Short Communication

Oncogene (2011) 30, 4757–4764; doi:10.1038/onc.2011.191; published online 30 May 2011

Tp53 deletion in B lineage cells predisposes mice to lymphomas with oncogenic translocations

M A W Rowh1,2,3, A DeMicco2,3,4, J E Horowitz1,2,3, B Yin2,3,4, K S Yang-Iott2,3, A M Fusello2,3, E Hobeika5, M Reth5 and C H Bassing1,2,3,4

  1. 1Immunology Graduate Group, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
  2. 2Division of Cancer Pathobiology, Department of Pathology and Laboratory Medicine, Center for Childhood Cancer Research, Children's Hospital of Philadelphia, Philadelphia, PA, USA
  3. 3Abramson Family Cancer Research Institute, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
  4. 4Cell and Molecular Biology Graduate Group, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
  5. 5Centre for Biological Signaling Studies, BIOSS, Albert-Ludwigs-Universität Freiburg and Max Planck Institute of Immunobiology, Freiburg, Germany

Correspondence: Dr CH Bassing, Department of Pathology, CHOP/University Pennsylvania, 4054 Colket Translational Research Building, 421 Curie Blvd, 3501 Civic Center Boulevard, Philadelphia, PA 19104, USA. E-mail: bassing@email.chop.edu

Received 27 August 2010; Revised 3 April 2011; Accepted 16 April 2011; Published online 30 May 2011.

Top

Abstract

Inactivating Tp53 mutations are frequent genetic lesions in human tumors that harbor genomic instability, including B lineage lymphomas with IG translocations. Antigen receptor genes are assembled and modified in developing lymphocytes by RAG/AID-initiated genomic rearrangements that involve the induction of DNA double strand breaks (DSBs). Although TP53 inhibits the persistence of DSBs and induces apoptosis to protect cells from genomic instability and transformation, the development of spontaneous tumors harboring clonal translocations has not been reported in mice that only lack wild-type Tp53 protein or express Tp53 mutants. Tp53-deficient (Tp53/) mice succumb to T lineage lymphomas lacking clonal translocations but develop B lymphoid tumors containing immunoglobulin (Ig) translocations upon combined inactivation of DSB repair factors, RAG mutation or AID overexpression; mice expressing apoptosis-defective Tp53 mutants develop B cell lymphomas that have not been characterized for potential genomic instability. As somatic rather than germline inactivating mutations of TP53 are typically associated with human cancers and Tp53 deletion has cellular context dependent effects upon lymphocyte transformation, we generated mice with conditional Tp53 deletion in lineage-committed B lymphocytes to avoid complications associated with defective Tp53 responses during embryogenesis and/or in multi-lineage potential cells and, thereby, directly evaluate the potential physiological role of Tp53 in suppressing translocations in differentiated cells. These mb1-cre:Tp53flox/flox mice succumbed to lymphoid tumors containing Ig gene rearrangements and immunophenotypes characteristic of B cells from various developmental stages. Most mb1-cre:Tp53flox/flox tumors harbored clonal translocations, including Igh/c-myc or other oncogenic translocations generated by the aberrant repair of RAG/AID-generated DSBs. Our data indicate that Tp53 serves critical functions in B lineage lymphocytes to prevent transformation caused by translocations in cell populations experiencing physiological levels of RAG/AID-initiated DSB intermediates, and provide evidence that the somatic TP53 mutations found in diffuse large B-cell lymphoma and Burkitt's lymphoma may contribute to the development of these human malignancies.

Keywords:

p53; B lineage lymphomas; Igh/c-myc translocations

Top

Introduction

The diversity of antigen receptors expressed on the surface of B and T cells enables adaptive immune systems to recognize and respond to an enormous variety of pathogens. Antigen-recognition diversity is established in developing lymphocytes through the assembly of immunoglobulin (Ig) and T-cell receptor (TCR) genes from germline variable (V), diversity (D) and joining (J) gene segments (Soulas-Sprauel et al., 2007). V(D)J recombination is initiated by the RAG endonuclease, which cleaves Ig/TCR loci adjacent to gene segments in G1 phase cells, and completed by non-homologous end-joining (NHEJ) and DNA damage response proteins, which process and join RAG-generated DNA ends. The combination of joining events and the imprecise processing of V(D)J ends generates a vast repertoire of antigen receptor genes. During immune responses, Ig genes are diversified further in mature B cells through class switch recombination (CSR) of Ig heavy-chain constant (CH) region exons and somatic hypermutation (SHM) of IgH, Igκ and Igλ variable region exons (Soulas-Sprauel et al., 2007). CSR is initiated by the AID protein, which deaminates cytosines and leads to double strand breaks (DSBs) within IgH switch (S) regions in G1 phase cells, and is completed by NHEJ and DNA damage response proteins, which join AID-liberated DNA ends. SHM also is initiated by AID, but does not involve DSB intermediates. CSR creates IgH chains with different effector functions and SHM selects for higher-affinity antibodies. Although V(D)J recombination and CSR are essential for adaptive immunity, these processes impose hazards upon lymphocytes and host organisms due to their obligate DSB intermediates.

