC1 indicates homology to the glutathione transferase family of proteins. It is hypothesized that soluble CLICs may become activated as anion channels or channel regulators when "autoinserted" into intracellular membranes (6).

Among the CLIC family proteins, the biological functions of CLIC4 have been most thoroughly studied. CLIC4 is expressed in many cell types. In skin keratinocytes, CLIC4 was first localized to mitochondria and cytoplasm and later was localized specifically to the inner mitochondrial membrane by immunogold electron microscopy (7, 8). Other reports have localized CLIC4 in the trans-Golgi network in pancreatic cells, endoplasmic reticulum in rat hippocampal HT-4 cells, and large dense core vesicles in neurosecretory cells (9–11). CLIC4 has also been associated with the actin cytoskeleton in membrane ruffles and lamellipodia. Electrophysiologic analysis suggests that CLIC4 has Cl– selective channel activity (4, 10, 12, 13). CLIC4 is highly conserved in different species with nearly 95% identity in amino acid sequence indicating an important functional role in cellular physiology (8). CLIC4 associates with dynamin I, actin, tubulin, and 14:3:3 isoforms in neuronal cells, suggesting it may also play a role in cell signaling (14). This is consistent with the recently reported induction of CLIC4 in transforming growth factor-ß and serum-activated human breast fibroblasts, where it was associated with transdifferentiation to myofibroblasts (15).

CLIC4 is a direct response gene for p53 transactivation, and the up-regulation of CLIC4 is strongly associated with p53-mediated apoptosis (7). CLIC4 overexpression induces apoptosis characterized by changes in the intrinsic mitochondrial apoptotic pathway such as loss of mitochondrial membrane potential, cytochrome c release, and caspase activation (7). CLIC4 also translocates to the nucleus in cells induced to undergo apoptosis by a variety of stress inducers (16), and nuclear-targeted CLIC4 is strongly proapoptotic even when the mitochondrial death pathway is inhibited by genetic deletion of Apaf1 (16). Exposure to tumor necrosis factor {alpha} (TNF{alpha}) also increases CLIC4 transcripts and protein and causes CLIC4 to translocate to the nucleus independent of p53 (8, 16).

TNF{alpha} induces apoptosis in some cell types and is in clinical trials for the treatment of certain cancers (17). The interaction of TNF{alpha} with its receptor can activate a death pathway through caspase-8 and caspase-3 leading to a cytochrome c–independent apoptotic response (18). However, TNF{alpha} can simultaneously induce an antiapoptotic response through its activation of the downstream transcription factor NF{kappa}B and subsequent induction of inhibitors of apoptosis and other NF{kappa}B response genes to blunt the apoptotic response (19). In experimental settings, this can be overcome by inhibiting the TNF{alpha}-mediated nuclear translocation of NF{kappa}B using the mutant form of the NF{kappa}B cytoplasmic binding partner I{kappa}B (20, 21). The mutant I{kappa}B (I{kappa}Bsr) cannot be phosphorylated and degraded and thus does not dissociate from NF{kappa}B to allow nuclear translocation and DNA binding. Whereas this has been an effective tool to understand the antiapoptotic activity of NF{kappa}B, this antiapoptotic pathway could compromise the clinical effectiveness of TNF{alpha} as an antitumor agent (22).

The death receptor pathway together with inhibition of NF{kappa}B is considered the major route through which TNF{alpha} induces apoptosis in experimental settings, but other pathways, such as p53 and mitogen-activated protein kinases, have also been implicated in TNF{alpha}-mediated apoptosis (23). These pathways may contribute to cell killing by TNF{alpha} independently of NF{kappa}B (24, 25). Because expression and nuclear translocation of proapoptotic CLIC4 are induced by TNF{alpha}, we embarked on a study to determine where CLIC4 might fit into the TNF{alpha} proapoptotic response. We considered this an important undertaking because CLIC4 could be a collateral target in biological approaches to cancer therapy with TNF{alpha}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture. Tet-On U2OS cell lines were purchased from Clontech (Palo Alto, CA). Both Tet-On U2OS, p53 Tet-On SaOS cell line (26), and their derivatives were maintained in DMEM/10% fetal bovine serum. SP1 keratinocytes and its derivatives were maintained as described previously (7). Recombinant human and murine TNF{alpha} were obtained from Calbiochem (San Diego, CA).

Immunoblot Analysis. Cells were lysed into 100 µL M-Per (Pierce, Rockford, IL), and 30 µg of proteins were resolved by SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore, Bedford, MA). Antibodies against the COOH terminus of CLIC4 (8) were used at a 1:10,000 dilution. The following antibodies were also used: rabbit polyclonal anti-NF{kappa}B, Bax, Bcl-2, and anti-I{kappa}B{alpha} antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA); anti-ß-actin mouse monoclonal antibody was from Chemicon International, Inc. (Temecula, CA); peroxidase-conjugated secondary antibodies were obtained from Amersham Biosciences (Piscataway, NJ). Monospecific polyclonal antibodies recognizing CLIC1 and CLIC5 as well as recombinant CLIC1, CLIC4, and CLIC5 proteins were generous gifts from Dr. Mark Berryman (College of Osteopathic Medicine, Ohio University; ref. 27). Immunoblots were developed with SuperSignal chemiluminescent substrates (Pierce). Total Image software (Amersham Pharmacia, Sunnyvale, CA) was used to determine densitometry for protein bands.

