Cytofluorometric detection of mitochondrial alterations in early CD95/Fas/APO-1-triggered apoptosis of Jurkat T lymphoma cells. Comparison of seven mitochondrion-specific fluorochromes.
It is commonly accepted that mitochondria undergo major changes early during the apoptotic process and that these alterations are critical for the death/life decision. Here we report that Jurkat T cell leukemia cells exhibit a perturbed incorporation of potential-sensitive fluorochromes. After 6 h of CD95/Fas/APO-1 crosslinking, a significant fraction of still normal-sized Jurkat cells exhibit a decreased incorporation of three different cationic lipophilic dyes commonly used for the quantitation of the mitochondrial transmembrane potential (deltapsi(m)): DiOC6(3), chloromethyl-X-rosamine, and tetramethylrhodaminemethylester. In contrast, upon induction of apoptosis, cells tend to exhibit an increase in the fluorescence obtained with rhodamine 123. The increased rhodamine 123 fluorescence into cells undergoing apoptosis is not affected by labeling in the presence of the protonophore m-chlorophenylhydrazone and thus cannot be attributed to a change in the deltapsi(m). Six hours after CD95 ligation no changes are found among normal-sized cells in the incorporation of mitotracker green and nonylacridine orange, which both measure mitochondrial mass. However, a fraction of cells exhibit an increased staining with the Apo2.7 antibody which detects a mitochondrial antigen generated during apoptosis. These findings underline the importance of using adequate fluorochromes for the quantitation of mitochondrial changes occurring during early apoptosis. Moreover, they cast doubts on those studies that, using rhodamine 123, hypothesized that apoptosis would be associated with a stable or increased deltapsi(m).
Métivier
D
D
Centre National de la Recherche Scientifique, Unité Propre de Recherche 420, Villejuif, France.
Dallaporta
B
B
Zamzami
N
N
Larochette
N
N
Susin
S A
SA
Marzo
I
I
Kroemer
G
G
D000447:Aldehydes; D000609:Aminoacridines; D000911:Antibodies, Monoclonal / Q000378:metabolism; D017209:Apoptosis; D002232:Carbocyanines; D005434:Flow Cytometry / Q000379:methods*; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D019169:Jurkat Cells; D008564:Membrane Potentials; D008928:Mitochondria / Q000502:physiology*; D009930:Organic Chemicals; D020112:Rhodamine 123; D012235:Rhodamines; D019014:fas Receptor / Q000494:pharmacology / Q000502:physiology*
D000447:Aldehydes; D000609:Aminoacridines; D000911:Antibodies, Monoclonal; D002232:Carbocyanines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D012235:Rhodamines; D019014:fas Receptor; C111472:mitotracker green FM; C107472:red dye CMXRos; D020112:Rhodamine 123; C007392:3,3'-dihexyl-2,2'-oxacarbocyanine; C005358:tetramethylrhodamine; C041691:10-N-nonylacridinium orange