E mu N- and E mu L-myc cooperate with E mu pim-1 to generate lymphoid tumors at high frequency in double-transgenic mice.

Transgenic mice that contain the L-myc gene under the control of the immunoglobulin heavy-chain enhancer (E mu) express the transgene preferentially in T cells, develop thymic hyperplasia and are predisposed to T-cell lymphomas. An analogous E mu N-myc transgene is expressed preferentially in pre-B and B cells and provokes the development of B-cell neoplasias. Animals with an E mu pim-1 construct express the transgene in both B and T cells, but succumb to T-cell lymphomas. Complementation of the E mu N- and L-myc transgenic mice by breeding with E mu pim-1 animals leads to much more rapid development and a dramatically higher incidence of lymphoid malignancies, but the lineage specificity prescribed by the E mu N- and L-myc transgenes is maintained. The different oncogenic potential of myc genes is illustrated by the average latency period of tumor manifestation in double transgenics. Whereas c-myc/pim-1 animals develop pre-B-cell leukemia prenatally, the mean latency period for N-myc/pim-1 and L-myc/pim-1 mice is 36 and 94 days respectively. The N- and L-myc transgenes are expressed at high levels in tumors from double transgenic mice, but expression of the endogenous c- and N-myc genes is undetectable, directly implicating the myc transgenes in the tumor formation process.     

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.     

The N-ras proto-oncogene can suppress the malignant phenotype in the presence or absence of its oncogene

ras proto-oncogenes have traditionally been associated with the regulation and promotion of cell growth. We have induced thymic lymphomas in N-ras(-/-) mice and in transgenic mice that overexpress wild-type N-ras and found that the lack of wild-type N-ras alleles favors the development of thymic lymphomas,whereas overexpression of wild-type N-ras protects against thymic lymphomagenesis in the presence or absence of its oncogene. To investigate the inhibitory role of wild-type N-ras in in vitro transformation, we introduced wild-type N-ras in N-ras-deficient tumor cells that lack ras activating mutations and found decreased growth in both low serum and soft agar. Taken together, our results indicate that wild-type N-ras has "tumor suppressor" activity, even in the absence of its oncogenic allele.     

Induction of neurogenic tumors in C3HeB-FeJ mice by nitrosourea derivatives: observations by light microscopy, tissue culture, and electron microscopy

Neurogenic tumors were induced in 32.3% of C3HeB/FeJ mice treated transplacentally with ethylnitrosourea (ENU) and in 10.5% of mice treated intravenously (IV) with methylnitrosourea (MNU). Earlier reports on other strains of mice treated with nitrosourea derivatives indicated that non-neural tumors, particularly lymphomas, were readily induced. However, tumors of the nervous system did not develop or occurred only in a low percentage of treated mice. Genetic strain of mice appears to influence susceptibility of mice to nitrosoureas. Two brain tumors, an oligodendroglioma in the diencephalon and a mixed neuroblastoma-glioma of the cerebellum, occurred in 19 adult male mice treated IV every 4 weeks with 25 mg/kg MNU to a total of 175 mg/kg. Neurinomas of cranial nerves developed in 9 of 31 offspring of pregnant mice treated with 20 mg/kg ENU IV on the 19th day of gestation. One MNU-induced oligodendroglioma and 1 ENU-induced neurinoma were propagated in tissue culture. Cells of both tumors produced intracerebral (IC) and subcutaneous (SC) tumors following injection into syngeneic mice. Tissue samples from both IC transplanted tumors contained abundant clusters of C-type RNA viruses in interstitial spaces. Budding virus particles were readily demonstrated on the plasma membranes of tumor cells.     

Overexpression of Frat1 in transgenic mice leads to glomerulosclerosis and nephrotic syndrome, and provides direct evidence for the involvement of Frat1 in lymphoma progression

The proto-oncogene Frat1 was originally identified as a common site of proviral insertion in transplanted tumors of Moloney murine leukemia virus (M-MuLV)-infected Emu-Pim1 transgenic mice. Contrary to most common insertion sites implicated in mouse T cell lymphomagenesis, retroviral insertional mutagenesis of Frat1 constitutes a relatively late event in M-MuLV-induced tumor development, suggesting that proviral activation of Frat1 contributes to progression of T cell lymphomas rather than their genesis. To substantiate this notion we have generated transgenic mice that overexpress Frat1 in various organs, including lymphoid tissues. Frat1 transgenic mice develop focal glomerulosclerosis and a nephrotic syndrome, but they do not exhibit an increased incidence of spontaneous lymphomas. Conversely, these mice are highly susceptible to M-MuLV-induced lymphomagenesis, and Frat1/Pim1 bitransgenic animals develop lymphomas with increased frequency compared to Pim1 transgenic littermates. These data support a role for Frat1 in tumor progression.     