Immunofluorescent Microscopy. Cells at the density of 6 x 104 per well were seeded in 60-mm dish or 8-chamber slides and incubated in the presence or absence of TNF{alpha} for 30 minutes with null, I{kappa}Bsr or Tet-Off adenovirus for the specified times. Immunostaining was done as described previously (16), and antibody dilutions were based on suggestions from the manufacturers. The stained cells were detected by Zeiss510 confocal microscope, and LSM browser was used for cropping images.

NF{kappa}B-DNA Binding Activity Assay. The activity of p65 binding to oligonucleotides containing a NF{kappa}B consensus binding site was measured by using TransAM NF{kappa}B p65 Transcription Factor Assay Kits according to the manufacturer's instructions (Active Motif, Inc., Carlsbad, CA).

Adenoviruses. I{kappa}Bsr (generous gift from Dr. Dennis, Guttridge of the University of North Carolina), Tet-On and Tet-Off adenoviruses (Clontech, Palo Alto, CA) were amplified in the Molecular Biology Core Facility of National Cancer Institute-Frederick Cancer Research and Development Center. Adenoviruses were purified over two CsCl gradients, dialyzed with a TGM buffer [Tris-HCl (pH 7.5), 1 mmol/L MgCl2, and 10% glycerol], and stored in aliquots at –70°C. Viral titer was determined by plaque assay, and the adenoviral vector without a recombinant insert was used as a viral control (Null virus).

Generation of Bovine Keratin 5–Driven Tet-Off CLIC4-Antisense Squamous Papilloma-1 Cell Line (Bk5/tTA SP1). To generate the pBk5/tTA transactivator plasmid, pBK5 (ref. 28; obtained from David Bol, MD Anderson, TX) was digested with SnaB1 and ligated with BamHI/EcoRI–digested tTA coding sequence from the plasmid pUHD15-1 (29). Recombinant constructs with correct orientation were identified by NotI and SalI digestion. SP1 cells were cotransfected with 10 µg of BK5/tTA DNA and 2 µg of the Hygromycin marker plasmids using lipofectamine (Invitrogen, San Diego, CA) for 6 hours. Hygromycin-resistant colonies were ring cloned and tested for induced regulation of a transfected Tet-On luciferase construct.

Generation of Tetracycline-Inducible CLIC4-Antisense (Tet-On or Tet-Off CLIC4-Antisense) Cell Lines. The cloning of CLIC4 and the construction of the sense CLIC4 plasmids have been described elsewhere (8). The recombinant plasmid was digested using NotI, and the resulting sense and antisense orientations of CLIC4 were subcloned into the NotI site of the expression vector pTRE2pur Vector (Clontech). To allow tetracycline regulation, ~8 x 105 Tet-On U2OS cells and p53 Tet-On SaOS cells were transfected with 0.4 µg of CLIC4-antisense pTRE2pur plasmid using Effectene Transfection Reagent (Qiagen, Chatsworth, CA). To generate Tet-inducible CLIC4-antisense SP1 cell lines, 1 x 107 Bk5/tTA SP1 cells were transfected with 1 µg of CLIC4-antisense pTRE2pur plasmid. Stable transfectants were selected by limiting dilution or cloning-ring method in a medium containing 1 µg/mL puromycin, and the selected clones were analyzed for inducible suppression of CLIC4 by immunoblotting. These p53 Tet-On SaOS cell, Tet-On U2OS, and SP1 cell derivatives are designated as CLIC4AS-SaOS, CLIC4AS-U2OS, and CLIC4AS-SP1 cells, respectively. The presence of the CLIC4-antisense region was confirmed by genomic PCR analysis. The CLIC4AS-SaOS cells or CLIC4AS-U2OS cells were treated with doxycycline (800 ng/mL) or infected with Tet-Off adenovirus without doxycycline for 17 hours to induce CLIC4-antisense before treatment with test agents. The Tet-Off adenovirus method was used to avoid doxycycline in experiments comparing CLIC4-antisense and the I{kappa}Bsr adenovirus.

Immunohistochemistry and Tissue BrdU and Terminal Deoxynucleotidyl Transferase (Tdt)–Mediated Nick End Labeling Staining. For tissue BrdU staining, mice were i.p. injected with 20 mg/mL of BrdU (Roche, Nutley, NJ) 1 hour before harvesting tumor tissues, and the proliferative cells from the harvested tissues were detected by 5-bromo-2'-deoxy-uridine Labeling and Detection Kit II (Roche). For tissue terminal deoxynucleotidyl transferase (Tdt)–mediated nick end labeling (TUNEL) staining, Apoptag (Intergen, Burlington, MA) was used as described by the manufacturer except metal-enhanced 3,3'-diaminobenzidine chromogenic substrate was used. Stained slides mounted and analyzed with bright-field microscopy using Leitz-DMRB (Leica, Bannockburn, IL) and OpenLab (Improvision, Lexington, MA) software.

