Differentiation between senescence (M1) and crisis (M2) in human fibroblast cultures.
Normal human fibroblasts undergo only a limited number of divisions in culture and eventually enter a nonreplicative state designated senescence or mortality stage 1 (M1). Expression of certain viral oncogenes, such as the SV40 large T antigen (SV40 T-Ag), can elicit a significant extension of replicative life span, but these cultures eventually also cease dividing. This proliferative decline has been designated crisis or mortality stage 2 (M2). BrdU incorporation assays are commonly used to distinguish between senescence (<5% labeling index) and crisis (>30% labeling index). It has not been possible, however, to ascertain whether the high labeling index, indicative of ongoing DNA replication, was caused by the presence of T-Ag. We used gene targeting to knock out both copies of the p21(CIP1/WAF1) gene in presenescent human fibroblasts. p21 -/- cells displayed an extended life span but eventually entered a nonproliferative state. In their terminally nonproliferative state both p21 +/+ and p21 -/- cultures were positive for the senescence-associated beta-galactosidase (SA-beta-gal) activity; in contrast, the labeling index of p21 +/+ cells was low (<5%) whereas the labeling index of p21 -/- cells was high (>30%). The observation that p21 -/- and SV40 T-Ag-expressing cells behave identically with respect to life span extension as well as the high labeling index in the terminally nonproliferative state indicates that crisis is not a phenomenon induced solely by viral oncogenes, but a physiological state resulting from the bypass of normal senescence mechanisms. The widely used biomarker for senescence, SA-beta-gal, cannot distinguish between senescence and crisis. We propose that all SA-beta-gal-positive cultures should be further examined for their BrdU labeling index.
Wei
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Department of Molecular Biology, Cell Biology and Biochemistry, Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912, USA.
Sedivy
J M
JM
D000963:Antimetabolites / Q000494:pharmacology; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000235:genetics*; D003584:Cytological Techniques / Q000592:standards; D005347:Fibroblasts / Q000166:cytology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects / Q000502:physiology; D006801:Humans; D008967:Molecular Biology / Q000379:methods / Q000592:standards; D015203:Reproducibility of Results; D001616:beta-Galactosidase / Q000235:genetics
D000963:Antimetabolites; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D001616:beta-Galactosidase; D001973:Bromodeoxyuridine