The late increase in intracellular free radical oxygen species during apoptosis is associated with cytochrome c release, caspase activation, and mitochondrial dysfunction

Abstract

Mitochondria play central roles in cellular metabolism and apoptosis and are a major source of reactive oxygen species (ROS). We investigated the role of ROS and mitochondria in radiation-induced apoptosis in multiple myeloma cells. Two distinct levels of ROS were generated following irradiation: a small increase observed early, and a pronounced late increase, associated with depletion of reduced glutathione (GSH) and collapse of mitochondrial membrane potential (deltapsi(m)). Exogenous ROS and caspase-3 induced deltapsi(m) drop and cytochrome c release from mitochondria, which could be prevented by molecular (dominant-negative caspase-9) and pharmacologic (zVAD-fmk) caspase inhibitors and overexpression of Bcl-2. Exogenous ROS also induced mitochondrial permeability transition (PT) pore opening and cytochrome c release in isolated mitochondria, which could be blocked by inhibition of PT with cyclosporin A. These results indicate that the late ROS production is associated with increased PT pore opening and decreased deltapsi(m), and GSH, events associated with caspase activation and cytochrome c release.

Figures

Figure 1

IR induces apoptosis, which is inhibited by Bcl-2. (a) Flow cytometric assays for DNA content were performed in parental IM-9 and derivative cells expressing ectopic Bcl-2 (Clone 1-1), as described in the ‘Materials and methods’ section. The sub G1 DNA content is indicative of apoptotic cells. (b) IM-9 and IM-9/Bcl-2 cells were irradiated in the presence or absence of the pancaspase inhibitor zVAD-fmk and prepared for immunoblotting for DFF45 as described in the ‘Materials and methods’ section. The DFF45 proteolytic cleavage was examined following irradiation (10 Gy) in the absence or presence of the pharmacologic caspase inhibitor zVAD-fmk and Bcl-2 overexpression. DFF-Δdesignates the truncated form of DFF45

Figure 2

IR induces distinct phases of ROS production during apoptosis. Control or irradiated (10 Gy) IM-9 or IM-9/Bcl-2 cells were harvested at the indicated times and washed with serum-free medium, then incubated with 5 mM DHE (a) or 1 mM DCFDA (b, c) in PBS at 37°C for 10 min. The cellular fluorescence levels (Fl-1, emission wavelength 620 or 530 nm) were analyzed by flow cytometry, after the cell debris and PI-positive population were gated out. P: percentage of PI-negative cells; x: relative fluorescence. ROS levels, determined by DHE (a) or H2DFCDA (b) staining, are presented as relative fluorescence of DCFDA or DHE. The dot plots shown (c) represent only the viable cell population and are presented gated as relative fluorescence of DCFDA. The mean value of Fl-1 fluorescence was normalized against untreated cells. Data represent mean values from duplicate values from three separate experiments

Figure 3

zVAD-fmk blocks DEVD cleavage activity and late ROS increase. (a) IM-9 cells were lysed at the indicated times following irradiation and assayed for caspase-3 activity toward the peptide substrate Ac-DEVD-p-nitroanilide, which was normalized to that of control cells. (b) The effect of IR on Δψm was examined in IM-9 and RPMI-8226 cells, irradiated in the absence or presence of zVAD-fmk. Δψm was measured using R123 as described in the ‘Materials and methods’ section. Data were normalized against untreated cells and represent the mean values of three separate experiments. Cells were stained with 1 μM CM-H2DCFDA (c) or 5 mM DHE (d) and cellular fluorescence levels were analyzed by flow cytometry, similar to Figure 2. The data shown represent the mean values of two duplicate experiments. The values shown are for control, zVAD, and YVAD-treated cells as represented by empty, shaded, and filled symbols, respectively. *P<0.05, as determined by t-test

Figure 4

Caspase-9-DN inhibits IR-induced apoptosis and late increase in ROS. (a) IM-9 cells were transfected and selected for expression of caspase-9-DN. Clone B-4, which abundantly expressed caspase-9 and parental IM-9 cells were then used to examine phosphatidylserine exposure, an early marker of apoptosis, by annexin V staining and flow cytometry. (b) Parental IM-9 or derivative cells ectopically expressing Bcl-2 or caspase-9-DN were subjected to DHE staining and flow cytometry at 24 h following irradiation, as described above. The data shown are mean values of three experiments with duplicate samples and are representative of three separate experiments. (c) Level of expression of caspase-9 as determined by immunoblotting with anti-caspase-9, and as a control, anti-β-actin primary antibodies. DN, dominant-negative

Figure 5

Effect of anti- and pro-oxidants on apoptosis and Δψm. (a) Morphological changes characteristic of apoptosis were determined by DAPI staining in cells pretreated with the antioxidants PDTC (50 μM) and NAC (10 mM) for 1 h before irradiaton (10 Gy). Apoptosis was then examined at 28 h after IR. The data represent mean values from three separate experiments. (b, c) The effect of xanthine (X) and xanthine oxidase (XO) on apoptosis and Δψm was examined in cells treated with X and XO, alone or in combination, or H2O2 for 30 min at 30°C and then subjected to R123 staining (c) or incubated in complete medium for 24 h before annexin V staining and flow cytometry (b)

Figure 6

Effect of ROS and caspase-3 on isolated mitochondria. (a) The effect of ROS generation from the reaction of X with XO on PT pore opening was examined in isolated mitochondria from mouse liver, as described in the ‘Materials and Methods’ section. XO was added at the indicated times with X being added as designated by the arrow. Control and CsA represent mitochondria alone or those treated with cyclosporin A (1 μM) in addition to X and XO. (b) The release of cyt c from X/XO-treated mouse liver mitochondria was determined by immunoblotting with an anti-cyt c antibody. Loading control represents a nonspecific antibody-reactive band. (c) P10 mitochondrial fractions from IM-9 cells were incubated with different concentrations of recombinant caspase-3 in the absence, or presence, of the pancaspase inhibitor zVAD-fmk at 30°C for 2.5 h. The reaction mixtures were then pelleted and examined for cyt c levels by immunoblotting with anti-cyt c mAb