Molecular and cytological features of the mouse B-cell lymphoma line iMycEμ-1

Background 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. Methods 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. Results 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. Conclusion 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.


Background
Gene-targeted iMyc Eµ mice contain a single-copy mouse Myc His (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 Myc His transgene is in the intervening region of the Igh joining gene locus, J H , and the intronic heavy-chain enhancer, Eµ. The inserted transgene encodes a C-terminal His 6 tag that is useful to distinguish message and protein encoded by Myc His and normal Myc [1]. The iMyc Eµ 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 [2]. Specifically, the iMyc Eµ mice mimic the type of t(8;14)(q24;q32) and T(12;15) translocation that is found in the endemic form of BL [3] and a subset (~20%) of IL-6 transgenic mouse plasmacytomas [4], respectively. We have recently shown that heterozygous transgenic iMyc Eµ 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) [1]. We now report on a newly established LBL-derived cell line, iMyc Eµ -1, which was developed to study the growth and survival requirements of LBL in vitro.

Features of iMyc Eµ -1 cells
The iMyc Eµ -1 cell line, which demonstrated the typical cytological features of mouse LBL (Fig. 1A top), was derived from a primary IgM + LBL (Fig. 1A bottom) that exhibited moderate plasmacytic differentiation potential in situ (not shown). FACS analysis using a panel of antibodies to B cell surface markers (Fig. 1B) showed that iMyc Eµ -1 cells were positive for CD40, CD48, CD54, Features of iMyc Eµ -1 cells Figure 1 Features of iMyc Eµ -1 cells. A, cytofuge specimen of cultured cells stained according to May-Grünwald-Giemsa (top). Tissue section of the LBL from which the cell line was derived after immunostaining for µ H-chain (bottom). B, B-cell surface marker expression evaluated by FACS in cells treated with specific antibodies (purple histograms) or isotype controls (green lines). C, H/L rearrangements and surface Ig expression. Southern blots of Igh (top left) and Igk (top right) rearrangements of the LBL from which the cell line was derived. Included as control is liver DNA from homozygous (Tg/Tg) or heterozygous (Tg/+) transgenic iMyc Eµ mice (left panel) or inbred C57BL/6 and 129SvJ mice (right panel). Recombination at the Igh locus was detected by the reduction of the normal, H chain-encoding upper fragment (upper arrowhead) in the face of comparable amounts of the mutated, Myc His -harboring lower fragment (lower arrowhead). Thus, the 6.2 kb long upper fragment was diminished in the LBL compared to the Tg/+ sample (and absent, as expected, in the Tg/Tg sample), whereas the Myc His -harboring lower fragment was comparable. The Myc His -bearing Igh locus cannot encode H chain because of the gene insertion. Recombination at the Igκ locus resulted in an enlarged fragment (~7.8 kb) compared to the germ line fragment that is indicated by the arrowhead pointing left. Detection of surface IgM hi IgD low using FACS analysis (bottom).
CD80/86 (B7-1/2) and CD138 (syndecan 1). Expression of class I and II MHC antigens and CD45 (B220) was detectable at low or very low levels, respectively, but CD95 (Fas) was absent. Treatment of iMyc Eµ -1 cells with antibody to CD40 led to the induction of Fas and upregulation of CD45 and CD54, activation markers CD80/86, and CD138, indicating that CD40 signaling was functional (Additional File 1). Southern blotting of genomic DNA from the LBL from which the cell line was derived demonstrated V(D)J rearrangement at the Ig heavy-chain and κ light chain loci (Fig. 1C top). The expression of sur-face IgM high IgD low by the derivative cell line was consistent with this and indicated that the rearrangement was productive ( Fig. 1C bottom). SKY analysis of metaphase chromosomes from iMyc Eµ -1 cells (Fig. 2) uncovered four chromosomal translocations that took the form of a reciprocal T(9;11) exchange and three non-reciprocal exchanges: T(13;16), T(14;13) and T(17;6). Although it is unclear whether these translocations occurred during tumor development or establishment of the cell line [5], repeat karyotyping showed that the present iMyc Eµ -1 line is cytogenetically stable.
Similar gene expression profile of iMyc Eµ -1 cells and LBL using comparative cDNA microarray measurements

Gene expression profile of iMyc Eµ -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 [6]. To compare the gene expression profile of LBL and iMyc Eµ -1 cells, RNA was obtained from primary B cells, fresh-frozen tumors and iMyc Eµ -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/.
Three independent RNA samples of iMyc Eµ -1 cells and ten primary LBL were analyzed together with a collection of normal, resting mouse B cells and T cells, mouse embryonic fibroblasts (MEF), and peritoneal plasmacytomas that arose in pristane-treated BALB/c mice. A total of 414 well-characterized array elements that clustered across these samples based on gene expression patterns (Fig. 3) demonstrated a clear distinction of lymphoid and nonlymphoid cell types (B and T cells versus MEF), lymphocyte lineages (B versus T cells), and transformationand development-associated differences within the B-cell lineage (normal B cells versus LBL and PCT). The gene expression profiles of LBL and iMyc Eµ -1 cells, which clustered in one tight group (Fig. 3 top, blue rectangle), exhibited a remarkable homogeneity. Compared to the plasmacytomas, LBL and iMyc Eµ -1 cells maintained many hallmark genes in the B cell signature (e.g., those encoding CD19, CD79 and µ heavy-chain) but under-expressed numerous genes in the plasma cell signature (e.g., Sec61, Ssr4 and DNAjc3) and matrix signature (e.g., those encoding vinculin, gelsolin and integrin B1) [7,8]. A more detailed analysis of the plasma cell signature revealed that in contrast to Xbp1 and its target genes, the iMyc Eµ -1 cells expressed Prdm1 (Blimp1). This suggested that the cells underwent neoplastic transformation at the early stage of plasmacytic differentiation (Additional File 2).