Xenograft and Tumor Development. CLIC4AS-SP1 cells were grafted as a skin graft on the back of nude mice, and tumor size was measured as described previously (30). One group of mice was given doxycycline-containing feed (200 µg/kg w/w, Bioserve, Laurel, MD) starting 1 week before grafting, whereas another group received standard mouse feed (Purina) to allow expression of the CLIC4-antisense. In one experiment, all recipient mice received doxycycline diet 1 week before grafting and subgroups were changed to control diets at 0, 1, and 3 weeks following the application of tumor grafts. In the second experiment, recombinant TNF{alpha} (1 µg per injection per mouse) was given by i.p. injection twice per week starting at week 5 after grafting, which then continued for four more weeks before termination.

Construction of Human CLIC4 Promoter Reporter and Luciferase Assays. The cloning of the 3.5-kb human CLIC4 promoter region was described elsewhere (7). Primer sets containing NheI sites (5'NheI-gtgtaccatgagctgtcctctgagccagg-3' and 5'NheI-ctgtgtttcaggctctgagctagcccttgg-3') were used to PCR amplify 2.5 kb of the human CLIC4 promoter in pGEM-T Easy (7) excluding the repeated segments. This region was subcloned into pGlow-Topo (Invitrogen), cut with NheI, and subcloned into a NheI-linearized pGL3 (Promega, San Luis Obispo, CA) vector. Osteosarcoma cells were transfected for 17 hours with either the 1 µg/mL of CLIC4 reporter or NF{kappa}B reporter plasmid (31; a gift from Dr. Zheng-Gang Liu, National Cancer Institute) using effectene transfection reagent (Qiagen) before treatment with I{kappa}Bsr and/or TNF{alpha}, and luciferase activity was measured by using the luciferase reporter assay kit (Clontech). Results were normalized to the total protein content.

Apoptosis Assays
Annexin V and TUNEL Fluorescence-Activated Cell Sorting Analysis. Control or treated cells (n = 2 x 105) were analyzed by allophycocyanin-conjugated recombinant human Annexin V (Caltag, Burlingame, CA) and In situ Cell Death assay (Roche) as described by the manufacturer. Analysis was done after 10,000 counting events. Data acquisition and analysis were done using Cell Quest software. The apoptosis data shown are one of representative results of at least three independent experiments.

Statistical Analysis. Comparisons of experimental data were analyzed by a two-tailed Student's t test. P < 0.05 was considered to indicate a statistically significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Suppressing NF{kappa}B Enhances TNF{alpha}-Mediated Apoptosis but Reduces CLIC4. Human osteosarcoma (p53 Tet-On SaOS and Tet-On U2OS) cells were treated with TNF{alpha} to activate NF{kappa}B and/or with I{kappa}Bsr (the dominant-negative mutant of I{kappa}B) adenovirus to inhibit NF{kappa}B activity selectively. NF{kappa}B translocated to the nucleus within 30 minutes of TNF{alpha} treatment in both osteosarcoma cells that are infected with empty (null) adenovirus (Fig. 1A), and I{kappa}B fluorescence was noticeably reduced after TNF{alpha} treatment (Fig. 1A). TNF{alpha} treatment also caused translocation of CLIC4 to the nucleus as reported previously (16). Expression of CLIC4-antisense (indicated by T in Fig. 1B) did not prevent nuclear translocation of CLIC4 or NF{kappa}B in response to TNF{alpha}. Introduction of I{kappa}Bsr (Fig. 1C), or the combination of both CLIC4-antisense and I{kappa}Bsr (Fig. 1D) did not prevent TNF{alpha}-mediated translocation of CLIC4 but did prevent NF{kappa}B translocation to the nucleus, suggesting CLIC4 and NF{kappa}B nuclear trafficking are mediated by independent mechanisms. I{kappa}Bsr prevented translocation of NF{kappa}B at multiplicity of infection (MOI) of 50 in both cell types (Fig. 1C), indicating that I{kappa}Bsr adenovirus was effective in blocking NF{kappa}B activation in these cells.



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  		Figure 1. I{kappa}Bsr or CLIC4-antisense expression does not inhibit TNF{alpha}-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) I{kappa}Bsr adenovirus (I), or in (D) combination of I{kappa}Bsr and Tet-Off adenoviruses (I+T) with 10 MOI for 17 hours, exposed to 25 ng/mL TNF{alpha} for 30 minutes (–TNF or +TNF), and fixed in 2% paraformaldehyde. NF{kappa}B, I{kappa}B, 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.

