Administration of an Antioxidant Prevents Lymphoma Development in Transmitochondrial Mice Overproducing Reactive Oxygen Species

Because of the difficulty to exclude possible involvement of nuclear DNA mutations, it has been a controversial issue whether pathogenic mutations in mitochondrial DNA (mtDNA) and the resultant respiration defects are involved in tumor development. To address this issue, our previous study generated transmitochondrial mice (mito-mice-ND6¹³⁹⁹⁷), which possess the nuclear and mtDNA backgrounds derived from C57BL/6J (B6) strain mice except that they carry B6 mtDNA with a G13997A mutation in the mt-Nd6 gene. Because aged mito-mice-ND6¹³⁹⁹⁷ simultaneously showed overproduction of reactive oxygen species (ROS) in bone marrow cells and high frequency of lymphoma development, current study examined the effects of administrating a ROS scavenger on the frequency of lymphoma development. We used N-acetylcysteine (NAC) as a ROS scavenger, and showed that NAC administration prevented lymphoma development. Moreover, its administration induced longevity in mito-mice-ND6¹³⁹⁹⁷. The gene expression profiles in bone marrow cells indicated the upregulation of the Fasl gene, which can be suppressed by NAC administration. Given that natural-killer (NK) cells mediate the apoptosis of various tumor cells via enhanced expression of genes encoding apoptotic ligands including Fasl gene, its overexpression would reflect the frequent lymphoma development in bone marrow cells. These observations suggest that continuous administration of an antioxidant would be an effective therapeutics to prevent lymphoma development enhanced by ROS overproduction.     

Enhanced glycolysis induced by mtDNA mutations does not regulate metastasis

We addressed the issue of whether enhanced glycolysis caused by mtDNA mutations independently induces metastasis in tumor cells using mtDNA transfer technology. The resultant trans-mitochondrial cybrids sharing the same nuclear background of poorly metastatic carcinoma P29 cells, P29mtA11 and P29mtDelta cybrids, possessed mtDNA with a G13997A mutation from highly metastatic carcinoma A11 cells and mtDNA with a 4696bp deletion mutation, respectively. The P29mtDelta cybrids expressed enhanced glycolysis, but did not express ROS overproduction and high metastatic potential, whereas P29mtA11 cybrids showed enhanced glycolysis, ROS overproduction, and high metastatic potential. Thus, enhanced glycolysis alone does not induce metastasis in the cybrids.     

Generation of trans-mitochondrial mito-mice by the introduction of a pathogenic G13997A mtDNA from highly metastatic lung carcinoma cells

To investigate the effects of respiration defects on the disease phenotypes, we generated trans-mitochondrial mice (mito-mice) by introducing a mutated G13997A mtDNA, which specifically induces respiratory complex I defects and metastatic potentials in mouse tumor cells. First, we obtained ES cells and chimeric mice containing the G13997A mtDNA, and then we generated mito-mice carrying the G13997A mtDNA via its female germ line transmission. The three-month-old mito-mice showed complex I defects and lactate overproduction, but showed no other phenotypes related to mitochondrial diseases or tumor formation, suggesting that aging or additional nuclear abnormalities are required for expression of other phenotypes.     

Reversible regulation of metastasis by ROS-generating mtDNA mutations

It has been controversial whether mtDNA mutations are responsible for oncogenic transformation (normal cells to develop tumors), and for malignant progression (tumor cells to develop metastases). To clarify this issue, we created trans-mitochondrial cybrids with mtDNA exchanged between mouse tumor cells that express different metastatic phenotypes. The G13997A mutation in the ND6 gene of mtDNA from high metastatic tumor cells reversibly controlled development of metastases by overproduction of reactive oxygen species (ROS), but did not control development of tumors. The mtDNA-mediated reversible control of metastasis reveals a novel function of mtDNA, and suggests that ROS scavengers may be therapeutically effective in suppressing metastasis.     

