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[Cancer Research 65, 8706-8714, October 1, 2005]
© 2005 American Association for Cancer Research

Molecular Biology, Pathobiology and Genetics

A Novel Isoform of TUCAN Is Overexpressed in Human Cancer Tissues and Suppresses Both Caspase-8– and Caspase-9–Mediated Apoptosis

Masaaki Yamamoto1,2, Toshihiko Torigoe1, Kenjiro Kamiguchi1, Yoshihiko Hirohashi1, Katsuya Nakanishi1, Chika Nabeta1, Hiroko Asanuma1, Tetsuhiro Tsuruma2, Takashi Sato3, Fumitake Hata2, Tousei Ohmura2, Koji Yamaguchi2, Takehiro Kurotaki2, Koichi Hirata2 and Noriyuki Sato1

Departments of 1 Pathology and 2 Surgery, Sapporo Medical University School of Medicine, Sapporo, Japan and 3 Hokkaido Prefecture Haboro Hospital, Haboro, Japan

Requests for reprints: Toshihiko Torigoe, Department of Pathology, Sapporo Medical University School of Medicine, South-1 West-17, Chuo-ku, Sapporo 060-8556, Japan. Phone: 81-11-611-2111; Fax: 81-11-643-2310; E-mail: torigoe{at}sapmed.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Caspase-associated recruitment domains (CARD) are protein-protein interaction modules found extensively in proteins that play important roles in apoptosis. One of the CARD-containing proteins, TUCAN (CARD8), was reported previously as an antiapoptotic protein with a molecular weight of 48 kDa, which was up-regulated in colon cancer cells. We identified a novel isoform of TUCAN with a molecular weight of 54 kDa. The new variant of TUCAN, termed TUCAN-54, was expressed in gastric, colon, and breast cancer tissues but was barely detected in normal noncancerous tissues, whereas 48-kDa TUCAN was detected in tumor tissues and noncancerous tissues. To know the function of TUCAN-54 in the apoptosis of cancer cells, TUCAN-54 was overexpressed in tumor cells by gene transfection. Its overexpression inhibited pro-caspase-9 activation, leading to the suppression of the cell death induced by a protein kinase inhibitor, staurosporine, or a chemotherapeutic reagent, etoposide (VP-16). In contrast, specific small interfering RNA–mediated suppression of TUCAN-54 expression in tumor cells increased the VP-16–induced cell death rate, indicating that expression of TUCAN-54 might be associated with chemoresistance of tumor cells. In addition, it inhibited caspase-8 activation as well, thereby suppressing Fas-induced cell death. It was revealed that Fas-associated death domain was physically associated with TUCAN-54 but not with 48-kDa TUCAN. Thus, TUCAN-54 might be a novel tumor-specific antiapoptotic molecule expressed in a variety of human cancer tissues, which might aggravate malignant potential of cancer cells, such as chemoresistance and immunoresistance.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Apoptosis or programmed cell death is an indispensable process for normal development and homeostasis (1, 2). Although the death signals that initiate the apoptotic program can originate from several sources, in most cases they lead to the activation of a family of cysteine proteases known as caspases (3–5), which execute the apoptotic program. It has been shown in various tumors that dysregulation of apoptosis was closely correlated with oncogenesis and malignant phenotype of tumor cells. For example, expression of antiapoptotic molecules, such as BCL-2 and inhibitor of apoptosis proteins, is up-regulated, whereas function of proapoptotic molecules, such as Apaf-1 and caspases, is impaired in certain tumor cells.

Recently, several caspase-associated recruitment domain (CARD)–containing proteins have been identified and their roles for the regulation of apoptotic signals have been clarified. In mammals, eight CARD-containing caspases have been identified, such as pro-caspase-1, -2, -4, -5, -9, -11, -12, and -13, and non-caspase CARD-containing proteins include Apaf-1, Nod1 (CARD4), NAC (DEPCAP), RAIDD (CRADD), Cardiak (Rip2, RICK), BCL-10 (CIPER), ARC (Nop30), Asc, CARD6, TUCAN (CARD8), CARD9, CARD10, CARD11, CARD14, cIAP1, cIAP2, and CLAN. The CARD is a protein module that participates in apoptotic signaling through protein-protein interactions (6). CARD domains consist of six or seven antiparallel {alpha}-helices that form highly specific homophilic interactions between signaling partners. Confirming the selectivity of CARD-CARD interactions, several CARD protein family members have been found to assemble into discrete signaling complexes. For example, Apaf-1 and caspase-9 assemble together in the presence of cytochrome c and dATP, resulting in caspase oligomerization and activation (7). Other CARD proteins that segregate with discrete binding partners include caspase-1 with RICK and Ipaf/CARD12 (8, 9), CARD4 with RICK, RAIDD with caspase-2, and CARD9 with BCL-10 (10–12). The mechanisms by which upstream stimuli activate and/or assemble these CARD-CARD signaling complexes are not presently understood.

