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

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:Tp53(flox/flox) mice succumbed to lymphoid tumors containing Ig gene rearrangements and immunophenotypes characteristic of B cells from various developmental stages. Most mb1-cre:Tp53(flox/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.     

N-methyl-N-nitrosourea-induced and spontaneous AKR/J thymic lymphomas express distinct differentiation antigen phenotypes

Spontaneous and N-methyl-N-nitrosourea (MNU)-induced AKR/J thymic lymphomas were characterized for expression of several lymphocyte differentiation antigens. The majority (53%) of spontaneous lymphomas expressed both Lyt-2 and L3T4 antigens, similar to the predominant normal thymocyte subset. In contrast, 63% of the thymic lymphomas induced by the chemical carcinogen MNU, expressed an Lyt-2+ L3T4- antigenic profile. Although this profile suggested that MNU-induced lymphomas are phenotypically similar to a mature thymocyte subset, the presence of ThB antigen on Lyt-2+ L3T4- lymphomas did not support this notion. Diagonal gel electrophoresis of 125I-labeled membrane extracts and immunoprecipitates revealed that 17 of 29 Lyt-2+ L3T4-MNU-induced lymphomas expressed cell surface T-cell receptor heterodimer components. Northern blot analyses confirmed that the T-cell receptor material was composed of alpha and beta polypeptide chains. The results from this study indicate a distinct origin or differentiation potential of the target cells involved in viral and chemical induced lymphomagenesis of AKR/J mice.     

p53 mutations in C57BL/6J murine thymic lymphomas induced by gamma-irradiation and N-methylnitrosourea.

Genomic DNA from thymus tissue obtained from 47 C57BL/6J animals treated with the DNA alkylating agent N-methylnitrosourea or gamma-irradiation were screened for the presence of p53 mutations by using the single strand conformation polymorphism assay. Mutations were detected in 13% (4 of 30) of primary thymic lymphomas but none of 17 early stage lymphomas. The frequency of p53 mutations was the same in tumors induced by N-methylnitrosourea (2 of 15) or by gamma-irradiation (2 of 15). Mutations occurred in the highly conserved regions of the p53 gene in exons 5, 7, and 8. G:C to A:T transitions were commonly observed. One of 4 of the tumors analyzed contained two p53 mutations in exons 7 and 8. A previous study of the same tumors showed that ras mutations occurred with high frequency (greater than 50%) (E. W. Newcomb et al., Cancer Res., 48:5514-5521, 1988). Our data suggest that p53 mutations do not play a major role in carcinogen-induced thymic lymphomas studied here.     

Transgenic expression of human MGMT blocks the hypersensitivity of PMS2-deficient mice to low dose MNU thymic lymphomagenesis.

Mice deficient in the DNA mismatch repair (MMR) gene, PMS2, develop spontaneous thymic lymphomas and sarcomas. We have previously shown that PMS2(-/-) mice were hypersensitive to a single i.p. injection of 50 mg/kg of N-methyl-N-nitrosourea (MNU) for thymic lymphoma induction. We postulated that MNU sensitivity was due to formation of O(6)-methylguanine (O(6)-mG), which, if unrepaired by O(6)-alkylguanine DNA alkyltransferase (AGT), leads to apoptosis in MMR competent cells and O(6)-mG:T mismatches in MMR deficient cells. Tumor induction is less in MMR(+/+) mice because cells with residual DNA adducts die, whereas mutagenized cells survive in MMR(-/-) mice. Overexpression of AGT (encoded by the methylguanine DNA methyltransferase-MGMT-gene) is known to block MNU induced tumorigenesis in mice with functional MMR. To further determine the sensitivity of PMS2(-/-) mice to MNU and the protective effect of hAGT overexpression, a low dose of MNU (25 mg/kg) was studied in PMS2(-/-) mice and PMS2(-/-)/hMGMT(+) mice. No thymic lymphomas were found in MNU-treated PMS2(+/+) and PMS2(+/-) mice. At 1 year, 46% of the MNU-treated PMS2(-/-) mice developed thymic lymphoma, compared with an incidence of 25% in both untreated PMS2(-/-) mice and MNU treated PMS2(-/-)/hMGMT(+) mice. In addition, a significantly shorter latency in the onset of thymic lymphomas was seen in MNU-treated PMS2(-/-) mice. K-ras mutations were detected almost equally in the thymic lymphomas induced by MNU in both PMS2(-/-) and PMS2(-/-)/hMGMT(+) mice, but not in the spontaneous lymphomas. These data suggest that PMS(-/-) mice are hypersensitive to MNU, that there are different pathways responsible for spontaneous and MNU induced thymic lymphomas in PMS2(-/-) mice, and that overexpression of hMGMT protects the mice by blocking non-K-ras pathways.     

