The replicative life span of human fibroblasts is heterogeneous, with a fraction of cells senescing at every population doubling. To find out whether this heterogeneity is due to premature senescence, i.e. driven by a nontelomeric mechanism, fibroblasts with a senescent phenotype were isolated from growing cultures and clones by flow cytometry. These senescent cells had shorter telomeres than their cycling counterparts at all population doubling levels and both in mass cultures and in individual subclones, indicating heterogeneity in the rate of telomere shortening. Ectopic expression of telomerase stabilized telomere length in the majority of cells and rescued them from early senescence, suggesting a causal role of telomere shortening. Under standard cell culture conditions, there was a minor fraction of cells that showed a senescent phenotype and short telomeres despite active telomerase. This fraction increased under chronic mild oxidative stress, which is known to accelerate telomere shortening. It is possible that even high telomerase activity cannot fully compensate for telomere shortening in all cells. The data show that heterogeneity of the human fibroblast replicative life span can be caused by significant stochastic cell-to-cell variation in telomere shortening.Heterogeneity is a hallmark of aging. Even genetically identical organisms in a controlled homogeneous environment age at different rates and have vastly different life spans. This is also true for aging of human somatic cells in vitro (1). Replicative life spans differ widely between subclones (2), and there is an ever increasing number of senescent cells in dividing cultures (3). Senescence of human fibroblasts is telomere-driven, i.e. induced by the uncapping of telomeres (4). However, it is often assumed that only the life span of the longest surviving clone(s) is governed by telomere shortening, whereas termination of growth in the early senescing clones might be premature, i.e. caused by stress via telomere-independent mechanisms (5). In fact, a senescent phenotype can be generated in response to a variety of stresses that are telomere-independent and inhibit cell growth, for instance, in response to generalized DNA damage (6), oncogene activation (7), histone acetylation (8), or by induction of the p16 pathway through so far not well characterized mechanisms (9).An alternative to this idea of premature senescence is the proposal that stochastic telomere uncapping can cause heterogeneity in replicative life span (10). The probability of telomere uncapping is dependent on a number of factors, including the presence of active telomerase, the integrity of the singlestranded telomeric G-rich overhang and of the higher order structure of telomeres, the functionality of a number of telomere-binding proteins, and telomere length (for review see Ref. 4). Finally, there is evidence for a substantial interaction between stress and specifically mild oxidative stress and telomeres in cell replicative senescence. Stress accelerates telomere shortening ...

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