TUCAN was first reported in 2001 as a tumor–up-regulated CARD-containing protein with a molecular weight of 48 kDa (13). In that report, it was shown that 48-kDa TUCAN was overexpressed in colon cancer tissues and associated with poor prognosis of the colon cancer patients. It was also shown that the overexpression of 48-kDa TUCAN inhibited apoptosis and caspase activation induced by Apaf-1/caspase-9–dependent stimuli (13). To the contrary, it was reported in 2002 that 48-kDa TUCAN (14) could induce apoptosis, thereby having a proapoptotic function in certain cells. In addition, we have found that 48-kDa TUCAN is expressed in noncancerous tissues as well as in cancer tissues. Therefore, there are controversial findings as to the expression and function of this CARD family protein.

Here, we report a novel isoform of TUCAN, termed TUCAN-54, which is overexpressed preferentially in cancer tissues. Overexpression of TUCAN-54 in tumor cells resulted in the inhibition of caspase-9 activation and conferred resistance against staurosporine-induced or etoposide (VP-16)–induced cell death, whereas small interfering RNA (siRNA)–mediated decreases of TUCAN-54 expression increased VP-16-induced death rate. In addition, TUCAN-54 was associated with Fas-associated death domain (FADD), and its overexpression inhibited the Fas-induced caspase-8 activation and cell death. The present study highlights the distinct function of TUCAN-54 conferring apoptosis resistance on tumor cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Expression plasmids and antibodies. The plasmid encoding 48-kDa TUCAN and pro-caspase-9 was kindly provided by Dr. John C. Reed (The Burnham Institute, La Jolla CA). The open reading frame, NH2-terminal myc epitope-tagged 54- or 48-kDa TUCAN was amplified by using the specific forward and reverse primers, including BamHI and NotI restriction sites, respectively. The PCR product was purified and cloned into pcDNA3 mammalian expression vector (Invitrogen, Carlsbad, CA). The nucleotide sequence of each insert was analyzed using an ABM analyzer PRISM 310 and AmpliCycle Sequencing kit (Perkin-Elmer, Norwalk, CT).

The mouse anti-myc-tag monoclonal antibody (mAb) and anti-Flag-tag mAb were purchased from Invitrogen and MBL (Watertown, MA), respectively. The mouse anti-caspase-9 mAb, mouse anti-caspase-8 mAb (p18), mouse anti-cleaved poly(ADP-ribose) polymerase (PARP; D214) mAb, and mouse anti-ß-actin mAb used were purchased from MBL, Biosource International (Camarillo, CA), Cell Signaling (Beverly, MA), and Sigma (St. Louis, MO), respectively.

Establishment of anti-TUCAN antibody. To characterize the expression of endogenous TUCAN, we established rabbit antisera against TUCAN as described previously (15). Briefly, we immunized a New Zealand white rabbit with a synthetic peptide (residues 99-115 of TUCAN-54 conjugated with carrier protein keyhole limpet hemocyanin; PIERCE, Rockford, IL). The specificity of the polyclonal antibody was examined by Western blotting.

Reverse transcription-PCR. A set of cDNA from normal human adult tissues (multiple tissue cDNA panels) was purchased from BD Biosciences, San Jose, CA. Total RNA was isolated from cultured cells or tissues by using a RNeasy Mini kit (Qiagen, Valencia, CA) following the manufacturer's protocol. The cDNA mixture was synthesized from 1 µg total RNA by reverse transcription using SuperScript II and oligo(dT) primer (Life Technologies, Inc., Gaithersburg, MD) according to the manufacturer's protocol. For the analysis of mRNA expression, we did reverse transcription-PCR (RT-PCR) as described previously (16). Briefly, PCR amplification was done in 50 µL PCR mixture containing 1 µL from the cDNA mixture, KOD Plus DNA polymerase (Toyobo, Osaka, Japan), and 50 pmol primers. The PCR mixture was initially incubated at 94°C for 2 minutes followed by 30 cycles of denaturation at 94°C for 15 seconds, annealing at 60°C for 30 seconds, and extension at 68°C for 1 minute. For specific detection of TUCAN-54, primer pairs used for RT-PCR analysis were 5'-TCCCAGTGTATCAGAAGAGC-3' and 5'-GGAGACGTCCACCTCACCTG-3' as forward and reverse primers, respectively. The expected size of the PCR product for TUCAN-54 was 428 bp. As an internal control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was detected by using forward primer 5'-ACCACAGTCCATGCCATCAC-3' and reverse primer 5'-TCCACCACCCTGTTGCTGTA-3' with an expected PCR product of 452 bp. The PCR products were visualized with ethidium bromide staining under UV light after electrophoresis on 1.0% agarose gel. Nucleotide sequences of the PCR products were confirmed by direct sequencing using an ABI Genetic analyzer PRIM 310 and an AmpliCycle sequencing kit (Perkin-Elmer, Foster City, CA).