High incidence of T-cell lymphomas in mice deficient in the retinoid-related orphan receptor RORgamma

Nuclear receptors are critical regulators of many physiological processes and have been shown to be involved in a variety of disease processes, including malignant neoplasms. Our laboratory is investigating the function of the retinoid-related orphan receptor gamma (RORgamma) and its possible role in disease. Studies of mice deficient in the expression of RORgamma demonstrated that this receptor plays a crucial role in the regulation of thymopoiesis and lymph node organogenesis. In this study, we show that changes in homeostasis in the thymus of RORgamma-/- mice are associated with a high incidence of T-cell lymphomas. Over 50% of the deficient mice of mixed genetic background die within the first 4 months as a result of thymic lymphomas. A high incidence of lymphomas was also observed in RORgamma-/- 129/SvEv mice. The lymphoblastic cells metastasized frequently to spleen and liver. No other tumor types were detected in any of RORgamma-/- mice that died during the course of the experiment, and none of the heterozygous mice developed thymic lymphomas. Lymphoma formation was associated with increased cellular proliferation and an increase in the number of apoptotic cells. When placed in culture, the RORgamma-/- lymphoblastic cells underwent accelerated "spontaneous" apoptosis at a rate similar to that of RORgamma-/- thymocytes. Upon prolonged culture, several lymphoblastic cell lines could be established. Analysis of the immunophenotype of the lymphoblastic cells showed that the CD4 and CD8 subpopulations varied substantially among different lymphomas. The established cell lines consisted mostly of CD44-CD25+CD4-CD8- cells. Our studies indicate that loss of RORgamma disturbs homeostasis in the thymus by enhancing apoptosis and cellular proliferation. The latter may enhance the probability of individual cells to acquire genetic alterations that make them escape negative selection and normal differentiation programs and as a consequence lead to increased susceptibility to the development of T-cell lymphoma.     

Tumorigenic activity of p21Waf1/Cip1 in thymic lymphoma

The cell cycle inhibitor p21Waf1/Cip1 is among the most important mediators of the tumor suppressor p53. However, there is increasing evidence indicating that p21 could favor tumorigenesis in specific cell types. In particular, the absence of p21 delays the development of thymic lymphomas induced either by ataxia-telangiectasia mutated deficiency or by ionizing irradiation. Here, we extend these observations to the context of p53-deficient mice. The absence of p21 results in a significant extension of the lifespan of p53-null and p53-haploinsufficient mice, and this effect can be attributed exclusively to a decrease in the incidence of spontaneous thymic lymphomas. Specifically, despite the occurrence of a variety of tumor types in the context of p53 deficiency, the only tumors that were significantly impaired by the absence of p21 were thymic lymphomas. Moreover, the absence of p21 also delays the incidence of radiation-induced thymic lymphomas in p53-deficient mice. Interestingly, p21-deficient lymphomas have a higher apoptotic rate than p21-proficient lymphomas, and this could be on the basis of the delayed incidence of thymic lymphomas in the absence of p21. Together, our results indicate that p21 plays an oncogenic role restricted to thymic lymphomas that is mechanistically independent of p53 and associated to a lower tumor apoptotic rate.     

Mice defective in the DNA mismatch gene PMS2 are hypersensitive to MNU induced thymic lymphoma and are partially protected by transgenic expression of human MGMT.