Mitochondrial DNA Mutations in Mutator Mice Confer Respiration Defects and B-Cell Lymphoma Development

Mitochondrial DNA (mtDNA) mutator mice are proposed to express premature aging phenotypes including kyphosis and hair loss (alopecia) due to their carrying a nuclear-encoded mtDNA polymerase with a defective proofreading function, which causes accelerated accumulation of random mutations in mtDNA, resulting in expression of respiration defects. On the contrary, transmitochondrial mito-miceΔ carrying mtDNA with a large-scale deletion mutation (ΔmtDNA) also express respiration defects, but not express premature aging phenotypes. Here, we resolved this discrepancy by generating mtDNA mutator mice sharing the same C57BL/6J (B6J) nuclear background with that of mito-miceΔ. Expression patterns of premature aging phenotypes are very close, when we compared between homozygous mtDNA mutator mice carrying a B6J nuclear background and selected mito-miceΔ only carrying predominant amounts of ΔmtDNA, in their expression of significant respiration defects, kyphosis, and a short lifespan, but not the alopecia. Therefore, the apparent discrepancy in the presence and absence of premature aging phenotypes in mtDNA mutator mice and mito-miceΔ, respectively, is partly the result of differences in the nuclear background of mtDNA mutator mice and of the broad range of ΔmtDNA proportions of mito-miceΔ used in previous studies. We also provided direct evidence that mtDNA abnormalities in homozygous mtDNA mutator mice are responsible for respiration defects by demonstrating the co-transfer of mtDNA and respiration defects from mtDNA mutator mice into mtDNA-less (ρ⁰) mouse cells. Moreover, heterozygous mtDNA mutator mice had a normal lifespan, but frequently developed B-cell lymphoma, suggesting that the mtDNA abnormalities in heterozygous mutator mice are not sufficient to induce a short lifespan and aging phenotypes, but are able to contribute to the B-cell lymphoma development during their prolonged lifespan.     

Influence of murine leukemia proviral integrations on development of N-methyl-N-nitrosourea-induced thymic lymphomas in AKR mice.

The AKR mouse strain is characterized by a high incidence of spontaneous thymic lymphoma that appears in older animals (greater than 6 months of age) and is associated with novel provirus integrations of ecotropic and recombinant murine leukemia viruses (MuLVs). Treatment of 4- to 6-week-old AKR/J mice with the carcinogen N-methyl-N-nitrosourea (MNU) results in thymic lymphomas that arise as early as 3 to 4 months of age and contain novel somatically acquired MuLV provirus integrations. The AKR/J strain develops MNU-induced lymphoma with a higher incidence and shorter latency than has been observed for other inbred mouse strains. To determine whether provirus integrations of endogenous MuLV account for the enhanced susceptibility of the AKR strain, the incidence and latency of MNU-induced lymphoma development was compared in AKR/J and AKR.Fv-1b mice. The restrictive b allele of the Fv-1 locus restricts integration and replication of endogenous N-tropic MuLV; therefore, AKR-Fv-1b mice have a very low incidence of spontaneous lymphoma. In contrast, AKR.Fv-1b mice develop MNU-induced lymphomas with an incidence and latency similar to those of the AKR/J strain. Furthermore, thymic lymphomas from both strains express an immature CD4-8+ phenotype, indicating neoplastic transformation of the same thymocyte subset. Southern blot analysis confirmed that lymphoma DNA from AKR.Fv-1b mice did not contain somatically acquired provirus integrations. These results demonstrate that provirus integration does not contribute to the predisposition of AKR mice to develop a high incidence of early MNU-induced lymphomas. Nevertheless, MNU treatment stimulated high-level expression of infectious ecotropic MuLV in AKR.Fv-1b as well as in AKR/J mice, suggesting that viral gene products might enhance lymphoma progression.     

Polymorphic mutations in mouse mitochondrial DNA regulate a tumor phenotype

To examine whether polymorphic mtDNA mutations that do not induce significant respiration defects regulate phenotypes of tumor cells, we used mouse transmitochondrial tumor cells (cybrids) with nuclear DNA from C57BL/6 (B6) strain and mtDNA from allogenic C3H strain. The results showed that polymorphic mutations of C3H mtDNA in the cybrids induced hypoxia sensitivity, resulting in a delay of tumor formation on their subcutaneous inoculation into B6 mice. Therefore, the effects of polymorphic mutations in normal mtDNA have to be carefully considered, particularly when we apply the gene therapy to the embryos to replace their pathogenic mtDNA by normal mtDNA.     

