Purpose: A major obstacle in chemotherapy is treatment failure due to anticancer drug resistance. The emergence of acquired resistance results from host factors and genetic or epigenetic changes in the cancer cells. The purpose of this study was to identify differentially expressed genes associated with acquisition of resistance in human gastric cancer cells.

Experimental Design: We performed global gene expression analysis in the acquired drug-resistant gastric cancer cell lines to the commonly used drugs 5-fluorouracil, doxorubicin, and cisplatin using Affymetrix HG-U133A microarray. The gene expression patterns of 10 chemoresistant gastric cancer cell lines were compared with those of four parent cell lines using fold-change and Wilcoxon’s test for data analysis.

Results: We identified over 250 genes differentially expressed in 5-fluorouracil-, cisplatin-, or doxorubicin-resistant gastric cancer cell lines. Our expression analysis also identified eight multidrug resistance candidate genes that were associated with resistance to two or more of the tested chemotherapeutic agents. Among these, midkine (MDK), a heparin-binding growth factor, was overexpressed in all drug-resistant cell lines, strongly suggesting that MDK might contribute to multidrug resistance in gastric cancer cells.

Conclusions: Our investigation provides comprehensive gene information associated with acquired resistance to anticancer drugs in gastric cancer cells and a basis for additional functional studies.

Gastric cancer is one of the most common cancers worldwide. Although the occurrence rate of gastric cancer has decreased, Asian countries such as Korea, China, and Japan, and some European and South American countries still have a high incidence of the disease (1, 2). Many chemotherapeutic agents have been used to treat gastric cancer patients, but the emergence of drug resistance has prevented successful treatment in many cases. The two major forms of drug resistance are intrinsic resistance, in which previously untreated tumor cells are inherently insensitive to the chemotherapeutic agent, and acquired resistance, in which treated tumor cells become insensitive after drug exposure (3). To date, many research groups have studied the various mechanisms of drug resistance, hoping to overcome this major obstacle in chemotherapy. Researchers have determined that acquired drug resistance is multifactorial, in that it involves host factors and genetic and epigenetic changes, as well as numerous molecular events (4). The resistance itself may be due to decreased drug accumulation, alteration of intracellular drug distribution, reduced drug-target interaction, increased detoxification response, cell-cycle deregulation, increased damaged-DNA repair, and reduced apoptotic response (5). However, although researchers believe that multiple factors participate in chemoresistance, most studies have focused on a limited number of candidate genes. For example, it has been well known that overexpression of the multidrug resistance gene (MDR1) is associated with cancer cells that have drug resistance. However, little is known about the genes differentially expressed in a variety of drug-resistant cancer cells, especially in gastric cancer (6). It is hoped that the recently developed techniques for genome-wide expression analysis will provide additional information, novel candidate genes associated with cancer drug resistance, and perhaps new therapeutic targets.

Microarray technologies have been widely used for comprehensive gene expression analysis as well as mutation and single nucleotide polymorphism detection (7, 8, 9, 10, 11, 12, 13, 14). In particular, large-scale microarray analysis of gene expression enables researchers to analyze simultaneous changes in thousands of genes and identify significant patterns. Because the genome-wide expression analysis in a variety of drug-resistant gastric cancer cells has not yet been performed, we have used the recently developed Affymetrix HG-U133A high-density oligonucleotide microarray for analysis of the global gene expression.

The drugs 5-fluorouracil (5-FU), doxorubicin, and cisplatin are widely used in the treatment of various malignancies: 5-FU is a well-known antimetabolite that acts as a thymidylate synthase inhibitor (3); doxorubicin targets topoisomerase II by interfering with the catalytic cycle (15); and cisplatin intercalates into DNA, leading to DNA damage in the cancer cells (16). In this study, we have examined genes that are differentially expressed in 5-FU-, doxorubicin-, or cisplatin-induced chemoresistant gastric cancer cell lines, as compared with their drug-sensitive parent cell lines. We identified genes showing altered expression in resistant cell lines, as well as several potential multidrug resistance candidate genes that were associated with resistance to two or more of the chemotherapeutic agents.

Cell Lines and Cell Culture.

Four 5-FU-resistant gastric cancer cell lines (SNU-620R-5-FU/1000; SNU-638R-5-FU/50000; SNU-668R-5-FU/4000; SNU-719R-5-FU/600), 3 doxorubicin-resistant cell lines (SNU-620R-DOX/300; SNU-668R-DOX/50; SNU-719R-DOX/100), and 3 cisplatin-resistant cell lines (SNU-620R-CIS/2000; SNU-638R-CIS/400; SNU-668R-CIS/400) were created from four different gastric carcinoma cell lines (SNU-620; SNU-638; SNU-668; SNU-719) established by Park et al.(17). All 14 cell lines were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 20 mm HEPES, and 100 units/ml penicillin-streptomycin (Invitrogen, Carlsbad, CA) and the indicated concentrations of drugs in Table 1 (5-FU; doxorubicin; cisplatin) in a humidified incubator at 37°C in an atmosphere of 5% CO2 until 80–90% confluence was achieved.

Drugs and Selection of Drug-Resistant Cells.

5-FU was purchased from Choongwae Pharma Corp. (Gyeonggi, Korea), doxorubicin from Dong-A Pharmaceutical Co. Ltd. (Seoul, Korea), and cisplatin from Ildong Pharmaceutical Co. Ltd. (Seoul, Korea). For production of the resistant cell lines, the four parent gastric cancer cells were initially exposed to the various drugs at concentrations indicated by the respective IC50 values determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (described below). The drug concentration was increased 2–4-fold after at least 8 weeks of continuous drug exposure. Fresh drugs were added by gradually increasing to the final concentrations shown in Table 1. Stable, drug-resistant cell lines were selected and cultured in the presence of the final drug concentrations. The establishment period of the drug-resistant cell lines varied from 11 months to 2 years. Before microarray experiments, cells were maintained for 1 week without drugs to eliminate acute effects.

MTT Assay.

Sensitivities of the drug-resistant and parent cell lines to 5-FU, doxorubicin, and cisplatin were determined by MTT assay as described previously (18). Briefly, single-cell suspensions were obtained by pipette disaggregation of the floating cells or by trypsinization of monolayer cultures. The number of cells plated into 96 wells was determined after preliminary cell growth studies so that untreated cells were in exponential growth phase at the time of initial harvest and at the end of the 4-day incubation. Equal number of cells was inoculated into each well in RPMI 1640 supplemented with 10% fetal bovine serum. For each drug, 5–10 concentrations were used, covering a 3–5-log concentration range that was chosen to span the 50% inhibitory concentration determined by preliminary assays. After 4 days of culture, MTT (Sigma Chemical Co., St. Louis, MO) was added to each well and was incubated at 37°C for an additional 4 h. The medium was aspirated from plates leaving about 30 μl of medium in each well. Care was taken not to disturb the formazan crystals at the bottom of the wells. One hundred fifty μl of DMSO was added to each well, and the plates were placed on a shaker for 15 min to solubilize the formazan crystals. The plates were then read immediately at 540 nm on a scanning multiwell spectrophotometer (ELISA reader; Biotek Instruments Inc., Burlington, VT). All of the data points represent the mean value of a minimum of six wells. Table 1 shows the IC50 representing the drug concentration resulting in 50% growth inhibition and the relative resistance calculated from the ratio of the IC50 of the drug-resistant cell lines versus that of the parent cell lines.

RNA Preparation and Affymetrix GeneChip Hybridization.

Total RNA was extracted using the Trizol reagent (Life Technologies, Inc. Carlsbad, CA) according to the manufacturer’s instructions. Genes expressed in 10 drug-resistant cell lines and their parent cell lines were analyzed on a high-density oligonucleotide microarray (HG-U133A; Affymetrix, Santa Clara, CA) containing 22,282 transcripts. Target preparation and microarray processing procedures were performed as described in the Affymetrix GeneChip Expression Analysis Manual (Affymetrix, Santa Clara, CA). Briefly, the extracted total RNA was purified with an RNeasy kit (Qiagen, Valencia, CA). Twenty μg of total RNA was used to synthesize double-strand cDNA with SuperScript II reverse transcriptase (Life Technologies, Inc. Rockville, MD) and a T7-(dT)24 primer (Metabion, Germany). Then, biotinylated cRNA was synthesized from the double-stranded cDNA using the RNA Transcript Labeling kit (Enzo Life Sciences, Farmingdale, NY) and was purified and fragmented. The fragmented cRNA was hybridized to the oligonucleotide microarray, which was washed and stained with streptavidin-phycoerythrin. Scanning was performed with an Agilent Microarray Scanner (Agilent Technologies, Palo Alto, CA).

Data Analysis.

GeneChip analysis was performed based on the Affymetrix GeneChip Manual (Affymetrix Inc., Santa Clara, CA) with Microarray Analysis Suite (MAS) 5.0, Data Mining Tool (DMT) 2.0, and Microarray Database software. All of the genes represented on the GeneChip were globally normalized and scaled to a signal intensity of 500. Fold changes were calculated by comparing transcripts between parent and acquired drug-resistant cell lines. The Microarray Analysis Suite software used the Wilcoxon’s test to generate detected (present or absent) and changed (increased or decreased) calls, and used the calls to statistically determine whether a transcript was expressed or not, and whether it was relatively increased, decreased, or unchanged. After being filtered through a “present” call (p < 0.05), a transcript was considered differentially expressed when it satisfied one of the following two conditions: 1) by fold change, transcripts increased or decreased >1.5-fold; 2) by one-sided Wilcoxon’s rank test, transcripts’ average fold change exceeded 1.5, with an “increased” (p < 0.003) or “decreased” (p > 0.997) call. In the case of 5-FU, all transcripts meeting the above conditions in at least three of four cell lines were considered differentially expressed. Hierarchical clustering and dendrogram figures were generated using Cluster and TreeView software (http://rana.stanford.edu). A three-dimensional graph of the multidimensional scaling was created using SPSS (SPSS Inc., Chicago, IL) and SigmaPlot (SPSS Inc.). To evaluate the statistical significance of eight genes differentially expressed in two or three of the drug subsets, paired t test was performed using SPSS (SPSS Inc.).

Quantitative and Semiquantitative RT-PCR.

