Hypermethylation of XIAP-Associated Factor 1, a Putative Tumor Suppressor Gene from the 17P13.2 Locus, in Human Gastric Adenocarcinomas1

[CANCER RESEARCH 63, 7068–7075, November 1, 2003] Advances in Brief

Hypermethylation of XIAP-associated Factor 1, a Putative Tumor Suppressor Gene from the 17p13.2 Locus, in Human Gastric Adenocarcinomas1

Do-Sun Byun, Kyucheol Cho, Byung-Kyu Ryu, Min-Goo Lee, Min-Ju Kang, Hak-Ryul Kim, and Sung-Gil Chi2 Department of Pathology, School of Medicine, Kyung Hee University, Seoul 130-701 [D-S. B., K. C., B-K. R., M-G. L., M-J. K., S-G. C.], and Graduate School of Life Science and Biotechnology, Korea University, Seoul 136-701 [D-S. B., H-R. K.], Korea

Abstract gested to play a key role in the aberrantly increased cell viability and resistance to the anticancer therapy in human cancers, whereas over- X-linked inhibitor of apoptosis (XIAP) is the most potent member of the expression seems to suppress apoptosis against a large variety of IAP family that exerts antiapoptotic effects by interfering with the activ- ities of caspases. Recently, XIAP-associated factor 1 (XAF1) and two triggers (4, 5). The human IAP family includes cIAP-1, cIAP-2, mitochondrial proteins, Smac/DIABLO and HtrA2, have been identified XIAP, NAIP, survivin, apollon, ILP2, and livin, and several members to negatively regulate the caspase-inhibiting activity of XIAP. To explore of the human IAP family including XIAP, c-IAP-1, and c-IAP-2 have the candidacy of XAF1, Smac/DIABLO, and HtrA2 as a tumor suppressor been shown to be potent inhibitors of caspase-3, -7, and -9 (2, 6, 7). in gastric tumorigenesis, we investigated the expression and mutation Of the eight known human IAP proteins, XIAP is the most potent status of the genes in 123 gastric tissues and 15 cancer cell lines. Whereas and versatile inhibitor of caspases and apoptosis. XIAP demonstrates Smac/DIABLO and HtrA2 transcripts were normally expressed in all significant inhibition of apoptosis induced by many triggers, including cancer specimens we examined, XAF1 transcript was not expressed or serum withdrawal and etoposide exposure (8, 9). XIAP possesses present at extremely low levels in 40% (6 of 15) of cancer cell lines and in 23% (20 of 87) of primary carcinomas. Abnormal reduction of XAF1 three BIR domains and a COOH-terminal RING zinc finger and expression showed a strong correlation with stage and grade of tumors, inhibits both the initiator caspase-9 and the effectors caspase-3 and -7. and a tumor-specific down-regulation of XAF1 was observed in 45% (9 of XIAP mRNA levels are relatively high in the majority of cancer cell 20) of matched sets. Unlike XAF1, XIAP expression exhibited no detect- lines, and high levels of XIAP protein are generally found in renal able alteration in cancers. Whereas loss of heterozygosity within the XAF1 cancer and melanoma cell lines (10, 11). region or somatic mutations of the gene was not detected, expression of The caspase-inhibiting effects of IAPs are antagonized by apoptosis- XAF1 transcript was reactivated in all nonexpressor cell lines after 5-aza- promoting proteins. Two mitochondrial proteins, termed Smac/DIABLO 2-deoxycytidine treatment. The 5؅ upstream region of the XAF1 gene and HtrA2, have been identified to promote caspase activation by encompasses no gastric cell-rich region that rigorously satisfies the formal criteria for CpG islands. However, bisulfite DNA sequencing analysis for antagonizing the caspase-inhibitory activity of XIAP (12–14). Smac/ 34 CpG sites in the promoter region revealed a strong association between DIABLO is released from mitochondria together with cytochrome c hypermethylation and gene silencing. Moreover, transcriptional silencing during mitochondria-induced apoptosis. The binding of Smac/DIABLO of XAF1 was tightly associated with hypermethylation of seven CpGs to XIAP is proposed to destabilize the XIAP-caspase interaction by located in the 5؅ proximal region (nucleotides ؊23 to ؊234). Additionally, steric hindrance, resulting in disruption of the XIAP-caspase complex loss or abnormal reduction of XAF1 expression was found to inversely (15, 16). HtrA2 is a mammalian serine protease homologous to the correlate with p53 mutations, suggesting that epigenetic inactivation of bacterial HtrA endoprotease and directly binds to the BIR3 domain of XAF1 and mutational alteration of p53 might be mutually exclusive events XIAP and activates caspases. In a similar manner to Smac/DIABLO, in gastric tumorigenesis. Collectively, our study suggests that epigenetic silencing of XAF1 by aberrant promoter methylation may contribute to HtrA2 is released from mitochondria into the cytosol and forms the malignant progression of human gastric tumors. complexes with XIAP at high stoichiometry as a result of apoptosis stimulus. These observations demonstrate that Smac/DIABLO and Introduction HtrA2 sensitize cells to apoptosis in response to a number of stimuli by binding to and antagonizing XIAP. Apoptosis is essential for elimination of defective or potentially Very recently, a novel negative regulator of XIAP termed XAF1 dangerous cells and provides a defense against malignant transforma- was isolated based on its ability to bind XIAP (17). Transient over- tion and autoimmunity (1). Several genes critical in the regulation of expression of XAF1 sensitizes tumor cells to the proapototic effects of 3 apoptosis have been identified, including the IAP family (2). The IAP etoposide and reverses the XIAP-mediated inhibition of caspase-3 proteins are a new class of intrinsic cellular regulators of apoptosis activity. In contrast to Smac/DIABLO and HtrA2, XAF1 resides in that are structurally defined by the presence of the evolutionary the nucleus and can effect a marked relocalization of XIAP protein conserved BIR domain (2, 3). Deregulation of IAPs has been sug- from the cytoplasm to the nucleus. The XAF1 gene is located at 17p13.2, approximately 3 cM telomeric to the p53 tumor suppressor Received 4/1/03; revised 8/19/03; accepted 8/27/03. gene, and encodes M 33,100 protein with seven zinc fingers with high The costs of publication of this article were defrayed in part by the payment of page r charges. This article must therefore be hereby marked advertisement in accordance with amino acid similarity with the zinc finger domains of both FLN29 and 18 U.S.C. Section 1734 solely to indicate this fact. TRAF3 (10, 17). XAF1 mRNA is expressed ubiquitously in all normal 1 Supported by a grant (R02-2001-000-00052-0) from the Basic Research Program of the Korea Science and Engineering Foundation and an intramural grant-in-aid from the adult and fetal tissues but is present at very low or undetectable levels Kyung Hee University (2002), Republic of Korea. in various cancer cell lines. Expression analysis using the NCI 60 cell 2 To whom requests for reprints should be addressed, at Department of Pathology, line panel also revealed that cancer cell lines exhibit very low levels College of Medicine, Kyung Hee University, 130-701 Seoul, Republic of Korea. Phone: 82-2-961-0920; Fax: 82-2-961-0277; E-mail: [email protected]. of XAF1 mRNA, whereas the majority of cancer cell lines express 3 The abbreviations used are: IAP, inhibitor of apoptosis; BIR, baculovirus IAP repeat; relatively high levels of XIAP mRNA, suggesting that deregulation of XIAP, X-linked IAP; LOH, loss of heterozygosity; SSCP, single-strand conformation polymorphism; obs/exp CpG, observed/expected CpG; XAF1, XIAP-associated factor 1; apoptosis through the loss of XAF1 expression might be important for GAPDH, glyceraldehyde-3-phosphate dehydrogenase. malignant cell survival, and a high level of XIAP to XAF1 expression 7068