Transfections and cell culture. 293T cells were cultured at 37°C in 5% CO2 in DMEM with 10% heat-inactivated fetal bovine serum (FBS), 1 mmol/L L-glutamine, and antibiotics. Jurkat cells were maintained in culture in RPMI 1640 supplemented with 10% FBS and 1 mmol/L L-glutamine. For transient transfection, 293T cells were transfected with the plasmid encoding full-length 48- or 54-kDa TUCAN, pro-caspase-9, FADD, or other proteins by using LipofectAMINE 2000 reagent (Invitrogen). For stable transfection, Jurkat cells were transfected by using LipofectAMINE 2000 reagent. Two days after the transfection, cells were replated at 106/mL in a medium containing 1.5 mg/mL geneticin (Invitrogen). The culture medium was replaced twice weekly until colonies of stably transfected clones arose. Multiple clones were pooled and expanded in culture.

Western blotting. Cultured cells were washed in ice-cold PBS, lysed by incubation on ice in a lysis buffer [50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 1% NP40, protease inhibitor cocktail; Complete, Roche Diagnostics, Inc., Basel, Switzerland], and clarified by centrifugation at 15,000 rpm for 20 minutes at 4°C. The whole-cell lysates were boiled for 5 minutes in the presence of SDS sample buffer, resolved by 10% SDS-PAGE, and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore, Billerica, MA). The membranes were then incubated with blocking buffer (5% nonfat dry milk in PBS) for 1 hour at room temperature and then incubated for 40 minutes with the rabbit anti-TUCAN antibody, mouse anti-c-myc antibody, mouse anti-caspase-9 antibody, mouse anti-caspase-8 antibody, mouse anti-cleaved PARP (D214) antibody, or mouse anti-ß-actin antibody followed by incubation with the horseradish peroxidase–conjugated anti-rabbit IgG antibody or anti-mouse IgG antibody (KPL, Gaithersburg, MD). Finally, the reaction was made visible with an enhanced chemiluminescence kit (Amersham Biosciences Corp., Piscataway, NJ) according to the manufacturer's protocol. Signal intensities were quantified by using Image J software. The relative expression ratio of cleaved caspase-9 was calculated as: relative expression ratio of cleaved caspase-9 = (cleaved caspase-9 signal density / ß-actin signal density).

Annexin V labeling assay. First, 2 x 106 Jurkat-pcDNA3-myc cells, Jurkat-pcDNA3-myc-48-kDa TUCAN cells, and Jurkat-pcDNA3-myc-TUCAN-54 cells were plated on six-well tissue culture dishes with a complete medium. Apoptosis was induced by incubating cells with various concentrations (0.01, 0.1, and 1 µmol/L) of staurosporine (WAKO, Japan) or various concentrations (0.05, 0.1, and 0.2 µg/mL) of anti-Fas antibody 2D1 (17) for 24 hours. Following the induction of apoptosis, apoptotic cells were labeled with Annexin V using an Annexin V-FLUOS Staining kit (Roche Diagnostics). Briefly, the cell pellet was washed with PBS twice and resuspended in 100 µL of a staining solution containing Annexin V-FLUOS and propidium iodide. After 15-minute incubation at room temperature, cells were analyzed using a fluorescence-activated cell sorter (FACSCalibur and Cell Quest software, BD Biosciences).

Caspase activity assay. Cells were plated at 1 x 104 per well in flat-bottomed 96-well tissue culture plates and then treated with 0.1 µg/mL antibody 2D1 or 0.1 µmol/L staurosporine (n = 3). Caspase-3/7 or -8 activity was measured by using a Caspase-Glo-3/7 or -8 Assay kit (Promega Corp., Madison, WI) following the manufacturer's protocol (Technical Bulletin TB295 and TB323, respectively). Briefly, substrate reagents were added directly to the cell culture plates that had been preincubated at room temperature. The plates were shaken at 500 rpm for 30 seconds and measured for luminescence output at various time points (0.5, 1, 2, 3, or 4 hours) using a WALLAC ARVO SX 1420 multilabel counter with a 1.0-second read time (relative light units factor = 10.0).

Small interfering RNA–mediated knockdown of endogenous TUCAN-54. Synthetic ready-to-use siRNA (21 nucleotides) complementary to a region of TUCAN-54-specific domain and nonsilencing control siRNA targeting to green fluorescence protein gene were custom synthesized by Qiagen (Tokyo, Japan). HSC2 human oral cancer cells, which had an endogenous expression of TUCAN-54 but not 48-kDa TUCAN, were transfected with siRNA using the LipofectAMINE 2000 reagent (Invitrogen). Briefly, 100 pmol siRNA and 5 µL LipofectAMINE 2000 reagent were diluted with Opti-MEM (Invitrogen) to a volume of 250 µL, mixed, and added to cells in six-well plates that had been grown to ~60% confluency. After 4 hours, the cells were washed and replated onto six-well plate in fresh medium followed by incubation for 72 hours. The cells were then harvested for Western blotting analysis or cultured in medium containing the indicated concentrations of VP-16 for 4.5 hours. Live cell numbers were assessed by Annexin V labeling assay.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Structure of TUCAN-54. To identify novel tumor antigens involved in the survival potential of tumor cells, a BLAST search of the public database was done by searching for sequences homologous to the CARD domain of TUCAN as a query. We identified an expressed sequence tag (EST) clone with an open reading frame encoding a novel isoform of TUCAN (Fig. 1A). The CARD domain of TUCAN-54 was identical to that of 48-kDa TUCAN in a ClustalW search and had a homology with that of caspase-1 (35%), CARD12 (29%), CARD5 (29%), cIAP1 (25%), CARD6 (24%), Apaf-1 (20%), RICK (20%), and caspase-9 (17%). TUCAN-54 contained several candidate phosphorylation sites, including protein kinase C ([S/T]X[R/K]) sites at amino acids 127, 342, 368, and 472, casein kinase II ([S/T]XX[D/E]) sites at amino acids 11, 61, 67, 345, 432, 454, and 472, and mitogen-activated protein kinase (MAPK)/cyclin-dependent kinase (CDK; [S/T]P) sites at 243, 312, and 345. On the other hand, 48-kDa TUCAN contained four protein kinase C phosphorylation sites, four casein kinase II sites, and two MAPK/CDK sites as reported previously (13). TUCAN-54 also contained a candidate caspase cleavage site (DEED) at residues 299 to 302.