DNA mismatch repair (MMR) stabilizes the cellular genome. Mice defective in the MMR gene PMS2 are susceptible to spontaneous thymic lymphoma and sarcomas. To determine the sensitivity of PMS2 knockout mice to environmental carcinogens and the protective effect of O6-methylguanine DNA methyltransferase (MGMT), heterozygous PMS2 knockout mice and human MGMT (hMGMT) transgenic mice were mated and the PMS2-/- and PMS2+/+ with or without hMGMT offspring were treated at 5 weeks of age with 50 mg/kg N-methyl-N-nitrosourea (MNU). MNU produces carcinogenic O6-methylguanine (O6-meG) adducts, resulting in thymic lymphoma in mice, which can be prevented in normal mice by overexpression of hMGMT. A significantly higher incidence of thymic lymphomas was observed in MNU-treated PMS2-/- mice, compared to wildtype PMS2+/+ mice (100 vs 52%; P < 0.001). The mean latency of lymphomas was also significantly shortened in PMS2-/- mice (81 vs 102 days, P < 0.01). Transgenic expression of hMGMT significantly but incompletely blocked MNU lymphomagenesis in PMS2-/- mice. The incidence of lymphomas in PMS2-/-/hMGMT+ mice was reduced to 80% (P < 0.01) and mean latency increased to 91 days (P < 0.05). Thymic lymphomagenesis was efficiently blocked in PMS2+/+/hMGMT+ mice with rapid repair of O6-meG. Since O6-meG:T mismatches in MMR+ cells may trigger mismatch repair resulting in abortive repair and cell death whereas in the absence of MMR, these mismatches are converted to A:T, we predicted that G to A point mutations in codon 12 of the K-ras gene would occur. In this study, we found G to A point mutations in codon 12 of the K-ras gene in many tumors. Thus, in MMR deficient tissues, methylating agents induce point mutations in cells with a higher rate of cell survival which together are potently carcinogenic in the thymus. These data suggest that PMS2 defective lymphomas may arise by the concerted action of environmental and perhaps endogenous methylation of DNA coupled to genomic instability.     

Candidate mutator genes in mismatch repair-deficient thymic lymphomas: no evidence of mutations in the DNA polymerase delta gene

DNA mismatch repair (MMR) proteins recognize nucleotides that are incorrectly paired. Deficiencies in MMR lead to increased genomic instability reflected in an increased mutation frequency and predisposition to tumorigenesis. Mice lacking the MMR gene, Msh2, develop thymic lymphomas that exhibit much higher mutational frequencies than other Msh2(-/-) tumours and Msh2(-/-) normal thymic tissue, suggesting that an additional mutator may have been acquired in a tissue-specific manner. Clustered mutations observed exclusively in the thymic lymphomas suggests that a gene(s) associated with the replication machinery might have become altered during tumorigenesis. Based on mutation studies in Saccharomyces cerevisiae lacking Msh2 and DNA polymerase delta (DNA pol delta), we hypothesized that the acquisition of mutations in DNA pol delta could contribute to the hypermutator phenotype and tumorigenesis in Msh2(-/-) thymic tissue. Furthermore, previous reports have suggested that genes containing mononucleotide repeats are non-random mutational targets in the absence of MMR. Therefore, we sequenced all 26 exons of the DNA pol delta catalytic subunit, including the six exons containing mononucleotide repeats of >5 bp, from nine Msh2(-/-) thymic lymphomas and two wild-type controls. No DNA pol delta pathogenic mutations were found in the thymic lymphomas, although several DNA base differences compared with published DNA pol delta sequences were observed. We conclude, therefore, that inactivating mutations in DNA pol delta are not a contributing factor in the development of the hypermutator phenotype in MMR-deficient murine thymic lymphomas.     

Analysis of Early Initiating Event(s) in Radiation-induced Thymic Lymphomagenesis

Since the T cell receptor rearrangement is a sequential process and unique to the progeny of each clone, we investigated the early initiating events in radiation-induced thymic lymphomagenesis by comparing the oncogenic alterations with the pattern of γ T cell receptor (TCR) rearrangements. We reported previously that after leukemogenic irradiation, preneoplastic cells developed, albeit infrequently, from thymic leukemia antigen-2⁺ (TL-2⁺) thymocytes. Limited numbers of TL-2⁺ cells from individual irradiated B10.Thy 1.1 mice were injected into B10.Thy 1.2 mice intrathymically, and the common genetic changes among the donor-type T cell lymphomas were investigated with regard to p53 gene and chromosome aberrations. The results indicated that some mutations in the p53 gene had taken place in these lymphomas, hut there was no common mutation among the donor-type lymphomas from individual irradiated mice, suggesting that these mutations were late-occurring events in the process of oncogenesis. On the other hand, there were common chromosome aberrations or translocations such as trisomy 15, t(7F;10C), t(1A;13D) or t(6A;XB) among the donor-type lymphomas derived from half of the individual irradiated mice. This indicated that the aberrations/translocations, which occurred in single progenitor cells at the early T cell differentiation either just before or after γ T cell receptor rearrangements, might he important candidates for initiating events. In the donor-type lymphomas from the other half of the individual irradiated mice, microgenetic changes were suggested to be initial events and also might take place in single progenitor cells just before or right after γ TCR rearrangements.     