The TP53 tumor suppressor protein responds to DSBs and other many other cellular stresses to activate cell cycle checkpoints, induce senescence or trigger apoptosis and, thereby, prevent cellular transformation (Vousden and Prives, 2009). Upon G1 phase DSBs, TP53 functions to prevent G1/S transition until DNA breaks are repaired or trigger apoptosis if the damage is un-repairable (Vousden and Prives, 2009). Somatic inactivation of TP53 is observed in human tumors containing genomic instability including B lineage lymphomas with clonal IG translocations generated through aberrant V(D)J recombination or CSR (Cheung et al., 2009), indicating that TP53 may prevent genomic instability such as RAG/AID-initiated translocations from forming and/or driving transformation. Multiple independent observations in mice support these notions. First, Tp53 induces apoptosis of NHEJ-deficient lymphocytes with un-repaired RAG DSBs and inhibits persistence of RAG DSBs outside of G1 phase in NHEJ-sufficient thymocytes (Gao et al., 1998; Dujka et al., 2010). Second, germline NHEJ/Tp53-deficient mice succumb to pro-B lymphomas with RAG-dependent Igh/c-myc or Igh/N-myc translocations (Difilippantonio et al., 2002; Zhu et al., 2002; Gladdy et al., 2003; Rooney et al., 2004). Third, Tp53/ mice expressing a RAG1 mutant that exhibits defects in DNA end joining develop thymic lymphomas with clonal antigen receptor locus translocations (Giblin et al., 2009). Fourth, Tp53/ mice containing a genetic block in thymocyte development succumb to thymic lymphomas with RAG-dependent genomic instability (Haines et al., 2006). Fifth, Tp53 deficiency leads to or unmasks the oncogenic potential of RAG-dependent Igh/c-myc and Tcrα/c-myc translocations and AID-initiated Igh/c-myc translocations in lymphocytes lacking histone H2AX (Bassing et al., 2003; Celeste et al., 2003a; Bassing et al., 2007). Sixth, Tp53 suppresses the frequency of non-malignant B cells with AID-initiated Igh/c-myc translocations (Ramiro et al., 2006; Santos et al., 2010). Seventh, Tp53/ mice with inactivation of NHEJ in mature B cells succumb to tumors with oncogenic Igh/c-myc translocations arising through CSR errors, or clonal translocations involving aberrant Igκ and Igλ V(D)J recombination (Wang et al., 2008, 2009). Finally, Tp53/ mice with transgenic AID overexpression develop B cell lymphomas containing clonal miR142/c-myc or Igh/c-myc translocations formed by ‘off-target’ AID activity (Robbiani et al., 2009).

Despite the role of Tp53 in suppressing B lineage lymphomas with Ig translocations in cell populations experiencing elevated frequencies of RAG/AID-initiated DSB intermediates, Tp53/ mice succumb to thymic lymphomas that lack clonal translocations involving Tcr or other genomic loci and do not develop B lymphoid tumors (Liao et al., 1998; Bassing et al., 2003; Celeste et al., 2003a). Mice expressing apoptosis-defective Tp53 mutants in all cells beginning in early embryos develop B lineage lymphomas (MacPherson et al., 2004; Slatter et al., 2009); however, whether these malignancies contain Ig translocations or other types of genomic instability was not reported. The cellular origin, developmental timing and molecular mechanism of oncogene activation and tumor suppressor gene inactivation can influence cancer predisposition and tumor phenotype. For example, wild-type and Rag1/ mice with C-MYC expression initiating in B and T lymphocyte lineages succumb at similar ages to B lymphoid tumors (Adams et al., 1985; Nepal et al., 2008); whereas, mice with C-MYC expression starting in hematopoietic stem cells develop T cell lymphomas, but succumb at later ages to both B and T lineage tumors when on a Rag1/ background (Smith et al., 2005). In addition, mice with H2ax and Tp53 deletion initiating in lineage-committed T cells develop immature T-cell lymphomas more rapidly and of later developmental stages than germline H2ax/Tp53/ mice (Yin et al., 2010). Accordingly, the mortality of Tp53/ mice from thymic lymphomas involving oncogenic lesions that arise during embryogenesis and/or in cells before lineage-commitment, might mask the role of Tp53 in suppressing B lymphoid tumors with Ig translocations formed by physiological levels of RAG/AID-initiated DSB intermediates. Thus, to directly evaluate the potential role of Tp53 in suppressing oncogenic translocations in differentiated cells, we generated and analyzed mice with conditional Tp53 deletion in lineage-committed B lymphocytes.

