Role of the intermediate filament protein vimentin in delaying senescence and in the spontaneous immortalization of mouse embryo fibroblasts

Because knockout of the vimentin gene in mice did not produce an immediatel y obvious, overt, or lethal specific phenotype, the conjecture was made tha t the mutation affects some subtle cellular functions whose loss manifests itself only when the mutant animals are exposed to stress. In order to subs tantiate this idea in a tractable in vitro system, primary embryo fibroblas ts from wildtype (V+/+) and vimentin-knockout (V-/-) mice were compared wit h regard to their growth behavior under the pseudophysiologic conditions of conventional cell culture. Whereas in the course of serial transfer, the V +/+ fibroblasts progressively reduced their growth potential, passed throug h a growth minimum around passage 12 (crisis), and, as immortalized cells, resumed faster growth, the V-/- fibroblasts also cut down their growth rate but much earlier, and they either did not immortalize or did so at an almo st undetectable rate. Cells withdrawing from the cell cycle showed increase d concentrations of reactive oxygen species and signs of oxidative damage: enlarged and flattened morphology, large nuclear volume, reinforced stress fiber system as a result of increased contents of actin and associated prot eins, prominent extracellular matrix, and perinuclear masses of pathologica l forms of mitochondria with low membrane potential. The differences in the cell cycle behavior of the V+/+ and V-/- cells in conjunction with the mor phologic changes observed in mitotically arrested cells suggests a protecti ve function of vimentin against oxidative cell damage. Because vimentin exh ibits affinity for and forms crosslinkage products with recombinogenic nucl ear as well as mitochondrial DNA in intact cells, it is credible to postula te that vimentin plays a role in the recombinogenic repair of oxidative dam age inflicted on the nuclear and mitochondrial genome throughout the cells' replicative lifespan. Recombinational events mediated by vimentin also app ear to take place when the cells pass through the genetically unstable stat e of crisis to attain immortality. The residual immortalization potential o f V-/- fibroblasts might be attributable to their capacity to synthesize, i n place of vimentin, the tetrameric form of a lacZ fusion protein carrying, in addition to a nuclear localization signal, the N-terminal 59 amino acid s of vimentin and thus its DNA-binding site. On the basis of these results and considerations, a major biologic role or vimentin may be to protect ani mals during development and postnatal life against genetic damage and, beca use of its contribution to the plasticity of the genome, to allow them to r espond to environmental challenges.