Caspase-independent cytochrome c release is a sensitive measure of low-level apoptosis in cell culture models.
Age-associated loss of tissue function and several chronic diseases may derive in part from the cumulative effects of subtle changes in the level of apoptotic cell death. Because apoptosis is rapid and undetectable once complete, small changes in its incidence are difficult to detect, even in well-controlled cell cultures. We describe a new apoptosis assay that provides greater sensitivity than conventional assays because it measures the accumulation of apoptotic cells. Human and mouse fibroblasts and human mammary epithelial cells that initiated apoptosis were preserved for 3 days by inhibiting caspase activity using the chemical inhibitor Q-VD-OPH (QVD). Cells suspended in the process of apoptosis were scored by immunostaining for cytochrome c, which redistributed from mitochondria in healthy cells to the cytoplasm in dying cells. This caspase-independent cytochrome c release (CICR) assay was more sensitive than several conventional assays when apoptosis was induced by actinomycin D, and detected cumulative background levels of apoptosis over a 3-day interval. Using this assay, we show that normal fibroblasts undergo very little apoptosis upon X-irradiation, indicating dominance of the senescence response in this cell type. Further, apoptosis increased subtly but measurably when human mammary epithelial and skin fibroblast cells entered crisis, indicating that cell death during crisis is largely non-apoptotic.
Goldstein
Joshua C
JC
Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
Rodier
Francis
F
Garbe
James C
JC
Stampfer
Martha R
MR
Campisi
Judith
J
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D061945:Caspase Inhibitors; D020169:Caspases / Q000378:metabolism*; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism*; D004249:DNA Damage; D053938:DNA Fragmentation; D005347:Fibroblasts / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D006801:Humans; D042361:Mammary Glands, Human / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D051379:Mice
D061945:Caspase Inhibitors; D045304:Cytochromes c; D020169:Caspases