Genes Genet. Syst. (2008) 83, p. 339–345 A comparison of gene expression profiles between primary human AML cells and AML cell line Jinseok Lee1, Junmo Hwang1, Hyung-Soo Kim1, Seonggon Kim1, Young Hun Kim1, So-Young Park2, Kil Soo Kim3, Zae Young Ryoo1, Kyu-Tae Chang4 and Sanggyu Lee1* 1School of Life Science and Biotechnology, Kyungpook National University, Daegu, Republic of Korea 2Environmental Toxico-Genomic & Proteomic Center, College of Medicine, Korea University, 5 Anam-dong, Sungbukgu, Seoul 136-705, Korea 3Department of Veterinary Medicine, Kyungpook National University, Daegu, Republic of Korea 4National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon, Republic of Korea (Received 20 June 2008, accepted 5 August 2008) In acute myeloid leukemia (AML), hematologic malignancies are characterized by recurring chromosomal abnormalities. Chromosome translocation t(9;11)(p22;q23) is one of the most common genetic aberrations and results in the formation of the MLL-AF9 fusion gene that functions as a facilitator of cell growth directly. In order to study this type of AML, the cell lines with cytogenetically diagnosed t(9;11)(p22;q23), such as Mono Mac 6 (MM6), have been widely used. To examine whether there is any difference in gene expression between the primary human t(9;11) AML cells and MM6 cell line, genome-wide transcriptome analysis was performed on MM6 cell line using SAGE and the results were compared to the profile of primary human t(9;11) AML cells. 884 transcripts which were alternatively expressed between MM6 cells and primary human t(9;11) cells were identified through statistical analysis (P < 0.05) and 4-fold expression change. Of these transcripts, 830 (94%) matched to known genes or EST were classified by functional categories (http://david.abcc.ncifcrf.gov/). The majority of alternatively expressed genes in MM6 were involved in biosynthetic and metabolic processes, but HRAS, a protein that is known to be associated with leukemogenesis, was expressed only in MM6 cells and several other genes involved in Erk1/Erk2 MAPK pathway were also over-expressed in MM6. Therefore, since MM6 cell line has a similar expression profile to primary human t(9;11) AML in general and expresses uniquely a strong Erk1/Erk2 MAPK pathway including HRAS, it can be used as a model for HRAS-positive t(9;11) AML. Key words: AML, gene expression, SAGE, translocation al., 1996). Nearly 40 different partner genes, many of INTRODUCTION which are transcription factors, have been identified as Recurring chromosome rearrangements are a common being involved in this translocation (Daser and Rabbitts, feature of hematopoietic malignancies. In acute myeloid 2004). Of these genes, the AF9 gene located at human leukemia (AML), the MLL (mixed-lineage leukemia) gene chromosome band 9p22 is known as one of the most located at human chromosome band 11q23 is frequently common partner genes with MLL. Chromosome involved in reciprocal chromosome translocation with translocation t(9;11)(p22;q23) leads to form the MLL-AF9 other genes, which results in a break in MLL and the fusion protein that functions as a facilitator of cell growth partner genes, and leads to formation of a new fusion directly (Nakamura et al., 1993; Pession et al., 2003). gene. The fusion gene contains 5’ MLL joined to the 3’ The availability of human leukemia cell lines as a self- part of the partner gene (Thirman et al., 1993; Rubnitz et renewing resource of accessible and manipulable living cells has contributed significantly to a better under- Edited by Hiroshi Iwasaki standing of the pathophysiology of hematopoietic tumors * Corresponding author. E-mail: [email protected] (Drexler et al., 2000). For researching acute myeloid 340 J. LEE et al. leukemia carrying t(9;11)(p22;q23), the cell lines with Bioinformatics and Statistical Analysis SAGE tags cytogenetically diagnosed t(9;11)(p22;q23) have been were matched to SAGEmap database updated on Febru- widely used in laboratories. Mono Mac 6 (MM6), one of ary 19, 2008 (http://www.ncbi.nlm.nih.gov/SAGE/). For these cell lines, is a human acute monocytic leukemia cell the SAGE tags shared by multiple genes, only the single- line (AML-M5; FAB-classification) which was originally matched SAGE tags were selected. To determine the dif- established from the peripheral blood of a 64-year-old ferentially expressed genes between the primary human male patient with monoblastic leukemia in 1988 (Ziegler- t(9;11) and MM6 cells, SAGE data were analyzed using Heitbrock et al., 1988). The MM6 cell line have a IDEG6 (http://telethon.bio.unipd.it/bioinfo/IDEG6_form/). complex karyotype like hypotetraploid, including 2 copies For statistical analysis, a general Chi-squared test, with of normal chromosome 9 and 11 as