Distinct Pathways for Stimulation of Cytochrome c Release by Etoposide*

The present study examined the ability of different doses of etoposide to stimulate the release of cytochrome c from mitochondria in Jurkat T-lymphocytes or a reconstituted cell-free system, as well as mitochondria isolated from rat liver. The results indicate that 10 m M etoposide stimulates cytochrome c release in Jurkat cells and in a cell-free system that is significantly reduced by z-VAD.fmk or z-VDVAD.fmk. However, caspase inhibition was not able to prevent the release of cytochrome c in response to a higher dose (50 m M) of etoposide. At the same time, 50 m M, and not 10 m M, etoposide significantly diminished mitochondrial Ca 2+ buffering capacity in digitonin-permeabilized cells and stimulated the release of cytochrome c from liver mitochondria.


INTRODUCTION
DNA damage brought about by the exposure of cells to any number of cytotoxic stimuli, including oxidants, ultraviolet radiation, x-rays, environmental toxicants and chemotherapeutic drugs, can stimulate the onset of a series of intracellular changes characteristic of a form of cell death known as apoptosis (1)(2)(3)(4)(5). Numerous studies have reported the ability of specific DNA-damaging agents to stimulate well-known changes at the mitochondrial level that are key initiative steps in the apoptotic process (6)(7)(8)(9). However, the signaling mechanism responsible for linking DNA damage with downstream mitochondrial events is unknown. It is also unclear whether these reputedly specific DNA-damaging agents, such as etoposide, are capable of exerting their toxicity, at least in part, by directly damaging mitochondria.
Etoposide is a topoisomerase II poison that is routinely prescribed for the treatment of cancer (1). Topoisomerase II contains three primary domains and is involved in various aspects of DNA metabolism (10,11). Normally, this enzyme binds to DNA, and in the presence of a divalent cation (12), as well as ATP (13), generates a transient, double-stranded break through which an entire intact helix is passed in order to prevent intertwining of DNA. Etoposide does not altogether inhibit the activity of topoisomerase II. Instead, it selectively exploits the catalytic activity of this enzyme by increasing the frequency and duration of DNA cleavage sites, resulting ultimately in permanent double-stranded breaks that are lethal to the cell (14).
Recently, it was reported that etoposide-induced cytochrome c release is a caspaseindependent event and that caspase-9 is the most apical caspase in chemical-induced apoptosis (6,15,16). While the precise mechanism controlling cytochrome c release from mitochondria remains obscure, several models have been proposed that largely focus on the role that mitochondrial permeability transition (MPT) and/or a loss of membrane potential may serve in the process (17)(18)(19)(20). According to Sun et al. (15), the general caspase inhibitor z-VAD.fmk failed to inhibit decreases in both mitochondrial membrane potential and cell size in etoposide-treated (50 mM) Jurkat T-lymphocytes. Moreover, z-VAD.fmk did not abrogate cytochrome c release, whereas it was able to inhibit caspase-9 and executioner caspases. In contrast, Tepper  Hepes, pH 7.5) supplemented with 1 mM EDTA, and homogenized with a glass Dounce homogenizer and Teflon pestle. Homogenates were centrifuged at 600 x g for 8 min at 4°C. The supernatant was decanted and recentrifuged at 5,500 x g for 15 min to form a mitochondrial pellet that was resuspended in MSH buffer without EDTA and centrifuged again at 5,500 x g for 15 min.