Replicative advantage and tissue-specific segregation of RR mitochondrial DNA between C57BL/6 and RR heteroplasmic mice

To investigate the interactions between mtDNA and nuclear genomes, we produced heteroplasmic maternal lineages by transferring the cytoplasts between the embryos of two mouse strains, C57BL/6 (B6) and RR. A total of 43 different nucleotides exist in the displacement-loop (D-loop) region of mtDNA between B6 and RR. Heteroplasmic embryos were reconstructed by electrofusion using a blastomere from a two-cell stage embryo of one strain and an enucleated blastomere from a two-cell stage embryo of the other strain. Equivalent volumes of both types of mtDNAs were detected in blastocyst stage embryos. However, the mtDNA from the RR strain became biased in the progeny, regardless of the source of the nuclear genome. The RR mtDNA population was very high in most of the tissues examined but was relatively low in the brain and the heart. An age-related increase of RR mtDNA was also observed in the blood. The RR mtDNAs in the reconstructed embryos and in the embryos collected from heteroplasmic mice showed a different segregation pattern during early embryonic development. These results suggest that the RR mtDNA has a replicative advantage over B6 mtDNA during embryonic development and differentiation, regardless of the type of nuclear genome.     

Trading mtDNA uncovers its role in metastasis

It has been controversial for many years of whether mtDNA mutations are involved in phenotypes related to cancer due to the difficulty in excluding possible involvement of nuclear DNA mutations in these phenotypes. We addressed this issue by complete trading of mtDNAs between tumor cells expressing different metastatic phenotypes. Resultant trans-mitochondrial cybrids share the same nuclear background, but possess mtDNA from tumor cells expressing different metastatic phenotypes, and thus can be used to uncover the role of mtDNA in these phenotypes. The results showed that mtDNA controls development of metastasis in tumor cells, while tumor development is controlled by nuclear genome.Key words: pathogenic mtDNA mutations, respiration defects, enhanced glycolysis, ROS overproduction, rho-zero cells, mtDNA transfer technology, metastasisHuman mtDNAs with pathogenic mutations inducing significant respiration defects have been shown to be closely associated with mitochondrial diseases.^1,2 Although mitochondrial respiratory function is controlled by both nuclear and mitochondrial genomes, the pathogenicity of these mtDNA mutations has been proven by co-transmission of the mutant mtDNAs and mitochondrial respiration defects to mtDNA-less (ρ⁰) human cells: the resultant trans-mitochondrial cybrids sharing the same nuclear backgrouond showed respiration defects, only when they accumulated the mutated mtDNA from the patients.^3–6 Moreover, we generated transmitochondrial mito-mice sharing the same nuclear background, but carrying various proportions of mtDNA with a pathogenic mutation, and provided model systems for studying exactly how mtDNAs with pathogenic mutations are transmitted and distributed in tissues resulting in the pathogenesis of mitochondrial diseases that show various clinical phenotypes.^7–9With respect to the involvement of mtDNA in tumor phenotypes, it has been proposed that most chemical carcinogens bind preferentially to mtDNA rather than to nuclear DNA in mammalian cells,^10–12 and thus, mtDNA should be the major cellular target of chemical carcinogens, and resultant creation of mutations in mtDNAs is responsible for expression of tumor phenotypes.¹²Although, there has been no direct evidence for creation of mtDNA mutations by chemical carcinogens, and for their contribution to tumor development in mammalian cells, recent studies showed that somatic mtDNA mutations accumulated in human colorectal tumors¹³ and in various tumor types¹⁴ rather than in normal cells of the same subjects, probably by the clonal expansion of the mutated mtDNAs along with the repeated division of tumor cells. Many subsequent studies supported preferential accumulation of mutated mtDNAs in tumor cells,^15–18 suggesting that mutated mtDNAs in tumor cells have acquired replication advantages to be homoplasmic. However, these studies did not address the fundamental question of whether the mutated mtDNAs are involved in tumor development.Our previous studies directly addressed this issue using transmitochondrial cybrids obtained by mtDNA trading between normal and tumor cells, and provided convincing evidence that mutations in nuclear DNA, but not in mtDNA were involved in tumor development in the mouse^19,20 and in human cultured cells.