mitotracker-green-fm and Osteosarcoma

Reactive oxygen species (ROS) that are produced by mitochondria are released toward the mitochondrial matrix or the intermembrane space. Each ROS pool is likely involved in different cellular mechanisms and damage. Unfortunately, it is difficult to distinguish the provenance and effects of ROS. Here we introduce a method to semiquantitate the steady-state levels of superoxide produced in the matrix of mitochondria. Superoxide produced during cellular respiration is capable of oxidizing hydroethidine, a probe that is membrane permeant. The poor membrane permeability of the hydroethidine oxidation products causes accumulation of these fluorescent products within the mitochondria. After isolation of mitochondria, a method based on the capillary electrophoretic separation of individual organelles and their detection by laser-induced fluorescence detection is used to determine their fluorescent contents. Use of this method for the analysis of organelle fractions obtained from cells treated with antimycin A or rotenone confirms that the detected fluorescence is associated with superoxide produced by mitochondria. Furthermore, using this method the superoxide levels in the mitochondrial matrix of a cytoplasmic hybrid (cybrid) cell line (DeltaH2-1) and one of its parent cell lines (143B) were compared.

Topics: Aldehydes; Bone Neoplasms; Cell Fractionation; Cell Line, Tumor; DNA, Mitochondrial; Humans; Kinetics; Mitochondria; Osteosarcoma; Phenanthridines; Superoxides

Severe disruption of mitochondrial function is generally considered to provide a powerful trigger for apoptosis in mammalian cells. We report here that intact cells may undergo the mitochondrial permeability transition and mitochondria swell in a fully reversible manner, without inducing cell death. Cultured human osteosarcoma cells (143B TK-) stained with JC-1, MitoTracker dyes, or calcein plus Co2+ were imaged by confocal microscopy to visualize changes of mitochondrial membrane potential (DeltaPsim), morphology, and permeability transition, respectively, during treatment with a protonophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP). Cells rapidly exhibited mitochondrial permeability transition and swelling after addition of CCCP, but the swelling subsided within hours, leaving mitochondria that appeared in punctate form, not filamentous as before CCCP treatment. Cyclosporin A impeded the permeability transition and swelling, although complete inhibition was not observed. Cells survived the dissipation of DeltaPsim by CCCP for up to 6 h without developing any obvious cell damage or signs of apoptosis. With the restoration of DeltaPsim after removal of CCCP (following 6 h of CCCP treatment), permeability transition pores were closed. These results suggest that none of the following events represent a point of no return in the process of apoptotic cell death: loss of DeltaPsim, mitochondrial permeability transition, or mitochondrial swelling.

Topics: Aldehydes; Apoptosis; Benzimidazoles; Carbocyanines; Carbonyl Cyanide m-Chlorophenyl Hydrazone; Cell Survival; Cyclosporine; Fluoresceins; Fluorescent Dyes; Humans; Intracellular Membranes; Ionophores; Membrane Potentials; Microscopy, Confocal; Mitochondria; Mitochondrial Swelling; Organic Chemicals; Osteosarcoma; Permeability; Staining and Labeling; Tumor Cells, Cultured