Top

Results and discussion

The mb1-cre mouse provides an experimental approach to obtain deletion of ‘floxed’ genes in B lineage cells initiating at the early pro-B cell stage (Hobeika et al., 2006). Thus, we generated and characterized a cohort of 14 mb1-cre:Tp53flox/flox mice. We frequently observe mb1-cre-mediated deletion of ‘floxed’ genes in non-lymphoid cells when the transgene is inherited maternally, but not paternally. Consequently, to avoid complications associated with transgene activation in female germ cells and bi-allelic copies of the transgene, mb1-cre:Tp53flox/flox mice were generated by breeding mb1-cre males with Tp53flox/flox females (Jonkers et al., 2001; Hobeika et al., 2006) and their mb1-cre:Tp53flox/wt male offspring with Tp53flox/flox females. Cohort mb1-cre:Tp53flox/flox mice survived tumor-free between 81–177 days with a median age of mortality at 128 days (Figure 1a). All mice developed malignancies that were either disseminated throughout peripheral lymphoid tissues or restricted to one or more lymph nodes (data not shown). mb1-cre:Tp53flox/flox cohort mice did not develop other malignancies. In addition, no tumors arose in mb1-cre, mb1-cre:Tp53flox/+ or Tp53flox/flox control mice aged over the same time. These data reveal that cre-mediated deletion of Tp53 initiating in pro-B cells predisposes mice to spontaneous lymphoid malignancies.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

mb1-cre:Tp53flox/flox mice succumb to B lineage lymphomas. (a) Kaplan–Meier curve showing the tumor-free survival of the 14 mb1-cre:Tp53flox/flox cohort mice over time. These mice were of a mixed C57BL6 and 129SvEv background. (b) Schematic of the DQ52-JH locus with gene segments represented by open boxes, recombination signal sequences depicted by open triangles, the location of the 3′JH probe indicated by a black bar, and the relative locations of the EcoRI restriction sites shown. (c) Southern blot analysis using the 3′JH (top panel) or 3′Vβ14 (bottom panel) probe as a loading control on EcoRI-digested genomic DNA isolated from mb1-cre:Tp53flox/flox tumors and 129SvEv or C57BL6 kidneys, conducted as previously described (Bassing et al., 2003; Wu et al., 2003). The position of the germline (GL) band is indicated. The images were cropped from a larger blot. (d) Schematic of the Jκ locus with gene segments represented by open boxes, recombination signal sequences depicted by open triangles, the location of the 3′Jκ probe indicated by a black bar and the relative locations of the BamHI restriction sites are shown. (e) Southern blot analysis using the 3′Jκ (top panel) or 3′Vβ14 (bottom panel) probe as a loading control on BamHI-digested genomic DNA isolated from mb1-cre:Tp53flox/flox tumors and 129SvEv or C57BL6 kidneys, conducted as previously described (Bassing et al., 2003; Wu et al., 2003). The position of the germline (GL) band is indicated. The images were cropped from a larger blot. (f-h) FACS analysis with anti-B220 and anti-CD43 antibodies (f), anti-IgM and anti-IgD antibodies (g) or anti-Igκ and anti-Igλ antibodies (h) on single cell suspensions of the indicated mb1-cre:Tp53flox/flox lymphomas and wild-type (WT) lymph nodes, conducted as previously described (Bassing et al., 2003).

Full figure and legend (188K)

Mature B cells develop through a differentiation program that involves the developmental stage-specific assembly of Ig genes. To determine the developmental stage(s) from which mb1-cre:Tp53flox/flox lymphomas arose, we analyzed potential Ig rearrangements in these tumors. Germline JH segments reside on a 6.0-kb EcoRI fragment (Figure 1b) that changes size upon Igh rearrangements in pro-B cells. Southern blot analysis of EcoRI-digested DNA using a 3′JH probe revealed that most tumors contained non-germline JH loci (Figure 1c). Notably, tumor #160 contained three non-germline sized fragments where the signal intensity of one was greater than the other two (Figure 1c), analogous to Igh rearrangements in pro-B lymphomas of NHEJ/Tp53-deficient and H2AX/Tp53-deficient mice. These tumors harbor a t(12;15) translocation with 3′JH sequences and a t(15;12) translocation with amplified 3′JH sequences, which arise following replication of a chromosome 12 harboring un-repaired Igh DSBs, as well as a normal chromosome 12 with 3′JH sequences (Difilippantonio et al., 2000; Gao et al., 2000; Bassing et al., 2003; Celeste et al., 2003b). Germline Jκ segments reside on an 8.9-kb BamHI fragment (Figure 1d) that changes size upon Vκ-to-Jκ rearrangements or is deleted upon Vκ rearrangements to the 3′Igκ deleting element, each of which occurs in pre-B cells. Southern blotting of BamHI-digested DNA using a 3′Jκ probe and then a loading control probe revealed that at least seven tumors contained non-germline or deleted Jκ loci (Figure 1e). In addition, at least four of the tumors with Igκ rearrangements (#55, 29, 99, and 603) also harbored non-germline S regions (data not shown). These Southern data demonstrate that cre-mediated Tp53 deletion initiating in pro-B cells results in the malignant transformation of B lineage-committed cells at various developmental stages.

