The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

The Journal of Immunology, 2007, 179, 6033 -6042
Copyright © 2007 by The American Association of Immunologists, Inc.
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Related articles in The JI
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Truffinet, V.
Right arrow Articles by Denizot, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Truffinet, V.
Right arrow Articles by Denizot, Y.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene

The 3' IgH Locus Control Region Is Sufficient to Deregulate a c-myc Transgene and Promote Mature B Cell Malignancies with a Predominant Burkitt-Like Phenotype1

Véronique Truffinet*, Eric Pinaud*, Nadine Cogné*, Barbara Petit*,{dagger}, Laurence Guglielmi2,*, Michel Cogné* and Yves Denizot3,*

* Unité Mixte de Recherche, Centre National de la Recherche Scientifique 6101, Université de Limoges, Limoges, France; and {dagger} Service d’Anatomie Pathologique, University Hospital, Limoges, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Burkitt lymphoma (BL) features translocations linking c-myc to an Ig locus. Breakpoints in the H chain locus (IgH) stand either close to JH or within switch regions and always link c-myc to the 3' IgH locus control region (3' LCR). To test the hypothesis that the 3' LCR alone was sufficient to deregulate c-myc, we generated mice carrying a 3' LCR-driven c-myc transgene and specifically up-regulating c-myc in B cells. Splenic B cells from mice proliferated exaggeratedly in response to various signals had an elevated apoptosis rate but normal B220/IgM/IgD expression. Although all Ig levels were lowered in vivo, class switching and Ig secretion proved normal in vitro. Beginning at the age of 12 wk, transgenic mice developed clonal lymphoblastic lymphomas or diffuse anaplastic plasmacytomas with an overall incidence of 80% by 40 wk. Lymphoblastic lymphomas were B220+IgM+IgD+ with the BL "starry sky" appearance. Gene expression profiles revealed broad alterations in the proliferation program and the Ras-p21 pathway. Our study demonstrates that 3' IgH enhancers alone can deregulate c-myc and initiate the development of BL-like lymphomas. The rapid and constant occurrence of lymphoma in this model makes it valuable for the understanding and the potential therapeutic manipulation of c-myc oncogenicity in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Expression of c-myc is tightly linked to the early G1 phase of the cell cycle and plays a critical role in cell proliferation, differentiation, metabolism, and apoptosis (1). Burkitt lymphoma (BL)4 is characterized by specific chromosomal translocations involving the c-myc gene and one of the Ig loci (Ig{kappa}, Ig{lambda}, and IgH) (2). Once translocated and having lost its normal control, c-myc is constitutively expressed throughout the cell cycle in B-lineage cells, the oncogene deregulation being considered the result from juxtaposition to the Ig gene enhancers. In 80% of cases, the translocation involves the IgH locus (2), itself regulated by a complex interplay of regulatory elements responsible for tissue- and stage-specific regulation of both transcription and rearrangements. Early B cell-specific events such as the germline transcription initiated at the DQ52 promoter and the initiation of VDJ recombination are regulated by upstream elements including the Eµ enhancer (3, 4). This cis-acting element was the first candidate proposed for c-myc deregulation in BL because c-myc transgenes linked to Eµ were shown to promote B cell malignancies, although with a pro-B phenotype (5). Whether Eµ was included, mice carrying yeast artificial chromosomes linking the c-myc gene to a 5' portion of the IgH locus (VH to C{delta}) developed rather immature B cell malignancies expressing a variable degree of CD43 (6, 7). Altogether, 5' elements of the locus induced malignancies with features of pre-B, immature B, or transitional B cells rather than mature B cells as in BL. These results suggest that downstream IgH elements must deregulate c-myc in BL.

Several lymphoid-specific transcriptional enhancers (hs3a, hs1, 2, hs3b, and hs4 in the mouse) have been identified within a 3' regulatory region lying downstream of the IgH locus. The hs4 distal element is active from the pre-B cell stage and throughout B cell ontogeny. A larger region with a global "palindromic" structure encompassing the hs1, 2 central enhancer flanked by inverted repeats, including hs3a and hs3b elements, is active at late B cell stages (8). Altogether, the four elements constitute a potent locus control region (LCR) conferring position-independent and copy-dependent expression to transgenes (9). These elements display specific activity during terminal B cell differentiation, with a major role in class switch recombination (CSR) (10, 11, 12). A construct containing the four 3' IgH enhancers confers, when surrounded by insulators, an expression from pre-B cells to mature B cells that is strictly confined to the B cell lineage (13).

In BL, IgH locus breakpoints are located either in the VDJ (endemic BL) or the switch region (sporadic BL). When upstream of Eµ, the chromosomal breakage may result from an abnormal somatic hypermutation event in early germinal center B cells. Breakpoints located within switch regions are rather initiated by an erroneous class switching and link c-myc to a downstream portion of the locus lacking Eµ (2). By contrast, 3' IgH enhancers are always conserved on the c-myc-translocated chromosome and constitute candidates for oncogene deregulation.

In 20% of cases, c-myc translocation involved the Ig L chain (Ig{kappa} and Ig{lambda}) loci. A BL-like model has been previously generated by using the c-myc gene fused to elements of the {lambda} L chain locus (14). However, in 80% of cases, c-myc translocation involved the IgH locus. For now, two mouse models have closely mimicked c-myc translocation into the IgH locus. In one case, a c-myc insertion downstream of JH imitates the translocation observed in endemic BL. Aged animals develop B cell and plasma cell neoplasms resulting from c-myc activation (15, 16). Deregulation of c-myc cannot be attributed to any precise cis-responsive elements because the transgene is integrated in the whole IgH locus chromatin environment. In the second model, a truncated 3' IgH cassette (lacking hs3a) was integrated upstream of the c-myc-coding region on mouse chromosome 15 and induced B cell lymphomas in aged animals (17). This latter model demonstrated the important role of the 3' IgH LCR on c-myc deregulation, although it is not excluded that the knockin might also have disrupted upstream and/or downstream c-myc regulatory elements (18, 19). To test the hypothesis that the complete 3' IgH enhancer combination in its palindromic configuration, and independently of any other cis-acting element, might be sufficient to deregulate c-myc (and more efficient that an incomplete 3' LCR cassette), we generated mice harboring a single copy of a (c-myc-3' IgH LCR) transgene. Interestingly, mice developed lymphomas within a few weeks. BL-like proliferations occurred in 75% of cases while the remaining 25% were characterized as diffuse anaplastic lymphomas. The observed spectrum of tumors provides strong evidence that the 3' LCR alone, insulated from the chromatin environment, strongly deregulates c-myc and mimics the critical features promoting human BL.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Vector construction