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2003 American Association for Cancer Research. EPIGENETIC INACTIVATION OF XAF1 IN GASTRIC CARCINOMA in cancer cells might provide a survival advantage through the relative Quantitation was achieved by densitometric scanning of the ethidium bromide- increase of XIAP antiapoptotic function (10). stained gels. Absolute area integrations of the curves representing each spec- Gastric cancer is one of the most commonly diagnosed malignan- imen were then compared after adjustment for GAPDH. Integration and cies worldwide and a leading cause of cancer mortality in certain analysis were performed using the Molecular Analyst software program (Bio- areas, such as Korea, Japan, South America, and Eastern Europe (18). Rad, Hercules, CA). Quantitative PCR was repeated at least three times for each specimen, and the mean was obtained. Although evidence has accumulated indicating the involvement of the Western Blot Assay. Cells were lysed in a lysis buffer containing 20 mM alterations of multiple genes such as p53, K-ras, c-erbB2, K-sam, and Tris (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, 2.5 mM E-cadherin, the underlying molecular events that drive the neoplastic ␤ ␮ sodium phosphate, 1 mM -glycerolphosphate, 1 mM Na3VO4,1 g/ml leu- process in gastric cancer are largely undefined (19). In the present peptin, and 1 mM phenylmethylsulfonyl fluoride. The cell lysate was clarified study, we investigated the expression and mutation status of XAF1, by centrifugation, and 20 ␮g of total protein were supplemented with Laemmli Smac/DIABLO, and HtrA2 in a series of primary gastric adenocarci- buffer and loaded on a 10% SDS-polyacrylamide gel for electrophoresis. XIAP nomas and cell lines to explore the candidacy of these genes as a was detected by immunoblot using an anti-XIAP monoclonal antibody (BD suppressor in gastric carcinogenesis. Our data demonstrate that XAF1 Biosciences, San Diego, CA). Antibody binding was detected by enhanced mRNA expression is lost or significantly down-regulated in a consid- chemiluminescence (Amersham Biosciences, Piscataway, NJ) using a second- erable fraction of gastric cell lines and primary tumors by aberrant ary antibody conjugated to horseradish peroxidase. For stripping, the blots were incubated in a stripping buffer [0.2 M glycine (pH 2.2), 0.1% SDS, and promoter CpG hypermethylation, whereas none of tumors show de- 1% Tween 20] at room temperature for 60 min. -tectable abnormality of Smac/DIABLO and HtrA2. Moreover, loss or 5-Aza-2؅-Deoxycytidine Treatment. To assess reactivation of XAF1 ex down-regulation of XAF1 expression because of hypermethylation pression, cells were plated in 6-well tissue plates 24 h before treatment. was strongly associated with advanced stage and higher grade of 5-Aza-2Ј-deoxycytidine (Sigma Chemical Co., St. Louis, MO) was added to tumor, suggesting that epigenetic inactivation of XAF1 may play a the fresh medium at concentrations of 5 ␮M in duplicate, and cells were critical role in the malignant progression of gastric cancers. harvested after 4 days. Bisulfite DNA Sequencing. One microgram of genomic DNA was incu- Materials and Methods bated with 3 M sodium bisulfite (pH 5.0), and 50 ng of bisulfite-modified DNA were subjected to PCR amplification of the XAF1 promoter region using Tissue Specimens and Human Cell Lines. A total of 138 gastric tissues primer sets: MS12 (sense, 5Ј-GTTTAGGTTGGAGTGTAGTGG-3Ј) and MS2 including 87 primary adenocarcinomas, 3 adenomas, 6 hamartomas, 7 hyper- (antisense, 5Ј-CATATTCTACTCTCTACAAAC-3) for nucleotides ϩ3to plastic polyps, and 20 normal gastric tissues were obtained from 87 gastric Ϫ1852 (1855 bp) and MS3 (sense, 5Ј-TGTTAGTTTTAGGGAGGTAGA-3Ј) cancer patients and 36 noncancer patients by surgical resection in the Kyung and MS2 (antisense; see above) for nucleotides ϩ3toϪ257 (260 bp). The II Hee University Medical Center (Seoul, Korea). Tissue specimens were snap- PCR products were cloned into pCR vectors (Invitrogen, Carlsbad, CA), and Ϫ 10 clones of each specimen were sequenced by automated fluorescence-based frozen in liquid N2 and stored at 70°C until used. Tissue slices were subjected to histopathological review, and tumor specimens composed of at DNA sequencing to determine the methylation status. least 80% carcinoma cells were chosen for molecular analysis. Fifteen human LOH Analysis. LOH was determined using three polymorphic CA markers gastric cancer cell lines (SNU1, SNU5, SNU16, SNU216, SNU484, SNU601, (D17S796, D17S1832, and D17S1828) localized at chromosome 17p13.1– SNU620, SNU638, SNU719, MKN1, MKN28, MKN45, MKN74, AGS, 13.2. PCR amplification was performed on each tumor and normal DNA and KATO-III) were obtained from the Korea Cell Line Bank (Seoul sample pair obtained from 87 patients using primers 796-S (sense, 5Ј-CAAT- National University, Seoul, Korea) or American Type Culture Collection GGAACCAAATGTGGTC-3Ј) and 796-AS (antisense, 5Ј-AACAACCATT- (Manassas, VA). TACTTACTAG-3Ј) for D17S796, 1832-S (sense, 5Ј-CTTGACATAGTTGC- Quantitative PCR Analysis. One microgram of DNase1-treated RNA was CCACAG-3Ј) and 1832-AS (antisense, 5Ј-CTTTAGGTTTGGATCCAGCC- converted to cDNA by reverse transcription using random hexamer primers 3Ј) for D17S1832, and 1828-S (sense, 5Ј-GCAGGTATACAGCCACACAC- and MoMuLV reverse transcriptase (Life Technologies, Inc., Gaithersburg, 3Ј) and 1828-AS (antisense, 5Ј-TGGATTCAGCCATACCTGAA-3Ј) for MD). PCR was performed initially over a range of cycles (24, 26, 28, 30, 32, D17S1828. Ten microliters of the PCR products were electrophoresed on 34, 36, and 38 cycles), and 2 ␮l of 1:4 diluted cDNA (12.5 ng/50 ␮l PCR standard denaturing 8% polyacrylamide gels. If the two alleles appeared in the reaction) undergoing 28–36 cycles was observed to be within the logarithmic normal tissue DNA, the patient was considered an informative case for the phase of amplification with primers Xaf1–3 (sense, 5Ј-ATGGAAGGAGACT- particular marker. Signal intensity of fragments and the relative ratio of both TCTCGGT-3Ј) and Xaf1–4 (antisense, 5Ј-TTGCTGAGCTGCATGTCCAG- tumor and normal allele intensities were determined by scanning densitometry. 3Ј) for XAF1, Smac-1 (sense, 5Ј-GAGCAGTGTCTTTGGTAACA-3Ј) and Because certain numbers of noncancerous cells might be present in tumor Smac-2 (antisense, 5Ј-ACCTGCAGTTTCACCAGCTG-3Ј) for Smac/DIA- tissues, LOH was assigned when the intensity ratio of the two tumor alleles BLO, HtrA2–1 (sense, 5Ј-AGATCCTGGACCGGCACCCT-3Ј) and HtrA2–2 differed by at least 50% from that observed on its corresponding normal DNA. (antisense, 5Ј-TCCAGAGTTTCCAAAATCAA-3Ј) for HtrA2, Xiap-3 (sense, The same PCR products were also subjected to nonisotopic SSCP analysis for 5Ј-GATTATGAAGCACGGATC-3Ј) and Xiap-4 (sense, 5Ј-GACTTGACT- verification of LOH. CATCTTGCA-3Ј) for XIAP, and G2 (sense, 5Ј-CATGTGGGCCATGAG- Nonisotopic RT-PCR–SSCP Analysis. To screen the presence of somatic GTCCACCAC-3Ј) and G3 (antisense, 5Ј-AACCATGAGAAGTATGACAA- mutations, RT-PCR–SSCP analysis of XAF1 was performed. The XAF1 transcript CAGC-3Ј) for an endogenous expression standard gene, GAPDH. PCR was amplified with seven sets of primers that were designed to cover the entire primers for Smac/DIABLO and HtrA2 were designed to amplify the common coding region of the gene. Sequences of the primers used will be obtained on exonic regions of the transcripts (exons 3–6ofSmac/DIABLO and exons 1–4 request. The PCR products of over 300 bp in lengths were digested with endo- of HtrA2) to cover all known splicing variants, and primers for XIAP RT-PCR nuclease(s) to increase the sensitivity of SSCP analysis. Twenty microliters of were designed to specifically amplify XIAP transcript but not the XIAP PCR products were mixed with 10 ␮l of 0.5 N NaOH, 10 mM EDTA, and 15 ␮l pseudogene. PCR was performed for 32–36 cycles at 95°C (1 min), 56–60°C of denaturing loading buffer (95% formamide, 20 mM EDTA, 0.05% bromphenol blue, and 0.05% xylene cyanol). After heating at 95°C for 5 min, samples were (0.5 min), and 72°C (1 min) in 1.5 mM MgCl2-containing reaction buffer (PCR buffer II; Perkin-Elmer). For quantitative genomic PCR, the exon 6 region of loaded in wells precooled to 4°C and run using 8% nondenaturating acrylamide XAF1, exon 8 region of p53, and intron 5 region of GAPDH were amplified gels containing 10% glycerol at 4–8°C and 18–22°C. separately with intron-specific primers Xaf1-I1 (sense, 5Ј-TCACAATTG- CAGGGTAAATG-3Ј) and Xaf1-I2 (antisense, 5Ј-TAAGCAGAGAACA- Results GAGGCTG-3Ј), SG85 (sense, 5Ј-TCCTTACTGCCTCTTGCTTCTCTTT-3Ј) and SG83 (antisense, 5Ј-TCTCCTCCACCGCTTCTTGT-3Ј), and G3 (see Expression of XAF1, Smac/DIABLO, and HtrA2 in Normal and above) and G5 (antisense, 5Ј-GAGTCCTTCCACGATACCAAAG-3Ј), re- Benign Tumor Tissues. To investigate the candidacy of XAF1, spectively. Ten microliters of PCR products were resolved on 2% agarose gels. Smac/DIABLO, and HtrA2 as a tumor suppressor in gastric tumori- 7069

