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Establishment and characterization of a rare atypical chronic myeloid leukemia cell line NT-1
Leukemia Research 38 (2014) 1111–1116
Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Establishment and characterization of a rare atypical chronic myeloid leukemia cell line NT-1 Juan Qian a,1 , Qin-Rong Wang b,1 , Jie Liu a , Sheng-Hua Jiang a , Xiao-Qing Ni a , Zeng-Hua Lin a , Ya-Ping Zhang a , Hong Liu a,∗ a b
Department of Hematology, Affiliated Hospital of Nantong University, Nantong, China The First Affiliated Hospital of Soochow University, Jiangsu Institute of Hematology, Suzhou, China
a r t i c l e
i n f o
Article history: Received 23 February 2014 Received in revised form 9 June 2014 Accepted 17 June 2014 Available online 25 June 2014 Keywords: Atypical chronic myeloid leukemia Cell line Translocation Tumorigenicity
a b s t r a c t Human leukemia cell lines are of great value in leukemia research. In this study, we established and described the biological characteristics of a rare atypical chronic myeloid (aCML) leukemia cell line (NT1). Mononuclear cells were isolated from the bone marrow of a patient with atypical chronic myeloid leukemia (Ph− /bcr− /abl− ), and were passaged by liquid culture. Cells were maintained without any cytokines for over 1 year, and named NT-1. This cell line was extensively characterized using morphological assays, flow cytometry, cytogenetic analysis, clonogenic culture, quantitative fluorescent PCR, short tandem repeating sequence PCR (STR-PCR) and array-CGH. Its tumorigenic capacity was also examined in nude mice. The NT-1 cell line had morphological features of chronic myeloid leukemia and major myeloid markers (CD13, CD33, CD11b). Additionally, NT-1 expressed progenitor cells and natural killer cell-related antigens such as CD34, CD117, CD56. Cytogenetic analysis initially demonstrated two abnormalities: 47, xx, +8 and 47, xx, +8 accompanied by t(5;12)(q31;p13) translocation. The one-year passage process did not alter the karyotype. NT-1 cells maintained the same morphology, immunophenotyping and cytogenetic features as primary leukemia cells, which was strongly supported by STR-PCR results. Neither Epstein–Barr virus nor mycoplasma was detected in the NT-1 line. In addition, NT-1 cells showed high tumorigenic capacity in nude mice. NT-1 is a new atypical chronic myeloid leukemia cell line with the +8 and t(5,12) translocation, and exhibits high tumorigenicity in nude mice. This new cell line provides a useful tool for the study of leukemogenesis. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction In the past five decades, since the establishment of the Raji cell line, at least 1500 human leukemia lymphoma cell lines have been developed [1,2]. The majority of those lines belong to lymphoidoriginated cell lines, while myeloid-originated cell lines account for only 19.5% [3]. Since 2005, Suning Chen [4], Huiying Qiu [5], and Jinlan Pan [6] have reported at least four acute myeloid leukemia lines. Various methods have been used to identify and characterize these cell lines, including morphological, cytogenetic analysis, fluorescence in situ hybridization (FISH), multiplexFISH
∗ Corresponding author at: Department of Hematology, Affiliated Hospital of Nantong University, Nantong, China. Tel.: +86 13951300660. E-mail address: [email protected] (H. Liu). 1 Both Juan Qian and Qin-Rong Wang are co-authors who contributed equally to this manuscript. http://dx.doi.org/10.1016/j.leukres.2014.06.008 0145-2126/© 2014 Elsevier Ltd. All rights reserved.
(M-FISH), reverse transcriptase polymerase chain reaction (RTPCR), multiplex RT-PCR, short tandem repeat (STR)-PCR, direct sequencing of DNA, clonogenic assay and tumorigenicity in nude mice. These cells have been broadly used in leukemia and oncogenic research, as well as drug development. However, due to the large variation and complexity of leukemic cell lines, new cell lines are always needed for research and drug screening. In this study, we report a new myeloid leukemia cell line named NT-1, which was established from the bone marrow of a atypical chronic myeloid leukemia (aCML) patient in our hospital. Atypical chronic myeloid leukemia is an uncommon myelodysplastic/myeloproliferative (MDS/MPN) neoplasm, with a relative incidence estimated at 1 to 2 cases for every 100 patients with BCR-ABL1-positive chronic myeloid leukemia (CML) [7]. The cell line had 47, xx, +8 and 47, xx, idem, t (5;12)(q31;p13), and showed high tumorigenicity in nude mice. The phenotypic, genetic and functional properties of this cell line were described following the guidelines for characterization and publication of human malignant hematopoietic cell lines [8].
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2. Materials and methods 2.1. Case report In June 2011, a 67-year-old female was admitted to our hospital because of fever and fatigue that had persisted for 1 month. Physical examination revealed splenomegaly (5 cm below the left costal margin) and pleural effusion. Peripheral blood (PB) examination showed a white blood cell (WBC) count of 79 × 109 L−1 , monocytosis <1 × 109 L−1 , basophils <2%, a hemoglobin (Hb) level of 55 g/L, a platelet level of 63 × 109 L−1 , immature circulating precursors >10%, and bone marrow blast count <20%. G-banding analysis of cells in the bone marrow showed 47, xx, +8/47, xx, idem, t(5;12)(q31;p13), in the absence of the Ph chromosome. Meanwhile RTPCR showed the absence of BCR/ABL fusion gene, and platelet derived growth factor receptors ␣ and  (PDGFRA/B) and fibroblast growth factor receptor 1 (FGFR1) were both negative. The patient was diagnosed with atypical chronic myeloid leukemia according to French-American-British criteria. The patient was treated with hydroxycarbamide for 1 week. A full blood count showed a WBC of 8 × 109 L−1 , an Hb level of 67 g/L, and a platelet level of 40 × 109 L−1 . On the eighth day of therapy, the patient discontinued the treatment for personal reasons [9]. The patient died of leukemia 15 days after diagnosis without reaching complete remission. 2.2. Cell culture On June 28, 2011, a bone marrow sample was collected from the patient at the time of initial diagnosis and prior to therapy. Mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation and cultured in 25-cm2 flasks at a density of 1.5 × 106 cells/mL in Iscove’s Modified Dulbecco’s Medium (GIBCO BRL, Grand Island, NY, USA) supplemented with 20% heat-inactivated fetal calf serum (GIBCO BRL) without any cytokines. Cells were incubated at 37 ◦ C in a humidified incubator with 5% CO2 . The medium was exchanged every 3 days by replacing half of the medium with fresh medium. Two months after initiation of the culture, the cells proliferated slowly. However, after 6 months, cell proliferation was continuous. 2.3. Morphological and cytochemical studies
Table 1 The immunoprofiles of the NT-1 cell line (%). Antigen (CD) T/NK cell markers CD2 CD3 CD4 CD5 CD7 CD8 CD56 B cell markers CD10 CD19 CD20 CD22 Myelomonocytic markers CD13 CD15 CD33 MPO Progenitor/activation markers CD34 HLA-DR Adhesion markers CD11b Megakaryocytic markers CD41 CD61 Cytokine receptors CD117
NT-1 cell line (%) 1 7 1 2 3 3 39 4 1 0 14 97 5 99 9 38 9 33 4 2 76
Note: Positive markers are highlighted.
