CYTOFLUOROMETRIC DETECTION OF MITOCHONDRIAL ALTERATIONS IN EARLY CD95FAS/APO-1-TRIGGERED APOPTOSIS OF JURKAT T-LYMPHOMA-CELLS - COMPARISONOF 7 MITOCHONDRION-SPECIFIC FLUOROCHROMES/

It is commonly accepted that mitochondria undergo major changes early during the apoptotic process and that these alterations are critical f or the death/life decision. Here we report that Jurkat T cell leukemia cells exhibit a perturbed incorporation of potential-sensitive fluoro chromes. After 6 h of CD95/Fas/APO-1 crosslinking, a significant fract ion of still normal-sized Jurkat cells exhibit a decreased incorporati on of three different cationic lipophilic dyes commonly used for the q uantitation of the mitochondrial transmembrane potential (Delta psi(m) ): DiOC(6)(3), chloromethyl-X-rosamine, and tetramethylrhodaminemethyl ester. In contrast, upon induction of apoptosis, cells tend to exhibit an increase in the fluorescence obtained with rhodamine 123. The incr eased rhodamine 123 fluorescence into cells undergoing apoptosis is no t affected by labeling in the presence of the protonophore m-chlorophe nylhydrazone and thus cannot be attributed to a change in the Delta ps i(m). Six hours after CD95 ligation no changes are found among normal- sized cells in the incorporation of mitotracker green(TM) and nonylacr idine orange, which both measure mitochondrial mass. However, a fracti on of cells exhibit an increased staining with the Apo2.7 antibody whi ch detects a mitochondrial antigen generated during apoptosis. These f indings underline the importance of using adequate fluorochromes for t he quantitation of mitochondrial changes occurring during early apopto sis. Moreover, they cast doubts on those studies that, using rhodamine 123, hypothesized that apoptosis would be associated with a stable or increased Delta psi(m). (C) 1998 Elsevier Science B.V. All rights res erved.