High-resolution analysis of aberrant regions in autosomal chromosomes in human leukemia THP-1 cell line
- Naoki Adati†1,
- Ming-Chih Huang†1, 3,
- Takahiro Suzuki2,
- Harukazu Suzuki2 and
- Toshio Kojima1Email author
© Kojima et al; licensee BioMed Central Ltd. 2009
Received: 24 April 2009
Accepted: 27 July 2009
Published: 27 July 2009
THP-1 is a human monocytic leukemia cell line derived from a patient with acute monocytic leukemia. The cell line differentiates into macrophage-like cells by stimulation with phorbol myristate acetate (PMA). Although it has been used frequently as a model for macrophage differentiation in research including the FANTOM4/Genome Network Project, there are few reports on its genomic constitution. Therefore, we attempted to reveal the genomic aberrations in these cells with the microarray-based comparative genomic hybridization (aCGH) technique.
We report large aberrations, including deletions 6p, 12p, 17p, and trisomy 8, and revealed breakpoints in the MLL and MLLT3 genes. Moreover, we found novel genomic aberrations such as a hemizygous narrow deletion partially containing the TP73 gene and homozygous deletions, including the CDKN2A, CDKN2B and PTEN genes.
In this study, we identified 119 aberrant regions in autosomal chromosomes, and at least 16 of these aberrations were less than 100 kb, most of which were undetectable in the previous works. We also revealed a total of 4.6 Mb of homozygous deleted regions. Our results will provide a base to precisely understand studies involving the THP-1 cell line, especially the huge amount of data generated from the FANTOM4/Genome Network Project.
As models for the study of myeloid differentiation and hematopoietic cell differentiation, several human leukemia cell lines are available . Although these myeloid leukemia cell lines are blocked at certain steps in the maturation and differentiation process, they can be induced to differentiate into macrophage-like cells by several stimuli [1, 2].
THP-1 is a human monocytic leukemia cell line that was cultured from the blood of a 1-year-old male with acute monocytic leukemia . On stimulation with phorbol 12-myristate 13-acetate (PMA), THP-1 cells cease proliferation, become adherent, and differentiate into macrophage-like cells. They resemble native monocyte-derived macrophages with respect to numerous criteria [4, 5]. In comparison with other human myeloid cell lines such as HL-60, U937, KG-1 or HEL cells, differentiated THP-1 cells behave more like native monocyte-derived macrophages . Because of these characteristics, the THP-1 cell line is a valuable model for studying the mechanisms involved in macrophage differentiation. Therefore, THP-1 has been used not only as a clinical model of a leukemic cell, but also as a scientific model of differentiation in response to various stimuli.
Chromosome rearrangements are commonly associated with multiple disease states such as cancer. The identification and analysis of these genomic rearrangements have been fundamental for the advancement of research in these diseases. Cell lines are mostly established from such disordered tissues, and in the case of some cultured cells, their genomic constitutions and characteristics continuously alter through passages. Heterogeneity of cells and its derivative cell lines along with different characteristics were also reported in the case of THP-1 [4, 6, 7]. In the present study, we adopted microarray-based comparative genomic hybridization (aCGH) techniques and attempted to provide a comprehensive and detailed understanding of the genomic aberrations in THP-1 cells.
Materials and methods
The THP-1 cell line was subcloned by the limiting dilution technique and 1 clone (#5) was selected for its ability to differentiate relatively homogeneously in response to PMA . THP-1 cells were cultured in RPMI, containing 10% fetal bovine serum (FBS), penicillin/streptomycin, 10 mM HEPES, 1 mM sodium pyruvate and 50 μM 2-mercaptoethanol. Genomic DNA was extracted from 5 × 106 cells according to the manufacturer's instructions with the illustra GenomicPrep Cells and Tissue DNA Isolation Kit (GE Healthcare UK Ltd., Buckinghamshire, England) and quantified spectrophotometrically. Human Genomic DNA: Female (Promega Corporation, Madison, WI, USA) was purchased as a reference sample.