 
For both cell lines, flow cytometry data showed a small increase in Annexin-positive apoptotic cells after treatment with 25 ng/mL TNF{alpha} (Fig. 2A and B). This increase in apoptosis is independent of p53 because the p53 Tet-On SaOS cells lack p53 in the absence of doxycycline. TNF{alpha} also induced CLIC4 protein (Fig. 2C and D) and caused a modest increase in CLIC4 promoter activity (Fig. 2E and F) in both cell lines. Combined treatment of these cells with TNF{alpha} plus I{kappa}Bsr caused a substantial increase in apoptotic cells but decreased CLIC4 protein levels (Fig. 2A-D). CLIC4 promoter activity was not significantly changed from TNF{alpha} alone with the addition of I{kappa}Bsr (Fig. 2E and F).



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  		Figure 2. I{kappa}Bsr enhances TNF{alpha}-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 I{kappa}Bsr adenovirus for 24 hours prior to 25 ng/mL TNF{alpha}. Annexin V fluorescence-activated cell sorting analysis was done 24 hours after TNF{alpha} (A and B). Columns, mean of three independent experiments; bars, ±SE. CLIC4 protein levels were determined by Western blot analysis in p53 Tet-On SaOS cells (C) and Tet-On U2OS cells (D). Numbers between CLIC4 and actin blots (C) and (D): CLIC4 band intensity relative to the corresponding actin band as determined by densitometry using ImageMaster software. The cells were pretreated with I{kappa}Bsr adenovirus for 24 hours before the treatment with TNF{alpha} for 6 hours. CLIC4 promoter transcriptional activity was determined by luciferase reporter gene assay (E and F). Columns, mean (n = 3); bars, ±SD. *, P < 0.01, when compared with TNF{alpha} alone.

 
Expression of Stably Integrated Conditional CLIC4- Anti-sense Enhances TNF{alpha}-Mediated Apoptosis. To determine if reduction of CLIC4 enhanced TNF{alpha}-induced apoptosis as seen for inhibition of NF{kappa}B activation, CLIC4AS-SaOS and CLIC4AS-U2OS cells were infected with the Tet-Off adenovirus to induce the antisense vector in the presence or absence of TNF{alpha}. Treatment with TNF{alpha} or Tet-Off adenovirus alone caused apoptosis in CLIC4AS-SaOS cells, and TNF{alpha} alone induced apoptosis in CLIC4AS-U2OS cells (Fig. 3A and B). The Annexin V binding was associated with condensed chromatin and nuclear fragmentation as detected by Hoechst staining (data not shown). Immunoblots of the duplicate experiments showed that TNF{alpha} alone increased the level of CLIC4 protein in both cell types (Fig. 3C and D). The combination of TNF{alpha} and antisense expression enhanced apoptosis in both cell types, and antisense expression prevented the increase in TNF{alpha}-mediated CLIC4 protein expression in both cell types (Fig. 3B and D). Apoptosis measured in this setting was dependent on the adenoviral MOI used, suggesting that TNF{alpha} and CLIC4-antisense expression synergize to enhance apoptosis. In CLIC4AS-U20S cells, apoptosis induced by TNF{alpha} progressed more slowly (48 hours) than in CLIC4AS-SaOS cells, and more adenovirus (5 MOI of Tet-Off adenovirus) was needed to achieve an apoptotic response in 65% of treated cells (Fig. 3B).



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  		Figure 3. Reduction of CLIC4 by antisense expression enhances TNF{alpha}-mediated apoptosis. Osteosarcoma cell lines with stably integrated tetracycline regulated CLIC4-antisense vector were treated with TNF{alpha} and Tet-Off Ad and examined for apoptosis (A and B) and CLIC4 protein levels (C and D). Numbers between CLIC4 and actin blots (C) and (D): CLIC4 band intensity relative to the corresponding actin band as determined by densitometry using ImageMaster software. Cells were pretreated with Tet-Off Ad for 17 hours before 25 ng/mL TNF{alpha} treatment. Apoptosis was examined by flow cytometric analysis for Annexin V binding after treatment with TNF{alpha} for 24 hours in CLIC4AS-SaOS cells (A) and for 48 hours in CLIC4AS-U2OS cells (B). Columns, of three independent experiments mean; bars, ±SE. Western blotting was done for CLIC4 after treatment with TNF{alpha} for 24 hours in both cell lines. *, P < 0.01, when compared with TNF{alpha} alone.

 
CLIC4-antisense induced by the Tet-Off adenovirus was not influencing NF{kappa}B nuclear translocation (Fig. 1B), but as an independent approach, NF{kappa}B binding activity assays were done after TNF{alpha} treatment of CLIC4AS-SaOS and CLIC4AS-U2OS cells (Fig. 4). NF{kappa}B in the nucleus as detected by binding to its consensus DNA binding site and NF{kappa}B transcriptional function as measured by luciferase reporter activity were not significantly affected by the Tet-Off adenovirus, but were completely inhibited by the I{kappa}Bsr adenovirus (Fig. 4A-D). In both cell lines, NF{kappa}B nuclear activity was enhanced by TNF{alpha}.