well as 2 copies of the the significance threshold set as 0.05, was performed. t(9;11) translocated forms (MacLeod et al., 1993; Super et The differentially expressed genes between the primary al., 1995). In addition, the MM6 cell line exhibits a human and MM6 cells were selected based on p values phagocytosis of antibody-coated erythrocytes in 80% of < 0.05 and over 4-fold expression change. the cells, and it is known to exhibit the phenotypic and functional features of mature monocytes (Ziegler- Acquisition of the primary human t(9;11) cells Heitbrock et al., 1988). Therefore, the MM6 cell line has SAGE data For the comparison with MM6, the SAGE been applied for various experiments as a model of data of primary human t(9;11) cells were obtained from monocytes and t(9;11) AML cells. the paper published by Lee et al. (2006). The data of In addressing the genome response to various stimuli three t(9;11) AML-M5/5a (FAB classification) cells were on cell lines, microarray-based approaches have been integrated and extracted randomly to make one widely used. The microarray-based approaches can only expression profile. As a result, total 65,307 tags having detect the known genes or ESTs. However, SAGE unique 36,465 tags were constructed. (Serial Analysis of Gene Expression) can detect not only relatively rare transcripts but also novel transcripts, Functional Classification of the differentially regardless of the expression level. expressed genes For the functional classification of In this study, SAGE was performed to determine the the differentially expressed genes between the primary gene expression profile of the MM6 cell line. This study human t(9;11) and MM6 cells, EASE (version 2.0) soft- is an attempt to determine whether the gene expression ware (http://david.niaid.nih.gov/david/ease.htm) was used pattern of the cell line is different from the primary for gene ontology analysis. EASE can perform a statis- human t(9;11) cells that have a same chromosome tical analysis of gene categories in a gene list to find those translocation, in an aim to provide a new insight into the that are most overrepresented, either because of under- or AML related studies. over-expression. This enabled the ‘‘biological process’’ for the analyzed genes able to be defined. MATERIALS AND METHODS RESULTS AND DISCUSSION Mono Mac 6 cell line Mono Mac 6 cell line was obtained from DSMZ (Braunschweig, Germany). Mono Distribution of the SAGE tags from Mono Mac 6 Mac 6 Cells were cultured in an RPMI 1640 medium cells 58,472 SAGE tags were collected from the MM6 supplemented with 10% FBS, 100 unit/ml penicillin, and cells and 14,661 unique SAGE tags were identified. 100 μg/ml streptomycin in a humidified 5% CO2 These unique SAGE tags were matched to the reference atmosphere. database (SAGEmap), which showed that 74% of the tags were matched to known transcripts (Table 1). Generally, SAGE library construction and SAGE tag collec- the tags of high copies accounted for high percentage in tion SAGE libraries were constructed following SAGE protocol (Lee et al., 2001). Briefly, total RNA and mRNA were purified from MM6 cells. Double-strand cDNA Table 1. Distribution of the SAGE tags from MM6 cells were synthesized, and 3' cDNA were purified using NlaIII ≥ 100 99 to 10 9 to 5 4 to 2 1 Total digestion. SAGE tags were released from 3' cDNA for Unique tags 68 623 762 2,720 10,488 14,661 cancatemerization and cloning into the pZero vector. % 0 4 5 19 72 100 Sequencing reactions for SAGE clones were performed Matched tags 66 613 726 2,437 6,991 10,833 with ABI Big-Dye 3.1 kit. SAGE tag sequences were col- % 97 98 95 90 67 74 lected with an ABI3730 sequencer. Sequences passed Novel tags 2 10 36 283 3,497 3,828 Phred20 were used for SAGE tag extraction. SAGE tags were extracted from the sequences using SAGE 2000 soft- % 3 2 5 10 33 26 ware. ※ Total SAGE tags: 58,472. A comparison of gene expression profiles 341 Table 2. Genes highly expressed in MM6 cells Tag Copies UniGene ID Symbol Title TGCACGTTTT 1459 HS.265174 RPL32 Ribosomal protein L32 CCCATCGTCC 1262 HS.694507 Transcribed locus, strongly similar to NP_976229.1 GAGGGAGTTT 1048 HS.523463 RPL27A Ribosomal protein L27a CAATAAATGT 767 HS.558601 RPL37 Ribosomal protein L37 GGATTTGGCC 754 HS.437594 RPLP2 Ribosomal protein, large, P2 GCATAATAGG 702 HS.381123 RPL21 Ribosomal protein L21 AGCACCTCCA 678 HS.515070 EEF2 Eukaryotic translation elongation factor 2 CCTAGCTGGA 659 HS.356331 PPIA Peptidylprolyl isomerase A (cyclophilin A) CTCATAAGGA 566 HS.406683 RPS15 Ribosomal protein S15 ATTCTCCAGT 525 HS.406300 RPL23 Ribosomal protein L23 ATAATTCTTT 492 HS.156367 RPS29 Ribosomal protein S29 GTGAAACCCC 479 HS.527778 CD82 CD82 molecule AGGAAAGCTG 434 HS.408018 RPL36 Ribosomal protein L36 GAAAAATGGT