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[Cancer Research 65, 1306-1315, February 15, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

Insertion of c-Myc into Igh Induces B-Cell and Plasma-Cell Neoplasms in Mice

Sung Sup Park1, Joong Su Kim1, Lino Tessarollo4, James D. Owens1, Liangping Peng1, Seong Su Han1, Seung Tae Chung1, Ted A. Torrey3, Wan C. Cheung5, Roberto D. Polakiewicz6, Nicole McNeil2, Thomas Ried2, J. Frederic Mushinski1, Herbert C. Morse, III3 and Siegfried Janz1

1 Laboratory of Genetics, 2 Genetics Branch, Center for Cancer Research, National Cancer Institute, and 3 Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland; 4 Laboratory for Neural Development, Center for Cancer Research, National Cancer Institute, Frederick, Maryland; and 5 Cell Signaling Technology, Beverly, Massachusetts

Requests for reprints: Siegfried Janz, Laboratory of Genetics, Center for Cancer Research, National Cancer Institute, Building 37, Room 3140A, Bethesda, MD 20892-4256. Phone: 301-496-2202; Fax: 301-402-1031; E-mail: sj4s{at}nih.gov.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We used gene targeting in mice to insert a His6-tagged mouse c-Myc cDNA, MycHis, head to head into the mouse immunoglobulin heavy-chain locus, Igh, just 5' of the intronic enhancer, Eµ. The insertion of MycHis mimicked both the human t(8;14)(q24;q32) translocation that results in the activation of MYC in human endemic Burkitt lymphomas and the homologous mouse T(12;15) translocation that deregulates Myc in certain mouse plasmacytomas. Beginning at the age of 6 months, MycHis transgenic mice developed B-cell and plasma neoplasms, such as IgM+ lymphoblastic B-cell lymphomas, Bcl-6+ diffuse large B-cell lymphomas, and CD138+ plasmacytomas, with an overall incidence of 68% by 21 months. Molecular studies of lymphoblastic B-cell lymphoma, the most prevalent neoplasm (50% of all tumors), showed that the lymphomas were clonal, overexpressed MycHis, and exhibited the P2 to P1 promoter shift in Myc expression, a hallmark of MYC/Myc deregulation in human endemic Burkitt lymphoma and mouse plasmacytoma. Only 1 (6.3%) of 16 lymphoblastic B-cell lymphomas contained a BL-typical point mutation in the amino-terminal transactivation domain of MycHis, suggesting that most of these tumors are derived from naive, pregerminal center B cells. Twelve (46%) of 26 lymphoblastic B-cell lymphomas exhibited changes in the p19Arf-Mdm2-p53 tumor suppressor axis, an important pathway for Myc-dependent apoptosis. We conclude that MycHis insertion into Igh predictably induces B-cell and plasma-cell tumors in mice, providing a valuable mouse model for understanding the transformation-inducing consequences of the MYC/Myc-activating endemic Burkitt lymphoma t(8;14)/plasmacytoma T(12;15) translocation.

Key Words: Chromosomal translocations activating human c-MYC or mouse c-Myc • human t(8;14)(q24;q32) and mouse T(12;15) translocations • human Burkitt's lymphoma and mouse plasmacytoma • gene targeting in mice


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Reciprocal chromosomal translocations that activate the cellular oncogene MYC are widely accepted as the initiating events in the natural history of human endemic Burkitt lymphoma, an aggressive form of mature, postgerminal center, non-Hodgkin lymphoma. The most common translocation found in endemic Burkitt lymphoma is t(8;14)(q24;q32), which recombines MYC at 8q24 with the immunoglobulin heavy-chain gene locus, IGH, at 14q32 (1, 2). The breakpoints of t(8;14) usually occur in the joining gene region of the IGH locus, JH, and in the 5' flank of MYC. The resulting exchanges allocate the intact MYC gene, including the noncoding regulatory first exon with the two major MYC promoters, P1 and P2, to the MYC-deregulated product of translocation, where MYC is apposed to the intronic heavy-chain enhancer, Eµ, in opposite transcriptional orientation (Fig. 1). Mouse plasmacytoma, a malignant tumor of terminally differentiated B cells, is thought to be initiated, just like human endemic Burkitt lymphoma, by Myc-activating chromosomal translocations (3). The most common translocation observed in plasmacytoma is the T(12;15), the direct counterpart of the human t(8;14) translocation. A recent molecular analysis of T(12;15) in IL-6 transgenic plasmacytomas and their precursors has shown that 8 (18%) of 45 translocation breakpoint regions exhibited the endemic Burkitt lymphoma t(8;14)-typical Myc-Eµ juxtaposition on the Myc-deregulated product of translocation (Fig. 1; ref. 4). Thus, homologous MYC/Myc-Eµ rearrangements lead to B-cell and plasma-cell neoplasms in humans and mice.



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  		Figure 1. Targeted transgenic insertion of MycHis in iMycEµ mice. Shown is a scheme of the normal mouse Igh locus (top) and the targeted Igh locus (bottom) with the inserted MycHis in iMycEµ mice (red). The Eµ and E{alpha} enhancers are depicted by blue diamonds. The transcriptional orientations of Igh and Myc are indicated by black and red arrows, respectively. The gene insertion site is also the preferred recombination site of human t(8;14)(q24;q32) IGH-MYC exchanges in human endemic Burkitt lymphoma and mouse T(12;15) Igh-Myc exchanges in certain mouse plasmacytoma.

 
The mechanism by which human endemic Burkitt lymphoma t(8;14) and mouse plasmacytoma T(12;15) deregulate the expression of MYC/Myc and, thereby, promote the malignant transformation of B lymphocytes and plasma cells has not been shown (5). Further insights into this mechanism might be possible if a mouse model that accurately reproduces the key features of the endemic Burkitt lymphoma t(8;14)/plasmacytoma T(12;15) could be produced. Previous attempts to develop such a model used mice that harbor multiple copies of human MYC or mouse Myc under control of the human or mouse Eµ enhancer (6–9). Clearly, the Eµ-driven MYC/Myc genes are incomplete models of the endemic Burkitt lymphoma t(8;14)/plasmacytoma T(12;15) because they reproduce just one aspect of this translocation, the juxtaposition of Eµ and MYC/Myc (5). Another approach used mice that carry a 240-kb single-copy yeast artificial chromosome (YAC), which consists of a human germ line VDJ-Cµ-C{delta} locus and the MYC gene from the BL cell line Raji (10, 11). Serious shortcomings of the YAC-MYC approach include the absence of the E{alpha} enhancer, which is believed to be the critical element for MYC up-regulation in tumors of late B and plasma cells (12), the unusual location of MYC in the IGH locus (3' of C{delta}) and the head-to-tail orientation of MYC relative to IGH, features that are not observed in human endemic Burkitt lymphoma or mouse plasmacytoma. Consistent with the apparent inability of the YAC-MYC and Eµ-MYC/Myc transgenes to faithfully recreate the endemic Burkitt lymphoma t(8;14)/plasmacytoma T(12;15), the transgenic mice develop predominantly lymphomas with features of pre-B, immature B, or transitional B cells, rather than mature B cells (human endemic Burkitt lymphoma) or plasma cells (mouse plasmacytoma).