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  		Figure 4. I{kappa}Bsr, but not CLIC4-antisense, inhibits NF{kappa}B-DNA binding and transcriptional activity. CLIC4AS-SaOS cells (A and C) and CLIC4AS-U2OS cells (B and D) were pretreated with Tet-Off, I{kappa}Bsr, or Null Ad for 24 hours before TNF{alpha} treatment, and then NF{kappa}B binding and reporter gene assays were done. For the binding assay, both cell types (A and B) were treated with 25 ng/mL TNF{alpha} for 1 hour, and cell lysates were used for ELISA assay. For reporter gene assay, both cell types (C and D) were transiently transfected with NF{kappa}B-luciferase plasmid for 17 hours before the indicated MOI of adenoviral infection. Both cell types were treated with 25 ng/mL TNF{alpha} for 10 hours, and cell lysates were used for luciferase and protein assays. Columns, mean (n = 3) of one of two independent experiments; bars, ±SD. *, P < 0.01, when compared with TNF{alpha} alone.

 
CLIC4-Antisense Reduces the Expression of Other CLIC Proteins. The extensive sequence homology among the CLIC family proteins (32) prompted us to examine if our reverse orientation full-length CLIC4-antisense could also reduce the expression of other CLIC proteins. Antibodies to CLIC1, CLIC4, and CLIC5 detected single bands on immunoblots of the respective recombinant proteins, although there was slight cross-reactivity of COOH terminus CLIC4 antibody with CLIC5-recombinant protein (Fig. 5A). However, CLIC5 protein is in extremely low abundance in the cell lines used in these studies and requires ultra sensitive reagents for detection (see legend of Fig. 5B); thus, essentially each antibody is monospecific for cell lysates. Upon induction of CLIC4-antisense with addition of doxycycline in the cell culture medium, the levels of all three CLIC proteins decreased indicating that CLIC4-antisense has the broader capability of reducing expression of multiple CLIC family members. To determine if CLIC4 antisense had a more general influence on reducing cellular protein expression, the blot shown in Fig. 5B was reprobed for Bax, another proapoptotic protein associated with mitochondria and Bcl-2, an antiapoptotic protein. In neither case was the protein level reduced by CLIC4-antisense expression (Fig. 5C).



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  		Figure 5. CLIC4-antisense expression down-regulates other CLIC family members. A, bacterially expressed recombinant CLIC1, CLIC4, and CLIC5 were resolved by SDS-PAGE, and the recombinant proteins were detected by silver staining. Solid black arrows, recombinant CLIC proteins. A parallel gel was used to probe the membrane with CLIC1, CLIC4, and CLIC5 antibodies sequentially after stripping. B, CLIC4AS-U2OS cells (AS) and the parental U2OS (Par) cells were treated with doxycycline for 2 days and analyzed by immunoblotting using CLIC monospecific antibodies. Because of the low abundance of CLIC5 protein in these cells, the highly sensitive enhanced chemiluminescence (Dura, Pierce) reagent and longer exposure time were used to detect CLIC 5, whereas the more conventional enhanced chemiluminescence (Pico, Pierce) was used to detect CLIC1 and CLIC4. C, immunoblot from B was reprobed for Bax and Bcl-2.

 
Reduction of CLIC Proteins Is as Effective as Inhibition of NF{kappa}B for Enhancing TNF{alpha}-Induced Apoptosis in Osteosarcoma Cell Lines. To compare the effects of inhibiting NF{kappa}B activity and suppressing CLIC4 expression on TNF{alpha}-mediated apoptosis, we treated CLIC4AS-SaOS cells with either I{kappa}Bsr or Tet-Off adenovirus together with TNF{alpha}. After incubation with 25 ng/mL TNF{alpha} for 12 or 24 hours in combination with I{kappa}Bsr, 32% and 55% of the cells underwent apoptosis, whereas a 30% and 66% apoptotic response was measured for Tet-Off adenovirus and TNF{alpha} treatment at these time points (Fig. 6A). Thus, reduction of CLIC4 (together with other CLIC family members) is as effective for enhancing TNF{alpha}-mediated apoptosis as inhibiting NF{kappa}B in this cell line. In TNF{alpha} dose-response studies, reduction of CLIC4 was more effective than suppression of NF{kappa}B (Fig. 6B) in CLIC4AS-SaOS cells. In CLIC4AS-U2OS cells, both NF{kappa}B inhibition and CLIC reduction enhanced TNF{alpha} apoptosis at all TNF{alpha} doses tested, and suppression of NF{kappa}B may be more effective in these cells (Fig. 6C).



