Deregulated expression of RasGRP1 initiates thymic lymphomagenesis independently of T-cell receptors

RasGRP1 is a Ras-specific exchange factor, which is activated by T-cell receptor (TCR) and promotes TCR-dependent positive selection of thymocytes. RasGRP1 is highly expressed on most T lymphocytic leukemias and is a common site of proviral insertion in retrovirus-induced murine T-cell lymphomas. We used RasGRP1 transgenic mice to determine if deregulated expression of RasGRP1 has a causative role in the development of T-cell malignancies. Thymic lymphomas occurred in three different RasGRP1 transgenic mouse lines. Thymocyte transformation correlated with high transgene expression in early stage lymphomas, indicating that deregulated RasGRP1 expression contributed to the initiation of lymphomagenesis. Expression of the positively selectable H-Y TCR accelerated lymphomagenesis in RasGRP1 transgenic mice. However, the transformed thymocytes lacked markers of positive selection and lymphomas occurred when positive selection was precluded by negative selection of the H-Y TCR. Therefore, initiation of lymphomagenesis via RasGRP1 was not associated with TCR-dependent positive selection of thymocytes. Thymic lymphomas occurred in RasGRP1 transgenic/Rag2-/- mice, demonstrating that neither TCR nor pre-TCR were required for RasGRP1-driven lymphomagenesis. The RasGRP1 transgene conferred pre-TCR-independent survival and proliferation of immature thymocytes, suggesting that deregulated expression of RasGRP1 promotes lymphomagenesis by expanding the pool of thymocytes which are susceptible to transformation.     

Carcinogen-induced lymphomagenesis in pim-1 transgenic mice: dose dependence and involvement of myc and ras

Transgenic mice overexpressing the pim-1 oncogene in their lymphoid compartments are predisposed to T-cell lymphomagenesis but only to the extent that approximately 10% of the transgenic mice develop lymphomas within 34 weeks after birth. Recently, we have shown that lymphomagenesis in pim-1 transgenic mice can be accelerated by infecting pim-1 transgenic mice with murine leukemia viruses or by treating the mice with a relatively low dose of 60 mg of the carcinogen N-ethyl-N-nitrosourea (ENU) per kg of body weight. Here we describe the incidence of tumors as a function of the dose of ENU. Either 200, 15, 4, 1, or 0.1 mg/kg ENU was injected into transgenic and control mice and the tumor incidence was monitored. T-cell lymphomas developed in 100 and 70% of the pim-1 transgenic mice treated with 200 and 15 mg/kg ENU, respectively. Approximately 20% of the Emu-pim-1 transgenic mice developed lymphomas after treatment with either 4, 1, or 0.1 mg/kg ENU. The nontransgenic mice developed lymphomas only after injection with 200 mg/kg (45%). The data show that Emu-pim-1 transgenic mice are approximately 25-fold more susceptible to ENU-induced lymphomagenesis than control mice. In most tumors the expression of c-myc was strongly elevated, probably as a direct or indirect effect of ENU. Analysis of the lymphomas for ras mutations revealed that approximately 10% of the lymphomas bear a ras mutation. The data indicate that at least some of these mutations are not the direct result of alkylation by ENU but rather represent spontaneous mutations that occurred later in the tumorigenic process.     

Expression of Frat1 correlates with expression of β-catenin and is associated with a poor clinical outcome in human SCC and AC

Overexpression of frequently rearranged in advanced T-cell lymphomas 1 (Frat1) has been reported in several human malignant tumors, but the relationship between Frat1 and ??-catenin in lung cancer is still unclear. Our goal was to investigate the correlation between Frat1 and ??-catenin in patients with lung cancers. Immunohistochemistry was performed in 110 cases of non-small cell lung cancer (NSCLC) with clinical follow-up. Results showed that both Frat1 and ??-catenin were overexpressed in NSCLC. The expression of Frat1 and ??-catenin was significantly correlated with tumor differentiation, TNM stage, and lymph node metastasis. Interestingly, the overexpression of ??-catenin was positively correlated with the overexpression of Frat1 (correlation coefficient?=?0.285; P?=?0.003). In addition, overexpression of Frat1 and abnormal expression of ??-catenin were found to represent a poor prognosis for the patients. Furthermore, based on the transfection of Frat1 and ??-catenin, we found that Frat1 can upregulate the expression of ??-catenin in BE1 cells.     

Frat1与TCF4在非小细胞肺癌(NSCLC)中表达的相关性及意义

目的:研究Frat1与TCF4在NSCLC中的表达及其意义。方法:用免疫组化SP法对95例原发性NSCLC患者手术标本中Frat1和TCF4的表达进行检测。结果:在NSCLC中Frat1表达与肺癌组织的低分化、淋巴结转移、TNM分期相关(P<0.05);TCF4的表达与淋巴结转移、TNM分期相关(P<0.05);Frat1的表达与TCF4的表达有相关性(P<0.05)。结论:Frat1和TCF4在NSCLC中与肺癌的侵袭和转移等恶性表型有关,Frat1和TCF4在癌组织中的表达有相关性。     

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.     

