Intrinsic Pathway of Hydroquinone Induced Apoptosis Occurs via Both

Feb 25, 2005 - Molecular Toxicology and Environmental Health Sciences Program, Department of Pharmaceutical Sciences, School of Pharmacy, University o...
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Articles Intrinsic Pathway of Hydroquinone Induced Apoptosis Occurs via Both Caspase-Dependent and Caspase-Independent Mechanisms Salmaan H. Inayat-Hussain*,† and David Ross Molecular Toxicology and Environmental Health Sciences Program, Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver, Colorado 80262 Received August 24, 2004

The role of mitochondria and apical caspases in apoptosis induced by the benzene metabolite hydroquinone (HQ) remains to be elucidated. Here, we investigated the involvement of mitochondria and activation of the apical caspases-8 and -9 in HQ induced apoptosis in myeloperoxidase (MPO)-rich HL-60 and MPO-deficient Jurkat T cells. Treatment of HL-60 and Jurkat cells with HQ resulted in apoptosis as assessed by phosphatidyl serine (PS) exposure, loss of mitochondrial transmembrane potential (MTP), release of cytochrome c, and processing of apical caspases-8 and -9 and executioner caspase-3. In HQ-treated HL-60 cells, pretreatment with the pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (ZVAD), which did not inhibit PS exposure, also failed to abrogate the loss of MTP and release of cytochrome c. However, complete processing of caspase-9 was inhibited in the presence of ZVAD. In marked contrast, in HQ-treated Jurkat cells, ZVAD significantly abrogated PS exposure, loss of MTP, and caspase-9 processing but not release of cytochrome c. Although ZVAD-sensitive caspase-8 processing occurred in both cell types, pretreatment with either fasreceptor blocking ZB4 or fas-ligand NOK1 neutralizing antibodies did not inhibit HQ-induced apoptosis. In conclusion, our results demonstrate that HQ induced apoptosis in Jurkat cells occurs via a ZVAD-inhibitable, caspase-dependent process, while in HL-60 cells, apoptosis occurs predominantly via caspase-independent mechanisms. Our results emphasize that both caspasedependent and independent mechanisms should be considered in the intrinsic apoptotic pathway induced by HQ.

Introduction Activation of caspases, cysteine proteases that cleave substrates after aspartate residues, has been shown to play a vital role in the initiation and execution phases of many models of apoptosis (1). Upstream or initiator caspases such as caspase-8 and -9 are believed to participate in instigating the caspase activation cascade leading to further orchestration of downstream caspases such as caspase-3 and -7. This results in morphological and biochemical hallmarks of apoptosis (2). In the past decade, several studies have demonstrated the involvement of extrinsic (receptor-mediated) pathways initiated by the Fas/Fas ligand (Fas L) system leading to caspase-8 activation, which occurs in many types of toxic stimuli including anticancer drugs and other toxicants (3-6). In contrast to receptor mediated signaling, a central role for the mitochondria (intrinsic pathway) in regula* Corresponding author. Tel: (303) 315-6077. Fax: (303) 315-0274. E-mail: [email protected]. † Permanent address: Department of Biomedical Science, Faculty of Allied Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia.

tion of chemical induced apoptosis has been demonstrated (reviewed in refs 7 and 8). With the development and use of potentiometric fluorochromes, many models of apoptosis show the loss of the mitochondrial transmembrane potential (MTP) mediated by the opening of the megachannel (permeability transition pore), which precedes caspase activation (9, 10). Cytochrome c has been demonstrated to be released once mitochondria lose integrity, and a cytoplasmic complex with cytochrome c, dATP, APAF-1, and caspase-9 called the apoptosome is formed, which initiates the apical caspase cascade (1, 2, 7). Although caspase involvement in the classical intrinsic apoptotic pathway has largely been considered postmitochondrial, recent work has suggested that some caspases, such as caspase 2, may be involved in signaling the initiation of apoptosis and release of mitochondrial mediators (11). One proposed mechanism underlying benzene induced myelotoxicity involves the benzene metabolite hydroquinone (HQ), which is bioactivated to benzoquinone (BQ) in myeloperoxidase (MPO) rich cells (12, 13). Further evidence showed that HQ induced apoptosis in MPO rich

10.1021/tx049762o CCC: $30.25 © 2005 American Chemical Society Published on Web 02/25/2005

