Genes Genet. Syst. (2008) 83, p. 339–345 A comparison of 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. 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 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 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 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 419 HS.449909 RPSA Ribosomal protein SA TTCATACACC 382 HS.631498 Transcribed locus, strongly similar to NP_002286.2 TTGGTCCTCT 379 HS.632703 RPL41 Ribosomal protein L41 TCAGATCTTT 371 HS.446628 RPS4X Ribosomal protein S4, X-linked CCACTGCACT 353 HS.107003 CCNB1IP1 Cyclin B1 interacting protein 1 GGCAAGCCCC 347 HS.546269 RPL10A Ribosomal protein L10a GGGCTGGGGT 347 HS.425125 RPL29 Ribosomal protein L29 AGGGCTTCCA 330 HS.534404 RPL10 Ribosomal protein L10 CACCTAATTG 301 HS.591538 Transcribed locus, strongly similar to NP_536849.1 TTGTAATCGT 284 HS.446427 OAZ1 Ornithine decarboxylase antizyme 1 TTGGCAGCCC 275 HS.523463 RPL27A Ribosomal protein L27a CTCCTCACCT 268 HS.523185 RPL13A Ribosomal protein L13a TGTACCTGTA 263 HS.705373 TUBA1B Tubulin, alpha 1b TAGGTTGTCT 260 HS.374596 TPT1 Tumor protein, translationally-controlled 1 TACCATCAAT 249 HS.544577 GAPDH Glyceraldehyde-3-phosphate dehydrogenase CGCTGGTTCC 244 HS.388664 RPL11 Ribosomal protein L11 CCCCAGTTGC 240 HS.515371 CAPNS1 Calpain, small subunit 1 AATAGGTCCA 239 HS.512676 RPS25 Ribosomal protein S25 CCAGAACAGA 229 HS.400295 RPL30 Ribosomal protein L30 GTGAAACCCT 222 HS.213088 CHST12 Carbohydrate (chondroitin 4) sulfotransferase 12 GCAAGCCAAC 204 HS.631498 Transcribed locus, strongly similar to NP_002286.2 TAATAAAGGT 203 HS.512675 RPS8 Ribosomal protein S8 ACTTACCTGC 183 HS.431668 COX6B1 Cytochrome c oxidase subunit Vib polypeptide 1 (ubiquitous) AAGACAGTGG 183 HS.433701 RPL37A Ribosomal protein L37a GACTCTGGTG 179 HS.370504 RPS15A Ribosomal protein S15a CTGTTGATTG 175 HS.447506 RP11-78J21.1 Heterogeneous nuclear ribonucleoprotein A1-like TTACCTCCTT 171 HS.469925 FAM128B Family with sequence similarity 128, member B GTGAAGGCAG 170 HS.356572 RPS3A Ribosomal protein S3A AGGCTACGGA 166 HS.523185 RPL13A Ribosomal protein L13a GTAAGTGTAC 165 Novel Novel Novel CACAAACGGT 159 HS.546291 RPS27 Ribosomal protein S27 (metallopanstimulin 1) GTTGTGGTTA 154 HS.534255 Transcribed locus, strongly similar to NP_004039.1 GCGACAGCTC 150 HS.649475 RPL24 Ribosomal protein L24 ACATCATCGA 149 HS.408054 RPL12 Ribosomal protein L12 ATTGTTTATG 146 HS.181163 HMGN2 High-mobility group nucleosomal binding domain 2 CCCCAGCCAG 141 HS.546286 RPS3 Ribosomal protein S3 GACTCACTTT 135 HS.434937 PPIB Peptidylprolyl isomerase B (cyclophilin B) GGCCACGTAG 134 HS.155597 CFD Complement factor D (adipsin) CTAAGACTTC 134 HS.534061 Transcribed locus, strongly similar to XP_982612.1 AGCTCTCCCT 132 HS.374588 RPL17 Ribosomal protein L17 GCCTCCTCCC 130 HS.314359 EIF3K Eukaryotic translation initiation factor 3, subunit K TCTCCATACC 130 Novel Novel Novel AAAACATTCT 128 HS.694999 RP11-217H1.1 Implantation-associated protein GCTCCGAGCG 127 HS.397609 RPS16 Ribosomal protein S16 TGGGCAAAGC 124 HS.144835 EEF1G Eukaryotic translation elongation factor 1 gamma TTGGGGTTTC 120 HS.524910 FTH1 Ferritin, heavy polypeptide 1 GTGACCTCCT 117 HS.433901 COX8A Cytochrome c oxidase subunit 8A (ubiquitous) GCGTGCTCTC 116 HS.337766 RPL18A Ribosomal protein L18a GCAAAACCCC 115 HS.308074 C9orf5 Chromosome 9 open reading frame 5 ATCACGCCCT 115 HS.694507 Transcribed locus, strongly similar to NP_976229.1 CCTGTAATCC 114 HS.650174 HLA-E Major histocompatibility complex, class I, E TTGGAGATCT 110 HS.50098 NDUFA4 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 4, 9 kDa ATGGCGATCT 106 HS.356794 RPS24 Ribosomal protein S24 ATCCTTGCTG 102 HS.518198 CSTA Cystatin A (stefin A) GCCTGCTGGG 101 HS.433951 GPX4 Glutathione peroxidase 4 (phospholipid hydroperoxidase) 342 J. LEE et al. matching tags and the majority of the novel tags were variety of genes among the remaining 26 genes. Some concentrated in the low-abundance class. were the housekeeping genes such as GAPDH and FTH1 and some were functional genes related with Genes highly expressed in Mono Mac 6 cells To chemotaxis such as PPIA and PPIB (Xu et al., 1992; check highly expressed genes in MM6 cells, the tags with Yurchenko et al., 2001) (Table 2). Although the highly more than 100 copies were arranged (Table 2). There expressed genes were helpful for determining the charac- were 68 SAGE tags, and all the tags except two were ter of the MM6 cells, because the SAGE tag data of MM6 matched to known genes or EST. Of these, 33 tags were only show the expression level of the individual tran- represented to the ribosomal and 7 were tran- script, in order to obtain more meaningful data, further scribed loci similar to protein sequences. There were a analysis is required. Accordingly, we performed the

