Vol. 11, 473–482, January 15, 2005 Clinical Cancer Research 473 Identification of Gastric Cancer–Related Genes Using a cDNA Microarray Containing Novel Expressed Sequence Tags Expressed in Gastric Cancer Cells Jeong-Min Kim,1,5 Ho-Yong Sohn,4 overexpressed in z68% of tissues and the MT2A gene Sun Young Yoon,1 Jung-Hwa Oh,1 Jin Ok Yang,1 was down-expressed in 72% of the tissues. Western blotting and immunohistochemical analyses for CDC20 and SKB1 Joo Heon Kim,2 Kyu Sang Song,3 Seung-Moo Rho,2 1 1 5 showed overexpression and localization changes of the Hyan Sook Yoo, Yong Sung Kim, Jong-Guk Kim, corresponding protein in human gastric cancer tissues. 1 and Nam-Soon Kim Conclusions: Novel genes that are related to human 1Genome Research Center, Korea Research Institute of Bioscience and gastric cancer were identified using cDNA microarray Biotechnology; 2Department of Pathology, Eulji University School of 3 developed in our laboratory. In particular, CDC20 and Medicine; and Department of Pathology, College of Medicine, MT2A represent a potential biomarker of human gastric Chungnam National University, Daejeon, Korea; 4Department of Food and Nutrition, Andong National University, Andong, Korea; and cancer. These newly identified genes should provide a 5Department of Microbiology, College of Natural Sciences, Kyungpook valuable resource for understanding the molecular mecha- National University, Daegu, Korea nism associated with tumorigenesis of gastric carcinogenesis and for the discovery of potential diagnostic markers of gastric cancer. ABSTRACT Purpose: Gastric cancer is one of the most frequently INTRODUCTION diagnosed malignancies in the world, especially in Korea and Japan. To understand the molecular mechanism associated Gastric cancer is one of the most frequently diagnosed with gastric carcinogenesis, we attempted to identify novel malignancies in the world (1). It is particularly prevalent in gastric cancer–related genes using a novel 2K cDNA micro- Korea and Japan and is one of the leading causes of cancer death array. in these regions (2). Although the incidence and mortality have Experimental Design: A 2K cDNA microarray was been decreasing during the last several years, gastric cancer still fabricated from 1,995 novel expressed sequence tags (ESTs) has a notorious position, with the first incidence and the second showing no hits or a low homology with ESTs in public cause of mortality in Korea (3). databases from our 143,452 ESTs collected from gastric Advances in diagnostic and treatment technologies have cancer cell lines and tissues. An analysis of the gene expression enabled us to offer excellent long-term survival results for early for human gastric cancer cell lines to a normal cell line was gastric cancer, but the prognosis of advanced gastric cancer still done using this cDNA microarray. Data for the different remains poor (4). Recent molecular analyses revealed that gastric expressed genes were verified using semiquantitative reverse cancers are closely related to genetic alterations in several genes, transcription-PCR, Western blotting, and immunohistochem- such as p53, APC, E-cadherin, b-catenin, TGF-a, c-met, trefoil ical staining in the gastric cell lines and tissues. factor 1, and Runx3 (5–7). However, the common pathways of Results: Forty genes were identified as either up- carcinogenesis and the subsequent progression of gastric cancer regulated or down-regulated genes in human gastric cancer remained to be elucidated. cells. Among these, genes such as SKB1, NT5C3, ZNF9, A cDNA microarray was used to simultaneously study p30, CDC20, and FEN1, were confirmed to be up-regulated the expression profiles of a number of genes at specific genes in nine gastric cell lines and in 25 pairs of tissue conditions in a single hybridization (8, 9). Many reports on samples from patients by semiquantitative reverse tran- gene expression profiles of various cancers and diseases using scription-PCR. On the other hand, genes such as MT2A cDNA microarray techniques have been reported (10–14). and CXX1 were identified as down-regulated genes. In Among them, changes in gene expression in gastric cancer particular, the SKB1, CDC20,andFEN1 genes were cell lines and malignant tissues have been reported. In gastric adenocarcinomas, genes such as S100A4, CDK4, MMP1, and b-catenin genes have been reported as being up-regulated genes, the GIF gene was reported to be a down- Received 4/20/04; revised 9/25/04; accepted 10/5/04. regulated gene (15). Ji et al. (16) has also reported the first Grant support: 21C Frontier Functional Human Genome Project from comprehensive review of gene expression patterns in gastric the Ministry of Science and Technology of Korea. cancer cell lines on a genomic scale. In this study, they The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked analyzed global gene expression patterns of 27 human cell advertisement in accordance with 18 U.S.C. Section 1734 solely to lines, including 12 gastric