Loss of mutant mitochondrial DNA harboring the MELAS A3243G mutation in human cybrid cells after cell-cell fusion with normal tissue-derived fibroblast cells

Abstract

Mutant mitochondrial (mt) DNA variants are related to human disease and have been investigated using cytoplasmic hybrid (cybrid) cells generated from human tumor cells in which mutant mt maintenance depends on the cell line. It is, however, unclear whether human intercellular fusion of non-tumorous cells influences the maintenance of disease-related mutant mt. A preliminary experiment of cell-cell fusion between a human skin fibroblast cell line from a Lesch-Nyhan syndrome patient and an osteosarcoma cybrid cell line harboring the mitochondrial tRNALeu(UUR)A3243G mutation showed a decrease of A3243G mutant mtDNA in fused cells during passages. In order to confirm the decrease of mutant mtDNA, we performed cell-cell fusion experiments using another human lung fibroblastic cell line. When the hygromycin-resistant osteosarcoma cybrid cell line was fused with the fibroblasts without any A3243G mtDNA mutations, the proportion of A3243G mutant mtDNA in the hybrid cells gradually decreased during cell culture and almost completely disappeared in all hybrid clones at the end of 15 passages. These results indicated that A3243G mutant specific mtDNA decreases in the hybrid background when normal fibroblast-derived cell contents, including the nucleus and mt, were introduced. Thus, we are hypothesizing that the non-tumorigenic fibroblast cellular components induce a difference in replication efficacy between the mtDNAs with and without the A3243G mutant sequence, which may be related to the decrease of disease-related mutant mtDNA in the hybrid cells.