^21,22 The possibility that these observations may represent some specific tumor cases can be excluded since there has been no statistical evidence for association of tumor development and pathogenic mtDNA mutations in the patients with mitochondrial diseases expressing respiration defects caused by pathogenic mutations in mtDNA. The possibility that some polymorphic mtDNA mutations that do not induce respiration defects, but somehow contribute to tumor development also can be excluded, because there has been no statistical evidence for the presence of maternal inheritance of tumor development in spite of the strictly maternal inheritance of mammalian mtDNA.^23,24Nonetheless, it was still possible that mtDNA mutations are involved in other processes than oncogenic transformation of normal cells to develop tumors, such as in malignant progression of tumor cells to develop a metastatic potential. Recent studies demonstrated that mitochondrial respiration defects in TCA-cycle enzymes caused by nuclear DNA mutations controls tumor phenotypes as a consequence of induction of a pseudo-hypoxic pathway under normoxia.^25–27 Thus, some mtDNA mutations also induce the pseudo-hypoxic pathway under normoxia by inducing mitochondrial respiration defects. However, there has been no direct evidence for involvement of mtDNA mutations in malignant progression or in the regulation of the pseudo-hypoxic pathway under normoxia, because of the difficulty in excluding possible contribution of nuclear DNA mutations in these processes.²⁸Recently, we addressed this issue using trans-mitochondrial cybrids²⁹ obtained by complete trading of mtDNAs between highly and poorly metastatic mouse lung carcinoma cells (Fig. 1). By this approach, we could provide convincing evidence for the control of malignant progression of tumor cells to develop metastatic potential by mtDNA:²⁹ all the trans-mitochondrial cybrids with mtDNA from highly metastatic tumor cells expressed high metastatic potential, while those with mtDNA from poorly metastatic tumor cells expressed low metastatic potential, irrespective of whether their nuclear genome was derived from highly or poorly metastatic tumor cells. The findings in our study²⁹ can be summarized as follows: (1) A missense G13997A mutation in the ND6 gene of mtDNA from highly metastatic lung tumor cells induces a complex I defect, and reversibly controls malignant progression of tumor cells to develop metastatic potential, but does not control oncogenic transformation of normal cells to develop tumors; (2) The complex I defect simultaneously induces enhanced glycolysis and ROS overproduction, but induction of metastasis is due to ROS overproduction; (3) ROS overproduction induces metastasis not by acceleration of genetic instability as usually proposed, but by reversible upregulation of nuclear-coded genes related to metastasis, such as Mcl-1; (4) ROS scavengers are therapeutically effective in suppressing mtDNA-mediated metastasis.Open in a separate windowFigure 1Scheme for the isolation of the trans-mitochondrial cybrids with completely exchanged mtDNA between parental cells expressing different metastatic phenotypes. Trading mtDNA shown in this scheme uncovered a role of mtDNA in metastasis. For trading mtDNA, parental P29 and A11 cells were treated with ditercalinium, an antitumor bis-intercalating agent, to isolate ρ⁰P29 and ρ⁰A11 cells (*), which have no mtDNA. Complete depletion of mtDNA was confirmed by PCR analysis. Enucleated cells of the mtDNA donors were prepared by their pretreatment with cytochalasin B and centrifugation. Resultant cytoplasts were fused with ρ⁰ cells by polyethylene glycol to obtain trans-mitochondrial cybrids. High metastatic potential is transferred to the P29mtA11 cybrids with the transfer of mtDNA from the A11 cells, and poor metastatic potential is transferred to the A11mtP29 cybrids with the transfer of mtDNA from the P29 cells. Involvement of cytoplasmic factors other than mtDNA from the A11 cells in expression of the high metastatic potential in the P29mtA11 cybrids can be ruled out by the observations that the A11mtP29 cybrids lost their high metastatic potential, even though they always contain cytoplasmic factors transcribed by the nuclear genes derived from the A11 cells.Thus, our study partly resolves the controversial issue on the relevance or irrelevance of mtDNA mutations in tumor development and/or tumor phenotypes by showing that mutations in mtDNA control development of metastasis in tumor cells.²⁹ Considering that complex I defects simultaneously induce enhanced glycolysis under normoxia (the Warburg effect) and ROS overproduction,²⁹ it remains possible that the Warburg effect alone can control metastasis independently from ROS overproduction. More recently, we examined this possibility by generating trans-mitochondrial cybrids with the deletion mutant mtDNA, which can be expected to induce overall respiration defects, and express enhanced glycolysis under normoxia, but not express ROS overproduction. The results showed that the Warburg effect alone did not control metastasis.     