carcinoma cell lines and compared indicate this fact. heterogeneity between gastric cancer cell lines. In addition, a Requests for reprints: Nam-Soon Kim, Laboratory of Human Genomics, comparison of the gastric cancer–related genes using gastric Genome Research Center, Korea Research Institute of Bioscience and Biotechnology, P.O. Box 115, Yusong, Daejeon, Korea. Phone: 82-42- cancer tissues and surrounding gastric mucosa tissues has been 879-8112; Fax: 82-42-879-8119; E-mail: [email protected]. reported, as well as a connection between the clinical D2005 American Association for Cancer Research. phenotypes of patients (17). Downloaded from clincancerres.aacrjournals.org on September 26, 2021. © 2005 American Association for Cancer Research. 474 Identification of Gastric Cancer–Related Novel Genes In a previous study, we collected an entire set of genes that The novelty of these ESTs were reanalyzed by a BLAST are expressed in gastric cancer cell lines or tissues using full- search against human mRNA (Genbank release 138.0, down- length enriched cDNA libraries, subtracted cDNA libraries, and loaded on Dec. 2003), RefSeq (downloaded on Dec. 2003) under normalized cDNA libraries from gastric cancer cell lines and conditions of an identity of >90% for >50 bp with E V 1 Â tissues from Korean patients and identified the genes associated 10À20. The remaining ESTs were analyzed by a BLAT search with gastric cancer by examining their expression profiles (18). against the human genome database (University of California Using this process for identifying novel gastric cancer-related Santa Cruz6 Golden Path genome database build 15) under the genes in which there were no hits or a low homology with known above conditions. Analysis of the ESTs that were not included in genes in public databases, we isolated 1,995 novel genes from the the above searches were done under conditions of an identity of collected gastric expressed sequence tags (ESTs) and fabricated a >90% for >50 bp with E =1Â 10À20 to 1 Â 10À3 against human cDNA microarray containing these genes. However, some of the mRNA and RefSeq databases and with E V 1 Â 10À1 against the ESTs were identified as known genes in recent updated public NR database (downloaded on Dec. 2003). databases. Using the cDNA microarray, a gene expression anal- ysis of these genes in gastric cancer cell lines and tissues was Fabrication and Hybridization of cDNA Microarray done. Here, we report on the identification of novel genes that are Clones containing the novel ESTs were grown in 96-well differentially expressed in gastric cancer cell lines and tissues. culture plates and plasmid DNAs were purified using a Millipore plasmid kit (Millipore Co., Bedford, MA). The inserts of cDNAs MATERIALS AND METHODS using purified plasmid DNAs were amplified by PCR with the sense primer 5V-GCAGAGCTCTCTGGCTAAC-3V, which is Cell Culture, Tissues, and RNA Preparation localized in the vector region and the antisense primer 5V- Human gastric cancer cell lines, SNU-1, SNU-16, SNU- CGTGCGGCCGCT21(G/A/C)-3V. After purifying the PCR 216, SNU-484, SNU-601, SNU-638, SNU-668, and SNU-719 products on Sephadex G-50 Superfine (Amersham Pharmacia were cultured in RPMI 1640 (Life Technologies, Grand Island, Biotech AB, Uppsala, Sweden), they were suspended in a NY) and human normal gastric cell lines Hs 677.St (ATCC CRL- Microspotting solution (ArrayItTM Brand Products, TeleChem, 7407) in DMEM (Life Technologies) supplemented with 10% Sunnyvale, CA) and spotted on CSS-100 Silyated Slides inactivated fetal bovine serum, 2 mg/mL sodium bicarbonate, (Aldehyde; CEL Associates, Pearland, TX) using a Carte- and 1% antibiotic-antimycotic solution (Invitrogen Life Tech- sian Prosys 5510 robot (Cartesian, Inc., Irvine, CA) with 32 nologies, Carlsbad, CA). The Hs 677.St cell line was derived printing tips. Our cDNA microarray contained a total of 6,912 from normal fetal stomach tissue and had a morphology similar spots in one slide including triplicates of 1,995 cDNA, control j to a fibroblast. All cultured cells were incubated at 37 Cina genes of GAPDH and b-actin, and empty spots for negative humidified incubator maintained with a 5% CO2 atmosphere controls. (19, 20). When the cells were about 80% to 90% confluent, they Twenty micrograms of total RNA from a normal cell line or were harvested and used for total RNA isolation. Fifty gastric cancer cell lines, respectively, were used in the cDNA micro- tissues containing the tumor and normal regions of 25 gastric array analysis. RNA of the normal cell line, labeled with Cy3, cancer patients were obtained from the College of Medicine, was used as a reference versus RNA with Cy5 from each of eight Chungnam National University, Korea with informed consent. cancer cell lines as a sample. Probe labeling and hybridization The tumors were staged according to tumor-node-metastasis were done using a 3DNA Array 50 kit (Genisphere, Inc., classification of Union Internationale Contre le Cancer.