Ablation of a peptidyl prolyl isomerase Pin1 from p53-null mice accelerated thymic hyperplasia by increasing the level of the intracellular form of Notch1

Tumor suppressor p53 is essential for checkpoint control in response to a variety of genotoxic stresses. DNA damage leads to phosphorylation on the Ser/Thr-Pro motifs of p53, which facilitates interaction with Pin1, a pSer/pThr-Pro-specific peptidyl prolyl isomerase. Pin1 is required for the timely activation of p53, resulting in apoptosis or cell cycle arrest. To investigate the physiological relationship between Pin1 and p53, we created Pin1-/-p53-/- mice. These p53-deficient mice spontaneously developed lymphomas, mainly of thymic origin, as well as generalized lymphoma infiltration into other organs, including the liver, kidneys and lungs. Ablation of Pin1, in addition to p53, accelerated the thymic hyperplasia, but the thymocytes in these Pin1-/-p53-/- mice did not infiltrate other organs. The thymocytes in 12-week-old Pin1-/-p53-/- mice were CD4(-)CD8(-) (double negative) and had significantly higher levels of the intracellular form of Notch1 (NIC) than the thymocytes of p53-/- or wild-type mice. Presenilin-1, a cleavage enzyme for NIC generation from full-length Notch1 was increased in the thymocytes of Pin1-/-p53-/- mice. Pin1 depletion also inhibited the degradation of NIC by proteasomes. These results suggest that both Pin1 and p53 control the normal proliferation and differentiation of thymocytes by regulating the NIC level.     

Suppression of thymic lymphomas and increased nonthymic lymphomagenesis in Trp53-deficient mice lacking inducible nitric oxide synthase gene

Trp53-deficient mice spontaneously develop lymphomas, mainly of thymic origin, although the molecular mechanism remains largely unknown. As several interaction effects between p53 and iNOS have been reported, we hypothesized that iNOS activity in the thymus is causally linked to lymphomagenesis in Trp53-deficient mice. We therefore created mouse strains with different combinations of the Trp53 and iNOS genes. Western blot and histologic analyses showed that the iNOS protein was constitutively expressed in the thymus independently of Trp53 status and its expression was enhanced in Trp53+/- and Trp53-/- mice compared to Trp53+/+ mice. Homozygous disruption of iNOS decreased the incidence of thymic lymphomas by almost 40% (p=0.087) and 90% (p<0.05) in Trp53-/- and Trp53+/- mice, respectively, compared to the respective iNOS wild-type mice but significantly (p<0.05) increased the development of nonthymic lymphomas in Trp53-/- and Trp53+/- mice. Although iNOS gene disruption did not affect the phenotype of thymic lymphomas, absence of the iNOS gene shifted the spectrum of nonthymic lymphoma from the B-cell to the T-cell lineage. RT-PCR analysis revealed enhanced expression of IL-10, which could have a promoting effect on lymphomagenesis, even without any stimulation, in the spleen of aging mice with the gene combinations Trp53-/-iNOS-/- and Trp53+/-iNOS-/- but not Trp53-/-iNOS+/+ or Trp53+/-iNOS+/+. These results suggest that iNOS could increase the development of thymic lymphomas in Trp53-deficient mice. While iNOS may have protective effects against nonthymic lymphomagenesis, the regulation of cytokine production by iNOS may be involved in the underlying mechanism of antilymphomagenesis effects in the peripheral lymphoid organ.     

Prevention of thymic lymphoma development in Atm-/- mice by dexamethasone

We have reported (M. Yan et al., FASEB J., 15: 1132-1138, 2001) that spontaneous DNA synthesisis markedly increased in the thymocytes from the atrophied thymi of young Atm-/- mice. We, therefore, set out to determine whether this elevated DNA synthesis is responsible for the development of thymic lymphomas in all Atm-/- mice by 4-5 months of age. We show here that in Atm-/- mice: (a) increased DNA synthesis occurs, especially in the immature CD4(-) CD8(-) (dominant negative) and CD8(+) thymocyte populations; (b) the relative percentage of dominant negative cells increases significantly during postnatal development, with a sharp peak at 4 weeks of age; and (c) dexamethasone suppresses DNA synthesis in these thymocytes and prevents thymic lymphoma development. These observations suggest that ataxia telangiectasia mutated (ATM) down-regulates the proliferation of thymocytes, allowing T-cell development and differentiation. The results also show that dexamethasone, like ATM, checks DNA synthesis in developing thymocytes. Finally, the data document for the first time that dexamethasone prevents or slows thymic lymphoma development in Atm-/- mice.     