Termination of the B cell lymphoma dormant state in thymectomized AKR mice.

AKR mice are highly susceptible to spontaneous T cell lymphomagenesis and thymus removal at the age of 1 to 3 mo greatly reduces its development. Twelve-mo-old AKR mice thymectomized at young age were shown previously to carry potential lymphoma cells that could be triggered to develop into B cell lymphomas (80 to 100%) after removal from their host "restrictive" environment into young histocompatible hosts. Additional attempts were made to terminate the potential lymphoma cell dormant state in 12-mo-old thymectomized AKR mice. Replenishment of some deficiencies caused by thymectomy at a young age, including a s.c. syngeneic thymus graft or a single injection of the dual tropic recombinant virus isolates DTV-71 or MCF-247 into 12-mo-old thymectomized AKR mice resulted in Ly-1+ pre-B or B cell lymphoma development in 80 to 98% of these treated mice. In vivo elimination of T cell subsets by administration of cyclosporin A or by mAb expressed on Th cells (anti-CD4) or cytotoxic T cells (anti-CD8) stimulated the progression of dormant potential lymphoma cells towards B cell lymphoma development. The most striking results were observed after administration of anti-CD8 mAb: 90 to 100% of these treated mice developed Ly-1+ B cell lymphomas within 80 days. The effect of rIL-2 on dormant PLC was also tested. Administration of rIL-2 to 12-mo-old thymectomized mice terminated tumor dormancy in 94% of the treated mice within 66 days. Tests of the resulting B lymphomas for dual tropic recombinant virus/mink cell focus-inducing virus infection indicated that the breakdown of tumor dormancy did not result from development of pathogenic class I mink cell focus-inducing viruses. These results suggest that T cell subsets and/or their products are involved in the proliferation arrest of potential lymphoma cells present in thymectomized AKR mice.     

Effects of FVB/NJ and C57Bl/6J strain backgrounds on mammary tumor phenotype in inducible nitric oxide synthase deficient mice

The ability to genetically manipulate mice has led to rapid progress in our understanding of the roles of different gene products in human disease. Transgenic mice have often been created in the FVB/NJ (FVB) strain due to its high fecundity, while gene-targeted mice have been developed in the 129/SvJ-C57Bl/6J strains due to the capacity of 129/SvJ embryonic stem cells to facilitate germline transmission. Gene-targeted mice are commonly backcrossed into the C57Bl/6J (B6) background for comparison with existing data. Genetic modifiers have been shown to modulate mammary tumor latency in mouse models of breast cancer and it is commonly known that the FVB strain is susceptible to mammary tumors while the B6 strain is more resistant. Since gene-targeted mice in the B6 background are frequently bred into the polyomavirus middle T (PyMT) mouse model of breast cancer in the FVB strain, we have sought to understand the impact of the different genetic backgrounds on the resulting phenotype. We bred mice deficient in the inducible nitric oxide synthase (iNOS) until they were congenic in the PyMT model in the FVB and B6 strains. Our results reveal that the large difference in mean tumor latencies in the two backgrounds of 53 and 92 days respectively affect the ability to discern smaller differences in latency due to the Nos2 genetic mutation. Furthermore, the longer latency in the B6 strain enables a more detailed analysis of tumor formation indicating that individual tumor development is not stoichastic, but is initiated in the #1 glands and proceeds in early and late phases. NO production affects tumors that develop early suggesting an association of iNOS-induced NO with a more aggressive tumor phenotype, consistent with human clinical data positively correlating iNOS expression with breast cancer progression. An examination of lung metastases, which are significantly reduced in PyMT/iNOS^−/− mice compared with PyMT/iNOS^+/+ mice only in the B6 background, is concordant with these findings. Our data suggest that PyMT in the B6 background provides a useful model for the study of inflammation-induced breast cancer.     

