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

The innate immune system in host mice targets cells with allogenic mitochondrial DNA

Tumors or embryonic stem cells bearing foreign mitochondrial DNA are rejected by the innate immune system via a mechanism that depends on MyD88

Mitochondrial DNA mutations regulate metastasis of human breast cancer cells.

Mutations in mitochondrial DNA (mtDNA) might contribute to expression of the tumor phenotypes, such as metastatic potential, as well as to aging phenotypes and to clinical phenotypes of mitochondrial diseases by induction of mitochondrial respiration defects and the resultant overproduction of reactive oxygen species (ROS). To test whether mtDNA mutations mediate metastatic pathways in highly metastatic human tumor cells, we used human breast carcinoma MDA-MB-231 cells, which simultaneously expressed a highly metastatic potential, mitochondrial respiration defects, and ROS overproduction. Since mitochondrial respiratory function is controlled by both mtDNA and nuclear DNA, it is possible that nuclear DNA mutations contribute to the mitochondrial respiration defects and the highly metastatic potential found in MDA-MB-231 cells. To examine this possibility, we carried out mtDNA replacement of MDA-MB-231 cells by normal human mtDNA. For the complete mtDNA replacement, first we isolated mtDNA-less (ρ(0)) MDA-MB-231 cells, and then introduced normal human mtDNA into the ρ(0) MDA-MB-231 cells, and isolated trans-mitochondrial cells (cybrids) carrying nuclear DNA from MDA-MB-231 cells and mtDNA from a normal subject. The normal mtDNA transfer simultaneously induced restoration of mitochondrial respiratory function and suppression of the highly metastatic potential expressed in MDA-MB-231 cells, but did not suppress ROS overproduction. These observations suggest that mitochondrial respiration defects observed in MDA-MB-231 cells are caused by mutations in mtDNA but not in nuclear DNA, and are responsible for expression of the high metastatic potential without using ROS-mediated pathways. Thus, human tumor cells possess an mtDNA-mediated metastatic pathway that is required for expression of the highly metastatic potential in the absence of ROS production

Genetic characteristics of genome donors and selection of the trans-mitochondrial cybrids.

a<p>ρ⁰ MDA-MB-231 cells were used as nuclear DNA donors and mtDNA recipients. As mtDNA donors, we used HeLamt231 and HeLamtFt cybrids sharing the HeLa nuclear genome background to exclude the influence of variations in nuclear-coded cytoplasmic factors on the phenotypes. HeLamt231 cybrids carrying nuclear DNA from HeLa cells and mtDNA from MDA-MB-231 cells were isolated by the fusion of ρ∼ HeLa cells with enucleated MDA-MB-231 cells and subsequent cultivation in selection medium with 6-thioguanine medium without uridine and pyruvate (6-tg+UP⁻). The 6-tg eliminates unenucleated MDA-MB-231 cells and UP⁻ eliminates unfused ρ⁰ HeLa cells, which require uridine and pyruvate due to their complete respiration defects caused by mtDNA depletion. HeLamtFt cybrids carrying nuclear DNA from HeLa cells and mtDNA from human fetal skin fibroblasts were obtained previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023401#pone.0023401-Hayashi4" target="_blank">[17]</a> by the fusion of ρ⁰ HeLa cells with enucleated human fetal skin fibroblasts and subsequent cultivation in the 6-tg+UP⁻ medium.</p>b<p>en represents enucleated.</p>c<p>The mtDNA donors sharing the HeLa nuclear genome background and expressing 6-tg resistance cannot survive in the presence of hypoxanthine/aminopterin/thymidine (HAT). Nuclear donor ρ⁰ MDA-MB-231 cells can grow in the HAT selection medium, but not in UP^- selection medium. Thus, only transmitochondrial cybrids can survive in the selection medium.</p

Genotyping of mtDNA in the isolated transmitochondrial cybrids.

<p>The ρ⁰ MDA-MB-231 cells have had their mtDNA removed and were used as mtDNA recipients and nuclear DNA donors; HeLamt231 and HeLamtFt correspond to mtDNA donors sharing the HeLa nuclear genome, but possessing mtDNA from the MDA-MB-231 cells and human fetal fibroblasts, respectively (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023401#pone-0023401-t002" target="_blank">Table 2</a>). For identification of the C12084T mutation exclusively present in mtDNA of MDA-MB-231 cells (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023401#pone-0023401-t001" target="_blank">Table 1</a>), the PCR products were digested with <i>Ear</i>I. Because of the gain of an <i>Ear</i>I site due to the C12084T mutation, 231mt231 cybrids carrying mtDNA from MDA-MB-231 cells produce 113- and 33-bp fragments, whereas 231mtFt cybrids carrying normal mtDNA without the mutation produce a 146-bp fragment.</p

Characterization of the phenotypes of the isolated transmitochondrial cybrids.

<p>Using 231mtFt and 231mt231 cybrids, we examined the effects of transferring normal mtDNA from fetal fibroblasts into ρ⁰ MDA-MB-231 on (<b>A</b>) mitochondrial respiratory function, (<b>B</b>) lactate production, (<b>C</b>) ROS production, and (<b>D</b>) experimental metastatic potential. Bars represent the mean ± S.D. (<i>n</i> = 3). *<i>P</i><0.05; **<i>P</i><0.01.</p