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2003 American Association for Cancer Research. EPIGENETIC INACTIVATION OF XAF1 IN GASTRIC CARCINOMA genesis, we initially characterized mRNA expression status of the compared with noncancerous tissues (P Ͻ 0.0001). Collectively, 40% genes in 20 normal gastric tissues and 16 benign tumors including 3 (6 of 15) of human gastric carcinoma cell lines exhibited loss or adenomas, 6 hamartomas, and 7 hyperplastic polyps. For validation of significantly reduced levels of XAF1 mRNA expression. our quantitative PCR approach, serially diluted cDNA was subjected Frequent Reduction of XAF1 mRNA Expression in Primary to PCR amplification of XAF1, Smac/DIABLO, HtrA2, and GAPDH Gastric Cancers. Next, we evaluated the expression of XAF1, Smac/ over a range of cycles. Linearity of the cDNA dilution experiments DIABLO, and HtrA2 in 87 primary gastric carcinomas including 20 demonstrated the ability of our PCR procedure to discriminate the matched sets. Whereas Smac/DIABLO and HtrA2 transcripts were various levels of transcripts (data not shown). As shown in Fig. 1A, expressed at similar levels in all tumor tissues examined, marked mRNA expression of XAF1, Smac/DIABLO, and HtrA2 was easily reductions of XAF1 expression were identified in a substantial fraction detectable in all normal and benign tumor tissues examined, and no of tumors (Fig. 1C). Moreover, tumor-specific reduction of XAF1 was significant variations in expression levels were observed among spec- detected in 9 of 20 (45%) matched sets from the same patients. As imens (XAF1/GAPDH, 0.92–1.37; Smac/GAPDH, 0.98–1.42; HtrA2/ shown in Fig. 2A, XAF1, Smac/DIABLO, and HtrA2 transcript levels GAPDH, 0.74–1.06). Quantitative RT-PCR was repeated at least in primary carcinomas were determined in the ranges of 0.30–1.39, three times for each specimen, and the mean expression level of 0.94–1.48, and 0.66–1.17, respectively. Unlike Smac/DIABLO and XAF1, Smac/DIABLO, and HtrA2 in noncancerous tissues was deter- HtrA2, expression levels of XAF1 in primary carcinomas were sig- mined as 1.16, 1.22, 0.92, respectively. nificantly low compared with noncancerous tissues (P Ͻ 0.0001). We Loss of XAF1 mRNA Expression in Gastric Cancer Cell Lines. arbitrarily set expression levels less than a half (XAF1, Ͻ0.58; Smac/ We next investigated mRNA expression of XAF1, Smac/DIABLO, and DIABLO, Ͻ0.61; HtrA2, Ͻ0.46) of normal means as abnormally low. HtrA2 in 15 human gastric carcinoma cell lines. As shown in Fig. 1B, On this basis, 23% (20 of 87) of primary tumors were classified as expressions of Smac/DIABLO and HtrA2 transcripts were easily de- abnormally low XAF1 expressors whereas none of the tumors were tectable in all cell lines, and its levels (Smac/DIABLO, 1.00–1.44; identified as abnormal expressors of Smac/DIABLO and HtrA2. Fur- HtrA2, 0.76–1.12) were comparable with those of noncancerous tis- thermore, loss or abnormal reduction of XAF1 was significantly high sues. In contrast, the XAF1 transcript was not detected in four cell in advanced tumors (17 of 62, 27%) compared with early stage tumors lines (SNU1, SNU5, SNU484, and SNU719) and was extremely low (3 of 25, 12%; P ϭ 0.005) and more frequent in poorly differentiated in two cell lines (MKN45 and MKN74). The mRNA expression status tumors (14 of 50, 28%) than well or moderately differentiated tumors of XAF1 in cancer cell lines was further verified by Northern blot [16% (5 of 31) and 17% (1 of 6), respectively; P ϭ 0.043; Fig. 2B]. assay (Fig. 1B). The mean expression level of XAF1 mRNA in gastric However, XAF1 alteration was not associated with histological types cancer cell lines was determined as 0.70, which is significantly low of tumors [diffused, 25% (13 of 52); intestinal, 20% (7 of 35)]. Expression levels of Smac/DIABLO and HtrA2 showed no correlation with histopathological characteristics of tumors (data not shown). Collectively, our results indicate that loss or abnormal reduction of XAF1 is a frequent event in gastric tumorigenesis and may contribute to the malignant progression of human gastric cancers. No Alteration of XIAP Expression in Primary Gastric Carci- nomas and Cell Lines. It was reported previously that XIAP mRNA levels are relatively high in the majority of cancer cell lines, suggesting that high expression of XIAP mRNA coupled with low expression of XAF1 mRNA is a very common characteristic in cancer cells and may provide a survival advantage through the relative increase of XIAP antiapoptotic function (10). This prompted us to investigate whether abnormal overexpression of XIAP is implicated in gastric tumorigenesis. We first analyzed mRNA levels of XIAP in 15 gastric cancer cell lines, but no detectable variation was recognized among specimens (Fig. 3). Moreover, XIAP mRNA levels in cancer cell lines (1.20–1.66) and primary carcinomas (1.18–1.55) were not significantly high compared with normal and benign tumor tissues (1.12– 1.48). To explore the possibility that XIAP is regulated at the post- transcriptional level, XIAP protein levels were analyzed in cancer cell lines using Western blot assay. Consistent with mRNA expression, all of the 15 gastric cell lines exhibited similar levels of XIAP protein, suggesting that overexpression of XIAP might not be a predominant mechanism for the relative increase of XIAP activity in human gastric cancers. Absence of Allelic Deletion of the XAF1 Gene in Gastric Car- Fig. 1. Expression of XAF1, Smac/DIABLO, and HtrA2 transcripts in gastric carcinoma cinomas. The 17p13 region, where the XAF1 gene is located, under- cell lines and tissue specimens. A, quantitative RT-PCR analysis of XAF1, Smac/DIABLO, goes frequent allelic losses in a variety of human malignancies, and HtrA2 expression in normal and benign tumor tissues. PCR was performed using exon-specific primers, and 10 ␮l of the PCR products were resolved on a 2% agarose gel. including gastric cancer (20). Recent microsatellite analysis using the GAPDH was used as an endogenous control. N1–N11, normal gastric tissues; Ad1–Ad3, NCI 60 cell line panel revealed significantly decreased heterozygosity adenomas; Ha1–Ha3, harmatomas; HP1–HP3, hyperplastic polyps. B, expression status at the XAF1 locus, suggesting that allelic loss of XAF1 is prevalent in of XAF1, Smac/DIABLO, and HtrA2 in 15 gastric cancer cell lines. Expression levels of XAF1 mRNA were also confirmed by Northern blot analysis. C, quantitative RT-PCR cancer cell lines (10). To elicit whether loss or abnormal reduction of analysis of XAF1, Smac/DIABLO, and HtrA2 in primary gastric carcinomas. Expression XAF1 mRNA expression in gastric cancers is associated with allelic levels in cancer and adjacent noncancerous tissues (1–5) were compared using matched tissue sets obtained from the same cancer patients. N1–N5, normal tissues; T1–T15, deletion of the gene, we examined the XAF1 gene level using quan- primary tumor tissues. titative genomic PCR. XAF1 is located at the 17p13.2, approximately 7070