The final mitochondrial pellet was resuspended in MSH buffer at a protein concentration of 80-100 mg/ml. (1 mg/ml) were incubated in a buffer containing 150 mM KCl, 1 mM KH 2 PO 4 , 5 mM succinate, and 5 mM Tris, pH 7.4 at 25°C. Rotenone (2 µM) was added to maintain pyridine nucleotides in a reduced form. Estimation of Dy was performed using an electrode sensitive to the lipophilic cation tetraphenylphosphonium (TPP + ). Energized mitochondria rapidly accumulate TPP + from the incubation buffer and release this cation as Dy decays. Ca 2+ fluxes across the inner mitochondrial membrane were monitored using a Ca 2+ -sensitive electrode (model 97-20, Orion Research, Inc, Beverly, MA). Mitochondrial swelling was monitored continuously as changes in OD 540 . Oxygen consumption by isolated rat liver mitochondria was measured using a Clark-type oxygen electrode (Yellow Spring Instrument Co., OH, USA) at 25°C. Mitochondria with a respiratory control ratio (defined as the rate of respiration in the presence of ADP divided by the rate obtained following the expenditure of ADP) above 4 were used for all experiments. Fresh mitochondria were prepared for each experiment and used within 4 h.

Digitonin-permeabilized Cells and Estimation of Mitochondrial Ca 2+ Accumulation-
Jurkat cells (2.5´10 6 ) were washed in PBS, resuspended in 500 ml of buffer (150 mM KCl, 5 mM KH 2 P0 4 , 1 mM MgSO 4 , 5 mM succinate, 5 mM Tris, pH 7.4) and added to the incubation chamber. Following a 2 min stabilization period, cells were permeabilized with 0.005 % digitonin and 5 mM rotenone was added in order to maintain pyridine nucleotides in a reduced form. MPT was induced by sequential additions of Ca 2+ and changes in the level of this cation were monitored using a Ca 2+ -selective electrode. Samples were incubated at 37°C for up to 3 h. Nuclei and mitochondria were removed by centrifugation at 12,500 x g for 10 min at 4°C and the supernatants stored at -20°C until used for western-blot analysis.
Western-blot analysis-Samples were mixed with Laemmli's loading dye, boiled for 5 min and subjected to 15% SDS-PAGE at 130 V followed by electroblotting to nitrocellulose for 2 h at 100 V. Membranes were blocked for 1 h with 5% nonfat milk in phosphate-buffered saline at room temperature and subsequently probed overnight with an anti-cytochrome c (1:2500) or anti-G3PDH (1:5000) antibody. The membranes were rinsed and incubated with a horseradish-peroxidaseconjugated secondary antibody (1:10,000). Following the secondary antibody incubation, the membranes were rinsed and bound antibodies were detected using enhanced chemiluminescence according the manufacturer's instructions.

Etoposide Induces Cytochrome c Release and DEVDase Activity in Jurkat cells-
Treatment of Jurkat T-lymphocytes with etoposide stimulates the release of cytochrome c (Fig. 1A).
To investigate whether this effect is dose-and/or caspase-dependent, cells were treated with either  (Fig. 1B). A smaller ~1.5-fold difference was observed between the two treatments at 6 h, which appeared to be due to a flattening out of DEVDase activity in response to the higher dose of etoposide.
Etoposide Induces Cytochrome c Release in a Cell-free System-To investigate the potential relationship between caspases and cytochrome c release in the presence of 10 mM etoposide, experiments were performed using a reconstituted cell-free system. Isolated rat liver nuclei (5 x 10 6 ) were incubated with mitochondria (15 mg of protein) and/or Jurkat cytosol (25 mg 8 of protein) in the presence or absence of etoposide for 3 h at 37°C (Fig. 2A). Western-blot analysis for cytochrome c was first performed on isolated nuclei and Jurkat cytosol in order to verify that these fractions were free from mitochondrial contamination (data not shown). Results indicated that 10 mM etoposide was sufficient to stimulate cytochrome c release from mitochondria incubated in the presence of nuclei and Jurkat cytosol ( Fig. 2A, lanes 3 versus 4). Furthermore, this effect was not dependent on the presence of cytosol as evidenced by the similar results that were obtained when cytosol was absent ( Fig. 2A, lanes 1 versus 2). To ensure that 10 mM etoposide was not exerting a direct effect on mitochondria, these organelles were incubated under the same conditions as described for Fig. 2A except nuclei were excluded (Fig. 2B). However, when mitochondria were incubated with a higher dose (25 mM) of the drug, cytochrome c release was observed as compared to vehicle alone (Fig. 2C).
