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

The induction of apoptosis from different intracellular sites was studied b y exposing V79 Chinese hamster fibroblasts to photodynamic therapy (PDT) wi th various porphyrins and light. The effects of two lipophilic, intracellul ar membrane-localized porphyrins, tetra(3-hydroxyphenyl)porphyrin (3THPP) a nd Photofrin, were compared with that of two sulphonated meso-tetraphenylpo rphines (TPP2a 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) as say. Cell fragmentation was measured in parallel, while the nuclear morphol ogy of apoptotic cells was studied by fluorescence microscopy. Different ki netics 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 lysosom es, apoptotic cells were not detected until more than 12 h after PDT. Furth ermore, apoptotic bodies were not observed after PDT-induced damage to intr acellular membranes, whereas apoptosis induced from lysosomal sites was cha racterized by extensive cell fragmentation. Cell fragmentation occurred in combination with or in the absence of nuclear fragmentation. The results su pport the idea that the degradation phase of apoptosis can consist of a seq uence of independent steps rather than a common final pathway.