Comparison of methods used to study cell death in an adherent tumoral cell line with moderate clonogenic radiosensitivity.
Our objective was to compare different methods for studying programmed cell death in adherent H460 non-small lung cancer cells of moderate clonogenic radiosensitivity. The major effect of gamma-radiation was found to be the release of cells from the substratum. The different methods gave complementary and unexpected information: a) with the TUNEL method, a few non-apoptotic cells were found in the culture medium; b) with the flow cytometry after propidium iodide labeling, some hypodiploid cells which remained attached to the substratum were apoptotic, as demonstrated by the effect of a caspase inhibitor; c) with the annexin V labeling, the detached cells were demonstrated either necrotic or very late apoptotic; d) the mitochondria transmembrane potential (deltapsim), measurements demonstrated that the mitochondria were implicated in cell death induced by gamma-radiation. These data illustrate the need to use several complementary methods in the study of apoptosis in adherent cells exposed to gamma-radiation.
Broquet
P
P
INSERM U189, Faculté de Médecine Lyon-Sud, Oullins, France. broquet@lyon-sud.univ-lyon1.fr
Rodriguez-Lafrasse
C
C
Alphonse
G
G
Aloy
M T
MT
Louisot
P
P
Rousson
R
R
D017304:Annexin A5 / Q000494:pharmacology; D002448:Cell Adhesion; D002453:Cell Cycle / Q000528:radiation effects; D016923:Cell Death; D004171:Diploidy; D004307:Dose-Response Relationship, Radiation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005434:Flow Cytometry; D005720:Gamma Rays; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008564:Membrane Potentials; D008928:Mitochondria / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D011419:Propidium / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D017304:Annexin A5; D004791:Enzyme Inhibitors; D010718:Phosphatidylserines; D011419:Propidium