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  		Figure 6. Relative activity of I{kappa}Bsr and CLIC4-antisense for enhancing TNF{alpha}-mediated apoptosis. Apoptosis was induced in CLIC4AS-SaOS (A and B) and CLIC4AS-U2OS cells (C) by TNF{alpha} in the presence of Null, I{kappa}Bsr, or Tet-Off Ad and examined by flow cytometric analysis of Annexin V binding. Cells were pretreated with I{kappa}Bsr or Tet-Off Ad for 17 hours before TNF{alpha} treatment. Apoptosis was induced with indicated dose of TNF{alpha} for 12 and 24 hours in CLIC4AS-SaOS cells (A), 24 hours in B and 48 hours in CLIC4AS-U2OS cells (C). Columns, mean of three independent experiments; bars, ±SD.

 
CLIC4-Antisense Expression Inhibits Tumor Growth In vivo. The reduction in viability associated with CLIC4-antisense expression in osteosarcoma tumor cell lines prompted us to test the possibility that reduction in CLIC family proteins might be inhibitory to tumor growth in vivo. We created a mouse tumor cell line CLIC4AS-SP1 that expresses abundant CLIC4 in the presence of doxycycline, but substantially reduced the level of CLIC4 when doxycycline is withdrawn or the dose is reduced (Fig. 7A and B). CLIC4AS-SP1 cells were then grafted to nude mice that had been primed with doxycycline diet for 1 week or maintained on a standard diet. Tumor growth was markedly retarded on mice receiving standard diet (Fig. 7C). Withdrawal of doxycycline from the diet at 3 weeks when tumors were well established produced tumors of intermediate size compared with control mice maintained on doxycycline and immediate doxycycline withdrawal, suggesting CLIC4-antisense expression affected growth even in well-established tumors. Three independent grafting experiments showed identical results. All tumors were poorly differentiated squamous cell carcinomas independent of expression of the CLIC4-antisense. Analysis of CLIC4 protein in a series of tumors excised from the experimental groups indicated that tumors expressing the antisense had significantly reduced CLIC4 levels (Fig. 8A and B), whereas the parental tumor and the CLIC4AS-SP1 tumors from mice maintained on doxycycline had CLIC4 levels similar to normal skin keratinocytes. Immunostaining of tumor sections revealed that CLIC4 was relatively abundant in the cytoplasm of tumors not expressing the antisense but substantially reduced in tumors in which doxycycline was withdrawn (Fig. 9A). Furthermore, proliferation in the antisense expressing tumors was reduced by 65% when assayed by BrdU staining (Fig. 9B), although a substantial number of proliferating cells were detected. The number of apoptotic cells detected by TUNEL staining was 3-fold higher in antisense expressing tumors than in tumors in which antisense was suppressed by doxycycline (Fig. 9C). Together, these changes are consistent with a suppressed tumor expansion, reduced proliferation and increased apoptosis when CLIC proteins are reduced.



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  		Figure 7. CLIC4-antisense expression retards tumor growth in vivo. Inducible CLIC4AS-SP1 keratinocytes were cultured in medium without doxycycline for the indicated times (A) or in medium with various amounts (µg/mL) of doxycycline for 2 days (B) before harvest and analyzed for CLIC4 levels by immunoblotting. (C) two million CLIC4AS-SP1 cells were mixed with 5 million BALB/c dermal fibroblasts, and the cell mixture was grafted onto the backs of the nude mice being fed either a diet containing doxycycline (–AS, solid triangle on black line), control diet (+AS, solid circle on dotted line) or changed from doxycycline to control diet at 3 weeks (+AS at 3 weeks, solid square on gray line; n = 10 per group). Tumor size was measured weekly for 7 weeks and presented as mean volume ± SE. Similar results were obtained from three independent grafting experiments.

 


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  		Figure 8. Tumors expressing CLIC4-antisense have reduced levels of CLIC4. Normal mouse skin (N), parental Tet-Off SP1 cells (P), and individual tumors from CLIC4AS-SP1 (solid black bar) grafts with or without antisense expression were dissected, homogenized, and protein lysates were assayed for CLIC4 levels by immunoblotting and further quantified by densitometry (A and B) using Image Master software.

 


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  		Figure 9. Expression of CLIC4-antisense in tumors inhibits cell proliferation and increases apoptosis. Tissue sections from the tumors with (+AS) or without (–AS) CLIC4-antisense expression were immunostained for CLIC4 and examined by light microscopy (A). BrdU was injected i.p. 1 hour before sacrifice, and BrdU incorporation in tumors was detected by anti-BrdU antibody and immunohistochemistry (B). BrdU positive cells (+BrdU) were counted from eight random fields from +AS and –AS tumor sections and plotted (solid black bar). Serial sections from B were TUNEL stained (C), and positive cells were quantified as described in B and plotted. Sections without secondary antibody were used as the negative control (C), and the DNase-treated (+DNase) sections were used as the positive control.

