Differentiation between Senescence (M1) and Crisis (M2) in Human Fibroblast Cultures

Abstract

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 p21CIP1/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 β-galactosidase (SA-β-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-β-gal, cannot distinguish between senescence and crisis. We propose that all SA-β-gal-positive cultures should be further examined for their BrdU labeling index.