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  		Figure 1. Amino acid sequence and domain structure of 54-kDa TUCAN isoform. A, amino acid sequence of 54-kDa TUCAN isoform (TUCAN-54). The CARD domain in the COOH terminus of the protein is underlined boldfaced. Unique amino acid sequence in the NH2 terminus (distinct from 48-kDa TUCAN) is indicated by the gray box in the top box and italic boldfaced in the amino acid sequence. NALP homology region is indicated by the dotted box in the top box and underlined face in the amino acid sequence. The amino acid sequence of NALP homology region in TUCAN-54 is aligned with that of COOH-terminal region in NALP1 (CARD7) in the bottom. B, gene structures of TUCAN-54 and 48-kDa TUCAN. BLAST search revealed that the gene encoding TUCAN-54 resided on chromosome 19 and was expected to contain 13 exons spreading over 27 kb. This TUCAN isoform is transcribed from an alternative starting codon (exon 1) and subjected to alternative splicing (exon 6).

 
The NH2-terminal region of TUCAN-54 protein contained no known functional domain structure, except for the segment (amino acids 111-290) sharing 66% amino acid sequence similarity with the region (amino acids 1,079-1,256) of the other CARD family protein NALP1/NAC/DEFCAP (CARD7; Fig. 1A). The BLAST search revealed that the gene encoding TUCAN-54 resided on chromosome 19 and was expected to contain 13 exons spreading over 27 kb (Fig. 1B).

Expression of TUCAN-54 mRNA and protein in various tumor cell lines, tumor tissues, and adult normal tissues. We first defined the expression profile of TUCAN-54 using panels of normal tissue mRNA and tumor cell line mRNA. As shown in Fig. 2A, RT-PCR analysis revealed that TUCAN-54 mRNA was widely expressed in a variety of tumor cell lines with modest tissue specificity. Except for the cell lines derived from stomach and liver cancer, almost all the tumor cell lines derived from various tissues expressed TUCAN-54. In normal adult human tissues, there was only a trace of mRNA expression in various tissues, whereas high levels of mRNA expression were detected in leukocytes and spleen.



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  		Figure 2. Expression of TUCAN-54 mRNA and protein in tumor cell lines and normal human adult tissues. A, cDNA samples from tumor cell lines and normal human adult tissues were analyzed for the expression of TUCAN-54 and GAPDH by RT-PCR with specific primers. A plasmid encoding TUCAN-54 was used as a control template for the reaction. B, Western blot analysis detecting TUCAN-54 and 48-kDa TUCAN proteins expressed in various human tumor cell lines. Proteins were extracted from cultured cells and protein (10 µg) from each extract was resolved by 10% SDS-PAGE, transferred to a PVDF membrane, and probed with the anti-TUCAN antibody. C, expression of TUCAN-54 protein was determined in tissue pairs of adenocarcinoma tissues (T) and noncancerous tissues (N) derived from surgical specimens of the same patients with breast (I), gastric, or colon (N) cancer. The lysate of colo205 or SW480 cells was used as a positive control sample. D, several paired samples of adenocarcinoma tissues and noncancerous tissues were analyzed for TUCAN-54 mRNA expression by RT-PCR analysis. The patient case numbers are identical to those of (C). ß-actin mRNA was detected as an internal control.

 
Some antiapoptotic proteins are produced at high levels in cancers, conferring apoptosis resistance (18). To gain preliminary insights into the expression of TUCAN-54 protein in cancers, 26 human tumor cell lines were analyzed by immunoblotting with an anti-TUCAN polyclonal antibody (Fig. 2B). For this study, we established rabbit antisera against TUCAN protein. An immunoreactive band of ~48 or 54 kDa was detected in 293T cells transfected with a myc-48-kDa TUCAN or myc-TUCAN-54 expression vector, respectively, but not in mock-transfected cells when stained with this anti-TUCAN antibody (data not shown). Cell lysates were normalized for total protein content before the analysis. Relative levels of TUCAN-54 protein varied widely among the tumor lines tested, and the expression rates were 100% (6 of 6) in colon cancer cell lines, 60% (3 of 5) in pancreas cancer cell lines, and 63.6% (7 of 11) in oral cancer cell lines. TUCAN-54 protein could not be detected in any of breast cancer cell lines, although mRNA was detected by RT-PCR. However, 48-kDa TUCAN protein was detected in two of them. It is likely that TUCAN-54 expression may have post-transcriptional regulation.