Microarray-based CGH Analysis
Oligonucleotide microarray experiment using the Human Genome CGH Microarray Kit 244A (Agilent Technologies, Inc., Santa Clara, CA, USA) was conducted according to manufacturer's protocol (version 5.0). The microarray used for this study was a 1× 244 K slide format printed using Agilent's 60-mer SurePrint technology, and it has 236385 biological features. Its probes span both the coding and noncoding regions for comprehensive genome-wide representation, and the overall median probe spacing is 8.9 kb (7.4 kb in RefSeq genes). THP-1 and human female genomic DNA (1 μg each) were labeled with Cy5 and Cy3, respectively. The hybridized and washed array slide was scanned with an Agilent MicroArray Scanner G2505A (Agilent Technologies, Inc.) and the obtained TIFF image data was processed with Agilent Feature Extraction software (version 18.104.22.168) by the CGH-v4_95_Feb07 protocol (Agilent Technologies, Inc.). Extracted data was analyzed with Agilent DNA Analytics 4.0 software (version 4.0.81) (Agilent Technologies, Inc.) and the Aberration Detection Method 2 (ADM-2) algorithm  was used to identify contiguous genomic regions that corresponded to chromosomal aberrations. Following parameters were used in this analysis: Threshold of ADM-2: 6.0; Centralization: ON (Threshold: 6.0, Bin Size: 10); Fuzzy Zero: ON; Aberration Filters: ON (minProbes = 3 AND minAvgAbsLogRatio = 0.5 AND maxAberrations = 10000 AND percentPenetrance = 0); Feature Level Filters: ON (gIsSaturated = true OR rIsSaturated = true OR gIsFeatNonUnifOL = true OR rIsFeatNonUnifOL = true). At a minimum, 3 contiguous suprathreshold probes were required to define a change. To find an obvious homozygous deletion, aberrant regions with a signal log ratio of less than -3.0 were searched. Genomic positions were based on the UCSC March 2006 human reference sequence (hg18) (NCBI build 36.1 reference sequence).
Microarray data generated from this study has been deposited in the Center for Information Biology gene EXpression database (CIBEX) in DNA Data Bank of Japan (DDBJ) (CIBEX accession number: CBX82)  and is also available from the Genome Network Platform .
Results and discussion
THP-1 cells were established by Tsuchiya et al., and they were reported to have a diploid (46, XY) chromosome number by karyotype analysis . It is important to examine the genomic constitution of these cells in order to interpret the characteristics of THP-1, but there is little information on the whole-genome analysis of THP-1 cells. Odero et al. conducted cytogenetic analyses to detect chromosome changes using G-banding, fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY) . They reported that these cells have a near-diploid karyotype and contain a number of aberrations. The disadvantage of their techniques, however, was the low-resolution genomic mapping along the chromosome, permitting the identification of only large chromosomal aberrations.
Recently, the microarray-based CGH (aCGH) technique, in particular, the high-density oligonucleotide array-based technique, was established for whole-genome aberration analysis, and this increased the mapping resolution. A high mapping resolution enables precise definition of aberration boundaries and identification of small homozygous deletions, pinpointing the possible locations of tumor-suppressor genes. At present, microarray-based CGH methods focus on detecting copy number changes, but rearrangements, such as balanced translocations or inversions, are not detected by this method.
Regions of homozygous deletion in THP-1 cells.
163997028 – 164101976
69138837 – 69166014
182336611 – 182614113
21271776 – 22236965
IFNA5, KLHL9, IFNA6, IFNA13, IFNA2, IFNA8,
IFNA1, IFNE1, MTAP, CDKN2A, CDKN2B, hsa-mir-31
89654755 – 89680176
41631875 – 44807102
CASK, MAOA, MAOB, NDP, EFHC2, FUNDC1, DUSP21, UTX
153194224 – 153211806
The FANTOM4/Genome Network Project used THP-1 cells as the model system to understand the transcriptional network underlying growth arrest and differentiation in mammalian cells. A large amount of data, including the genome-wide transcription start site (TSS) and systematic siRNA knockdown of key transcription factors, was generated in this project . We analyzed the identical THP-1 clone used in the FANTOM4/Genome Network Project. The results of this study will provide a useful base for studies in general cell biology, and they would help to precisely understand the data generated by the FANTOM4/Genome Network Project.
We thank Ms. Yuko Sawada, Agilent Technologies Japan, Ltd., for providing technical support. This work was supported by the in-house budget of RIKEN.