To model endemic Burkitt lymphoma t(8;14)/plasmacytoma T(12;15) more precisely, we inserted a His6-tagged mouse Myc cDNA in the JH-Eµ intervening region of mouse Igh and developed a mouse strain, designated iMycEµ, in which Myc is juxtaposed head to head with Eµ, just as in human endemic Burkitt lymphoma and mouse plasmacytoma (Fig. 1). Between the age of 6 and 21 months, nearly 70% of the heterozygous iMycEµ mice developed lymphoblastic B-cell lymphomas with a BL-like morphology, diffuse large B-cell lymphomas and plasmacytomas. All these are tumors of mature B cells. The observed spectrum of tumors provided strong empirical evidence that the iMycEµ transgene mimicked the critical features of the endemic Burkitt lymphoma t(8;14)/plasmacytoma T(12;15) translocation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Generation of iMycEµ Mice. Gene targeting (13) and cre-loxP recombination (14) were used to insert a mouse Myc gene into the mouse germ line Igh locus. The inserted Myc consisted of an intronless cDNA clone, the noncoding first exon with the natural P1/P2 promoter, 1.5 kb of genomic 5' flank containing the normal transcription-regulatory region, and a short stretch of 3' untranslated region harboring the Myc major polyadenylation site. The untranslated region downstream of the polyadenylate signal, which has been shown to be dispensable in vivo (15), was not present in the construct. The Myc also contained the coding sequence for an artificial histidine tag, His6, added in frame at the 3' end of the gene. The tag made it possible to distinguish Myc mRNA and Myc protein encoded by the inserted MycHis gene on chromosome 12 from the endogenous Myc gene (no tag) on chromosome 15. MycHis was inserted in the intervening region between JH4 and Eµ, ~ 0.5 kb 5' of Eµ, in opposite transcriptional orientation to Igh. The construction of the inserted gene, MycHisNeoloxP, the assembly of the targeting vector, and the generation of Neo-less iMycEµ mice are described in the Supplementary Materials and Methods section and illustrated in Supplementary Fig. 1. This study used heterozygous transgenic mice harboring one targeted Igh allele, iMycEµ, and one wild-type Igh allele. The targeted and wild-type Igh alleles were from strains 129SvJ (129) and C57BL/6 (B6), respectively. The genetic background of the iMycEµ colony was initially a variable, segregating mixture of 129 and B6 alleles. The mice used for the studies reported here were from generations three to six of an ongoing backcross of the iMycEµ transgene onto B6, resulting in an average content of B6 alleles from 93.8% (N3) to 99.2% (N6).

Turnover of "Premalignant" B Splenocytes. Cell surface marker expression was assessed using a FACSCalibur flow cytometer (BD Biosciences Pharmingen, San Diego, CA) and a panel of reagents useful for distinguishing subsets of hematopoietic cells. Total Myc mRNA levels, encoded by Myc and MycHis, were determined in fluorescence-activated cell-sorting (FACS)-purified subpopulations of bone marrow B cells using the quantitative PCR assay on demand kit from Applied Biosystems (Foster City, CA). The turnover of MACS-purified B220+ splenic B cells was measured by bromodeoxyuridine (BrdUrd) incorporation in vivo using the kit from BD Biosciences Pharmingen. For cell cycle analysis, B cells were stained with 50 µg/mL propidium iodide in buffer containing 0.1% sodium citrate and 0.1% Triton X-100 followed by FACS analysis. For determination of apoptosis, the FITC-conjugated monoclonal active caspase-3 antibody kit I was used (BD Bioscience). Proliferation of B cells in vitro was measured with the assistance of the colorimetric 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay (CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay) from Promega (Madison, WI). To that end, B220+ splenic cells were purified by MACS (Miltenyi, Auburn, CA) and grown for 48 hours in RPMI 1640 supplemented with 25 µg/mL lipopolysaccharide (LPS) from Escherichia coli 055:B5 (Sigma, St. Louis, MO), 10% fetal calf serum, 200 mmol/L L-glutamine, 50 µmol/L 2-mercaptoethanol, and penicillin/streptomycin (Life Technologies, Inc., Gaithersburg, MD). The immunologic competence of iMycEµ mice was assessed by s.c. injection of 100 µg of the synthetic peptide CLQLKEQKNAGTRTNEG that had been conjugated to keyhole limpet hemocyanin and emulsified in complete Freund's adjuvant. This was followed by two i.p. booster injections of keyhole limpet hemocyanin-peptide in incomplete Freund's adjuvant spaced 3 weeks apart. Total immunoglobulin levels and peptide-specific antibodies were measured by ELISA as previously described (16).

Histological and Immunohistochemical Examination of Tumors. Histological features of B-cell and plasma-cell tumors were assessed in 5-µm-thick sections of paraffin-embedded tissues stained with H&E, Giemsa according to Lennert, or periodic acid-Schiff. Apoptosis was determined by using the terminal deoxynucleotidyl transferase–mediated nick end labeling (TUNEL) assay (17). Briefly, tissue sections were digested with proteinase K and placed in a reaction buffer containing digoxigenin-labeled dUTP and deoxynucleotidyl transferase (15 units/µL). Sections were incubated with anti-digoxigenin antibody, developed with New Fuchsin substrate, and counterstained with hematoxylin. To measure proliferation of tumor cells in vivo, mice were pulsed with BrdUrd (200 mg/m2 i.p., 8 hours), followed by immunostaining of tissue sections with antibody to BrdUrd. To visualize surface antigen expression in tumor cells, tissue sections were deparaffinized, treated with hot steam or microwaving to unmask antigen, incubated with 1% hydrogen peroxide to eliminate endogenous peroxidase activity, blocked with 5% goat serum, incubated with primary antibody overnight at 4°C, and detected by horseradish peroxidase–conjugated secondary antibody (1:200) and 3,3'-diaminobenzidine. Bcl-6 expression in diffuse large B-cell lymphomas was detected with a rabbit polyclonal antibody (N-3, Santa Cruz Biotechnology, Santa Cruz, CA) as previously described (18).

