Commitment to cell death measured by loss of clonogenicity is separable from the appearance of apoptotic markers.
Kinetic analysis of dexamethasone-induced apoptosis in the human lymphoblastoid cell line CCRF CEM C7A has revealed a point when cells, morphologically indistinguishable from untreated cells, have irreversibly engaged a program leading to death, measured by a loss of clonogenicity. Since all cells that fail to clone eventually died through apoptosis, measurements of clonogenicity in this system provide an accurate measure of commitment to apoptotic death. Inhibition of caspases by peptide inhibitors blocked proteolysis of endogenous substrates and reduced nuclear condensation yet did not alter either dexamethasone-induced changes in clonogenicity or mitochondrial membrane potential. In contrast to the results with caspase inhibitors, expression of BCL-2 in CCRF CEM C7A cells proved sufficient to block all changes associated with apoptosis, including loss of both clonogenicity and changes in mitochondrial membrane potential. These results demonstrate that commitment to cell death can precede the key biochemical or morphological features of apoptosis by several hours and indicate that separate regulators govern cellular commitment to clonogenic death and the subsequent execution phase characterised as apoptosis.
Brunet
C L
CL
School of Biological Sciences, University of Manchester, Stopford Building G.38, Oxford Road, Manchester M13 9PT, UK.
Gunby
R H
RH
Benson
R S
RS
Hickman
J A
JA
Watson
A J
AJ
Brady
G
G
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D015415:Biomarkers; D020169:Caspases / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D002999:Clone Cells; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003593:Cytoplasm / Q000737:chemistry / Q000201:enzymology; D003907:Dexamethasone / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008214:Lymphocytes / Q000166:cytology* / Q000201:enzymology / Q000648:ultrastructure; D008854:Microscopy, Electron; D008928:Mitochondria / Q000737:chemistry / Q000201:enzymology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000201:enzymology / Q000648:ultrastructure
D000590:Amino Acid Chloromethyl Ketones; D000893:Anti-Inflammatory Agents; D015415:Biomarkers; D002843:Chromatin; D015853:Cysteine Proteinase Inhibitors; D019253:Proto-Oncogene Proteins c-bcl-2; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D003907:Dexamethasone; D020169:Caspases