 
To determine if the addition of TNF{alpha} would further reduce tumor size, mice carrying tumor grafts on doxycycline or standard diets were treated with recombinant mTNF{alpha} after appearance of tumors at 5 weeks after grafting (Fig. 10A). Whereas TNF{alpha} and CLIC4-antisense both had a substantial antitumor effect as a single agent, combination therapy using TNF{alpha} and antisense modestly improved the tumor response for at least 3 weeks after TNF{alpha} was given (Fig. 10A). CLIC4-antisense alone had a more powerful tumor inhibitory influence than TNF{alpha} as a single therapeutic agent in vivo in this model. In contrast, the apoptotic response of CLIC4AS-SP1 cells to combined antisense expression and TNF{alpha} treatment in vitro was synergistic (Fig. 10B), similar to that seen with the human osteosarcoma tumor cell lines.



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  		Figure 10. Combined CLIC4-antisense and TNF{alpha} enhances CLIC4AS-SP1 regression in vivo and is synergistic for apoptosis in vitro. CLIC4AS-SP1 cells were grafted as described in Fig. 7 and treated with recombinant mTNF{alpha} by twice weekly intraperitoneal injections (1 µg per injection per mouse) starting at 5 weeks postgrafting when tumors were established (A). Mice were fed either a diet containing doxycycline with (+TNF/–AS, solid square with dotted line) or without TNF{alpha} injection (PBS/–AS, solid diamond on black line), and control diet with (TNF/+AS, solid triangle on dotted line) or without TNF{alpha} injection (PBS/+AS, solid circle on black line; n = 10 per group). Solid black arrow, start of TNF{alpha} administration. Tumor size was measured weekly for 9 weeks and presented as mean volume ± SE. Similar results were obtained from three independent grafting experiments. CLIC4AS-SP1 cells in vitro were treated with (+) or without (–) TNF{alpha} (25 µ/mL) at 0 hour (white bar), 24 hours (gray bar), and 48 hours (black bar) in the presence (–AS) or absence (+AS) of doxycycline to control CLIC4-antisense expression (B). TUNEL-positive cells were detected by fluorescence-activated cell sorting analysis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study was designed to determine if induction of CLIC4 by TNF{alpha} was an essential component of the TNF{alpha}-mediated apoptotic response, as we have previously shown for p53-mediated apoptosis (7). Based on the results of the CLIC4 promoter-reporter construct, CLIC4 does seem to be a direct transcriptional target for TNF{alpha} independent of NF{kappa}B stimulation. However, the up-regulation of CLIC4 is not required for TNF{alpha}-mediated apoptosis, and in contrast, reduction of CLIC4 enhances TNF{alpha}-mediated apoptosis. This conclusion is tempered somewhat since our antisense approach involved reduction of several CLIC family proteins whereas not affecting other cellular proteins that we have tested. Thus, until more selective reagents for individual CLIC family proteins are available, we conclude that reductions in CLIC proteins together can enhance TNF{alpha}-mediated apoptosis independent of p53.

A critical amount of CLIC proteins is needed to maintain cell survival. If the CLIC family proteins are required to maintain ionic balance, pH, and volume in cellular organelles, one would expect precise regulation of protein levels to be required. The inhibition of NF{kappa}B in concert with TNF{alpha} treatment reduces CLIC4 protein without altering CLIC4 transcription. This suggests that NF{kappa}B-regulated factors may contribute to the stability of CLIC proteins, and this could be a component of NF{kappa}B antiapoptotic activity. This possibility will require additional studies because the processing of CLIC proteins has not yet been explored.

We considered that suppressing CLIC4 by antisense expression could have an influence on TNF{alpha}-induced NF{kappa}B activation. However, suppressing CLIC4 did not inhibit TNF{alpha}-induced translocation of NF{kappa}B to the nucleus, DNA binding activity of NF{kappa}B, or NF{kappa}B-mediated transcriptional activation, indicating that inhibition of NF{kappa}B activation is not one of the mechanisms by which CLIC4 suppression sensitizes cells to TNF{alpha}-mediated apoptosis. Reduction in CLIC proteins is as potent as inhibition of NF{kappa}B function for enhancing TNF{alpha}-mediated apoptosis in human osteosarcoma cells. In CLIC4AS-SP1 mouse cells, the combined influence of TNF{alpha} and reduction in CLIC proteins seems to be synergistic for apoptosis.

Whereas our use of antisense methods to study reduction in CLIC4 results in a broader reduction in CLIC family proteins, CLIC4 itself is perhaps the most ubiquitous and abundantly expressed family member and likely to be central to the results reported here. Nevertheless, the development of selective CLIC4 modulating agents will be essential to sorting out specific action of individual family members. A previous report (7) and current results consistently indicate that an increase or reduction of CLIC4 impairs cell viability through an apoptotic pathway. This may be unusual but is not unique. For example, C-MYC up-regulation induces apoptosis in fibroblasts, whereas down-modulation induces apoptosis in hematopoietic cells (33, 34).

The physiologic significance of CLIC4 induction and nuclear translocation in TNF{alpha}-treated cells is still unclear. It is possible that induction and translocation of CLIC4 might contribute to another physiologic function of TNF{alpha} such as differentiation or growth inhibition. The development of the Caenorhabditis elegans excretory canal requires the function of EXC-4, the worm orthologue of mammalian CLIC proteins (35), suggesting CLIC proteins may also have a role in development or tissue remodeling.