Molecular and Cytogenetic Studies with Lymphoblastic B-Cell Lymphoma. For Southern hybridization of clonotypic V(D)J rearrangements, genomic DNA obtained from tumor tissues was digested with EcoRI (for Igh) or EcoRI and BamHI (for Igk), fractionated by electrophoresis on 0.7% agarose gels, transferred onto a nitrocellulose membrane, and hybridized to a [32P]dCTP-labeled 1.5-kb HindIII/EcoRI Igh probe (pJ11) spanning JH3 and Eµ or a 1.1-kb C{kappa} probe. The latter probe was generated by PCR using a primer pair (forward: 5'-GAT GCT GCA CCA ACT GTA TCC A; reverse: 5'-GGG GTG ATC AGC TCT CAG CTT) and method developed by Dr. Michael Kuehl (National Cancer Institute, NIH). For spectral karyotyping, metaphase chromosomes were prepared from primary tumor cells ex vivo or tumor cells cultured in vitro for 1.5 hours in 20 µg/mL Colcemid (Life Technologies). Cells were lysed in hypotonic KCl solution; chromosomes were fixed in methanol-acetic acid (3:1); and spectral karyotyping analysis was done as previously described (19). For each tumor, at least 10 complete metaphase plates were analyzed.

Detection of MycHis by Immunoblotting and Total Myc mRNA by Northern Blotting. Tissue samples were homogenized in 50 mmol/L TRIS, 150 mmol/L NaCl, 0.5% Triton X-100, 0.5% SDS, 0.5% sodium deoxycholate, and 10% glycerol. Proteins from clarified lysates (40 µg) were resolved electrophoretically in denaturing 10% SDS-PAGE gels and transferred by electroblotting to nitrocellulose membranes. Membranes were probed with rabbit anti-His6-tag antibody (1:5,000, Clontech, Palo Alto, CA). The position of the MycHis protein was visualized with horseradish peroxidase–conjugated goat anti-rabbit antibody (1:5,000; Amersham, Arlington Heights, IL) using the chemiluminescence detection kit from Amersham. To confirm equal loading, the membrane was stripped and reprobed using an antibody specific for ß-actin (Sigma). For Northern blot analysis of Myc mRNA, polyadenylated RNA was extracted from tumor nodules, fractionated by electrophoresis on formaldehyde-containing 1.0% agarose gels, transferred to Hybond N membranes, and hybridized to a Myc exon 1 probe (pMmyc54) that was nick translated to a specific activity of 2 x 108 cpm/µg (20).

Determination of Myc and MycHis mRNA Levels by Allele-Specific Reverse Transcription–PCR. MycHis and Myc expression was determined by semiquantitative reverse transcription–PCR (RT-PCR) using a common 5' primer (5'-TCT CCA CTC ACC AGC ACA AC-3') but 3' primers specific either for MycHis (5'-CCT CGA GTT AGG TCA GTT TA-3') or wild-type Myc (5'-ATG GTG ATG GTG ATG ATG AC-3'). cDNA was synthesized using the AMV reverse transcriptase kit (Roche, Indianapolis, IN) and amplified by PCR using the following thermal cycling conditions: 95°C for 5 minutes (initial template denaturation) followed by 20 cycles of amplification at 62°C (primer annealing), 72°C (extension) and 95°C (melting), each for 1 minute. PCR amplification of the housekeeping gene Gapd (glyceraldehyde-3-phosphate dehydrogenase) was done for each sample as a control using the following primer pair: 5'-GGT GGA GCC AAA CGG GTC ATC ATC C-3' and 5'-CAC ATT GGG GGT AGG AAC ACG GAA GG-3'.

Analysis of Myc Promoter Shift by RNase Protection Analysis. RNA was extracted from B220+ MACS-purified splenic B cells and tumor tissues of iMycEµ mice with the TRIZOL reagent (Invitrogen, San Diego, CA) and then treated with DNase I. RNA samples from mouse plasmacytomas TEPC 1165 and ABPC 20, pooled thymus glands from twenty 4- to 6-weeks-old BALB/c mice and splenic B cells from C57BL/6 mice were included as controls. A 1,144-base antisense RNA probe was synthesized by T7 RNA polymerase using a MAXIscript in vitro transcription kit from Ambion (Austin, TX). The probe was synthesized in the presence of [{alpha}-32P]UTP at 800 Ci/mmol, 40 mCi/mL (PerkinElmer Life Sciences, New England Nuclear, Boston, MA) and purified on a 6% acrylamide-urea gel. The Myc probe covered sequences from an EcoRI star site 626 bp upstream of the P1 promoter to the SstI site near the 3' end of exon 1. Ribonuclease protection was done with the Ambion RPA III kit following the instructions of the manufacturer. Briefly, 10 µg of total RNA was hybridized with about 1 ng of Myc probe at 58°C for 16 hours, followed by digestion with 0.4 units of RNase A and 15 units of RNase T1 at 37°C for 30 minutes. The protected products were resolved on a 6% acrylamide-urea gel. Transcripts derived from the Myc P1 promoter protected 518 bases of the probe and transcripts derived from the P2 promoter protected 355 bases of the probe. Signals were quantitated with a Storm phosphor imager (Molecular Dynamics, Sunnyvale, CA) and normalized to correct signal strength for the number of uridine residues in the protected probe fragments. RNAs with known P1/P2 ratios of about 2 (TEPC 1165) and 0.4 (ABPC 20), respectively, were used as controls (21).

Detection of Point Mutations in Myc Amino-Terminal Transactivation Domain. Genomic DNA was purified from BLL using the Trizol reagent (Life Technologies), removing contaminating mRNA by digestion with Rnase A. MycHis was amplified by PCR using the following thermal cycling conditions: 95°C for 5 minutes followed by 30 cycles of amplification at 62°C for 20 seconds, 72°C for 60 seconds, and 95°C for 20 seconds. PCR primer sequences were 5'-ACA ATC TGC GAG CCA GGA CAG GAC T-3' (forward) and 5'-TCC TCA TCT TCT TGC TCT TCT TCA GAG T-3' (reverse). The resultant amplicon was 942 bp long and contained the protein-encoding exons 2 and 3 of MycHis together with the regulatory exon 1, which harbors the P1/P2 promoter. Normal Myc sequence was not amplified because the primers were separated by ~2.5 kb of intron 1 and 2 sequence, which was not present in the MycHis cDNA. PCR products were sequenced using equipment and reagents from Applied Biosystems.