Light microscopy examination was performed on Wright’s stained cytospin preparations. Cytochemical staining for POX, ␣-naphthyl acetate esterase, and NaF inhibition test were also performed by standard methodology. Ultrastructural analysis was carried out as described previously [10,11].
previous description, and the size of amplified products was determined using a DNA capillary electrophoresis apparatus (ABI PRISM 310 Genetic Analyzer; Applied Biosystems, USA) at a single base definition [12].
2.4. Cell surface marker analysis
2.8. Tumorigenicity in nude mice
Surface immunotyping of the primary leukemia cells and NT-1 cells was performed by flow cytometry using a broad panel of monoclonal antibodies (mAb). The cells were analyzed on an Epicx XL-1 flow cytometer (Beckman Coulter, France) for fluorescence intensity using fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies. All of the mAb used were purchased from Immunotech (Marseille, France) and are listed in Table 1.
Six 4-week old Balb/c nude mice were injected subcutaneously with the NT1 cell line (1 × 107 cells/injection). Mice were humanely killed after developing solid tumors. Tumors were isolated and fixed in 10% buffered formalin for histological staining. Tumors were also used to prepare viable single cell suspensions for karyotype analysis and pathology assays.
2.5. Cytogenetic analysis Chromosomes of both the primary leukemia cells and the NT-1 cells were analyzed. Chromosomes were prepared by a standard method and banded by the R-banding technique. Karyotypic abnormalities were identified according to the International System for Human Cytogenetic Nomenclature (ISCN 1995). 2.6. Detection of Epstein–Barr virus (EBV) and mycoplasma Real-time PCR was performed according to the manufacturer’s instructions to detect EBV genomic DNA (Daan Gene Company, Guangzhou, China). In brief, genomic DNA was extracted from the cell line and amplified using EBV-specific primer pairs in an ABI PRISM 7700 Real-time thermal cycler (Applied Biosystems, USA). The following oligonucleotide primer sequences were used: sense primer: 5 CAG GCT TCC CTG CAA TTT TAC AAG CGG-3 ; antisense primer: 5 -CCC AGA AGT ATA CGT GGT GAC GTA GA-3 . The PCR was started with an initial denaturation of 5 min at 94 ◦ C, 2 min at 57 ◦ C, 2 min at 72 ◦ C, followed by 35 cycles of 1 min at 94 ◦ C, 1 min at 57 ◦ C and 2 min at 72 ◦ C. Two classical detection methods—DNA fluorochrome staining with Hoechst 33258 and microbiological colony assay—were used to determine contamination by mycoplasma.
3. Results Two months after beginning the culture, the cells proliferated slowly and grew as single cells in suspension without any cytokines. Six months later, the cells were growing rapidly and were maintained in continuous culture for more than 1 year. Thus, the stable cells was regarded as a continuous cell line and designated as NT1. The NT-1 cell line had a doubling time of about 72 h at a cell density of 5 × 105 . Cell cycle analysis by flow cytometry showed 77.79% of cells in G0/G1 phase, 20.28% in S phase, and 1.93% in G2/M phase. NT-1 cells were tolerant to freezing in defined medium (70% medium, 20% FCS, 10% DMSO), storage in liquid nitrogen, thawing (with viabilities of 80% or higher), and subsequently expanding [13]. During the passage culture period, NT-1 cells maintained stable growth, and thus, have been identified as a continuous cell line. 3.1. Morphology of NT-1 cells
2.7. Authentication of the cell line The identity of the NT-1 cell line was checked using DNA fingerprinting against a BM cell sample taken from the patient during progression of her disease. DNA was prepared from whole blood or whole BM using the QIAamp Blood Kit (Qiagen Company, Germany) according to the instructions provided by the manufacturer. The following 16 highly polymorphic short tandem repeat (STR) loci were tested by a multiplex PCR reaction: D8S1179, D21S11, D18S51, CSF1PO, D3S1358, TH01, D13S317, D16S539, D7S820, D2S1338, D19S433, Vwa, TPOX, X/Y, D5S818, and FGA. The multiplex PCR reaction conditions and primers were chosen according to the
The morphology of the NT-1 cells resembled the characteristics of primary leukemia cells. The NT-1 cells were round or oval shaped and intermediate in size, and had folded nuclei, partly reticular chromatin, and blue-gray cytoplasm with finely sand granules (Fig. 1A). Fifty-six percent of the NT-1 cells were positive for peroxide staining (Fig. 1B), 26% were positive for ␣-naphthyl acetate esterase staining, while the NaF inhibition test was negative.
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Fig. 1. Morphology of NT-1 cells. Light microscopy images (×1000) of NT-1 cells with (A) Wright’s stain and (B) peroxide stain. (C) Electron microscopic photograph of NT-1 cells showing numerous mitochondria, lysosomes, vacuoles, glycogens and ribosomes (×5000).
Transmission electron microscopy showed that most cells had numerous mitochondria, lysosomes, vacuoles, glycogens and ribosome, which were consistent with those of myeloid lineage cells (Fig. 1C).
3.2. Immunophenotypic profiles The immunoprofile of the NT-1 cell line is shown in Table 1. The NT-1 cell line expressed myeloid antigens, including CD13 (97%), CD33 (99%), CD34 (38%), CD11b (33%) and CD117 (76%). The cell line also expressed natural killer (NK)-related antigen CD56 (39%). However, T and B lymphocytic lineage antigens were all negative (Table 1).