To further define the developmental stage(s) from which mb1-cre:Tp53flox/flox lymphomas arose, we sought to determine their cell surface immunophenotype. FACS analysis of 11 mb1-cre:Tp53flox/flox tumors revealed that all expressed B220, CD43 and CD19 (Figures 1f–h and Table 1), confirming they arose from B lineage lymphocytes. Tumors #160, 323, 953 and 972 lacked expression of IgM, IgD, Igκ and Igλ (Figures 1f–h and Table 1), which along with their Ig rearrangement patterns indicate that they arose from pro-B cells; tumors #29, 57, 61 and 333 each expressed Igκ, IgM and/or IgD (Figures 1f–h and Table 1), which along with their rearrangement patterns indicate that they arose from mature B cells. Notably, although tumors #55, 99 and 603 lacked expression of IgM, IgD, Igκ or Igλ (Figures 1f–h and Table 1), their Ig rearrangement patterns indicate that they arose from ‘switched’ mature B cells. These FACS data along with our Southern blot data demonstrate that cre-mediated Tp53 deletion initiating in pro-B cells predisposes mice to the malignant transformation of pro-B cells, mature B cells and mature B cells that had attempted CSR.


We next conducted spectral karyotyping of mb1-cre:Tp53flox/flox B lymphoid tumors to assay for potential translocations. We analyzed metaphase spreads prepared from 11 lymphomas and found that nine contained at least one clonal translocation (Table 1). Six of these tumors also contained one or more non-clonal translocation (Table 1). Three B lineage lymphomas (#55, 99 and 603) contained clonal t(12;15) translocations with configurations suggesting that aberrant V(D)J recombination or CSR juxtaposed the Igh locus on chromosome 12 and the c-myc locus on chromosome 15 (Figures 2a and b and Table 1). Tumor #972 contained a similar non-clonal t(12;15) translocation (Figure 2b and Table 1). Another tumor (#57) contained clonal t(16;9) and t(11;16) translocations (Figure 2c and Table 1), which could have arisen from aberrant V(D)J recombination or SHM involving the Igλ locus on chromosome 16. Five mb1-cre:Tp53flox/flox B lymphoid malignanices (#29, 333, 931, 953 and 983) harbored clonal translocations between chromosomes lacking antigen receptor loci (Table 1); these lesions could have developed from DSBs generated by ‘off-target’ RAG/AID activity and/or other factors such as DNA replication errors. Consistent with the former notion, tumor #29 harbored a clonal t(11;15) translocation (Figure 2d and Table 1) similar to the AID-dependent translocations that fuse miR142 on chromosome 11 to the c-myc locus on chromosome 15 creating fusion genes that drive c-myc overexpression (Robbiani et al., 2009). These spectral karyotyping data demonstrate that cre-mediated deletion of Tp53 in developing B cells leads to lymphomas with clonal and non-clonal translocations.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

mb1-cre:Tp53flox/flox B lineage lymphomas harbor clonal translocations. (a) Spectral (left), DAPI (middle) and classified karyotype (right) images of a metaphase prepared from mb1-Tcre:p53flox/flox B lineage lymphoma #55 are shown. The clonal t(12;15) translocation of this tumor is circled within each image. (b) Spectral and classified images of the clonal t(12;15) translocations of mb1-cre:Tp53flox/flox tumors #99 and #603 and the non-clonal t(12;15) translocation in tumor #972. (c) Spectral and classified images of the clonal t(9;16) and t(16;11) translocations in mb1-cre:Tp53flox/flox tumor #57. (d) Spectral and classified images of the clonal t(11;15) translocation in tumor #29. Metaphase spreads were prepared from tumors as described (Bassing et al., 2003). Cytogenetic analyses were performed by manufacturer's instructions (Applied Spectral Imaging (ASI); Vista, CA, USA). Slides were examined under a BX61 microscope (magnification: × 600) from Olympus (Center Valley, PA, USA), controlled by a LAMBDA 10-B Smart Shutter from Sutter Instrument (Novato, CA, USA). Images were captured using a LAMBDA LS light source from Sutter Instrument, and a COOL-1300QS camera ASI (ASI, Vista, CA, USA) then analyzed and managed through Case Data Manager Version 5.5 configured by ASI.