The human CLIC4 gene is mapped to chromosome 1p36.11, a region that is frequently altered in cancers, and previous studies have shown aberrations at this locus in lymphomas, leukemias, invasive ductal and lobular breast carcinomas, metastasizing squamous cell carcinomas, and lung cancer (comparative genomic hybridization and loss of heterozygosity database, National Center for Biotechnology Information). The generation of human tumor cell lines where we can conditionally regulate CLIC expression has provided evidence that CLIC proteins could be useful targets for tumor therapy. This prediction is further supported by the in vivo grafting model of a mouse squamous cell tumor line where conditional expression of CLIC4-antisense reduces CLIC4, inhibits tumor proliferation and expansion of tumor size, and increases tumor cell apoptosis. These results indicate that a pan-CLIC knockdown could serve as a novel anticancer therapy. In vitro studies suggest that lowering CLIC levels could significantly enhance the apoptotic activity of TNF{alpha}, independent of NF{kappa}B or 53 pathways. The antitumor response to combined treatment was not as potent as the proapoptotic response in vitro; although there was a modest combined effect in vivo. Because only one regimen of TNF{alpha} administration was tested, additional modification of the protocol may produce a more pronounced result. This preclinical model suggests that reduction of CLIC levels, physiologically translated to reduced chloride channel activity, could enhance biological or cytotoxic drug–mediated antitumor therapy, but further testing is required to confirm this possibility.

Ion channels and transporters have been among the most successful therapeutic targets (i.e., Ca2+ channel blocker for heart disease and Na+ transport/exchanger inhibitors for diuretics) for pharmaceuticals. Previous experimental studies indicate that a chloride channel blocker (i.e., tamoxifen), calcium channel inhibitor (i.e., verapamil) or sodium/hydrogen exchanger inhibitor (i.e., amiloride) enhance the effectiveness of cancer chemotherapy by interfering with the activity of multidrug resistance proteins that influence the ability of chemotherapeutic agents to accumulate in cancer cells in sufficient concentrations. From a therapeutic point of view, ion transport inhibitors such as intracellular chloride channel inhibitors can serve as modifiers of drug traffic across the plasma membrane. Recent reports suggest that multiple classes of intracellular chloride channel proteins are central to cell viability and thus should be considered as potential therapeutic targets in cancer. Voltage-gated chloride channels CLC-2, CLC-3, and CLC-5 channels are expressed at high levels in acute patient biopsies from low- and high-grade malignant gliomas (36). Glycine inhibits the growth of B16 melanoma tumors in vivo through a glycine-gated Cl–channel in endothelial cells that blocks the effect of vascular endothelial growth factor on blood vessel growth (37), suggesting that chloride channels may influence angiogenesis. CLIC4 expression is increased in myofibroblasts that form the stroma in breast cancers, participating in the regulation of the tumor microenvironment (15). Calcium-activated chloride channels, CLCA1 and CLCA2, are significantly down-regulated in ~80% of colorectal carcinomas and >90% of highly proliferating tumor cells, suggesting a tumor suppressor activity (38, 39). Reconstitution of CLCA2 expression in highly malignant cell lines reduced Matrigel invasion in vitro and prevented the growth or metastasis of tumor cells transplanted s.c. in nude mice (40, 41). It is anticipated that reduction of CLIC levels could provide an advantage in increasing the sensitivity of tumor cells to drug-mediated anticancer therapy, and further clarification of CLIC functions will be needed for a development of small molecules to modulate CLIC activities. These molecular characteristics of intracellular chloride channels suggest that chloride gradients and flux influence a variety of important cellular controls for proliferation, migration, adhesion, and viability that are involved in cancer pathogenesis, and CLIC proteins should be considered as potential molecular targets for cancer.


   Acknowledgments
 
Grant support: NIH JSPS Research Fellow in Biomedical and Behavioral Research, 2003 to 2005 (M. Mutoh).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Karen Vousden and Kevin Ryan of the National Cancer Institute for supplying the SaOS cell lines; Dr. Zheng-Gang Liu for supplying the NF{kappa}B-luciferase plasmid DNA; Dr. Dennis Guttridge of the University of North Carolina for supplying the I{kappa}Bsr adenovirus; Dr. Narayan Bhat of the Science Applications International Co., Inc., Frederick, National Cancer Institute, Molecular Biology, Gene Expression Laboratory for adenoviral amplification; Barbara Taylor of the CCR FACS Core Facility; Dr. Mark Berryman for the generous contribution of antibodies against CLIC1 and CLIC5 and recombinant CLIC proteins; and Bettie Sugar for the excellent editorial assistance.


   Footnotes
 
Note: K.S. Suh and M. Mutoh contributed equally to this work.

Received 7/26/04. Revised 10/ 4/04. Accepted 11/11/04.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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