3.4. Authentication of the cell line Short tandem repeat (STR)-PCR was used to confirm the derivation of the NT-1 cell line. Fifteen STR loci in the cell line corresponded with the patient’s leukemia cells, except the vWA locus, which was lacking in the NT-1 cells, but present in the patient’s BM cells. Thus, STR-PCR provided powerful evidence for the derivation of NT-1 cell line from the patient’s leukemia cells [6]. 3.5. Direct sequencing Sequence analysis of the DNA fragments of the p53 gene amplified by PCR showed heterozygous mutation of C to G in exon 4 that changed codon 215 from CCC to CGC (Fig. 3). This point mutation resulted in the substitution of proline for arginine in the p53 protein.
3.3. Cytogenetics 3.6. Detection of Epstein–Barr virus and mycoplasma The karyotype of the patient’s BM cells at diagnosis was 47, xx, +8[18]/47, xx, idem, t(5;12)(q31;p13) [2]. After 1 year, NT-1 cells in the culture had the following karyotype: 47, xx, +8[3]/47, xx, idem, t(5;12)(q31;p13) [17] (Fig. 2). Thus, the chromosome abnormalities of NT-1 cells remained the same, confirming that the NT-1 cell line was derived from this patient. However, during the passage cultures, the cells with the 47, xx, +8 karyotype decreased, while the cells with the 47, xx, idem, t(5;12)(q31;p13) karyotype gradually increased and became the dominant cells in the population.
NT-1 cells were negative for Epstein–Barr virus and mycoplasma as detected by RT-PCR, indicating that the NT-1 cells were free of Epstein–Barr virus or mycoplasma contamination after the long period of in vitro culture. 3.7. Expression of fusion genes in leukemia None of the 29 types of fusion genes linked to leukemia were detected using a Multiplex RT-PCR Assay, including
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Fig. 2. Karyotype analysis. An R-banded karyotype of the NT-1 cell line showed 47, xx, +8/47, xx, idem, t(5;12)(q31;p13).
AML1/ETO, BCR/ABL, CBF/MYH11, DEK/CAN, E2A/HLF, E2A/PBX1, EVI1/MDS1/EVI1, MLL/AF1P, MLL/AF4, MLL/AF9, MLL/AF10, MLL/AF17, MLL/AF19, MLL/AFX, MLL/ELL, MLL/ENL, NPM/ALK, NPM/MLF1, NPM/RARA, PLZF/RARA, PML/RARA, SET/CAN, SIL/TAL1, TEL/ABL, TEL/AML1, TLS/ERG and TEL/PDGFR [14]. 3.8. Analysis of breakpoints by array CGH To confirm the identification of chromosomal breakpoints in NT1 cells, we performed array CGH (aCGH) analysis (Fig. 4A and 4B), and found a whole chromosome 8 addition and 0.2 Mb amplification at 12p13 including the DDX12 gene. 3.9. Tumorigenicity in nude mice Tumor masses were found in hypodermic tissue in four of six nude mice 25 days after the NT-1 cells were injected (Fig. 5A). The sizes of the tumor masses ranged from 0.7 × 0.8 cm to 1.1 × 1.8 cm (Fig. 5B). Histopathology examination showed that the tumor masses were composed of leukemia cells, and blood vessels could be seen (Fig. 5C). Their morphological features resembled the NT-1
cell line. POX staining was strongly positive (Fig. 5D). Chromosome analysis was performed on mononuclear cells isolated from the tumor masses, and these cells had the same chromosomal abnormalities as the NT-1 cell line: 47, xx, +8/47, xx, +8, t(5;12)(q31;p13). The immunophenotyping profile of the cells isolated from tumor masses was the same as the NT-1 cell line (data not shown). All major features of the NT-1 cell line are summarized in Table 2.
4. Discussion In the present study, we describe a new cell line, NT-1, established from a patient with atypical CML. The NT-1 cell line has been grown in suspension without any cytokines for over 12 months. NT-1 cells remained stable during the passage culture period and after repeated freezing and thawing cycles. The cells were negative for Epstein–Barr virus and mycoplasma, making them suitable for research and drug screening. NT-1 cells had tumorigenic capacity in nude mice, confirming their malignant and invasive characteristics. Therefore, these results indicated that NT-1 is a new continuous human leukemia cell line.
Fig. 3. Sequence analysis of p53 gene. Direct sequence analysis of the PCR products showed the heterozygous mutation of C to G in exon 4 that changed codon 215 from CCC to CGC. The arrow indicates the position of base mutations.
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Fig. 4. Whole-genome array CGH. Image shows array CGH (244 K) analysis in the NT-1 cell line, which revealed a whole chromosome 8 addition (A) and 0.2 Mb amplification at 12p13 including the DDX12 gene (B).
The NT-1 cell line retained the morphologic, immunologic, cytogenetic and molecular characteristics of the donor patient’s primary leukemia cells. We propose that the expression of the disordered antigens reflects the immature nature of the NT-1 cells,
suggesting that these cells were situated in earlier myeloid differentiation. NT-1 cells and the patient’s primary leukemia cells were identical in terms of karyotypic abnormality, as demonstrated by conventional karyotyping. STR-PCR results confirmed that the
Fig. 5. Tumorigenicity in nude mice. (A) Tumor masses were found in the hypodermic tissue of nude mice. (B) The size of the tumor masses ranged from 0.7 × 0.8 cm to 1.1 × 1.8 cm. (C) Histopathology examination showed that the tumor masses were composed of leukemia cells and blood vessels. (D) The morphological features of cells from the tumor mass.
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Table 2 Synopsis of NT-1 features. Parameter
Conflict of interest statement NT-1
Clinical characterization 67-year-old female Patient Diagnosis aCML At diagnosis Treatment status Bone marrow Specimen site 2011 Year of establishment Cell culture characterization 5% CO2 , 37 ◦ C, in 25-cm2 flasks Culture conditions Culture medium 80% IMDM + 20% FBS Doubling time 72 h Subcultivation 1:2 every 3–4 days 1 × 106 cells/mL Maximum cell density Single cells in suspension Growth pattern 70% medium + 20% FBS + 10% DMSO Cryoperservation Morphology Myeloblasts Negative for EBV Viral status Negative for mycoplasma Contamination Yes (by STR-PCR, cytogenetic characteristics) Authentication Immunophenotypical characterization CD16/56+ NK cell Negative T/B cell CD13+, CD33+ Myelocytic Negative Monocytic CD34+ Progenitor/activation CD11b+ Adhesion CD117 Cytokine receptors Genetic characterization 47, xx, +8/47, xx, idem, t(5;12)(q31;p13) Cytogenetic profile Molecular profile Negative for 29 types of fusion genes Functional characterization Tumorigenicity in nude mice Tumor masses in 4/6 nude mice Cell line available by request to Dr. Juan Qian.