Immunoblotting of p19Arf, Mdm2, and p53 and Detection of Deletions in Ink4a (p19ARF). For immunoblotting, whole-cell protein extracts were prepared from primary tumors in ice-cold modified radioimmunoprecipitation assay buffer buffer [50 mmol/L Tris-HCl (pH 7.4), 1% NP40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L phenylmethylsulfonylfluoride, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 1 mmol/L Na3VO4]. Undissolved material was sedimented in a microfuge (4°C, 7 minutes, 14,000 rpm) followed by measurement of protein in the supernatant. Protein (30 µg/lane) was separated on 10% SDS-PAGE gels, transferred to Protran nitrocellulose membranes (Schleicher & Schuell, Keene, NH), probed with antibody to mouse p19ARF (Ab-80, Abcam, Cambridge, MA), p53 (FL-393, Santa Cruz), and Mdm2 (C-18, Santa Cruz) and detected by horseradish peroxidase–conjugated secondary antibody using the enhanced chemiluminescence kit from Amersham. For detection of deletions of exon 1 in the Ink4a locus, PCR was done. PCR-amplified indicator fragments of Trp53 (p53) and Myc were included as controls. Genomic DNA was isolated from primary tumors using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN) followed by amplification with the Expand High-Fidelity PCR kit (Roche). Thermal cycling conditions were as follows: denaturation at 94°C for 1 minute, primer annealing at 61°C for 30 seconds in case of Ink4a (p19ARF) and Myc, or 58°C and 45 seconds in case of Trp53, followed by extension at 72°C for 45 seconds for the first 10 cycles and a progressive 5-second increase per cycle for the remaining 20 cycles. The following primer pairs were used: Ink4a (exon 1): 5'-ACT GAA TCT CCG CGA GGA AAG CGA ACT-3' (forward) and 5'-GAA TCG GGG TAC GAC CGA AAG AGT-3' (reverse), Trp53 (exon 3) 5'-CTT TGA GGT TCG TGT TTG TGC CC-3' (forward) and 5'-CCC TGA AGT CAT AAG ACA GCA AGG-3' (reverse), and Myc (exon 2-3) 5'-GAC GAC GAG ACC TTC ATC AAG AAC-3' (forward) and 5'-TCA CGC AGG GCA AAA AAG C-3' (reverse).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MycHis Is Expressed throughout B-Cell Development. Because onset and level of MycHis expression during B-cell development are likely to play an important role in determining incidence and types of lymphomas that might arise in iMycEµ mice, we assessed the expression of MycHis protein in lysates of FACS-sorted B cells at different maturation stages using immunoblotting with antibody to the His6 tag (Fig. 2A). B220+CD43+ pro-B cells (fraction ABC according to Hardy; refs. 22, 23) and B220+CD43– pre-B cells (DEF) collected from the bone marrow exhibited a 1.7-fold difference in MycHis levels after normalization to the actin loading controls (experiment 1). Further dissection of the pre-B cell compartment DEF into fractions D (early pre-B; B220+CD43–IgM–) and EF (late pre-B; B220+CD43–IgM+) showed that the MycHis levels varied again in a similar range (~2-fold) in these fractions (experiment 2). MycHis expression was also comparable in transitional and mature B splenocytes, including T1 B cells (IgMbrightIgDdull), T2 B cells (IgMbrightIgDbright), mature (M) B cells (IgMdullIgDbright), and sIg+{kappa}+ B cells further separated based on CD138 expression (experiment 3). These results showed that MycHis is overexpressed throughout B-cell development, presumably driven by the Eµ enhancer, which is thought to be active at relatively constant levels throughout B-cell development (24).



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  		Figure 2. Overexpression of MycHis throughout B-cell development in iMycEµ mice. A, expression of MycHis at different stages of B-cell development. Samples with MycHis/actin ratios set at 1.0 were used as internal standards in the three independent experiments. MycHis expression is not strictly limited to B cells and is, therefore, leaky. This is shown in Supplementary Fig. 2, which shows transgene expression in splenic T cells, but not in splenic monocytes. B, elevated Myc mRNA expression in pro-B/pre-B cells (B220lowIgM–), transitional B cells (B220low–IgM+), and mature B cells (B220highIgM+) obtained from bone marrow of iMycEµ mice and nontransgenic littermates (Control). Average levels and SDs based on three independent measurements.

 
To determine the elevation of Myc mRNA caused by insertion of MycHis in the vicinity of Eµ, we did quantitative PCR in three major B-cell fractions collected by FACS based on expression of B220 and IgM (Fig. 2B). Total Myc mRNA was ~10-fold higher in B220lowIgM– pro-B/pre-B cells (columns 1 and 4), B220lowIgM+ transitional B cells (columns 2 and 5), and B220hiIgM+ mature B cells (columns 3 and 6) obtained from the bone marrow of iMycEµ mice, compared with the same cell populations in normal littermates. These findings were consistent with Eµ controlling MycHis expression and suggested that the constitutive overexpression of MycHis leads to chronic changes in the B-cell compartment of iMycEµ mice before malignant transformation occurs.

Increased Turnover of Premalignant MycHis Transgenic B cells In vivo. To evaluate whether constitutive expression of MycHis leads to chronic activation and expansion of B cells before their malignant transformation, we studied proliferation and apoptosis in B cells from 4- to 6-months-old, tumor-free iMycEµ mice. FACS analysis of MACS-sorted B220+ splenocytes stained with propidium iodide showed that, on average, 4.73% MycHis B cells were in S phase of the cell cycle, a 3.5-fold increase compared with B cells from nontransgenic littermates (1.36%; Fig. 3A). To better quantify the apparent increase in MycHis-dependent proliferation, we labeled B cells with BrdUrd in vivo followed by FACS analysis. The MycHis-transgenic B cells (8.79% BrdUrd+) proliferated, on average, 3.8 times faster than controls (2.34% BrdUrd+; Fig. 3B). Increased proliferation of MycHis B cells was accompanied by elevated apoptosis in these cells (7.46 ± 2.18%) compared with normal B cells (0.14 ± 0.02%), using reactivity for activated caspase 3 as the FACS label (Fig. 3C). The increased proliferation of MycHis B cells in vivo noted above was also observed in cell culture upon examination of the LPS response using the MTS assay. MACS-purified B splenocytes from iMycEµ mice proliferated more vigorously than B cells from wild-type littermates after stimulation with three different doses of LPS (Fig. 3D).



