Increased Mitochondrial Gene Deletions and ROS Production in Transgenic Mice Overexpressing a Human 8-Oxoguanine DNA-Glycosylase Gene: A Causal Association with the Development of Hematopoietic Neoplasms

Alterations of nuclear genes in human diseases including neoplasms have been well investigated in past several decades and unequivocally established their predominant role in the pathogenesis. However, the relationship of mitochondrial genome alteration with human diseases remains largely unknown. Mitochondria are dynamic organelles involved in oxidative phosphorylation and production of reactive oxygen species (ROS). Accumulated evidence supports that mitochondrial DNA damage and dysfunction play vital roles in the development of a wide array of mitochondria-related human diseases, such as obesity, diabetes, infertility, neurodegenerative disorders and malignant tumors. We previously described the development of a transgenic (TG) mouse model for mitochondrial DNA damage by overexpressing human mitochondrial isoform of 8-oxoguanine DNA Glycosylase 1 (hOGG1) gene. Over-expression of this gene produced a wide range of adverse biological phenotypes, manifesting early-onset obesity, metabolic disturbance, female infertility, high frequency of B-cell lymphoma and human essential thrombocythemia like myeloproliferative disorder, involving the lymph node, bone marrow, spleen, liver and other extranodal sites. Development of these hematopoietic neoplasms appeared to be age-dependent. In the current study, we focused on the pathogenesis of the hematopoietic neoplasms by characterization of the neoplasms via pathologic, biochemical and molecular approaches. While expression of mOGG1 was very similar in parallel organs from transgenic and wild-type control mice, the hOGG1 TG mice expressed very high levels of human OGG1 transgene mRNA, being 6.8- and 112-fold as high as the endogenous mouse OGG, in the spleen and bone marrow. By contrast, hOGG1 transgene mRNA were not detected at all in the above two organs from control mice, indicating that the transgene is highly expressed in the hematopoietic organs in TG mice. We then measured mitochondrial NADH dehydrogenase 1 (ND1) gene expression as an indirect measure of mitochondrial respiratory function. ND1 mRNA levels in the spleen (4) and lymphoma (4) of TG mice were 83% and 58% higher, respectively, than those in the spleen (4) of wild-type control mice (P < 0.01), indicative of increased mitochondrial respiration in the lymphoma and spleen of hOGG1 TG mice. We next measured the levels of intracellular H₂O₂ production in the lymphoma and spleen of hOGG1 transgenic (4) and the spleen from wild-type control (4) mice. The amount of H₂O₂ produced in the lymphoma and the spleen of hOGG1 transgenic mice was ~166% and ~66% higher, respectively, than that in the spleen from wild-type control mice (P < 0.001). The amount of H₂O₂ produced in the lymphoma was ~60% higher than that in the spleen from hOGG1 transgenic mice (P < 0.05). Finally, we examined mitochondrial DNA alterations in TG mice. Mitochondrial DNA samples were extracted from various organs and lymphoma tissues from hOGG1 TG and age-matched non-TG control animals and subjected to PCR analysis using specific primer sets franking the breakpoints of 7 major mitochondrial DNA deletions. Six deletions (3.7, 3.82, 3.86, 4.2, 4.9 and 5.2 kilobase in length) have been previously reported in the literatures. One novel deletion of 15.kilobase was identified in hOGG1 TG mouse in our laboratory. Among 7 major mitochondrial DNA deletion analyzed, Five (3.7, 3.86, 4.2, 5.2 and 15 kilobase in length) deletions were detected in higher frequency in various organs of hOGG1 TG but not in non-TG control mice, suggesting that those deletions might be resulted from overexpression of the transgene hOGG1. Notably, 3 deletions (del3729, del3868, and del15139) were present in significantly higher frequencies in spleen with myeloproliferative disorders or lymphoma from TG mice in comparison to the spleen of the age-matched wild type animals (P<0.01). While the precise mechanisms leading to the development of hematopoietic neoplasms remain elusive, we hypothesized that major mitochondrial gene deletions and increased mitochondrial respiration, as a result of overexpressed hOGG1 gene in the mitochondria, may contribute significantly to the increased intracellular ROS in hematopoietic progenitor cell populations, which, in turn, causes further genetic mutation and the development of lymphoma and myeloproliferative disorder seen in these TG mice.