The expression of TUCAN-54 protein and mRNA was determined in tissue pairs of adenocarcinoma tissues and noncancerous tissues derived from surgical specimens of the same cancer patient. The lysate of colo205 or SW480 cells was used as a positive control sample. TUCAN-54 protein was detected as a single band of MW 54,000 Da. As shown in Fig. 2C, TUCAN-54 protein was detected in three of five cases of breast cancer (patients 2-4), four of six cases of gastric cancer (patients 2 and 4-6), and three of six cases of colon cancer (patients 3, 5, and 6). The TUCAN-54 expression was highly specific in tumor tissues of the breast and stomach, although a minimum level of the expression was detected in noncancerous tissue of cases 3 and 5 of colon cancer. It is possible that microinfiltration of tumor cells into the noncancerous tissue might have been the source of this band.

Several paired samples were analyzed for TUCAN-54 mRNA expression by RT-PCR analysis (Fig. 2D). The patient case numbers of Fig. 2D are identical to those of Fig. 2C. It was shown that the expression of TUCAN-54 was tumor tissue specific at the RNA level as well.

Overexpression of 48-or 54-kDa TUCAN suppressed caspase-9 activation. It has been reported that 48-kDa TUCAN is capable of inhibiting caspase-9 activation by binding to the CARD region of pro-caspase-9 (13), thereby suppressing the formation of the Apaf-1-caspase-9 apoptotic complex and apoptosis (14). To confirm the antiapoptotic function of 48-kDa TUCAN, we tested the effect of transfected 48-kDa TUCAN on the caspase-9 activation in 293T cells.

293T cells were cultured in six-well dishes and transfected with the plasmid encoding pro-caspase-9, 48-kDa TUCAN, or a mock transfectant in various combinations as indicated in Fig. 3A. Twenty-four hours after the transfection, cellular proteins were extracted from the cells and analyzed for the levels of 48-kDa TUCAN, pro-caspase-9, cleaved caspase-9, cleaved PARP (a substrate of activated caspases; ref. 19), and ß-actin by Western blotting. Overexpression of pro-caspase-9 produced cleaved caspase-9 and cleaved PARP (Fig. 3A, lane 3), indicating that caspase-9 and downstream caspases were activated in the cells. In 48-kDa TUCAN-transfected cells, the levels of cleaved caspase-9 and cleaved PARP were suppressed almost to background levels in a dose-dependent manner (Fig. 3A, lanes 5-7). The relative expression ratio of cleaved caspase-9 was calculated and is shown in Fig. 3A (bottom). Our study showed that overexpression of 48-kDa TUCAN inhibited the activation of caspase-9. The present results were consistent with a previous report as to the antiapoptotic function of 48-kDa TUCAN (13).



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  		Figure 3. Overexpression of 48-kDa TUCAN or TUCAN-54 suppresses caspase-9 activation in 293T cells. 293T cells were transfected with a plasmid encoding pro-caspase-9, 48-kDa TUCAN (A) or TUCAN-54 (B) in various combinations as indicated in the figure. Cleaved caspase-9, 48-kDa TUCAN (A), TUCAN-54 (B), cleaved PARP, and ß-actin were detected by Western blotting. Relative expression level of cleaved caspase-9 was calculated as: caspase-9 relative activation ratio = (cleaved caspase-9 signal density / ß-actin signal density).

 
For the antiapoptotic function of 48-kDa TUCAN, the CARD domain, which resides in the COOH-terminal region, is important. Because the COOH-terminal region is conserved between 48-kDa TUCAN and TUCAN-54, we investigated whether TUCAN-54 had the same antiapoptotic effect on the caspase-9 activation. 293T cells were transfected with the plasmid encoding TUCAN-54, pro-caspase-9, or a mock transfectant in various combinations as indicated in Fig. 3B. The protein levels of TUCAN-54, pro-caspase-9, cleaved caspase-9, cleaved PARP, and ß-actin were detected by Western blotting. In TUCAN-54-transfected cells, the levels of cleaved caspase-9 and cleaved PARP were suppressed significantly in a dose-dependent manner (Fig. 3B, lanes 3-5). Relative expression ratios of cleaved caspase-9 in TUCAN-54-transfected cells were reduced to 19.1% in the 4 µg transfectant (Fig. 3B, lane 4) and 6.0% in the 8 µg transfectant (Fig. 3B, lane 5) compared with 30% in the mock transfectant (Fig. 3B, lane 6). Our results clearly showed that overexpression of TUCAN-54 suppressed activation of caspase-9 and downstream caspases as in 48-kDa TUCAN.

Stable overexpression of TUCAN-54 suppresses Fas-induced apoptosis in Jurkat cells. To further explore the role of TUCAN-54 as an antiapoptotic molecule, we next focused on death receptor–mediated apoptosis. Jurkat cells express Fas molecules on the cell surface and are sensitive to Fas-mediated apoptotic stimulation. We established Jurkat cell transfectants stably overexpressing 48-kDa TUCAN or TUCAN-54. Immunoblot analysis of the established stable transfectants is shown in Fig. 4A, indicating that both 48- and 54-kDa TUCAN-transfected cells expressed comparable amounts of TUCAN proteins, whereas vector-transfected cells expressed no TUCAN protein.



