Changes in intramitochondrial cardiolipin distribution in apoptosis‐resistant HCW‐2 cells, derived from the human promyelocytic leukemia HL‐60

Abstract

Using a cytofluorimetric approach, we studied intramitochondrial cardiolipin (CL) distribution in HCW-2 cells, an apoptosis-resistant clone of human HL-60 cells. In HL-60, about 50% of total CL is distributed in the outer leaflet of mitochondrial inner membrane, while in HCW-2 a significantly higher amount of CL (about 65%) is in that site. In basal conditions, HSW-2 cells also show a reduced mitochondrial membrane potential even if they are able to proliferate as the parental line. Taking into account the complex functions that CL plays in the regulation of mitochondrial activity, it is likely that HCW-2 could produce ATP utilizing more glycolytic pathways rather than mitochondrial respiratory chain.

Mitochondrial and nonmitochondrial reduction of MTT: Interaction of MTT with TMRE, JC‐1, and NAO mitochondrial fluorescent probes

“…Moreover, this hypothesis is in agreement with the observation that MTT decreased the binding of NAO to mitochondrial membranes. This dye is known to bind to cardiolipin in the mitochondrial membrane in a potential-independent fashion (22)(23)(24). Because the concentration of NAO used in our experiments was low (0.01 mM), one may assume that most of the probe resided at the outer surface of the membrane (23,35) and was displaced by MTT.…”

“…Mitochondria in viable HepG2 human hepatoma cells were visualized using TMRE and JC-1, whose accumulation is dependent on the mitochondrial transmembrane electric potential (17)(18)(19)(20)(21). We also used NAO, which, due to its affinity to mitochondrial lipid cardiolipin, labels these organelles in a potential-independent fashion (22)(23)(24).…”

Stress-induced activation of the p53 tumor suppressor in leukemia cells and normal lymphocytes requires mitochondrial activity and reactive oxygen species

“…NAO binds specifically to mitochondrial cardiolipin, and its binding affinity and fluorescence properties depend on the oxidation state of cardiolipin. [49][50][51] NAO binds with high affinity to a nonoxidized cardiolipin in a 2:1 ratio, and its fluorescence shifts toward longer wavelength from 525 nm (green) to about 640 nm (red) as a result of NAO dimerization on the site of the mitochondrial membrane. 49,50 In the case of oxidized cardiolipin, NAO has been reported to bind cardiolipin with a decreased affinity reflected by lower levels of red fluorescence.…”

“…[49][50][51] NAO binds with high affinity to a nonoxidized cardiolipin in a 2:1 ratio, and its fluorescence shifts toward longer wavelength from 525 nm (green) to about 640 nm (red) as a result of NAO dimerization on the site of the mitochondrial membrane. 49,50 In the case of oxidized cardiolipin, NAO has been reported to bind cardiolipin with a decreased affinity reflected by lower levels of red fluorescence. 49 Therefore, oxidative stress localized to mitochondria can be assessed by red fluorescence measurement of NAO.…”

“…49,50 In the case of oxidized cardiolipin, NAO has been reported to bind cardiolipin with a decreased affinity reflected by lower levels of red fluorescence. 49 Therefore, oxidative stress localized to mitochondria can be assessed by red fluorescence measurement of NAO. 51 Using this approach we observed a 26% increase of the NAO red fluorescence in MOLT-3 cells in the presence of oligomycin compared with cells cultured in the medium (P Ͻ .001, t test; Figure 6A).…”