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Glutathione depletion enforces the mitochondrial permeability transition and causes cell death in Bcl-2 overexpressing HL60 cells¹

Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia, USA

²Correspondence: Department of Biochemistry, Emory University School of Medicine, 1510 Clifton Road, 4123 Rollins Research Center, Atlanta, GA 30322-3050, USA. E-mail: jeff30322{at}lycos.com

SPECIFIC AIM

Bcl-2, a protein that blocks apoptosis by inhibiting the mitochondrial permeability transition (MPT) and release of cytochrome c, appears to affect normal mitochondrial function by altering electron flow and increasing rates of reactive oxygen species (ROS) production. The aim of this study was to investigate the control of Bcl-2 function by glutathione (GSH). HL60 cells were depleted of GSH using L-buthionine S,R- sulfoximine (BSO) or diethyl maleate (DEM). BSO inhibits the rate-controlling enzyme for GSH synthesis, -glutamyl cysteine synthetase (GCS), and DEM is a GSH-depleting agent. The effects of GSH depletion on apoptotic potential in HL60 overexpressing Bcl-2 and neo vector control cells were investigated by determining biochemical indices of apoptosis including ROS production, mitochondrial membrane potential (mt), mitochondrial cytochrome c release, caspase 3 activation, and DNA fragmentation. The mitochondrial site of ROS production was determined using site-specific inhibitors of electron transport.

PRINCIPAL FINDINGS

1. Endogenously generated ROS were constitutively increased in Bcl-2 overexpressing cells compared to neo control cells and correlated with GSH redox status
Mean dichlorofluorescein (DCF) fluorescence (an indicator of intracellular ROS production) was increased in Bcl-2 overexpressing cells vs. neo control cells, indicating that these cells constitutively produced increased levels of ROS compared to neo vector controls. BSO or DEM increased levels of ROS, and Bcl-2 failed to block the ROS increase (Fig. 1 ). In contrast, ROS increase was effectively blocked in cells cocultured with the thiol donor dithiothreitol (DTT). GSH redox status (Nernst redox) was determined in HL60 neo vector control and Bcl-2 overexpressing cells, and correlated with ROS production (Fig. 1) .

View larger version (30K): [in this window] [in a new window]   Figure 1. A) Representative flow cytometric analysis of HL60 cells (neo vector control and Bcl-2 overexpressing) stained with DCFDA and analyzed using FL1 channel. HL60 cells (2x106/ml) were washed and suspended in PBS containing 10 mM glucose. Cells were loaded with DCFDA (50 µM) for 15 min and immediately analyzed. In each analysis, 10,000 events were recorded. B) Cell aliquots (4x106/ml) were treated with BSO (500 µM) or RPMI (control) for 20 h. GSH levels were determined on these samples by HPLC assay. The GSH concentration was plotted as nmol/mg protein. Data are expressed as mean ± SE (n=3). C) Bar graph showing mean DCF fluorescence intensity (arbitrary units) of HL60 cells (neo vector control and Bcl-2 overexpressing) treated with BSO (500 µM) or RPMI (control) for 20 h. After treatment, cells were loaded with DCFDA (50 µM) for 15 min and green fluorescence was measured by flow cytometry using the FL-1 setting. Data are expressed as mean ± SE (n=3). D) HL60 cells overexpressing Bcl-2 and neo vector controls were treated with 500 µM BSO for 0, 12, 24, and 36 h. GSH/GSSG redox (Eh) was calculated using the Nernst equation with E0 adjusted to cell pH. Data are expressed as mean ± SE (n=3).

2. GSH depletion with BSO or DEM in HL60 cells overexpressing Bcl-2 induced selective cell death, which was mediated by the MPT
After BSO or DEM treatment, HL60 cells overexpressing Bcl-2 lost mt whereas neo vector control cells did not lose mt [compared to protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) -treated cells]. Bcl-2 overexpressing cells selectively lost viability after depletion of GSH with BSO or DEM compared to neo vector controls. mt and cell viability were preserved in a dose-dependent fashion with bongkrekic acid (BK) and DTT, implicating dependence on thiols and the involvement of the adenine nucleotide translocator (ANT) on MPT (Fig. 2 ).

View larger version (52K): [in this window] [in a new window]   Figure 2. A) HL60 cells overexpressing Bcl-2 and neo vector controls were treated with CCCP (10 µM) for 24 h, staurosporine (1 µM) for 24 h, diamide (250 µM) for 24 h, BSO (500 µM) for 40 h, and DEM (1 mM) for 4 h. Percentage of PI-stained apoptotic cells (±SE) after treatments indicated above. Data are expressed as mean ± SE (n=3). B) Representative flow cytometric cell cycle analysis of HL60 cells overexpressing Bcl-2 and neo vector controls treated with BSO for 20 and 40 h. Apoptosis was measured as percentage of PI-stained cells containing hypodiploid amounts of DNA (sub-G1). Shown is a representative example of at least 5 experiments. In each analysis, 10,000 events were recorded. C) Representative flow cytometric analysis of mt in HL60 cells overexpressing Bcl-2 treated with BSO (500 µM), BSO (500 µM) + DTT (250 µM) or BSO (500 µM) + BK (25 µM) for 40 h. Cells were loaded with TMRM (250 nM) for 15 min and red fluorescence was immediately measured by flow cytometry using the FL-2 setting. A representative example of at least 5 experiments is shown. In each analysis, 10,000 events were recorded. D) HL60 cells overexpressing Bcl-2 were treated with BSO (500 µM) for 40 h in the presence of varying concentrations of DTT and BK. Cell viability was determined by trypan blue analysis. Data are expressed as mean ± SE (n=3).

