Molecular and cytological features of the mouse B-cell lymphoma line iMycEμ-1
© Su Han et al; licensee BioMed Central Ltd. 2005
Received: 31 August 2005
Accepted: 09 November 2005
Published: 09 November 2005
Myc-induced lymphoblastic B-cell lymphoma (LBL) in iMycEμ mice may provide a model system for the study of the mechanism by which human MYC facilitates the initiation and progression of B cell and plasma cell neoplasms in human beings. We have recently shown that gene-targeted iMycEμ mice that carry a His6-tagged mouse Myc cDNA, MycHis, just 5' of the immunoglobulin heavy-chain enhancer, Eμ, are prone to B cell and plasma cell tumors. The predominant tumor (~50%) that arose in the iMycEμ mice on the mixed genetic background of segregating C57BL/6 and 129/SvJ alleles was LBL. The purpose of this study was to establish and characterize a cell line, designated iMycEμ-1, for the in-depth evaluation of LBL in vitro.
The morphological features and the surface marker expression profile of the iMycEμ-1 cells were evaluated using cytological methods and FACS, respectively. The cytogenetic make-up of the iMycEμ-1 cells was assessed by spectral karyotyping (SKY). The expression of the inserted MycHis gene was determined using RT-PCR and qPCR. Clonotypic immunoglobulin gene arrangements were detected by Southern blotting. The global gene expression program of the iMycEμ-1 cells and the expression of 768 "pathway" genes were determined with the help of the Mouse Lymphochip© and Superarray© cDNA micro- and macroarrays, respectively. Array results were verified, in part, by RT-PCR and qPCR.
Consistent with their derivation from LBL, the iMycEμ-1 cells were found to be neoplastic IgMhighIgDlow lymphoblasts that expressed typical B-cell surface markers including CD40, CD54 (ICAM-1), CD80 (B7-1) and CD86 (B7-2). The iMycEμ-1 cells harbored a reciprocal T(9;11) and three non-reciprocal chromosomal translocations, over-expressed MycHis at the expense of normal Myc, and exhibited gene expression changes on Mouse Lymphochip© microarrays that were consistent with MycHis-driven B-cell neoplasia. Upon comparison to normal B cells using eight different Superarray© cDNA macroarrays, the iMycEμ-1 cells showed the highest number of changes on the NFκB array.
The iMycEμ-1 cells may provide a uniquely useful model system to study the growth and survival requirements of Myc-driven mouse LBL in vitro.
Gene-targeted iMycEμ mice contain a single-copy mouse MycHis (c-myc) cDNA that has been inserted in opposite transcriptional orientation in the mouse immunoglobulin heavy-chain gene cluster, Igh. The specific insertion site of the MycHis transgene is in the intervening region of the Igh joining gene locus, JH, and the intronic heavy-chain enhancer, Eμ. The inserted transgene encodes a C-terminal His6 tag that is useful to distinguish message and protein encoded by MycHis and normal Myc . The iMycEμ mice provide a model system for the study of the molecular and oncogenic consequences of the human MYC- and mouse Myc-deregulating chromosomal t(8;14)(q24;q32) and T(12;15) translocations that are widely accepted as the crucial initiating oncogenic events in the great majority of human Burkitt lymphomas (BL) and mouse plasmacytomas, respectively . Specifically, the iMycEμ mice mimic the type of t(8;14)(q24;q32) and T(12;15) translocation that is found in the endemic form of BL  and a subset (~20%) of IL-6 transgenic mouse plasmacytomas , respectively. We have recently shown that heterozygous transgenic iMycEμ mice on the mixed genetic background of segregating C57BL/6 and 129/SvJ alleles are genetically prone to mature B cell and plasma cell neoplasms, ~50% of which are IgM+ lymphoblastic B-cell lymphomas (LBL) . We now report on a newly established LBL-derived cell line, iMycEμ-1, which was developed to study the growth and survival requirements of LBL in vitro.