Fig. 2. Expression levels of XAF1, Smac/DIABLO, and HtrA2 in primary gastric carcinomas. A, expression levels of three gene transcripts in gastric tissues and carcinoma cell lines. Quanti- tation was achieved by densitometric scanning of RT-PCR products in ethidium bromide-stained gels, and absolute area integrations of the curves representing each specimen were compared after adjustment for GAPDH. Quantitative PCR was repeated at least three times for each specimen, and the means were obtained. Bar, mean expression level of each specimen group; N, normal gastric tissue; BT, benign tumor; T, primary tumor; CL, cell line. B, comparison of XAF1 expression levels between early (E) and advanced (A) tumors, well differentiated (WD), moderately differentiated (MD), and poorly differentiated (PD) tumors, and intestinal (I) and diffused (D) types of tumors.

3cMtothep53 tumor suppressor. Consistent with the previously XAF1 gene. These results, thus, suggest that genomic deletion of reported allelic status of p53, absence or low gene levels of p53 were XAF1 might be infrequent and not associated with abnormal down- detected in nine cell lines harboring homozygous deletion or LOH of regulation of XAF1 mRNA in human gastric cancers. p53 (Fig. 4A; Refs. 21 and 22). However, in contrast to p53, none of To define further the allelic status of the XAF1 gene, we surveyed the 15 cell lines showed detectable reduction of the XAF1 gene level. 48 tumors for LOH of the 17p13.1–13.2 region using three polymor- Moreover, whereas 22 of 87 (25.3%) primary tumors showed marked phic markers: D17S796, D17S1832, and D17S1828. Among 48 reduction of the p53 gene level, none of the tumors, including 20 matched sets tested, 12 (25.0%) were informative at the centromeric abnormal mRNA expressors of XAF1, exhibited reduction of the marker D17S796. LOH at D17S796, which is located approximately 2.5 cM telomeric of the p53 locus, was observed in 4 of 12 (33.3%) informative cases (Fig. 4B). In contrast, all of the 11 tumors that are informative for at least one of two telomeric markers (D17S1832 and D17S1828) were found to retain heterozygosity. Consistent with these results, our quantitative PCR analysis of three marker DNAs demonstrated that the four LOH tumors have relatively low genomic levels of D17S796, compared with the eight retention of heterozygosity tumors, and all tumor specimens examined have normal genomic levels of D17S1832 and D17S1828 (Fig. 4B). Interestingly, however, Fig. 3. Expression status of XIAP in human gastric carcinoma cell lines. For analysis of XIAP mRNA expression, quantitative RT-PCR was performed using exon-specific all four LOH tumors were identified to have normal genomic and primers. GAPDH was used as an endogenous control. For analysis of XIAP protein expression levels of XAF1, whereas two of four tumors exhibited low expression, 30 ␮g of total protein were fractionated using 10% SDS-PAGE and XIAP protein was detected using an anti-XIAP monoclonal antibody and enhanced chemilumi- genomic and mRNA levels of p53, suggesting that LOH at D17S796 nescence. Actin was used as a loading control. might be associated with allelic deletion of p53 but not with XAF1. 7071

82–88% (28–30 sites) and 71–77% (24–26 sites) of the 34 CpGs were partially or completely methylated in four nonexpressor and two low expressor cell lines, respectively, whereas only 18–44% (6–15 sites) of the CpGs were methylated in nine normal expressor cell lines. In addition, completely methylated CpG sites were more frequently observed in four nonexpressors (66 of 136, 48%) than two low expressors (10 of 68, 15%), indicating that methylation extent corre- lates with mRNA expression status. The methylation rate of the 5Ј proximal region (nucleotides Ϫ23 to Ϫ692) was approximately 4-fold higher in abnormal expressors (88.9%, 80 of 90) compared with normal expressors (23.0%, 31 of 135), whereas the regions I and II in abnormal expressors showed only 1.8-fold higher methylation compared with normal expressors (75.4%, 86 of 114 versus 41.5%, 71 of 171). Intriguingly, the methylation status of seven CpGs (numbers 1–7 in Fig. 5B) within nucleotides Ϫ23 to Ϫ234 was most tightly associated with mRNA expression. Whereas all of the seven CpGs were methylated in six abnormal expressor cell lines, none of these CpGs was methylated in nine normal expressor cell lines, suggesting that hypermethylation of CpG sites located in the 5Ј proximal region might be critical for the transcriptional silencing of XAF1. A tight correlation of gene silencing with methylation of the seven CpGs was also recognized in primary tumors. Eighteen of 20 (90%) carcinoma tissues with abnormal expression showed methylation of the seven CpGs, whereas none of the adjacent normal tissues exhibited methyl-

Fig. 4. Quantitative genomic PCR and LOH analyses of XAF1 in gastric carcinoma cell ation (Fig. 6). The seven CpGs were unmethylated in 15 primary lines and tissues. A, genomic levels of XAF1 in gastric cancer cell lines and tumor tissues. tumors with normal expression and five noncancerous tissues in- Exon 6 of XAF1 and exon 8 of p53 were amplified by intron-specific primers. N1–N5, cluded for comparison (data not shown). normal gastric tissues; T1–T15, primary carcinomas. B, no association of LOH at D17S796 with genomic and expression status of XAF1. Microsatellite marker regions Absence of XAF1 Mutations in Gastric Cancers. To evaluate the were amplified using genomic PCR and resolved on 8% polyacrylamide gels. At centro- mutational status of XAF1, we performed RT-PCR–SSCP analysis for meric marker D17S796, four tumor specimens (T7, T11, T19, and T22) exhibited LOH in the tumor DNA (T) compared with its corresponding normal DNA (N). Genomic and 11 XAF1-expressing cell lines and 45 primary carcinomas. The entire mRNA expression levels of XAF1 and p53 were evaluated using quantitative DNA-PCR coding region of XAF1 transcript was amplified using seven sets of and RT-PCR, respectively. exon-specific primers. To improve mutation detection sensitivity, the same RT-PCR products were digested using a different restriction endonuclease(s) and SSCP was performed using two different running Collectively, these observations showed that allelic deletion at the conditions. However, we failed to find any types of mutation leading centromeric region of the XAF1 locus occurs in a subset of gastric to amino acid substitutions or frameshifts in the XAF1 transcripts cancers but might rarely extend into the XAF1 gene. expressed, although 33% (15 of 45) of the same set of primary tumors Aberrant Hypermethylation at the CpG Sites in the XAF1 and 67% (10 of 15) of cancer cell lines were found to carry homozy- Promoter. To investigate whether altered expression of XAF1 is gous deletions or mutations of p53, indicating that somatic mutation associated with promoter hypermethylation of the gene, 15 gastric cell of XAF1 is infrequent in gastric cancers. Ј lines were treated with the demethylating agent 5-aza-2 -deoxycyti- Inverse Correlation between Down-Regulation of XAF1 and dine. As shown in Fig. 5A, XAF1 mRNA expression was reactivated p53 Mutations. Recent studies demonstrated that methylation of in all four nonexpressor cell lines (SNU1, SNU5, SNU484, and p14ARF is associated with the absence of p53 mutations in human SNU719) and significantly increased in two low expressor cell lines colon cancer and p53 acts as an upstream controller of the DNA (MKN45 and MKN74), indicating that XAF1 is transcriptionally methylation apparatus, raising the possibility that hypermethylation in silenced in these cells by DNA hypermethylation. To explore the cancer cells could be associated with the mutational status of p53 (24, relationship between aberrant CpG methylation and gene silencing, 25). In this context, we explored whether epigenetic silencing of we performed bisulfite DNA sequencing analysis of the XAF1 gene XAF1 is associated with p53 alterations in gastric cancer. Mutation promoter. The 5Ј upstream region of the XAF1 gene is not highly enriched in CpGs, and only two short regions (regions I and II in Fig. analysis of p53 revealed that 10 (SNU5, SNU16, SNU216, SNU484, 5B), which encompass only 200 bp (region I, nucleotides Ϫ1248 to SNU601, SNU620, SNU638, MKN1, MKN28, and KATOIII) of 15 Ϫ1447; obs/exp CpG, 0.60; 51.9% GϩC) and 380 bp (region II, cell lines and 15 of 45 primary cancers harbor homozygous deletions nucleotides Ϫ1558 to Ϫ1937; obs/exp CpG, 0.68; 50.3% GϩC), or mutations of p53. Interestingly, loss or down-regulation of XAF1 satisfy the formal criteria for CpG islands (23). Thus, we analyzed the expression was found in 4 (SNU1, SNU719, MKN45, and MKN74) of methylation status of 34 CpGs including 19 CpGs located in regions 5 (80%) cell lines with wild-type p53 but only in 2 (SNU5 and I and II and 14 CpGs located in the 5Ј proximal region (nucleotides SNU484) of 10 (20%) cell lines harboring homozygous deletions or Ϫ23 to Ϫ692). The sequence region (nucleotides Ϫ23 to Ϫ1831) mutations of p53. Similarly, whereas 7 of 9 (78%) primary tumors spanning these 34 CpG sites was amplified by PCR using sodium with altered expression of XAF1 carried wild-type p53,13of15 bisulfite-modified DNA as templates, and 10 PCR clones were se- (87%) primary tumors with mutant p53 showed normal expression of quenced to determine methylation frequency at individual CpG sites XAF1, suggesting that epigenetic silencing of XAF1 by hypermethyl- (complete methylation, 70–100%; partial methylation, 10–60%; un- ation and mutational alteration of p53 might be mutually exclusive methylation, 0%; Fig. 5C). As shown in Fig. 5D, approximately events in human gastric tumorigenesis. 7072