EBV Latent Membrane Protein 1 Activates Akt,NFκB,and Stat3 in B Cell Lymphomas

Latent membrane protein 1 (LMP1) is the major oncoprotein of Epstein-Barr virus (EBV). In transgenic mice, LMP1 promotes increased lymphoma development by 12 mo of age. This study reveals that lymphoma develops in B-1a lymphocytes, a population that is associated with transformation in older mice. The lymphoma cells have deregulated cell cycle markers, and inhibitors of Akt, NFκB, and Stat3 block the enhanced viability of LMP1 transgenic lymphocytes and lymphoma cells in vitro. Lymphoma cells are independent of IL4/Stat6 signaling for survival and proliferation, but have constitutively activated Stat3 signaling. These same targets are also deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition. These results suggest that Akt, NFκB, and Stat3 pathways may serve as effective targets in the treatment of EBV-associated B cell lymphomas.     

Phenylhydrazine stimulates lymphopoiesis and accelerates Abelson murine leukemia virus-induced pre-B cell lymphomas

Infection of bone marrow or fetal liver cells with Abelson murine leukemia virus (A-MuLV) results in the transformation of pre-B cells and the development of erythroid colonies, indicating that the abl oncogene can affect the growth characteristics of immature cells in both the B cell and erythroid lineages. By comparison, infection of mice with A-MuLV results primarily in the development of pre-B cell lymphomas. To determine whether A-MuLV could induce erythroid disease in vivo, NFS/N mice were pretreated with phenylhydrazine (PHZ) to stimulate erythropoiesis and increase the frequency of potential target cells for A-MuLV. No erythroleukemias developed in mice treated with PHZ. Instead, the latency for pre-B cell lymphomas was reduced by half. This acceleration of disease could be attributed to a marked increase in pre-B cells as targets for transformation by A-MuLV in the bone marrows but not the spleens of treated mice. Increases in the frequencies of T cells in bone marrow and spleen also followed treatment with PHZ. These results show that although PHZ-induced anemia stimulates the production of T and B cells as well as erythroid progenitors, PHZ-treated mice do not develop erythroleukemia or T cell lymphomas. It was also found that the genetically determined resistance of adult C57BL/6 mice to lymphoma induction by A-MuLV could not be overcome by pretreatment with PHZ even though the frequency of pre-B cells in bone marrow was greatly increased by this treatment.     

EBV latent membrane protein 1 activates Akt, NFkappaB, and Stat3 in B cell lymphomas

Latent membrane protein 1 (LMP1) is the major oncoprotein of Epstein-Barr virus (EBV). In transgenic mice, LMP1 promotes increased lymphoma development by 12 mo of age. This study reveals that lymphoma develops in B-1a lymphocytes, a population that is associated with transformation in older mice. The lymphoma cells have deregulated cell cycle markers, and inhibitors of Akt, NFkappaB, and Stat3 block the enhanced viability of LMP1 transgenic lymphocytes and lymphoma cells in vitro. Lymphoma cells are independent of IL4/Stat6 signaling for survival and proliferation, but have constitutively activated Stat3 signaling. These same targets are also deregulated in wild-type B-1a lymphomas that arise spontaneously through age predisposition. These results suggest that Akt, NFkappaB, and Stat3 pathways may serve as effective targets in the treatment of EBV-associated B cell lymphomas.     

A phenotypic spectrum of sexual development in Dax1 (Nr0b1)-deficient mice: consequence of the C57BL/6J strain on sex determination