Mechanisms of Hydroquinone Induced Apoptosis

bone marrow progenitor cells, target cells for induction of aplastic anemia and leukemia and in HL-60 myeloid leukemia cells (12, 14). HL-60 cells have often been used as a surrogate for studying myeloid progenitor cells in vitro (15). HQ, produced from pyrolysis of tobacco, has also been demonstrated to inhibit proliferation of human T-lymphocytes, and the antiproliferative effect of HQ has also been shown in the human Jurkat T cell line (16). Recently, we have shown that hydroquinone can induce apoptosis in both human HL-60 myeloid and MPOdeficient Jurkat T-lymphoblastic leukemia cells to a similar extent, suggesting that MPO is not a prerequisite for hydroquinone induced apoptosis (17). However, HL60 and Jurkat cells exhibit differential pathways of apoptosis where the externalization of phosphatidyl serine (PS) occurs independent of inhibition of caspases by ZVAD in HL-60 but not in Jurkat cells (17), suggesting a caspase-independent process in MPO-rich HL-60 cells. The purpose of this study was to investigate the precise role of mitochondrial and apical/initiator caspases involved in HQ induced apoptosis in both HL-60 and Jurkat cells and to define caspase-dependent and independent mechanisms of apoptosis.

Experimental Procedures Chemicals. The pan-caspase inhibitor ZVAD (Z-Val-Ala-Aspfluoromethyl ketone) and the caspase-2 inhibitor ZVDVAD (ZVal-Asp-Val-Ala-Asp-fluoromethyl ketone) were obtained from Enzyme Systems (Dublin, CA). Annexin V was from Oncogene Research, and RIPA buffer was from Boehringer Mannheim (GmBh, Germany). The mitochondrial membrane dye 3,3′dihexyloxacarbocyanine iodide (DIOC6(3)) and tetramethylrhodamine ethyl ester (TMRE) were obtained from Molecular Probes (Eugene, OR), whereas clone CH-11 agonistic anti-Fas antibody and the mouse antagonistic anti-Fas monoclonal antibody, ZB4, were from Upstate. The monoclonal antibody that reacts with Fas ligand NOK-1 was from Pharmingen. The remoxipride metabolites (NCQ344 and NCQ436) were from AstraZeneca, Sodertalje, Sweden. All other reagents including HQ and N-acetylcysteine (NAC) were from Sigma Chemical (St. Louis, MO). Jurkat and HL-60 Cell Culture. The human Jurkat T lymphoblastic leukemic cell line and human promyelocytic HL60 cell line were obtained from ATCC (Rockville, MD). The cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 1% penicillin/streptomycin. Media and serum were obtained from GIBCO (Grand Island, NY). Flow Cytometric Analysis of Apoptosis. Apoptosis in HL60 and Jurkat cells (1 × 106 cells) was induced by HQ (75 and 50 µM, respectively) for 14 h as described previously (17). In some experiments, the cells were preincubated for 1 h with ZVAD (100 µM), ZVDVAD (50 µM), NAC (5 mM), ZB4 (1 µg/ mL), or NOK-1 (1 µg/mL) prior to HQ treatment. In experiments using the remoxipride metabolites, cells were treated with 100 µM NCQ344 and 100 µM NCQ436 for 14 h. Apoptosis was assessed using flow cytometry essentially as described previously using the annexin-V/propidium iodide (PI) and DIOC6(3) methods (18, 19). Cells (0.5 × 106) were resuspended in media binding buffer containing annexin-V and incubated for 12 min in the dark at room temperature. Subsequently, PI was added, and the cell suspension was immediately analyzed by flow cytometry using a FACScan (Becton Dickinson). Loss of mitochondrial transmembrane potential (MTP) during apoptosis was assessed using the cationic lipophilic fluorochrome DIOC6(3), as outlined previously (19). In some experiments, TMRE was used to confirm the loss of MTP in HQ treated HL60 cells. Briefly, 50 nM DIOC6(3) or 50 nM TMRE was added to 1 × 106 cells in 1 mL of medium for 15 min at 37 °C, and immediate analysis by flow cytometer was carried out.