To further characterize this effect, nuclei alone were incubated with 10 mM etoposide for 1 h at 37°C. Following the 1 h incubation, nuclei were removed by centrifugation and the resulting supernatant was collected and used to treat isolated mitochondria for an additional 2 h. As shown in Fig. 2D (lanes 1 and 2), the supernatant from etoposide-treated nuclei stimulated significant cytochrome c release as compared to control supernatants, suggesting that etoposide stimulated the release of some factor(s) from nuclei which was (were) capable of targeting mitochondria and triggering the release of cytochrome c. Next, to determine if the factor(s) released from nuclei in the presence of etoposide was (were) a protein(s), supernatants were heat-inactivated at 70°C for 30 min prior to being added to mitochondria. Heat-inactivation successfully mitigated the ability of the supernatant to stimulate cytochrome c release (Fig. 2D, lane 3 versus 2), indicating that the factor(s) released from etoposide-treated nuclei is (are) likely to be a protein(s). Moreover, it would appear that this factor is present constitutively since pre-treatment of intact Jurkat cells with the protein synthesis inhibitor cycloheximide (1 mg/ml) for 1 h failed to block etoposide-mediated cytochrome c release (data not shown).
The potential inhibitory effect of z-VAD.fmk was tested to see whether caspase activity was associated with cytochrome c release in this system. Nuclei and mitochondria were co-treated with 25 mM z-VAD.fmk and 10 mM etoposide for 3 hours. This was sufficient to block cytochrome c release induced by etoposide (Fig. 2E, lane 3 versus 2), suggesting that caspase activity is involved in this response. When the supernatants of isolated nuclei that had been treated with etoposide and z-VAD.fmk for 1 h were subsequently added to mitochondria, cytochrome c release was also inhibited (Fig. 2F, lane 3 versus 2), whereas treatment of nuclear supernatants with z-VAD.fmk at the end of etoposide treatment, but before addition to mitochondria, was unable to retard release of cytochrome c (Fig. 2F, lane 4 versus 2). Taken together, this suggests that z-VAD.fmk most likely inhibits pro-apoptotic nuclear, and not mitochondrial, events in this system.
Experiments with more specific caspase inhibitors indicated that only z-VDVAD.fmk, which primarily inhibits caspase-2, and to a lesser extent -3 and -7, mimicked the effect observed with z-VAD.fmk (Fig. 2G, lanes 6 versus 3). Neither z-DEVD.fmk (caspase-3 and -7) nor z-LEHD.fmk (caspase-9) was able to inhibit cytochrome c release when nuclei and mitochondria were treated with 10 mM etoposide for 3 h (Fig. 2G, lanes 4 and 5). Moreover, enzymatic studies revealed that caspase-2 activity was detected in advance of caspase-9 in Jurkat cells treated with 10 mM etoposide ( Table I), suggesting that the nuclear signal responsible for stimulating cytochrome c release in response to etoposide involves caspase-2.

Etoposide Stimulates Cytochrome c Release via MPT in Isolated Mitochondria-Since
25 mM etoposide stimulated the release of cytochrome c from mitochondria alone in our cell-free system, the next step was to test whether this effect might be due to an induction of MPT. It is wellknown that mitochondrial Ca 2+ accumulation is obligatory for MPT induction, although the sensitivity of MPT to Ca 2+ can be significantly enhanced by different factors. Among these factors are the depletion of adenine nucleotides, an elevated level of inorganic phosphate and oxidative stress.