Fig. 5. Epigenetic silencing of XAF1 by promoter hypermethylation. A, reexpression of XAF1 mRNA after treatment with 5-aza- 2Јdeoxycytidine. Six gastric cancer cell lines with no or low XAF1 expression were treated with 5-aza-2Ј-deoxycytidine (5 ␮M) for 4 days, and expression of XAF1 was evaluated by quantitative RT-PCR. C, untreated control; T, treated. B, a map of the CpG sites of the 5Ј upstream region of the XAF1 gene. Thirty-four CpGs (nucleotides Ϫ23 to Ϫ1831) analyzed by bisulfite DNA sequencing are represented by bold vertical lines and numbered 1–34. The first nucleotide of the ATG start codon is indicated by an arrow at ϩ1. C, representative examples of bisulfite genomic sequencing in cancer cell lines. The region comprised 34 CpGs of a XAF1-expressing cell line (SNU638), and a XAF1-nonexpressing cell line (SNU5) was amplified by PCR. The PCR products were cloned, and 10 plasmid clones were sequenced for each cell line. f, methylated CpG; Ⅺ, unmethylated CpG. D, methylation status of 34 CpGs in the XAF1 promoter in 15 gastric cancer cell lines. The percentage of methylation was determined from the number of alleles containing a methylated CpG at each position relative to the total number of alleles analyzed. f, complete methylation (70–100%); u, partial methylation (10–60%); Ⅺ, unmethylation.

Discussion polymorphic markers tested, suggesting that allelic loss of the XAF1 gene might be prevalent in human cancers. However, no matched In the present study, we demonstrate first that a substantial fraction controls for these cell lines were available for LOH assay and none of of gastric cancer cell lines and primary carcinomas express no or the cell lines tested showed any gross rearrangements of the XAF1 extremely low levels of XAF1 transcript whereas two other IAP gene by Southern blot analysis. Therefore, the question remains as to antagonists, Smac/DIABLO and HtrA2, are normally expressed in all whether loss or reduction of XAF1 mRNA expression in human cancer specimens examined. Loss or down-regulation of XAF1 ex- cancer cells is caused by other transcriptional regulatory mechanisms pression was strongly associated with aberrant CpG methylation in the promoter region. Moreover, abnormal expression of XAF1 correlated rather than homozygous or allelic deletion of the gene. In the present with advanced stage and high grade of tumors, suggesting that epi- study, our quantitative genomic PCR and LOH analyses indicated that genetic silencing of XAF1 because of promoter hypermethylation allelic deletion at the XAF locus rarely extends into the XAF1 gene. might contribute to the malignant progression of human gastric Interestingly, we found that XAF1 expression is reactivated in all cancers. nonexpressor cell lines and significantly elevated in low expressor cell Despite the possible role for XAF1 in the suppression of malig- lines after 5Ј-aza-2-deoxycytine treatment, suggesting that loss or nancy, the mechanism by which XAF1 expression is down-regulated down-regulation of XAF1 expression in cancer cells might be caused in human cancers remains to be characterized. Recent microsatellite by an epigenetic mechanism such as aberrant DNA methylation. Our analysis using the NCI 60 cell line panel revealed significantly de- bisulfite DNA sequencing analysis revealed that methylation extent in creased heterozygosity within the XAF1 region at 17p13.2 (10). Of the the promoter region is closely associated with the mRNA expression 58 cell lines tested, 22 were shown to be homozygous at all three status of the XAF1 gene in both cancer cell lines and tumor tissues. 7073