We selected two genes for real-time quantitative reverse transcription (RT)-PCR and seven genes for semiquantitiative RT-PCR for validation of the microarray data. Five μg of total RNA was used for creation of single-stranded cDNA using the SuperScript Preamplification System for First Strand cDNA Synthesis (Life Technologies, Inc., Rockville, MD). The cDNA was diluted and quantitatively equalized for PCR amplification. For real-time quantitative RT-PCR of MDK (midkine) and BIRC5, TaqMan PCR method using a 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA) was performed according to the manufacturer’s instructions. We used the primers and probes provided as Assays-on-Demand Gene Expression Products (Applied Biosystems). The expression of seven genes (MDK, FOSB, NQO1, DDB2, ITGB4, ABCC1, and IGFBP2) was verified by semiquantitative RT-PCR. The primer sets for PCR amplification were as follows: MDK forward 5′-ATGCAGCACCGAGGCTTCCT-3′, reverse 5′-ATCCAGGCTTGGCGTCTAGT-3′; FOSB forward 5′-GAGAGGGGAAGAGACAAAGT-3′, reverse 5′-CTTCATCCTCACACAGGACT-3′; NQO1 forward 5′-TGGAGAATATTTGGGATGAG-3′, reverse 5′-AATCCAGGCTAAGGAATCTC-3′; DDB2 forward 5′-GGAGATATCATGCTCTGGAA-3′, reverse 5′-GGCTACTAGCAGACACATCC-3′; ITGB4 forward 5′-TTCCAAATCACAGAGGAGAC-3′, reverse 5′-CTTGAGGTTGTCCAGATCAT-3′; ABCC1 forward 5′-CTGACAAGCTAGACCATGAATGT-3′, reverse 5′-TCACACCAAGCCGGCGTCTTT-3′; and IGFBP2 forward 5′- TTCCAGTTCTGACACACGTA-3′, reverse 5′-GACACAGGGGTTCAAAAATA -3′.

PCR was carried out with 1 μl of cDNA as follows: initial denaturation at 94°C for 5 min followed by 25–30 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 1 min, followed by a final elongation at 72°C for 7 min.

Western Blot Analysis.

To investigate the correspondence between mRNA and protein of MDK, we performed Western blot analysis in SNU-620, SNU-620R-5-FU/1000, SNU-620R-CIS/2000, SNU-638, and SNU-638R-CIS/400. MDK protein was detected with an MK antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Western blot analysis was performed as described previously (19).

Gene Selection from Microarray Data Analysis.

Because the high-density oligonucleotide microarray contains a large number of probes, two different statistical analysis methods were used in parallel to select genes that were differentially expressed in drug-resistant gastric cancer cells. First, we investigated genes that showed altered expression patterns in drug-specific cell line subsets composed of the four 5-FU-, three doxorubicin-, and three cisplatin-resistant cell lines. In the 5-FU-resistant gastric cancer cell lines, a total of 38 genes were selected as having significant fold-change and Wilcoxon’s test results. Twenty-two genes were up-regulated and 16 were down-regulated in at least three of the four 5-FU-resistant cell lines. In the doxorubicin-resistant cell lines, we found over 200 differentially expressed genes, many of which were overexpressed. In the cisplatin-resistant cell lines, we identified 27 differentially expressed genes, 19 of which were up-regulated and 8 of which were down-regulated. After the individual gene selection in each drug subset, we next screened for genes that were differentially expressed in more than one of the drug subsets, i.e. multidrug resistance genes. We identified eight genes that were differentially expressed in more than one drug subset; only one of the eight was differentially expressed in all three subsets.

Validation of Microarray Results.

To verify the expression of the genes identified in microarray experiments, either real-time quantitative RT-PCR or semiquantitative RT-PCR was performed using the same RNA as that used in the microarray analysis. We tested two genes (MDK and BIRC5) for real-time quantitative RT-PCR and seven genes (MDK, FOSB, NQO1, DDB2, ITGB4, ABCC1, and IGFBP2) for semiquantitative RT-PCR, and found that the results were in good agreement with those from the microarray data, in that the observed differences were not significant.

Differentially Expressed Genes in 5-FU-Resistant Gastric Cancer Cells.

In 5-FU-resistant gastric cancer cell lines, we identified 38 differentially expressed genes (Table 2), most of which are involved in cell proliferation, metabolic pathways, cell growth, and cell organization. In addition, a subset of differentially expressed genes was associated with signaling, responses to external stimuli, and cell adhesion. In particular, we observed up-regulation of cell growth regulators (NOV and IGFBP2) and genes involved in nucleobase, nucleotide, and nucleic acid metabolism (HOXB6, SF3B3, TYMS, DGUOK, and DDB2). Also, the overexpression of TYMS (thymidylate synthetase) was consistent with the action of 5-FU as a TYMS inhibitor. The signal transducers sorcin (SRI), MDK, and NQO1 were up-regulated, and the calcium-binding protein SRI was up-regulated in all of the 5-FU-resistant cell lines. A novel hypoxia-inducible factor 1 (HIF-1)-responsive gene, RTP801, recently identified as involved in both pro- and antiapoptotic activities (20), was down-regulated, as were several calcium ion-binding molecules (S100P, CALB2, and LDLR).

Differentially Expressed Genes in Doxorubicin-Resistant Gastric Cancer Cells.

Table 3 shows the top 54 up-regulated genes among >200 genes and 20 down-regulated in doxorubicin-resistant gastric cancer cells. Many of the up-regulated genes are associated with the cell cycle, including genes involved with regulation of the cell cycle (FOSB, CDC2, CDC20, CDKN3, and MKI67), control of the mitotic cell cycle (BUB1, BUB1B, RRM1, and RRM2), DNA replication (TOP2A and MCM4), and antiapoptosis (BIRC5-survivin). Among the ATP-binding cassette (ABC) transporters, ABCC1 was up-regulated and ATP2B1 was down-regulated. In our microarray data, the expression of the MDR1 gene, which encodes a P-glycoprotein, was not detected. The chloride transporter CLIC4, responders to external stimulus (TBL1X and MBP), and transcription factor NFAT5 were all down-regulated in doxorubicin-resistant gastric cancer cells.

Differentially Expressed Genes in Cisplatin-Resistant Gastric Cancer Cells.

We identified 27 genes that were differentially expressed in cisplatin-resistant gastric cancer cells, 19 of which were up-regulated and 8 of which were down-regulated (Table 4). The differentially expressed genes in cisplatin-resistant cells showed the lowest fold change levels among three different drug subsets. The up-regulated genes included cell proliferation regulators (IGFBP6, FTH1, and GRN), genes associated with cell cycle (CDKN1A), stress responders (HSPA1B), transporters (ATP7A), cell adhesion molecules (PCDHGB7 and JUP), and metabolic factor (SF3B3). In contrast, the DNA repair and cell cycle checkpoint gene NBS1, and the ubiquitin-conjugate enzyme and apoptosis suppressor HIP2 were down-regulated.

Genes Differentially Expressed in Two or Three of the Drug Subsets.

We identified eight genes that were differentially expressed in more than one of the three drug subsets. For example, MDK (p = 0.0007) was overexpressed by >1.5-fold in 9 of 10 drug-resistant gastric cancer cell lines. The remaining cell line (SNU-719R-DOX/100) showed a 1.4-fold MDK overexpression, which was slightly below the 1.5-fold cutoff value used to determine significance. CDKN1A (p = 0.088), SF3B3 (p = 0.029), and LZTFL1 (p = 0.00074) were overexpressed in both 5-FU- and cisplatin-resistant gastric cancer cells. NQO1 (p = 0.044), TYMS (p = 0.079), and IGFBP2 (p = 0.012) were overexpressed in both 5-FU-resistant and doxorubicin-resistant cell lines, whereas CALB2 (p = 0.051) was down-regulated in these cells.

Recently, several studies on the drug-sensitivity and drug-resistance in either untreated human cancer cell lines or drug-exposed cells have been performed using microarray technologies (7, 9, 11, 21, 22). They have revealed the correlations between gene expression and drug activity, as well as identification of genes differentially expressed in drug-sensitive and drug-resistant cancer cells. Also, several microarray studies on the identification of genes with altered expression in human gastric cancers have been performed (8, 10, 12). From these studies, numerous genes have been identified as being associated with gastric cancer development and progression, some of which will be used as novel chemotherapeutic targets for the treatment or prevention of gastric cancer. However, although the emergence of drug resistance is recognized as a major burden in all cancer treatment, few microarray studies have sought to identify candidate genes associated with drug resistance in gastric cancer. Here, we established 12 drug-resistant gastric cancer cell lines from 4 different gastric cancer cell lines (SNU-620, SNU-638, SNU-668, and SNU-719) by repeated exposure to the chemotherapeutic drugs 5-FU, doxorubicin, and cisplatin. The resistant cell lines have acquired drug-resistance over the long-term period from 11 months to 2 years, by increasing drug dosage, and confirmed their stable resistance by repeated drug sensitivity assays (MTT assay). It has previously been shown that gastric cancer cells are more sensitive to doxorubicin and cisplatin than to 5-FU (23). In support of this, we found that the relative resistances of 5-FU-resistant cell lines measured by MTT assay were higher than those of doxorubicin- and cisplatin-resistant cell lines. Of the 12 drug-resistant cell lines, two (the doxorubicin-resistant cell line from SNU-638 and the cisplatin-resistant cell line from SNU-719) were excluded from our microarray analysis because MTT assay determined that their relative resistances were <2-fold, which was regarded as a low degree of resistance. Thus, we used a total of 14 cell lines (10 drug-resistant and 4 parent cell lines) for genome-wide expression analysis to identify drug resistance candidate genes in drug-resistant gastric cancer cell lines.

It was suggested that each cancer cell represents a different pattern of drug-resistance gene expression even within cells clonally derived from the same cancer, and may be expected to exhibit a considerable amount of heterogeneity with respect to drug resistance (4). Because our four gastric cancer parent cell lines were genetically different (17), the overall gene expression pattern of each resistant cell line tended to be more similar to the same-parent-originated resistant cell lines than to the same-drug-treated resistant cell lines, as shown in multidimensional scaling (Fig. 1 A).

Our microarray data provided information on genes that were differentially associated with resistance to specific chemotherapeutic drugs, as well as those differentially associated with two or more of the drugs. In this study, cells were maintained without drugs before microarray experiments to avoid making note of transcriptional changes caused by the insult of drugs themselves. Thus, this precaution may not be compatible to detect dynamic changes in response to the drugs themselves, like acquired transcriptional activation. Although 5-FU, doxorubicin, and cisplatin exert their chemotherapeutic effects in different ways, we found eight genes that were differentially expressed in association with resistance to more than one of the tested drugs, and one that was overexpressed in association with resistance to all three tested drugs.

The doxorubicin-resistant cell lines provided the most differentially expressed genes. This suggests that the doxorubicin-resistant cells, although derived from different gastric cancer patients, contain more consistent molecular changes than the other drug-resistant cells. When we performed clustering analysis between all drug-resistant cell lines and the differentially expressed genes, we observed that the doxorubicin-resistant cell lines were clustered together (Fig. 1 B). A previous study demonstrated that at different time points, etoposide-resistant melanoma cells showed stable gene deregulation, whereas cisplatin- and fotemustine-resistant cells showed substantial variation (11). Together with our results, this suggests that anticancer drugs targeting topoisomerase II, such as doxorubicin and etoposide, exhibit coherent resistance in different cell types and during different periods.