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  		Figure 3. Increased turnover of premalignant B cells in tumor-free iMycEµ mice. A, FACS analysis indicates increased cell cycle progression in MycHis-transgenic B cells (bottom) compared with normal B cells (top). Freshly isolated B220+ splenocytes were stained with propidium iodide (PI) and cell cycle profiles determined by FACS. Means and SDs (%) of cells in three different cell cycle stages. The results are based on five independent determinations. B, increased proliferation in MycHis-transgenic B cells (right) compared with normal B cells (left). B220+ splenocytes were labeled with antibody to BrdUrd and analyzed by FACS to determine the fraction of dividing cells that had incorporated BrdUrd in vivo (B). Means and SDs (%) of BrdUrd+ cells are shown. The results are based on three independent determinations. C, elevated apoptosis in MycHis-transgenic B cells (bottom) relative to normal B cells (top). Splenic B cells were sorted using magnetic beads followed by labeling with antibody to activated caspase 3 and FACS analysis. The average FACS reactivity to activated caspase 3 was markedly increased in transgenic B cells (7.46%) compared with controls (0.14%) in three independent determinations. D, enhanced LPS response in iMycEµ B cells. MACS-purified B220+ splenocytes from transgenic mice and nontransgenic littermates (Control) were cultured for 24 hours in the absence of LPS (background control) and presence of 25, 50, or 100 µg/mL LPS. Proliferation was measured using the MTS assay. Average results of three independent measurements.

 
We next evaluated whether Myc-transgenic B cells exhibit changes in surface marker expression compared with wild-type controls. FACS analysis using a panel of antibodies to B cell–associated differentiation and activation antigens showed that B cells from 4- to 6-months-old iMycEµ mice expressed heightened levels of costimulatory receptor CD40 (Fig. 4A, top) and activation markers CD38, CD86, and MHC class II (results not shown). In contrast, CD48 and CD95 (Fas) were down regulated (Fig. 4A, center and bottom). The down-regulation of Fas may be an adaptive change to counterbalance Myc-induced apoptosis (25), but this has not been shown here.



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  		Figure 4. Lymphoid hyperplasia and normal immune response in tumor-free iMycEµ mice. A, up-regulation of CD40 and down-regulation of CD48 and CD95 (Fas) on the cell surface of premalignant B220+ splenocytes obtained from iMycEµ transgenic mice (pink) and nontransgenic littermates (green). Shown are representative FACS histograms with isotype-specific antibody controls depicted in purple. B and C, normal immune response of iMycEµ mice after immunization with a thymus-dependent peptide antigen. Antigen-specific (F) and overall IgG and IgM titers (G) were determined by ELISA in transgenic mice and wild-type littermate controls. Means and SDs based on four mice in each group. D, lymphoid hyperplasia in the spleen of a 6-month-old tumor-free iMycEµ mouse. The enlarged white pulp (blue) occupies more than 90% of the tissue section stained with H&E.

 
To assess whether the changes in the B-cell compartment of iMycEµ mice compromise immune responsiveness, we immunized the mice with the T cell–dependent antigen, CLQLKEQKNAGTRTNEG-keyhole limpet hemocyanin. The response of transgenics and wild-type controls was indistinguishable, based on antigen-specific titers of IgM and the four IgG isotypes (Fig. 4B). Transgenic mice had also normal overall serum IgG and IgM levels compared with wild-type littermates, which served as controls (Fig. 4C). Stained sections of lymphoid tissues of 4- to 6-months-old mice revealed the long-term consequences of MycHis-driven B-cell proliferation at the morphologic level. Spleen (Fig. 4D) and lymph nodes (not shown) of transgenic mice were enlarged relative to nontransgenic controls because of progressive, follicular hyperplasia indicative of B-cell accumulation in vivo. This finding indicated that in B cells of tumor-free iMycEµ mice, the balance of Myc-dependent proliferation and apoptosis is tipped in favor of proliferation, leading to characteristic prelymphomatous changes in these mice.

Development of Mature B-Cell and Plasma-Cell Neoplasms in iMycEµ Mice. To examine the possibility that MycHis-dependent lymphomas might arise in iMycEµ mice, we monitored 109 mice for tumor development. The overall tumor incidence was 68% at 21 months of age (Fig. 5A). Histological examination of 76 tumor-bearing mice revealed three major tumor types (Fig. 5B), which appeared with similar latency. The most prevalent tumors were lymphoblastic B-cell lymphomas with a Burkitt-like "starry sky" morphology due to tingible body macrophages (ref. 26; Fig. 5C, top). Immunostaining showed that the tingible body macrophages engulfed TUNEL-positive cells (not shown). Lymphoblastic B-cell lymphomas were IgM+IgD+B220+CD19+CD5– using flow cytometry and had a high proliferative index (>70%) after BrdUrd labeling in vivo (not shown). Nearly one quarter of the lymphomas were diagnosed as diffuse large B-cell lymphomas (26) strongly positive for Bcl-6 by immunohistochemistry (Fig. 5C, center). Another fifth of the lymphomas were CD19+CD138+ plasmacytomas (Fig. 5C, bottom) that exhibited varying degrees of differentiation, including plasmablastic, anaplastic, and plasmacytic variants. Plasmacytoma produced monoclonal immunoglobulin of {gamma}, {alpha} (shown in Fig. 5C, bottom), and µ isotypes. Although the morphologic characteristics of lymphoblastic B-cell lymphoma, diffuse large B-cell lymphoma, and plasmacytoma will be described in depth in a subsequent article, the findings presented here clearly indicated that MycHis induced a spectrum of mature B-cell and plasma-cell tumors. Because lymphoblastic B-cell lymphoma was the most common lymphoma iMycEµ mice, it was chosen for further study described below.



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  		Figure 5. B-cell and plasma-cell tumors in iMycEµ mice. A, tumor incidence and survival of mice. B, graphical representation of the observed tumor spectrum: LBL, lymphoblastic B-cell lymphoma; DLBCL, diffuse large B-cell lymphoma; PCT, plasmacytoma. Tumor classification was difficult in 5% of cases, designated as Other. C, representative histologic tissue sections of the three main tumor types observed in the iMycEµ mice, either stained with H&E (top) or immunostained with antibody to Bcl-6 (middle) or IgA (bottom).