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  		Figure 4. Overexpression of TUCAN-54 suppresses staurosporine-, VP-16-, and Fas-induced apoptosis in Jurkat cells. A, Jurkat cells were transfected with pcDNA3-myc-48-kDa TUCAN or pcDNA3-myc-TUCAN-54 expression vector. The transfected cells were characterized by Western blotting with an anti-myc-tag mAb or anti-TUCAN antibody. ß-actin was detected as an internal control. B-D, Jurkat transfectants (2 x 106) with pcDNA3-myc, pcDNA3-myc-48-kDa TUCAN, or pcDNA3-myc-TUCAN-54 were plated in a six-well tissue culture dish. Apoptosis was induced by incubating cells with various concentrations of staurosporine (B), VP-16 (C), or anti-Fas antibody 2D1 (D). Following induction of apoptosis, cell pellets were resuspended in 100 µL of a staining solution containing Annexin V-FLUOS and propidium iodide. Apoptotic cell numbers were analyzed by flow cytometry. Columns, averages of triplicate samples; bars, SD.

 
A protein kinase inhibitor, staurosporine, has been shown to induce apoptosis through an Apaf-1-caspase-9–dependent mechanism (20–22). As shown in Fig. 4B, significantly fewer apoptotic cells were induced by staurosporine in either 48-kDa TUCAN-overexpressing or TUCAN-54-overexpressing Jurkat cells compared with control transfectant cells. This was consistent with the former finding that both TUCAN isoforms could equally suppress the activation of the caspase-9 pathway in the apoptotic signaling. Similar results were obtained from the experiment using a chemotherapeutic reagent, VP-16, to induce apoptosis (Fig. 4C). Both 48- and 54-kDa TUCAN conferred chemoresistance on Jurkat cells.

Antibody-mediated cross-linking of CD95 (APO-1/Fas) results in the recruitment of a set of proteins that includes FADD/MORT1 (23, 24) and caspase-8 (FLICE/MACH/Mch5; refs. 25–27) to the receptor leading to the formation of death-inducing signaling complex (25, 28) and apoptosis. We used anti-Fas mAb 2D1 for inducing apoptosis via caspase-8 activation. Jurkat transfectants were treated with various concentrations of the anti-Fas mAb for 24 hours, and apoptotic cell numbers were determined by Annexin V labeling assay. As shown in Fig. 4D, >50% of 48-kDa TUCAN overexpressing cells and control transfectants fell into apoptosis after the anti-Fas mAb treatment (0.2 µg/mL). In contrast, the apoptotic cell ratio of TUCAN-54-overexpressing cells was approximately half of them (27 ± 4%) after the same treatment. Therefore, it was suggested that TUCAN-54, but not 48-kDa TUCAN, might suppress the caspase-8 pathway in the apoptotic signaling. Considering the structural difference between these TUCAN isoforms, it was speculated that the NH2-terminal unique region of TUCAN-54 should have a distinct function in the apoptotic signaling.

Effect of overexpression of 48-or 54-kDa TUCAN on the caspase-3/7 or -8 activation in Jurkat cells. Caspase-3/7 are downstream caspases in both the staurosporine-induced caspase-9-mediated apoptotic pathway and the Fas-induced caspase-8-mediated apoptotic pathway. To determine the intracellular signaling events in the TUCAN-overexpressing cells, we detected caspse-3/7 and -8 activity in Jurkat transfectants after stimulation with staurosporine (Fig. 5A) or the anti-Fas mAb (Fig. 5B and C). In control transfectants, caspase-3/7 activity rose up to 7-fold the background level after the staurosporine stimulation. On the other hand, increases of caspase-3/7 activity were suppressed to the levels of 3.5- and 4.5-fold the background level in the 48-kDa TUCAN-expressing cells and TUCAN-54-expressing cells, respectively. These data indicated that both 48- and 54-kDa TUCAN could suppress the activation of downstream caspase-3/7 induced by staurosporine in Jurkat cells.



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  		Figure 5. Overexpression of TUCAN-54 suppresses caspase-8 and -3/7 activation in Jurkat cells. A-C, Jurkat transfectants were treated with 0.1 µmol/L staurosporine (A) or 0.1 µg/mL anti-Fas antibody 2D1 (B and C). Following induction of apoptosis, caspase-3/7 or -8 activity was measured by using a Caspase-Glo-3/7 (A and C) or Caspase-Glo-8 (B) assay kit. Jurkat cells transfected with pcDNA3-myc ({bullet}), pcDNA3-myc-48-kDa TUCAN ({blacksquare}), or pcDNA3-myc-TUCAN-54 ({blacktriangleup}). Points, averages of triplicate samples; bars, SD. D, Jurkat transfectants were cultured in the presence or absence of 0.1 µg/mL anti-Fas antibody 2D1 for 2 hours. Following the incubation, cell lysates were collected, and pro-caspase-8, cleaved caspase-8, and ß-actin were detected by Western blotting.