Accumulation of aberrant Y chromosomes in gamma-ray-induced thymic lymphomas lacking p53

Male F(1) hybrids between MSM mice carrying a deficient p53 allele and BALB/c mice were irradiated with gamma-rays, and 80 thymic lymphomas were obtained, 46 of which developed in mice carrying the deficient p53 allele. Because the Y chromosome contributes little to cellular function, the stability of the Y chromosome in the tumors was assessed by polymerase chain reaction by examining three genes: Smcy and Sry on the short arm and Sts in the pseudoautosomal region of the long arm of the Y chromosome. Twenty-one lymphomas had lost one or two genes, probably as a result of mitotic recombination or interstitial deletion, whereas no lymphomas had lost all three genes. The p53 status of the lymphomas was determined by genotyping and allelic loss analysis; 34 had retained two wild-type p53 alleles, suggesting normal function; 34 had lost both alleles, indicating loss of function; and the other 12 had at least one wild-type p53 allele, so their p53 status was unclear. Compilation of these data revealed that changes in the Y chromosome were detected in only two of the 34 lymphomas retaining functional p53 but in 18 of the 34 lymphomas lacking p53 function, suggesting that p53 deficiency leads to an increase in the accumulation of radiation-induced aberrant chromosomes. This is consistent with our previous result from analysis of the inactive X chromosome. In contrast, a decrease in the fidelity of mitotic transmission in p53-deficient lymphomas was not noted for the Y chromosome.     

Proviral structure and differentiation antigen phenotype of spontaneous and chemically induced AKR lymphomas

AKR mice develop spontaneous thymomas after 6 months of age due to a novel class of murine leukemia viruses that are generated by a series of genetic recombinations between endogenous proviral loci. AKR mice also are more susceptible to N-methyl-N-nitrosourea (MNU)-induced thymomas than are low-leukemia-incidence mouse strains. To determine whether virally and chemically induced lymphomagenesis proceeds by similar pathways in AKR mice, spontaneous and MNU-induced thymic lymphomas were analyzed for a DNA restriction linkage generated during spontaneous tumor development by recombination between envelope genes of endogenous murine leukemia proviral loci. DNA from spontaneous thymic lymphomas invariably contained a restriction fragment characteristic of recombinant murine leukemia virus etiology, while four of five MNU-induced thymic lymphomas did not show this restriction linkage. In addition, analysis of lymphocyte differentiation antigen profiles indicated that MNU-induced lymphomas represent a more immature stage of T-cell differentiation than the majority of spontaneous lymphomas. These data suggest that there are fundamental differences in the mechanisms of induction of virally and chemically induced thymic lymphomas in AKR mice.     

The role of p53 in suppression of KSHV cyclin-induced lymphomagenesis

Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a cyclin D homolog, K cyclin, that is thought to promote viral oncogenesis. However, expression of K cyclin in cultured cells not only triggers cell cycle progression but also engages the p53 tumor suppressor pathway, which probably restricts the oncogenic potential of K cyclin. Therefore, to assess the tumorigenic properties of K cyclin in vivo, we transgenically targeted expression of K cyclin to the B and T lymphocyte compartments via the E micro promoter/enhancer. Around 17% of E micro -K cyclin animals develop lymphoma by 9 months of age, and all such lymphomas exhibit loss of p53. A critical role of p53 in suppressing K cyclin-induced lymphomagenesis was confirmed by the greatly accelerated onset of B and T lymphomagenesis in all E micro -K cyclin/p53(-/-) mice. However, absence of p53 did not appear to accelerate K cyclin-induced lymphomagenesis by averting apoptosis: E micro -K cyclin/p53(-/-) end-stage lymphomas contained abundant apoptotic cells, and transgenic E micro -K cyclin/p53(-/-) lymphocytes in vitro were not measurably protected from DNA damage-induced apoptosis compared with E micro -K cyclin/p53(wt) cells. Notably, whereas aneuploidy was frequently evident in pre-lymphomatous tissues, end-stage E micro -K cyclin/p53(-/-) tumors showed a near-diploid DNA content with no aberrant centrosome numbers. Nonetheless, such tumor cells did harbor more restricted genomic alterations, such as single-copy chromosome losses or gains or high-level amplifications. Together, our data support a model in which K cyclin-induced genome instability arises early in the pre-tumorigenic lymphocyte population and that loss of p53 licenses subsequent expansion of tumorigenic clones.     

