Identification of Differentially Expressed Genes in Pancreatic Cancer Cells Using Cdna Microarray1

[CANCER RESEARCH 62, 2890–2896, May 15, 2002] Identification of Differentially Expressed Genes in Pancreatic Cancer Cells Using cDNA Microarray1 Haiyong Han, David J. Bearss, L. Walden Browne, Robert Calaluce, Raymond B. Nagle, and Daniel D. Von Hoff2 Arizona Cancer Center [H. H., D. J. B., R. C., R. B. N., D. D. V. H.] and Department of Pathology [W. B., R. C., R. B. N.], University of Arizona, Tucson, Arizona 85724 ABSTRACT pressor genes that are altered in pancreatic cancer include BRCA2 (10), ALK-5 (11), MKK4 (12), and STK11 (13). One oncogene that is To identify new diagnostic markers and drug targets for pancreatic commonly mutated in pancreatic cancer is K-ras.K-ras mutations cancer, we compared the gene expression patterns of pancreatic cancer have been found in 90% of the pancreatic cancers, with most of these cell lines growing in tissue culture with those of normal pancreas using cDNA microarray analysis. Fluorescently (cyanine 5) labeled cDNA being point mutations on codon 12 (7, 14). Some other cancer-related probes, made individually from mRNA samples of nine pancreatic cell genes such as Her-2/neu, COX-2, and VEGF have also been reported lines, were each combined with fluorescently (cyanine 3) labeled universal to be overexpressed in pancreatic cancer cells (15–17). Microsatellite reference mRNA. The mixed probes of each sample were then hybridized instability in cancer cells usually suggests a defective mismatch repair with 5760 cDNA arrays (5289 unique cDNA sequences) printed on indi- system (18). Yamamoto et al. (19) have reported recently that 26 of vidual microscope slides. Fluorescently (cyanine 5) labeled normal pan- 100 sporadic pancreatic tumors showed microsatellite instability, in- creas mRNA was also compared with the same universal reference mRNA dicating mutations in mismatch repair genes in those tumors. reference pool. The expression ratios of neoplastic versus normal pancreas The identification and characterization of these cancer-related cells were then calculated by multiplying the ratio of cancer versus the genes have increased our understanding of pancreatic cancer devel- universal reference mRNA and the ratio of the universal reference mRNA opment, but unfortunately the treatment of pancreatic cancer has not cell versus normal pancreas. For 5289 different genes interrogated by the arrays, 30 of them showed an expression ratio 2 SD from the mean in at advanced as much in the past 20 years. This is mainly attributable to least three of the nine pancreatic cell lines studied. To confirm the the lack of early diagnosis and effective chemotherapeutic treatments. expression profiles of these genes, quantitative reverse transcription-PCR To increase the survival rate of pancreatic cancer patients, better and Northern blot were carried out for 25 of the overexpressed genes. To tumor markers for diagnosis and new molecular targets for drug verify the overexpression in patient samples, two of the overexpressed development are desperately needed. genes, c-Myc and Rad51, were selected to undergo analysis by reverse Because the development and progression of pancreatic cancer is a transcription-PCR in frozen tumor tissues and by immunostaining in very complicated process, we hypothesized that many other genes, as paraffin-embedded tissue section microarrays. The results of these exper- yet undiscovered, are potential tumor markers or drug targets. How- iments are in agreement with the microarray data. Potential up-regulated ever, identifying these genes by conventional methods such as North- targets of note from this study include urokinase-type plasminogen acti- ern blots, differential display, and serial analysis of gene expression vator receptor, serine/threonine kinase 15, thioredoxin reductase, and CDC28 protein kinase 2, as well as several others. has been either labor intensive or nonsystematic (20, 21). Proteomics have provided some promise for massive protein analysis, but tech- niques involved are still in the stage of early development (22). In this INTRODUCTION study, we have used a high-density cDNA microarray technique to assess the gene expression levels in neoplastic versus normal pancre- The development and growth of cancer is a multistep process atic cells. This DNA microarray technique allows simultaneous com- including initiation, progression, invasion, and ultimately establish- parison of a large number of genes in two samples in a quantitative ment of metastatic disease. Each of these steps involves multiple and expeditious way (21). We used a cDNA microarray slide con- genetic alterations that give cancer cells a selective advantage over taining 5289 unique cDNA sequences and compared the mRNA levels normal cells (1). Similar to most tumor types, the development of of nine pancreatic cell lines with that of normal pancreas. We char- malignant adenocarcinoma of the pancreas is thought to be driven by