Normal human cells have a limited replicative potential and inevitably reach replicative senescence in culture. Replicatively senescent cells show multiple molecular changes, some of which are related to the irreversible growth arrest in culture, whereas others resemble the changes occurring during the process of aging in vivo. Telomeres shorten as a result of cell replication and are thought to serve as a replicometer for senescence. Recent studies show that young cells can be induced to develop features of senescence prematurely by damaging agents, chromatin remodeling, and overexpression of ras or the E2F1 gene. Accelerated telomere shortening is thought to be a mechanism of premature senescence in some models. In this work, we test whether the acquisition of a senescent phenotype after mild-dose hydrogen peroxide (H2O2) exposure requires telomere shortening. Treating young HDFs with 150 muM H2O2 once 75 muM H2O2 twice in 2 weeks causes long-term,growth arrest, an enlarged morphology, activation of senescence-associated beta -galactosidase, and elevated expression of collagenase and clusterin mRNAs. No significant telomere shortening was observed with H2O2 at doses ranging from 50 to 200 muM. Weekly treatment with 75 muM H2O2 also failed to induce significant telomere shortening. Failure of telomere shortening correlated with an inability to elevate p16 protein or mRNA in H2O2-treated cells. In contrast, p21 mRNA was elevated over 40-fold and remained at this level for at least 2 weeks after a pulse treatment of H2O2. The role of cell cycle checkpoints centered on p21 in premature senescence induced by H2O2 is discussed here. (C) 2001 Academic Press.