3. Mitochondrial ROS production occurs principally at respiratory chain complex III
To determine the respiratory chain site involved in ROS production, we used the pharmacological inhibitors rotenone (ROT), myxothiazol (MYX), antimycin A (AA), and stigmatellin (STIG). Inhibitors were added to cells after BSO treatment for 20 h or at the time of DEM addition 0 h. AA and STIG inhibited ROS production (determined by DCF fluorescence measurements), blocked MPT, inhibited caspase 3 activation, and preserved cell viability. ROT and MYX did not block ROS production or reduce cell death. Inhibition of respiratory chain complex III activity with AA and STIG after GSH depletion preserved the redox status of protein and nonprotein thiols compared to cells treated with BSO or DEM alone or in the presence of MYX or ROT.

CONCLUSIONS AND SIGNIFICANCE

Bcl-2 is a prototypical member of a family of mammalian proteins that regulate the activation of the caspase cascade of apoptosis. The family consists of both anti-apoptotic members such as Bcl-2 and proapoptotic members such as Bax. Although the proteins probably have multiple modes of action, a central function appears to be associated with mitochondria and control of mitochondrial permeability to ions.

Our data show that GSH depletion with BSO or DEM increases endogenous ROS production sufficient to induce MPT and apoptosis in Bcl-2 overexpressing HL60 cells. The increase in ROS is likely to be the result of significant depletion of the mitochondrial pool of GSH since this pool is required for the enzymatic metabolism of mitochondrially generated peroxides. We found that levels of GSH were significantly lower in HL60 cells overexpressing Bcl-2 and that GSH was rapidly depleted and more oxidized in these cells than in neo control cells. Bcl-2 overexpressing cells constitutively produced increased levels of ROS compared to neo control cells (determined by DCF fluorescence) and were more sensitive to cell death mediated by ROS.

Although the classical idea of the MPT is a regulated polyprotein complex consisting of ANT, VDAC, and other mitochondrial proteins, including cyclophilin D, our data suggest that ANT is the principal player in cell death induced by mitochondrial ROS.

ROT, which inhibits respiratory chain complex I, did not block ROS production or prevent cell death, suggesting that this site is relatively unimportant in ROS production due to Bcl-2. On the other hand, respiratory chain complex III appears to play a key role in the generation of ROS and induction of MPT in response to Bcl-2. This conclusion is supported by the observation that both AA and STIG, but not MYX, blocked the formation of ROS after GSH depletion and prevented MPT, cytochrome c release, caspase 3 activation, and cell death. Respiratory chain complex III has two ubiquinone-reactive sites: Qo, where ubiquinol is oxidized by redox active centers cytochrome c1 and the ‘Rieske’ [2Fe-2S] protein (ISP), and Qi, where ubiquinone is reduced by the redox center cytochrome b. Recently it has been suggested that the ISP is a mobile structure and its mobility may facilitate rapid electron transfer between cytochrome b and ISP. MYX and STIG both inhibit the Qo site, although they appear to exert different effects on the mobility of the [2Fe-2S] cluster and, perhaps, the whole extra-membrane domain of ISP. STIG increases the midpoint potential of ISP and decreases the motion of ISP, whereas binding of the MYX causes a red shift in the optical spectrum of cytochrome b566 and an increase in the motion of ISP. STIG blocked ROS production and toxicity whereas MYX appeared to enhance ROS production and toxicity. These results suggest that ROS production via respiratory chain complex III may be linked to the electron transfer kinetics in complex III, which is controlled in part by the motion of the ISP. Thus, STIG, which effectively decreases the rate of electron transfer through complex III, protects against ROS production and cell death, whereas MYX, which does not affect electron transfer through ISP, fails to protect against ROS increase after GSH depletion. AA, which also protected against ROS production, inhibits complex III and reduces the electron transfer rate through complex III by exerting negative control on cytochrome c1. These results suggest that the rate of electron transfer through complex III activity is a controlling factor for ROS production and suggest a link between complex III activity and MPT.

Although it is widely believed that MPT is composed of proteins that include the ANT and VDAC, this has yet to be verified. In our study, AA and STIG, but not ROT or MYX, blocked MPT after GSH depletion, suggesting that complex III may be functionally linked to the MPT (Fig. 3 ). Our data suggest a new model for MPT in which the activity of mitochondrial respiratory complex III is intimately associated with MPT by its ability to generate ROS, which convert ANT to a MPT pore overriding the anti-apoptotic function of Bcl-2.

View larger version (51K): [in this window] [in a new window]   Figure 3.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0097fje; to cite this article, use FASEB J. (June 7, 2002) 10.1096/fj.02-0097fje

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