Table 3. Classification of the alternatively expressed genes

(A) Over-expressed Genes in MM6 cells (491) Term Count* %* P-Value* cellular biosynthetic process 79 16.1 6.90E-16 macromolecule biosynthetic process 66 13.4 2.30E-14 protein metabolic process 146 29.7 2.20E-09 ribonucleoprotein complex biogenesis and assembly 23 4.7 1.00E-08 cellular macromolecule metabolic process 138 28.1 2.10E-08 cellular catabolic process 34 6.9 2.30E-05 rRNA processing 10 2 3.20E-05 macromolecule catabolic process 27 5.5 6.70E-05 mRNA processing 18 3.7 3.60E-04 macromolecular complex assembly 28 5.7 1.10E-03 cellular component assembly 29 5.9 1.60E-03 RNA splicing 15 3.1 2.00E-03 electron transport 24 4.9 2.80E-03 regulation of protein metabolic process 17 3.5 5.40E-03 regulation of programmed cell death 25 5.1 6.30E-03

(B) Under-expressed Genes in MM6 cells (136) Term Count % P-Value cellular biosynthetic process 30 22.1 5.60E-09 macromolecule biosynthetic process 25 18.4 6.50E-08 protein metabolic process 43 31.6 2.10E-03 locomotory behavior 6 4.4 1.70E-02 chemotaxis 5 3.7 2.70E-02 negative regulation of biological process 16 11.8 3.30E-02 homeostatic process 8 5.9 4.00E-02 cell motility 8 5.9 4.10E-02 negative regulation of cellular process 15 11 4.70E-02 cell death 12 8.8 5.10E-02 macromolecule catabolic process 8 5.9 5.10E-02 oxidative phosphorylation 4 2.9 5.70E-02 cell differentiation 21 15.4 5.90E-02 * Count indicates the number of genes. * The percentage(%) represents the ratio of gene numbers per the total numbers of genes. * P-Value means the measure of gene-enrichment in annotation terms. Briefly, the smaller, the more enriched.[http://david.abcc.ncifcrf.gov/helps/functional_annotation.html#fisher]. A comparison of gene expression profiles 343 comparison analysis between MM6 cells and the primary analyzed by the DAVID Functional Annotation Tool human t(9;11) AML cells that have the same chromosome (http://david.abcc.ncifcrf.gov/). 491 out of 644 over- translocation t(9;11). expressed genes had DAVID ID. Majority of the genes were related with biosynthetic and metabolic processes on A comparison of Gene Expression profiles between the GOTERM_BP_3 category (Table 3A). On the other Mono Mac 6 cells and the primary human t(9;11) hand, 136 out of the 186 under-expressed genes were cells To examine whether the pattern of gene matched to DAVID IDs, and the result of grouping the expression of the Mono Mac 6 cell line is different from genes showed that many genes were involved in that of primary cells from bone marrow samples with biosynthetic and metabolic processes, similar to the over- t(9;11) AML patients (the SAGE tags data were from Lee expressed genes (Table 3B). Collectively, although there et al., 2006), the SAGE tags from both cells were were a number of the alternatively