 
Lymphoblastic B-Cell Lymphomas Are Monoclonal Tumors and Express MycHis Predominantly from the P1 Promoter. Southern analysis of V(D)J recombination showed that lymphoblastic B-cell lymphomas had undergone clonotypic Ig{kappa} rearrangements (Fig. 6A, left). Rearrangements of Igh were also readily detected, but these were limited to the B6-derived locus (Fig. 6A, right). Western blotting with anti-His6 antibody showed that lymphoblastic B-cell lymphoma invariably expressed MycHis, although the expression levels varied (Fig. 6B, left). MycHis protein was clearly elevated in lymphoblastic B-cell lymphoma compared with premalignant B splenocytes from iMycEµ mice and normal B splenocytes (negative controls) using immunocytochemistry (Figure 6B, right). Northern blotting showed that total Myc mRNA (MycHis plus Myc) was overexpressed compared with controls (Fig. 6C). Estimation of the abundance of MycHis and Myc mRNA by allele-specific RT-PCR showed that the bulk of the Myc message was encoded by the inserted transgene (Fig. 6D). A characteristic feature of Myc deregulation in human BL and mouse plasmacytoma is a shift in promoter usage from the second Myc promoter, P2, which initiates ~80% to 90% of Myc mRNA in normal B cells, to the first promoter, P1, which initiates ~10% to 20% of normal Myc mRNA. To evaluate whether mouse lymphoblastic B-cell lymphoma exhibited a shift in the primacy of promoter usage, RNase protection assays were done with primary lymphoblastic B-cell lymphoma. Two mouse plasmacytoma cell lines that displayed a pronounced promoter shift (TEPC 1165) or did not (ABPC 20) were included as positive and negative controls (21). The analysis (summarized in Fig. 7A and presented in detail in Supplementary Fig. 2) showed that lymphoblastic B-cell lymphoma exhibited the full P2 to P1 promoter shift in Myc expression. In contrast, B220+ B cells from iMycEµ mice did not show a promoter shift relative to B220+ B cells from normal B6 mice, BALB/c thymus, and ABPC 20. These findings suggested that the juxtaposition of Myc to Eµ is, on its own, not sufficient to cause the promoter shift. The shift seems to require additional changes associated with acquisition of the malignant phenotype of lymphoblastic B-cell lymphoma.



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  		Figure 6. Lymphoblastic B-cell lymphomas are monoclonal tumors overexpressing MycHis. A, clonotypic VDJ rearrangements. Shown are Southern blots that contain DNA from six tumors after EcoRI restriction digestion and hybridization to an Ig{kappa} (left) or JH probe (right). Lymphoblastic B-cell lymphoma arose in mice that harbored one normal C57BL/6 (B6)-derived Igh allele and one mutant 129SvJ(129)-derived Igh allele with the MycHis. The B6 allele is indicated by one fragment (6.3 kb), whereas the 129 allele is indicated by two fragments (5.3 and 5.1 kb) that are poorly separated on the gel. VDJ rearrangements occurred only on the B6 allele, indicated by a reduction of the fragment relative to the germ line fragment in iMycEµ liver (Tg liver) or liver from normal B6 mice. B, lymphoblastic B-cell lymphomas express MycHis protein using Western blotting (left) and immunocytochemistry (right). Left, two immunoblots that contain lysates from 13 randomly chosen, primary lymphoblastic B-cell lymphomas. MycHis` was detected with an anti-His antibody. Anti-actin was used as loading control. Right, cytofuge specimens of lymphoblastic B-cell lymphoma cells (bottom) next to FACS-purified premalignant B cells from iMycEµ mice (middle), and normal splenic B cells from C57BL/6 mice (control, top) after immunostaining with an antibody to the histidine tag. C, lymphoblastic B-cell lymphomas overexpress Myc mRNA. Shown is a Northern blot that contains RNA samples from six primary lymphoblastic B-cell lymphoma. RNA samples obtained from two lymph nodes and the BALB/c plasmacytoma line, TEPC 1165, were included as negative and positive controls, respectively. The ethidium bromide–stained agarose gel showing the 28S rRNA band (bottom) is included as loading control. D, lymphoblastic B-cell lymphomas express Myc mRNA mainly from the inserted MycHis gene. Shown are ethidium bromide–stained agarose gels with RT-PCR products that reflect the abundance of Myc mRNA (top), MycHis mRNA (middle), and Gapd mRNA (bottom) in 16 primary lymphoblastic B-cell lymphomas and B220+ C57BL/6 splenocytes (control sample). White asterisks in the top, presence of faint PCR indicator fragment. Size markers are in the left-hand lane.

 


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  		Figure 7. Lymphoblastic B-cell lymphomas exhibit the P2 to P1 promoter shift of Myc expression and distortions of the p19Arf-Mdm2-p53 tumor suppressor axis. A, lymphoblastic B-cell lymphomas exhibit the P2 to P1 promoter shift of Myc expression. Summary of the experimental results presented in Supplementary Fig. 2. Mean values of normalized P1/P2 ratios and SDs of the mean are plotted. The normalization was based on the P1/P2 ratio in TEPC 1165, which was arbitrarily set at 1. The P1/P2 ratio was significantly elevated in lymphoblastic B-cell lymphoma relative to splenic B cells from iMyc mice (two-sided Student's t test; P = 0.0007). B, tandem point mutation in Myc homology box I, a primary lymphoblastic B-cell lymphoma. Top, scheme of the MycHis protein that contains the two Myc homology boxes MbI and MbII, the basic region (B), the helix-loop-helix domain (HLH), the leucine zipper (LZ), and the histidine tag (His). The MbI domain spans residues 47 to 62. Bottom, histograms of DNA sequencing reactions of MbI nucleotides encoding residues 53 to 59 of normal Myc and mutated Myc found in two independent specimens in 1 of 16 primary lymphoblastic B-cell lymphomas. The mutated Myc gene harbored point mutations in two neighboring codons, resulting in L56F and P57S substitutions. C, p19ARF, Mdm2, and p53 expression and biallelic Ink4a deletions in primary lymphoblastic B-cell lymphomas. Protein levels of p19ARF, Mdm2 and p53 were compared by immunoblotting in 12 lymphomas (LBL 1-12) and B splenocytes from nontransgenic littermates (Normal), using ß-actin as loading control. Two of three major isoforms of Mdm2 are discernable in some lanes (e.g., LBL 3 and 4). Potential deletions of the genes encoding p19ARF and p53 were assessed by genomic PCR for exon 1 of Ink4a and exon 2 of Trp53, respectively. Apparent biallelic deletions of Ink4a were found in LBL 5-10. Deletions of Trp53 were not found.