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Figure 1. Differential effects of ZVAD on HQ induced loss of MTP in HL-60 cells (A) and Jurkat cells (B). Cells were treated as described in Experimental Procedures, and the loss of MTP was determined using DIOC6(3). The data represents the mean ( SE from five separate experiments. *Significant difference from control (CON, p < 0.05), and †significant difference from HQ treatment (p < 0.05) by ANOVA and Scheffe F test. Western Blotting. Cell pellets (1 × 106) were resuspended in 20 µL of RIPA buffer and kept on ice for 15 min prior to addition of 2x sample buffer (20 µL). Finally, the cells were subjected to sonication, and 20 µL of the whole cell lysates was resolved on 12% SDS-PAGE gel. The gels were then blotted onto nitrocellulose membrane, and Western blotting was carried out as outlined previously (20, 21). Mouse monoclonal procaspase-9 and rabbit polyclonal procaspase-8 antibodies were kindly provided by Drs. Yuri Lazebnik and Gerald M. Cohen, respectively. Caspase-3 antibody was from Cell Signaling. For detection of cytochrome c, the cell pellet was resuspended in cytosol extraction buffer, and cytosolic extracts were prepared as described previously (20). Cytochrome c antibody was from BD Pharmingen. Proteins were detected by Enhanced ChemiLuminescence staining. Statistical Analysis. All the data were expressed as the mean ( standard error of mean (SE). The difference in means was determined by Scheffe F-test. A significance level was established when the P value was less than 0.05 (n g 3).

Results Differential Effects of ZVAD on HQ-Induced Apoptosis. We have recently demonstrated that the pancaspase inhibitor ZVAD inhibits the externalization of phosphatidyl serine (PS) in MPO-deficient Jurkat cells but not in MPO-rich HL-60 cells (17). This suggested a differential involvement of caspase(s) during HQ induced apoptosis. In this study, we wished to further investigate the upstream events involved in HQ induced apoptosis in both HL-60 and Jurkat cells. Control HL-60 and Jurkat cells (CON), respectively, showed a background level of 12.5 and 9.2% loss of mitochondrial transmembrane potential (MTP) (Figure 1). Our results show that in both cell lines, treatment with HQ for 14 h resulted in mitochondrial damage where 80.3 and 81.5% of HL60 and Jurkat cells, respectively, lost their MTP (Figure 1). This is in agreement with our previous study (17), where we found a similar extent of PS exposure during

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Figure 2. Flow cytometric histogram of the effects of ZVAD on HQ-induced apoptosis in HL-60 cells. HL-60 cells were treated as described in Experimental Procedures, and PS exposure was determined using annexin V method. The x-axis determines the fluorescence of annexin V-FITC, which was measured on FL-1 using flow cytometry.

Figure 3. Effects of ZVAD on the loss of MTP during apoptosis induced by remoxipride metabolites. HL-60 cells were treated as shown in Figure 1. Data represent mean ( SE from four separate experiments. *Indicates significant difference from control.

apoptosis as measured by the annexin-V/propidium iodide method (Figure 2, also see Figure 9). As expected, ZVAD significantly inhibited the loss of mitochondrial transmembrane potential (MTP) induced by HQ in Jurkat cells (22.9%, Figure 1B). However, ZVAD at this concentration did not significantly inhibit the loss of MTP in HL-60 cells with approximately 58.3% cells still showing HQ-induced loss of MTP in the presence of ZVAD (Figure 1A). Consistent with the loss of MTP data, ZVAD also failed to inhibit HQ-induced apoptosis in HL60 cells as assessed by PS exposure as shown in the flow histogram (Figure 2). This is in agreement with our previous work (17), where in HL-60 cells, PS exposure occurred in the presence of ZVAD, but significant inhibition by ZVAD was observed in HQ-treated Jurkat cells. To further confirm this finding, HL-60 cells were also treated with the hydroquinone and catechol metabolites (NCQ344 and NCQ436, respectively) of the antipsychotic drug remoxipride. These polyphenolic metabolites of remoxipride were selected for study since they are known to induce apoptosis in MPO-rich HL-60 cells, and remoxipride, like benzene, has been associated with induction of aplastic anemia (18, 22). When HL-60 cells were treated with NCQ344, the percentage of cells with loss of MTP increased significantly from 13% (control cells) to 51% (Figure 3). Similarly, NCQ436 also caused a significant increase in cells that had lost MTP (57%). Pretreatment with ZVAD did not significantly effect loss of MTP; 31.5 and 38.6% of HL-60 cells still showed loss