Rapid induction of uterine endometrial proliferative lesions in transgenic mice carrying a human prototype c-Ha-ras gene (rasH2 mice) given a single intraperitoneal injection of N-ethyl-N-nitrosourea

In our previous study, uterine endometrial stromal sarcomas and atypical hyperplasias of the endometrial glands were induced in heterozygous p53 deficient mice (p53 (+/-) mice) of the CBA strain given a single dose of N-ethyl-N-nitrosourea (ENU). In order to clarify whether uterine tumors can be induced in transgenic mice carrying a human prototype c-Ha-ras gene (rasH2 mice) that are very susceptible to genotoxic carcinogens, rasH2 mice and their wild-type littermates received an intraperitoneal injection of 120 or 0mg/kg body weight of ENU followed by no further treatment for 22 weeks. Eighteen and 94% of ENU-treated rasH2 mice had uterine endometrial adenocarcinomas and atypical hyperplasias, respectively. Other malignant and benign tumors such as lung alveolar/bronchiolar adenomas and carcinomas, forestomach squamous cell papillomas and carcinomas, splenic hemangiomas/sarcomas, skin papillomas, malignant lymphomas and harderian gland adenomas were also observed in ENU-treated rasH2 mice. The result in the present study suggests that female rasH2 mice are very susceptible to uterine carcinogenesis, providing a useful model for ENU-induced uterine epithelial tumors.     

Rearrangement of c-myc, pim-1 and Mlvi-1 and trisomy of chromosome 15 in MCF- and Moloney-MuLV-induced murine T-cell leukemias

Provirus insertion near the c-myc, pim-1 or Mlvi-1 genes occurred in 7 out of 59 virally induced T-cell leukemias. C-myc was exclusively rearranged in approximately 10% of MCF247-induced tumors while Mlvi-1 was rearranged to a similar frequency in Moloney-virus-induced lymphomas. Out of 25 karyotyped tumors, 9 (36%) showed trisomy of chromosome 15. Provirus insertion near c-myc, pim-1 or Mlvi-1 occurred both in diploid lymphomas and in tumors with trisomy 15. These results suggest that the molecular and cytogenetic changes observed in murine T-cell leukemias are independent tumor-associated events and that trisomy of chromosome 15 is a common tumor-progression-related event.     

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.     

Transgenic overexpression of a dominant negative mutant of FADD that, although counterselected during tumor progression, cooperates in L-myc-induced tumorigenesis

Activation of the so-called death receptors, e.g., CD95/Fas/Apo-1, is a potent stimulus to trigger apoptosis. Overexpression of the C-terminal FADD deletion mutant FADD-DN blocks death receptor-induced apoptosis, but despite this antiapoptotic activity, lck FADD-DN transgenic mice do not develop lymphomas. To analyze whether functional inactivation of FADD cooperates with Myc overexpression in tumorigenesis, lck FADD-DN transgenic mice were crossed with Emicro L-myc transoncogenic animals. While no tumors were detected in single transgenic FADD-DN or L-myc mice within 15 months, 5 of 17 (29%) FADD-DN/L-myc double transgenic animals developed lymphomas with an average latency period of 47 weeks. Protein analysis of FADD-DN/L-myc tumors showed, however, undetectable levels of FADD-DN protein. FADD-DN protein expression was again lost in 16 of 17 FADD-DN/p53 k.o. T-cell lymphomas, though no significant acceleration of tumorigenesis in P53-deficient lck FADD-DN mice compared to p53 k.o. animals was observed. These data suggest a strong counterselection against the FADD-DN protein during tumor progression, which could be explained by the cell cycle inhibitory activity of FADD-DN. Such counterselection would have to be compensated for by other antiapoptotic mutations, and indeed, strong upregulation of the antiapoptotic Bcl-2 family member Bcl-xL was found in one of the tumors. This in vivo mouse model demonstrates that an antiapoptotic protein involved in the onset of tumorigenesis is selected against and consequently lost during tumor progression because of its additional antiproliferative activity.     

Staging of T-cell receptor beta chain gene rearrangements and ras oncogene mutations in the development of murine thymic lymphomas.

While it is known that the T-cell receptor beta chain gene is rearranged in fully developed murine thymic lymphomas induced by N-nitrosomethylurea and that the ras gene is activated in approximately 50% of these tumors (L. E. Diamond et al., Mol. Cell. Biol., 8: 2233-2236, 1988), it is unknown when these events occur or where the cells committed to a malignant phenotype are first located. We have studied these questions by treating mice with N-nitrosomethylurea, extracting thymocytes and bone marrow cells from the treated mice before they would have developed tumors, transferring the cells into recipient mice, monitoring those mice until they developed lymphoid tumors, and analyzing those tumors. This analysis showed that the initial cells committed to becoming malignant can be located in either the bone marrow or thymus and that both activation of the ras oncogene and rearrangements of the T-cell receptor gene can occur earlier than 30 days after N-nitrosomethylurea treatments. Furthermore, these results suggest that the T-cell receptor beta chain gene can undergo additional rearrangements during progression of a tumor.     