A schematic representation of the c-myc-3' LCR transgene is presented in Fig. 1A. The hs1, 2 is a 0.6-kb StuI-EcoRV murine genomic fragment (20). Two 2.1-kb EcoRI-HindIII fragments, corresponding to hs3a and hs3b, were cloned with symmetric orientations on both sides of the hs1, 2 enhancer, according to their endogenous arrangement (21). hs4 is a 1.38-kb PstI-HindIII fragment (22). The chicken beta-globin HS4 insulator was a 250-bp NotI fragment. The c-myc gene was a 7-kb SmaI-KpnI murine genomic fragment including P1 and P2 promoters.


Figure 1
View larger version (35K):
[in this window]
[in a new window]

  		FIGURE 1. Generation of c-myc-3' LCR-transgenic mice. A, Schematic diagrams of the murine IgH locus and of the c-myc-3' LCR transgene. The hs4 probe used for Southern analysis is shown. BH1, BamHI. B, Southern analysis of DNA from ES cells, wt, and transgenic mice. Analysis of BamHI-digested genomic DNA with a hs4 probe. The genomic DNA is from ES cells (left) or mouse (middle). The wt allele is 3 kb. When the transgene is integrated, the allele is 10 kb. Analysis of XhoI plus BamHI digested DNA from wt and transgenic mice with an {alpha} probe (right). C, Survival curves of wt and c-myc-3' LCR mice. Upper panel, Twenty-four wt mice and 37 transgenic mice were followed over a period of 34 wk. Lower panel, Pictures of c-myc-3' LCR mice with lymphomas. Arrows showed lymph node-derived tumors.

 
Generation of transgenic mice

The studies have been reviewed and approved by Centre National de la Recherche Scientifique (CNRS) and university review committee. Cells of the embryonic stem (ES) cell line E14 were cotransfected by electroporation with the linearized vector and the neomycin-resistance gene (molar ratio 10:1) and selected using geneticin (330 µg/ml; Sigma-Aldrich) (12, 13). ES clones were injected into C57BL/6 blastocysts, and the resulting male chimeras were mated with C57BL/6 females. Germline transmission was assessed by coat color and the presence of the transgene was verified by Southern blot.

Southern blot analysis

Genomic DNA was extracted from ES clones and mice organs. Ten micrograms of DNA was digested with BamHI, loaded on 0.7% agarose gels, transferred on nylon sheets (Amersham), hybridized with a 32P-labeled hs4 probe (a 1.3-kb PstI fragment) and autoradiographed. In another set of experiments, DNA was digested with XhoI + BamHI prior hybridization with a 32P-labeled 3' end C{alpha} probe (a 0.5-kb EcoRI-HindIII fragment) and autoradiographed.

B cell purification

For studies investigating cell cycle, proliferation and apoptosis rate and mRNA expression splenic B cells were purified using CD43-coupled beads from Miltenyi Biotec according to the manufacturer’s recommendations.

mRNA expression

Total RNA was extracted from fresh tissues and cell suspensions using Tri-reagent (Ambion). RNA was reverse-transcribed into cDNA by addition of reverse transcriptase to 2 µg of total RNA in a final volume of 20 µl. Quantitative PCR was performed in duplicate by using TaqMan assay reagents and analyzed on an ABI Prism 7000 system (Applied Biosystems). Product reference for c-myc was Mm00487803-m1 and for the 18S RNA used for normalization of gene expression levels was Hs99999901-s1 (Applied Biosystems). Primers and probe for the P1 promoter were: 5'-GAGGGATCCTGAGTCGCAGTAT-3', 5'-CTCTGCACACACGGCTCTTC-3' and 5'-(6-FAM)TCTCCTGCCTGATAAACAACAACTTGGGATG-3'.

Flow cytometry analysis

Single-cell suspensions from thymus, spleen, liver, and bone marrow were labeled with various Abs (Southern Biotechnology Associates): anti-B220-conjugated with Spectral Red (PC5), anti-CD19, anti-CD117, anti-IgM, and anti-IgD conjugated with PE. Control experiments included irrelevant isotype-matched Abs conjugated with PC5 or PE. Cells were analyzed on a Coulter XL apparatus (Beckman Coulter).

Proliferation and cell cycle analysis

Splenic B cells (2.5 x 105 cells/well) were cultured (six times) in 96-well plates, either alone or in the presence of 10 µg/ml LPS from Salmonella typhimurium (Sigma-Aldrich) or anti-CD40 (5 µg/ml) for 72 h. Proliferation was assessed by [3H]thymidine incorporation into DNA.

Incubation with a hypotonic solution of propidium iodide of cells taken after 24 h of stimulation resulted in disruption of the cell membrane and staining of the nuclear chromatin, then allowing cytometric counting of cells in the G0/G1, S, and G2/M phase.

Apoptosis assay

Freshly isolated splenocytes (106 cells/ml) were cultured in growth medium in 24-well plates. Immediately after isolation and after 24 h of growth, cells were incubated for 15 min at 4°C with 7-aminoactinomycin D and FITC-labeled annexin V Abs (BD Biosciences), and analyzed by flow cytometry.

Clonality assay

Genomic DNA prepared from splenic cells or lymph nodes was digested with EcoRI and analyzed by Southern blot with a 32P-labeled JH probe.

Blood sampling

Blood samples were recovered from transgenic mice and wild-type (wt) controls with heparinized needles. Circulating white blood cells (WBC) were counted. Plasma samples were recovered by centrifugation and stored at –20°C until used.

Histology and immunohistochemistry

Morphological analysis was realized on 5-mm-thick sections stained with hematin, eosin, and safranine. Lymphomas were classified according to criteria proposed by Morse et al. (23). Ki67 protein was detected in lymphomas with a rat polyclonal Ab (Ki67 clone TEC-3; DakoCytomation).