Cell cycle deregulation is an important molecular event in the acquisition of drug resistance (5). Most of the genes we identified as overexpressed in doxorubicin-resistant gastric cancer cells were involved in the cell cycle, including the mitotic cell cycle-associated genes BUB1, BUB1B, CDKN3, RRM1, and RRM2. It was previously shown that cell cycle genes had an interesting expression pattern in microarray experiment after exposure to doxorubicin in MCF-7 cells and a subset of these genes was also constitutively overexpressed in MCF-7 doxorubicin-resistant cells (7). Thus, these results suggest that cell cycle genes might significantly contribute to the doxorubicin resistance. In our microarray result, the up-regulations of BIRC5 were observed in doxorubicin-resistant cells. BIRC5, also termed survivin, is a protein responsible for inhibiting apoptosis and preventing cell death (24). Negative correlation between BIRC5 (survivin) and 5-FU derivatives in untreated human cancer cell lines was suggested (9), implying that the overexpression of BIRC5 might be associated with drug-resistance. RRM2, one of two ribonucleotide reductase subunits, is a rate-limiting enzyme in DNA synthesis and repair (25). Previously, it was reported that the overexpression of RRM2 mRNA and protein was found in gemcitabine-resistant cells, implicating the gene in drug resistance (25). Finally, the ATP-binding cassette (ABC) transporter ABCC1 (MRP1) was up-regulated in all doxorubicin-resistant gastric cancer cells, as compared with ABCC1 expressions in the parent cells. Drug resistance through drug efflux pumps has been well described, and the ABC transporters are also known as energy-dependent drug efflux pumps (4). In both our doxorubicin-resistant cells and their parent gastric cancer cells, ABCB1 (MDR1) expressions were not detectable. It was previously demonstrated that the levels of MDR1 RNA was relatively low in gastric cancer cell lines, whereas intermediate or high levels were present in colorectal carcinoma cell lines (6, 18).

A total of 38 genes were differentially expressed in 5-FU-resistant gastric cancer cell lines. The relationship between the overexpression of TYMS and 5-FU resistance is well-characterized (3), and was noted in this investigation except in one case (SNU-638R–5-FU/50000). Although SNU-638R–5-FU/50000 represented the highest degree of resistance to 5-FU (>12,500-fold), TYMS expression was slightly decreased in these cells per our microarray data. This may be explained by the proposition that high doses of 5-FU reduce the activity of TYMS (26). Because the SNU-638R–5-FU/50000 cells were cultured at high 5-FU concentrations (380 μm) over almost 2 years, it is possible that TYMS transcription levels had been down-regulated, and that the acquired 5-FU resistance was not dependent on the TYMS mechanism. A more possible explanation for the TYMS down-regulation in SNU-638R-5-FU/50000 cells is that TYMS may be mutated and has a decreased affinity for 5-fluoro-dUMP. In our microarray data, DYPD expression was not significant in 5-FU resistant cells, although a previous report suggested that DYPD expression was negatively correlated with 5-FU sensitivity in cancer cells (21). Another up-regulated gene is that for SRI, a soluble resistance-related calcium-binding protein known to be overexpressed in various drug-resistant cancer cells (27, 28), although this is the first identification of its association with 5-FU resistance. All four of the 5-FU-resistant cell lines showed up-regulated SRI expression, and, although increased expression of SRI is believed to result from gene amplification (28), it is not yet known how SRI confers multidrug resistance. CLU (clusterin), also overexpressed in all of the 5-FU-resistant cell lines, is a ubiquitous glycoprotein that is highly overexpressed in some normal and malignant tissues undergoing apoptosis (29). The overexpression of CLU has been shown to inhibit apoptotic cell death in cisplatin-treated renal cell carcinoma, suggesting that CLU confers a chemoresistance phenotype through its antiapoptotic activity (29, 30). Moreover, it has been demonstrated that suppression of CLU expression by an antisense oligonucleotide had a chemosensitizing effect (29, 30). From these results, it can be suggested that CLU is a potent candidate gene for gastric cancer cell drug resistance.

In cisplatin-resistant gastric cancer cells, the differentially expressed genes were mainly associated with stress response, transport, cell cycle, and metabolism. ATP7A, a copper-transporting P-type adenosine triphosphatase, was overexpressed in cisplatin-resistant gastric cancer cells. The closely related ATP7B is well known for conferring resistance to cisplatin (31), but little is known about the association of ATP7A with cisplatin resistance. ATP7A is highly homologous with ATP7B and has a similar export function for copper. A recent report (32) showed ATP7A overexpression in a cisplatin-resistant ovarian cancer cell line. These results suggest a potential role for ATP7A in cisplatin resistance, as well as ATP7B, although the mechanism has not yet been elucidated (32). The Fos gene family members, FOS and FOSB, showed up-regulation in two of three cisplatin-resistant cells (not listed in Table 4), consistent with previous reports suggesting a relationship between Fos gene expression and drug resistance (33).

In this study, we have identified eight genes whose differential expression was associated with resistance to more than one of the three chemotherapeutic drugs tested. Of these, the MDK gene was consistently overexpressed in all 5-FU-, doxorubicin-, and cisplatin-resistant gastric cancer cells compared with the expressions in their parent cells, strongly suggesting that it may contribute to multidrug resistance. The correspondence between mRNA and protein of MDK was observed in SNU-620R-5-FU/1000 and SNU-638R-CIS/400 by Western blot analysis. MDK, a heparin-binding growth factor, is associated with promoting neuronal survival, inducing neurite outgrowth (34, 35, 36), and has been implicated in carcinogenesis and angiogenesis. Found at low levels in normal adult tissue, MDK is frequently overexpressed in esophageal, gastric, colon, pancreatic, hepatocellular, lung, breast, urinary bladder carcinoma, neuroblastoma and Wilms’ tumors (35, 36). Transfection of an MDK antisense oligodeoxynucleotide into CMT-93 mouse rectal carcinoma cells markedly suppressed tumor growth, suggesting that MDK could be a potent target for cancer therapy (36, 37). MDK has cytoprotective activity and has rescued cisplatin-induced apoptotic cell death in both murine kidney and cultured G401 cells, implying that MDK confers a cellular growth advantage by functioning as an antiapoptotic factor (34). Recently, the overexpression of HB-EGFGF (heparin-binding epidermal growth factor-like growth factor) was observed in the cisplatin-resistant and 5-FU-resistant gastric tumors by microarray analysis, suggesting that HB-EGFGF is a candidate chemoresistant-related gene of gastric cancer (38). Interestingly, these results support that heparin-binding growth factors such as MDK or HB-EGFGF might be one of the important genes contributing to chemoresistance in gastric cancers. Accordingly, we hypothesize that the overexpressed MDK can contribute to the growth progression of gastric cancer cells with an acquired multidrug resistance and may be a potent target to restore chemosensitivity. To clarify the relationship between MDK overexpression and multidrug resistance, it will be necessary to further investigate whether the inhibition of MDK overexpression in drug-resistant cancer cells leads to recovered sensitivity.

In summary, we have identified genes that are differentially expressed in 5-FU-, doxorubicin-, and cisplatin-resistant gastric cancer cells. Some of the identified genes were previously known to be associated with drug-resistance. To date, most studies on drug resistance in gastric cancer have focused on candidate gene work targeting a limited number of drug resistance genes identified in other cancers. In contrast, our use of microarray technology for a genome-wide screen of 10 drug-resistant gastric cancer cell lines has provided useful information on differentially expressed genes and possible new candidate multidrug resistance genes in gastric cancer cells. Although further in vivo validations for the identified genes are required because the materials used in the study were induced drug-resistant cells in vitro, this important information may lead to the discovery of new drug resistance targets, and perhaps to the development of better cancer chemotherapy strategies.

Grant support: Research Grant 2002 from the National Cancer Center, Korea, and the BK21 (Brain Korea 21) Project for Medicine, Dentistry, and Pharmacy.

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.

Notes: Drs. H. C. Kang, I-J. Kim, and J-H. Park contributed equally to this work.

Requests for reprints: Jae-Gahb Park, National Cancer Center, 809 Madu-dong, Ilsan-gu, Goyang, Gyeonggi, 411-764, Korea. Phone: 82-31-920-1501; Fax: 82-31-920-1511; E-mail: park@ncc.re.kr

Fig. 1.

Multidimensional scaling and cluster analysis of acquired drug-resistant gastric cancer cells. A, three-dimensional graph of multidimensional scaling generated by SPSS and SigmaPlot. Using the 22,282 transcripts contained in the Affymetrix HG-U133A oligonucleotide microarray, we arranged the whole expression pattern of each drug-resistant gastric cancer cell line in the three-dimensional graph. The overall expression patterns follow the parent cell specificity rather than drug specificity. 5-FU, 5-fluorouracil; DOX, doxorubicin; CIS, cisplatin. B, cluster analysis of the drug-resistant gastric cancer cell lines (vertical axis) and genes selected for fold-change (>1.5-fold) and Wilcoxon’s test in three of four 5-fluorouracil (5-FU)-resistant, or all doxorubicin (DOX)- and cisplatin (CIS)-resistant cell lines (horizontal axis).

Fig. 1.

Multidimensional scaling and cluster analysis of acquired drug-resistant gastric cancer cells. A, three-dimensional graph of multidimensional scaling generated by SPSS and SigmaPlot. Using the 22,282 transcripts contained in the Affymetrix HG-U133A oligonucleotide microarray, we arranged the whole expression pattern of each drug-resistant gastric cancer cell line in the three-dimensional graph. The overall expression patterns follow the parent cell specificity rather than drug specificity. 5-FU, 5-fluorouracil; DOX, doxorubicin; CIS, cisplatin. B, cluster analysis of the drug-resistant gastric cancer cell lines (vertical axis) and genes selected for fold-change (>1.5-fold) and Wilcoxon’s test in three of four 5-fluorouracil (5-FU)-resistant, or all doxorubicin (DOX)- and cisplatin (CIS)-resistant cell lines (horizontal axis).