The addition of etoposide to mitochondria induced a concentration-dependent sub-maximal shift of Dy that was not preventable by CsA, which was added to mitochondria prior to etoposide (Fig. 3A, trace 5 versus 3). The addition of Ca 2+ to mitochondria in the presence of different concentrations of etoposide stimulated an additional and more prominent drop in Dy and a release of accumulated Ca 2+ (Fig. 3A and B, traces 1 and 2); effects that were completely abolished by CsA ( Fig. 3A and B, trace 3), indicating that the second phase of the drop of Dy was due to MPT induction. Importantly, Ca 2+ alone was not sufficient to induce a drop in Dy or a release of Ca 2+ ( Fig. 3A and B, trace 4). Similarly, etoposide treatment alone did not induce a complete collapse of Dy (Fig. 3A, trace 5). Etoposide-induced drops in Dy and the release of Ca 2+ from mitochondria were accompanied by mitochondrial swelling, an indicator of mitochondrial permeability transition (Fig.   3C, traces 1 and 2 versus 4). Swelling only occurred in Ca 2+ -loaded mitochondria and led to the rupture of the outer membrane and the release of cytochrome c (Fig. 3D). CsA (1 mM) or EGTA (1 mM) completely prevented swelling of Ca 2+ -loaded mitochondria (Fig. 3C, trace 3) and the release of cytochrome c (Fig. 3D). Mitochondria accumulated sequential additions of Ca 2+ until MPT was induced and the accumulated Ca 2+ released ( Fig. 4A and B). Treatment with 10 mM etoposide for 3 h did not significantly alter either the rate of Ca 2+ accumulation or the threshold level of Ca 2+ , which is necessary for MPT induction (Fig. 4C and D). In contrast, 50 mM etoposide markedly suppressed both parameters by 3 h (Fig. 4B-D). At 6 h, the Ca 2+ buffering capacity of mitochondria was significantly impaired at both concentrations of etoposide. CsA, which was added to the cells at the same time as etoposide, did not offer protection, indicating that MPT was not involved in etoposide-mediated damage to these organelles (data not shown).

DISCUSSION
Currently, there is some confusion and uncertainty in the literature as to the mechanism controlling cytochrome c release in response to specific DNA-damaging agents. In particular, what is the signal linking DNA damage to downstream mitochondrial events? Are these reputedly specific DNA-damaging agents, in fact, "specific" or can some of them exert their toxicity by directly targeting mitochondria? Which, if any, of these effects is/are caspase-dependent?
Numerous reports have described the ability of various specific and non-specific DNAdamaging agents to stimulate the release of mitochondrial cytochrome c (6,15,16,24). In contrast to death receptor-mediated apoptosis, during which caspase-8 activity is often responsible for the cleavage of a cytosolic substrate, e.g., Bid, which targets mitochondria triggering the release of cytochrome c, this event is traditionally accepted as caspase-independent in chemical-and/or DNAdamage-induced apoptosis (16,18,25). Moroever, recent results from our laboratory indicate that neither is caspase-8 activated nor is Bid cleaved in response to etoposide up to 3 h (J.D. Robertson, B. Zhivotovsky, and S. Orrenius, unpublished data). In this respect, the general caspase inhibitor z-VAD.fmk is believed to protect against chemical-induced apoptosis downstream of mitochondria by preventing the activation of caspase-9 (15), which is activated within the apoptosome complex (26,27). However, a more recent report proposes that z-VAD.fmk is able to inhibit the second of a two-stage cytochrome c release process that involves caspase activation, a reduction in ATP levels and decreases in Dy (28). In this study, we examined the nature of cytochrome c release in response to two different, and frequently cited, doses of the topoisomerase II inhibitor etoposide. We demonstrate that z-VAD.fmk is effective at mitigating early cytochrome c release in response to a low (10 mM), and not a high (50 mM), dose of etoposide in Jurkat Tlymphocytes. These results were subsequently extended using a cell-free system where it was determined that etoposide-mediated (10 mM) release of cytochrome c was dependent upon the presence of nuclei, but not Jurkat cytosol. Moreover, studies performed with isolated liver mitochondria revealed the inability of this same dose of etoposide to exert a direct effect on mitochondria, whereas 25 mM etoposide prominently diminished the ability of mitochondria to accumulate Ca 2+ , suggesting that higher doses of the drug are able to directly target and damage these organelles. Taken together, the current data clearly separate two different pathways for etoposide-induced cytochrome c release that are largely dose-dependent (Fig. 5).