expressed genes compared via statistical and bioinformatical analysis. between MM6 and primary cells, the majority of the Among the 884 unique SAGE tag obtained from the genes belonged to similar biological process. That analysis, p values < 0.05 and 4-fold expression change, implies the gene expression of MM6 cells is generally 693 (78%) tags were increased in MM6 cells and 191 similar to that of primary AML cells. (22%) were decreased. And 830 (94%) were matched to Then, in order to find out the additional feature of the SAGE map database and 54 (6%) had no match. To MM6, we performed an in-depth study. To examine classify the 830 matched genes, the SAGE tags were which biological pathways were strengthened or weak-

Table 4. The genes involved in Erk1/Erk2 MAPK signaling pathway

Genes UniGene ID SAGE Tag SAGE tag copy ratio* P value Primary MM6 HRAS HS.37003 GCGCTGTACT 0 86 0.018118 PDGFRA HS.74615 TGATTGGTGG 153 975 0.000000 MKNK2 HS.515032 TGTTAATGTT 15 103 0.041441 RPS6KA1 HS.149957 GCCTTTCTAA 46 308 0.000412 ※ The genes obtained from statistical analysis (p < 0.05).

NRAS HS.486502 GCACTGTACT 15 17 0.937659 KRAS HS.505033 AACTGTACTA 0 17 0.290586 KRAS HS.505033 GTCACTCTCC 15 17 0.937659 KRAS HS.505033 TGCTTGTTTT 0 17 0.290586 KRAS HS.505033 TTTGTGGAAG 0 17 0.290586 ※ Other RAS family genes.

GRB2 HS.444356 AAGCCTTGCT 214 359 0.130427 SOS2 HS.291533 GGAAGTGCAT 0 17 0.290586 RAF1 HS.159130 TGTTCTGCCT 15 17 0.937659 RAF1 HS.159130 TGTATACAAG 15 17 0.937659 ARAF HS.446641 CCATCCCGGA 0 17 0.290586 MAP2K1 HS.145442 AGTTGCTTCA 0 34 0.135019 MAPK1 HS.431850 GCACCTTATT 31 17 0.629487 MAPK1 HS.431850 TTTCAAACAC 0 17 0.290586 MAPK1 HS.431850 GTGTGCTCTG 0 17 0.290586 HS.202453 ATCAAATGCA 15 17 0.937659 ELK4 HS.497520 TAACCAAAAA 15 17 0.937659 SRF HS.520140 GTCACAGTCC 31 120 0.066477 ※ Other genes involved in the pathway.

* SAGE tag copy ratio indicates the number of tags per total 1 million copies in both cells. 344 J. LEE et al. ened in MM6 cells, the over-expressed or under-expressed over-expressed in MM6 cells. Therefore, the results genes were applied to the biocarta pathway category on indicate that MM6 cell line can be a good model for the DAVID Functional Annotation Tool. Interestingly, researching HRAS- positive t(9;11) AML. Based on this four genes involved in Erk1/Erk2 MAPK pathway were result, MM6 cell line is expected to be applied to the exhibited to be over-expressed in MM6 cells. The path- correlation study between the expression of HRAS and way is known to govern the growth, proliferation, differ- leukemia. entiation and survival of many, if not all, cell types and cause various diseases, including cancer when deregu- This study was supported by a research grant from Ministry lated (Orton et al., 2005). 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