Myc expression in LBL and iMyc Eµ -1 cells
The expression levels of Myc, as measured by the arrays, was clearly elevated in LBL and iMyc Eµ -1, intermediate in "premalignant" B cells from tumor-free iMyc Eµ mice, and absent, as expected, in resting lymphocytes from normal mice (Fig. 4A). Because the inserted Myc cDNA in iMyc Eµ mice also encodes a C-terminal His 6 tag, it is possible to distinguish message and protein encoded by Myc His and normal Myc. Allele-specific RT-PCR analysis of Myc His and Myc mRNA demonstrated that, in common with LBL, iMyc Eµ -1 cells expressed predominantly the transgene (Fig. 4B top, lanes 2-3). This pattern of suppression of the normal Myc gene [9] is also a feature of human B-cell lymphomas containing constitutively deregulated MYC [10]. Western blotting with an anti-Myc antibody detecting both Myc His and normal Myc proteins (Fig. 4B bottom) showed that LBL and iMyc Eµ -1 cells over-expressed Myc at comparable levels (lanes 2-3) relative to B splenocytes from non-transgenic littermates (lane 1). To compare the levels of Myc in LBL and iMyc Eµ -1 cells more precisely, we performed qPCR using Aktb mRNA levels as internal standard. The iMyc Eµ -1 cells expressed nearly twice as much Myc as the LBL (Fig. 4C). The levels of Myc also correlated with the expression of genes from the proliferation cluster when the gene expression from the proliferation signature, as defined in Figure 3, was averaged for each cell type. Proliferation gene expression was low in unstimulated cells (MEF and B/T cell samples), intermediate in pre-malignant B cells from iMyc Eµ mice, and upregulated in LBL and iMyc Eµ -1 (Fig. 4D). Figure 3 were significantly differential in their expression when B cells were compared to iMyc Eµ -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 upregulated in the LBL and iMyc Eµ -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 iMyc Eµ -1 as Myc-driven B-cell tumors and firmly established the similarity of LBL and iMyc Eµ -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 iMyc Eµ -1 cells
To further compare the gene expression profiles of LBL and iMyc Eµ -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, iMyc Eµ -1 and normal B cells. Included in the analysis were RNA samples of iMyc Eµ -1 and LBL previously analyzed on the microarray. Freshly

Concordant gene expression changes in iMyc Eµ -1 cells and LBL compared to normal B cells Figure 5 Concordant gene expression changes in iMyc Eµ -1 cells and LBL compared to normal B cells.
A, gene expression changes were assessed by comparative filter cDNA macroarray measurements using a representative apoptosis array of normal B cells (left), LBL (center) and iMyc Eµ -1 cells (right) as the example. Compared to B cells, LBL and iMyc Eµ -1 cells underexpressed Bcl2a1d (array position D2), Birc2 (G3), Cflar (G4), Ripk1 (G8) and Traf5 (E12; indicated by green squares). Additional File 3 shows four additional LBL arrays that exhibit the same changes. B, expression changes of 5 up-regulated and 11 down-regulated genes in iMyc Eµ -1 cells compared to normal B cells (see Additional File 6 for names, functions and groupings of these genes). Similar changes were seen when LBL and B cells were compared using cDNA macroarrays (not shown) or when iMyc Eµ -1 cells and/or LBL were compared to B cells using cDNA microarrays ( Fig. 3; results not shown). C, verification of gene array results using RT-PCR. Shown are ethidium bromide-stained PCR fragments of the differentially regulated genes plotted in panel B except Myc, which was verified in the experiments presented in Figure 4A-C. The iMyc Eµ -1 and B-cell samples are shown in the right and left lane, respectively. Up and down regulated genes are depicted on the pink and green background, respectively.
prepared RNA from normal, MACS purified, B220 + splenocytes were used as control. RNA samples were labeled with 32 P-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.
The changes on the macroarrays were remarkably consistent in LBL and iMyc Eµ -1 cells compared to B cells. This is illustrated in Figure 5A, using the apoptosis array as the example. Among a total of 768 genes present on eight different macroarrays, 121 (16%) genes were concordantly up-or down-regulated in LBL and iMyc Eµ -1 cells relative to B cells. The NFκB array showed the highest number of changes (n = 22) among the eight different macroarrays, followed by the MAPK (n = 19), cell cycle (n = 18) and apoptosis arrays (n = 17). The stress and toxicity array contained the lowest number of changes (n = 9). Altogether, down-regulated genes (83/121, 69%) outnumbered upregulated genes (38/121, 31%) by a factor of 2.2. The presence of some genes on two or more arrays afforded an opportunity for additional quality control. Genes of that sort exhibited the same trend on different arrays, either up or down relative to normal B cells, thus adding confidence in the results. This is illustrated in Additional File 4 using one representative array each of iMyc Eµ -1 and normal B cells.
Among the differentially regulated genes that exhibited concordant changes in LBL and iMyc Eµ -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 iMyc Eµ -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 iMyc Eµ -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).

Conclusion
This study reports the molecular, cytogenetic and morphological features of a stable cell line, designated iMyc Eµ -1. The iMyc Eµ -1 cells are surface IgM high IgD low and cytoge-netically stable using SKY. The cells express high levels of the inserted Myc His transgene and exhibit a global gene expression profile consistent with that of Myc His -driven Bcell neoplasia. The iMyc Eµ -1 cells may be useful for indepth studies on the growth and survival requirements of Myc His -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 [1]. 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 iMyc Eµ -1 cells
Transgenic iMyc Eµ 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 [11]. 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 iMyc Eµ -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).