- Koeffler HP: Human acute myeloid leukemia lines: models of leukemogenesis. Semin Hematol. 1986, 23: 223-236.PubMed
- Åbrink M, Gobl AE, Huang R, Nilsson K, Hellman L: Human cell lines U-937, THP-1 and Mono Mac 6 represent relatively immature cells of the monocyte-macrophage cell lineage. Leukemia. 1994, 8: 1579-1584.PubMed
- Tsuchiya S, Yamabe M, Yamaguchi Y, Kobayashi Y, Konno T, Tada K: Establishment and characterization of a human acute monocytic leukemia cell line (THP-1). Int J Cancer. 1980, 26: 171-176. 10.1002/ijc.2910260208.View ArticlePubMed
- Tsuchiya S, Kobayashi Y, Goto Y, Okumura H, Nakae S, Konno T, Tada K: Induction of maturation in cultured human monocytic leukemia cells by a phorbol diester. Cancer Res. 1982, 42: 1530-1536.PubMed
- Auwerx J: The human leukemia cell line, THP-1: a multifacetted model for the study of monocyte-macrophage differentiation. Experientia. 1991, 47: 22-31. 10.1007/BF02041244.View ArticlePubMed
- Odero MD, Zeleznik-Le NJ, Chinwalla V, Rowley JD: Cytogenetic and molecular analysis of the acute monocytic leukemia cell line THP-1 with an MLL-AF9 translocation. Genes Chromosomes Cancer. 2000, 29: 333-338. 10.1002/1098-2264(2000)9999:9999<::AID-GCC1040>3.0.CO;2-Z.View ArticlePubMed
- Tominaga T, Suzuki M, Saeki H, Matsuno S, Tachibana T, Kudo T: Establishment of an activated macrophage cell line, A-THP-1, and its properties. Tohoku J Exp Med. 1998, 186: 99-119. 10.1620/tjem.186.99.View ArticlePubMed
- Suzuki H, Forrest ARR, van Nimwegen E, additional 157 co-authors: The transcriptional network that controls growth arrest and differentiation in a human myeloid leukemia cell line. Nat Genet. 2009, 41: 553-562. 10.1038/ng.375.View ArticlePubMed
- Lipson D, Aumann Y, Ben-Dor A, Linial N, Yakhini Z: Efficient calculation of interval scores for DNA copy number data analysis. J Comput Biol. 2006, 13: 215-228. 10.1089/cmb.2006.13.215.View ArticlePubMed
- Center for Information Biology gene EXpression database. [http://cibex.nig.ac.jp/cibex2/index.jsp]
- Genome Network Platform. [http://genomenetwork.nig.ac.jp/index_e.html]
- Langer T, Metzler M, Reinhardt D, Viehmann S, Borkhardt A, Reichel M, Stanulla M, Schrappe M, Creutzig U, Ritter J, Leis T, Jacobs U, Harbott J, Beck JD, Rascher W, Repp R: Analysis of t(9;11) chromosomal breakpoint sequences in childhood acute leukemia: almost identical MLL breakpoints in therapy-related AML after treatment without etoposides. Genes Chromosomes Cancer. 2003, 36: 393-401. 10.1002/gcc.10167.View ArticlePubMed
- Ogawa S, Hirano N, Sato N, Takahashi T, Hangaishi A, Tanaka K, Kurokawa M, Tanaka T, Mitani K, Yazaki Y, Hirai H: Homozygous loss of the cyclin-dependent kinase 4-inhibitor (p16) gene in human leukemias. Blood. 1994, 84: 2431-2435.PubMed
- Guo S-X, Taki T, Ohnishi H, Piao H-Y, Tabuchi K, Bessho F, Hanada R, Yanagisawa M, Hayashi Y: Hypermethylation of p16 and p15 genes and RB protein expression in acute leukemia. Leuk Res. 2000, 24: 39-46. 10.1016/S0145-2126(99)00158-7.View ArticlePubMed
- Yin Y, Shen WH: PTEN: a new guardian of the genome. Oncogene. 2008, 27: 5443-5453. 10.1038/onc.2008.241.View ArticlePubMed
- Dahia PLM, Aguiar RCT, Alberta J, Kum JB, Caron S, Sill H, Marsh DJ, Ritz J, Freedman A, Stiles C, Eng C: PTEN is inversely correlated with the cell survival factor Akt/PKB and is inactivated via multiple mechanisms in haematological malignancies. Hum Mol Genet. 1999, 8: 185-193. 10.1093/hmg/8.2.185.View ArticlePubMed
- Yilmaz ÖH, Valdez R, Theisen BK, Guo W, Ferguson DO, Wu H, Morrison SJ: Pten dependence distinguishes haematopoietic stem cells from leukaemia-initiating cells. Nature. 2006, 441: 475-482. 10.1038/nature04703.View ArticlePubMed
- Pluta A, Nyman U, Joseph B, Robak T, Zhivotovsky B, Smolewski P: The role of p73 in hematological malignancies. Leukemia. 2006, 20: 757-766. 10.1038/sj.leu.2404166.View ArticlePubMed
- Peters UR, Tschan MP, Kreuzer KA, Baskaynak G, Lass U, Tobler A, Fey MF, Schmidt CA: Distinct expression patterns of the p53-homologue p73 in malignant and normal hematopoiesis assessed by a novel real-time reverse transcription-polymerase chain reaction assay and protein analysis. Cancer Res. 1999, 59: 4233-4236.PubMed
- Tschan MP, Grob TJ, Peters UR, De Laurenzi V, Huegli B, Kreuzer K-A, Schmidt CA, Melino G, Fey MF, Tobler A, Cajot J-F: Enhanced p73 expression during differentiation and complex p73 isoforms in myeloid leukemia. Biochem Biophys Res Commun. 2000, 277: 62-65. 10.1006/bbrc.2000.3627.View ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.