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Table 1

Drug-resistant gastric cancer cell lines, drug concentrations, IC50, and relative resistance used in the study

DrugsResistant cell linesDrug concentrations (μg/ml)IC50 (μg/ml)Relative resistance
ParentResistant
5-Fluorouracil (5-FU) SNU-620R-5-FU/1000 0.03 ± 0.01 5.64 ± 2.57 188 
 SNU-638R-5-FU/50000 50 0.04 ± 0.05 >500 >12,500 
 SNU-668R-5-FU/4000 2.38 ± 0.12 164 ± 36.5 69 
 SNU-719R-5-FU/600 0.6 0.40 ± 0.06 2.59 ± 0.14 6.5 
Doxorubicin (DOX) SNU-620R-DOX/300 0.3 0.45 ± 0.19 3.21 ± 0.95 7.1 
 SNU-668R-DOX/50 0.05 0.20 ± 0.01 0.68 ± 0.25 3.4 
 SNU-719R-DOX/100 0.1 0.04 ± 0.01 0.26 ± 0.06 6.5 
Cisplatin (CIS) SNU-620R-CIS/2000 4.30 ± 0.30 25.779 ± 0.939 
 SNU-638R-CIS/400 0.4 0.81 ± 0.21 4.339 ± 1.466 5.4 
 SNU-668R-CIS/400 0.4 6.52 ± 0.84 22.384 ± 7.655 3.4 
DrugsResistant cell linesDrug concentrations (μg/ml)IC50 (μg/ml)Relative resistance
ParentResistant
5-Fluorouracil (5-FU) SNU-620R-5-FU/1000 0.03 ± 0.01 5.64 ± 2.57 188 
 SNU-638R-5-FU/50000 50 0.04 ± 0.05 >500 >12,500 
 SNU-668R-5-FU/4000 2.38 ± 0.12 164 ± 36.5 69 
 SNU-719R-5-FU/600 0.6 0.40 ± 0.06 2.59 ± 0.14 6.5 
Doxorubicin (DOX) SNU-620R-DOX/300 0.3 0.45 ± 0.19 3.21 ± 0.95 7.1 
 SNU-668R-DOX/50 0.05 0.20 ± 0.01 0.68 ± 0.25 3.4 
 SNU-719R-DOX/100 0.1 0.04 ± 0.01 0.26 ± 0.06 6.5 
Cisplatin (CIS) SNU-620R-CIS/2000 4.30 ± 0.30 25.779 ± 0.939 
 SNU-638R-CIS/400 0.4 0.81 ± 0.21 4.339 ± 1.466 5.4 
 SNU-668R-CIS/400 0.4 6.52 ± 0.84 22.384 ± 7.655 3.4 
Table 2

Genes differentially expressed in 5-fluorouracil (5-FU)-resistant gastric cancer cells

Affymetrix identificationSymbolDescriptionFunctionCriteriaa620R-5-FU/1000638R-5-FU/50000668R-5-FU/4000719R-5-FU/600
F.C.bpF.C.pF.C.pF.C.p
Genes overexpressed              
 202589_at TYMS Thymidylate synthetase dTMP biosynthesis • ○ 56.1 0.00002 −2.6 0.99998 11.2 0.00002 1.9 0.000438 
 214321_at NOV Nephroblastoma overexpressed gene Cell growth • ○ 10.3 0.000046 −1.6 0.934434 2.3 0.000389 16.0 0.001651 
 212877_at KNS2 Kinesin 2 (60–70 kD) Cell organization and biogenesis •  2.0 0.000046 1.9 0.004481 14.7 0.000078 1.0 0.532344 
 201467_s_at NQO1 NAD(P)H dehydrogenase, quinone 1 Detoxification response • ○ −1.4 0.99998 4.1 0.00225 3.4 0.000027 10.6 0.00002 
 202718_at IGFBP2 Insulin-like growth factor binding protein 2 (36 kD) Cell growth • ○ 1.5 0.00002 9.6 0.00002 4.3 0.000865 −1.1 0.5 
 202284_s_at CDKN1A Cyclin-dependent kinase inhibitor 1A (p21, Cip1) Cell cycle • ○ 2.6 0.000023 10.0 0.000865 2.3 0.000023 1.3 0.033304 
 212614_at  Homo sapiens mRNA; cDNA DKFZp586N012 Unknown • ○ 3.8 0.330589 5.1 0.00002 5.2 0.000114 1.9 0.000273 
 218437_s_at LZTFL1 Leucine zipper transcription factor-like 1 Unknown •  2.4 0.002753 1.6 0.000101 8.0 0.016731 1.9 0.013078 
 201141_at GPNMB Glycoprotein (transmembrane) nmb Regulation of cell proliferation • ○ 5.5 0.000865 2.1 0.000101 4.3 0.00004 1.9 0.252851 
 203816_at DGUOK Deoxyguanosine kinase Guanosine metabolism • ○ 2.5 0.000023 1.0 0.5 5.9 0.000046 2.2 0.000346 
 203409_at DDB2 Damage-specific DNA binding protein 2 (48 kD) DNA repair • ○ 2.0 0.000273 3.3 0.00002 3.2 0.000692 1.2 0.284967 
 208791_at CLU Clusterin (complement lysis inhibitor, SP-40,40, sulfated glycoprotein 2, testosterone-repressed prostate message 2, apolipoprotein J) Lipid metabolism, apoptosis • ○ 4.1 0.00002 1.7 0.002032 2.1 0.000114 1.7 0.002032 
 209674_at CRY1 Cryptochrome 1 (photolyase-like) G-protein coupled photoreceptor • ○ 2.0 0.000027 1.4 0.002032 3.9 0.000966 2.2 0.263341 
 212957_s_at  Homo sapiens mRNA; cDNA DKFZp434F172 Unknown •  3.6 0.094279 1.8 0.002753 2.2 0.493524 1.9 0.002753 
 205366_s_at HOXB6 Homeo box B6 Transcription factor •  2.2 0.48058 3.6 0.00002 1.7 0.000692 2.0 0.094279 
 209035_at MDK Midkine (neurite growth-promoting factor 2) Cytokine, growth factor •  2.4 0.00002 3.1 0.00002 1.8 0.033304 1.8 0.004073 
 208920_at SRI Sorcin Cell motility, cell-cell signaling •  2.0 0.057676 1.8 0.005409 2.7 0.004073 1.9 0.033304 
 209627_s_at OSBPL3 Oxysterol binding protein-like 3 Lipid metabolism, transport • ○ 1.9 0.00003 2.9 0.00002 1.5 0.000618 1.9 0.000865 
 200687_s_at SF3B3 Splicing factor 3b, subunit 3, 130 kD mRNA splicing •  1.6 0.035785 1.6 0.000068 2.8 0.000966 1.8 0.018128 
 209120_at  Homo sapiens cDNA: FLJ22189 fis, clone HRC01043 Unknown • ○ 1.7 0.000078 2.3 0.00002 1.8 0.008511 1.9 0.001201 
 205548_s_at BTG3 BTG family, member 3 Cell cycle • ○ 1.8 0.00002 1.5 0.001486 2.4 0.00002 1.5 0.033304 
 211998_at H3F3B H3 histone, family 3B (H3.3B) DNA binding • ○ 1.5 0.000023 1.6 0.000241 2.5 0.000023 1.6 0.003355 
Genes down-regulated              
 204351_at S100P S100 calcium binding protein P Calcium binding • ○ 1.5 0.999448 25.5 0.99998 63.1 0.99998 2.6 0.99997 
 217867_x_at BACE2 β-site APP-cleaving enzyme 2 Protein catabolism • ○ 1.7 0.99997 1.8 0.999693 22.0 0.997247 1.5 0.996645 
 205428_s_at CALB2 Calbindin 2, (29 kD, calretinin) Calcium binding •  4.0 0.996645 12.6 0.999922 −1.2 0.088938 1.7 0.999833 
 218677_at LOC57402 S100-type calcium binding protein A14 Unknown • ○ 2.1 0.99998 5.7 0.99998 3.2 0.999508 1.5 0.999948 
204990_s_at ITGB4 Integrin, β 4 Cell adhesion • ○ 2.2 0.99998 6.1 0.99998 1.8 0.999853 2.2 0.99998 
Affymetrix identificationSymbolDescriptionFunctionCriteriaa620R-5-FU/1000638R-5-FU/50000668R-5-FU/4000719R-5-FU/600
F.C.bpF.C.pF.C.pF.C.p
Genes overexpressed              
 202589_at TYMS Thymidylate synthetase dTMP biosynthesis • ○ 56.1 0.00002 −2.6 0.99998 11.2 0.00002 1.9 0.000438 
 214321_at NOV Nephroblastoma overexpressed gene Cell growth • ○ 10.3 0.000046 −1.6 0.934434 2.3 0.000389 16.0 0.001651 
 212877_at KNS2 Kinesin 2 (60–70 kD) Cell organization and biogenesis •  2.0 0.000046 1.9 0.004481 14.7 0.000078 1.0 0.532344 
 201467_s_at NQO1 NAD(P)H dehydrogenase, quinone 1 Detoxification response • ○ −1.4 0.99998 4.1 0.00225 3.4 0.000027 10.6 0.00002 
 202718_at IGFBP2 Insulin-like growth factor binding protein 2 (36 kD) Cell growth • ○ 1.5 0.00002 9.6 0.00002 4.3 0.000865 −1.1 0.5 
 202284_s_at CDKN1A Cyclin-dependent kinase inhibitor 1A (p21, Cip1) Cell cycle • ○ 2.6 0.000023 10.0 0.000865 2.3 0.000023 1.3 0.033304 
 212614_at  Homo sapiens mRNA; cDNA DKFZp586N012 Unknown • ○ 3.8 0.330589 5.1 0.00002 5.2 0.000114 1.9 0.000273 
 218437_s_at LZTFL1 Leucine zipper transcription factor-like 1 Unknown •  2.4 0.002753 1.6 0.000101 8.0 0.016731 1.9 0.013078 
 201141_at GPNMB Glycoprotein (transmembrane) nmb Regulation of cell proliferation • ○ 5.5 0.000865 2.1 0.000101 4.3 0.00004 1.9 0.252851 
 203816_at DGUOK Deoxyguanosine kinase Guanosine metabolism • ○ 2.5 0.000023 1.0 0.5 5.9 0.000046 2.2 0.000346 
 203409_at DDB2 Damage-specific DNA binding protein 2 (48 kD) DNA repair • ○ 2.0 0.000273 3.3 0.00002 3.2 0.000692 1.2 0.284967 
 208791_at CLU Clusterin (complement lysis inhibitor, SP-40,40, sulfated glycoprotein 2, testosterone-repressed prostate message 2, apolipoprotein J) Lipid metabolism, apoptosis • ○ 4.1 0.00002 1.7 0.002032 2.1 0.000114 1.7 0.002032 
 209674_at CRY1 Cryptochrome 1 (photolyase-like) G-protein coupled photoreceptor • ○ 2.0 0.000027 1.4 0.002032 3.9 0.000966 2.2 0.263341 
 212957_s_at  Homo sapiens mRNA; cDNA DKFZp434F172 Unknown •  3.6 0.094279 1.8 0.002753 2.2 0.493524 1.9 0.002753 
 205366_s_at HOXB6 Homeo box B6 Transcription factor •  2.2 0.48058 3.6 0.00002 1.7 0.000692 2.0 0.094279 
 209035_at MDK Midkine (neurite growth-promoting factor 2) Cytokine, growth factor •  2.4 0.00002 3.1 0.00002 1.8 0.033304 1.8 0.004073 
 208920_at SRI Sorcin Cell motility, cell-cell signaling •  2.0 0.057676 1.8 0.005409 2.7 0.004073 1.9 0.033304 
 209627_s_at OSBPL3 Oxysterol binding protein-like 3 Lipid metabolism, transport • ○ 1.9 0.00003 2.9 0.00002 1.5 0.000618 1.9 0.000865 
 200687_s_at SF3B3 Splicing factor 3b, subunit 3, 130 kD mRNA splicing •  1.6 0.035785 1.6 0.000068 2.8 0.000966 1.8 0.018128 
 209120_at  Homo sapiens cDNA: FLJ22189 fis, clone HRC01043 Unknown • ○ 1.7 0.000078 2.3 0.00002 1.8 0.008511 1.9 0.001201 
 205548_s_at BTG3 BTG family, member 3 Cell cycle • ○ 1.8 0.00002 1.5 0.001486 2.4 0.00002 1.5 0.033304 
 211998_at H3F3B H3 histone, family 3B (H3.3B) DNA binding • ○ 1.5 0.000023 1.6 0.000241 2.5 0.000023 1.6 0.003355 
Genes down-regulated              
 204351_at S100P S100 calcium binding protein P Calcium binding • ○ 1.5 0.999448 25.5 0.99998 63.1 0.99998 2.6 0.99997 
 217867_x_at BACE2 β-site APP-cleaving enzyme 2 Protein catabolism • ○ 1.7 0.99997 1.8 0.999693 22.0 0.997247 1.5 0.996645 
 205428_s_at CALB2 Calbindin 2, (29 kD, calretinin) Calcium binding •  4.0 0.996645 12.6 0.999922 −1.2 0.088938 1.7 0.999833 
 218677_at LOC57402 S100-type calcium binding protein A14 Unknown • ○ 2.1 0.99998 5.7 0.99998 3.2 0.999508 1.5 0.999948 
204990_s_at ITGB4 Integrin, β 4 Cell adhesion • ○ 2.2 0.99998 6.1 0.99998 1.8 0.999853 2.2 0.99998 
Table 2A