Spleen cell cultures

Single-cell suspensions of splenocytes were cultured for 5 days at 5 x 105 cells/ml in RPMI 1640 medium supplemented with 10% FCS and 20 µg/ml LPS, with or without addition of cytokines: 1 ng/ml mouse rIL4 (PeproTech), 1 ng/ml TGF-beta (PeproTech), or 100 U/ml murine rIFN-{gamma} (PeproTech). Supernatants (harvested after 5 days of stimulation) were frozen at –20°C until used.

ELISAs

Culture supernatants and sera from c-myc-3' LCR and wt mice were analyzed for the presence of the various Ig classes (IgM, IgG1, IgG2b, IgG3; IgE and IgA) by ELISA as previously described (12). Repeated ELISA determinations were submitted to a statistical analysis by using the Student t test.

Sequence analysis of expressed VH genes

Genomic DNA extracted from tumors was amplified by PCR. Forward primers: VHJ558 5'-GCGAAGCTTARGCCTGGGRCTTCAGTGAAG-3' and VHQ52 5'-GCGAAGCTTCTCACAGAGCCTGTCCATCAC-3'; backward primer: JH4 5'-AGGCTCTGAGATCCCTAGACAG-3'. The PCR product was cloned into the pCRII-TOPO vector (Invitrogen Life Technologies) and sequenced with an automated laser fluorescent DNA ABI-PRISM 310 sequencer (PerkinElmer).

Microarray analysis

Gene expression profiles of BL-like lymphoma and plasmacytoma tumors derived from lymph nodes were compared with lymph node B cells from wt animals. Experiments were done in triplicate (each sample consisted of RNA from two tumors or wt B cells, respectively). cDNA labeling, dual color microarray hybridization, and microarray data analysis were done in the Montpellier Genopole Microarray Facility essentially as previously described (24).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Generation of c-myc-3' LCR-transgenic mice

The transgene consisted in a cassette carrying a mouse c-myc gene under the control of the four 3' IgH enhancers assembled according to their endogenous palindromic configuration (Fig. 1A). The SmaI-KpnI c-myc genomic fragment contained the mouse c-myc exons and its 5' untranslated regulatory region encompassing P1 and P2 promoters. The cassette was flanked by two copies of the chicken beta-globin HS4 insulator. Previous reports demonstrated that when flanked with insulators, the combined 3' IgH regulatory elements altogether behave as a true LCR noticeably displaying position independent and copy-dependent expression (13, 25, 26). E14 ES cells were transfected with the c-myc-3' LCR transgene to obtain ES clones. Transgenic animals bearing a single copy of the integrated cassette were generated (Fig. 1B, middle). A control hybridization with a probe located at the 3' end of the C{alpha} gene was performed on XhoI plus BamHI digested DNA from wt and transgenic mouse highlighting a unique wild-type band of 8 kb that includes the endogenous hs3a and arguing against a potential insertion of the transgene within the endogenous 3' IgH LCR (Fig. 1B, right).

Lifespan of c-myc-3' LCR-transgenic mice

Twenty-four wt littermates and 37 c-myc-3' LCR-transgenic mice were followed to record their lifespan. Transgenics showed dramatically higher mortality than wt (Fig. 1C, upper panel). The overall tumor incidence was 80% at 34 wk of age. The mean age of death for c-myc-3' LCR-transgenic mice was ~23 wk. Beginning at the age of 12 wk, transgenic mice progressively developed profound enlargement of lymph nodes (Fig. 1C, lower panel). Mice exhibiting obvious tumors or presenting signs of illness were sacrificed.

Expression of c-myc transcripts in young c-myc-3' LCR transgenic

We first used real-time PCR to analyze tissue c-myc RNA expression in 6- to 8 wk-old c-myc-3' LCR-transgenic mice. At this time and although apparently healthy, premalignant mice showed markedly elevated levels of c-myc transcripts in the spleen (p < 0.05, Mann-Whitney U test) (relative expression of 3.74 ± 0.37, mean ± SEM of three animals) as compared with controls (relative expression of 0.93 ± 0.05, mean ± SEM of three animals). In agreement with the known B cell specificity of the 3' IgH LCR cassette (13, 26), increased c-myc transcription was also found in sorted B splenocytes but not in non-B splenocytes (mostly monocytes/macrophages and T cells), nor in lung, liver, thymus, heart, and kidney of transgenic mice (data not shown).

B cell development and Ig secretion in young c-myc-3' LCR-transgenic mice

We analyzed B cells in young transgenic mice (6–8 wk) before any manifestation of disease. Spleens were of normal sizes, with germinal centers of normal morphologies (data not shown). Numbers of B cells in spleen and surface expression of IgM were normal (data not shown). The number of circulating leukocytes in young transgenic mice did not significantly differ from those of wt animals (14.97 ± 3.2 x 103 cells/µl vs 10.8 ± 0.4 x 103 cells/µl, mean ± SEM of five transgenic mice and five wt mice, respectively). In contrast Ig production was impaired in transgenic mice, as shown by analysis of plasma Ig isotypes (Table I). Because Ig CSR is a key event of B cell maturation in peripheral lymphoid organs, we determined whether elevated c-myc expression could affect CSR in transgenic B cells. In response to LPS stimulation with or without cytokines, we analyzed the percentage of B cell-expressing membrane-switched Ig isotypes. No difference was found between c-myc-3' LCR-transgenic animals and wt mice (data not shown). Concomitantly, to determine the effect of c-myc overexpression on the expression of class-switched genes, Ig secretion was assessed in vitro on the same splenocyte culture supernatants and did not show any alterations of Ig secretion in young c-myc-3' LCR mice by comparison to wt (Table II).