 
Point Mutations in Myc Transactivation Domain Are Rare in Lymphoblastic B-Cell Lymphoma. More than 60% of human BL and AIDS-associated B-cell lymphomas carrying MYC-activating chromosomal translocations harbor mutations in the rearranged MYC gene that alter the biological properties of the expressed MYC protein. The clustering of the great majority of these mutations in a highly conserved region of MYC amino-terminal transactivation domain (MYC homology box 1, MbI) suggests that these mutations were selected during tumor development. The underlying reason for this selection and the mechanism of mutagenesis in MbI are not known, but some circumstantial evidence points to VDJ hypermutation in germinal center B cells as the underlying mutagenic principle (27). To evaluate whether mouse lymphoblastic B-cell lymphomas contain point mutations in MycHis, we sequenced the entire MycHis gene (2.2 kb for both DNA strands) in 34 tumors obtained from 16 lymphoblastic B-cell lymphoma–bearing mice. 74.8 kb of DNA sequence analysis resulted in the detection of just one mutation, a tandem missense mutation, L56F/P57S, which was found in two tumors harvested from different anatomic sites in the same mouse (Fig. 7B). A recent tabulation of data from the literature (28) indicates that the P57S mutation is the second most common base substitution mutation in human BL, comprising 8 (14.5%) of 55 mutations in MbI. Interestingly, biological studies with the P57S allele have showed that this mutation results in lower oncogenic activity than wild-type MYC when assayed for neoplastic transformation of rat embryo cells in cooperation with the H-rasG12V oncogene (28). The significance of the L56F mutation, which has not been observed thus far in human BL, is not known. It may be an innocent "bystander" mutation in the present case of mouse lymphoblastic B-cell lymphoma.

Interruption of the Arf-Mdm-p53 Tumor Suppressor Axis in Lymphoblastic B-Cell Lymphoma. Myc-induced B-cell transformation and lymphomagenesis in Eµ-Myc transgenic mice (29) is limited by Myc-dependent apoptosis (30). One important mechanism of curtailing Myc-dependent apoptosis in Eµ-Myc mice is inactivation of the p19Arf-Mdm2-p53 tumor suppressor pathway (31, 32). When activated, this pathway involves the up-regulation of p19Arf, which results, in turn, in sequestration of Mdm2 and activation of the p53 checkpoint (33). To determine whether interruption of this pathway also plays a role in lymphoma development in the iMycEµ mice, we studied 26 lymphoblastic B-cell lymphomas using immunoblotting. Applying stringent assay conditions aimed at detecting pronounced alterations, we found changes in 12 (46.2%) of 26 tumors. Four (33%) of the 12 tumors with alterations overexpressed p19Arf (Fig. 7C, LBL 1-4). Loss of p19Arf protein expression, apparently due to biallelic deletions in the Ink4a locus, occurred in 6 (50%) of 12 tumors, as detected by Western blot and genomic PCR (LBL 5-10). Overexpression of Mdm2 was found in 2 tumors (17%, LBL 11-12). Elevation of (presumably dysfunctional) p53 was seen in 3 tumors (25%; LBL 2, 4, and 9, indicated by red squares), however, only in combination with overexpression of p19Arf (LBL 2 and 4) or loss of p19Arf (LBL 9). Irrespective of the underlying reasons, which may be complex, all of the above alterations are likely to result in the crippling of the p53 response (cell cycle arrest, apoptosis). Considering that loss of p53 (32, 34, 35) or p19Arf (31, 32) accelerates Eµ-Myc-induced lymphoma development, our data suggest that the interruption of the p19Arf-Mdm2-p53 pathway also plays a significant role in lymphomagenesis in iMycEµ mice (31).

Lymphoblastic B-Cell Lymphomas Contain Clonal Cytogenetic Alterations. Because MycHis-dependent genomic instability (36) may be one mechanism by which the p53 pathway is interrupted during tumor progression, we used spectral karyotyping to analyze three randomly chosen lymphoblastic B-cell lymphomas. One of them exhibited a translocation/deletion at chromosome 11B (Fig. 8, top) and another one showed a large deletion in chromosome 13 (Fig. 8, center). The third tumor had undergone tetraploidization and contained an extra copy of chromosome 18 (Fig. 8, bottom). Thus, three of three lymphoblastic B-cell lymphomas contained cytogenetic aberrations that were readily detectable by spectral karyotyping. Because these markers or extra chromosomes were found in 10 metaphase plates of each tumor, this provides additional evidence for the clonality of these tumors.



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  		Figure 8. Chromosomal aberrations in lymphoblastic B-cell lymphoma detected by spectral karyotyping. Shown are a nonreciprocal T(11;3) translocation and a large internal deletion of chromosome 13B found in two primary tumors. Aberrant chromosomes are depicted in spectral karyotyping display colors (middle) and classification colors (right) next to inverted 4',6-diamidino-2-phenylindole images (left). Normal chromosomes (Chr.) 11 and 13 are included for comparison. The T(11;3) was highly variable in different metaphases, presumably reflecting ongoing genomic instability of the chromosome. Three variants of T(11;3) from different metaphase spreads of LBL 1 are depicted to the right of the complete spectral karyotyping karyotype of this tumor. An image of Del 13B found in LBL 2 is also shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Gene insertion was used to recreate as a germ line mutation in mice the MYC/Myc-activating Igh translocations seen in human endemic Burkitt lymphomas and certain mouse plasmacytomas. The newly developed mouse model, designated iMycEµ, has several noteworthy features. First is the precise reconstruction of the translocation breakpoint region on human der(14) and mouse der(12), that is, the head-to-head juxtaposition of MycHis and Eµ. Second is the potential for MycHis to interact with the complete set of correctly spaced Igh enhancers, designed to create the same complex interplay of promoter and enhancer interactions that govern the expression of translocated MYC/Myc in human endemic Burkitt lymphoma/mouse plasmacytoma. Third is the insertion of MycHis in the Igh chromatin domain, which presumably subjects the gene to the same higher-order regulatory influences (e.g., those imposed by chromatin remodeling and positional effects in the interphase nucleus) that affect MYC/Myc involved in the t(8;14)/T(12;15) exchange. Our finding that many of the heterozygous iMycEµ mice spontaneously developed lymphoblastic B-cell lymphoma, diffuse large B-cell lymphomas, and plasmacytomas suggests that the iMycEµ transgene reproduced the key features of the t(8;14)/T(12;15) translocation that initiate the development of endemic Burkitt lymphoma in humans and plasmacytoma in mice.

