Photodynamic therapy with toluidine blue in Jurkat cells: cytotoxicity, subcellular localization and apoptosis...

Toluidine blue (TBO) is a cationic thiazine dye with an affinity for neoplastic tissues in vivo. The objective of this study was to explore the in vitro photosensitizing potential of TBO and its capacity to induce apoptosis in human leukaemic T cells. Jurkat cells were incubated with TBO for one hour followed by exposure to 11 J cm(-2) of visible light from a slide projector. Cytotoxicity was assessed at 24 hours using a MTT assay. DNA fragmentation was examined at different intervals after photodynamic treatment using a DNA elution-filtration assay with [14C]-thymidine labelled cells. Caspase-3 like activation induced by photodynamic treatment was studied by measuring AC-DEVD-AMC peptide hydrolysis. The MTT assay showed a 97% decrease in optical density 24 hours following photodynamic therapy with 0.15 microg ml(-1) of TBO. Dark toxicity was absent under these conditions. DNA fragmentation was detected as early as 2 hours after photodynamic therapy and reached 68% at 6 hours. At higher TBO concentrations less DNA fragmentation and more dark toxicity was observed. An increase in caspase-3 like activity was also induced by photodynamic therapy with TBO. At the time of light exposure TBO was present in the endoplasmic reticulum and Golgi regions. In conclusion, TBO-based photodynamic therapy has a potent phototoxic effect and induces apoptosis in Jurkat cells.