Continued

Affymetrix identificationSymbolDescriptionFunctionCriteriaa620R-5-FU/1000638R-5-FU/50000668R-5-FU/4000719R-5-FU/600
F.C.bpF.C.pF.C.pF.C.p
 202887_s_at RTP801 HIF-1 responsive RTP801 Apoptosis • ○ 2.3 0.99998 2.4 0.99998 4.1 0.999973 3.3 0.99998 
 204109_s_at NFYA Nuclear transcription factor Y, α Transcription factor •  1.5 0.777251 1.7 0.994067 2.4 0.98579 5.5 0.955851 
 202847_at PCK2 Phosphoenolpyruvate carboxykinase 2 (mitochondrial) Glucose metabolism • ○ 3.5 0.99998 2.0 0.99997 1.9 0.999727 2.7 0.99998 
 208813_at GOT1 Glutamic-oxaloacetic transaminase 1, soluble Amino acid metabolism • ○ 1.6 0.999886 2.5 0.999932 1.6 0.989865 4.1 0.99998 
 222025_s_at OPLAH 5-Oxoprolinase (ATP-hydrolyzing) ATP hydrolysis • ○ 2.9 0.99998 2.6 0.999948 3.0 0.99998 1.2 0.997247 
 201250_s_at SLC2A1 Solute carrier family 2 (facilitated glucose transporter), member 1 Glucose transport • ○ 1.7 0.998514 2.0 0.999759 1.3 0.964215 4.2 0.99997 
 202068_s_at LDLR Low density lipoprotein receptor (familial hypercholesterolemia) Lipid metabolism, endocytosis • ○ 1.5 0.999654 2.8 0.999911 1.9 0.99998 2.3 0.99994 
 202267_at LAMC2 Laminin, gamma 2 [nicein (100 kD), kalinin (105 kD), BM600 (100 kD), Herlitz junctional epidermolysis bullosa)] Cell adhesion • ○ 2.5 0.999508 2.0 0.999948 1.2 0.5 2.8 0.99998 
 208613_s_at FLNB Filamin B, β (actin binding protein 278) Cytoskeletal anchoring • ○ 2.4 0.998664 4.7 0.999965 −1.0 0.242585 2.0 0.999932 
 204394_at POV1 Prostate cancer overexpressed gene 1 Oncogenesis • ○ 1.8 0.999654 1.8 0.999382 3.1 0.977068 1.5 0.999382 
205455_at MST1R Macrophage stimulating 1 receptor (c-met- related tyrosine kinase) Protein modification •  1.6 0.925732 1.80 0.999226 2.6 0.999308 1.8 0.993497 
Affymetrix identificationSymbolDescriptionFunctionCriteriaa620R-5-FU/1000638R-5-FU/50000668R-5-FU/4000719R-5-FU/600
F.C.bpF.C.pF.C.pF.C.p
 202887_s_at RTP801 HIF-1 responsive RTP801 Apoptosis • ○ 2.3 0.99998 2.4 0.99998 4.1 0.999973 3.3 0.99998 
 204109_s_at NFYA Nuclear transcription factor Y, α Transcription factor •  1.5 0.777251 1.7 0.994067 2.4 0.98579 5.5 0.955851 
 202847_at PCK2 Phosphoenolpyruvate carboxykinase 2 (mitochondrial) Glucose metabolism • ○ 3.5 0.99998 2.0 0.99997 1.9 0.999727 2.7 0.99998 
 208813_at GOT1 Glutamic-oxaloacetic transaminase 1, soluble Amino acid metabolism • ○ 1.6 0.999886 2.5 0.999932 1.6 0.989865 4.1 0.99998 
 222025_s_at OPLAH 5-Oxoprolinase (ATP-hydrolyzing) ATP hydrolysis • ○ 2.9 0.99998 2.6 0.999948 3.0 0.99998 1.2 0.997247 
 201250_s_at SLC2A1 Solute carrier family 2 (facilitated glucose transporter), member 1 Glucose transport • ○ 1.7 0.998514 2.0 0.999759 1.3 0.964215 4.2 0.99997 
 202068_s_at LDLR Low density lipoprotein receptor (familial hypercholesterolemia) Lipid metabolism, endocytosis • ○ 1.5 0.999654 2.8 0.999911 1.9 0.99998 2.3 0.99994 
 202267_at LAMC2 Laminin, gamma 2 [nicein (100 kD), kalinin (105 kD), BM600 (100 kD), Herlitz junctional epidermolysis bullosa)] Cell adhesion • ○ 2.5 0.999508 2.0 0.999948 1.2 0.5 2.8 0.99998 
 208613_s_at FLNB Filamin B, β (actin binding protein 278) Cytoskeletal anchoring • ○ 2.4 0.998664 4.7 0.999965 −1.0 0.242585 2.0 0.999932 
 204394_at POV1 Prostate cancer overexpressed gene 1 Oncogenesis • ○ 1.8 0.999654 1.8 0.999382 3.1 0.977068 1.5 0.999382 
205455_at MST1R Macrophage stimulating 1 receptor (c-met- related tyrosine kinase) Protein modification •  1.6 0.925732 1.80 0.999226 2.6 0.999308 1.8 0.993497 
a

Criteria shows how the genes were selected by data analysis. • indicates that the gene was selected by fold-change. ○ indicates that the gene was selected by Wilcoxon’s test. • ○ indicates that the gene was selected by both fold-change and Wilcoxon’s test.

b

F.C., fold change.

Table 3

Genes differentially expressed in doxorubicin (DOX)-resistant gastric cancer cells