Figure 4. Flow histogram of the effects of caspase inhibitors in HQ treated HL-60 cells. HL-60 cells were treated as described in Experimental Procedures, and loss of MTP was determined using TMRE. The x-axis determines the fluorescence of TMRE, which was measured on FL-2 using flow cytometry.

of MTP in NCQ344 and NCQ436 treated cells in the presence of ZVAD (Figure 3). Effects of a Caspase-2 Inhibitor and ROS Scavenger on HQ Induced Loss of MTP in HL-60 Cells. To confirm the findings observed in HL-60 cells, we utilized another lipopholic cationic fluorescent mitochondrial dye TMRE. During apoptosis, dissipation of the membrane potential will cause a leakage of TMRE from the matrix leading to a decrease of fluorescence intensity as measured by flow cytometry. Our results are in agreement with the DIOC6(3) data, where HQ treated HL-60 cells resulted in loss of MTP (85%), and ZVAD was unable to block dissipation of MTP (90%) (Figure 4). Caspase-2 has been suggested to play an upstream role in the initiation of apoptosis and release of mitochondrial

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Figure 5. Effects of ZVAD in cytochrome c release in HL-60 and Jurkat cells. Cytosolic cell extracts were prepared from HL60 and Jurkat cells after 14 h treatment. The cell extracts were immunoblotted with either cytochrome c antibody as described in Experimental Procedures.

pro-apoptotic proteins (11). To identify a role for caspase-2 in HQ-induced loss of MTP in HL-60 cells, we employed the irreversible caspase-2 inhibitor ZVDVAD, and as shown in Figure 4, this inhibitor alone failed to abolish HQ-induced loss of MTP (69%). Additionally, when this inhibitor was used in combination with ZVAD, no protection against the loss of MTP induced by HQ was observed (92%). This suggests that the signal leading to mitochondrial damage in HL-60 cells could be directly due to MPO catalyzed HQ to BQ with concomitant ROS production rather than being modulated by caspase-2 or other caspases. To confirm this, we used NAC, which can function as an antioxidant and as a protective agent against reactive quinones such as BQ. As shown in Figure 4, when NAC was preincubated for 1 h prior to addition of HQ, HL-60 cells were essentially unaffected, and only 8% of cells showed loss of MTP with a similar histogram profile as the control cells. These data implicate ROS and/or reactive 1,4 BQ generated from HQ in the loss of MTP in HL-60 cells. Release of Cytochrome c in HQ-Induced Apoptosis. In HQ-induced apoptosis in both HL-60 and Jurkat cells, cytochrome c was released into the cytosol as compared to control as shown in Figure 5. As expected, cytochrome c release was not blocked by the presence of ZVAD in HQ treated HL-60 cells (Figure 5A). However, in Jurkat cells, ZVAD that inhibited loss of MTP also failed to block release of cytochrome c (Figure 5B). The lack of effect of ZVAD on cytochrome c release in Jurkat cells suggests the effect of ZVAD is mediated downstream at the level of caspase activation. Processing of Caspase-9 and -8 in HQ-Induced Apoptosis. We have demonstrated the processing of caspase-3 and -7 in HQ induced apoptosis in both HL-60 and Jurkat cells leading to PARP and DNA cleavage (17). To investigate the role of initiator caspase(s) in HQ induced apoptosis, we investigated processing of caspase-9 and -8 by Western blotting. As shown in Figure 6, we found that the intact 46 kDa pro-caspase-9 was totally cleaved in HQ treated HL-60 cells to lower molecular weight products that have been reported to include 37, 35, and 18 kDa fragments (20, 23) (Figure 6A). Moderate processing of caspase-9 into its 37 and 35 kDa subunits was observed in Jurkat cells treated with HQ (Figure 6B). While complete inhibition of caspase-9 processing with ZVAD was observed in Jurkat cells, this pan-caspase inhibitor only blocked the processing of caspase-9 to the

Figure 6. Effects of ZVAD on caspase-9 processing in HQ induced apoptosis. Whole cell lysates were immunoblotted with the caspase-9 antibody. The intact protein is 46 kDa, and the cleaved products are p37 and p35 kDa.