NT-1 cell line was derived from the patient’s leukemia cells, thus ruling out possible cross-contamination from other cell lines. Based on these observations, we concluded that the NT-1 cell line retained the originality of the donor patient’s primary leukemia cells. NT-1 cells have the identical karyotype abnormalities, consisting of t(5;12)(q31;p13)and 47, xx, +8. To our knowledge, NT-1 is the first human atypical CML cell line carrying +8 and t(5;12)(q31;p13). This translocation has not been reported in the literature previously. As a rule, complex chromosomal abnormalities usually result in novel fusion genes [15], however we did not detect any of the common 29 fusion genes associated with leukemia using a Multiplex RT-PCR Assay. Furthermore, aCGH analysis showed the chromosomal breakpoint is in 12p13 and +8, rather than in 5q31. These results indicated a new fusion gene. Mutation of the P53 gene is often tightly associated with various human malignancies [16]. Most human LL cell lines have point mutation in p53 [17], which is a tumor suppressor gene with many important biological functions including regulation of the cell cycle, DNA repair, cell differentiation and cell apoptosis [18]. Thus, mutations or polymorphisms, which lead to loss-of-function of p53 gene, might play a role in oncogenesis [19]. In fact, this patient died of leukemia only 15 days after diagnosis. It is possible that this Pro215 to Arg mutation in the P53 gene leads to high risk of leukemia. 5. Conclusions In summary, the NT-1 cell line is a useful tool for study the pathogenesis and treatment strategy of atypical CML. The NT-1 cell line has been deposited to the China Center for Type Culture Collection, and is available worldwide for non-commercial research.
There is no conflict of interest of any authors in relation to the submission. Acknowledgements This work was supported in part by grants from the National Natural Science Foundation of China (81070400), the Jiangsu Province Innovative Medical Team and Leading Talent Project (LJ201136), and the Xing Wei, Jiangsu Province Key Medical Personnel Fund Project (RC2007084). The authors would like to thank Suning Chen for his technical assistance. References [1] Drexler HG, Macleod RA. History of leukemia-lymphoma cell lines. Hum Cell 2010;23:75–82. [2] MacLeod RA, Nagel S, Scherr M, Schneider B, Dirks WG, Uphoff CC, et al. Human leukemia and lymphoma cell lines as models and resources. Curr Med Chem 2008;15:339–59. [3] Drexler HG, Matsuo Y. Malignant hematopoietic cell lines: in vitro models for the study of natural killer cell leukemia-lymphoma. Leukemia 2000;14:777–82. [4] Chen S, Xue Y, Zhang X, Wu Y, Pan J, Wang Y, et al. A new human acute monocytic leukemia cell line SHI-1 with t(6;11)(q27;q23), p53 gene alterations and high tumorigenicity in nude mice. Haematologica 2005;90:766–75. [5] Qiu H, Xue Y, Zhang J, Pan J, Dai H, Wu Y, et al. Establishment and characterization of a new human acute myelocytic leukemia cell line SH-2 with a loss of Y chromosome, a derivative chromosome 16 resulting from an unbalanced translocation between chromosomes 16 and 17, monosomy 17, trisomy 19, and p53 alteration. Exp Hematol 2008;36:1487–95. [6] Pan J, Xue Y, Chen S, Qiu H, Wu C, Jiang H, et al. Establishment and characterization of a new human acute myelomonocytic leukemia cell line JIH-3. Leuk Res 2012;36:889–94. [7] Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al. WHO classification of tumors of haematopoietic and lymphoid tissues. In: Vardiman JW, Bennett JM, Bain BJ, Brunning RD, Thiele J, editors. Atypical chronic myeloid leukaemia, BCR-ABL1 negative. Lyon, France: IARC Press; 2008. p. 80–1. [8] Drexler HG, Matsuo Y. Guidelines for the characterization and publication of human malignant hematopoietic cell lines. Leukemia 1999;13:835–42. [9] Gotou M, Hanamura I, Nagoshi H, Wakabayashi M, Sakamoto N, Tsunekawa N, et al. Establishment of a novel human myeloid leukemia cell line, AMUAML1, carrying t(12;22)(p13;q11) without chimeric MN1-TEL and with high expression of MN1. Genes Chromosomes Cancer 2012;51:42–53. [10] Takeuchi S, Sugito S, Uemura Y, Miyagi T, Kubonishi I, Taguchi H, et al. Acute megakaryoblastic leukemia: establishment of a new cell line (MKPL-1) in vitro and in vivo. Leukemia 1992;6:588–94. [11] Anderson WA, Trantalis J, Kang YH. Ultrastructural localization of endogenous mammary gland peroxidase during lactogenesis in the rat results after tannic acid-formaldehyde-glutaraldehyde fixation. J Histochem Cytochem 1975;23:295–302. [12] Nollet F, Billiet J, Selleslag D, Criel A. Standardisation of multiplex fluorescent short tandem repeat analysis for chimerism testing. Bone Marrow Transplant 2001;28:511–8. [13] Jiang H, Qiu H, Xue Y, Pan J, Wu Y, Zhang J, et al. Establishment and characterization of a novel acute myeloid leukemia cell line, JIH-4, carrying a t(16;21)(p11.2;q22) and expressing the FUS-ERG fusion. Cancer Genet 2011;204:219–23. [14] Pallisgaard N, Hokland P, Riishoj DC, Pedersen B, Jorgensen P. Multiplex reverse transcription-polymerase chain reaction for simultaneous screening of 29 translocations and chromosomal aberrations in acute leukemia. Blood 1998;92:574–88. [15] Pedersen-Bjergaard J, Rowley JD. The balanced and the unbalanced chromosome aberrations of acute myeloid leukemia may develop in different ways and may contribute differently to malignant transformation. Blood 1994;83:2780–6. [16] Imamura J, Miyoshi I, Koeffler HP. p53 in hematologic malignancies. Blood 1994;84:2412–21. [17] Drexler HG, Fombonne S, Matsuo Y, Hu ZB, Hamaguchi H. Uphoff CC. p53 alterations in human leukemia-lymphoma cell lines: in vitro artifact or prerequisite for cell immortalization. Leukemia 2000;14:198–206. [18] Mhawech P, Saleem A. Myelodysplastic syndrome: review of the cytogenetic and molecular data. Crit Rev Oncol Hematol 2001;40:229–38. [19] Smith ML, Fornace Jr AJ. Genomic instability and the role of p53 mutations in cancer cells. Curr Opin Oncol 1995;7:69–75.