Full figure and legend (148K)

As a means to determine if the clonal translocations of particular mb1-cre:Tp53flox/flox B lineage lymphomas might have arisen through the aberrant repair of RAG/AID initiated DSBs and/or activated the c-myc oncogene, we performed fluorescence in situ hybridization and c-myc Southern blot analysis. Fluorescence in situ hybridization analysis of tumor #57 with a probe that hybridizes to the 5′ end of the Igλ locus and a chromosome 16 paint indicated that the t(16;9) and t(11;16) chromosome 16 breakpoint occurred near or within Igλ (Figure 3a). Fluorescence in situ hybridization of tumors #99, 55, 603 and 972 using probes that hybridize centromeric of the CH genes or across the c-myc locus demonstrated that the t(12;15) translocations in these tumors juxtaposed Igh and c-myc (Figure 3b and data not shown). In addition, Fluorescence in situ hybridization analysis of tumor #29 using a probe that hybridizes centromeric of miR142 and a chromosome 15 paint indicated that miR142 was located at the t(11;15) breakpoint (Figure 3c). None of these translocations involved c-myc amplification. The c-myc gene resides on an ~20-kb EcoRI fragment (Figure 3d) that can change size upon translocations involving c-myc. Southern blot analysis of EcoRI-digested DNA using a 3′c-myc probe revealed disruption of the c-myc locus on one allele in tumors #99 and #29 (Figure 3e), providing evidence that the Igh and miR142 loci translocated into the 5′ end of the c-myc locus in these tumors similar to the RAG/AID-initiated translocations found in Burkitt's lymphoma (BL) and other human B lymphomas. Tumors #55, 99 and 603 with clonal Igh/c-myc translocations and tumor #29 with a clonal miR142/c-myc translocation contained Igκ and Sμ rearrangements (Figure 1e and data not shown), consistent with the notion that these oncogenic lesions formed because of ‘off-target’ AID activity (Robbiani et al., 2009). We were not able to generate metaphases from tumor #160; however, Southern analysis revealed substantial c-myc amplification (Figure 3e), indicating that this pro-B lymphoma likely arose from a RAG-generated Igh/c-myc translocation with co-amplification of Igh and c-myc as occurs in NHEJ/Tp53-deficient and H2AX/Tp53-deficient mice (Zhu et al., 2002; Bassing et al., 2003; Celeste et al., 2003a; Gladdy et al., 2003; Rooney et al., 2004). Tumor #61 contained a disrupted c-myc locus (Figure 3e) but lacked translocations detectable by spectral karyotyping (Table 1), suggesting that a genomic deletion involving the 5′ end of the c-myc locus occurred on one allele. Collectively, these data indicate that cre-mediated Tp53 deletion initiating in pro-B cells leads to B lineage lymphomas with recurrent Igh/c-myc or other clonal translocations generated through aberrant V(D)J recombination, CSR, or possibly SHM.

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

mb1-cre:Tp53flox/flox tumors contain translocations generated by aberrant V(D)J recombination and CSR. (a) Fluorescence in situ hybridization image of the t(11;16) and t(16;9) translocations in mb1-cre:Tp53flox/flox tumor #57 using a green 5′Igλ probe and a red chromosome 16 paint. Fluorescence in situ hybridization BAC probes were labeled using a Biotin-Nick Translation Mix kit (Roche, Indianapolis, IA, USA). Images were captured and analyzed as outlined in the Figure 2 legend. The 5′ Igλ RP23-3829P9 BAC was purchased from the Children's Hospital of Oakland Research Institute (CHORI; Oakland, CA, USA). (b) Fluorescence in situ hybridization images of the t(12;15) translocation in mb1-cre:Tp53flox/flox tumor #99 showing the red centromeric Igh probe, green c-myc probes, and the merged image. The Igh CH BAC199 has been described previously (Bassing et al., 2003). The c-myc centromeric 285N22, c-myc internal 307D14 and c-myc telomeric 454G15 BACs were provided by K. Mills of the Jackson Laboratory. (c) Fluorescence in situ hybridization images of the t(11;15) translocation in mb1-cre:Tp53flox/flox tumor #29 showing the red centromeric miR142 probe, green chromosome 15 paint and the merged image. The miR142 centromeric RP24-376D9 BAC was purchased from CHORI. (d) Schematic of the c-myc locus with exons represented by open boxes, the location of the 3′c-myc probe indicated by a black bar, and the relative locations of the EcoRI restriction sites shown. (e) Southern blot analysis using the 3′c-myc (top panel) or 3′Vβ14 (bottom panel) probe as a loading control on EcoRI-digested genomic DNA isolated from mb1-cre:Tp53flox/flox tumors and 129SvEv or C57BL6 kidneys, conducted as previously described (Bassing et al., 2003; Wu et al., 2003). The position of the germline (GL) band is indicated. The images were cropped from a larger blot.

