Use of Human Cancer Cell Lines Mitochondria to Explore the Mechanisms of BH3 Peptides and ABT-737-Induced Mitochondrial Membrane Permeabilization
Figure 1
Isolation and functional characterization of mouse liver and human tumor cell line mitochondria.
A. Ultrastructural analysis of isolated mitochondria and their ability to swell. Electron micrographs were obtained after incubation of mitochondria isolated from healthy mouse liver, or PC-3 tumor cell lines untreated (NT) or treated with Ca2+ (50 µM) or with a 5 min-preincubation with cyclosporin A (CsA; 10 µM) or ruthenium red (RR; 1 µM) before calcium addition. The percentage of swollen mitochondria was <10% in the control and >80% 30 min after Ca2+ addition (n = 3). Scale bars 1 µm. B. Oxidative properties of isolated liver and PC-3 mitochondria. Traces represent oxygen consumption by isolated mitochondria (100 µg) after addition of the indicated reagents. Numbers along the trace are nmoles of O2 consumed per minute per milligram of protein. The respiratory control index (RCI) is calculated for each type of mitochondria as indicated in Materials and Methods. C. To evaluate mitochondrial swelling and ΔΨm loss, mitochondria isolated from healthy mice liver or PC-3 cell line were distributed in 96-well microplates and incubated for 30 min either with Ca2+ (100 µM) in presence (yellow) or absence (pink) of CsA (10 µM), with mClCCP (turquoise; 50 µM) or with t-Bid (purple; 1 nM). For quantitation of cytochrome c release, isolated mitochondria were treated with increasing concentrations of the t-Bid recombinant protein and mitochondrial supernatant was subjected to ELISA assays, given in percentage of release compared to 20 µg/ml alamethicin (Ala; 100% of cytochrome c release) (n = 3 independent experiments).