Affymetrix identificationSymbolDescriptionFunctionCriteriaaSNU-620R-DOX/300SNU-668R-DOX/50SNU-719R-DOX/100
F.C.bpF.C.pF.C.p
Genes overexpressed            
 219918_s_at FLJ10517 Hypothetical protein FLJ10517 Unknown • ○ 7.6 0.000114 3.1 0.000046 49.9 0.00002 
 202768_at FOSB FBJ murine osteosarcoma viral oncogene homolog B Regulation of transcription • ○ 4.3 0.000027 16.9 0.000865 22.6 0.00002 
 202805_s_at ABCC1 ATP-binding cassette, sub-family C (CFTR/MRP), member 1 Transport, drug resistance • ○ 34.5 0.00002 5.7 0.00006 1.7 0.00002 
 218663_at HCAP-G Chromosome condensation protein G Unknown •  24.8 0.00006 10.4 0.005933 3.4 0.00002 
 213007_at FLJ10719 Hypothetical protein FLJ10719 Unknown  ○ 15.5 0.00002 19.7 0.000052 1.4 0.001651 
 218355_at KIF4A Kinesin family member 4A Microtubule motor •  21.0 0.000068 3.4 0.015426 7.9 0.00004 
 203967_at CDC6 CDC6 cell division cycle 6 homolog (Saccharomyces cerevisiaeCell cycle regulator •  5.3 0.000147 17.5 0.000023 8.3 0.024755 
 202870_s_at CDC20 CDC20 cell division cycle 20 homolog (S. cerevisiaeRegulation of cell cycle • ○ 5.7 0.000101 20.4 0.000101 3.5 0.000046 
 204886_at STK18 Serine/threonine kinase 18 Regulation of cell cycle •  4.0 0.000492 19.6 0.011045 3.6 0.002032 
 219588_s_at FLJ20311 Hypothetical protein FLJ20311 Unknown • ○ 6.8 0.000027 16.4 0.000167 3.0 0.000035 
 202095_s_at BIRC5 Baculoviral IAP repeat-containing 5 (survivin) Anti-apoptosis • ○ 4.8 0.00013 8.2 0.00004 12.1 0.00002 
 218741_at MGC861 Hypothetical protein MGC861 Unknown • ○ 16.2 0.000214 6.1 0.001201 2.2 0.001486 
 204023_at RFC4 Replication factor C (activator 1) 4, 37 kDa DNA replication • ○ 2.4 0.000023 19.3 0.00004 1.9 0.000346 
 219000_s_at MGC5528 Hypothetical protein MGC5528 Unknown •  7.0 0.038415 7.4 0.061522 7.9 0.000101 
 218726_at DKFZp762E1312 Hypothetical protein DKFZp762E1312 Unknown •  4.9 0.000114 7.3 0.004925 10.2 0.001832 
 212621_at KIAA0286 KIAA0286 protein Unknown • ○ 4.2 0.000273 13.9 0.000389 3.1 0.000147 
 203276_at LMNB1 Lamin B1 Structural molecule •  13.5 0.001077 5.1 0.069813 2.0 0.001486 
 209773_s_at RRM2 Ribonucleotide reductase M2 polypeptide DNA synthesis • ○ 4.6 0.00002 12.9 0.00002 3.1 0.00002 
 204441_s_at POLA2 Polymerase (DNA-directed), α (70 kD) Unknown •  11.0 0.00712 2.1 0.5 7.4 0.030967 
 209053_s_at WHSC1 Wolf-Hirschhorn syndrome candidate 1 Oncogenesis • ○ 7.3 0.00004 10.4 0.000147 2.5 0.00003 
 204962_s_at CENPA Centromere protein A, 17 kDa Chromosome organigenesis •  12.9 0.002753 3.7 0.041201 3.5 0.000023 
 203214_x_at CDC2 Cell division cycle 2, G1 to S and G2 to M Cell cycle • ○ 5.5 0.000035 12.0 0.00002 2.3 0.000023 
 209642_at BUB1 BUB1 budding uninhibited by benzimidazoles 1 homolog Mitotic checkpoint • ○ 3.7 0.000346 11.5 0.00003 4.4 0.000023 
 201467_s_at NQO1 NAD(P)H dehydrogenase, quinone 1 Detoxification response  ○ 1.4 0.002032 7.6 0.00002 9.7 0.00002 
 213599_at OIP5 Opa-interacting protein 5 Unknown • ○ 3.3 0.00003 9.0 0.000273 5.0 0.000114 
 219510_at POLQ Polymerase (DNA directed), theta DNA repair •  10.7 0.213188 4.3 0.583567 2.1 0.010135 
 203755_at BUB1B BUB1 budding uninhibited by benzimidazoles 1 homolog beta Mitotic checkpoint • ○ 7.6 0.00002 4.6 0.000189 3.4 0.000027 
202503_s_at KIAA0101 KIAA0101 gene product Unknown • ○ 6.1 0.00002 6.4 0.00002 2.9 0.00002 
Affymetrix identificationSymbolDescriptionFunctionCriteriaaSNU-620R-DOX/300SNU-668R-DOX/50SNU-719R-DOX/100
F.C.bpF.C.pF.C.p
Genes overexpressed            
 219918_s_at FLJ10517 Hypothetical protein FLJ10517 Unknown • ○ 7.6 0.000114 3.1 0.000046 49.9 0.00002 
 202768_at FOSB FBJ murine osteosarcoma viral oncogene homolog B Regulation of transcription • ○ 4.3 0.000027 16.9 0.000865 22.6 0.00002 
 202805_s_at ABCC1 ATP-binding cassette, sub-family C (CFTR/MRP), member 1 Transport, drug resistance • ○ 34.5 0.00002 5.7 0.00006 1.7 0.00002 
 218663_at HCAP-G Chromosome condensation protein G Unknown •  24.8 0.00006 10.4 0.005933 3.4 0.00002 
 213007_at FLJ10719 Hypothetical protein FLJ10719 Unknown  ○ 15.5 0.00002 19.7 0.000052 1.4 0.001651 
 218355_at KIF4A Kinesin family member 4A Microtubule motor •  21.0 0.000068 3.4 0.015426 7.9 0.00004 
 203967_at CDC6 CDC6 cell division cycle 6 homolog (Saccharomyces cerevisiaeCell cycle regulator •  5.3 0.000147 17.5 0.000023 8.3 0.024755 
 202870_s_at CDC20 CDC20 cell division cycle 20 homolog (S. cerevisiaeRegulation of cell cycle • ○ 5.7 0.000101 20.4 0.000101 3.5 0.000046 
 204886_at STK18 Serine/threonine kinase 18 Regulation of cell cycle •  4.0 0.000492 19.6 0.011045 3.6 0.002032 
 219588_s_at FLJ20311 Hypothetical protein FLJ20311 Unknown • ○ 6.8 0.000027 16.4 0.000167 3.0 0.000035 
 202095_s_at BIRC5 Baculoviral IAP repeat-containing 5 (survivin) Anti-apoptosis • ○ 4.8 0.00013 8.2 0.00004 12.1 0.00002 
 218741_at MGC861 Hypothetical protein MGC861 Unknown • ○ 16.2 0.000214 6.1 0.001201 2.2 0.001486 
 204023_at RFC4 Replication factor C (activator 1) 4, 37 kDa DNA replication • ○ 2.4 0.000023 19.3 0.00004 1.9 0.000346 
 219000_s_at MGC5528 Hypothetical protein MGC5528 Unknown •  7.0 0.038415 7.4 0.061522 7.9 0.000101 
 218726_at DKFZp762E1312 Hypothetical protein DKFZp762E1312 Unknown •  4.9 0.000114 7.3 0.004925 10.2 0.001832 
 212621_at KIAA0286 KIAA0286 protein Unknown • ○ 4.2 0.000273 13.9 0.000389 3.1 0.000147 
 203276_at LMNB1 Lamin B1 Structural molecule •  13.5 0.001077 5.1 0.069813 2.0 0.001486 
 209773_s_at RRM2 Ribonucleotide reductase M2 polypeptide DNA synthesis • ○ 4.6 0.00002 12.9 0.00002 3.1 0.00002 
 204441_s_at POLA2 Polymerase (DNA-directed), α (70 kD) Unknown •  11.0 0.00712 2.1 0.5 7.4 0.030967 
 209053_s_at WHSC1 Wolf-Hirschhorn syndrome candidate 1 Oncogenesis • ○ 7.3 0.00004 10.4 0.000147 2.5 0.00003 
 204962_s_at CENPA Centromere protein A, 17 kDa Chromosome organigenesis •  12.9 0.002753 3.7 0.041201 3.5 0.000023 
 203214_x_at CDC2 Cell division cycle 2, G1 to S and G2 to M Cell cycle • ○ 5.5 0.000035 12.0 0.00002 2.3 0.000023 
 209642_at BUB1 BUB1 budding uninhibited by benzimidazoles 1 homolog Mitotic checkpoint • ○ 3.7 0.000346 11.5 0.00003 4.4 0.000023 
 201467_s_at NQO1 NAD(P)H dehydrogenase, quinone 1 Detoxification response  ○ 1.4 0.002032 7.6 0.00002 9.7 0.00002 
 213599_at OIP5 Opa-interacting protein 5 Unknown • ○ 3.3 0.00003 9.0 0.000273 5.0 0.000114 
 219510_at POLQ Polymerase (DNA directed), theta DNA repair •  10.7 0.213188 4.3 0.583567 2.1 0.010135 
 203755_at BUB1B BUB1 budding uninhibited by benzimidazoles 1 homolog beta Mitotic checkpoint • ○ 7.6 0.00002 4.6 0.000189 3.4 0.000027 
202503_s_at KIAA0101 KIAA0101 gene product Unknown • ○ 6.1 0.00002 6.4 0.00002 2.9 0.00002 
Table 3A