Full figure and legend (109K)

The mb1-cre:Tp53flox/flox cohort mice in this study developed B lymphoid tumors harboring clonal translocations at a similar rate as published Tp53/ cohort mice succumbed to thymic lymphomas with aneuploidy, but lacking genomic instability (Donehower et al., 1992; Jacks et al., 1994; Bassing et al., 2003; Celeste et al., 2003a). This finding indicates that the development of aneuploid thymic lymphomas masks B lineage lymphomas in Tp53/ mice and/or the onset of B lymphoid tumors with clonal translocations is accelerated in mb1-cre:Tp53flox/flox mice as compared with in Tp53/ mice. We recently showed that mice with cre-mediated deletion of H2ax and Tp53 initiating in thymocytes succumb to spontaneous thymic lymphomas at later ages than germline H2ax/Tp53-deficient mice (Yin et al., 2010). In this context, oncogenic lesions that arise from stresses other than DSBs in Tp53/ cells before lymphocyte lineage commitment and/or in developing T lineage lymphocytes might drive thymocyte transformation more effectively than translocations that arise in B lineage cells. Although cre expression in cultured mouse embryonic cells causes genomic instability (Loonstra et al., 2001; Silver and Livingston, 2001), constitutive cre expression initiating in thymocytes neither leads to a detectable increase in genomic instability nor accelerates the development of thymic lymphomas in Tp53/ mice (Cheung et al., 2002). Still, it is possible that mb1-cre expression causes oncogenic translocations or other lesions that cooperate with RAG/AID-initiated translocations to accelerate the transformation of Tp53-deficient B lineage cells. Alternatively, Tp53 deletion in B lineage cells, theoretically, could preclude compensatory genetic and/or epigenetic changes associated with germline Tp53-deficiency. Comparison of spontaneous lymphoma predisposition among Tp53/ and mb1-cre:Tp53/ mice with and without genetic or surgical ablation of thymocytes would be needed to distinguish among these possibilities. Regardless, as mb1-cre mice do not develop B lymphoid tumors, our data demonstrate unequivocally that Tp53 serves critical functions in B lineage cells to prevent transformation caused by Igh/c-myc or other oncogenic translocations in cell populations experiencing physiological levels of RAG/AID DSB intermediates.

B lineage lymphomas account for a significant percentage of human cancers. These malignancies frequently contain oncogenic IG translocations. Inactivating somatic mutations of TP53 correlate with IGH translocations in diffuse large B-cell lymphoma (DLBCL) and BL, as well as chemotherapy resistance, rapid disease progression and poor prognosis in patients with B lymphoid tumors (Kuppers, 2005; Cheung et al., 2009). Our analysis of mb1-cre:Tp53flox/flox mice provides evidence that the somatic TP53 mutations found in DLBCL and BL may contribute to the development of these malignancies. The molecular pathogenesis of human B lineage lymphomas with IG translocations remains largely unknown, which hinders the development of more effective and less toxic treatments. Thus, mb1-cre:Tp53flox/flox mice may provide a useful pre-clinical model for conducting genome-wide screens to identify genetic/epigenetic changes that cooperate with TP53 inactivation and activation of C-MYC or other oncogenes to enable DLBCL and BL. This animal model also could be used to evaluate the potential efficacy of more specific and less toxic treatments for DLBCL and BL tumors with TP53 inactivation, such as inhibitors of CHK1 or p38 MAPK (Reinhardt et al., 2007; Fishler et al., 2010). Finally, our results suggest that conditional Tp53 deletion in different lineages and developmental stages may provide the basis for pre-clinical models of other human hematopoietic malignancies that contain TP53 inactivation.

Top

Conflict of interest

The authors declare no conflict of interest.