Results and Discussion
Features of iMycEμ-1 cells
Gene expression profile of iMycEμ-1 cells on cDNA microarray
The Mouse Lymphochip, a microarray of hematopoietic mouse cDNA clones, provides a powerful tool to evaluate the similarity of primary mouse B cells, B-cell tumors, and tumor-derived cell lines at the level of global gene expression . To compare the gene expression profile of LBL and iMycEμ-1 cells, RNA was obtained from primary B cells, fresh-frozen tumors and iMycEμ-1 cells. The RNA was labeled with Cy5-dUTP, and hybridized to the cDNA microarray spotted on a glass slide. An RNA control pool labeled with Cy3-dUTP was co-hybridized to the same array and used as a common denominator by which all samples were compared to one another. Further information on microarray make-up, analysis and data interpretation is available at: http://lymphochip.nih.gov/ShafferPCfactors/.
Myc expression in LBL and iMycEμ-1 cells
Myc target genes in LBL and iMycEμ-1 cells
To further examine the contribution of the MycHis transgene to the gene expression profile of LBL and iMycEμ-1, we performed a statistical analysis (Student's T test) of genes differentially expressed between normal B cells versus LBL and iMycEμ-1 cells. A total of 122 array elements from Figure 3 were significantly differential in their expression when B cells were compared to iMycEμ-1 cells and LBL (p < 0.015, 1.5-fold minimal difference in average expression). The vast majority (97%) of these elements, many of them previously identified as proliferation-associated Myc targets http://www.myc-cancer-gene.org, behaved similarly in both the cell line and LBL (Fig. 4E). Twenty-four known Myc targets were up-regulated in the LBL and iMycEμ-1 cells (Ahcy, Apex1, Cbfb, Cdk4, Ctps, Eef2, Gapd, Hdgf, Hnrpa1, Hnrpd, Idh1, Myc, Ncl, Nme1, Npm1, Pa2g4, Pcna, Pim1, Pkm2, Ppia, Sfrs2Slc7a5, Tfdp1), two were down-regulated (Btg1, Igk), and five were undetermined as to the effect of Myc on their expression (Aldh2, Hint1, Mcl1, Rheb, Slc1a4). These findings were in accordance with the nature of LBL and iMycEμ-1 as Myc-driven B-cell tumors and firmly established the similarity of LBL and iMycEμ-1at the level of a single gene (Fig. 4A), a gene expression signature (Fig. 4D), and globally (Fig. 3).
Validation of gene expression changes in iMycEμ-1 cells
To further compare the gene expression profiles of LBL and iMycEμ-1, and validate the cDNA microarray results with an independent method, we used cDNA macroarrays on nylon membranes to assess the expression of selected "pathway" genes in LBL, iMycEμ-1 and normal B cells. Included in the analysis were RNA samples of iMycEμ-1 and LBL previously analyzed on the microarray. Freshly prepared RNA from normal, MACS purified, B220+ splenocytes were used as control. RNA samples were labeled with 32P-dUTP and individually hybridized to the macroarrays. Individual expression profiles were determined and compared with each other. Reproducible two-fold or higher changes in hybridization signal intensity were used as threshold for gene expression changes. Eight different macroarrays, each containing 96 genes involved in cell cycle regulation, apoptosis, cancer, signal transduction, stress and toxicity responses, and the NFκB and MAPK pathways, were used. The primary data set is depicted in Additional File 3.
Among the differentially regulated genes that exhibited concordant changes in LBL and iMycEμ-1 cells relative to normal B cells on both the gene micro- and macroarrays were 16 genes that were selected for further confirmation using semi-quantitative RT-PCR (Additional File 5). These genes were of particular interest to us because of possible follow-up studies on signaling pathways in iMycEμ-1 cells. Eleven of the 16 genes were down regulated (Bcl2a1, Birc2, Cflar, Cdkn1b, Grb2, Irf1, Jun, Map2k1, Rb1, Ripk1, Traf5) and five genes were up regulated (Ccna2, Ccnb, Myc, Nfkb1, Odc). Figure 5B presents average quantitative changes on the macroarrays when iMycEμ-1 cells were compared with normal B cells. These changes were readily confirmed by RT-PCR in all cases (Fig. 5C) except Myc, which was not included because it was confirmed in previous work (Fig. 4).