acterized gene-expression profiles that molecularly discriminate pan- the accumulation of genetic alterations. Over the past decade, many creatic cancer cell lines from normal pancreas cells. Moreover, we studies involving pancreatic cancer have searched for cancer-causing identified genes that may be involved in pancreatic tumorigenesis as genes (2, 3). As a result, several cancer-related genes have been well as genes that are potential clinical biomarkers that may lead to an identified in pancreatic tumors. These genes can be categorized into improved early diagnosis for this disease. Several of these genes may three groups including: (a) tumor suppressor genes; (b) oncogenes; also constitute potential novel therapeutic targets. and (c) mismatch repair genes (2, 4, 5). DPC4, p53, and p16 are the three most frequently inactivated tumor suppressor genes in sporadic pancreatic adenocarcinomas (6). Accumulating data suggest that p53 MATERIALS AND METHODS function is inactivated in ϳ75% of pancreatic tumors, whereas p16 is lost in ϳ95% of all pancreatic cancers (7, 8). DPC4, also known as Cell Culture. Pancreatic cell lines AsPC-1, BxPC-3, Capan-1, CFPAC-1, SMAD4, is a tumor growth factor-␤ signaling pathway member and is HPAF II, MIA PaCa-2, PANC-1, and SU.86.86 were purchased from Amer- ican Type Tissue Culture Collection. Pancreatic cell line Mutj (UACC-462) inactivated in ϳ50% of all pancreatic cancers (9). Other tumor sup- was established at the Arizona Cancer Center (23). All pancreatic cell lines were cultured in RPMI 1640 supplemented with fetal bovine serum (10% for Received 9/21/01; accepted 3/7/02. AsPC-1, BxPC-3, HPAF II, MIA PaCa-2 PANC-1, and Mutj; 15% for CF- The costs of publication of this article were defrayed in part by the payment of page PAC-1 and SU.86.86; 20% for Capan-1), penicillin, and streptomycin. HeLa charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. cells were grown in DMEM supplemented with 10% fetal bovine serum. All 1 Supported in part by a grant from the National Foundation for Cancer Research and cells were harvested when they were about 80–90% confluent and were the Arizona Cancer Center. D. V. H. is a fellow of National Foundation for Cancer directly subjected to RNA isolation. Research. Microarray Sample Preparation and Hybridization. cDNA microarray 2 To whom requests for reprints should be addressed, at Arizona Cancer Center, University of Arizona, P. O. Box 245024, Tucson, AZ 85724-5024. Phone: (520) 626- slides used in this study were fabricated in the microarray core facilities at the 7925; Fax: (520) 626-6898; E-mail: [email protected]. Arizona Cancer Center (24). Briefly, each slide has 5760 spots divided into 2890 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 2002 American Association for Cancer Research. PANCREATIC CANCER DIFFERENTIAL GENE EXPRESSION four blocks, with each containing eight identical ice plant genes from Mesem- RT-PCR. Two ␮g of total RNA isolated from pancreatic cancer cell pellets bryanthemum crystallinum and 23 different housekeeping genes as references or frozen pancreatic tumor tissues were used for reverse transcriptase reactions for data normalization. Each slide had 5289 unique human cDNA sequences. (20 ␮l in total volume), which were carried out using the Omniscript RT kit Poly(A)ϩ RNA was directly isolated from cell pellets using the FastTrack (Qiagen, Inc.), following the manufacturer’s protocol. The PCRs were then 2.0 kit (Invitrogen, Carlsbad, CA), following the instruction manual provided carried out by mixing 2 ␮l of reverse transcriptase reaction mixture, 5 ␮lof ϩ by the manufacturer. Normal pancreas poly(A)ϩ RNA was isolated from total 10ϫ PCR buffer containing 15 mM Mg2 ,1␮lof10mM deoxynucleotide RNA, which was purchased from Clontech Laboratories (Palo Alto, CA) using triphosphate mixture, 2.5 ␮lof5␮M PCR primer pair for specific gene, 1 ␮l the Oligotex Direct mRNA kit (Qiagen, Inc., Valencia, CA). This “normal of ␤-actin primer pair, 1 ␮lof␤-actin competimers (Ambion, Inc., Austin, ␮ ␮ ␮ pancreata” consisted of a pool of two tissue specimens donated by two male TX), 37 lofH2O, and 0.5 l of 5 units/ l Taq polymerase (Promega Corp., Caucasians 18 and 40 years of age. Labeling and purification of cDNA probes Madison, WI). The amplification cycle (94°C for 30 s; 56°C for 45 s; and 72°C were carried out using the MICROMAX direct cDNA microarray system for 1 min) was repeated 29 times. PCR primers for individual genes were ϳ (NEN Life Science Products, Boston, MA). Two to 4 ␮g of the poly(A)ϩ RNA designed to generate a DNA fragment 600 bp in length (if the mRNA itself samples were used for each labeling. Probes for each pancreatic cell line were is less than 600 bases, PCR products were generated in maximal length) using labeled with Cy5,3 and probes for HeLa cells were labeled with Cy3. For HeLa the Primer3 program (25). cell versus normal pancreas hybridization, a normal pancreas sample was Northern Blot.

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