Original ContributionPhoto-Oxidative Disruption of Lysosomal Membranes Causes Apoptosis of Cultured Human Fibroblasts
Introduction
Cultured cells which are stained intravitally with the lysosomotropic weak base acridine orange (AO)1, 2, 3become photosensitized by this metachromatic fluorophore. Afterwards, they show a rapid relocalization of AO from lysosomes to the cytosol, later followed by cellular degeneration, upon exposure to blue light.4, 5Similarly, oxidative stress, e.g., in the form of exposure to hydrogen peroxide, results in the same type of relocalization, as a consequence of intralysosomal, iron-catalyzed, oxidative reactions.6, 7, 8, 9, 10, 11Irrespectively of the inducing mechanism, a moderate degree of lysosomal rupture seems to result in cell damage of the apoptotic type, with pyknotic nuclei and shrunken cytoplasm, while more extensive lysosomal damage results in cellular swelling and necrosis.6, 9Finally, the exposure of cells to only minor lysosomal damage results in reparative autophagocytotic activity and only limited cell death.[10]Initially, the AO-relocalization is due to a destruction of the proton gradient over the lysosomal membranes. Later, however, this consequence of peroxidative damage seems to be accompanied by general leakage of lysosomal contents, including hydrolytic enzymes, such as cathepsin-D, and previously endocytosed large molecules, such as lucifer yellow.8, 9
It is generally believed that apoptosis involves the activity of a host of enzymes, including endonucleases. The latter enzymes initially degrade cellular DNA into nucleosomal, 180 base-pair multiples, which produce the typical “laddering pattern” on agarose-gels after DNA electrophoresis.12, 13, 14, 15
The aim of the present study was to analyze whether or not the cellular degeneration that occurs after a selective, photo-oxidative damage to lysosomal membranes, produced by exposing living cells in culture with acridine orange-loaded lysosomes to blue light, is of true apoptotic nature, and if such degeneration follows upon the relocalization of lysosomal hydrolases.
Section snippets
Chemicals
Glutamine, pencillin-G and streptomycin were obtained from Flow (Rickmansworth, UK), EMEM and dialysed FCS were from GIBCO (Paisley, UK), while uranyl acetate, lead nitrate, acridine orange and Giemsa solution were purchased from Merck (Darmstadt, Germany), and paraformaldehyde and Epon-812 from Fluka AG (Buchs, Switzerland). Glutaraldehyde was from Bio-Rad (Cambridge, MA, USA), osmium tetroxide from Johnson Matthey (Roystone, UK) and goat anti-rabbit IgG-Texas Red conjugate from Vector
Results
The control- and AO-loaded cells were irradiated with blue light in PBS for 0, 2, 4, or 8 min and then returned to ordinary culture conditions. Subsequently, they were examined over the next 12 h with respect to: (1) AO-distribution by microfluorometry and confocal laser scanning microscopy; (2) altered morphology, by inverted light microscopy and Giemsa staining; and (3) viability, by the trypan blue dye-exclusion method. After appropriate aldehyde fixation, cells, during the initial 4 h after
Discussion
When AO-loaded cells were irradiated with blue light, AO leaked from the lysosomes in a dose dependent manner, presumably due to photo-oxidation with peroxidation of the membranes surrounding the acidic vacuolar apparatus that causes disruption of their proton-gradients.1, 2The AO-relocalization, which was manifested by an increased green, cytosolic- and nuclear fluorescence, was also accompanied by leakage of cathepsin-D from the acidic vacuolar apparatus into the cytosol, as shown by light
Acknowledgements
This work was supported by the Swedish Medical Research Council (grant 4481).
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