Contents lists available at ScienceDirect
Leukemia Research journal homepage: www.elsevier.com/locate/leukres
Establishment and characterization of a rare atypical chronic myeloid leukemia cell line NT-1 Juan Qian a,1 , Qin-Rong Wang b,1 , Jie Liu a , Sheng-Hua Jiang a , Xiao-Qing Ni a , Zeng-Hua Lin a , Ya-Ping Zhang a , Hong Liu a,∗ a b
Department of Hematology, Affiliated Hospital of Nantong University, Nantong, China The First Affiliated Hospital of Soochow University, Jiangsu Institute of Hematology, Suzhou, China
a r t i c l e
i n f o
Article history: Received 23 February 2014 Received in revised form 9 June 2014 Accepted 17 June 2014 Available online 25 June 2014 Keywords: Atypical chronic myeloid leukemia Cell line Translocation Tumorigenicity
a b s t r a c t Human leukemia cell lines are of great value in leukemia research. In this study, we established and described the biological characteristics of a rare atypical chronic myeloid (aCML) leukemia cell line (NT1). Mononuclear cells were isolated from the bone marrow of a patient with atypical chronic myeloid leukemia (Ph− /bcr− /abl− ), and were passaged by liquid culture. Cells were maintained without any cytokines for over 1 year, and named NT-1. This cell line was extensively characterized using morphological assays, flow cytometry, cytogenetic analysis, clonogenic culture, quantitative fluorescent PCR, short tandem repeating sequence PCR (STR-PCR) and array-CGH. Its tumorigenic capacity was also examined in nude mice. The NT-1 cell line had morphological features of chronic myeloid leukemia and major myeloid markers (CD13, CD33, CD11b). Additionally, NT-1 expressed progenitor cells and natural killer cell-related antigens such as CD34, CD117, CD56. Cytogenetic analysis initially demonstrated two abnormalities: 47, xx, +8 and 47, xx, +8 accompanied by t(5;12)(q31;p13) translocation. The one-year passage process did not alter the karyotype. NT-1 cells maintained the same morphology, immunophenotyping and cytogenetic features as primary leukemia cells, which was strongly supported by STR-PCR results. Neither Epstein–Barr virus nor mycoplasma was detected in the NT-1 line. In addition, NT-1 cells showed high tumorigenic capacity in nude mice. NT-1 is a new atypical chronic myeloid leukemia cell line with the +8 and t(5,12) translocation, and exhibits high tumorigenicity in nude mice. This new cell line provides a useful tool for the study of leukemogenesis. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction In the past five decades, since the establishment of the Raji cell line, at least 1500 human leukemia lymphoma cell lines have been developed [1,2]. The majority of those lines belong to lymphoidoriginated cell lines, while myeloid-originated cell lines account for only 19.5% [3]. Since 2005, Suning Chen [4], Huiying Qiu [5], and Jinlan Pan [6] have reported at least four acute myeloid leukemia lines. Various methods have been used to identify and characterize these cell lines, including morphological, cytogenetic analysis, fluorescence in situ hybridization (FISH), multiplexFISH
∗ Corresponding author at: Department of Hematology, Affiliated Hospital of Nantong University, Nantong, China. Tel.: +86 13951300660. E-mail address: [email protected] (H. Liu). 1 Both Juan Qian and Qin-Rong Wang are co-authors who contributed equally to this manuscript. http://dx.doi.org/10.1016/j.leukres.2014.06.008 0145-2126/© 2014 Elsevier Ltd. All rights reserved.
(M-FISH), reverse transcriptase polymerase chain reaction (RTPCR), multiplex RT-PCR, short tandem repeat (STR)-PCR, direct sequencing of DNA, clonogenic assay and tumorigenicity in nude mice. These cells have been broadly used in leukemia and oncogenic research, as well as drug development. However, due to the large variation and complexity of leukemic cell lines, new cell lines are always needed for research and drug screening. In this study, we report a new myeloid leukemia cell line named NT-1, which was established from the bone marrow of a atypical chronic myeloid leukemia (aCML) patient in our hospital. Atypical chronic myeloid leukemia is an uncommon myelodysplastic/myeloproliferative (MDS/MPN) neoplasm, with a relative incidence estimated at 1 to 2 cases for every 100 patients with BCR-ABL1-positive chronic myeloid leukemia (CML) [7]. The cell line had 47, xx, +8 and 47, xx, idem, t (5;12)(q31;p13), and showed high tumorigenicity in nude mice. The phenotypic, genetic and functional properties of this cell line were described following the guidelines for characterization and publication of human malignant hematopoietic cell lines [8].
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2. Materials and methods 2.1. Case report In June 2011, a 67-year-old female was admitted to our hospital because of fever and fatigue that had persisted for 1 month. Physical examination revealed splenomegaly (5 cm below the left costal margin) and pleural effusion. Peripheral blood (PB) examination showed a white blood cell (WBC) count of 79 × 109 L−1 , monocytosis <1 × 109 L−1 , basophils <2%, a hemoglobin (Hb) level of 55 g/L, a platelet level of 63 × 109 L−1 , immature circulating precursors >10%, and bone marrow blast count <20%. G-banding analysis of cells in the bone marrow showed 47, xx, +8/47, xx, idem, t(5;12)(q31;p13), in the absence of the Ph chromosome. Meanwhile RTPCR showed the absence of BCR/ABL fusion gene, and platelet derived growth factor receptors ␣ and  (PDGFRA/B) and fibroblast growth factor receptor 1 (FGFR1) were both negative. The patient was diagnosed with atypical chronic myeloid leukemia according to French-American-British criteria. The patient was treated with hydroxycarbamide for 1 week. A full blood count showed a WBC of 8 × 109 L−1 , an Hb level of 67 g/L, and a platelet level of 40 × 109 L−1 . On the eighth day of therapy, the patient discontinued the treatment for personal reasons [9]. The patient died of leukemia 15 days after diagnosis without reaching complete remission. 2.2. Cell culture On June 28, 2011, a bone marrow sample was collected from the patient at the time of initial diagnosis and prior to therapy. Mononuclear cells were separated by Ficoll-Hypaque density gradient centrifugation and cultured in 25-cm2 flasks at a density of 1.5 × 106 cells/mL in Iscove’s Modified Dulbecco’s Medium (GIBCO BRL, Grand Island, NY, USA) supplemented with 20% heat-inactivated fetal calf serum (GIBCO BRL) without any cytokines. Cells were incubated at 37 ◦ C in a humidified incubator with 5% CO2 . The medium was exchanged every 3 days by replacing half of the medium with fresh medium. Two months after initiation of the culture, the cells proliferated slowly. However, after 6 months, cell proliferation was continuous. 2.3. Morphological and cytochemical studies
Table 1 The immunoprofiles of the NT-1 cell line (%). Antigen (CD) T/NK cell markers CD2 CD3 CD4 CD5 CD7 CD8 CD56 B cell markers CD10 CD19 CD20 CD22 Myelomonocytic markers CD13 CD15 CD33 MPO Progenitor/activation markers CD34 HLA-DR Adhesion markers CD11b Megakaryocytic markers CD41 CD61 Cytokine receptors CD117
NT-1 cell line (%) 1 7 1 2 3 3 39 4 1 0 14 97 5 99 9 38 9 33 4 2 76
Note: Positive markers are highlighted.
