Spontaneously immortalized mouse embryo fibroblasts: growth behavior of wild-type and vimentin-deficient cells in relation to mitochondrial structure and activity.
Dependent on the presence or absence of vimentin, primary mouse embryo fibroblasts exhibit different growth characteristics in vitro. While most Vim(+/+) fibroblasts stop dividing and die via apoptosis, a substantial fraction of cells immortalize and proliferate almost normally. Vim(-/-) fibroblasts cease to divide earlier, immortalize in vanishingly small numbers and thereafter proliferate extremely slowly. Early after immortalization, Vim(+/+) (imm) fibroblasts appear structurally almost normal, whereas Vim(-/-) (imm) fibroblasts equal postmitotic "crisis" cells, which are characterized by increased cell size, altered cell ultrastructure, nuclear enlargement, genome destabilization, structural degeneration of mitochondria, and diminution of mitochondrial respiratory activity. The differences between immortalized Vim(+/+) (imm) and Vim(-/-) (imm) fibroblasts persist during early cell cloning but disappear during serial subcultivation. At high cell passage, cloned, immortalized vim(-) fibroblasts grow nearly as fast as their cloned vim(+) counterparts, and also resemble them in size, ultrastructure, nuclear volume, and mitochondrial complement; they very likely employ redundancy to cope with the loss of vimentin function when adjusting structure and behavior to that of immortalized vim(+) fibroblasts. Reduction in nuclear size occurs via release of large amounts of filamentous chromatin into extracellular space; because it is complexed with extracellular matrix proteins, it tends to form clusters and to tightly stick to the surface of other cells, thus providing a potential for horizontal gene transfer. On the other hand, cloned vim(+) and vim(-) fibroblasts are equal in showing contact inhibition at young age and becoming anchorage-independent during serial subcultivation, as indicated by the formation of multilayered and -faceted cell sheets and huge spheroids on top of or in soft agar. With this, immortalized vim(-) fibroblasts reduce their adhesiveness to the substratum which, in their precrisis state and early after cloning, is much higher than that of their vim(+) counterparts. In addition, the coupling between the mitochondrial respiratory chain and oxidative phosphorylation is stronger in vim(+) than vim(-) fibroblasts. It appears from these data that after explantation of fibroblasts from the mouse embryo the primary cause of cell and mitochondrial degeneration, including genomic instability, is the mitochondrial production of reactive oxygen species in a vicious circle, and that vimentin provides partial protection from oxidative damage. As a matrix protein with specific in vitro and in vivo affinities for nuclear and mitochondrial, recombinogenic DNA, it may exert this effect preferentially at the genome level via its influence on recombination and repair processes, and in this way also assist the cells in immortalizing. Additional protection of mitochondria by vimentin may occur at the level of mitochondrial fatty acid metabolism.
Tolstonog
Genrich V
GV
Max-Planck-Institut für Zellbiologie, Rosenhof, Ladenburg, Germany. Genrich.Tolstonog@hpi.uni-hamburg.de
Belichenko-Weitzmann
Irina V
IV
Lu
Jian-Ping
JP
Hartig
Roland
R
Shoeman
Robert L
RL
Traub
Ulrike
U
Traub
Peter
P
D000818:Animals; D002448:Cell Adhesion / Q000502:physiology*; D002460:Cell Line; D049109:Cell Proliferation; D019069:Cell Respiration / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D003260:Contact Inhibition / Q000502:physiology; D004622:Embryo, Mammalian / Q000166:cytology; D005347:Fibroblasts / Q000166:cytology* / Q000502:physiology / Q000648:ultrastructure; D051379:Mice; D018345:Mice, Knockout; D046529:Microscopy, Electron, Transmission; D008928:Mitochondria / Q000502:physiology* / Q000648:ultrastructure; D017382:Reactive Oxygen Species / Q000378:metabolism; D018874:Spheroids, Cellular / Q000166:cytology; D014746:Vimentin / Q000235:genetics / Q000502:physiology*
D017382:Reactive Oxygen Species; D014746:Vimentin