Nuclear receptor subfamily 0, group B, member 1 (Nr0b1; hereafter referred to as Dax1) is an orphan nuclear receptor that regulates adrenal and gonadal development. Dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (Dax1) mutations in the mouse are sensitive to genetic background. In this report, a spectrum of impaired gonadal differentiation was observed as a result of crossing the Dax1 knockout on the 129SvIm/J strain onto the C57BL/6J strain over two generations of breeding. Dax1-mutant XY mice of a mixed genetic background (129;B6Dax1(-/Y) [101 total]) developed gonads that were predominantly testislike (n = 61), ovarianlike (n = 27), or as intersex (n = 13). During embryonic development, Sox9 expression in the gonads of 129;B6Dax1(-/Y) mutants was distributed across a wide quantitative range, and a threshold level of Sox9 (>0.4-fold of wild-type) was associated with testis development. Germ cell fate also varied widely, with meiotic germ cells being more prevalent in the ovarianlike regions of embryonic gonads, but also observed within testicular tissue. Ptgds, a gene associated with Sox9 expression and Sertoli cell development, was markedly downregulated in Dax1(-/Y) mice. Stra8, a gene associated with germ cell meiosis, was upregulated in Dax1(-/Y) mice. In both cases, the changes in gene expression also occurred in pure 129 mice but were amplified in the B6 genetic background. Sertoli cell apoptosis was prevalent in 129;B6Dax1(-/Y) gonads. In summary, Dax1 deficiency on a partial B6 genetic background results in further modulation of gene expression changes that affect both Sertoli cell and germ cell fate, leading to a phenotypic spectrum of gonadal differentiation.     

Different human copper-zinc superoxide dismutase mutants, SOD1G93A and SOD1H46R, exert distinct harmful effects on gross phenotype in mice

Amyotrophic lateral sclerosis (ALS) is a heterogeneous group of fatal neurodegenerative diseases characterized by a selective loss of motor neurons in the brain and spinal cord. Creation of transgenic mice expressing mutant Cu/Zn superoxide dismutase (SOD1), as ALS models, has made an enormous impact on progress of the ALS studies. Recently, it has been recognized that genetic background and gender affect many physiological and pathological phenotypes. However, no systematic studies focusing on such effects using ALS models other than SOD1(G93A) mice have been conducted. To clarify the effects of genetic background and gender on gross phenotypes among different ALS models, we here conducted a comparative analysis of growth curves and lifespans using congenic lines of SOD1(G93A) and SOD1(H46R) mice on two different genetic backgrounds; C57BL/6N (B6) and FVB/N (FVB). Copy number of the transgene and their expression between SOD1(G93A) and SOD1(H46R) lines were comparable. B6 congenic mutant SOD1 transgenic lines irrespective of their mutation and gender differences lived longer than corresponding FVB lines. Notably, the G93A mutation caused severer disease phenotypes than did the H46R mutation, where SOD1(G93A) mice, particularly on a FVB background, showed more extensive body weight loss and earlier death. Gender effect on survival also solely emerged in FVB congenic SOD1(G93A) mice. Conversely, consistent with our previous study using B6 lines, lack of Als2, a murine homolog for the recessive juvenile ALS causative gene, in FVB congenic SOD1(H46R), but not SOD1(G93A), mice resulted in an earlier death, implying a genetic background-independent but mutation-dependent phenotypic modification. These results indicate that SOD1(G93A)- and SOD1(H46R)-mediated toxicity and their associated pathogenic pathways are not identical. Further, distinctive injurious effects resulted from different SOD1 mutations, which are associated with genetic background and/or gender, suggests the presence of several genetic modifiers of disease expression in the mouse genome.     

Radiation-induced lung response of AcB/BcA recombinant congenic mice

Lemay, A-M. and Haston, C. K. Radiation-Induced Lung Response of AcB/BcA Recombinant Congenic Mice. Radiat. Res. 170, 299-306 (2008).The genetic factors that influence the development of radiotherapy-induced lung disease are largely unknown. Herein we identified a strain difference in lung response to radiation wherein A/J mice developed alveolitis with increased levels of pulmonary mast cells and cells in bronchoalveolar lavage while the phenotype in C57BL/6J mice was fibrosis with fewer inflammatory cells. To identify genomic loci that may influence these phenotypes, we assessed recombinant congenic (RC) mice derived from the A/J and C57BL/6J strains for their propensity to develop alveolitis or fibrosis after 18 Gy whole-thorax irradiation. Mouse survival, lung histopathology and bronchoalveolar lavage cell types were recorded. Informative strains for each of mast cell influx, bronchoalveolar cell numbers, alveolitis and fibrosis were identified. In mice with the A/J strain background, the severity of alveolitis correlated with increased mast cell numbers while in C57BL/6J background strain mice fibrosis was correlated with the percentage of neutrophils in lavage. The data for RC mice support the association of specific inflammatory cells with the development of radiation-induced lung disease and provide informative strains with which to dissect the genetic basis of these complex traits.