Rare Activation of the Human c-Ha-ras Transgene of Mice in Hemangioendothelial Sarcomas and Liver Tumors Induced by Glu-P-1

A transgenic mouse (Tg), having the human c-Ha- ras proto-oncogene, has been demonstrated to develop hemangioendothelial sarcomas (HESs) which are associated with the transgene mutation, but not to develop liver tumors. In this study, we examined the effects of 2-amino-6-methyldipyrido [1,2- a :3',2'- d ] imidazole (Glu-P-1), a food-borne carcinogen, which has been demonstrated to induce HESs and liver tumors in CDF₁ mice, on Tg mice. Chronic administration of 0.05% Glu-P-1 in the diet induced HESs in Tg (7/17), but not in 18 non-transgenic mice (N-Tg). With basal diet, two out of 17 Tg but none of 22 N-Tg, developed HESs. In contrast, Glu-P-1 administration induced liver tumors, both in Tg and in N-Tg; 16/17 in Tg and 13/18 in N-Tg. The incidence of hepatocellular carcinomas in Tg was higher than that in N-Tg (8/17 versus 3/18). With basal diet, only one out of 17 Tg and none of 22 N-Tg developed liver tumors. The Ha- ras mutation in tumors developed by the groups administered Glu-P-1, was examined. No mutations were detected in the transgene and mouse c-Ha- ras genes in all three HESs examined. In contrast, when 29 liver tumors taken from Tg were examined, two mutations of the transgene were detected in two HCCs. No mouse c-Ha- ras gene mutations were detected in any of the 47 liver tumors examined, which had developed in Tg and N-Tg mice. These results suggest that the transgene plays a role in the development of HESs induced by Glu-P-1, but not as a result of its mutation. Futher, the transgene plays no significant role in the development of liver tumors induced by Glu-P-1, but does play a role in the malignant conversion of some liver tumors, as a result of its mutation.     

Oncogenic response of rats with x-ray-induced microcephaly to transplacental ethylnitrosourea.

The relation of congenital malformations to tumor development was examined. Pregnant Sprague-Dawley rats were given 200 rads of X-rays on the 15th or 16th day of gestation and injections of 10 mg ethylnitrosourea (ENU)/kg 1-4 days later, or they were irradiated or injected only. Surviving weanlings that had been irradiated had micrencephaly and other malformations. Offspring exposed to ENU only had no external deformities. By 15 months of age 16.7% of the offspring exposed to X-rays and ENU prenatally had developed neurogenic tumors, whereas 62.2% of those exposed to ENU alone had developed tumors. Those only irradiated had no tumors. Both of the former groups developed oligodendrogliomas, mixed gliomas, ependymomas, and schwannomas, but the first manifestations of tumors occurred later in the group receiving the combined treatment. This delay persisted furing the subsequent period of the study.     

Chemically induced forestomach papillomas in transgenic mice carry mutant human c-Ha-ras transgenes.

Forestomach papillomas and skin papillomas were induced very efficiently by a single dose administration of the chemical carcinogen methylnitrosourea (MNU) in transgenic mice (rasH2 line) carrying human hybrid c-Ha-ras genes, which encode the prototype p21 gene product. The incidence of forestomach papillomas was dose dependent; when 50 mg/kg of MNU were administered i.p., all of the transgenic mice (56 of 56) developed forestomach papillomas within 12 weeks after administration, whereas 5 and 0.5 mg/kg of MNU induced papillomas in 2 of 19 and 1 of 19 mice, respectively. Nine of 56 transgenic mice (16%) also developed skin papillomas at sites wounded by bites or scratches. Only 1 of 77 nontransgenic littermates developed forestomach papillomas after administration of 50 mg/kg of MNU, and no skin papillomas appeared within 12 weeks after MNU administration. The transgenes (integrated copy number, 5-6) in the tumors developed in 55 of 56 affected transgenic mice (98%) contained at least 1 copy of the transgene that was activated by somatic point mutation at the 12th codon, from GGC (Gly) to GAC (Asp). Because somatic point mutations at the 12th or 61st codon of transgenes have never been detected in normal tissues of transgenic mice thus far examined, these mutational activations of transgenes are tumor-specific events. RNA expression of these activated transgenes was also detected. From these results, it is suggested that somatic mutational activation of the human c-Ha-ras transgene plays a causative role in the occurrence of forestomach and skin papillomas induced by MNU administration in these transgenic mice. This transgenic mouse provides a unique screening system for chemicals that induce or suppress papillomagenesis.     