Low doses of etoposide primarily exert their effect at the nuclear level, stimulating changes that ultimately lead to the release of protein factor(s) that target, and interact with, mitochondria to stimulate the release of cytochrome c. Since caspase inhibition by either z-VAD.fmk or z-VDVAD.fmk blocked this effect, and given that caspase-2 was the most apical caspase activated in intact Jurkat cells in response to etoposide, it is tempting to conclude that cytochrome c release induced by a low dose of etoposide requires active caspase-2. This conclusion is further supported by other work reporting the early activation of this caspase during apoptosis (29,30) and its nuclear localization (30,31). Additional studies designed to characterize caspase involvement in this process are currently ongoing in our laboratory.
The second pathway accounting for etoposide's toxic effect involves a direct targeting of mitochondria (Fig. 5). In this case, higher doses (³ 25 mM) of the drug elicit a caspase-independent release of cytochrome c, as well as earlier and more robust DEVDase activity, as compared to low doses, which signal through the nucleus and hence require more time to impart an effect at the mitochondrial level. Results with isolated liver mitochondria demonstrate the ability of etoposide to facilitate Ca 2+ -dependent MPT and cytochrome c release, which were preventable by CsA or EGTA. Because EGTA was able to block cytochrome c release, these results might seem to be in conflict with our original cell-free observation that a higher dose of etoposide directly targets mitochondria and stimulates cytochrome c release since the buffer used for those experiments contained EGTA. However, mitochondria were incubated in the presence of etoposide for 3 h in our cell-free system, whereas etoposide facilitated Ca 2+ -dependent MPT within several minutes in our isolated mitochondria model. Nonetheless, given that cytochrome c release was demonstrated in the presence of EGTA, a known inhibitor of MPT, suggests that there is more than one mechanism by which etoposide directly targets and damages mitochondria, resulting in cytochrome c release. It should be mentioned that the addition of etoposide to energized isolated mitochondria resulted in a sub-maximal decrease in Dy, which was not CsA-or EGTA-preventable. Therefore, this effect was not due to MPT but more likely due to a dissipation of Dy induced by futile cycling of either protons (indicative of mitochondrial uncoupling) or potassium ions (indicative of potassium entry into mitochondria).
A high dose of etoposide induced a decrease in the Ca 2+ -buffering capacity of mitochondria in permeabilized cells. This might be due to both an induction of MPT, at least in a sub-population of mitochondria, and a decrease in mitochondrial membrane potential, the main driving force for etoposide, indicating that MPT is not likely to be a dominating factor. Nonetheless, since Jurkat, and other, cells in culture normally maintain a lower rate of mitochondrial respiration (Crabtree effect) (32), and hence a lower Dy, etoposide-induced dissipation of Dy observed in isolated mitochondria may be more severe in these cells as even a slight disruption in Dy could significantly increase the likelihood of MPT.
It is important to add that the two pathways are unlikely to be mutually exclusive. In other words, it does not seem to be the case that higher doses of etoposide only target mitochondria, whereas lower doses of the drug only damage DNA. This could account for the apparent inability of z-VAD.fmk to block the release of cytochrome c induced by 10 mM etoposide in Jurkat cells at 6 h. Here, the lower dose of the drug ultimately targets mitochondria, exerting a similar effect as a higher dose at 3 h, and hence its ability to elicit cytochrome c release at this time-point is not caspase-dependent. This was observed not only for cytochrome c release in Jurkat cells but also in the decreased, and similar, abilities of Jurkat mitochondria treated with either a high or low dose of etoposide to accumulate Ca 2+ in a permeabilized model ( Fig. 5C and D).
In summary, we have demonstrated the ability of etoposide to stimulate cytochrome c release via two distinct pathways. On the one hand, low doses of the drug predominantly target the nucleus where damage to DNA triggers subsequent caspase-dependent events that converge on the mitochondria and elicit cytochrome c release. In contrast, higher doses of etoposide are directly toxic to mitochondria, altering their ability to accumulate Ca 2+ , increasing their sensitivity to MPT and stimulating the release of cytochrome c; an event that is not diminished by caspase inhibition.