Figure 7. ZVAD blocked caspase-3 processing in HQ-induced apoptosis. Whole cell lysates were prepared from cells treated with HQ and immunoblotted with a caspase-3 antibody. The intact caspase-2 is 32 kDa, and the cleaved products are p20, p19, and p17 kDa.

level of 37/35 kDa subunits in HL-60 cells. Nevertheless, 37/35 kDa subunits have been reported to be inactive in the presence of ZVAD (19, 23, 24). As further confirmation of the activity of the 37/35 kDa subunits in the presence of ZVAD, we examined cleavage of the downstream executioner caspase-3. The precursor form of caspase-3 is a substrate for activated caspase-9 (1). Caspase-3 processing to its p20, p19, and p17 subunits was inhibited to the level of the p20 subunit in both HL60 and Jurkat cells in the presence of ZVAD (Figure 7). We and others have shown that ZVAD inhibition of

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Figure 8. Effects of ZVAD on caspase-8 processing in HQ induced apoptosis. Whole cell lysates were immunoblotted with an antibody to caspase-8. The intact protein is a p55/53 kDa, and cleaved products are p43 and p41 kDa.

caspase-3 processing to the level of its p20 kDa subunit is inactive as cleavage of the downstream substrates PARP and DNA are inhibited in the presence of ZVAD (18-20). Furthermore, significant increases in caspase-3 activity in goniothalamin induced apoptosis in HL-60 and Jurkat cells as early as 4 h can be completely inhibited by ZVAD using the fluorogenic Z-DEVD‚AMC substrate (unpublished data), and this is associated with blockade of caspase-3 cleavage to its p20 subunit (19). The initiator caspase-8 has been shown to be recruited and activated upon engagement of the Fas/CD95 receptor leading to the downstream activation of executioner caspases. In this study, we also investigated if the Fas/FasL signaling pathway plays a role in HQ induced apoptosis. As shown in Figure 8, we found that HL-60 and Jurkat cells treated with HQ resulted in cleavage of intact procaspase-8 into the 43 and 41 forms. In both cell lines, ZVAD completely inhibited processing of caspase-8. The processing of caspase-8 in HQ induced apoptosis in both cell types suggests the possibility of the involvement of an autocrine Fas/FasL system. To investigate this possibility, we treated these leukemic cell lines with HQ with and without pretreatment with fas (ZB4) and fas-ligand (NOK-1) blocking monoclonal antibodies. Our results show that apoptosis induced by HQ in both cell lines was not inhibited by ZB4 or NOK-1 as assessed by annexin V/PI (Figure 9). In Jurkat cells, 56% of the cells underwent Fas-induced apoptosis, and this could be significantly inhibited by ZB4 (23%). However, we did not observe Fas induced apoptosis in HL-60 cells, suggesting that apoptotic effects are not likely to be mediated by Fas in HL-60 cells. These monoclonal antibodies were not toxic when treated in both cell lines, and apoptosis was essentially similar to control levels (data not shown).

Discussion The present study shows that apoptosis induced by HQ in HL-60 and Jurkat cells was accompanied by the loss of MTP and processing of caspase-9 and caspase-8 (Figures 1-4). There is now a wealth of evidence demonstrating that the dissipation of MTP occurs early in the commitment phase of apoptosis, which results in the release of mitochondrial pro-apoptotic proteins including cytochrome c and the apoptotic inducing factor (AIF)

Figure 9. Effects of ZB4 and NOK-1 on HQ induced apoptosis. Cells were treated as shown in Figure 1, and apoptosis was determined using annexin-V/PI. Panels A and B represent HL60 and Jurkat cells, respectively. *Indicates significant difference from control.