 
Similarly, we compared the caspase-8 and -3/7 activities after anti-Fas stimulation in the Jurkat transfectant cells (Fig. 5B and C). At 1 hour after the stimulation, there was no significant difference in the caspase-8 activity among control-, 48-kDa TUCAN-, and TUCAN-54-transfected Jurkat cells. However, the caspase-8 activity differed significantly at 2 hours after the stimulation. It was mostly suppressed in the TUCAN-54-overexpressing cells (Fig. 5B). Similar suppression was observed in the activity of the downstream caspase-3/7 after the anti-Fas stimulation, although the kinetics was different from that of caspase-8 (Fig. 5C). In TUCAN-54-overexpressing cells, caspase-3/7 activity was strongly inhibited over 6 hours after the stimulation, whereas it was increased to almost the half level of control transfectants in 48-kDa TUCAN-expressing cells.

To confirm further the inhibitory effect of TUCAN-54 on the caspase-8 activation, Western blotting analysis detecting active caspase-8 was done using the Jurkat transfectants. At 2 hours after the stimulation with anti-Fas mAb, 18-kDa cleaved caspase-8 was detected in the mock-transfected Jurkat cells and 48-kDa TUCAN-expressing cells, whereas it was not in the TUCAN-54-overexpressing cells (Fig. 5D). The result was almost consistent with the result of caspase-8 activity assay (Fig. 5B).

These data clearly show that TUCAN-54 had antiapoptotic functions in both caspase-9- and -8-mediated pathways.

TUCAN-54 is physically associated with Fas-associated death domain. Previously, it was shown that 48-kDa TUCAN could interact with caspase-9 through its CARD domain. To know the inhibitory mechanism of TUCAN-54 for the Fas-mediated apoptosis signaling, TUCAN-54-associated molecules were examined within the Fas-mediated signaling molecules. 293T cells were transfected with myc-48-kDa TUCAN plasmid or myc-TUCAN-54 plasmid in combination with Flag-tagged FADD expression vector. After immunoprecipitation of TUCAN proteins with anti-myc mAb, coprecipitation of FADD protein was tested by Western blotting with anti-Flag mAb. The result indicated that FADD was physically associated with TUCAN-54 but not with 48-kDa TUCAN (Fig. 6). Because FADD is constitutively associated with pro-caspase-8 in 293T transfected cells (data not shown), it was suggested that TUCAN-54 forms molecular complex with FADD/pro-caspase-8, thereby inhibiting the activation of pro-caspase-8.



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  		Figure 6. FADD is coimmunoprecipitated with TUCAN-54 but not with 48-kDa TUCAN. 293T cells were transfected with a plasmid encoding TUCAN-54-kDa (lane 1), 48-kDa TUCAN (lane 2), or a mock plasmid (lane 3) in combination with FADD-Flag expression plasmid. Twenty-four hours after the transfection, cell lysates were collected and subjected to immunoprecipitation (IP) with anti-myc mAb and/or Western blotting (WB) with anti-myc antibody or anti-Flag antibody.

 
Down-regulation of endogenous TUCAN-54 increases chemosensitivity of tumor cells. Next, we examined if decreases of TUCAN-54 protein levels in tumor cells might alter the cell death sensitivity against chemotherapeutic reagents. HSC2 cells were tested because these cells express endogenous TUCAN-54 but not 48-kDa TUCAN (Fig. 2B). Two kinds of TUCAN-54-specific siRNA could successfully decrease the endogenous TUCAN-54 protein level in HSC2 cells (Fig. 7A). After the culture of siRNA-transfected cells in the medium containing various concentrations of VP-16 for 4.5 hours, live cell rates were assessed by Annexin V labeling assay. It was shown that decreases of endogenous TUCAN-54 protein level could lead to the increased VP-16-sensitivity by 100-fold in HSC2 cells (Fig. 7B), suggesting that TUCAN-54 might be associated with chemoresistance of tumor cells and might become a suitable target for the molecular targeting therapy of cancer.



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  		Figure 7. siRNA–mediated knockdown of endogenous TUCAN-54 expression and its effect on VP-16-sensitivity of cancer cells. A, two kinds of synthetic siRNA (21 nucleotides) complementary to a region of TUCAN-54-specific domain (lanes 1 and 2) and nonsilencing control siRNA targeting to green fluorescence protein gene (lane 3) were transfected into HSC2 oral cancer cells, which had an endogenous TUCAN-54 expression. Forty-eight hours after the transfection, cell lysates were collected, and TUCAN-54 and ß-actin were detected by Western blotting. B, 72 hours after the siRNA transfection, cells were cultured in the medium containing the indicated concentrations of VP-16 for 4.5 hours. Then, cells were harvested, and living cell rates were assessed by Annexin V labeling assay. Columns, averages of triplicate samples; bars, SD.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have identified and characterized a new member of the human CARD family, TUCAN-54. This novel isoform of TUCAN protein was 487 amino acids longer at its NH2 terminus than 48-kDa TUCAN protein, although no known protein motifs were recognized in this region. In addition, the NH2-terminal 80 amino acids of TUCAN-54 protein were different from those of 48-kDa TUCAN. This TUCAN isoform was presumed to arise as a result of an alternative start codon and alternative splicing (Fig. 1B).

