Apoptosis or senescence-like growth arrest: influence of cell-cycle position, p53, p21 and bax in H2O2 response of normal human fibroblasts.
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.
Chen
Q M
QM
Department of Pharmacology, Skaggs Pharmaceutical Sciences Building, Room 130, University of Arizona, 1703 E. Mabel Street, Tucson, AZ 85721, USA. chen@pharmacy.arizona.edu
Liu
J
J
Merrett
J B
JB
D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000187:drug effects / Q000378:metabolism; D016922:Cellular Senescence / Q000187:drug effects*; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005434:Flow Cytometry; D016193:G1 Phase / Q000187:drug effects; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology*; D018384:Oxidative Stress / Q000187:drug effects; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D014343:Trypan Blue / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*; D051028:bcl-2-Associated X Protein
C494826:BAX protein, human; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; D004247:DNA; D006861:Hydrogen Peroxide; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D014343:Trypan Blue