Ϫ234) most tightly correlate with gene silencing. This observation leads to the conjecture that hypermethylation of the regions I and II does not act so critical for gene silencing as CpG island hypermethylation but has an inhibitory effect on XAF1 transcription. This is in line with a recent study demonstrating that silencing of caspase 8 expression in cancer cells is related to hypermethylation of the 5Ј non-CpG island region of the gene (28). Collectively, our findings suggest that hypermethylation of the 5Ј non-CpG island region of the gene is critical for the transcriptional silencing of the XAF1 gene in human gastric cancers. It was reported previously that XIAP mRNA levels are relatively high in the majority of cancer cell lines, raising the hypothesis that the relative increase of XIAP to XAF1 expression in cancer cells may contribute to the development of the transformed phenotype by sup- pressing apoptotic signaling (10). In this study, however, we could not observe any detectable increase of XIAP mRNA expression in gastric cancer cell lines and primary tumors compared with normal tissues. Western blot analysis of XIAP in 15 gastric cancer cell lines also showed that protein levels of XIAP are not variable among specimens and are comparable with mRNA expression patterns. This observation, thus, suggest that overexpression of XIAP mRNA is not a frequent event and may not be implicated in the relative increase of XIAP to XAF1 in human gastric cancers. Very recently, XAF1 was identified as a novel IFN-stimulated gene that contributes to IFN-␤-dependent sensitization of cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis (29). XAF1 mRNA was up-regulated by IFN-␣ and -␤, and high levels of XAF1 protein were induced predominantly in cell lines sensitive to the proapoptotic effects of IFN-␤. It was also observed that A375 melanoma cells expressing XAF1 constitutively are more sensitive to tumor necrosis factor-related apoptosis-inducing ligand-induced apo- Fig. 6. Methylation status of CpG sites in the XAF1 promoter region of primary gastric ptosis compared with empty vector-transfected cells and the degree of carcinomas. A, representative bisulfite DNA sequencing data of primary cancers. The sensitization by XAF1 is correlated with the level of XAF1 expressed. promoter region (nucleotides Ϫ23 to Ϫ234) comprised seven CpGs of T3 (no expression) ␣ ␤ ␥ and T28 (low expression) and their adjacent normal tissues (N3 and N28) was amplified Previous studies demonstrated that IFN- ,- , and - induce apoptosis by PCR. The PCR products were cloned, and 10 plasmid clones were sequenced for each and/or growth arrest of gastric cancer cells and a combination of tissue specimen. B, methylation status of the seven CpGs in primary gastric cancers and IFN-␣ or -␤ with 5-fluorouracil resulted in an additive or synergistic their matched normal tissues. N, normal tissues; T, tumor tissues; f, complete methylation (70–100%); u, partial methylation (10–60%); Ⅺ, unmethylation. effect against clinical gastric carcinomas (30, 31). However, some gastric cancer cells are resistant to IFN-induced growth arrest and apoptosis, leading to the conjecture that a defective response to Aberrant CpG methylation was also found in many different types of growth suppression effect of IFNs may give the tumors a selective human cell lines including lung, bladder, kidney, and prostate cancers, advantage and abet escape from T-cell antitumor response. In this indicating that transcriptional silencing of XAF1 by promoter hyper- context, it could be suspected that gastric cancers with loss or abnor- methylation is not a gastric cancer-specific phenomenon (data not mal reduction of XAF1 might be more resistant to IFN therapy than shown). cancers with normal XAF1 expression. On this basis, it will be It has been demonstrated extensively that hypermethylation in valuable to examine that expression status of XAF1 could be a CpG-rich promoter or exonic region is strongly associated with tran- clinically useful marker for cancer treatment including IFN therapy. scriptional silencing. CpG islands are more methylated in cancers In conclusion, our data presented here clearly demonstrate that compared with the non-CpG island region, and hypermethylation at XAF1 undergoes epigenetic silencing in a considerable proportion of CpG islands in the transcription regulatory region is a critical event gastric cancer cell lines and primary carcinomas by aberrant CpG site leading to the epigenetic inactivation of tumor suppressor genes in hypermethylation of the gene promoter. Loss or abnormal reduction of human tumorigenesis (26). In this context, it should be noted that the XAF1 mRNA expression showed a strong correlation with tumor 5Ј upstream region of the XAF1 gene is not highly enriched in CpGs. stage and grade, suggesting that XAF1 inactivation might contribute to Within the 5-kb upstream region, only two short regions (regions I and the malignant progression of human gastric cancers. Although addi- II in Fig. 5B; 200 bp; obs/exp CpG, 0.60; 51.9% GϩC; and 380 bp; tional studies are required to characterize the biological significance obs/exp CpG, 0.68; 50.3% GϩC, respectively) were identified to of XAF1 inactivation in gastric tumorigenesis, our study suggests that satisfy the formal criteria for CpG islands (23). However, these aberrant hypermethylation of the XAF1 gene could be a molecular regions (Ϫ1867 to Ϫ1343; 54.4% GϩC; obs/exp CpG, 0.60) do not marker for detection and treatment of human gastric cancers. fulfill the more rigorous description of a CpG island (Ն500 bp; GϩC, Ͼ55%; obs/exp CpG, Ͼ0.65) proposed by Takai and Jones (27). In References addition, in cancer cells lacking XAF1 expression, the 5Ј upstream region (nucleotides Ϫ23 to Ϫ692) are more significantly methylated 1. Thompson, C. B. Apoptosis in the pathogenesis and treatment of disease. Science (Wash. DC), 267: 1456–1462, 1995. compared with regions I and II, and aberrant methylation of the seven 2. Deveraux, Q. L., and Reed, J. C. IAP family proteins-suppressors of apoptosis. Genes CpG sites located in the 5Ј proximal region (nucleotides Ϫ23 to Dev., 13: 239–252, 1999. 7074

