3-3--dihexyl-2-2--oxacarbocyanine and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

Cellular viability is usually determined by measuring the capacity of cells to exclude vital dyes such as 4',6-diamidino-2-phenylindole (DAPI), or by assessing nuclear morphology with chromatinophilic plasma membrane-permeant dyes, such as Hoechst 33342. However, a fraction of cells that exclude DAPI or exhibit normal nuclear morphology have already lost mitochondrial functions and/or manifest massive activation of apoptotic caspases, and hence are irremediably committed to death. Here, we developed a protocol for the simultaneous detection of plasma membrane integrity (based on DAPI) or nuclear morphology (based on Hoechst 33342), mitochondrial functions (based on the mitochondrial transmembrane potential probe DiOC6(3)) and caspase activation (based on YO-PRO®-3, which can enter cells exclusively upon the caspase-mediated activation of pannexin 1 channels). This method, which allows for the precise quantification of dead, dying and healthy cells, can be implemented on epifluorescence microscopy or flow cytometry platforms and is compatible with a robotized, high-throughput workflow.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Benzimidazoles; Carbocyanines; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Cell Membrane Permeability; Cell Survival; Connexins; Drug Synergism; Flow Cytometry; Fluorescent Dyes; High-Throughput Screening Assays; Humans; Indoles; Lung Neoplasms; Membrane Potential, Mitochondrial; Microscopy, Fluorescence; Nerve Tissue Proteins; Organoplatinum Compounds; Oxaliplatin; Robotics; Staining and Labeling; Staurosporine; Workflow

Downstream mediators of p53 in apoptosis induction remain to be elucidated. We report that p53-induced apoptosis occurred in the absence of cytochrome c release into the cytosol. Although Bax was upregulated, it remained largely in the cytosol and there was no detectable translocation to the mitochondria. Bid was not activated as no cleavage could be detected. Thus, the absence of cytochrome c release may be due to the lack of Bax translocation to mitochondria and/or Bid inactivation. Nevertheless, p53-induced apoptosis is still caspase dependent because it could be abolished by z-VAD-fmk. To search for alternative downstream targets of p53, we detected production of reactive oxygen species (ROS) as well as mitochondrial membrane potential (Deltapsi). p53 induced ROS generation, which then caused a transient increase of Deltapsi followed by a decrease. Antioxidants could inhibit the alterations of Deltapsi, thereby preventing apoptosis. z-VAD-fmk was unable to abrogate Deltapsi elevation but inhibited Deltapsi decrease, indicating that Deltapsi elevation and its decrease are two independent events. Bcl-2 may abolish elevation as well as decrease of Deltapsi without interfering with ROS levels. Thus, the ROS-mediated disruption of Deltapsi constitutes a pivotal step in the apoptotic pathway of p53, and this pathway does not involve cytochrome c release.

Topics: Amino Acid Chloromethyl Ketones; Antioxidants; Apoptosis; bcl-2-Associated X Protein; BH3 Interacting Domain Death Agonist Protein; Carbocyanines; Carrier Proteins; Caspases; Cytochrome c Group; Enzyme Activation; Gene Expression Regulation; HeLa Cells; Humans; Membrane Potentials; Mitochondria; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction; Transfection; Tumor Suppressor Protein p53