SUBCELLULAR LOCALIZATION OF AND PHOTOSENSITIZATION BY PROTOPORPHYRIN IX IN HUMAN KERATINOCYTES AND FIBROBLASTS CULTIVATED WITH 5-AMINOLEVULINIC ACID |
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Authors: | Jean-Michel Gaullier Marc Gèze René Santus Teresa Sa e Melo Jean-Claude Mazière Marc Bazin Patrice Morlière Louis Dubertret |
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Institution: | Muséum National d'Histoire Naturelle, Laboratoire de Physico-Chimie de l'Adaptation Biologique (INSERM U 312), 43 rue Cuvier, 75231 Paris Cedex, 05 France;Instituto Superior Tecnico, Complexo I, Quimica-Fisica Moleculare, 1096 Lisboa Codex, Portugal, France;Laboratoire de Dermatologie (INSERM U 312), Hôpital St. Louis, 1 Avenue Claude Vellefaux, 75010 Paris, France |
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Abstract: | Abstract— The subcellular localization of protoporphyrin (PP) has been studied by microspectrofluo-rometric techniques in NCTC 2544 keratinocytes incubated with 5-aminolevulinic acid (ALA) for times up to 42 h. Whereas the plasma membrane shows strong staining, fluorescent spots are observed within the cytoplasm especially in the perinuclear region. Although the topographic pattern of the PP distribution does not change much with the incubation time with ALA, the fluorescence spectra suggest that the PP microenvironments are quite different at short and long incubation times. Addition of 18 uJW desferoxamine almost doubles the ALA-induced PP concentration. Colocalization experiments with rhodamine 123, a mitochondrial probe, and lucifer yellow (LY) or neutral red (NR), two lysosome probes, demonstrate that at least some of these spots are of lysosomal origin. Study of the time evolution of the NR fluorescence under irradiation with visible light in the presence and absence of ALA demonstrates that lysosomes are damaged in cells that have synthesized PP. No PP fluorescence can be detected in mitochondria after incubation with ALA. However, photosensitization of mitochondria occurs under irradiation with visible light. Very little formation of lipofuscins by photosensitization with exogenous PP or ALA-induced PP is observed with the NCTC 2544 keratinocytes, as compared to normal human fibroblasts. |
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