Distinct chromosomal abnormalities in murine leukemia virus-induced T- and B-cell lymphomas

We performed a cytogenetic study on 16 murine mature B-cell lymphomas and 10 T-cell lymphomas, using G-banding techniques. All tumors, with the exception of 3 spontaneous B-cell tumors, were induced by various slowly transforming murine leukemia viruses (MuLV). Metaphases were obtained from primary (10 B-cell tumors) and first or second transplant generation lymphomas (6 B-cell and 10 T-cell tumors), all of which were well characterized with respect to phenotypic, histologic and genotypic features. In the T-cell tumors we found relatively simple karyotypic abnormalities, including various numerical aberrations, such as trisomy 15, in line with many earlier reports. However, the majority of B-cell tumors showed a great variety of both structural and numerical chromosomal anomalies. Three B-cell lymphomas had an apparently normal karyotype. No single cytogenetic abnormality occurred commonly in the B-cell lymphomas, but some structural abnormalities were found in more than one stemline, in particular, ins (II) (A1; A2) in 3 tumors, and deletions involving the D-region of chromosome 14 in 3 other lymphomas. These cytogenetic results clearly indicate that the pathogenic mechanisms involved in MuLV-induced (long latency) B-cell lymphomagenesis and (short latency) T-cell lymphomagenesis differ considerably.     

Oncogenic potential of cyclin E in T-cell lymphomagenesis in transgenic mice: evidence for cooperation between cyclin E and Ras but not Myc

To study the oncogenic activity of cyclin E in an in vivo system we generated transgenic mice expressing high levels of cyclin E in T-lymphocytes by using a construct containing the CD2 locus control region. These animals were neither predisposed to develop any tumors spontaneously nor showed an increased incidence when crossbred with Emu L-myc transgenic mice but developed hyperplasia in peripheral lymphoid organs at later age with an incidence of 27%. When treated with the DNA methylating carcinogen N-methylnitrosourea (MNU) that provokes the development of T-cell lymphomas, CD2-cyclin E transgenic animals came down with T-cell neoplasia showing a significant higher incidence (54%) than normal non transgenic controls (31%). In one of eight tumors that arose in normal MNU treated mice we could find an expected activating point mutation in the Ki-ras gene (12.5%). In contrast, the same mutation occurred in five of 16 tumors from CD2-cyclin E transgenic mice (31.2%). Whereas cyclin E overexpression alone did not lead to an increased CDK2 activity we observed in all tumors that emerged from either MNU treated normal mice or treated CD2-cyclin E transgenics a downregulation of p27KIP1 and a higher histone H1 kinase activity in CDK2 immunoprecipitates compared to normal tissue. These findings demonstrate that high level expression of cyclin E can predispose T-cells for hyperplasia and malignant transformation. However, the results also suggest that this activity of cyclin E is manifest only when other cooperating oncogenes in particular ras genes are present and activated. This would be consistent with our previous finding that cyclin E and Ha-Ras cooperate in focus formation assays in rat embryo fibroblasts.     

DNA mismatch repair deficiency stimulates N-ethyl-N-nitrosourea-induced mutagenesis and lymphomagenesis

The primary role of the mismatch repair (MMR) system is the avoidance of mutations caused by replication and recombination errors. Furthermore, the lethality of methylating agents has been attributed to the processing of O(6)-methylguanine lesions in DNA by MMR. Loss of the MSH2 protein completely abolishes repair function and results in reduced cell killing by methylating agents and accelerated accumulation of methylation-damage-induced mutations. This has raised the question as to whether MMR is also involved in the cellular response to other genotoxic insults. Here we describe that in mice deficient for Msh2, lymphomagenesis was strongly accelerated by an ethylating agent, N-ethyl-N-nitrosourea (ENU), given at a dose that did not induce lymphomas in wild-type mice. This suggests that MMR deficiency and ENU-induced mutagenesis synergistically collaborate in inducing tumorigenesis. To study the interaction between MMR and ENU-induced DNA damage, we compared the lethality and mutagenicity of ENU in MSH2-proficient and -deficient mouse embryonic stem cells. Although MSH2-deficiency only slightly reduced the lethality of ENU, it strongly enhanced the mutagenicity of ENU. Mutation analysis of ENU-induced Hprt mutants revealed that base substitutions occurred predominantly at A-T base-pairs. These results suggest that MMR modulates the processing of ethylation damage at AT base-pairs.     

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.