View this table:
[in this window]
[in a new window]

  		Table I. Ig plasma levels in premalignant c-myc-3' LCR-transgenic mice and wt controlsa

 

View this table:
[in this window]
[in a new window]

  		Table II. ELISA analysis of Ig secretion by LPS (and/or appropriate cytokine)-stimulated B splenocytes from premalignant-transgenic mice and wt controlsa

 
Elevated proliferation and apoptosis of B cells from premalignant c-myc-3' LCR-transgenic mice

As reported in Fig. 2, upper panel, the proliferation of B cells from transgenic mice was markedly elevated (p < 0.05, Mann-Whitney U test) in response to anti-CD40 or LPS as compared with B cells from wt animals. Changes in the cell cycle in response to activation signals confirmed results obtained with [3H]thymidine uptake: cells in S and G2/M phases were markedly increased after 24 h stimulation by anti-CD40 or LPS in c-myc-3' LCR B cells compared with wt B cells (Fig. 2, lower panel). The rate of spontaneous apoptosis was also significantly higher (p < 0.05, Mann-Whitney U test) in freshly isolated B splenocytes from c-myc-3' LCR mice (14.86 ± 2.20%, mean ± SEM of three mice) as compared with B cells from wt animals (6.35 ± 3.31%, mean ± SEM of three mice). Similar results were found after 24 h in growth medium without survival factors or proliferation signals (37.30 ± 5.60% of apoptotic cells vs 18.82 ± 0.32% for c-myc-3' LCR mice and wt mice, respectively). Thus, B splenocytes from premalignant mice had elevated proliferation capacities in response to activation and elevated apoptosis rates.


Figure 2
View larger version (21K):
[in this window]
[in a new window]

  		FIGURE 2. Increased B cell proliferation of premaligant c-myc-3' LCR mice in response to several activation signals. Upper panel, Measurement of [3H]thymidine incorporation by purified B cells from wt or c-myc-3' LCR-transgenic mice (aged of 8 wk) in response to different stimuli. B cells were treated with LPS (10 µg/ml) or anti-CD40 (5 µg/ml) for 3 days. Results (mean dpm values from samples replicated six times) from three independent experiments are shown. Significance was determined with the Mann-Whitney U test. Lower panel, Cell cycle analysis of B cells from wt or young c-myc-3' LCR-transgenic mice. Splenic B cells were culture for 24 h with medium alone (control), LPS (10 µg/ml), or anti-CD40 (5 µg/ml). Cells were stained with propidium iodine and cell cycle progression was analyzed with XL.2 software to assess the percentage of cells in the G0/G1, S, and G2/M phase.

 
Analysis of B cell lymphomas in c-myc-3' LCR mice

Analysis of 29 animals revealed two tumor types overexpressing c-myc, which appeared with different latency (Fig. 3A). The most prevalent tumors (75%, 22 of 29) were lymphoblastic B cell lymphomas (Fig. 3B). Morphology analysis revealed medium-sized cells with basophilic cytoplasm, profound enlargement of spleen (682 ± 69 mg vs 233 ± 26 mg for transgenic mice and nine wt animals, respectively) and lymph nodes (Fig. 1C). Histological examination revealed "starry sky" morphology due to tangible body macrophages (Fig. 3B), a histologic hallmark of BL. Four of these mice also exhibited tumors located respectively to intestine (three animals) and stomach (one animal). The intensive proliferative activity of these tumor cells was highlighted by the high expression of the nuclear proliferation-associated Ag Ki67 (80% of cells, range 70–90%, eight mice), a nuclear protein present during G1, S, G2, and M phase of the cell cycle (Fig. 3B). The phenotype of these Burkitt-like cells was strengthened by flow cytometry analysis. Lymph node-derived tumor cells displayed a mature phenotype expressing B220, IgM, and IgD (Fig. 3B) but were negative for CD117, CD43, Thy1, 2, CD4, CD8, CD11b, and CD138 surface markers (data not shown). Analysis of bone marrow of these animals revealed the presence of B220+IgM+IgD+ cells (data not shown) indicating invasion with lymphoma cells. The number of circulating WBC was markedly elevated (p < 0.05, Mann-Whitney U test) in Burkitt-like lymphoma mice (127.9 ± 57.2 x 103 cells/µl, mean ± SEM of five mice) as compared with wt (11.1 ± 0.5 x 103 cells/µl, mean ± SEM of seven mice) due to the massive presence of tumoral cells. The use of a JH4 probe on Southern blots including various tissues (spleen, lymph nodes) from the same lymphoma mice revealed similar rearranged bands in addition to the germline band indicating that spleen and lymph nodes were invaded by lymphoma cells from clonal origin (Fig. 3B). Finally, the Ig VH genes were sequenced in five tumors. Strikingly, all were essentially unmutated (data not shown).


Figure 3
View larger version (74K):
[in this window]
[in a new window]

  		FIGURE 3. Analysis of lymphomas in c-myc-3' LCR mice. A, Left panel, Analysis of 29 animals revealed two tumor types (i.e., Burkitt-like lymphoma and diffuse anaplastic plasmacytoma) which appeared with different latency. Right panel, Analysis by real-time RT-PCR of c-myc transcript levels in splenic B cells of BL-like lymphoma mice, anaplastic plasmacytoma mice, and wt mice. c-myc levels were normalized to 18S RNA. Statistical differences were investigated using the Mann-Whitney U test. B, Upper panel, left, A representative histologic tissue section (x100 magnitude) of spleen of BL-like lymphoma mice; right, flow cytometry analysis of lymphoma cells. Lower panel, left, Immunostaining with Abs against the Ki67 protein reveals a large amount (80%) of actively cycling cells; right, clonality of lymphoma in c-myc-3' LCR mice. Southern blot analysis was used to examine IgH gene configuration with a JH4 probe. Genomic DNA was prepared and digested with EcoRI from splenic cells of wt mice (lane 1), splenic cells (lanes 2, 4, 6) and lymph nodes (lanes 3, 5, 7) of three BL-like lymphoma mice. C, Left, A representative histologic tissue section (x100 magnitude) of spleen with diffuse anaplastic plasmacytoma. Right, Immunostaining with Abs against the Ki67 protein reveals a low amount (30%) of actively cycling cells.

 
One quarter of the lymphomas (7 of 29) were diagnosed as diffuse anaplastic plasmacytoma (Fig. 3C) according to the classification of Morse et al. (23). Histology of enlarged liver and spleen showed a diffuse population of medium to large cells with abundant cytoplasm without "starry sky" appearance. Thirty percent (range 20–30%, nine mice) of cells expressed the Ki67 Ag. Cells were B220–, IgMlow, CD138–, and according with the lack of CD138 Ag, no circulating monoclonal Ig was documented in the blood of these transgenic animals (data not shown). By comparison to wt, transgenic animals had a 4-fold enlargement of the spleen and liver (915 ± 139 mg vs 233 ± 26 mg, and 6.55 ± 0.64 g vs 1.55 ± 0.15 g, respectively) and a minor enlargement of the lymph nodes. Although significantly lower (p < 0.05) than in Burkitt-like lymphoma mice, the number of circulating WBC was elevated in these animal (18.3 ± 4.3 x 103 cells/µl, mean ± SEM of four mice) as compared with wt animals (p < 0.05) again due to the presence of numerous circulating tumoral cells. The use of a JH4 probe on Southern blot from spleen and liver highlighted clonality of these lymphomas (data not shown).