This study reports the molecular, cytogenetic and morphological features of a stable cell line, designated iMycEμ-1. The iMycEμ-1 cells are surface IgMhighIgDlow and cytogenetically stable using SKY. The cells express high levels of the inserted MycHis transgene and exhibit a global gene expression profile consistent with that of MycHis-driven B-cell neoplasia. The iMycEμ-1 cells may be useful for in-depth studies on the growth and survival requirements of MycHis-driven mouse B-cell tumors in vitro. Specifically, the cells may facilitate the elucidation of the signal transduction pathways that appear to maintain high Myc protein levels in mouse LBL . These studies may results in new approaches to treat and prevent MYC-induced B cell and plasma cell neoplasms in human beings.
Mouse lymphomas and derivation of iMycEμ-1 cells
Transgenic iMycEμ mice develop a high incidence of B cell and plasma cell tumors with LBL being the predominant phenotype (8). Tumor samples obtained at autopsy were fixed in formalin for later histopathology or frozen for later preparation of protein, DNA and RNA. Histological criteria used for diagnosing mouse LBL are detailed elsewhere . Highly enriched splenic B cells were prepared from C57BL/6 mice using CD45R (B220) microbeads and MACS separation columns (Miltenyi Biotec, Auburn, CA). All mice were maintained under Animal Study Protocol LG-028. The iMycEμ-1 cell line was derived from a LBL and maintained at 37°C and 5% carbon dioxide in RPMI 1640 medium supplemented with 10% fetal calf serum, 200 mM L-glutamine, 50 μM 2-mercaptoethanol and penicillin/streptomycin (Gibco-BRL, Rockville, MD).
Characterization of iMycEμ-1 cells
For cytological analysis, cytofuge specimens were stained according to May-Grünwald-Giemsa and inspected by microscopy. For detection of chromosomal aberrations, cells were analyzed by spectral karyotyping (SKY) as previously described . For flow cytometry, single-cell suspensions were stained and analyzed on a FACSort® using the CELLQuest™ software (BD Pharmingen, San Diego, CA). Rat anti-mouse CD16/CD32 was used to block FcγII and FcγIII receptors. Antibodies to mouse CD45 (catalog number 553076), CD80 (553766), Fas (CD90, 554255), CD86 (553689), CD40 (553787), I-Ab (MHC class II, 553551), H-2Kb (MHC class I, 553569), CD48 (557483), CD54 (553250), CD138 (553712), IgD (553438) and IgM (53519) were purchased from BD Biosciences. For the evaluation of surface marker changes upon ligation of CD40, cells were incubated with rat anti-mouse CD40 (553787) using 3.5 μg antiboy per 5 × 105 cells. For Southern blot hybridization of clonotypic V(D)J rearrangements, genomic DNA (20 μg) was digested with BamHI and EcoRI, fractionated on a 0.7 % agarose gel, transferred to a nylon membrane, and crosslinked under UV light. Following pre-hybridization (Hybrisol I, Intergen) at 42°C, the membrane was hybridized to a 1.5-kb HindIII/EcoRI fragment of Igh spanning J H 2 and Eμ or to a 1.1-kb Cκ probe, which was generated by PCR using a primer pair obtained from Dr. Michael Kuehl (NCI): 5'-GAT GCT GCA CCA ACT GTA TCC A-3' and 5'-GGG GTG ATC AGC TCT CAG CTT-3'. Probes were labeled with [32P]-CTP using a random priming kit.
Allele-specific RT-PCR of Myc and MycHis mRNA
For semi-quantitative determination of Myc and MycHis mRNA, total RNA was isolated using TRIzol (Sigma, St. Louis, MO, USA). Double stranded cDNA was synthesized from 1 μg of total RNA, using the AMV Reverse Transcriptase kit (Roche, Indianapolis, IN). A common 5' primer for both MycHis and Myc (5'-TCT CCA CTC ACC AGC ACA AC-3') was combined with a specific 3' primer for MycHis (5'-CCT CGA GTT AGG TCA GTT TA-3') and Myc (5'-ATG GTG ATG GTG ATG ATG AC-3') to distinguish the two messages. Thermal cycling conditions were as follows: 95°C for 5 min followed by 20 cycles of amplification at 57°C, 72°C and 95°C, each for 1 min. PCR amplification of Aktb cDNA was performed as control using the following primer pair: 5'-GCA TTG TTA CCA ACT GGG AC-3' and 5'-AGG CAG CTC ATA GCT CTT CT-3'. PCR products were analyzed by electrophoresis in 1% agarose gel and visualized by staining with ethidium bromide.
