We report that
chloromethyl-X-rosamine (MitoTracker Red), a mitochondrion-selective
fluorescent probe, has a strong photosensitising action. Photoirradiation of intact cells loaded with
chloromethyl-X-rosamine induces depolarisation of the inner mitochondrial membrane and swelling of mitochondria, subsequently resulting in apoptosis. We have studied human
osteosarcoma 143B TK-(rho+) cells and the derived (rho)0 206 cell line devoid of
mitochondrial DNA. Colony formation tests revealed that
chloromethyl-X-rosamine itself has no toxicity to either cell line in the concentration range 100-250 nM (unless photoirradiated).
Chloromethyl-X-rosamine has potent
phototoxicity such that almost quantitative cell killing was achieved at light doses of >2 J/cm2. These photodamaged cells initially showed swollen degenerative mitochondria and, later, uptake of
propidium iodide in their apoptotic nuclei was observed. When cells were loaded with
chloromethyl-X-rosamine (100 nM) and imaged by
laser scanning confocal microscopy, photoirradiation by the
laser beam under routine scanning conditions was sufficient to induce mitochondrial damage in both cell lines. This was evidenced by a rapid decrease of fluorescence intensity of co-loaded
rhodamine 123 (indicative of mitochondrial depolarisation). Globular swelling of mitochondria took place within 15 minutes, imaged by the residual fluorescence of
chloromethyl-X-rosamine itself, which also markedly decreased in intensity after imaging. Mitochondrial membrane depolarisation of cells loaded with
chloromethyl-X-rosamine after photoirradiation using a measured dose of visible light was independently confirmed in 143B TK- and (rho)0 206 cells, by the significant decrease in uptake into cells of [3H]
methyltriphenylphosphonium ions. Photoactivation of
chloromethyl-X-rosamine in 143B TK-(rho+) cells, whose mitochondria had previously been loaded with
calcein, caused rapid release of the mitochondrially entrapped
calcein into the cytosol and nucleus. This major change in permeability of the mitochondrial inner membrane could not be prevented by
cyclosporin A. Immunohistochemical study of
cytochrome c revealed its diffuse redistribution into the cytoplasm in
chloromethyl-X-rosamine-loaded cells after irradiation, as opposed to its specific mitochondrial localisation in non-irradiated cells. As a photosensitiser specifically targeted to mitochondria, and also a reporter of membrane potential and morphology,
chloromethyl-X-rosamine may provide versatile new applications in studies of mitochondrial roles in cell death.