Differentiation between senescence (M1) and crisis (M2) in human fibroblast cultures

Exp Cell Res. 1999 Dec 15;253(2):519-22. doi: 10.1006/excr.1999.4665.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antimetabolites / pharmacology
  • Bromodeoxyuridine / pharmacology
  • Cells, Cultured
  • Cellular Senescence / physiology*
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclins / genetics*
  • Cytological Techniques / standards
  • Fibroblasts / cytology
  • Gene Expression Regulation, Enzymologic / drug effects
  • Gene Expression Regulation, Enzymologic / physiology
  • Humans
  • Molecular Biology / methods
  • Molecular Biology / standards
  • Reproducibility of Results
  • beta-Galactosidase / genetics


  • Antimetabolites
  • CDKN1A protein, human
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclins
  • beta-Galactosidase
  • Bromodeoxyuridine