Photo-Oxidative Disruption of Lysosomal Membranes Causes Apoptosis of Cultured Human Fibroblasts

Abstract

Acridine orange (AO) is a lysosomotropic weak base, a metachromatic fluorochrome, and a photosensitizer, as well. Living cells that are exposed for a short period of time to this compound at low concentration, and under ordinary culture conditions, accumulate the drug within their acidic vacuolar compartment, giving rise to a mainly red, granular fluoresence upon excitation with blue light. When AO-loaded cells are irradiated with intense blue light, AO soon starts to leak from late endosomes and lysosomes, partially shifting the fluorescence to a green, nuclear and diffuse cytosolic, one. This AO-relocalization is a consequence of photo-oxidation of the lysosomal membranes, which initially results in disruption of their proton-gradients and later, in leakage into the cytosol of a host of hydrolytic enzymes—as was here demonstrated by immunocytochemistry—which are capable of causing cellular damage. Most fibroblasts survived minor photo-oxidation, with a period of reparative autophagocytosis. Severe photo-oxidation, which resulted in severe lysosomal damage, caused cellular necrosis; whereas moderate stress, resulting in only partial lysosomal leakiness lead to apoptosis with TUNEL-positive nuclei and shrunken cytoplasm. The findings of the present study show that photo-oxidative damage to the membranes that surround the acidic vacuolar compartment, is an event that results in release of proteolytic and DNA-fragmenting enzymes into the cytosol, which may induce either necrosis, apoptosis, or reparable sublethal damage, depending on the magnitude of lysosomal rupture. Furthermore, the results strongly suggest that proteases and endonucleases of lysosomal origin may induce apoptosis if relocalized from the acidic vacuolar compartment into the cytosol.

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.