Light microscopy examination was performed on Wright’s stained cytospin preparations. Cytochemical staining for POX, ␣-naphthyl acetate esterase, and NaF inhibition test were also performed by standard methodology. Ultrastructural analysis was carried out as described previously [10,11].
previous description, and the size of amplified products was determined using a DNA capillary electrophoresis apparatus (ABI PRISM 310 Genetic Analyzer; Applied Biosystems, USA) at a single base definition [12].
2.4. Cell surface marker analysis
2.8. Tumorigenicity in nude mice
Surface immunotyping of the primary leukemia cells and NT-1 cells was performed by flow cytometry using a broad panel of monoclonal antibodies (mAb). The cells were analyzed on an Epicx XL-1 flow cytometer (Beckman Coulter, France) for fluorescence intensity using fluorescein isothiocyanate (FITC)- or phycoerythrin (PE)-conjugated antibodies. All of the mAb used were purchased from Immunotech (Marseille, France) and are listed in Table 1.
Six 4-week old Balb/c nude mice were injected subcutaneously with the NT1 cell line (1 × 107 cells/injection). Mice were humanely killed after developing solid tumors. Tumors were isolated and fixed in 10% buffered formalin for histological staining. Tumors were also used to prepare viable single cell suspensions for karyotype analysis and pathology assays.
2.5. Cytogenetic analysis Chromosomes of both the primary leukemia cells and the NT-1 cells were analyzed. Chromosomes were prepared by a standard method and banded by the R-banding technique. Karyotypic abnormalities were identified according to the International System for Human Cytogenetic Nomenclature (ISCN 1995). 2.6. Detection of Epstein–Barr virus (EBV) and mycoplasma Real-time PCR was performed according to the manufacturer’s instructions to detect EBV genomic DNA (Daan Gene Company, Guangzhou, China). In brief, genomic DNA was extracted from the cell line and amplified using EBV-specific primer pairs in an ABI PRISM 7700 Real-time thermal cycler (Applied Biosystems, USA). The following oligonucleotide primer sequences were used: sense primer: 5 CAG GCT TCC CTG CAA TTT TAC AAG CGG-3 ; antisense primer: 5 -CCC AGA AGT ATA CGT GGT GAC GTA GA-3 . The PCR was started with an initial denaturation of 5 min at 94 ◦ C, 2 min at 57 ◦ C, 2 min at 72 ◦ C, followed by 35 cycles of 1 min at 94 ◦ C, 1 min at 57 ◦ C and 2 min at 72 ◦ C. Two classical detection methods—DNA fluorochrome staining with Hoechst 33258 and microbiological colony assay—were used to determine contamination by mycoplasma.
3. Results Two months after beginning the culture, the cells proliferated slowly and grew as single cells in suspension without any cytokines. Six months later, the cells were growing rapidly and were maintained in continuous culture for more than 1 year. Thus, the stable cells was regarded as a continuous cell line and designated as NT1. The NT-1 cell line had a doubling time of about 72 h at a cell density of 5 × 105 . Cell cycle analysis by flow cytometry showed 77.79% of cells in G0/G1 phase, 20.28% in S phase, and 1.93% in G2/M phase. NT-1 cells were tolerant to freezing in defined medium (70% medium, 20% FCS, 10% DMSO), storage in liquid nitrogen, thawing (with viabilities of 80% or higher), and subsequently expanding [13]. During the passage culture period, NT-1 cells maintained stable growth, and thus, have been identified as a continuous cell line. 3.1. Morphology of NT-1 cells
2.7. Authentication of the cell line The identity of the NT-1 cell line was checked using DNA fingerprinting against a BM cell sample taken from the patient during progression of her disease. DNA was prepared from whole blood or whole BM using the QIAamp Blood Kit (Qiagen Company, Germany) according to the instructions provided by the manufacturer. The following 16 highly polymorphic short tandem repeat (STR) loci were tested by a multiplex PCR reaction: D8S1179, D21S11, D18S51, CSF1PO, D3S1358, TH01, D13S317, D16S539, D7S820, D2S1338, D19S433, Vwa, TPOX, X/Y, D5S818, and FGA. The multiplex PCR reaction conditions and primers were chosen according to the
The morphology of the NT-1 cells resembled the characteristics of primary leukemia cells. The NT-1 cells were round or oval shaped and intermediate in size, and had folded nuclei, partly reticular chromatin, and blue-gray cytoplasm with finely sand granules (Fig. 1A). Fifty-six percent of the NT-1 cells were positive for peroxide staining (Fig. 1B), 26% were positive for ␣-naphthyl acetate esterase staining, while the NaF inhibition test was negative.
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Fig. 1. Morphology of NT-1 cells. Light microscopy images (×1000) of NT-1 cells with (A) Wright’s stain and (B) peroxide stain. (C) Electron microscopic photograph of NT-1 cells showing numerous mitochondria, lysosomes, vacuoles, glycogens and ribosomes (×5000).
Transmission electron microscopy showed that most cells had numerous mitochondria, lysosomes, vacuoles, glycogens and ribosome, which were consistent with those of myeloid lineage cells (Fig. 1C).
3.2. Immunophenotypic profiles The immunoprofile of the NT-1 cell line is shown in Table 1. The NT-1 cell line expressed myeloid antigens, including CD13 (97%), CD33 (99%), CD34 (38%), CD11b (33%) and CD117 (76%). The cell line also expressed natural killer (NK)-related antigen CD56 (39%). However, T and B lymphocytic lineage antigens were all negative (Table 1).
