Datensatz

Zusammenfassung

o extend previous observations demonstrating differences in number, morphology, and activity of mitochondria in spontaneously immortalized vim+ and vim fibroblasts derived from wild−type and vimentin knockout mice, some structural and functional aspects of mitochondrial genome performance and integrity in both types of cells were investigated. Primary Vim+/+ and Vim/ fibroblasts, which escaped terminal differentiation by immortalization were characterized by an almost twofold lower mtDNA content in comparison to that of their primary precursor cells, whereby the average mtDNA copy number in two clones of vim+ cells was lower by a factor of 0.6 than that in four clones of vim cells. However, during serial subcultivation up to high passage numbers, the vim+ and vim fibroblasts increased their mtDNA copy number 1.5− and 2.5−fold, respectively. While early−passage cells of the vim+ and vim fibroblast clones differed only slightly in the ratio between mtDNA content and mitochondrial mass represented by mtHSP70 protein, after ca. 300 population doublings the average mtDNA/mtmass ratio in the vim+ and vim cells was increased by a factor of 2 and 4.5, respectively. During subcultivation, both types of cells acquired the fully transformed phenotype. These findings suggest that cytoskeletal vimentin filaments exert a strong influence on the mechanisms controlling mtDNA copy number during serial subcultivation of immortalized mouse embryo fibroblasts, and that vimentin deficiency causes a disproportionately enhanced mtDNA content in high−passage vim fibroblasts. Such a role of vimentin filaments was supported by the stronger retention potential for mtDNA and mtDNA polymerase (ã) detected in vim+ fibroblasts by Triton X−100 extraction of mitochondria and agaroseembedded cells. Moreover, although the vim+ and vim fibroblasts were equally active in generating free radicals, the vim cells exhibited higher levels of immunologically detectable 8−oxoG and mismatch repair proteins MSH2 and MLH1 in their mitochondria. Because in vim fibroblasts only one point mutation was detected in the mtDNA D−loop control region, these cells are apparently able to efficiently remove oxidatively damaged nucleobases. On the other hand, a number of large−scale mtDNA deletions were found in high−passage vim fibroblasts, but not in low−passage vim cells and vim+ cells of both low and high passage. Large mtDNA deletions were also induced in young vim fibroblasts by treatment with the DNA intercalator ethidium bromide, whereas no such deletions were found after treatment of vim+ cells. These results indicate that in immortalized vim fibroblasts the mitochondrial genome is prone to large−scale rearrangements, probably due to insufficient control of mtDNA repair and recombination processes in the absence of vimentin