Real-time qPCR of Myc mRNA
For quantitative Taqman RT-PCR of Myc (Myc plus MycHis), total RNA was isolated from cells using TRIzol Reagent (Invitrogen). Serial dilutions of input RNA (100 ng - 1.56 ng) were analyzed in triplicates using the ABI PRISM 7900HT sequence detector system, primers, probes, and the Taqman One-Step RT-PCR Master Mix Reagents kit, all purchased from Applied Biosystems. The reaction mixture was held at 48°C for 30 min for reverse transcription of RNA into cDNA. This was followed by incubation at 95°C for 10 min to activate the Taq polymerase. PCR amplification of cDNA was performed for 40 cycles using the following cycling conditions: denaturing for 15 s at 95°C and annealing and extending for 1 min at 60°C. All samples were tested in triplicates, and average values were used for quantification. Analysis was performed using SDS v2.1 software (Applied Biosystems) according to the manufacturer's instruction. Aktb was used as internal reference gene. The comparative CT method (ΔΔCT) was used for quantification of gene expression.
Gene microarray hybridization and analysis
cDNA made from total RNA (50 μg) from each tumor, primary cell sample, or iMycEμ-1 cells was labeled with cyanine 5-conjugated dUTP (Cy5). cDNA made from pooled mouse cell line RNA (50 μg) was labeled with cyanine 3-conjugated dUTP (Cy3) and used as reference. Microarray hybridizations were performed on Mouse Lymphochip microarrays . After washing, the slides were scanned using an Axon GenePix 4.0 scanner (Axon Instruments Inc., Union City, CA). After normalization, those elements that failed to meet confidence criteria based on signal intensity and spot quality were excluded from analysis. In addition, data were discarded for any gene for which measurements were missing on >30% of the arrays or were not sequence-verified. The Cy5:Cy3 intensity ratios of the remaining spots were log2 transformed. To compare normal samples, hierarchical cluster analysis was performed using the Gene Cluster and Treeview programs .
Gene macroarray hybridization and analysis
The relative mRNA expression of genes involved in regulation of apoptosis, cell cycle progression, NFkB signaling, and cellular stress and toxicity responses was analyzed with GEArray (SuperArray Inc., Bethesda, MD) according to the manufacturer's protocol. Cells were treated for 24 hrs with 0.4 mM and 1 mM CDDO-Im, respectively, followed by preparation of total RNA using TriReagent (Sigma). Five μg from each sample were reverse transcribed into 32P-labeled cDNA using MMLV reverse transcriptase (Promega, Madison, WI) and 32P-dCTP (NEN, Boston, MA). The resulting cDNA probes were hybridized to gene-specific cDNA fragments spotted in quadruplicates on the GEArray membranes. After stringent washing of the arrays, the signal of the hybridized spots was measured with a STORM PhosphorImager (Molecular Dynamics, Sunnyvale, CA) and normalized to the signal of the housekeeping gene Gapd. Array results on six CDDO-Im inducible genes were validated using semi-quantitative RT-PCR.
Gene array validation using RT-PCR
For semi-quantitative determination of mRNA levels, total RNA was isolated and double stranded cDNA was synthesized as described above for Myc. Information on PCR primers and thermal cycling conditions is available in Additional File 6. PCR products were analyzed by electrophoresis in 1% agarose gel and visualized by staining with ethidium bromide.
We thank Wendy duBois, Nicole Wrice and Vaishali Jarral, NCI, for assistance with the in vivo studies; Michael Kuehl, NCI, for the Cκ probe; R. Eric Davis, NCI, for helpful scientific discussions; and Beverly A. Mock, NCI, for support. This research was supported by the Intramural Research Program of the NIH, NCI, CCR.
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