PHOTOOXIDATIVE DISRUPTION OF LYSOSOMAL MEMBRANES CAUSES APOPTOSIS OF CULTURED HUMAN FIBROBLASTS

Acridine orange (AO) is a lysomotropic weak base, a metachromatic fluo rochrome, and a photosensitizer, as well. Living cells that are expose d for a short period of time to this compound at low concentration, an d under ordinary culture conditions, accumulate the drug within their acidic vacuolar compartment, giving rise to a mainly red, granular flu oresence upon excitation with blue light, When AO-loaded cells are irr adiated with intense blue light, AO soon starts to leak from late endo somes and lysosomes, partially shifting the fluorescence to a green, n uclear and diffuse cytosolic, one. This AO-relocalization is a consequ ence of photo-oxidation of the lysosomal membranes, which initially re sults in disruption of their proton-gradients and later, in leakage in to the cytosol of a host of hydrolytic enzymes-as was here demonstrate d by immunocytochemistry-which are capable of causing cellular damage. Most fibroblasts survived minor photo-oxidation, with a period of rep arative autophagocytosis. Severe photo-oxidation, which resulted in se vere lysosomal damage, caused cellular necrosis; whereas moderate stre ss, resulting in only partial lysosomal leakiness lead to apoptosis wi th TUNEL-positive nuclei and shrunken cytoplasm. The findings of tile present study show that photo-oxidative damage to the membranes that s urround the acidic vacuolar compartment, Is an event that results in r elease of proteolytic and DNA-fragmenting enzymes into the cytosol, wh ich may induce either necrosis, apoptosis, or reparable sublethal dama ge, 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 c ompartment into the cytosol. (C) 1997 Elsevier Science Inc.