Top

References

  1. Adams JM, Harris AW, Pinkert CA, Corcoran LM, Alexander WS, Cory S et al. (1985). The c-myc oncogene driven by immunoglobulin enhancers induces lymphoid malignancy in transgenic mice. Nature 318: 533–538. | Article | PubMed | ISI | ChemPort |
  2. Bassing CH, Ranganath S, Murphy M, Savic V, Gleason M, Alt FW. (2007). Aberrant V(D)J recombination is not required for rapid development of H2ax/p53 deficient thymic lymphomas with clonal translocations. Blood 111: 2163–2169. | Article | PubMed | ISI |
  3. Bassing CH, Suh H, Ferguson DO, Chua KF, Manis J, Eckersdorff M et al. (2003). Histone H2AX: a dosage-dependent suppressor of oncogenic translocations and tumors. Cell 114: 359–370. | Article | PubMed | ISI | ChemPort |
  4. Celeste A, Difilippantonio S, Difilippantonio MJ, Fernandez-Capetillo O, Pilch DR, Sedelnikova OA et al. (2003a). H2AX haploinsufficiency modifies genomic stability and tumor susceptibility. Cell 114: 371–383. | Article | PubMed | ISI | ChemPort |
  5. Celeste A, Fernandez-Capetillo O, Kruhlak MJ, Pilch DR, Staudt DW, Lee A et al. (2003b). Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks. Nat Cell Biol 5: 675–679. | Article | PubMed | ISI | ChemPort |
  6. Cheung AMY, Hande MP, Jalali F, Tsao M-S, Skinnider B, Hirao A et al. (2002). Loss of Brca2 and p53 synergistically promotes genomic instability and deregulation of T-cell apoptosis. Cancer Res 62: 6194–6204. | PubMed | ISI | ChemPort |
  7. Cheung KJ, Horsman DE, Gascoyne RD. (2009). The significance of TP53 in lymphoid malignancies: mutation prevalence, regulation, prognostic impact and potential as a therapeutic target. Br J Haematol 146: 257–269. | Article | PubMed | ISI |
  8. Difilippantonio MJ, Petersen S, Chen HT, Johnson R, Jasin M, Kanaar R et al. (2002). Evidence for replicative repair of DNA double-strand breaks leading to oncogenic translocation and gene amplification. J Exp Med 196: 469–480. | Article | PubMed | ISI | ChemPort |
  9. Difilippantonio MJ, Zhu J, Chen HT, Meffre E, Nussenzweig MC, Max EE et al. (2000). DNA repair protein Ku80 suppresses chromosomal aberrations and malignant transformation. Nature 404: 510–514. | Article | PubMed | ISI | ChemPort |
  10. Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CA, Butel JS et al. (1992). Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356: 215–221. | Article | PubMed | ISI | ChemPort |
  11. Dujka ME, Puebla-Osorio N, Tavana O, Sang M, Zhu C.. (2010). ATM and p53 are essential in the cell-cycle containment of DNA breaks during V(D)J recombination in vivo. Oncogene 29: 957–965. | Article | PubMed | ISI |
  12. Fishler T, Li YY, Wang RH, Kim HS, Sengupta K, Vassilopoulos A et al. (2010). Genetic instability and mammary tumor formation in mice carrying mammary-specific disruption of Chk1 and p53. Oncogene 29: 4007–4017. | Article | PubMed | ISI |
  13. Gao Y, Ferguson DO, Xie W, Manis JP, Sekiguchi J, Frank KM et al. (2000). Interplay of p53 and DNA-repair protein XRCC4 in tumorigenesis, genomic stability and development. Nature 404: 897–900. | Article | PubMed | ISI | ChemPort |
  14. Gao Y, Sun Y, Frank KM, Dikkes P, Fujiwara Y, Seidl KJ et al. (1998). A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis. Cell 95: 891–902. | Article | PubMed | ISI | ChemPort |
  15. Giblin W, Chatterji M, Westfield G, Masud T, Theisen B, Cheng H-L et al. (2009). Leaky severe combined immunodeficiency and aberrant DNA rearrangements due to a hypomorphic RAG1 mutation. Blood 113: 2965–2975. | Article | PubMed | ISI |
  16. Gladdy RA, Taylor MD, Williams CJ, Grandal I, Karaskova J, Squire JA et al. (2003). The RAG-1/2 endonuclease causes genomic instability and controls CNS complications of lymphoblastic leukemia in p53/Prkdc-deficient mice. Cancer Cell 3: 37–50. | Article | PubMed | ISI | ChemPort |
  17. Haines BB, Ryu CJ, Chang S, Protopopov A, Luch A, Kang YH et al. (2006). Block of T cell development in P53-deficient mice accelerates development of lymphomas with characteristic RAG-dependent cytogenetic alterations. Cancer Cell 9: 109–120. | Article | PubMed | ChemPort |
  18. Hobeika E, Thiemann S, Storch B, Jumaa H, Nielsen PJ, Pelanda R et al. (2006). Testing gene function early in the B cell lineage in mb1-cre mice. Proc Natl Acad Sci USA 103: 13789–13794. | Article | PubMed | ChemPort |
  19. Jacks T, Remington L, Williams BO, Schmitt EM, Halachmi S, Bronson RT et al. (1994). Tumor spectrum analysis in p53-mutant mice. Curr Biol 4: 1–7. | Article | PubMed | ISI | ChemPort |
  20. Jonkers J, Meuwissen R, van der Gulden H, Peterse H, van der Valk M, Berns A. (2001). Synergistic tumor suppressor activity of BRCA2 and p53 in a conditional mouse model for breast cancer. Nat Genet 29: 418–425. | Article | PubMed | ISI | ChemPort |
  21. Kuppers R.. (2005). Mechanisms of B-cell lymphoma pathogenesis. Nat Rev Cancer 5: 251–262. | Article | PubMed | ISI | ChemPort |