F-MuLV acceleration of myelomonocytic tumorigenesis in SV40 large T antigen transgenic mice is accompanied by retroviral insertion at Fli1 and a novel locus, Fim4.

We describe here the development of a murine system for the identification of genes involved in myelomonocytic neoplasms. Transgenic C57BL/6J mice expressing SV40 early region under a myelomonocytic promoter develop histiocytic sarcomas with a latency of 167 days. We used retroviral proviral tagging to accelerate tumorigenesis and to uncover genetic changes that contribute to tumor development. Infection of transgenic mice with Friend murine leukemia virus (F-MuLV) shortened the latency of morbidity to 103 days (P< 0.001); this was associated with clonal proviral integrations in tumor DNA. As expected for F-MuLV, proviral insertions occurred at Fli1 in both transgenic and nontransgenic tumors. Four insertions were found at a novel locus, termed Fim4, on chromosome 6. This region is syntenic to human 7q32, a region that is commonly deleted in human myelodysplastic syndrome and acute myeloid leukemia. A murine BAC containing Fim4 was sequenced and analyzed, and while there was significant human-mouse homology in the area of the insertions, no candidate gene has been identified. Thus we have established a system to identify genes involved in myelomonocytic tumors, and have used it to identify Fim4, a new common site of proviral insertion. Study of this locus may provide insight into genes involved in AML-associated 7q32 deletions in humans.     

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.     

Insertional mutagenesis reveals progression genes and checkpoints in MYC/Runx2 lymphomas

In this study, we have exploited the power of insertional mutagenesis to elucidate tumor progression pathways in mice carrying two oncogenes (MYC/Runx2) that collaborate to drive early lymphoma development. Neonatal infection of these mice with Moloney murine leukemia virus resulted in accelerated tumor onset with associated increases in clonal complexity and lymphoid dissemination. Large-scale analysis of retroviral integration sites in these tumors revealed a profound bias towards a narrow range of target genes, including Jdp2 (Jundm2), D cyclin, and Pim family genes. Remarkably, direct PCR analysis of integration hotspots revealed that every progressing tumor consisted of multiple clones harboring hits at these loci, giving access to large numbers of independent insertion events and uncovering the contrasting mutagenic mechanisms operating at each target gene. Direct PCR analysis showed that high-frequency targeting occurs only in the tumor environment in vivo and is specific for the progression gene set. These results indicate that early lymphomas in MYC/Runx2 mice remain dependent on exogenous growth signals, and that progression can be achieved by constitutive activation of pathways converging on a cell cycle checkpoint that acts as the major rate-limiting step for lymphoma outgrowth.     

LMP1 signaling and activation of NF-kappaB in LMP1 transgenic mice

Transgenic mice expressing Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) under the control of an immunoglobulin heavy-chain promoter and enhancer develop lymphoma at a threefold higher incidence than LMP1-negative mice. In vitro, LMP1 activates numerous signaling pathways including p38, c-Jun N terminal kinase (JNK), phosphatidylinositol 3 kinase (PI3K)/Akt, and NF-kappaB through interactions with tumor necrosis receptor-associated factors (TRAFs). These pathways are frequently activated in EBV-associated malignancies, although their activation cannot be definitively linked to LMP1 expression in vivo. In this study, interactions between LMP1 and TRAFs and the activation of PI3K/Akt, JNK, p38, and NF-kappaB were examined in LMP1 transgenic mice. LMP1 co-immunoprecipitated with TRAFs 1, 2, and 3. Akt, JNK, and p38 were activated in LMP1-positive and -negative splenocytes as well as LMP1-positive and -negative lymphomas. Multiple forms of NF-kappaB were activated in healthy splenocytes from LMP1 transgenic mice, in contrast to healthy splenocytes from LMP1-negative mice. However, in both LMP1-positive and -negative lymphomas, only the oncogenic NF-kappaB c-Rel, was specifically activated. Similarly to EBV-associated malignancies, p53 protein was detected at high levels in the transgenic lymphomas, although mutations were not detected in the p53 gene. These data indicate that NF-kappaB is activated in LMP1-positive healthy splenocytes; however, NF-kappaB c-Rel is specifically activated in both the transgenic lymphomas and in the rare lymphomas that develop in negative mice. The LMP1-mediated activation of NF-kappaB may contribute to the specific activation of c-Rel and lead to the increased development of lymphoma in the LMP1 transgenic mice.     

