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Stress-induced premature senescence (SIPS) is induced 3 days after exposure of human diploid fibroblasts to subcytotoxic oxidative stress with H 2 O 2 , with appearance of several biomarkers of replicative senescence. In this work, we show that transforming growth factor-␤1 (TGF-␤1) regulates the induction of several of these biomarkers in SIPS: cellular morphology, senescence-associated ␤-galactosidase activity, increase in the steady-state level of fibronectin, apolipoprotein J, osteonectin, and SM22 mRNA. Indeed, the neutralization of TGF-␤1 or its receptor (TGF-␤ RII) using specific antibodies decreases sharply the percentage of cells positive for the senescent-associated ␤-galactosidase activity and displaying a senescent morphology. In the presence of each of these antibodies, the steady-state level of fibronectin, osteonectin, apolipoprotein J, and SM22 mRNA is no more increased at 72 h after stress. Results obtained on fibroblasts retrovirally transfected with the human papillomavirus E7 cDNA suggest that retinoblastoma protein (Rb) regulates the expression of TGF-␤1 in stressful conditions, leading to SIPS and overexpression of these four genes.Normal human diploid fibroblasts (HDFs) 1 exposed to various types of noncytotoxic oxidative stress display a senescentlike phenotype coined "stress-induced premature senescence" or SIPS (1, 2). Such stressful conditions include exposure to hydrogen peroxide (3, 4), tert-butylhydroperoxide (t-BHP) (5), hyperoxia (6), UV light (7), and radioactivity (8). Many biomarkers of replicative senescence appear in SIPS: typical cell morphology (5), irreversible growth arrest, lack of response to mitogenic stimuli (4), sharp decrease of the DNA synthesis, and an increase in cells positive for the senescent-associated ␤-galactosidase activity (SA ␤-gal) (9). A long term overexpression of the cyclin-dependent kinase inhibitor p21waf-1 was observed in SIPS induced by H 2 O 2 (10) or t-BHP (9). p21waf-1 inhibits the cyclin D/cyclin-dependent kinase 4 and 6 complexes, leading to hypophosphorylation of the retinoblastoma protein (Rb). A long term hypophosphorylation of Rb over several weeks was indeed observed in SIPS induced by H 2 O 2 or t-BHP, explaining the block of the cell cycle, through Rb-mediated inhibition of the E 2 F transcription factor (9, 10). Last, several genes overexpressed in senescent HDFs, such as fibronectin, osteonectin, SM22, and apolipoprotein J (clusterin), displayed a similar increase in mRNA level in SIPS induced by t-BHP or H 2 O 2 (9).In different experimental models, an overexpression of either SM22 (11), apolipoprotein J (12), osteonectin (13), or fibronectin (14) is induced by extracellular addition of transforming growth factor-␤1 (TGF-␤1). Moreover, incubation of HDFs with TGF-␤1 triggers the appearance of a senescent-like morphology (15, 16) and growth arrest (17).Two main arguments favor the hypothesis that oxidative stress-induced premature senescence could be triggered by a pRb-mediated TGF-␤1 overexpression. First, it has been shown that AT...

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No consensus exists so far on the definition of cellular senescence. The narrowest definition of senescence is irreversible growth arrest triggered by telomere shortening counting cell generations (definition 1). Other authors gave an enlarged functional definition encompassing any kind of irreversible arrest of proliferative cell types induced by damaging agents or cell cycle deregulations after overexpression of proto-oncogenes (definition 2). As stress increases, the proportion of cells in “stress-induced premature senescence-like phenotype” according to definition 1 or “stress-induced premature senescence,” according to definition 2, should increase when a culture reaches growth arrest, and the proportion of cells that reached telomere-dependent replicative senescence due to the end-replication problem should decrease. Stress-induced premature senescence-like phenotype and telomere-dependent replicatively senescent cells share basic similarities such as irreversible growth arrest and resistance to apoptosis, which may appear through different pathways. Irreversible growth arrest after exposure to oxidative stress and generation of DNA damage could be as efficient in avoiding immortalisation as “telomere-dependent” replicative senescence. Probabilities are higher that the senescent cells (according to definition 2) appearing in vivo are in stress-induced premature senescence rather than in telomere-dependent replicative senescence. Examples are given suggesting these cells affect in vivo tissue (patho)physiology and aging.

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WI-38 human diploid fibroblasts underwent accelerated telomere shortening (490 bp/stress) and growth arrest after exposure to four subcytotoxic 100 W WM tert-butylhydroperoxide (t-BHP) stresses, with a stress at every two population doublings (PD). After subcytotoxic 160 W WM H 2 O 2 stress or five repeated 30 W WM t-BHP stresses along the same PD, respectively a 322 þ 55 and 380 þ 129 bp telomere shortening was observed only during the first PD after stress. The percentage of cells resuming proliferation after stress suggests this telomere shortening is due to the number of cell divisions accomplished to reach confluence during the first PD after stress. ß