JC-1, a cationic fluorescent dye when added to living cells, is known to be localized exclusively in mitochondria, particularly in good physiological conditions characterized by sufficient mitochondrial membrane potential (ΔΨ). The accumulation of JC-1 in these organelles leads to the formation J-aggregates (with a specific red fluorescence emission maximum at 590 nm), which is in addition to the typical green fluorescence of J-monomers (emission maximum of ∼529 nm). The lack of mitochondrial ΔΨ leads to the depression of JC-1 mitochondrial accumulation and a decrease in J-aggregate formation. Therefore, the ratio between the red and green fluorescence of cells loaded with JC-1 is often used for the detection of the mitochondrial membrane potential. However, JC-1 represents a suitable substrate of the multidrug transporter P-glycoprotein (P-gp). Therefore, the depression of the JC-1 content in intracellular space and particularly in the mitochondria to a level that is inefficient for J-aggregate formation could be expected in P-gp-positive cells. In the current paper, we proved this behavior on parental P-gp-negative L1210 (S) cells and their P-gp-positive variants obtained by either selection with vincristine (R) or transfection with the human gene encoding P-gp (T). P-glycoprotein inhibitors cyclosporine A and verapamil fail to restore JC-1 loading of the R and T cells to an extent similar to that observed in S cells. In contrast, the noncompetitive high affinity P-gp inhibitor tariquidar fully restored JC-1 accumulation and the presence of the typical red fluorescence of J-aggregates. In the presence of tariquidar, measurement of the JC-1 fluorescence revealed similar levels of mitochondrial membrane potential in P-gp-negative (S) and P-gp-positive cells (R and T).

During the selection process in the thymus, most thymocytes are eliminated by apoptosis through signaling via TCR or glucocorticoids. The involvement of ceramide (Cer) and sphingosine (SP), important apoptotic mediators, remains poorly defined in glucocorticoid-induced apoptosis. We report that, in mouse thymocytes, apoptosis triggered by 10⁻⁶ M dexamethasone (DX) was preceded by a caspase-dependent Cer and SP generation, together with activation of acidic and neutral ceramidases. Apoptosis was drastically reduced by blocking either sphingolipid production (by acid sphingomyelinase inhibitor) or SP production (by ceramidase inhibitors), but not by inhibition of de novo Cer synthesis. Thus, SP generated through acid sphingomyelinase and ceramidase activity would contribute to the apoptotic effect of DX. Consistent with this hypothesis, SP addition or inhibition of SP kinase induced thymocyte apoptosis. DX induced a proteasome-dependent loss of mitochondrial membrane potential (Δψ_m) and caspase-8, -3, and -9 processing. Apoptosis was abolished by inhibition of Δψ_m loss or caspase-8 or -3, but not caspase-9. Δψ_m loss was independent of SP production and caspase-8, -3, and -9 activation. However, inhibition of SP production reduced caspase-8 and -3, but not caspase-9 processing. Proteasome inhibition impaired activation of the three caspases, whereas inhibition of Δψ_m loss solely blocked caspase-9 activation. These data indicate that DX-induced apoptosis is mediated in part by SP, which contributes, together with proteasome activity, to caspase-8-3 processing independently of mitochondria, and in part by the proteasome/mitochondria pathway, although independently of caspase-9 activation.