AID and RAG1 do not contribute to lymphomagenesis in Emu c-myc transgenic mice

DNA breaks caused by recombination-activating gene 1 (RAG1) and activation-induced cytidine deaminase (AID) induce c-myc/immunoglobulin (Ig) heavy chain chromosomal translocations and thereby stimulate lymphomagenesis. However, constitutive expression of c-myc alone is not sufficient to induce lymphomas. Because RAG1 and AID activity occurs outside of Ig genes, we assessed whether these enzymes provide the secondary genetic lesions in Emu c-myc transgenic mice to promote lymphoma development. We found that the tumor incidence and tumor phenotype in Emu c-myc transgenic mice is similar in AID+/+, AID+/- and AID-/- backgrounds in both specific pathogen-free and conventional animal facilities, indicating that AID does not contribute to lymphoma development in Emu c-myc transgenic mice. To examine the role of RAG proteins in Emu c-myc mice, we examined Emu c-myc transgenic mice that harbor the Ig-HEL heavy- and light-chain transgenes, and thus have reduced RAG expression in B cells. We found that tumor incidence was not affected by these Ig transgenes. However, we found that RAG1-/- Emu c-myc mice exhibited accelerated tumor development compared to controls. This data combined with our finding that Emu c-myc mice lived longer in the conventional facility than in the specific pathogen-free facility suggest an immune-mediated activity that suppresses lymphoma development.     

T-cell lymphomas mask slower developing B-lymphoid and myeloid tumours in transgenic mice with broad haemopoietic expression of MYC

Deregulation of MYC expression occurs in many haematological malignancies. Previous studies modelling MYC-induced lymphomagenesis in the mouse used transgenic vectors that directed MYC overexpression in a lineage-specific manner. Here, we describe a transgenic mouse strain in which constitutive MYC expression is driven broadly in haemopoiesis by a vector containing regulatory elements of the Vav gene. Healthy young VavP-MYC17 mice had multiple haemopoietic abnormalities, most notably increased size and numbers of B-lymphoid cells, monocytes and megakaryocytes. The mice rapidly developed tumours and, surprisingly, these were exclusively T-cell lymphomas, mostly of mature CD4(+) CD8(-) T cells, a tumour type that is seldom seen in mouse models. To examine tumour development in the absence of the susceptible T cells, we bred VavP-MYC17 mice lacking the Rag1 recombinase. They survived longer and succumbed to tumours of several different haemopoietic cell types: pre-T cells, pro-B cells, macrophages and unusual progenitor cells. Thus, although T-lineage cells have the shortest latent period to transformation, the VavP-MYC17 transgene drives malignant transformation of multiple cell types and VavP-MYC17 mice provide a new model for tumours of multiple haemopoietic lineages.     

Mutational analysis of transgenic mouse B cell lymphomas: indication of a Trp53-independent pathway in tumor progression

Deregulated myc, bcl-2 and/or TP53 gene expression is associated with non-Hodgkin's B cell lymphomas (B-NHLs). Emu-N-myc transgenic mice that misexpress N-myc protein and carry a non-disrupted bcl-2 gene develop indolent B cell lymphomas reminiscent of the B-NHL, follicular lymphoma. Tumors from mice with end-stage disease exhibited discrete, nodular lesions as well as areas of diffuse tumor likely due to coalescence of enlarged follicles. Tumor DNAs were screened for mutations in the Trp53 gene, the murine homologue of the TP53 gene, which participates in B cell differentiation and survival. By PCR-based sequence analyses, we determined there were no mutations in exons 5-8, the common sites of TP53 mutation in B-NHLs. These findings suggested that disease progression in our novel murine lymphoma model may proceed via a Trp53-independent pathogenetic pathway.     

Elevated Mdm2 expression induces chromosomal instability and confers a survival and growth advantage to B cells

Mdm2, a regulator of the p53 tumor suppressor, is frequently overexpressed in lymphomas, including lymphomas that have inactivated p53. However, the biological consequences of Mdm2 overexpression in lymphocytes are not fully resolved. Here, we report that increased expression of Mdm2 in B cells augmented proliferation and reduced susceptibility to p53-dependent apoptosis, which was due to inhibition of p53 and suppression of p21 expression. Notably, developing and mature B cells from Mdm2 transgenic mice had an increased frequency of chromosomal/chromatid breaks and/or aneuploidy. This Mdm2-mediated genome instability occurred at a similar frequency as that in B cells overexpressing the oncogene c-Myc, but the chromosomal instability was not further enhanced when Mdm2 and c-Myc were overexpressed together. Elevated Mdm2 expression alone increased the occurrence of B-cell transformation in vivo and cooperated with c-Myc overexpression, resulting in an acceleration of B-cell lymphomagenesis. In addition, the frequency of p53 mutations was reduced, but not eliminated, in lymphomas arising in Mdm2/Emu-myc double transgenic mice. Therefore, increased Mdm2 expression facilitated B-cell lymphomagenesis, in part, through regulation of p53 by altering B-cell proliferation and susceptibility to apoptosis, and by inducing chromosomal instability.