Relationships between c-myc transcripts and leucocytosis

As shown in Fig. 3A, c-myc transcripts were markedly elevated (p < 0.05, Mann-Whitney U test) in B cells of lymphoma mice as compared with wt animals (relative expression of 1.6 ± 0.35, mean ± SEM of four mice) with significantly (p < 0.05) higher c-myc expression in BL-like mice (relative expression of 15.11 ± 2.53, mean ± SEM of five mice) than in mice with anaplastic plasmacytoma (relative expression of 6.73 ± 3.3, mean ± SEM of four mice). Regression analysis showed a significant (p = 0.01; r = 0.679) link between the number of circulating WBC and c-myc transcript levels. Finally, overexpression of c-myc transcripts involved not only the conventional P2 promoter but also the P1 promoter known to be involved in oncogenesis (relative expression 7.40 ± 2.67, mean ± SEM of 11 transgenic animals as compared with 0.81 ± 4 in wt animals).

Gene expression profile of tumors in c-myc-3' LCR-transgenic mice

The gene expression profile of BL-like lymphomas and anaplastic plasmacytomas was compared with normal lymph node-derived B cells using an array of 16,643 genes. Among these, 2,125 genes significantly differed (1,179 up-regulated and 946 down-regulated) between BL as compared with controls (supplemental table I).5 Transgenic tumors showed differential expression of multiple genes involved in cell cycle regulation, cell growth, and differentiation (see selected genes in Table III). Noticeably, BL-like cells demonstrated a selective up-regulation of genes implicated in cell cycle progression including cyclin B1 (Ccnb1), cyclin B2 (Ccnb2), cyclin A2 (Ccna2), cyclin E1 (Ccne1), cyclin-dependent kinases (Cdk4, Cks1b), and several cell cycle-positive regulators (CDC20, CDCa5, CDCa3). In turn, a selective down-regulation was found in several genes implicated in cell cycle inhibition (Ccng2, Cdk2ap2, Gadd45g). In agreement with an elevated proliferation rate an up-regulation was noted for several genes implicated in DNA replication (Mcm6, McmD, Mcm10, Prim1, Prim2, Hdgh), DNA repair (Rad51, Rad51ap1), and protein folding (ppiL1, Hsp70-3, Hspa9a, Hsp60). In addition to c-myc, dramatic alterations in the ras-p21 signaling pathways were evidenced. Several genes positively implicated in the ras pathway were up-regulated (Rasl2-9, Rabl4, Shcbp1) while other negatively implicated in this pathway were down-regulated (Rras, Rasa1, Rasa2, Rasa3). Finally, several cytokine receptors (IL-10, IL-12, IL-17, and TGF-beta) and signaling molecules (IL-16, IL-4i, TGF-betai, Isgf3g, Stat2, and Stat4) were underexpressed in BL-like as compared with control B cells.


View this table:
[in this window]
[in a new window]

  		Table III. Various differentially regulated genes in Burkitt-like lymphoma tumors and control B cells

 
Two thousand genes were significantly altered (971 up-regulated and 1029 down-regulated) in anaplastic plasmacytomas as compared with controls (supplemental table II and selected genes in Table IV). Anaplastic plasmacytomas showed differential expression of important genes in cell cycle regulation (Cdk4, Ccnt1, Gadd45g, Cdc20, Cdc2a) and cell growth (Ccnb1, Ccnb2, Ccna2, Rbm5, Id2, Rasa2, Rasa3, stress-induced chaperones). Accordingly with their differentiation status, their anaplastic nature and the sustained c-myc expression, anaplastic plasmacytomas overexpressed some (Igj and Prdx1–4) but not all genes of the plasma cell signature (Blimp-1, Xbp-1). In turn, they underexpressed genes of the B cell signature (Igh6, CD79a, CD79b, CD19, IL17r, IL2r, Stat2, Stat4).


View this table:
[in this window]
[in a new window]

  		Table IV. Various differentially regulated genes in anaplastic plasmacytomas and control B cells

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Endemic BL are characterized by the translocation of c-myc into the IgH locus and c-myc deregulation seems to be the key step in lymphomagenesis (2). Consistent with this critical role of c-myc, mice model carrying insertion of this oncogene into the IgH locus develop, after a long latency (medium time 15 mo), B cell and plasma cell neoplasms (15, 16). The c-myc gene deregulation can be attributed to its juxtaposition to the IgH locus 5' and 3' transcriptional regulatory elements but the relative contribution of such cis-acting elements remains speculative. The Eµ element located between J and Cµ exons, the first enhancer discovered in the locus is involved in germline transcription and VDJ rearrangement in early B cell progenitors (3, 4). Besides Eµ, the four enhancers acting as a LCR downstream of the locus and able to act synergistically at long distances (27) are important for both BCR expression on resting B cells and germline transcription of constant gene before CSR (12). In the past decade, Eµ has been used to deregulate c-myc in the aim of mimicking human BL, but such transgenic mice in fact developed malignancies with a pro-B phenotype rather than the mature phenotype (IgM+IgD+) observed in BL (5). Recently, a model with 3' enhancer elements knocked-in in the 5' region of the c-myc gene has been generated (17). Those animals developed a BL-like lymphoma at a medium age of 13 mo suggesting that elements of the 3' IgH regulatory region deregulate c-myc at late stages of B cell differentiation. However, this model targeted the endogenous c-myc locus so that the 3' LCR fell associated with upstream and downstream c-myc regulatory elements that may somehow keep c-myc under control, although they would be apart from c-myc in the case of a translocation (18, 19). It is also important to notice that the 3' LCR used as a transgene was incomplete, lacking the hs3a element and its normal specific palindromic architecture (8). Indeed, while individually weak, the 3' enhancers have strong synergies when combined according to their normal palindromic arrangement (27). To test the hypothesis that the 3'IgH LCR alone was efficient to deregulate c-myc, we generated transgenic mice harboring an "insulated cassette" containing a c-myc transgene flanked by its 5' UTR region and driven by a 3' IgH enhancer combination mimicking the endogenous regulatory region. LCR are operationally defined by their ability to enhance the expression of linked transgenes in a tissue-specific, position-independent, and copy-number-dependent manner. We previously demonstrated using GFP-transgenic mice that constructs combining the four 3' IgH regulatory elements according to their endogenous architecture were strictly B cell specific and expressed from pre-B to mature B cells (13). Moreover, when insulated properly with flanking beta-globin insulators, such 3' IgH miniloci acquire copy-number dependence and position independence, i.e., behave as a true LCR (26). To be close to the situation in BL where one copy of c-myc is deregulated, we chose to generate transgenic mice carrying a single copy of the c-myc-3' LCR construct.