3.4. Authentication of the cell line Short tandem repeat (STR)-PCR was used to confirm the derivation of the NT-1 cell line. Fifteen STR loci in the cell line corresponded with the patient’s leukemia cells, except the vWA locus, which was lacking in the NT-1 cells, but present in the patient’s BM cells. Thus, STR-PCR provided powerful evidence for the derivation of NT-1 cell line from the patient’s leukemia cells [6]. 3.5. Direct sequencing Sequence analysis of the DNA fragments of the p53 gene amplified by PCR showed heterozygous mutation of C to G in exon 4 that changed codon 215 from CCC to CGC (Fig. 3). This point mutation resulted in the substitution of proline for arginine in the p53 protein.
3.3. Cytogenetics 3.6. Detection of Epstein–Barr virus and mycoplasma The karyotype of the patient’s BM cells at diagnosis was 47, xx, +8[18]/47, xx, idem, t(5;12)(q31;p13) [2]. After 1 year, NT-1 cells in the culture had the following karyotype: 47, xx, +8[3]/47, xx, idem, t(5;12)(q31;p13) [17] (Fig. 2). Thus, the chromosome abnormalities of NT-1 cells remained the same, confirming that the NT-1 cell line was derived from this patient. However, during the passage cultures, the cells with the 47, xx, +8 karyotype decreased, while the cells with the 47, xx, idem, t(5;12)(q31;p13) karyotype gradually increased and became the dominant cells in the population.
NT-1 cells were negative for Epstein–Barr virus and mycoplasma as detected by RT-PCR, indicating that the NT-1 cells were free of Epstein–Barr virus or mycoplasma contamination after the long period of in vitro culture. 3.7. Expression of fusion genes in leukemia None of the 29 types of fusion genes linked to leukemia were detected using a Multiplex RT-PCR Assay, including
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Fig. 2. Karyotype analysis. An R-banded karyotype of the NT-1 cell line showed 47, xx, +8/47, xx, idem, t(5;12)(q31;p13).
AML1/ETO, BCR/ABL, CBF/MYH11, DEK/CAN, E2A/HLF, E2A/PBX1, EVI1/MDS1/EVI1, MLL/AF1P, MLL/AF4, MLL/AF9, MLL/AF10, MLL/AF17, MLL/AF19, MLL/AFX, MLL/ELL, MLL/ENL, NPM/ALK, NPM/MLF1, NPM/RARA, PLZF/RARA, PML/RARA, SET/CAN, SIL/TAL1, TEL/ABL, TEL/AML1, TLS/ERG and TEL/PDGFR [14]. 3.8. Analysis of breakpoints by array CGH To confirm the identification of chromosomal breakpoints in NT1 cells, we performed array CGH (aCGH) analysis (Fig. 4A and 4B), and found a whole chromosome 8 addition and 0.2 Mb amplification at 12p13 including the DDX12 gene. 3.9. Tumorigenicity in nude mice Tumor masses were found in hypodermic tissue in four of six nude mice 25 days after the NT-1 cells were injected (Fig. 5A). The sizes of the tumor masses ranged from 0.7 × 0.8 cm to 1.1 × 1.8 cm (Fig. 5B). Histopathology examination showed that the tumor masses were composed of leukemia cells, and blood vessels could be seen (Fig. 5C). Their morphological features resembled the NT-1
cell line. POX staining was strongly positive (Fig. 5D). Chromosome analysis was performed on mononuclear cells isolated from the tumor masses, and these cells had the same chromosomal abnormalities as the NT-1 cell line: 47, xx, +8/47, xx, +8, t(5;12)(q31;p13). The immunophenotyping profile of the cells isolated from tumor masses was the same as the NT-1 cell line (data not shown). All major features of the NT-1 cell line are summarized in Table 2.
4. Discussion In the present study, we describe a new cell line, NT-1, established from a patient with atypical CML. The NT-1 cell line has been grown in suspension without any cytokines for over 12 months. NT-1 cells remained stable during the passage culture period and after repeated freezing and thawing cycles. The cells were negative for Epstein–Barr virus and mycoplasma, making them suitable for research and drug screening. NT-1 cells had tumorigenic capacity in nude mice, confirming their malignant and invasive characteristics. Therefore, these results indicated that NT-1 is a new continuous human leukemia cell line.
Fig. 3. Sequence analysis of p53 gene. Direct sequence analysis of the PCR products showed the heterozygous mutation of C to G in exon 4 that changed codon 215 from CCC to CGC. The arrow indicates the position of base mutations.
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Fig. 4. Whole-genome array CGH. Image shows array CGH (244 K) analysis in the NT-1 cell line, which revealed a whole chromosome 8 addition (A) and 0.2 Mb amplification at 12p13 including the DDX12 gene (B).
The NT-1 cell line retained the morphologic, immunologic, cytogenetic and molecular characteristics of the donor patient’s primary leukemia cells. We propose that the expression of the disordered antigens reflects the immature nature of the NT-1 cells,
suggesting that these cells were situated in earlier myeloid differentiation. NT-1 cells and the patient’s primary leukemia cells were identical in terms of karyotypic abnormality, as demonstrated by conventional karyotyping. STR-PCR results confirmed that the
Fig. 5. Tumorigenicity in nude mice. (A) Tumor masses were found in the hypodermic tissue of nude mice. (B) The size of the tumor masses ranged from 0.7 × 0.8 cm to 1.1 × 1.8 cm. (C) Histopathology examination showed that the tumor masses were composed of leukemia cells and blood vessels. (D) The morphological features of cells from the tumor mass.
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Table 2 Synopsis of NT-1 features. Parameter
Conflict of interest statement NT-1
Clinical characterization 67-year-old female Patient Diagnosis aCML At diagnosis Treatment status Bone marrow Specimen site 2011 Year of establishment Cell culture characterization 5% CO2 , 37 ◦ C, in 25-cm2 flasks Culture conditions Culture medium 80% IMDM + 20% FBS Doubling time 72 h Subcultivation 1:2 every 3–4 days 1 × 106 cells/mL Maximum cell density Single cells in suspension Growth pattern 70% medium + 20% FBS + 10% DMSO Cryoperservation Morphology Myeloblasts Negative for EBV Viral status Negative for mycoplasma Contamination Yes (by STR-PCR, cytogenetic characteristics) Authentication Immunophenotypical characterization CD16/56+ NK cell Negative T/B cell CD13+, CD33+ Myelocytic Negative Monocytic CD34+ Progenitor/activation CD11b+ Adhesion CD117 Cytokine receptors Genetic characterization 47, xx, +8/47, xx, idem, t(5;12)(q31;p13) Cytogenetic profile Molecular profile Negative for 29 types of fusion genes Functional characterization Tumorigenicity in nude mice Tumor masses in 4/6 nude mice Cell line available by request to Dr. Juan Qian.