(reviewed in ref 25). Furthermore, apical caspase activation or putative cytosolic substrate cleavage/activation have been recognized to promote the mitochondrial loss of membrane potential in a ZVAD inhibitable manner in both HL-60 and Jurkat cells (26-28). In this study, HQ treatment in HL-60 cells and Jurkat cells caused en masse loss of MTP. ZVAD was not able to significantly block HQ-induced loss of MTP in HL-60 cells using both DIOC6(3) and TMRE dyes, strengthening the argument for a ZVAD-insensitive/caspase-independent mechanism of HQ-induced apoptosis in MPO rich cells. In addition, the caspase-2 inhibitor, ZVDVAD either alone or in combination with ZVAD, also failed to block HQ-induced loss of MTP. When HL-60 cells were treated with NCQ344 and NCQ436, the hydroquinone and catechol metabolites of the anti-psychotic drug remoxipride, ZVAD, did not significantly block loss of MTP, further confirming the finding with HQ induced loss of MTP in MPO rich HL-60 cells (Figure 3). These data are in agreement with our previous studies where PS exposure in HL-60 cells treated with HQ, NCQ344, or NCQ436 occurred in a ZVAD-insensitive pathway(s) (17, 18). As a positive control, ZVAD has been demonstrated to abolish the loss of MTP and PS exposure as a result of treatment with the natural product goniothalamin in HL-60 cells (19), confirming the functionality of a caspase-dependent pathway leading to loss of MTP and PS exposure in this specific cell type. A caspase-independent or ZVAD-insensitive pathway resulting in the loss of MTP may be due to HQ perturbing mitochondria directly either because of a rapid metabolism of HQ to benzoquinone (BQ) and/or ROS generated as a result of HQ autoxidation or HQ/SQ•/BQ cycling.

Mechanisms of Hydroquinone Induced Apoptosis

MPO catalyzed oxidation of HQ to BQ can form thiol adducts, depletion/modification of GSH, or critical thiols that may occur in the mitochondria leading to irreparable damage (29-31). BQ has also been shown to depolarize the mitochondrial membrane directly (32), and this could result in the opening of mitochondrial permeability transition (PT) pores. The data obtained using NAC that blocked HQ-induced loss of MTP in HL-60 cells suggests that generation of ROS and reactive 1,4 BQ from HQ are responsible for mitochondrial damage. Mitochondrial damage and release of pro-apoptotic mitochondrial proteins could trigger PS exposure and morphological changes consistent with apoptosis in a ZVAD-insensitive manner (33). Interestingly, it has recently been suggested that apoptosis as indicated by PS exposure can occur through loss of MTP/cytochrome c in a ZVAD-independent manner (34). In our study, we found that release of cytochrome c occurs in both HQ treated HL-60 and Jurkat cells (Figure 5). Upon pretreatment with the pan-caspase inhibitor ZVAD, release of cytochrome c in these cell lines was not inhibited, suggesting that the inhibitory effects of ZVAD are mediated on downstream caspases rather than upstream events such as mitochondrial cytochrome c release, in agreement with previous work on chemicalinduced apoptosis (20). The identity of pro-apoptotic mitochondrial proteins released in HL-60 cells was not a focus of this study. However, AIF is a possible candidate since we have shown previously that in HL-60 cells, HQ in the presence of ZVAD still induces condensation of chromatin (17), and chromatin condensation has been well-characterized as one of the functions of AIF (7). However, other as yet unidentified proteins may also play a role in ZVAD insensitive apoptosis in HL-60 cells, and further work will be needed to characterize their identity. Two initiation pathways of apoptosis have been demonstrated, namely, the intrinsic pathway that perturbs the mitochondria resulting in release of proapoptotic proteins and the extrinsic pathway where the cell surface death ligand is recruited. Both pathways converge at a common point resulting in the execution of apoptosis (35). Release of caspase-9 from the mitochondria has been demonstrated to occur when there is a loss of MTP during apoptosis (33). After HQ treatment, we observed loss of intact caspase-9 in both HL-60 and Jurkat cells (Figure 6). Interestingly, after treatment of HL-60 cells for 14 h, only a small amount of p35/37 subunits was observed, but in the presence of ZVAD, caspase-9 cleavage was inhibited with concomitant accumulation of the p35/37 kDa. p35/37 subunits are inactive in the presence of ZVAD, and a concentration of this inhibitor as low as 100 nM has been shown to inhibit caspase-9 activity (LEHD‚ AFC) and generation of cleaved caspase-9 products (20, 23). In this study, we also confirmed that inhibition of cleavage of downstream caspase-9 substrates such as caspase-3 occurred. Inhibition of caspase-3 cleavage to its inactive p20 subunit in HQ treated HL-60 and Jurkat cells occurred in the presence of ZVAD. A similar inhibition profile of this pan-caspase inhibitor was also demonstrated by using NCQ344 and NCQ436 as well as in goniothalamin treated HL-60 cells (18, 19). Jurkat cells that showed a similar percentage of HQ-induced apoptosis as HL-60 cells had significant cleavage of caspase9, and inhibition by ZVAD completely inhibited the processing of this caspase, such that accumulation of the precursor 46 kDa form could be observed.