3. Miller, L. K. An exegesis of IAPs: salvation and surprises from BIR motifs. Trends 18. Fuchs, C. S., and Mayer, R. J. Gastric carcinoma. N. Engl. J. Med., 333: 32–41, 1995. Cell Biol., 9: 323–328, 1999. 19. Werner, M., Becker, K. F., Keller, G., and Hofler, H. Gastric adenocarcinoma: 4. Tamm, I., Wang, Y., Sausville, E., Scudiero, D. A., Vigna, N., Oltersdorf, T., and pathomorphology and molecular pathology. J. Cancer Res. Clin. Oncol., 127: 207– Reed, J. C. IAP-family protein survivin inhibits caspase activity and apoptosis 216, 2001. induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res., 58: 20. Yustein, A. S., Harper, J. C., Petroni, G. R., Cummings, O. W., Moskaluk, C. A., and 5315–5320, 1998. Powell, S. M. Allelotype of gastric adenocarcinoma. Cancer Res., 59: 1437–1441, 5. Sasaki, H., Sheng, Y., Kotsuji, F., and Tsang, B. K. Down-regulation of X-linked 1999. inhibitor of apoptosis protein induces apoptosis in chemoresistant human ovarian 21. Kim, J. H., Takahashi, T., Chiba, I., Park, J. G., Birrer, M. J., Roh, J. K., Lee, H., Kim, cancer cells. Cancer Res., 60: 5659–5666, 2000. J. P., Minna, J. D., and Gazdar, A. F. Occurrence of p53 gene abnormalities in gastric 6. Deveraux, Q. L., Takahashi, R., Salvesen, G. S., and Reed, J. C. X-linked IAP is a carcinoma tumors and cell lines. J. Natl. Cancer Inst., 83: 938–943, 1991. direct inhibitor of cell-death proteases. Nature (Lond.), 388: 300–304, 1997. 22. Yokozaki, H. Molecular characteristics of eight gastric cancer cell lines established in 7. Roy, N., Deveraux, Q. L., Takahashi, R., Salvesen, G. S., and Reed, J. C. The c-IAP-1 Japan. Pathol. Int., 50: 767–777, 2000. and c-IAP-2 proteins are direct inhibitors of specific caspases. EMBO J., 16: 6914– 6925, 1997. 23. Gardiner-Garden, M., and Frommer, M. CpG islands in vertebrate genomes. J. Mol. 8. Holcik, M., Gibson, H., and Korneluk, R. G. XIAP: apoptotic brake and promising Biol., 196: 261–282, 1987. therapeutic target. Apoptosis, 6: 253–261, 2001. 24. Shen, L., Kondo, Y., Hamilton, S. R., Rashid, A., and Issa, J. P. P14 methylation in 9. Holcik, M., and Korneluk, R. G. XIAP, the guardian angel. Nat. Rev. Mol. Cell. Biol., human colon cancer is associated with microsatellite instability and wild-type p53. 2: 550–556, 2001. Gastroenterology, 124: 626–633, 2003. 10. Fong, W. G., Liston, P., Rajcan-Separovic, E., St. Jean, M., Craig, C., and Korneluk, 25. Nasr, A. F., Nutini, M., Palombo, B., Guerra, E., and Alberti, S. Mutations of TP53 R. G. Expression and genetic analysis of XIAP-associated factor 1 (XAF1) in cancer induce loss of DNA methylation and amplification of the TROP1 gene. Oncogene, cell lines. Genomics, 70: 113–122, 2000. 22: 1668–1677, 2003. 11. Tamm, I., Kornblau, S. M., Segall, H., Krajewski, S., Welsh, K., Kitada, S., Scudiero, 26. Ehrlich, M., Jiang, G., Fiala, E., Dome, J. S., Yu, M. C., Long, T. I., Youn, B., Sohn, D. A., Tudor, G., Qui, Y. H., Monks, A., Andreeff, M., and Reed, J. C. Expression O. S., Widschwendter, M., Tomlinson, G. E., Chintagumpala, M., Champagne, M., and prognostic significance of IAP-family genes in human cancers and myeloid Parham, D., Liang, G., Malik, K., and Laird, P. W. Hypomethylation and hyper- leukemias. Clin. Cancer Res., 6: 1796–1803, 2001. methylation of DNA in Wilms tumors. Oncogene, 21: 6694–6702, 2002. 12. Du, C., Fang, M., Li, Y., Li, L., and Wang, X. Smac, a mitochondrial protein that 27. Takai, D., and Jones, P. A. Comprehensive analysis of CpG islands in human promotes cytochrome c-dependent caspase activation by eliminating IAP inhibition. chromosomes 21 and 22. Proc. Natl. Acad. Sci. USA, 99: 3740–3745, 2002. Cell, 102: 33–42, 2000. 28. Harada, K., Toyooka, S., Shivapurkar, N., Maitra, A., Reddy, J. L., Matta, H., 13. Verhagen, A. M., Ekert, P. G., Pakusch, M., Silke, J., Connolly, L. M., Reid, G. E., Miyajima, K., Timmons, C. F., Tomlinson, G. E., Mastrangelo, D., Hay, R. J., Moritz, R. L., Simpson, R. J., and Vaux, D. L. Identification of DIABLO, a Chaudhary, P. M., and Gazdar, A. F. Deregulation of caspase 8 and 10 expression in mammalian protein that promotes apoptosis by binding to and antagonizing IAP pediatric tumors and cell lines. Cancer Res., 62: 5897–5901, 2002. proteins. Cell, 102: 43–53, 2000. 29. Leaman, D. W., Chawla-Sarkar, M., Vyas, K., Reheman, M., Tamai, K., Toji, S., and 14. Suzuki, Y., Imai, Y., Nakayama, H., Takahashi, K., Takio, K., and Takahashi, R. A Borden, E. C. Identification of X-linked inhibitor of apoptosis-associated factor-1 as serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death. Mol. Cell, 8: 613–621, 2001. an interferon-stimulated gene that augments TRAIL Apo2L-induced apoptosis. 15. Wu, G., Chai, J., Suber, T. L., Wu, J. W., Du, C., Wang, X., and Shi, Y. Structural J. Biol. Chem., 277: 28504–28511, 2002. basis of IAP recognition by Smac/DIABLO. Nature (Lond.), 408: 1008–1012, 2000. 30. Shyu, R., Su, H., Yu, J., and Jiang, S. Direct growth suppressive activity of ␣ ␥ 16. Liu, Z., Sun, C., Olejniczak, E. T., Meadows, R. P., Betz, S. F., Oost, T., Herrmann, interferon- and - on human gastric cancer cells. J. Surg. Oncol., 75: 122–130, J., Wu, J. C., and Fesik, S. W. Structural basis for binding of Smac/DIABLO to the 2000. XIAP BIR3 domain. Nature (Lond.), 408: 1004–1008, 2000. 31. Kubota, T., Kase, S., Otani, Y., Watanabe, M., Teramoto, T., and Kitajima, M. 17. Liston, P., Fong, W. G., Kelly, N. L., Toji, S., Miyazaki, T., Conte, D., Tamai, K., Interferons ␣-2a and ␤ increase the antitumor activity, detected by MTT assay, of Craig, C. G., McBurney, M. W., and Korneluk, R. G. Identification of XAF1 as an 5-fluorouracil against experimental and clinical human gastrointestinal carcinomas. antagonist of XIAP anti-caspase activity. Nat. Cell Biol., 3: 128–133, 2001. Anticancer Res., 17: 725–728, 1997.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2003 American Association for Cancer Research. Hypermethylation of XIAP-associated Factor 1, a Putative Tumor Suppressor Gene from the 17p13.2 Locus, in Human Gastric Adenocarcinomas

Do-Sun Byun, Kyucheol Cho, Byung-Kyu Ryu, et al.

Cancer Res 2003;63:7068-7075.

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