Acridine orange (AO), a weakly basic
fluorescent dye, is permeable to plasma and vesicle membranes and preferentially remains in intracellular acidic regions. Using fluorescence microscopy, we observed dynamic changes in AO-loaded cultured
malignant melanoma cells during illumination with blue light. Immediately after the start of the illumination, the successive disruption of vesicles was observed as a flash of fluorescence, and shortly after that,
blebs were formed on the plasma membrane. These cells died within 5 min. Vesicle disruption was completely inhibited when cells were treated with the
vacuolar H(+)-ATPase inhibitor
bafilomycin A1 followed by loading with AO, but not when
bafilomycin A1 was treated after AO loading. Thus, the filling of AO in the vesicle, which is driven by
vacuolar H(+)-ATPase, is initially required for vesicle disruption. In contrast,
bafilomycin A1 did not prevent plasma membrane blebbing, indicating that the
blebs are formed independently of the vesicle disruption. Acute cell death was inhibited by treatment with
bafilomycin A1 before but not after AO loading. Thus, AO- and blue light-induced acute cell death is associated with vesicle disruption rather than
bleb formation. Both the vesicle disruption and the formation of plasma membrane
blebs were inhibited by removal of
oxygen from the cell environment and by
singlet oxygen scavengers,
sodium azide,
ascorbic acid, and
L-histidine, but not inhibited by the
hydroxyl radical scavenger dimethyl
thiourea. Acute cell death was also prevented by
singlet oxygen scavengers but not by dimethyl
thiourea. Thus, these phenomena are likely caused at least in part by the generation of
singlet oxygen. The photosensitive features of plasma and vesicle membranes observed in the present study may be based on the use of the photodynamic effect, such as
cancer therapy.