The sequence of TUCAN-54 was first disclosed as an EST clone similar to death effector filament-forming Ced-4-like apoptosis protein, clone IMAGE:4827771, from testis cDNAs in the Genbank EST database. The present study showed for the first time functional characterization of this protein.

TUCAN-54 contains a NH2-terminal unique domain and NALP homology domain (Fig. 1A), which shares amino acid similarity with a COOH-terminal segment of NALP1 (CARD7) protein, a CARD-carrying regulator of the Apaf-1 apoptosome (29–31). Although the function of these domains is presently unknown, it is likely that the domain might serve to regulate TUCAN in a variety of ways, such as controlling interactions with other proteins, affecting protein degradation, or altering subcellular location. Interestingly, the NH2-terminal (non-CARD) region of TUCAN-54 contains several candidate phosphorylation sites, including protein kinase C ([S/T]X[R/K]) sites at amino acids 127, 342, 368, and 472, casein kinase II ([S/T]XX[D/E]) sites at amino acids 11, 61, 67, 345, 432, 454, and 472, and MAPK/CDK ([S/T]P) sites at 243, 312, and 345. On the other hand, 48-kDa TUCAN contains four protein kinase C phosphorylation sites, four casein kinase II sites, and three MAPK/CDK sites (13). Three casein kinase II sites at amino acids 11, 61, and 67 are unique in 54-kDa TUCAN in comparison with 48-kDa TUCAN. It is probable that these distinct sites may lead to the functional difference between TUCAN-54 and 48-kDa TUCAN shown in the present study.

It was indicated in this study that TUCAN-54 protein suppressed both caspase-8- and -9-mediated apoptosis. In the antiapoptotic function of 48-kDa TUCAN, the CARD domain is the most important domain for the suppression of caspase-9-mediated apoptosis (13). The suppression of apoptosis by 48-kDa TUCAN is mediated by its ability to interacting with pro-caspase-9. Because TUCAN-54 had the same CARD domain structure, it was reasoned that the suppression of caspase-9-mediated apoptosis by TUCAN-54 arose from the same mechanism as that by 48-kDa TUCAN. In the present study, we clarified the distinct function of TUCAN-54. TUCAN-54 strongly suppressed the Fas-induced apoptosis and caspase-8 activation, but the 48-kDa TUCAN could not. The inhibitory action might be mediated by the physical interaction between FADD and TUCAN-54 as shown in Fig. 6. It is likely that the difference of the antiapoptotic function comes from the distinct NH2-terminal regions of both TUCAN proteins. Although there is no known functional domain structure in the NH2-terminal unique region of TUCAN-54, this region may directly or indirectly mediate the binding to FADD/pro-caspase-8 complex leading to the suppression of its activation. It is also possible that TUCAN-54 can bind and suppress the function of proapoptotic protein RICK (32), which has a CARD domain and promotes the apoptosis induced by anti-Fas antibodies. Further identification of TUCAN-54-binding proteins should reveal the precise mechanism of the antiapoptotic function.

In the present study, we showed the tumor-specific overexpression of this novel antiapoptotic protein. TUCAN-54 was barely detected in the noncancerous tissues, except for spleen and leukocytes. Taking into consideration that TUCAN-54 has wide and strong antiapoptotic functions in tumor cells, TUCAN-54 may contribute to the oncogenesis and/or malignant phenotype, such as chemoresistance and immunoresistance of tumor cells. Overexpression of TUCAN-54 might be functionally equivalent to the loss of Apaf-1 or pro-caspse-9. Therefore, elevated levels of TUCAN-54 might promote tumor pathogenesis or progression. It has been reported that expression of 48-kDa TUCAN or other CARD family proteins is correlated with poor prognosis in cancer patients (13). The anti-48-kDa TUCAN antibody developed by Drs. N. Pathan and John C. Reed reacted with the TUCAN-54 as well (data not shown). Thus, TUCAN-54 may also be a prognostic marker of various malignancies. In addition, the specific suppression of TUCAN-54 function may possibly become a potential novel chemosensitizing therapy when used in conjunction with conventional chemotherapy (33). It was reported that some proapoptotic proteins function as tumor suppressors in p53-dependent pathways and can be used for novel gene therapies (34). Therefore, TUCAN-54, a tumor-specific antiapoptotic protein, may become an important molecular target in cancer therapy.

In summary, we have identified and characterized a novel CARD-containing protein encoded in the TUCAN gene locus. This previously unstudied protein may have a significant role in the carcinogenesis, malignant phenotype, and chemoresistance of tumors.


   Acknowledgments
 
Grant support: Ministry of Education, Culture, Sports, Science and Technology of Japan.

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 Dr. John C. Reed for the generous gift of 48-kDa TUCAN cDNA and pro-caspase-9 cDNA.

Received 1/ 3/05. Revised 6/ 9/05. Accepted 8/ 2/05.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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