Photo-oxidative disruption of lysosomal membranes causes apoptosis of cultured human fibroblasts.
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.
Brunk
U T
UT
Department of Pathology, Faculty of Health Sciences, Linkoping University, Sweden.
Dalen
H
H
Roberg
K
K
Hellquist
H B
HB
D000165:Acridine Orange; D017209:Apoptosis; D002402:Cathepsin D / Q000032:analysis / Q000378:metabolism; D002460:Cell Line; D005347:Fibroblasts / Q000648:ultrastructure*; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry* / Q000502:physiology*; D008027:Light; D008247:Lysosomes / Q000648:ultrastructure*; D008297:Male; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D010084:Oxidation-Reduction
D002402:Cathepsin D; D000165:Acridine Orange