A biomarker that identifies senescent human cells in culture and in aging skin in vivo.

Normal somatic cells invariably enter a state of irreversibly arrested growth and altered function after a finite number of divisions. This process, termed replicative senescence, is thought to be a tumor-suppressive mechanism and an underlying cause of aging. There is ample evidence that escape from senescence, or immortality, is important for malignant transformation. By contrast, the role of replicative senescence in organismic aging is controversial. Studies on cells cultured from donors of different ages, genetic backgrounds, or species suggest that senescence occurs in vivo and that organismic lifespan and cell replicative lifespan are under common genetic control. However, senescent cells cannot be distinguished from quiescent or terminally differentiated cells in tissues. Thus, evidence that senescent cells exist and accumulate with age in vivo is lacking. We show that several human cells express a beta-galactosidase, histochemically detectable at pH 6, upon senescence in culture. This marker was expressed by senescent, but not presenescent, fibroblasts and keratinocytes but was absent from quiescent fibroblasts and terminally differentiated keratinocytes. It was also absent from immortal cells but was induced by genetic manipulations that reversed immortality. In skin samples from human donors of different age, there was an age-dependent increase in this marker in dermal fibroblasts and epidermal keratinocytes. This marker provides in situ evidence that senescent cells may exist and accumulate with age in vivo.

Association of a transglutaminase-related antigen with intermediate filaments.

A mouse monoclonal antibody, G92.1.2, raised against guinea pig liver transglutaminase (TGase) recognizes an antigen present in primary mouse dermal fibroblasts. A filamentous pattern, bearing remarkable similarity to the vimentin intermediate filament (IF) network, is seen when these cells are fixed and processed for indirect immunofluorescence with the antibody. Double-label immunofluorescence reveals that the antigen reacting with the antibody colocalizes precisely with vimentin IF and that this colocalization is retained after the treatment of fibroblasts with colchicine, which induces a redistribution of the majority of IFs into perinuclear aggregates. These morphological observations are further supported by the finding that the protein reacting with G92.1.2 is retained in IF-enriched cytoskeletal preparations made by using nonionic detergent-containing high ionic strength solutions. Western blots of the IF fraction show that G92.1.2 recognizes a major band of approximately 280 kDa and does not cross react with vimentin. Furthermore, when the antibody is microinjected into live dermal fibroblasts, it causes a collapse of the vimentin IF network in the majority of injected cells. The results suggest that a form of TGase, or a TGase-related antigen, is closely associated with the vimentin IF network of primary cultures of mouse dermal fibroblasts.