Continued

Affymetrix identificationSymbolDescriptionFunctionCriteriaaSNU-620R-DOX/300SNU-668R-DOX/50SNU-719R-DOX/100
F.C.bpF.C.pF.C.p
 202589_at TYMS Thymidylate synthetase dTMP biosynthesis  ○ 7.8 0.000052 6.2 0.00002 1.4 0.00004 
 202580_x_at FOXM1 Forkhead box M1 Transcription factor • ○ 7.6 0.00002 5.2 0.000023 2.5 0.00002 
 209714_s_at CDKN3 Cyclin-dependent kinase inhibitor 3 Cell cycle arrest • ○ 3.5 0.00002 2.3 0.000114 9.4 0.00002 
 202240_at PLK Polo-like kinase (Drosophila) Cell cycle •  7.1 0.000307 4.7 0.044149 2.9 0.000068 
 221520_s_at FLJ10468 Hypothetical protein FLJ10468 Unknown •  3.2 0.000618 8.4 0.003355 3.1 0.000027 
 210052_s_at C20orf1 Chromosome 20 open reading frame 1 Unknown • ○ 6.8 0.00002 2.8 0.00002 5.0 0.00002 
 205345_at BARD1 BRCA1 associated RING domain 1 Tumor suppressor • ○ 7.2 0.00225 4.5 0.001336 2.7 0.00013 
 204026_s_at ZWINT ZW10 interactor Kinetochore function • ○ 4.4 0.000027 7.0 0.000046 2.9 0.000046 
 203270_at DTYMK Deoxythymidylate kinase (thymidylate kinase) DNA metabolism  ○ 3.3 0.000618 9.6 0.000078 1.3 0.00249 
 218782_s_at PRO2000 PRO2000 protein Cell cycle •  6.1 0.00002 4.8 0.00002 3.1 0.005409 
 208079_s_at STK6 Serine/threonine kinase 6 Mitotic protein kinase • ○ 6.9 0.00002 3.5 0.000147 2.9 0.00002 
 212021_s_at MKI67 Antigen identified by monoclonal antibody Ki-67 Cell cycle regulator • ○ 5.7 0.000052 4.3 0.000346 3.2 0.000027 
 218009_s_at PRC1 Protein regulator of cytokinesis 1 Cytokinesis • ○ 6.8 0.00002 2.4 0.00002 3.7 0.00002 
 201292_at TOP2A Topoisomerase (DNA) II α 170 kDa DNA topoisomerase • ○ 6.5 0.00002 2.8 0.00002 3.4 0.00002 
 201477_s_at RRM1 Ribonucleotide reductase M1 polypeptide DNA synthesis • ○ 3.8 0.000068 7.1 0.000273 1.6 0.000189 
 222037_at MCM4 MCM4 minichromosome maintenance deficient 4 (S. cerevisiaeDNA replication • ○ 6.4 0.00002 3.9 0.000046 2.1 0.000492 
 202338_at TK1 Thymidine kinase 1, soluble Thymidine kinase • ○ 4.9 0.00006 4.9 0.000035 2.1 0.002032 
 218039_at ANKT Nucleolar protein ANKT Unknown • ○ 4.7 0.00002 3.7 0.000307 3.1 0.00002 
 203362_s_at MAD2L1 MAD2 mitotic arrest deficient-like 1 (yeast) Mitotic checkpoint • ○ 6.3 0.000027 2.8 0.000273 2.3 0.000114 
 204146_at PIR51 RAD51-interacting protein DNA repair • ○ 6.2 0.00002 2.5 0.000389 2.6 0.000346 
 204538_x_at NPIP Nuclear pore complex interacting protein Unknown • ○ 6.7 0.00002 2.5 0.00002 2.0 0.00002 
 218115_at FLJ10604 Hypothetical protein FLJ10604 Unknown • ○ 3.6 0.000147 3.8 0.000346 2.7 0.000035 
 206102_at KIAA0186 KIAA0186 gene product Unknown • ○ 4.1 0.00002 3.6 0.00002 2.2 0.00002 
 219148_at TOPK T-LAK cell-originated protein kinase Protein kinase • ○ 2.9 0.00003 3.7 0.00002 3.2 0.00013 
 207165_at HMMR Hyaluronan-mediated motility receptor (RHAMM) Cell motility •  2.9 0.000492 1.9 0.004481 4.8 0.000023 
 208808_s_at HMGB2 High-mobility group box 2 Transcription factor • ○ 3.9 0.00002 2.8 0.000052 2.9 0.00002 
Genes down-regulated            
 221705_s_at FLJ21168 Hypothetical protein FLJ21168 Unknown • ○ 11.2 0.999611 2.4 0.999308 3.2 0.998514 
 203803_at PCL1 Prenylcysteine lyase Protein catabolism • ○ 9.1 0.999922 2.4 0.99998 3.6 0.999899 
 208003_s_at NFAT5 Nuclear factor of activated T-cells 5, tonicity-responsive Transcription factor • ○ 5.7 0.999911 5.2 0.99998 3.6 0.99998 
 207755_at  Homo sapiens cDNA FLJ13892 fis, clone THYRO1001656, Unknown •  4.3 0.991489 5.4 0.999886 2.4 0.949447 
 215936_s_at KIAA1033 KIAA1033 protein Unknown • ○ 3.1 0.99987 3.8 0.99998 3.1 0.999654 
 207700_s_at NCOA3 Nuclear receptor coactivator 3 Transcription co-activator •  4.2 0.999833 2.7 0.999853 2.2 0.945978 
 201867_s_at TBL1X Transducin β-like 1X-linked Signal transduction • ○ 2.7 0.99998 3.4 0.99998 2.9 0.99987 
218659_at FLJ10898 KIAA1685 protein Unknown • ○ 3.1 0.99998 2.6 0.999948 3.2 0.99998 
Affymetrix identificationSymbolDescriptionFunctionCriteriaaSNU-620R-DOX/300SNU-668R-DOX/50SNU-719R-DOX/100
F.C.bpF.C.pF.C.p
 202589_at TYMS Thymidylate synthetase dTMP biosynthesis  ○ 7.8 0.000052 6.2 0.00002 1.4 0.00004 
 202580_x_at FOXM1 Forkhead box M1 Transcription factor • ○ 7.6 0.00002 5.2 0.000023 2.5 0.00002 
 209714_s_at CDKN3 Cyclin-dependent kinase inhibitor 3 Cell cycle arrest • ○ 3.5 0.00002 2.3 0.000114 9.4 0.00002 
 202240_at PLK Polo-like kinase (Drosophila) Cell cycle •  7.1 0.000307 4.7 0.044149 2.9 0.000068 
 221520_s_at FLJ10468 Hypothetical protein FLJ10468 Unknown •  3.2 0.000618 8.4 0.003355 3.1 0.000027 
 210052_s_at C20orf1 Chromosome 20 open reading frame 1 Unknown • ○ 6.8 0.00002 2.8 0.00002 5.0 0.00002 
 205345_at BARD1 BRCA1 associated RING domain 1 Tumor suppressor • ○ 7.2 0.00225 4.5 0.001336 2.7 0.00013 
 204026_s_at ZWINT ZW10 interactor Kinetochore function • ○ 4.4 0.000027 7.0 0.000046 2.9 0.000046 
 203270_at DTYMK Deoxythymidylate kinase (thymidylate kinase) DNA metabolism  ○ 3.3 0.000618 9.6 0.000078 1.3 0.00249 
 218782_s_at PRO2000 PRO2000 protein Cell cycle •  6.1 0.00002 4.8 0.00002 3.1 0.005409 
 208079_s_at STK6 Serine/threonine kinase 6 Mitotic protein kinase • ○ 6.9 0.00002 3.5 0.000147 2.9 0.00002 
 212021_s_at MKI67 Antigen identified by monoclonal antibody Ki-67 Cell cycle regulator • ○ 5.7 0.000052 4.3 0.000346 3.2 0.000027 
 218009_s_at PRC1 Protein regulator of cytokinesis 1 Cytokinesis • ○ 6.8 0.00002 2.4 0.00002 3.7 0.00002 
 201292_at TOP2A Topoisomerase (DNA) II α 170 kDa DNA topoisomerase • ○ 6.5 0.00002 2.8 0.00002 3.4 0.00002 
 201477_s_at RRM1 Ribonucleotide reductase M1 polypeptide DNA synthesis • ○ 3.8 0.000068 7.1 0.000273 1.6 0.000189 
 222037_at MCM4 MCM4 minichromosome maintenance deficient 4 (S. cerevisiaeDNA replication • ○ 6.4 0.00002 3.9 0.000046 2.1 0.000492 
 202338_at TK1 Thymidine kinase 1, soluble Thymidine kinase • ○ 4.9 0.00006 4.9 0.000035 2.1 0.002032 
 218039_at ANKT Nucleolar protein ANKT Unknown • ○ 4.7 0.00002 3.7 0.000307 3.1 0.00002 
 203362_s_at MAD2L1 MAD2 mitotic arrest deficient-like 1 (yeast) Mitotic checkpoint • ○ 6.3 0.000027 2.8 0.000273 2.3 0.000114 
 204146_at PIR51 RAD51-interacting protein DNA repair • ○ 6.2 0.00002 2.5 0.000389 2.6 0.000346 
 204538_x_at NPIP Nuclear pore complex interacting protein Unknown • ○ 6.7 0.00002 2.5 0.00002 2.0 0.00002 
 218115_at FLJ10604 Hypothetical protein FLJ10604 Unknown • ○ 3.6 0.000147 3.8 0.000346 2.7 0.000035 
 206102_at KIAA0186 KIAA0186 gene product Unknown • ○ 4.1 0.00002 3.6 0.00002 2.2 0.00002 
 219148_at TOPK T-LAK cell-originated protein kinase Protein kinase • ○ 2.9 0.00003 3.7 0.00002 3.2 0.00013 
 207165_at HMMR Hyaluronan-mediated motility receptor (RHAMM) Cell motility •  2.9 0.000492 1.9 0.004481 4.8 0.000023 
 208808_s_at HMGB2 High-mobility group box 2 Transcription factor • ○ 3.9 0.00002 2.8 0.000052 2.9 0.00002 
Genes down-regulated            
 221705_s_at FLJ21168 Hypothetical protein FLJ21168 Unknown • ○ 11.2 0.999611 2.4 0.999308 3.2 0.998514 
 203803_at PCL1 Prenylcysteine lyase Protein catabolism • ○ 9.1 0.999922 2.4 0.99998 3.6 0.999899 
 208003_s_at NFAT5 Nuclear factor of activated T-cells 5, tonicity-responsive Transcription factor • ○ 5.7 0.999911 5.2 0.99998 3.6 0.99998 
 207755_at  Homo sapiens cDNA FLJ13892 fis, clone THYRO1001656, Unknown •  4.3 0.991489 5.4 0.999886 2.4 0.949447 
 215936_s_at KIAA1033 KIAA1033 protein Unknown • ○ 3.1 0.99987 3.8 0.99998 3.1 0.999654 
 207700_s_at NCOA3 Nuclear receptor coactivator 3 Transcription co-activator •  4.2 0.999833 2.7 0.999853 2.2 0.945978 
 201867_s_at TBL1X Transducin β-like 1X-linked Signal transduction • ○ 2.7 0.99998 3.4 0.99998 2.9 0.99987 
218659_at FLJ10898 KIAA1685 protein Unknown • ○ 3.1 0.99998 2.6 0.999948 3.2 0.99998 
Table 3B

Continued

Affymetrix identificationSymbolDescriptionFunctionCriteriaaSNU-620R-DOX/300SNU-668R-DOX/50SNU-719R-DOX/100
F.C.bpF.C.pF.C.p
 209750_at NR1D2 Nuclear receptor subfamily 1, group D, member 2 Transcription factor • ○ 3.2 0.99998 3.0 0.999948 2.6 0.999973 
 222158_s_at LOC51029 CGI-146 protein Unknown • ○ 3.1 0.99998 3.3 0.999977 2.2 0.999932 
 212638_s_at WWP1 WW domain-containing protein 1 Protein interaction •  2.6 0.99998 3.8 0.99998 1.8 0.995519 
 208615_s_at PTP4A2 Protein tyrosine phosphatase type IVA, member 2 Dephosphorylation • ○ 3.3 0.99998 2.7 0.999977 1.7 0.999034 
 218930_s_at FLJ11273 Hypothetical protein FLJ11273 Unknown • ○ 3.3 0.99997 2.2 0.99997 2.0 0.99998 
 202551_s_at CRIM1 Cysteine-rich motor neuron 1 Insulin-like growth factor receptor • ○ 2.7 0.998923 2.1 0.999977 2.1 0.99997 
 220892_s_at PSA Phosphoserine aminotransferase Enzyme  ○ 1.3 0.99751 3.8 0.99997 1.5 0.999973 
 202033_s_at RB1CC1 RB1-inducible coiled-coil 1 Nuclear architecture •  1.8 0.999899 2.8 0.99997 1.6 0.952736 
 209281_s_at ATP2B1 ATPase, Ca++ transporting, plasma membrane 1 Transporter, ATP binding •  2.2 0.999693 1.8 0.958799 1.7 0.945978 
 201559_s_at CLIC4 Chloride intracellular channel 4 Chloride channel • ○ 1.5 0.999308 1.9 0.99775 1.9 0.997968 
 203973_s_at CEBPD CCAAT/enhancer binding protein (C/EBP), δ Transcription factor •  2.2 0.999954 1.5 0.998349 1.6 0.252851 
214168_s_at TJP1 Tight junction protein 1 (zona occludens 1) Intercellular junction assembly •  2.1 0.945978 1.7 0.916174 1.5 0.990708 
Affymetrix identificationSymbolDescriptionFunctionCriteriaaSNU-620R-DOX/300SNU-668R-DOX/50SNU-719R-DOX/100
F.C.bpF.C.pF.C.p
 209750_at NR1D2 Nuclear receptor subfamily 1, group D, member 2 Transcription factor • ○ 3.2 0.99998 3.0 0.999948 2.6 0.999973 
 222158_s_at LOC51029 CGI-146 protein Unknown • ○ 3.1 0.99998 3.3 0.999977 2.2 0.999932 
 212638_s_at WWP1 WW domain-containing protein 1 Protein interaction •  2.6 0.99998 3.8 0.99998 1.8 0.995519 
 208615_s_at PTP4A2 Protein tyrosine phosphatase type IVA, member 2 Dephosphorylation • ○ 3.3 0.99998 2.7 0.999977 1.7 0.999034 
 218930_s_at FLJ11273 Hypothetical protein FLJ11273 Unknown • ○ 3.3 0.99997 2.2 0.99997 2.0 0.99998 
 202551_s_at CRIM1 Cysteine-rich motor neuron 1 Insulin-like growth factor receptor • ○ 2.7 0.998923 2.1 0.999977 2.1 0.99997 
 220892_s_at PSA Phosphoserine aminotransferase Enzyme  ○ 1.3 0.99751 3.8 0.99997 1.5 0.999973 
 202033_s_at RB1CC1 RB1-inducible coiled-coil 1 Nuclear architecture •  1.8 0.999899 2.8 0.99997 1.6 0.952736 
 209281_s_at ATP2B1 ATPase, Ca++ transporting, plasma membrane 1 Transporter, ATP binding •  2.2 0.999693 1.8 0.958799 1.7 0.945978 
 201559_s_at CLIC4 Chloride intracellular channel 4 Chloride channel • ○ 1.5 0.999308 1.9 0.99775 1.9 0.997968 
 203973_s_at CEBPD CCAAT/enhancer binding protein (C/EBP), δ Transcription factor •  2.2 0.999954 1.5 0.998349 1.6 0.252851 
214168_s_at TJP1 Tight junction protein 1 (zona occludens 1) Intercellular junction assembly •  2.1 0.945978 1.7 0.916174 1.5 0.990708 
a

Criteria shows how the genes were selected by data analysis. • indicates that the gene was selected by fold-change. ○ indicates that the gene was selected by Wilcoxon’s test. • ○ indicates that the gene was selected by both fold-change and Wilcoxon’s test.

b

F.C., fold change.