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Figure 10. Schematic of the hypothesized mechanisms of mitochondrial mediated HQ induced apoptosis in MPO-rich HL60 and MPO-deficient Jurkat cells. Two pathways induced by HQ converge at a common point in the mitochondria leading to execution of apoptosis in caspase-dependent and independent manner.

The involvement of caspase-8 has been shown to be predominantly related to the extrinsic pathway of apoptosis. This caspase has been shown to be recruited and activated upon engagement of the Fas/CD95 receptor leading to the downstream activation of executioner caspases. Therefore, in this study, we wished to investigate the role of Fas/FasL signaling pathway in HQ induced apoptosis. As shown in Figure 8, we found that HL-60 and Jurkat cells treated with HQ resulted in cleavage of intact pro-caspase-8 into the 43 and 41 forms. In both cell lines, ZVAD completely inhibited processing of caspase-8. Although processing of caspase-8 could occur downstream of caspase-3 activation as a component of the intrinsic pathway as in PPARγ ligand triterpenoid CDDO induced apoptosis (36), the processing of caspase-8 in HQ induced apoptosis in HL-60 and Jurkat cells suggests the possibility of the involvement of an autocrine Fas/FasL system. Fas/fas ligand interactions have been demonstrated to mediate certain chemically induced apoptosis via caspase-8 activation (3-6, 37). To explore whether HQ treatment of HL-60 and Jurkat cells resulted in the activation of extrinsic pathways, we employed fas (ZB4) and fas-ligand (NOK-1) blocking monoclonal antibodies. Our results show that these inhibitors had little effect on HQ induced apoptosis in either HL-60 or Jurkat cells (Figure 9). That these inhibitors failed to inhibit apoptosis induced by HQ in both cell types suggests that the intrinsic mitochondrial pathway is responsible for HQ induced apoptosis. However, from this study, we cannot rule out the involvement of other receptor mediated apoptosis such as TRAIL or TNF-R. Although Jurkat and HL-60 cells cannot be directly compared since they are very different cell types, they have proved useful for defining caspase-dependent and caspase-independent mechanisms of apoptosis. Collectively, our results from the current work and our previous studies (17, 18) demonstrate that apoptosis induced by HQ can occur to a similar extent in both MPO-rich and MPO-deficient cells. Our data demonstrate that HQ treatment in both Jurkat and HL-60 cells is accompanied with downstream post-mitochondrial caspase-dependent

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processes leading to activation of caspase-9, -3, and -7; PARP; and DNA cleavage in a ZVAD-sensitive manner (Figure 10). In Jurkat cells, HQ-induced apoptosis occurs in a ZVAD-sensitive pathway. In HL-60 cells, however, the majority of apoptosis occurs via a ZVAD-insensitive mechanism, suggesting a caspase-independent pathway. Therefore, the conclusion of this work is that there are markedly different mechanisms of HQ-induced apoptosis in different leukemia cell types, and these mechanisms can either be caspase-dependent or independent. We propose that the caspase independent apoptotic mechanism occurs via MPO-catalyzed activation of HQ to highly reactive quinones and reactive oxygen species in MPOrich HL-60 cells, which directly damage mitochondria. Further definition of the role of MPO in caspaseindependent apoptosis induced by HQ needs to be pursued in well-defined genetic model systems. We speculate that mitochondrial damage then results in the release of apoptotic proteins such as AIF, Endo G, and as yet unidentified factors (25, 38) that can induce apoptosis in a caspase-independent manner. These data have implications for studies where caspase-activation has been used as a biomarker of benzene-induced apoptosis (39). Our results suggest that apoptosis may be underestimated by only measuring caspase activation and emphasize that both caspase-dependent and independent mechanisms should be considered in the intrinsic apoptotic pathway induced by benzene metabolites. Understanding the mechanisms of HQ induced apoptosis may have important implications in the pathophysiology of benzeneinduced myelotoxicity.

Acknowledgment. Work on benzene and its metabolites was supported by NIH RO1 ES09554 (D.R.). Work on remoxipride was supported by a grant from AstraZeneca, Sodertalje.

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