Tumors arising in DNA mismatch repair-deficient mice show a wide variation in mutation frequency as assessed by a transgenic reporter gene

We reported previously that thymic lymphomas arising in mice lacking the DNA mismatch repair (MMR) gene, Msh2(-/-), exhibited striking elevations in the mutation frequency of a transgenic lacI reporter gene when compared with normal Msh2(-/-) tissues. To investigate whether hypermutation was a feature of all tumors arising in MMR-deficient mice, lacI transgene mutation frequencies were obtained from several different mouse tumors deficient for PMS2 and/or MSH2. While lacI gene hypermutation was again clearly evident in Msh2 +/- ms2(-/-) and Msh2(-/-)Pms2(-/-) thymic lymphomas, three non-thymic MSH2-deficient tumors failed to show lacI gene mutation frequency elevations when compared with a normal tissue of MMR-deficient mice. The elevated mutation frequencies in the lymphoid tumors, and the finding of multiple clustered mutations in lacI genes rescued from these tumors, suggest that they are possibly generated by a lymphoma-specific hypermutational mechanism.     

A lack of DNA mismatch repair on an athymic murine background predisposes to hematologic malignancy

Inheritance of a germline mutation in one of the DNA mismatch repair genes predisposes human individuals to hereditary nonpolyposis colorectal cancer, characterized by development of tumors predominantly in the colon, endometrium, and gastrointestinal tract. Mice heterozygous for a mismatch repair-null mutation generally do not have an increased risk of neoplasia. However, mice constitutively lacking mismatch repair are prone to tumor development from an early age, particularly thymic lymphomas. Mismatch repair-deficient mice crossed to Apc(+/-) mice develop an increased spontaneous intestinal tumor incidence, demonstrating that the tumor spectrum can be genetically influenced. Here, we bred Msh2- and Msh6-deficient mice to athymic nude mice, hypothesizing that a broader tumor spectrum may be observed if mice are able to survive longer without succumbing to thymic lymphomas. However, Msh2(-/-);Foxn1(nu/nu) and Msh6(-/-);Foxn1(nu/nu) mice developed primarily early-onset lymphoblastic lymphomas. Using B-cell-specific markers, we found these tumors to be predominately B-cell in origin. The development of hematologic malignancy in the mouse, even in the absence of a thymus, parallels the development of B- and T-cell lymphoma and leukemia in the few rare mismatch repair-null human patients that have been identified. The persistent development of hematologic malignancy both in the mouse and in human patients deficient in mismatch repair leads us to implicate mismatch repair as an important repair mechanism in normal B- and T-cell development. Thus, mismatch repair-deficient mice may prove to be a good model to study human hematologic malignancy.     

Loss of one allele of ARF rescues Mdm2 haploinsufficiency effects on apoptosis and lymphoma development

The tumor suppressor p19ARF inhibits Mdm2, which restricts the activity of p53. Complicated feedback and control mechanisms regulate ARF, Mdm2, and p53 interactions. Here we report that ARF haploinsufficiency completely rescued the p53-dependent effects of Mdm2 haploinsufficiency on B-cell development, survival, and transformation. In contrast to Mdm2+/- B cells, Mdm2+/- B cells deficient in ARF were similar to wild-type B cells in their rates of growth and apoptosis and activation of p53. Consequently, the profoundly reduced numbers of B cells in Mdm2+/-Emu-myc transgenic mice were restored to normal levels in ARF+/-Mdm2+/-Emu-myc transgenics. Additionally, ARF+/-Mdm2+/-Emu-myc transgenics developed lymphomas at rates analogous to those observed for wild-type Emu-myc transgenics, demonstrating that loss of one allele of ARF rescued the protracted lymphoma latency in Mdm2+/-Emu-myc transgenics. Importantly, in ARF+/-Mdm2+/-Emu-myc transgenic lymphomas, p53 was inactivated at the frequency observed in lymphomas of wild-type Emu-myc transgenics. Collectively, these results support a model whereby the stoichiometry of Mdm2 and ARF controls apoptosis and tumor development, which should have significant implications in the treatment of malignancies that have inactivated ARF.