Apoptosis or senescence-like growth arrest: influence of cell-cycle position, p53, p21 and bax in H2O2 response of normal human fibroblasts

Biochem J. 2000 Apr 15;347(Pt 2):543-51. doi: 10.1042/0264-6021:3470543.

Abstract

Early-passage human diploid fibroblasts (HDFs) undergo senescence-like growth arrest in response to sublethal concentrations of H(2)O(2) [Chen and Ames (1994) Proc. Natl. Acad. Sci. USA. 95, 4130-4134]. We determine here whether H(2)O(2) can cause apoptosis in HDFs and the molecular changes that differ between apoptosis and senescence-like growth arrest. When exponentially growing early-passage IMR-90 cells were treated for 2 h with 50-200 microM (or 0.25-1 pmol/cell) H(2)O(2), a fraction of cells detached at 16-32 h after the treatment. The cells remaining attached were growth-arrested and developed features of senescence in 1 week. The detached cells showed caspase-3 activation and typical morphological changes associated with apoptosis. Caspase-3 activation was H(2)O(2) dose-dependent and preceded nuclear condensation or plasma membrane leakage. Apoptotic cells were mainly distributed in the S-phase of the cell cycle, while growth-arrested cells exhibited predominantly G1- and G2/M-phase distributions. H(2)O(2) pretreatment induced G1 arrest and prohibited induction of apoptosis by a subsequent H(2)O(2) challenge. The p53 protein showed an average 6.1-fold elevation in apoptotic cells and a 3.5-fold elevation in growth-arrested cells. Reduction of p53 levels with human papillomavirus E6 protein prohibited the activation of caspase-3 and decreased the proportion of apoptotic cells. Growth-arrested cells had elevated p21, while p21 was absent in apoptotic cells. Bcl-2 was elevated in both growth-arrested and apoptotic cells. Finally, although the overall level of bax did not change in growth-arrested or apoptotic cells, the solubility of bax protein increased in apoptotic cells. Our data suggest that in contrast with growth-arrested cells, apoptotic cells show an S-phase cell cycle distribution, a higher degree of p53 elevation, an absence of p21 protein and increased solubility of bax protein.

Publication types

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

MeSH terms

  • Apoptosis / drug effects*
  • Blotting, Western
  • Caspase 3
  • Caspases / metabolism
  • Cell Cycle* / drug effects
  • Cell Membrane / metabolism
  • Cell Nucleus / drug effects
  • Cell Nucleus / metabolism
  • Cellular Senescence / drug effects*
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclins / metabolism*
  • DNA / biosynthesis
  • Dose-Response Relationship, Drug
  • Enzyme Activation / drug effects
  • Fibroblasts / cytology
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism
  • Flow Cytometry
  • G1 Phase / drug effects
  • Humans
  • Hydrogen Peroxide / metabolism
  • Hydrogen Peroxide / pharmacology*
  • Oxidative Stress / drug effects
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-bcl-2 / metabolism
  • Trypan Blue / metabolism
  • Tumor Suppressor Protein p53 / metabolism*
  • bcl-2-Associated X Protein

Substances

  • BAX protein, human
  • CDKN1A protein, human
  • Cyclin-Dependent Kinase Inhibitor p21
  • Cyclins
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • Tumor Suppressor Protein p53
  • bcl-2-Associated X Protein
  • DNA
  • Hydrogen Peroxide
  • CASP3 protein, human
  • Caspase 3
  • Caspases
  • Trypan Blue