Our MycHis transgene model of the endemic Burkitt lymphoma t(8;14)/plasmacytoma T(12;15) effectively induces B-cell neoplasia even though it differs from the "natural" chromosomal translocations found in the human and mouse tumors in at least three potentially important aspects. First, the natural translocations are spontaneously occurring somatic mutations in tumor precursors, whereas MycHis is a germ line mutation present in all cells. Second, MycHis insertion reproduced only the MYC/Myc-activating product of translocation, not the reciprocal product of translocation. Third, in endemic Burkitt lymphoma t(8;14), the breakpoints on chromosome 8 are scattered over a considerable genomic distance in the 5' flank of MYC (37, 38), resulting in separation of MYC from Eµ by more than 340 kb (37) in some cases (class III translocations according to Cory 39). As shown here, these aspects of endemic Burkitt lymphoma t(8;14)/plasmacytoma T(12;15) are not critical to the development of B-cell and plasma-cell neoplasms following MYC/Myc-activating translocation. It follows that the juxtaposition of Myc and Eµ in the appropriate chromosomal context of the Igh chromatin domain defines the minimal, critical feature of the endemic Burkitt lymphoma t(8;14)/plasmacytoma T(12;15) translocation. The long latency of B-cell and plasma-cell tumors developing in iMycEµ mice suggests that secondary oncogenic events complement MycHis during lymphomagenesis. Alternatively, the oncogenic potential of MycHis may be offset in vivo by factors that inhibit Myc-induced transformation (e.g., Myc-induced apoptosis). The apparent occurrence of t(8;14)(q24;q32) in healthy blood donors (40), the repeated detection of T(12;15) in normal mice (41–43), and recent insights from mouse models of nonhematopoietic cancer driven by inducible Myc transgenes (44, 45) support this view.

The occurrence of plasmacytoma in ~20% of the tumor-bearing iMycEµ mice suggests that B cells bearing our model of Myc-activating translocation sometimes acquire the same subsequent genetic alterations that lead to the development of plasmacytoma following somatic acquisition of T(12;15). Plasmacytoma have not been observed in the Eµ-MYC/Myc and YAC-MYC mice. What mechanisms might be responsible for the unique property of the iMycEµ transgene to induce plasma cell tumors? One feature that distinguishes the iMycEµ mice from the Eµ-MYC/Myc and YAC-MYC mice is the genomic integration site of the Myc transgene. Whereas this site is ectopic in the Eµ-Myc and YAC-MYC, the insertion site of the MycHis transgene in iMycEµ corresponds precisely to the t(8;14)/T(12;15) translocation break site in endemic Burkitt lymphoma/T(12;15). The strategic location of MycHis in Igh may link timing and level of Myc activation to the developmentally controlled accessibility (46) and activity (47) of the Igh chromatin domain (48), generating thereby a temporal and quantitative pattern of MycHis activation that is conducive to plasma cell tumors. A second distinguishing feature of the iMycEµ is the potential for MycHis to interact with E{alpha} and E{delta} in addition to Eµ. The crucial importance of the Myc-E{alpha} interaction for Myc deregulation in plasma cell tumors has been clearly shown (12). The in vivo relevance of the Myc-E{delta} interaction has been recently noted by the surprising observation that Eµ is dispensable for MYC expression and lymphomagenesis in the YAC-MYC mouse (11), in which Eµ can apparently be substituted for by the E{delta} enhancer (49). Thus, the ability of the iMycEµ to promote plasma cell transformations may be created by the complex interplay of MycHis with all three Igh enhancers taking place in the appropriate genomic context of the Igh domain.

The iMycEµ mice were generated as part of a program to recreate and study in transgenic mice the molecular and oncogenic consequences of MYC/Myc-activating t(8;14)/T(12;15) translocations that characterize human and mouse B-cell and plasma-cell tumors, such as human endemic Burkitt lymphoma and mouse plasmacytoma. Among the new opportunities afforded by the iMycEµ is the identification of the secondary oncogenic events that complement Myc during neoplastic transformation, leading to lymphoblastic B-cell lymphoma in most cases, but diffuse large B-cell lymphomas and plasmacytoma in the others. Furthermore, although strain iMycEµ was not developed to model human endemic Burkitt lymphoma or any other specific type of human NHL in mice, it may provide guidance for possible future attempts to model human BL in mice more accurately. Human BL exhibits the phenotype of a germinal center (GC) B cell and contains somatic mutations in expressed immunoglobulin variable and MYC genes, indicating that the tumors have germinal center experience (50). Human BL acquire MYC translocations as spontaneous somatic mutations that are thought to be accidents of VDJ hypermutation (endemic Burkitt lymphoma) and isotype switching (sBL/iBL) in the germinal center (51). Human BL contains clonal EBV (52) and expresses the EBV-encoded EBNA-1 protein (53), which is consistent with malignant transformation occurring in an EBV-infected centroblast differentiating into a memory B cell (54). These features of human BL suggest that in order to model this neoplasm in mice more precisely, Myc activation should be delayed to the germinal center stage, for example, by placing Myc under control of a germinal center-specific promoter. Myc transgenes of this sort should then be combined with EBNA-1 transgenes (55) that could perhaps also be improved by targeting expression to germinal center B cells. Although these approaches may help to settle the long-standing thorny question of whether EBV plays an active role in the development of BL or is just a passive passenger in the tumor cells (56), the present study has shown that the iMycEµ transgene is a valuable model of endemic Burkitt lymphoma t(8;14) that predictably induces B-cell and plasma-cell tumors in mice.


   Acknowledgments
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank the following (from the National Cancer Institute): Lisa Craig, Tina Wellington, Nicole Wrice, and Wendy DuBois for genotyping and assistance with the mouse experiments; Eileen Southon for expert help with gene targeting; Elizabeth Mushinski for immunohistochemistry; Drs. Alexander L. Kovalchuk for generating the tumor incidence and survival curve; W. Michael Kuehl for the Ig{kappa} probe, reading an earlier version of the manuscript, and making helpful suggestions; Michael Potter for stimulating scientific discussion; and Beverly Mock for support; Drs. Hua Gu for the genomic clone (IgM DQ52) and Chen Feng Qi for Bcl-6 detection (both from the National Institute of Allergy and Infectious Diseases); and Dr. Georgina F. Miller (Veterinary Resources Program, NIH) for expert advice on tumor classification.


   Footnotes
 
Note: S.S. Park, J.S. Kim, and L. Tessarollo contributed equally to this article and are thus sharing first authorship. S.S. Park and J.S. Kim are currently in Laboratory of Functional Proteomics, Korea Research Institute of Bioscience and Biotechnology, Taejon, Korea.

Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Received 1/27/04. Revised 12/ 6/04. Accepted 12/13/04.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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