In such conditions, this c-myc-3' LCR construct displayed a strict B cell specific expression and a 3- to 4-fold increase of c-myc transcripts in B splenocytes of young mice. In most cell types, c-myc enforces S phase entry but also triggers the apoptotic programs (28). Using [3H]thymidine incorporation, cell cycle analysis, and annexin V labeling, we demonstrated that B splenocytes from young c-myc-3' LCR mice exaggeratedly proliferated upon activation, entered cell cycle, and underwent apoptosis. Increased apoptosis explains that exaggerated proliferation of B cells did not result in lymphocytosis in blood and spleen of premalignant mice. Despite reduced levels of all circulating Ig isotypes, B cells normally underwent CSR in vitro, suggesting that constitutive c-myc expression preserves responses of mature B cells but lowers their differentiation into Ab-secreting cells, consistent with the negative role of c-myc in plasma cell differentiation (29).

Interestingly, our model induced two kinds of B cell neoplasia comparable to those in the model mimicking the t(8:14) translocation of Park et al. (15, 16), with a major difference in the kinetic of apparition. The most prevalent neoplasm in our model arose at the mean age of 12 wk with features typical of human BL:clonal tumors of medium-sized cells with basophilic cytoplasms overexpressed c-myc, harbored a mature B cell phenotype (B220+CD19+CD117–IgM+IgD+), and a typical "starry sky" histological morphology. The lack of somatic mutations in VH genes in the current model is similar to that observed in mice exhibiting c-myc insertion downstream of JH (30) pointing to a common origin of these BL from pre-germinal center cells. A second type of neoplasms arose at the mean age of 20 wk, mostly involved liver and spleen, presented medium to large B220-negative cells with abundant cytoplasm and could be classified as diffuse anaplastic plasmacytomas (23). The absence of CD138 surface Ag and of an associated serum monoclonal component underlines that such tumors are not mature plasma cells. Similar neoplasms have already been observed in transgenic animal harboring a c-myc insertion into the IgH locus (15, 16) but not in animals carrying an incomplete 3' IgH LCR into the c-myc locus (17), and thus appear restricted to situations where c-myc is associated to a complete 3' IgH LCR, then mimicking a c-myc translocation into the IgH locus.

Although our model is sufficient to induce c-myc-driven BL-like neoplasia, spontaneous BL translocation has some additional features: first, the c-myc translocation and the reciprocal translocation product may potentially result in long-range effects; and second, the Eµ transcriptional enhancer may contribute to c-myc deregulation when the chromosomal breakpoint is in the JH region. However, it is also now well-established that Eµ does not play a crucial role at postmedullar stages of B cell development because the core region of this enhancer is dispensable for normal Ig surface expression, Ig CSR, and somatic hypermutation of H chain-rearranged regions (4). A minor role of Eµ in c-myc deregulation in BL seems reinforced by mouse models carrying yeast artificial chromosome transgenes: in such models, similar B cell malignancies were reported regardless of whether the portion of the IgH locus linked to c-myc contained Eµ (6, 7). Altogether, the "minilocus" approach seems to reproduce the key feature for the development of endemic BL: the 3' LCR-driven and B cell lineage-specific c-myc deregulation. Regarding the respective roles of the 3' LCR and the c-myc promoters in the B cell-specific overexpression of the transgene, several studies have demonstrated that the 3' LCR conferred B lineage specificity to various promoters and reporter genes (GFP, CAT, or c-myc) (9, 13, 27). Regarding c-myc promoters, the 3' LCR has been shown to strongly boost transcription in plasmacytoma and BL cells and also induced a promoter shift from P2 to P1 (9). The 3' LCR altered the distribution of RNA polymerase II complexes in these cell lines thus suggesting that it directly increased c-myc expression, at least in part, by abrogation of transcriptional elongation control mechanisms.

A major advantage of this simple transgenic model is its efficiency to induce mature B cell neoplasia with an average time of 6 mo as compared with 13–15 mo in other transgenic models (15, 16, 17). This faster kinetics of tumor apparition could be explained by the expression of the c-myc transgene in a locus independent context. Indeed, the insulated transgenic cassette restricts c-myc expression to the B cell lineage through the only influence of the 3' IgH regulatory region. In previously reported models, tumor outcome might be influenced by other cis-acting elements present on either the IgH or c-myc locus. In the model developed by Park et al. (15, 16), c-myc was directly apposed to Eµ to mimic the t(8:14) translocation. Because we previously showed the Eµ-3' IgH LCR cassette was at least as efficient as the 3' IgH LCR for the expression of an associated reported gene (13, 31), it is unlikely that the absence of Eµ in our transgene might explain its higher expression and the short latency of tumor cell development. Rather, the use of an intronless c-myc cDNA by Park et al. (15, 16) and the reported inhibition of JH rearrangements on the knockin allele may confine this artificially excluded allele into heterochromatin and finally result in poor expression of the c-myc protein. By contrast, Wang and Boxer (17) generated knockin mice with a truncated 3' LCR integrated into the 5' region of the c-myc gene, the knocked-in regulatory element might interfere with negative elements upstream of the murine c-myc gene (19), one of them displaying lymphoid cell specificity (32). In such a situation, c-myc may remain under a more or less normal regulation mediated by these elements and only result in tumors with a delayed outcome.

c-myc may have multiple effects on the cells, including a general growth-promoting effect via increased cell cycle progression, possible immortalization, but also increased apoptosis. In our model, we found a correlation between c-myc transcript levels and lymphocytosis highlighting the growth promoting effect of c-myc. The precise mechanism by which c-myc stimulates cell proliferation is not resolved. Yet, it is most likely transcriptional and indirect by taking advantage of up-regulation of numerous cyclin and cyclin-dependent kinase activators and alterations of the Ras-p21 pathway (15, 16, 17, 33, 34). Over half of the lymphomas arising in Eµ-Myc mice (35) or after insertion of the truncated 3' IgH upstream of the c-myc coding region (17) overexpressed the apoptotic molecules Bcl-2 and Bcl-xL. It is not the case in our model where Bcl-2 and Bcl-xL transcripts did not vary. Lymphoma cells for their survival might take advantage of the down-regulation of the p21 pathway. A number of studies have pointed out that in addition to being an inhibitor of cell proliferation, p21 possesses proapoptotic functions under certain conditions in a number of systems (36).

