Light-induced apoptosis involves a defined sequence of cytoplasmic and nuclear calcium release in AlPcS4-photosensitized rat bladder RR 1022 epithelial cells.

Oxidative stress induced by light activation of photosensitizers is regarded to have a role in triggering cell death pathways during photodynamic therapy (PDT). Reactive oxygen species have been proposed to act as signal transduction molecules activating downstream reactions that lead to apoptosis. Mainly debated is the cooperating role of other signaling systems like calcium or pH. The present work contributes to this discussion by studying PDT effects in cell cultures of rat bladder epithelial cells for the hydrophilic tetrasulfonated aluminum phthalocyanine (AlPcS4). Cells were coincubated with the photosensitizer and the calcium-sensitive probe Fluo-3. The light-induced reactions were analyzed with a confocal laser scanning microscope. The dynamics of the process during light activation was observed with subcellular resolution. A transient calcium elevation during the irradiation process was detected, especially in the cell's nuclei, followed by a more sustained increase. The evaluation of the energy-dose-dependent phototoxicity after an incubation time with the photosensitizer of 1 and 24 h, showed enhanced phototoxicity when the drug was present for 24 h. Surprisingly, stimulation of cell proliferation was observed at very low light doses (at 0.2 J/cm2) when the drug was incubated for 24 h (cell viability 160%). Induction of apoptosis could be observed after irradiation with fluences between 1 and 3 J/cm2. Apoptotic cells were identified with fluorescein isothiocyanate-labeled Annexin V, which binds to phosphatidylserine after its translocation to the outer plasma membrane. In the presence of the antioxidant pyrrolidinedithiocarbamate the transient calcium elevation was totally inhibited, as was the subsequent translocation of PS. In contrast, N-acetyl-L-cysteine did not suppress the transient calcium increase. Our data might be consistent with calcium regulated processes during AlPcS4-PDT and the involvement of oxygen radicals.

Nonlinear dynamics of intracellular methylene blue during light activation of cell cultures.

Methylene blue (MB+) is a well-known dye in medicine and has been discussed as an easily applicable drug for topical treatment in photodynamic therapy (PDT). Methylene blue can potentially be used as a redox indicator to detect the important redox reactions that are induced during PDT. The kinetics of this process was analyzed on a subcellular level with confocal laser scanning microscopy. BKEz-7 endothelial cells were incubated 4 h with 1 microM MB+. The fluorescence dynamics of MB+ during irradiation with 633 nm light was observed with subcellular resolution. Images were acquired at 0.5 s intervals (frame rate 1 image/0.5 s). Fluorescence was observed in the red channel of the laser scanning microscope. Synchronously, the phase-contrast image was visualized with the green channel. Morphological changes could therefore be correlated with the dynamics of MB+. In addition, the light-dose-dependent phototoxicity at 633 nm irradiation was determined by viable cell counting. After an induction period (phase I), fast fluorescent spikes could be observed in the whole cytoplasm, which decayed with a time constant of about 20 s (phase II), followed by a period of nearly constant fluorescence intensity (phase III) and exponential photobleaching (phase IV). Phase II exhibits highly nonlinear kinetics, which is hypothesized to correlate probably with a nonlinear quantal production of reactive oxygen species (ROS). Morphological cell changes were not observed during phase II. During phase III, a pycnotic cell nucleus developed. From the determination of viable cells we can conclude that a light dose applied within phase II was only sublethal in correlation with morphological observations. Overproduction of ROS leading finally to cell killing during phases III and IV is discussed.