Different apoptotic pathways are induced from various intracellular sites by tetraphenylporphyrins and light

Abstract

The induction of apoptosis from different intracellular sites was studied by exposing V79 Chinese hamster fibroblasts to photodynamic therapy (PDT) with various porphyrins and light. The effects of two lipophilic, intracellular membrane-localized porphyrins, tetra(3-hydroxyphenyl)porphyrin (3THPP) and Photofrin, were compared with that of two sulphonated meso-tetraphenylporphines (TPPS(2a) and TPPS4), which are taken up into lysosomes by endocytosis. Apoptotic fractions induced by the various dyes and light were quantified by flow cytometry using the terminal deoxynucleotidyl transferase (TdT) assay. Cell fragmentation was measured in parallel, while the nuclear morphology of apoptotic cells was studied by fluorescence microscopy. Different kinetics were found for the induction of DNA strand breaks characteristic of apoptotic cells. PDT-induced damage to membranes resulted in an increasing number of apoptotic cells for about 12 h after PDT. After damage to lysosomes, apoptotic cells were not detected until more than 12 h after PDT. Furthermore, apoptotic bodies were not observed after PDT-induced damage to intracellular membranes, whereas apoptosis induced from lysosomal sites was characterized by extensive cell fragmentation. Cell fragmentation occurred in combination with or in the absence of nuclear fragmentation. The results support the idea that the degradation phase of apoptosis can consist of a sequence of independent steps rather than a common final pathway.