  22. Liao MJ, Zhang XX, Hill R, Gao J, Qumsiyeh MB, Nichols W et al. (1998). No requirement for V(D)J recombination in p53-deficient thymic lymphoma. Mol Cell Biol 18: 3495–3501. | PubMed | ISI | ChemPort |
  23. Loonstra A, Vooijs M, Beverloo HB, Allak BA, van Drunen E, Kanaar R et al. (2001). Growth inhibition and DNA damage induced by Cre recombinase in mammalian cells. Proc Natl Acad Sci USA 98: 9209–9214. | Article | PubMed | ChemPort |
  24. MacPherson D, Kim J, Kim T, Rhee BK, van Oostrom CThM, DiTullio Jr RA et al. (2004). Defective apoptosis and B-cell lymphomas in mice with p53 point mutation at Ser 23. EMBO J 23: 3689–3699. | Article | PubMed | ISI | ChemPort |
  25. Nepal RM, Zaheen A, Basit W, Li L, Berger SA, Martin A. (2008). AID and RAG1 do not contribute to lymphomagenesis in Emu c-myc transgenic mice. Oncogene 27: 4752–4756. | Article | PubMed | ISI | ChemPort |
  26. Ramiro AR, Jankovic M, Callen E, Difilippantonio S, Chen HT, McBride KM et al. (2006). Role of genomic instability and p53 in AID-induced c-myc-Igh translocations. Nature 440: 105–109. | Article | PubMed | ISI |
  27. Reinhardt HC, Aslanian AS, Lees JA, Yaffe MB. (2007). p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell 11: 175–189. | Article | PubMed | ISI | ChemPort |
  28. Robbiani DF, Bunting S, Feldhahn N, Bothmer A, Camps J, Deroubaix S et al. (2009). AID produces DNA double-strand breaks in non-Ig genes and mature B cell lymphomas with reciprocal chromosome translocations. Mol Cell 36: 631–641. | Article | PubMed | ISI | ChemPort |
  29. Rooney S, Sekiguchi J, Whitlow S, Eckersdorff M, Manis JP, Lee C et al. (2004). Artemis and p53 cooperate to suppress oncogenic N-myc amplification in progenitor B cells. Proc Natl Acad Sci USA 101: 2410–2415. | Article | PubMed | ChemPort |
  30. Santos MA, Huen MS, Jankovic M, Chen HT, Lopez-Contreras AJ, Klein IA et al. (2010). Class switching and meiotic defects in mice lacking the E3 ubiquitin ligase RNF8. J Exp Med 207: 973–981. | Article | PubMed | ISI | ChemPort |
  31. Silver DP, Livingston DM. (2001). Self-excising retroviral vectors encoding the Cre recombinase overcome Cre-mediated cellular toxicity. Mol Cell 8: 233–243. | Article | PubMed | ISI | ChemPort |
  32. Slatter TL, Ganesan P, Holzhauer C, Mehta R, Rubio C, Williams G et al. (2009). p53-mediated apoptosis prevents the accumulation of progenitor B cells and B-cell tumors. Cell Death Diff 17: 540–550. | Article | ISI |
  33. Smith DP, Bath ML, Harris AW, Cory S. (2005). T-cell lymphomas mask slower developing B-lymphoid and myeloid tumours in transgenic mice with broad haemopoietic expression of MYC. Oncogene 24: 3544–3553. | Article | PubMed | ISI | ChemPort |
  34. Vousden KH, Prives C. (2009). Blinded by the Light: The growing complexity of p53. Cell 137: 413–431. | Article | PubMed | ISI | ChemPort |
  35. Wang JH, Alt FW, Gostissa M, Datta A, Murphy M, Alimzhanov MB et al. (2008). Oncogenic transformation in the absence of Xrcc4 targets peripheral B cells that have undergone editing and switching. J Exp Med 205: 3079–3090. | Article | PubMed | ISI | ChemPort |
  36. Wang JH, Gostissa M, Yan CT, Goff P, Hickernell T, Hansen E et al. (2009). Mechanisms promoting translocations in editing and switching peripheral B cells. Nature 460: 231–236. | Article | PubMed | ISI | ChemPort |
  37. Wu C, Bassing CH, Jung D, Woodman BB, Foy D, Alt FW. (2003). Dramatically increased rearrangement and peripheral representation of Vbeta14 driven by the 3′Dbeta1 recombination signal sequence. Immunity 18: 75–85. | Article | PubMed | ISI | ChemPort |
  38. Yin B, Yang-Iott KS, Chao L, Bassing CH. (2010). Cellular context dependent effects of H2ax and p53 deletion upon the development of thymic lymphoma. Blood Oct 117: 175–185. [Epub ahead of print]. | Article |
  39. Zhu C, Mills KD, Ferguson DO, Lee C, Manis J, Fleming J et al. (2002). Unrepaired DNA breaks in p53-deficient cells lead to oncogenic gene amplification subsequent to translocations. Cell 109: 811–821. | Article | PubMed | ISI | ChemPort |
Top

Acknowledgements

This work was supported by the Training Program Rheumatic Disease of the University of Pennsylvania (5T32-AR007442-23 to MAWR), the Training Grant TG GM-07229 of the University of Pennsylvania (AD), the Cancer Research Institute Pre-doctoral Emphasis Pathway in Tumor Immunology Training Grant awarded to the University of Pennsylvania (BY); the Department of Pathology and Laboratory Medicine and the Center for Childhood Cancer Research of the Children's Hospital of Philadelphia Research Institute, the Abramson Family Cancer Research Institute of the University of Pennsylvania School of Medicine, a grant from the Pennsylvania Department of Health, a Leukemia and Lymphoma Society Scholar Award (CHB), and the National Institutes of Health Grant R01 CA125195 (CHB).