In summary, we have shown with a simple transgenic model that a palindromic combination of the 3' IgH enhancer elements can by itself strongly deregulate c-myc expression in a B-specific manner. The overexpression of such a c-myc reporter gene allows in only few months the development of B cell lymphomas which resemble human BL. This new and original transgenic mouse model assesses the key role of the 3' LCR in the c-myc overexpression observed during endemic human BL. The rapid and constant occurrence of lymphoma in this model makes it an ideal tool for therapeutics testing.


   Acknowledgment
 
We thank S. Desforges for help with animal manipulation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by grants from Ligue Nationale contre le Cancer, Conseil Régional du Limousin, and "Lions Club de la Corrèze, Zone 33 district 103 Sud." Back

2 Current address: Institut National de la Santé et de la Recherche Médicale Unité 454, Centre Hospitalier de l’Université Arnaud de Villeneuve, Montpellier, France. Back

3 Address correspondence and reprint requests to Dr. Yves Denizot, Faculté Médecine, Laboratoire d’Immunologie, Unité Mixte de Recherche, Centre National de la Recherche Scientifique 6101, 2 rue Dr. Marcland, 87025, Limoges, France. E-mail address: yves.denizot{at}unilim.fr Back

4 Abbreviations used in this paper: BL, Burkitt lymphoma; LCR, locus control region; CSR, class switch recombination; ES, embryonic stem; wt, wild type; WBC, white blood cell. Back