Table 4

Genes differentially expressed in cisplatin (CIS)-resistant gastric cancer cells

Affymetrix identificationSymbolDescriptionFunctionCriteriaaSNU-620R-CIS/2000SNU-638R-CIS/400SNU-668R-CIS/400
F.C.bpF.C.pF.C.p
Genes overexpressed            
 218437_s_at LZTFL1 Leucine zipper transcription factor-like 1 Unknown •  2.1 0.019624 2.0 0.000438 5.5 0.145682 
 202284_s_at CDKN1A Cyclin-dependent kinase inhibitor 1A (p21, Cip1) Cell cycle •  3.6 0.37888 2.0 0.000023 1.5 0.003699 
 209035_at MDK Midkine (neurite growth-promoting factor 2) Cytokine, growth factor • ○ 1.9 0.00004 3.1 0.00002 2.0 0.000189 
 213258_at TFPI Tissue factor pathway inhibitor Plasma protein • ○ 2.2 0.00013 3.3 0.00002 1.6 0.00225 
 36711_at MAFF Homolog F (avian) Transcription activating factor  ○ 1.3 0.002825 1.3 0.000236 4.3 <0.000001 
 221766_s_at C6orf37 Chromosome 6 open reading frame 37 Unknown • ○ 1.7 0.000167 3.3 0.00002 1.7 0.00002 
 203571_s_at APM2 Adipose specific 2 Unknown  ○ 1.7 0.000273 1.3 0.000618 3.6 0.000027 
 215836_s_at PCDHGB7 Protocadherin γ subfamily B, 7 Cell adhesion •  1.9 0.5 3.2 0.00003 1.5 0.038415 
 213603_s_at HSPC022 HSPC022 protein Unknown • ○ 1.6 0.00003 2.5 0.000023 2.3 0.000023 
 203851_at IGFBP6 Insulin-like growth factor binding protein 6 Signal transduction  ○ 2.4 0.000307 1.3 0.000027 2.3 0.000023 
 216041_x_at GRN Granulin Positive regulation of cell proliferation  ○ 2.1 0.00002 2.2 0.00002 1.3 0.000052 
 212992_at LOC113146 Hypothetical protein BC011859 Unknown  ○ 2.5 0.00013 1.4 0.000214 1.4 0.00002 
 213861_s_at DKFZP586D0919 DKFZP586D0919 protein Unknown •  1.9 0.021224 1.6 0.000966 1.8 0.467656 
 200688_at SF3B3 Splicing factor 3b, subunit 3, 130 kDa mRNA splicing •  1.5 0.5 1.8 0.161038 2.0 0.131343 
 221934_s_at  Homo sapiens cDNA FLJ35885 fis, clone TESTI2009018 Unknown •  1.5 0.000552 2.2 0.000035 1.5 0.138386 
 201015_s_at JUP Junction plakoglobin Cell adhesion • ○ 1.5 0.000046 1.7 0.00004 1.8 0.000273 
 218078_s_at ZDHHC3 Zinc finger, DHHC domain containing 3 Unknown •  1.6 0.003041 1.9 0.000346 1.6 0.069813 
 205198_s_at ATP7A ATPase, Cu++ transporting, α polypeptide Copper ion transporter, ATP binding •  1.6 0.000167 1.7 0.003041 1.5 0.088938 
 218611_at IER5 Immediate early response 5 Unknown  ○ 1.4 0.003041 1.6 0.000241 1.7 0.000241 
 202581_at HSPA1B Heat shock 70 kDa protein 1B Heat shock response  ○ 1.3 0.000068 1.9 0.00002 1.3 0.00249 
 200748_s_at FTH1 Ferritin, heavy polypeptide 1 Iron binding, cell proliferation  ○ 1.6 0.000147 1.3 0.00002 1.7 0.000046 
Genes downregulated            
 218841_at MGC5540 Hypothetical protein MGC5540 Unknown •  2.0 0.994591 1.8 0.934434 4.1 0.973302 
 203788_s_at SEMA3C Domain, secreted, (semaphorin) 3C Drug resistance •  2.1 0.703911 2.2 0.971234 1.9 0.998799 
 201581_at DJ971N18.2 Hypothetical protein DJ971N18.2 Unknown •  1.9 0.98579 1.5 0.994067 2.0 0.999932 
 202906_s_at NBS1 Nijmegen breakage syndrome 1 (nibrin) DNA repair •  1.9 0.990708 1.6 0.98579 1.7 0.998923 
 202346_at HIP2 Huntingtin interacting protein 2 Ubiquitin protein enzyme •  2.0 0.999833 1.5 0.999948 1.5 0.99288 
 204632_at RPS6KA4 Ribosomal protein S6 kinase, 90 kDa, polypeptide 4 Protein kinase •  1.6 0.996301 1.8 0.998514 1.5 0.454766 
 222155_s_at FLJ11856 Putative G-protein coupled receptor GPCR41 Unknown • ○ 1.7 0.999135 1.7 0.999886 1.5 0.99994 
213622_at COL9A2 Collagen, type IX, α 2 Skeletal development •  1.6 0.5 1.7 0.925732 1.6 0.545234 
Affymetrix identificationSymbolDescriptionFunctionCriteriaaSNU-620R-CIS/2000SNU-638R-CIS/400SNU-668R-CIS/400
F.C.bpF.C.pF.C.p
Genes overexpressed            
 218437_s_at LZTFL1 Leucine zipper transcription factor-like 1 Unknown •  2.1 0.019624 2.0 0.000438 5.5 0.145682 
 202284_s_at CDKN1A Cyclin-dependent kinase inhibitor 1A (p21, Cip1) Cell cycle •  3.6 0.37888 2.0 0.000023 1.5 0.003699 
 209035_at MDK Midkine (neurite growth-promoting factor 2) Cytokine, growth factor • ○ 1.9 0.00004 3.1 0.00002 2.0 0.000189 
 213258_at TFPI Tissue factor pathway inhibitor Plasma protein • ○ 2.2 0.00013 3.3 0.00002 1.6 0.00225 
 36711_at MAFF Homolog F (avian) Transcription activating factor  ○ 1.3 0.002825 1.3 0.000236 4.3 <0.000001 
 221766_s_at C6orf37 Chromosome 6 open reading frame 37 Unknown • ○ 1.7 0.000167 3.3 0.00002 1.7 0.00002 
 203571_s_at APM2 Adipose specific 2 Unknown  ○ 1.7 0.000273 1.3 0.000618 3.6 0.000027 
 215836_s_at PCDHGB7 Protocadherin γ subfamily B, 7 Cell adhesion •  1.9 0.5 3.2 0.00003 1.5 0.038415 
 213603_s_at HSPC022 HSPC022 protein Unknown • ○ 1.6 0.00003 2.5 0.000023 2.3 0.000023 
 203851_at IGFBP6 Insulin-like growth factor binding protein 6 Signal transduction  ○ 2.4 0.000307 1.3 0.000027 2.3 0.000023 
 216041_x_at GRN Granulin Positive regulation of cell proliferation  ○ 2.1 0.00002 2.2 0.00002 1.3 0.000052 
 212992_at LOC113146 Hypothetical protein BC011859 Unknown  ○ 2.5 0.00013 1.4 0.000214 1.4 0.00002 
 213861_s_at DKFZP586D0919 DKFZP586D0919 protein Unknown •  1.9 0.021224 1.6 0.000966 1.8 0.467656 
 200688_at SF3B3 Splicing factor 3b, subunit 3, 130 kDa mRNA splicing •  1.5 0.5 1.8 0.161038 2.0 0.131343 
 221934_s_at  Homo sapiens cDNA FLJ35885 fis, clone TESTI2009018 Unknown •  1.5 0.000552 2.2 0.000035 1.5 0.138386 
 201015_s_at JUP Junction plakoglobin Cell adhesion • ○ 1.5 0.000046 1.7 0.00004 1.8 0.000273 
 218078_s_at ZDHHC3 Zinc finger, DHHC domain containing 3 Unknown •  1.6 0.003041 1.9 0.000346 1.6 0.069813 
 205198_s_at ATP7A ATPase, Cu++ transporting, α polypeptide Copper ion transporter, ATP binding •  1.6 0.000167 1.7 0.003041 1.5 0.088938 
 218611_at IER5 Immediate early response 5 Unknown  ○ 1.4 0.003041 1.6 0.000241 1.7 0.000241 
 202581_at HSPA1B Heat shock 70 kDa protein 1B Heat shock response  ○ 1.3 0.000068 1.9 0.00002 1.3 0.00249 
 200748_s_at FTH1 Ferritin, heavy polypeptide 1 Iron binding, cell proliferation  ○ 1.6 0.000147 1.3 0.00002 1.7 0.000046 
Genes downregulated            
 218841_at MGC5540 Hypothetical protein MGC5540 Unknown •  2.0 0.994591 1.8 0.934434 4.1 0.973302 
 203788_s_at SEMA3C Domain, secreted, (semaphorin) 3C Drug resistance •  2.1 0.703911 2.2 0.971234 1.9 0.998799 
 201581_at DJ971N18.2 Hypothetical protein DJ971N18.2 Unknown •  1.9 0.98579 1.5 0.994067 2.0 0.999932 
 202906_s_at NBS1 Nijmegen breakage syndrome 1 (nibrin) DNA repair •  1.9 0.990708 1.6 0.98579 1.7 0.998923 
 202346_at HIP2 Huntingtin interacting protein 2 Ubiquitin protein enzyme •  2.0 0.999833 1.5 0.999948 1.5 0.99288 
 204632_at RPS6KA4 Ribosomal protein S6 kinase, 90 kDa, polypeptide 4 Protein kinase •  1.6 0.996301 1.8 0.998514 1.5 0.454766 
 222155_s_at FLJ11856 Putative G-protein coupled receptor GPCR41 Unknown • ○ 1.7 0.999135 1.7 0.999886 1.5 0.99994 
213622_at COL9A2 Collagen, type IX, α 2 Skeletal development •  1.6 0.5 1.7 0.925732 1.6 0.545234 
a

Criteria shows how the genes were selected by data analysis. • indicates that the gene was selected by fold-change. ○ indicates that the gene was selected by Wilcoxon’s test. • ○ indicates that the gene was selected by both fold-change and Wilcoxon’s test.

b

F.C., fold change.

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