Normal cells can permanently lose the ability to proliferate when challenged by potentially oncogenic stress, a process termed cellular senescence. Senescence-associated beta-galactosidase (SA-betagal) activity, detectable at pH 6.0, permits the identification of senescent cells in culture and mammalian tissues. Here we describe first a cytochemical protocol suitable for the histochemical detection of individual senescent cells both in culture and tissue biopsies. The second method is based on the alkalinization of lysosomes, followed by the use of 5-dodecanoylaminofluorescein di-beta-D-galactopyranoside (C12FDG), a fluorogenic substrate for betagal activity. The cytochemical method takes about 30 min to execute, and several hours to a day to develop and score. The fluorescence methods take between 4 and 8 h to execute and can be scored in a single day. The cytochemical method is applicable to tissue sections and requires simple reagents and equipment. The fluorescence-based methods have the advantages of being more quantitative and sensitive.

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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...