NT-1 cell line was derived from the patient’s leukemia cells, thus ruling out possible cross-contamination from other cell lines. Based on these observations, we concluded that the NT-1 cell line retained the originality of the donor patient’s primary leukemia cells. NT-1 cells have the identical karyotype abnormalities, consisting of t(5;12)(q31;p13)and 47, xx, +8. To our knowledge, NT-1 is the first human atypical CML cell line carrying +8 and t(5;12)(q31;p13). This translocation has not been reported in the literature previously. As a rule, complex chromosomal abnormalities usually result in novel fusion genes [15], however we did not detect any of the common 29 fusion genes associated with leukemia using a Multiplex RT-PCR Assay. Furthermore, aCGH analysis showed the chromosomal breakpoint is in 12p13 and +8, rather than in 5q31. These results indicated a new fusion gene. Mutation of the P53 gene is often tightly associated with various human malignancies [16]. Most human LL cell lines have point mutation in p53 [17], which is a tumor suppressor gene with many important biological functions including regulation of the cell cycle, DNA repair, cell differentiation and cell apoptosis [18]. Thus, mutations or polymorphisms, which lead to loss-of-function of p53 gene, might play a role in oncogenesis [19]. In fact, this patient died of leukemia only 15 days after diagnosis. It is possible that this Pro215 to Arg mutation in the P53 gene leads to high risk of leukemia. 5. Conclusions In summary, the NT-1 cell line is a useful tool for study the pathogenesis and treatment strategy of atypical CML. The NT-1 cell line has been deposited to the China Center for Type Culture Collection, and is available worldwide for non-commercial research.
There is no conflict of interest of any authors in relation to the submission. Acknowledgements This work was supported in part by grants from the National Natural Science Foundation of China (81070400), the Jiangsu Province Innovative Medical Team and Leading Talent Project (LJ201136), and the Xing Wei, Jiangsu Province Key Medical Personnel Fund Project (RC2007084). The authors would like to thank Suning Chen for his technical assistance. References [1] Drexler HG, Macleod RA. History of leukemia-lymphoma cell lines. Hum Cell 2010;23:75–82. [2] MacLeod RA, Nagel S, Scherr M, Schneider B, Dirks WG, Uphoff CC, et al. Human leukemia and lymphoma cell lines as models and resources. Curr Med Chem 2008;15:339–59. [3] Drexler HG, Matsuo Y. Malignant hematopoietic cell lines: in vitro models for the study of natural killer cell leukemia-lymphoma. Leukemia 2000;14:777–82. [4] Chen S, Xue Y, Zhang X, Wu Y, Pan J, Wang Y, et al. A new human acute monocytic leukemia cell line SHI-1 with t(6;11)(q27;q23), p53 gene alterations and high tumorigenicity in nude mice. Haematologica 2005;90:766–75. [5] Qiu H, Xue Y, Zhang J, Pan J, Dai H, Wu Y, et al. Establishment and characterization of a new human acute myelocytic leukemia cell line SH-2 with a loss of Y chromosome, a derivative chromosome 16 resulting from an unbalanced translocation between chromosomes 16 and 17, monosomy 17, trisomy 19, and p53 alteration. Exp Hematol 2008;36:1487–95. [6] Pan J, Xue Y, Chen S, Qiu H, Wu C, Jiang H, et al. Establishment and characterization of a new human acute myelomonocytic leukemia cell line JIH-3. Leuk Res 2012;36:889–94. [7] Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, et al. WHO classification of tumors of haematopoietic and lymphoid tissues. In: Vardiman JW, Bennett JM, Bain BJ, Brunning RD, Thiele J, editors. Atypical chronic myeloid leukaemia, BCR-ABL1 negative. Lyon, France: IARC Press; 2008. p. 80–1. [8] Drexler HG, Matsuo Y. Guidelines for the characterization and publication of human malignant hematopoietic cell lines. Leukemia 1999;13:835–42. [9] Gotou M, Hanamura I, Nagoshi H, Wakabayashi M, Sakamoto N, Tsunekawa N, et al. Establishment of a novel human myeloid leukemia cell line, AMUAML1, carrying t(12;22)(p13;q11) without chimeric MN1-TEL and with high expression of MN1. Genes Chromosomes Cancer 2012;51:42–53. [10] Takeuchi S, Sugito S, Uemura Y, Miyagi T, Kubonishi I, Taguchi H, et al. Acute megakaryoblastic leukemia: establishment of a new cell line (MKPL-1) in vitro and in vivo. Leukemia 1992;6:588–94. [11] Anderson WA, Trantalis J, Kang YH. Ultrastructural localization of endogenous mammary gland peroxidase during lactogenesis in the rat results after tannic acid-formaldehyde-glutaraldehyde fixation. J Histochem Cytochem 1975;23:295–302. [12] Nollet F, Billiet J, Selleslag D, Criel A. Standardisation of multiplex fluorescent short tandem repeat analysis for chimerism testing. Bone Marrow Transplant 2001;28:511–8. [13] Jiang H, Qiu H, Xue Y, Pan J, Wu Y, Zhang J, et al. Establishment and characterization of a novel acute myeloid leukemia cell line, JIH-4, carrying a t(16;21)(p11.2;q22) and expressing the FUS-ERG fusion. Cancer Genet 2011;204:219–23. [14] Pallisgaard N, Hokland P, Riishoj DC, Pedersen B, Jorgensen P. Multiplex reverse transcription-polymerase chain reaction for simultaneous screening of 29 translocations and chromosomal aberrations in acute leukemia. Blood 1998;92:574–88. [15] Pedersen-Bjergaard J, Rowley JD. The balanced and the unbalanced chromosome aberrations of acute myeloid leukemia may develop in different ways and may contribute differently to malignant transformation. Blood 1994;83:2780–6. [16] Imamura J, Miyoshi I, Koeffler HP. p53 in hematologic malignancies. Blood 1994;84:2412–21. [17] Drexler HG, Fombonne S, Matsuo Y, Hu ZB, Hamaguchi H. Uphoff CC. p53 alterations in human leukemia-lymphoma cell lines: in vitro artifact or prerequisite for cell immortalization. Leukemia 2000;14:198–206. [18] Mhawech P, Saleem A. Myelodysplastic syndrome: review of the cytogenetic and molecular data. Crit Rev Oncol Hematol 2001;40:229–38. [19] Smith ML, Fornace Jr AJ. Genomic instability and the role of p53 mutations in cancer cells. Curr Opin Oncol 1995;7:69–75.