5 The online version of this article contains supplemental material. Back

Received for publication May 11, 2007. Accepted for publication August 13, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Pelengaris, S., M. Khan, G. Evan. 2002. c-Myc: more than just a matter of life and death. Nat. Rev. Cancer 2: 764-776. [Medline]
  2. Blum, K. A., G. Lozanski, J. C. Byrd. 2004. Adult Burkitt leukemia and lymphoma. Blood 104: 3009-3020. [Abstract/Free Full Text]
  3. Henderson, A., K. Calame. 1998. Transcriptional regulation during B cell development. Annu. Rev. Immunol. 16: 163-200. [Medline]
  4. Perlot, T., F. W. Alt, C. H. Bassing, H. Suh, E. Pinaud. 2005. Elucidation of IgH intronic enhancer functions via germ-line deletion. Proc. Natl. Acad. Sci. USA 102: 14362-14367. [Abstract/Free Full Text]
  5. Adams, J. M., A. W. Harris, C. A. Pinkert, L. M. Corcoran, W. S. Alexander, S. Cory, R. D. Palmiter, R. L. Brinster. 1985. The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature 318: 533-538. [Medline]
  6. Butzler, C., X. Zou, A. V. Popov, M. Bruggemann. 1997. Rapid induction of B-cell lymphomas in mice carrying a human IgH/c-mycYAC. Oncogene 14: 1383-1388. [Medline]
  7. Palomo, C., X. Zou, I. C. Nicholson, C. Butzler, M. Bruggemann. 1999. B-cell tumorigenesis in mice carrying a yeast artificial chromosome-based immunoglobulin heavy/c-myc translocus is independent of the heavy chain intron enhancer (Emu). Cancer Res. 59: 5625-5628. [Abstract/Free Full Text]
  8. Chauveau, C., M. Cogne. 1996. Palindromic structure of the IgH 3'locus control region. Nat. Genet. 14: 15-16. [Medline]
  9. Madisen, L., M. Groudine. 1994. Identification of a locus control region in the immunoglobulin heavy-chain locus that deregulates c-myc expression in plasmacytoma and Burkitt’s lymphoma cells. Genes Dev. 8: 2212-2226. [Abstract/Free Full Text]
  10. Laurencikiene, J., V. Tamosiunas, E. Severinson. 2007. Regulation of {epsilon} germline transcription and switch region mutations by IgH locus 3' enhancers in transgenic mice. Blood 109: 159-167. [Abstract/Free Full Text]
  11. Morvan, C. L., E. Pinaud, C. Decourt, A. Cuvillier, M. Cogne. 2003. The immunoglobulin heavy-chain locus hs3b and hs4 3' enhancers are dispensable for VDJ assembly and somatic hypermutation. Blood 102: 1421-1427. [Abstract/Free Full Text]
  12. Pinaud, E., A. A. Khamlichi, C. Le Morvan, M. Drouet, V. Nalesso, M. Le Bert, M. Cogne. 2001. Localization of the 3' IgH locus elements that effect long-distance regulation of class switch recombination. Immunity 15: 187-199. [Medline]
  13. Guglielmi, L., M. Le Bert, V. Truffinet, M. Cogne, Y. Denizot. 2003. Insulators to improve expression of a 3' IgH LCR-driven reporter gene in transgenic mouse models. Biochem. Biophys. Res. Commun. 307: 466-471. [Medline]
  14. Kovalchuk, A. L., C. F. Qi, T. A. Torrey, L. Taddesse-Heath, L. Feigenbaum, S. S. Park, A. Gerbitz, G. Klobeck, K. Hoertnagel, A. Polack, et al 2000. Burkitt lymphoma in the mouse. J. Exp. Med. 192: 1183-1190. [Abstract/Free Full Text]
  15. Park, S. S., J. S. Kim, L. Tessarollo, J. D. Owens, L. Peng, S. S. Han, S. Tae Chung, T. A. Torrey, W. C. Cheung, R. D. Polakiewicz, et al 2005. Insertion of c-Myc into Igh induces B-cell and plasma-cell neoplasms in mice. Cancer Res. 65: 1306-1315. [Abstract/Free Full Text]
  16. Park, S. S., A. L. Shaffer, J. S. Kim, W. duBois, M. Potter, L. M. Staudt, S. Janz. 2005. Insertion of Myc into Igh accelerates peritoneal plasmacytomas in mice. Cancer Res. 65: 7644-7652. [Abstract/Free Full Text]
  17. Wang, J., L. M. Boxer. 2005. Regulatory elements in the immunoglobulin heavy chain gene 3'-enhancers induce c-myc deregulation and lymphomagenesis in murine B cells. J. Biol. Chem. 280: 12766-12773. [Abstract/Free Full Text]
  18. Mautner, J., S. Joos, T. Werner, D. Eick, G. W. Bornkamm, A. Polack. 1995. Identification of two enhancer elements downstream of the human c-myc gene. Nucleic Acids Res. 23: 72-80. [Abstract/Free Full Text]
  19. Remmers, E. F., J. Q. Yang, K. B. Marcu. 1986. A negative transcriptional control element located upstream of the murine c-myc gene. EMBO J. 5: 899-904. [Medline]
  20. Pettersson, S., G. P. Cook, M. Bruggemann, G. T. Williams, M. S. Neuberger. 1990. A second B cell-specific enhancer 3' of the immunoglobulin heavy-chain locus. Nature 344: 165-168. [Medline]
  21. Matthias, P., D. Baltimore. 1993. The immunoglobulin heavy chain locus contains another B-cell-specific 3' enhancer close to the {alpha} constant region. Mol. Cell. Biol. 13: 1547-1553. [Abstract/Free Full Text]
  22. Michaelson, J. S., S. L. Giannini, B. K. Birshtein. 1995. Identification of 3' {alpha}-hs4, a novel Ig heavy chain enhancer element regulated at multiple stages of B cell differentiation. Nucleic Acids Res. 23: 975-981. [Abstract/Free Full Text]
  23. Morse, H. C., 3rd, M. R. Anver, T. N. Fredrickson, D. C. Haines, A. W. Harris, N. L. Harris, E. S. Jaffe, S. C. Kogan, I. C. MacLennan, P. K. Pattengale, J. M. Ward. 2002. Bethesda proposals for classification of lymphoid neoplasms in mice. Blood 100: 246-258. [Abstract/Free Full Text]
  24. Desagher, S., D. Severac, A. Lipkin, C. Bernis, W. Ritchie, A. Le Digarcher, L. Journot. 2005. Genes regulated in neurons undergoing transcription-dependent apoptosis belong to signaling pathways rather than the apoptotic machinery. J. Biol. Chem. 280: 5693-5702. [Abstract/Free Full Text]
  25. Guglielmi, L., V. Truffinet, C. Carrion, M. Le Bert, N. Cogne, M. Cogne, Y. Denizot. 2005. The 5'HS4 insulator element is an efficient tool to analyse the transient expression of an Em mu-GFP vector in a transgenic mouse model. Transgenic Res. 14: 361-364. [Medline]
  26. Guglielmi, L., V. Truffinet, M. Cogne, Y. Denizot. 2003. The beta-globin HS4 insulator confers copy-number dependent expression of IgH regulatory elements in stable B cell transfectants. Immunol. Lett. 89: 119-123. [Medline]
  27. Chauveau, C., E. Pinaud, M. Cogne. 1998. Synergies between regulatory elements of the immunoglobulin heavy chain locus and its palindromic 3' locus control region. Eur. J. Immunol. 28: 3048-3056. [Medline]
  28. Pelengaris, S., M. Khan. 2003. The many faces of c-Myc. Arch. Biochem. Biophys. 416: 129-136. [Medline]
  29. Lin, Y., K. Wong, K. Calame. 1997. Repression of c-myc transcription by Blimp-1, an inducer of terminal B cell differentiation. Science 276: 596-599. [Abstract/Free Full Text]
  30. Zhu, D., C. F. Qi, H. C. Morse, 3rd, S. Janz, F. K. Stevenson. 2005. Deregulated expression of the Myc cellular oncogene drives development of mouse "Burkitt-like" lymphomas from naive B cells. Blood 105: 2135-2137. [Abstract/Free Full Text]
  31. Guglielmi, L., M. Le Bert, I. Comte, M. L. Dessain, M. Drouet, C. Ayer-Le Lievre, M. Cogne, Y. Denizot. 2003. Combination of 3' and 5' IgH regulatory elements mimics the B-specific endogenous expression pattern of IgH genes from pro-B cells to mature B cells in a transgenic mouse model. Biochim. Biophys. Acta 1642: 181-190. [Medline]
  32. Weisinger, G., E. F. Remmers, P. Hearing, K. B. Marcu. 1988. Multiple negative elements upstream of the murine c-myc gene share nuclear factor binding sites with SV40 and polyoma enhancers. Oncogene 3: 635-646. [Medline]
  33. Lindstrom, M. S., K. G. Wiman. 2002. Role of genetic and epigenetic changes in Burkitt lymphoma. Semin. Cancer Biol. 12: 381-387. [Medline]
  34. Sanchez-Beato, M., A. Sanchez-Aguilera, M. A. Piris. 2003. Cell cycle deregulation in B-cell lymphomas. Blood 101: 1220-1235. [Abstract/Free Full Text]
  35. Eischen, C. M., D. Woo, M. F. Roussel, J. L. Cleveland. 2001. Apoptosis triggered by Myc-induced suppression of Bcl-xL or Bcl-2 is bypassed during lymphomagenesis. Mol. Cell. Biol. 21: 5063-5070. [Abstract/Free Full Text]
  36. Gartel, A. L., A. L. Tyner. 2002. The role of the cyclin-dependent kinase inhibitor p21 in apoptosis. Mol. Cancer Ther. 1: 639-649. [Abstract/Free Full Text]

Related articles in The JI:

IN THIS ISSUE

The JI 2007 179: 5615-5616. [Full Text]  




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Data Supplement
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Related articles in The JI
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Truffinet, V.
Right arrow Articles by Denizot, Y.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Truffinet, V.
Right arrow Articles by Denizot, Y.
Right arrowPubmed/NCBI databases
*Gene*GEO Profiles
*HomoloGene*UniGene

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS