Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

A Dual Role for Poly(ADP-Ribose) Polymerase-1 
During Caspase-Dependent Apoptosis

A Dual Role for Poly(ADP-Ribose) Polymerase-1 
During Caspase-Dependent Apoptosis Abstract 2,3,5-Tris(glutathion-S-yl)hydroquinone (TGHQ), a metabolite of benzene, catalyzes the generation of reactive oxygen species (ROS) and caspase-dependent apoptosis in human promyelocytic leukemia (HL-60) cells. We now report that TGHQ induces severe DNA damage, as evidenced by DNA ladder formation and H2AX phosphorylation. The subsequent activation of the DNA nick sensor enzyme, poly(ADP-ribose) polymerase-1 (PARP-1), leads to the rapid depletion of ATP and NAD and the concomitant formation of poly(ADP-ribosylated) proteins (PARs). PJ-34 (a PARP-1 inhibitor) completely prevented the formation of PARs, partially attenuated TGHQ-mediated ATP depletion, but had little effect on NAD depletion. Intriguingly, although z-vad-fmk (a pan-caspase inhibitor) attenuated TGHQ-induced apoptosis, cotreatment with PJ-34 led to a further decrease in apoptosis, suggesting that PARP-1 participates in caspase-dependent apoptosis. Indeed, PARP-1 inhibition reduced TGHQ-induced caspase-3, -7, and -9 activation, at least partially by attenuating cytochrome c translocation from mitochondria to the cytoplasm. In contrast, PJ-34 potentiated TGHQ-induced caspase-8 activation, suggesting that PARP-1 plays a dual role in regulating TGHQ-induced apoptosis via opposing effects on the intrinsic (mitochondrial) and extrinsic (death-receptor) pathways. PARP-1 knockdown in HL-60 cells confirmed that PARP-1 participates in effector caspase activation. Finally, PJ-34 also inhibited TGHQ-induced apoptosis-inducing factor (AIF) nuclear translocation, but neither c-jun NH(2)-terminal kinase nor p38 MAPK (p38 mitogen-activated protein kinase) activation was required for AIF translocation. In summary, TGHQ-induced apoptosis of HL-60 cells is accompanied by PARP-1, caspase activation, and AIF nuclear translocation. TGHQ-induced apoptosis appears to primarily occur via engagement of the mitochondrial-mediated pathway in a process amenable to PARP inhibition. Residual cell death in the presence of PJ-34 is likely mediated via the extrinsic apoptotic pathway. TGHQ, PARP-1, apoptosis, caspase, AIF, TGHQ Poly(ADP-ribose) polymerase-1 (PARP-1) is a member of a family of proteins that constitute a superfamily of at least 18 putative PARP proteins, based on protein domain homology and enzymatic function (Diefenbach and Burkle, 2005). PARP-1 
is activated by DNA strand nicks and breaks, which can be induced by a variety of environmental stimuli or by endogenous reactive oxygen species (ROS) (Andrabi et al., 2006; Virag and Szabo, 2002). In the absence of DNA damage, poly(ADP-ribosylation) is a rare event but increases over 100-fold upon DNA damage. PARP-1 catalyzes the formation of ADP-ribose polymers from nicotinamide adenine dinucleotide (NAD+) on various nuclear proteins, including histones (Adamietz and Rudolph, 1984), DNA polymerases (Yoshihara et al., 1985), topoisomerases (Scovassi et al., 1993), and PARP-1 itself (Erdelyi et al., 2005). Although PARP-1 may facilitate DNA repair and cell survival in response to mild genotoxic stimuli, it contributes to cell death in the face of severe DNA damage (Virag, 2005). Although PARP-1–dependent NAD+ consumption and energy failure are thought to contribute to cell death, the precise mechanisms by which PARP-1 regulates apoptotic cell death remain unclear. Apoptosis-inducing factor (AIF) is a flavin adenine dinucleotide–containing, NADH-dependent oxidoreductase residing in the mitochondrial intermembrane space (Sevrioukova, 2011). It is believed to play a central role in the regulation of PARP-induced cell death, because it is released into the cytoplasm after PARP-1 activation and ultimately translocates to the nucleus where it triggers chromatin condensation and large-scale DNA degradation in a caspase-independent manner (Siegel and McCullough, 2011). Moreover, AIF has emerged as a protein critical for cell survival, reflecting the complexity and multilevel regulation of AIF-mediated signal transduction (Sevrioukova, 2011). However, the relationship between AIF and PARP-1 in cell death remains unclear. Benzene is a ubiquitous chemical widely used in the manufacturing industry. However, benzene causes bone marrow suppression in rodents and is hematotoxic and leukemogenic in humans (Rinsky et al., 1981; Yin et al., 1987). Because benzene must be metabolized to mediate its harmful effects, a number of benzene metabolites have been implicated in benzene-mediated hematotoxicity (Eastmond et al., 1987; Snyder et al., 1993). 2,3,5-Tris(glutathion-S-yl)hydroquinone (TGHQ), a metabolite of benzene, causes hematotoxicity in rats and induces apoptosis in human promyelocytic leukemia (HL-60) cells in a ROS-dependent fashion (Bratton et al., 1997, 2000; Yang et al., 2005). Thus, in this study, we investigated the role of PARP-1 in TGHQ-mediated, ROS-dependent HL-60 cell apoptosis, and in particular the relationship between PARP-1 activation and caspase- and AIF-mediated cell death. Materials and Methods Reagents Caution. TGHQ is nephrotoxic and nephrocarcinogenic in rats and must therefore be handled with protective clothing and in a ventilated hood. Chemicals. Unless otherwise specified, all chemicals were purchased from Sigma Chemical Co. (St Louis, MO). All reagents were of the highest grade commercially available. TGHQ was synthesized in the laboratory according to established methodology (Lau et al., 1988). Antibodies and all other assay kits: GAPDH, c-jun NH(2)-terminal kinase (JNK), p-JNK, p38, p-p38, cytochrome c, caspase-3, -7, -8, cleaved caspase-9, β-actin, CoxIV, and AIF were all purchased from Cell Signaling Technology (San Diego, CA). The antibody for poly(ADP-ribose) was purchased from Biomol International (Plymouth Meeting, PA). The DNA fragmentation kit was purchased from Invitrogen (Carlsbad, CA). The pan-caspase inhibitor z-vad-fmk and the annexin-V/propidium iodide (PI) kit were obtained from BD Bioscience. Cell Culture Conditions The promyelocytic leukemia HL-60 cell line was obtained from the American Type Culture Collection (Manassas, VA). HL-60 cell suspensions were cultured in RPMI 1640 medium (Gibco BRL, Grand Island, NY) supplemented with 20% fetal bovine serum. No antibiotics were used in our experiments, and cells were incubated in a humidified atmosphere with 5% CO2 in air at 37°C. Exponentially growing cells were seeded at a cell density of 1.0 × 106 cells/ml. DNA Fragmentation Assay Apoptotic DNA fragmentation in HL-60 cells was determined using the Quick Apoptotic DNA Ladder Detection Kit (Invitrogen). Briefly, cells were treated with different concentrations of TGHQ for the indicated time periods. After the incubation, cells were harvested by centrifugation and washed with phosphate-buffered saline (PBS). DNA fragments were extracted according to the manufacturer’s instructions. Finally, DNA fragments were visualized following electrophoresis on a 1% agarose gel. Histone Extraction HL-60 cells were washed with 3 ml PBS and lysed in 1 ml ice-cold hypotonic lysis buffer (10mM Tris-HCl, pH 8.0, 1mM KCl, 1.5mM MgCl2, and 1mM DTT). Histones were extracted with 0.4N H2SO4 overnight at 4°C and then precipitated in trichloroacetic acid on ice for 0.5 h. The resulting pellet was washed with ice-cold acetone. Pellets were dried in air and subjected to Western blot analysis. Western Blot Analysis After specified treatments, HL-60 cells were washed twice with PBS and lysed in ice-cold RIPA (Radio-Immunoprecipitation Assay) lysis buffer. For nuclear protein extraction, the Nuclear Extraction Kit from Panomics, Inc. was used. After incubation for 15 min on a rotator at 4°C, cell lysates were centrifuged to remove cell debris. Protein concentrations were determined with detergent-compatible reagent (Bio-Rad Laboratories, Hercules, CA). Samples were incubated with sample buffer and boiled for 5 min at 100°C, and resolved in SDS polyacrylamide gels. After electrophoresis, proteins were transferred to a PVDF membrane (Millipore, Billerica, MA). After blocking with 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (TBST), membranes were incubated with appropriate primary antibodies (1:1000 dilution) overnight at 4°C. After washing in TBST, membranes were incubated with appropriate horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology). After thoroughly washing in TBST, bound antibodies were visualized using standard chemiluminescence on autoradiographic film. Preparation of Cytosolic and Mitochondrial Extracts After treatment of cells with TGHQ, cytosolic and mitochondrial extracts were prepared as described (Holden and Horton, 2009). Cells were harvested and centrifuged at 100 RCF at 4°C to pellet the cells. The supernatant was aspirated, and the cells were washed by gentle pipetting into 1 ml of ice-cold PBS. The cell suspension was again centrifuged at 4°C to pellet the cells, the supernatant aspirated and the cell pellet resuspended by gentle pipetting into 400 µl of ice-cold buffer 1 (150mM NaCl, 50mM HEPES, pH 7.4, 25 µg/ml digitonin, and 1mM PMSF). The cell suspension was gently mixed at 4°C for 10 min and then centrifuged at 2000 RCF to pellet the cells. The supernatants (cytosolic fraction) were saved. Cell pellets were resuspended by vortexing in 400 µl of ice-cold buffer 2 (150mM NaCl, 50mM HEPES, pH 7.4, and 1% NP40), incubated on ice for 30 min,and then centrifuged at 7000 RCF to pellet nuclei and cell debris. The supernatant, containing the mitochondrial proteins, was then collected for further analyses. Annexin-V/PI Staining Annexin-V/PI staining was used to detect early apoptotic cells during apoptotic progression. After treatment with TGHQ for 4 h alone or in combination with specific inhibitors, cells were collected and washed with cold PBS twice and then resuspended with 100 µl of binding buffer (10mM HEPES, 140mM NaCl, and 2.5mM CaCl2, pH 7.4) at a concentration of 1 × 106 cells/ml. The cell suspension (100 µl) was mixed with 5 µl of FITC-conjugated annexin-V and 5 µl of PI and incubated for 15 min at room temperature in the dark. Thereafter, cells were immediately added to 400 µl of binding buffer and analyzed by flow cytometry (Becton Dickinson, San Jose, CA). The dual-parametric dot plots were used for calculation of the percentage of nonapoptotic live cells in the lower left quadrant (annexin-V–/PI–), early apoptotic cells in the lower right quadrant (annexin-V+/PI–), late apoptotic cells in the upper right quadrant (annexin-V+/PI+), and necrotic cells in the upper left quadrant (annexin-V–/PI+). Cellular ATP Assay HL-60 cells were treated with test compounds. After the indicated time, cells were collected, resuspended in RPMI 1640, and seeded in opaque-walled 96-well plates. ATP content per well was determined using the CellTiter-Glo luminescent assay (Promega, Madison, WI) according to the manufacturer’s instructions. Determination of Total NAD Content Cells were seeded in 6-well plates at 2 × 106 cells/well and treated with 200µM TGHQ with or without PJ-34 (10µM) cotreatment for varying periods of time. NAD was extracted by addition of 1.0M HClO4 to dishes of cells on ice. Cell extracts were neutralized to pH 7.0 by addition of 2M KOH/0.66M KH2PO4. NAD concentrations were determined by enzymatic cycling assays as described (Jacobson and Jacobson, 1997). Small Interfering RNA Transfection Knockdown of PARP-1 was performed in HL-60 cells using predesigned ON-TARGETplus siRNA SMART pool purchased from Dharmacon. The transfections were performed by the electroporation method (Lonza, Nucleofector Program T-019), and the same numbers of the transfected cells were seeded in 6-well plates for treatment. ON-TARGETplus nontargeting siRNA pool was used as a control for nonsequence-specific effects. Statistical Analyses Data are expressed as mean ± SE. Statistical differences between the treated and control groups were determined by Student’s paired t-test. Differences between groups were assessed by one-way ANOVA using the SPSS software package for Windows. Differences between means were considered significant if p < 0.05. Results PARP-1 Is Activated in Response to TGHQ-Mediated DNA Damage TGHQ induced typical oligonucleosomal DNA ladder formation, confirming cell death in HL-60 cells is apoptotic in nature (Fig. 1A). To further confirm the DNA-damaging effect of TGHQ, cell lysates were analyzed for the presence of the phosphorylated form of the histone variant H2AX (γ-H2AX), an indicator of the presence of DNA double-strand breaks (Rogakou et al., 1998). TGHQ significantly induced the formation of γ-H2AX in HL-60 cells (Fig. 1B). PAR formation also increased in HL-60 cells following treatment with TGHQ and reached a peak 3 h after dosing (Fig. 2). FIG. 1. Open in new tabDownload slide TGHQ induces DNA damage in HL-60 cells. Cells were treated with various concentrations of TGHQ for the indicated periods of time. (A) Fragmented genomic DNA was extracted and resolved on 1% agarose gel. Apoptotic DNA was visualized by ethidium bromide staining. (B) Western blot analysis of H2AX and γ-H2AX levels. Cells were treated with TGHQ (200µM) for various periods of time and histones extracted and subjected to Western blot analysis. FIG. 1. Open in new tabDownload slide TGHQ induces DNA damage in HL-60 cells. Cells were treated with various concentrations of TGHQ for the indicated periods of time. (A) Fragmented genomic DNA was extracted and resolved on 1% agarose gel. Apoptotic DNA was visualized by ethidium bromide staining. (B) Western blot analysis of H2AX and γ-H2AX levels. Cells were treated with TGHQ (200µM) for various periods of time and histones extracted and subjected to Western blot analysis. FIG. 2. Open in new tabDownload slide TGHQ induces PARP-1 activation. HL-60 cells were treated with 200µM TGHQ for various periods of time, and PAR accumulation was determined by Western blot analysis. Equal loading was confirmed by Ponceau S staining. For densitometric analysis of Western blots, values are expressed as fold change relative to the appropriate controls. Values represent the mean ± SE (n = 3). Significantly different from controls at †p < 0.05; *p < 0.01. FIG. 2. Open in new tabDownload slide TGHQ induces PARP-1 activation. HL-60 cells were treated with 200µM TGHQ for various periods of time, and PAR accumulation was determined by Western blot analysis. Equal loading was confirmed by Ponceau S staining. For densitometric analysis of Western blots, values are expressed as fold change relative to the appropriate controls. Values represent the mean ± SE (n = 3). Significantly different from controls at †p < 0.05; *p < 0.01. TGHQ Induces ATP and NAD Depletion in HL-60 Cells The remarkable PARP activation led us to subsequently examine the effect of TGHQ on ATP and NAD+ concentrations in HL-60 cells. TGHQ caused a rapid depletion of both cellular ATP (Fig. 3A) and NAD+ (Fig. 3B) in HL-60 cells within 6 h of exposure, indicating the overactivation of PARP-1 in response to TGHQ-induced DNA damage. FIG. 3. Open in new tabDownload slide TGHQ causes ATP and NAD depletion in HL-60 cells. Cells were treated for up to 6 h with TGHQ in the presence or absence of a PARP inhibitor, PJ-34 (10µM). (A) ATP content was determined using the ATP luminescent assay. (B) NAD concentration was determined with the method described in Materials and Methods. Data represent the mean ± SE (n ± 3). •, control; ○, PJ-34; ■, TGHQ, and □, (TGHQ+PJ-34). Significantly different from controls at *p < 0.001. FIG. 3. Open in new tabDownload slide TGHQ causes ATP and NAD depletion in HL-60 cells. Cells were treated for up to 6 h with TGHQ in the presence or absence of a PARP inhibitor, PJ-34 (10µM). (A) ATP content was determined using the ATP luminescent assay. (B) NAD concentration was determined with the method described in Materials and Methods. Data represent the mean ± SE (n ± 3). •, control; ○, PJ-34; ■, TGHQ, and □, (TGHQ+PJ-34). Significantly different from controls at *p < 0.001. PARP-1 Modulates TGHQ-Induced HL-60 Cell Apoptosis To gain an insight into whether the activation of the pro apoptotic PARP pathway is involved in TGHQ-induced HL-60 cell apoptosis, we further determined by flow cytometry whether PJ-34, a specific PARP inhibitor, could ameliorate apoptosis. PJ-34 itself did not lead to any apoptosis in HL-60 cells (Fig. 4). Interestingly, however, apoptosis induced by TGHQ (200µM) was decreased from 25.7 to 18.9% (an ~30% reduction) in the presence of PJ-34, indicating that PARP-1 regulates TGHQ-induced apoptosis (with the caveat that PJ-34 is behaving exclusively as a PARP-1 inhibitor). To test the efficiency of PJ-34 as a PARP inhibitor, the effect of PJ-34 on PAR-modified proteins, as well as ATP and NAD concentrations in TGHQ-treated cells, was analyzed. Protein-bound PAR was evident in TGHQ-treated HL-60 cells (Fig. 5A) but was completely absent in PJ-34–treated cells. It is noteworthy that PAR formation in response to TGHQ-induced DNA damage decreases with increasing concentrations of TGHQ. Whether this occurs subsequent to the activation of caspase-3, and the concomitant cleavage (and inactivation) of PARP-1 (Kaufmann et al., 1993), remains to be determined. However, consistent with this view, the cleaved form of PARP-1 increased in parallel with increases in caspase-3 activation (Fig. 5B). Similarly, PJ-34 markedly attenuated TGHQ-mediated ATP depletion (Fig. 3A). In contrast, PJ-34 had little effect on TGHQ-mediated decreases in NAD concentrations (Fig. 3B). FIG. 4. Open in new tabDownload slide PJ-34 protects against TGHQ-induced HL-60 cell apoptosis. Cells were treated with various concentrations of TGHQ for 4 h, followed by costaining with PI and FITC-conjugated annexin-V, which specifically detects the translocation of phosphatidylserine to the cell surface. Cells were then analyzed by flow cytometry. FIG. 4. Open in new tabDownload slide PJ-34 protects against TGHQ-induced HL-60 cell apoptosis. Cells were treated with various concentrations of TGHQ for 4 h, followed by costaining with PI and FITC-conjugated annexin-V, which specifically detects the translocation of phosphatidylserine to the cell surface. Cells were then analyzed by flow cytometry. FIG. 5. Open in new tabDownload slide PJ-34 inhibits TGHQ-induced PARP-1 activation. Cells were treated with various concentrations of TGHQ in the presence or absence of PJ-34 for 3 h. (A) PAR accumulation was determined with a PAR antibody, SA-216. Equal loading was confirmed by Ponceau S staining. (B) Effect of TGHQ on PARP-1 cleavage and caspase-3 activation. For the quantitative analysis of Western blots by densitometry, values are expressed as fold changes relative to the appropriate controls. Values are given as mean ± SE (n = 3). †p < 0.05; *p < 0.001 compared with control. FIG. 5. Open in new tabDownload slide PJ-34 inhibits TGHQ-induced PARP-1 activation. Cells were treated with various concentrations of TGHQ in the presence or absence of PJ-34 for 3 h. (A) PAR accumulation was determined with a PAR antibody, SA-216. Equal loading was confirmed by Ponceau S staining. (B) Effect of TGHQ on PARP-1 cleavage and caspase-3 activation. For the quantitative analysis of Western blots by densitometry, values are expressed as fold changes relative to the appropriate controls. Values are given as mean ± SE (n = 3). †p < 0.05; *p < 0.001 compared with control. Caspase-Dependent HL-60 Cell Death Is Regulated by PARP-1 The broad-spectrum caspase inhibitor, z-vad-fmk, significantly inhibited TGHQ-induced HL-60 cell apoptosis (Fig. 6A), completely abrogating apoptosis at a concentration of 80µM. TGHQ-induced apoptosis in HL-60 cells is, thus, caspase dependent. Interestingly, the residual apoptosis occurring in HL-60 cells exposed to lower concentrations (< 80µM) of z-vad-fmk was further reduced when PJ-34 was added to the cell cultures, suggesting that PARP-1 can influence caspase(s) activation. To further verify this possibility, the PARP-1 gene was knocked down in HL-60 cells and caspase-3 (the effector caspase) activation in the presence of TGHQ was determined. Caspase-3 activation was greatly attenuated in PARP-1 siRNA-treated cells (Fig. 6B). FIG. 6. Open in new tabDownload slide Genetic or pharmacological inhibition of PARP-1 prevents TGHQ-mediated caspase activation and attenuates apoptosis. (A) Cells were treated with PJ-34 and/or different concentrations of z-vad-fmk in the presence or absence of 200µM of TGHQ for 4 h, followed by costaining with PI and FITC-conjugated annexin-V, which specifically detects the translocation of phosphatidylserine to the cell surface. Cells were then analyzed by flow cytometry. (B) Expression of PARP-1 was knocked down in HL-60 cells by using siRNA with Nucleofector Kits according to the manufacturer’s protocol. Western blot analysis was performed to determine PARP-1 and β-actin expression or caspase-3 activation. Data are representative of three independent experiments. For the densitometric analysis of the Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at †p < 0.01 and *p < 0.001 via one-way ANOVA. FIG. 6. Open in new tabDownload slide Genetic or pharmacological inhibition of PARP-1 prevents TGHQ-mediated caspase activation and attenuates apoptosis. (A) Cells were treated with PJ-34 and/or different concentrations of z-vad-fmk in the presence or absence of 200µM of TGHQ for 4 h, followed by costaining with PI and FITC-conjugated annexin-V, which specifically detects the translocation of phosphatidylserine to the cell surface. Cells were then analyzed by flow cytometry. (B) Expression of PARP-1 was knocked down in HL-60 cells by using siRNA with Nucleofector Kits according to the manufacturer’s protocol. Western blot analysis was performed to determine PARP-1 and β-actin expression or caspase-3 activation. Data are representative of three independent experiments. For the densitometric analysis of the Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at †p < 0.01 and *p < 0.001 via one-way ANOVA. PARP-1 Plays a Dual Role in TGHQ-Induced Caspase Activation To further clarify the role of PARP-1 in TGHQ-induced apoptosis, the activation of a series of caspases was investigated. Caspases-3, -7, -8, and -9 were all markedly activated in response to TGHQ (Fig. 7A,C) as revealed by the cleaved caspase(s) band(s) detection, suggesting that both the intrinsic and death-receptor pathways participate in TGHQ-induced apoptosis. Strikingly, PJ-34 significantly attenuated TGHQ-induced caspase-3, -7, and -9 cleavage, confirming that PARP-1 is involved in TGHQ-induced caspase activation. Furthermore, TGHQ-mediated cytochrome c translocation from mitochondria to the cytoplasm was attenuated following the inhibition of PARP-1 (Fig. 7B), suggesting that PARP-1 supports TGHQ-induced caspase-3, -7, and -9 activation by assisting in mitochondrial cytochrome c release. In contrast, inhibition of PARP-1 potentiated caspase-8 cleavage (Fig. 7C). These findings were replicated in experiments using an alternative PARP inhibitor, 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (data not shown). Thus, PARP-1 plays a dual role in regulating TGHQ-induced apoptosis via opposing effects on the intrinsic (mitochondrial) and extrinsic (death-receptor) pathways. FIG. 7. Open in new tabDownload slide PARP-1 plays a dual role in TGHQ-induced caspase activation. HL-60 cells were treated with various concentrations of TGHQ in the presence or absence of PJ-34 for 6 h. (A) Western blot analysis determined caspase-3, cleaved caspase-7, cleaved caspase-9, and β-actin expression. Data shown are representative of three independent experiments. (B) Western blot analysis determined cytochrome c, GAPDH, and CoxIV expression. (C) Western blot analysis determined caspase-8 and β-actin expression. For the densitometric analysis of the Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at #p < 0.05; †p < 0.01 and *p < 0.001 via one-way ANOVA. FIG. 7. Open in new tabDownload slide PARP-1 plays a dual role in TGHQ-induced caspase activation. HL-60 cells were treated with various concentrations of TGHQ in the presence or absence of PJ-34 for 6 h. (A) Western blot analysis determined caspase-3, cleaved caspase-7, cleaved caspase-9, and β-actin expression. Data shown are representative of three independent experiments. (B) Western blot analysis determined cytochrome c, GAPDH, and CoxIV expression. (C) Western blot analysis determined caspase-8 and β-actin expression. For the densitometric analysis of the Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at #p < 0.05; †p < 0.01 and *p < 0.001 via one-way ANOVA. PARP-1 Also Plays a Dual Role in Curcumin-Induced 
Caspase Activation Curcumin induces apoptosis in HL-60 cells by activating caspase-8 and caspase-3 (Anto et al., 2002). We, therefore, examined whether PARP-1 plays a similar dual role during curcumin-induced apoptosis of HL-60 cells. Indeed, inhibition of PARP-1 with PJ-34 attenuated curcumin-induced caspase-3 activation, while simultaneously potentiating caspase-8 activation, confirming that PARP-1 plays a dual role in regulating caspase activation in HL-60 cells (Fig. 8). FIG. 8. Open in new tabDownload slide PARP-1 plays a dual role in curcumin-induced caspase activation. HL-60 cells were treated with various concentrations of curcumin in the presence or absence of PJ-34 for 24 h. Western blot analysis determined caspase-3, caspase-8, and β-actin expression. For the densitometric analysis of the Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at *p < 0.001 via one-way ANOVA. FIG. 8. Open in new tabDownload slide PARP-1 plays a dual role in curcumin-induced caspase activation. HL-60 cells were treated with various concentrations of curcumin in the presence or absence of PJ-34 for 24 h. Western blot analysis determined caspase-3, caspase-8, and β-actin expression. For the densitometric analysis of the Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at *p < 0.001 via one-way ANOVA. Nuclear AIF Translocation Is PARP-1 Dependent In normal cells, AIF is confined to the mitochondrial intermembrane space. However, under certain conditions of stress, the outer mitochondrial membrane becomes permeable, and AIF can translocate to the nucleus, causing DNA condensation and fragmentation, and cell death. Because evidence s uggests that PARP-1 initiates a nuclear signal that propagates to mitochondria and triggers the release of AIF leading to cell death (Huber et al., 2004), we next investigated whether AIF nuclear translocation contributes to TGHQ-induced apoptosis of HL-60 cells. TGHQ induced marked nuclear AIF accumulation, which was significantly abrogated following PARP-1 inhibition (Fig. 9), suggesting that the DNA damage–induced PARP-1 activation plays an important role in mediating nuclear AIF translocation. FIG. 9. Open in new tabDownload slide PARP-1 activation mediates nuclear AIF translocation. HL-60 cells were treated with increasing concentrations of TGHQ in the presence or absence of PJ-34 for 5 h. Nuclear protein was extracted with the Nuclear Extraction Kit. Western blot analysis was performed to determine AIF, Cox IV, H3, and MEK1/2 levels in nucleus. Cox IV is used as the marker for mitochondrial protein, H3 is the marker for nuclear protein, and MEK1/2 is the marker for cytoplasmic protein. The membrane was stained by Ponceau S. For the densitometric analysis of Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at †p < 0.05; *p < 0.01. FIG. 9. Open in new tabDownload slide PARP-1 activation mediates nuclear AIF translocation. HL-60 cells were treated with increasing concentrations of TGHQ in the presence or absence of PJ-34 for 5 h. Nuclear protein was extracted with the Nuclear Extraction Kit. Western blot analysis was performed to determine AIF, Cox IV, H3, and MEK1/2 levels in nucleus. Cox IV is used as the marker for mitochondrial protein, H3 is the marker for nuclear protein, and MEK1/2 is the marker for cytoplasmic protein. The membrane was stained by Ponceau S. For the densitometric analysis of Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at †p < 0.05; *p < 0.01. PARP-1 Inhibition Potentiates JNK and p38 MAPK Activation It has been suggested that during ischemia/reperfusion injury, JNK and/or p38 MAPK lie downstream of PARP activation, and may be required for PARP-mediated AIF translocation (Song et al., 2008). We, therefore, examined the response of JNK and p38 MAPK in HL-60 cells triggered to undergo apoptosis following treatment with TGHQ. Exposure of HL-60 cells to TGHQ enhanced both JNK and p38 phosphorylation (Fig. 10). However, although inhibition of PARP-1 reduced TGHQ-induced AIF translocation (Fig. 9), this effect occurred in the context of potentiated JNK1/2 and p38 MAPK activation (Fig. 10). Thus, activation of JNK and p38 MAPK is not sufficient for PARP-1–mediated nuclear AIF translocation. FIG. 10. Open in new tabDownload slide PJ-34 potentiates TGHQ-mediated activation of JNK and p38 MAPKs. HL-60 cells were treated with increasing concentrations of TGHQ in the presence or absence of PJ-34 for 6 h. Western blot analysis was performed to determine JNK, p38, phosphorylated JNK, or phosphorylated p38 expression. Data are representative of three independent experiments. For the densitometric analysis of the Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at #p < 0.05; †p < 0.01 and *p < 0.001 via one-way ANOVA. FIG. 10. Open in new tabDownload slide PJ-34 potentiates TGHQ-mediated activation of JNK and p38 MAPKs. HL-60 cells were treated with increasing concentrations of TGHQ in the presence or absence of PJ-34 for 6 h. Western blot analysis was performed to determine JNK, p38, phosphorylated JNK, or phosphorylated p38 expression. Data are representative of three independent experiments. For the densitometric analysis of the Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at #p < 0.05; †p < 0.01 and *p < 0.001 via one-way ANOVA. Discussion In this study, we have shown that PARP-1 has the ability to regulate the nuclear translocation of AIF, and perhaps more importantly, to differentially regulate caspase activation during ROS-induced apoptosis of HL-60 cells. Despite the nuclear translocation of AIF during TGHQ-mediated apoptosis of HL-60 cells, DNA fragmentation is oligonucleosomal in nature (Fig. 1A) rather than the characteristic high-molecular-weight (MW) fragments associated with nuclear AIF (Siegel and McCullough, 2011). The ability of nuclear AIF to initiate DNA fragmentation has recently been linked to its ability to form a complex with the histone γ-H2AX and the nuclease cyclophilin A (Baritaud et al., 2010). Levels of γ-H2AX increase significantly in our model of HL-60 cell death, which is consistent with γ-H2AX being implicated in the caspase-3/caspase–activated DNAase pathway of apoptotic cell death (Lu et al., 2006; Rogakou et al., 2000). Perhaps additional posttranslational modifications of γ-H2AX might determine its relative affinity for either cyclophilin A or caspase-activated DNAase during cell death. Consistent with the oligonucleosomal nature of DNA fragmentation (Fig. 1), caspases are activated during TGHQ- induced HL-60 cell apoptosis (Fig. 7), and PARP-1 also modulates the caspase response. Thus, although PAR proteins are detected in TGHQ-treated cells and AIF is released from mitochondria and migrates to the nucleus, TGHQ-induced apoptosis in HL-60 cells is caspase dependent (Fig. 6A). Caspase- dependent apoptosis can be activated via two main pathways: the extrinsic pathway, which originates from the activation of cell-surface death-receptors, such as Fas and tumor necrosis factor- receptor 1, culminating in caspase-8 activation; and the intrinsic pathway, which originates from the mitochondrial release of cytochrome c and culminates in the activation of caspase-9 through the cytochrome c/apoptotic protease-activating factor-1 (Apaf-1)/procaspase-9 heptamer. Both the intrinsic (caspases-3, -7, and -9) and extrinsic (caspase-8) pathways are activated during TGHQ-induced apoptosis (Fig. 7). However, whereas inhibition of PARP-1 attenuates the activation of caspases-3, -7, and -9, it actually potentiates caspase-8 activation. Consistent with our findings, activation of caspases-3, -6, and -9 is also reduced in PARP-1–/– cells following H2O2 treatment (Blenn et al., 2011). The precise mechanisms by which PARP-1 differentially regulates the various caspases remain to be elucidated. The caspase-independent cell death pathway is associated with the activation of PARP-1 (Yu et al., 2002), and it has been proposed that either NAD+ depletion or PAR polymers themselves trigger AIF release (Alano et al., 2010; Siegel and McCullough, 2011; Yu et al., 2006). Pharmacological inhibition of PARP-1 significantly inhibited nuclear AIF translocation (Fig. 9), decreased PAR-positive proteins in TGHQ-treated HL-60 cells (Fig. 5A), but had only a nominal effect in restoring cellular NAD+ concentrations (Fig. 3B), whereas the nuclear translocation of AIF was completely abolished (Fig. 9). Thus, in HL-60 cells, the PARP-1–dependent mitochondrial release of AIF is more likely coupled to the PAR of mitochondrial proteins (Wang et al., 2009) than to NAD+ depletion. PARP-1 might, therefore, participate in regulating mitochondrial permeability by targeting mitochondrial proteins for poly(ADP-ribosylation). Direct binding of PAR to AIF may also be required for PARP-mediated cell death (Wang et al., 2011). Moreover, the restoration of cellular ATP concentrations following PARP inhibition, but not of NAD+ concentrations (Fig. 3), suggests that at least in HL-60 cells NAD+ is being consumed by mechanisms other than PARP. For example, SIRT1 consumes NAD+ during the deacetylation of SIRT1-target proteins, and inhibition of PARP-1 stimulates mitochondrial metabolism via the activation of SIRT1 (Bai et al., 2011). In addition, inhibition of SIRT1 induces apoptosis of breast cancer cells (Kalle et al., 2010) and SIRT1 deficiency attenuates MPP+-induced apoptosis of dopaminergic cells (Park et al., 2011). Maintaining SIRT1 function by preventing the complete depletion of NAD+ should, therefore, be expected to promote cell survival, consistent with the ability of PJ-34 to prevent HL-60 cell apoptosis (Fig. 4). That AIF undergoes nuclear translocation in concert with caspase activation in this model of apoptosis is unusual because AIF translocation is typically associated with caspase- independent cell death. Although this latter pathway is well recognized, mechanisms that trigger the release of AIF from the mitochondria and its translocation to the nucleus, where it initiates chromatin condensation and a characteristic high-MW (50 kbp) DNA fragmentation, and ultimately caspase-independent cell death are still unclear (Andrabi et al., 2006; Hong et al., 2004; Susin et al., 1999; Yu et al., 2002, 2006). In summary, TGHQ-induced apoptotic cell death of HL-60 cells is accompanied by PARP-1 and caspase activation and AIF nuclear translocation. The TGHQ-induced apoptosis appears to primarily occur via engagement of the mitochondrial-mediated pathway in a process amenable to PARP inhibition. The residual cell death in the presence of PJ-34 is likely mediated via the extrinsic apoptotic pathway. Unraveling the complex role of PARP-1 in cell death signaling pathways should help guide the therapeutic use of PARP inhibitors and assist in identifying novel targets for therapeutic intervention for diseases and conditions in which ROS-induced DNA damage play a critical role. Funding National Institute of Environmental Health Sciences to the Southwest Environmental Health Sciences Center (P30ES006694). REFERENCES Adamietz P., Rudolph A. 1984 ADP-ribosylation of nuclear proteins in vivo. Identification of histone H2B as a major acceptor for mono- and poly(ADP-ribose) in dimethyl sulfate-treated hepatoma AH 7974 cells J. Biol. Chem 259 6841 6846 Google Scholar PubMed OpenURL Placeholder Text WorldCat Alano C. C. Garnier P. Ying W. Higashi Y. Kauppinen T. M., Swanson R. A. 2010 NAD+ depletion is necessary and sufficient for poly(ADP-ribose) polymerase-1-mediated neuronal death J. Neurosci 30 2967 2978 Google Scholar Crossref Search ADS PubMed WorldCat Andrabi S. A. Kim N. S. Yu S. W. Wang H. Koh D. W. Sasaki M. Klaus J. A. Otsuka T. Zhang Z. Koehler R. C. et al. 2006 Poly(ADP-ribose) (PAR) polymer is a death signal Proc. Natl. Acad. Sci. U.S.A 103 18308 18313 Google Scholar Crossref Search ADS PubMed WorldCat Anto R. J. Mukhopadhyay A. Denning K., Aggarwal B. B. 2002 Curcumin (diferuloylmethane) induces apoptosis through activation of caspase-8, BID cleavage and cytochrome c release: Its suppression by ectopic expression of Bcl-2 and Bcl-xl Carcinogenesis 23 143 150 Google Scholar Crossref Search ADS PubMed WorldCat Bai P. Canto C. Oudart H. Brunyanszki A. Cen Y. Thomas C. Yamamoto H. Huber A. Kiss B. Houtkooper R. H. et al. 2011 PARP-1 inhibition increases mitochondrial metabolism through SIRT1 activation Cell Metab 13 461 468 Google Scholar Crossref Search ADS PubMed WorldCat Baritaud M. Boujrad H. Lorenzo H. K. Krantic S., Susin S. A. 2010 Histone H2AX: The missing link in AIF-mediated caspase-independent programmed necrosis Cell Cycle 9 3166 3173 Google Scholar Crossref Search ADS PubMed WorldCat Blenn C. Wyrsch P. Bader J. Bollhalder M., Althaus F. R. 2011 Poly(ADP-ribose)glycohydrolase is an upstream regulator of Ca2+ fluxes in oxidative cell death Cell Mol. Life Sci 68 1455 1466 Google Scholar Crossref Search ADS PubMed WorldCat Bratton S. B. Lau S. S., Monks T. J. 1997 Identification of quinol thioethers in bone marrow of hydroquinone/phenol-treated rats and mice and their potential role in benzene-mediated hematotoxicity Chem. Res. Toxicol 10 859 865 Google Scholar Crossref Search ADS PubMed WorldCat Bratton S. B. Lau S. S., Monks T. J. 2000 The putative benzene metabolite 2,3,5-tris(glutathion-S-yl)hydroquinone depletes glutathione, stimulates sphingomyelin turnover, and induces apoptosis in HL-60 cells Chem. Res. Toxicol 13 550 556 Google Scholar Crossref Search ADS PubMed WorldCat Diefenbach J., Burkle A. 2005 Introduction to poly(ADP-ribose) metabolism Cell Mol. Life Sci 62 721 730 Google Scholar Crossref Search ADS PubMed WorldCat Eastmond D. A. Smith M. T., Irons R. D. 1987 An interaction of benzene metabolites reproduces the myelotoxicity observed with benzene exposure Toxicol. Appl. Pharmacol 91 85 95 Google Scholar Crossref Search ADS PubMed WorldCat Erdelyi K. Bakondi E. Gergely P. Szabo C., Virag L. 2005 Pathophysiologic role of oxidative stress-induced poly(ADP-ribose) polymerase-1 activation: Focus on cell death and transcriptional regulation Cell Mol. Life Sci 62 751 759 Google Scholar Crossref Search ADS PubMed WorldCat Holden P., Horton W. A. 2009 Crude subcellular fractionation of cultured mammalian cell lines BMC Res. Notes 2 243 Google Scholar Crossref Search ADS PubMed WorldCat Hong S. J. Dawson T. M., Dawson V. L. 2004 Nuclear and mitochondrial conversations in cell death: PARP-1 and AIF signaling Trends Pharmacol. Sci 25 259 264 Google Scholar Crossref Search ADS PubMed WorldCat Huber A. Bai P. de Murcia J. M., de Murcia G. 2004 PARP-1, PARP-2 and ATM in the DNA damage response: Functional synergy in mouse development DNA Repair (Amst) 3 1103 1108 Google Scholar Crossref Search ADS PubMed WorldCat Jacobson E. L., Jacobson M. K. 1997 Tissue NAD as a biochemical measure of niacin status in humans Methods Enzymol 280 221 230 Google Scholar PubMed OpenURL Placeholder Text WorldCat Kalle A. M. Mallika A. Badiger J. Alinakhi Talukdar P., Sachchidanand 2010 Inhibition of SIRT1 by a small molecule induces apoptosis in breast cancer cells Biochem. Biophys. Res. Commun 401 13 19 Google Scholar Crossref Search ADS PubMed WorldCat Kaufmann S. H. Desnoyers S. Ottaviano Y. Davidson N. E., Poirier G. G. 1993 Specific proteolytic cleavage of poly(ADP-ribose) polymerase: An early marker of chemotherapy-induced apoptosis Cancer Res 53 3976 3985 Google Scholar PubMed OpenURL Placeholder Text WorldCat Lau S. S. Hill B. A. Highet R. J., Monks T. J. 1988 Sequential oxidation and glutathione addition to 1,4-benzoquinone: Correlation of toxicity with increased glutathione substitution Mol. Pharmacol 34 829 836 Google Scholar PubMed OpenURL Placeholder Text WorldCat Lu C. Zhu F. Cho Y. Y. Tang F. Zykova T. Ma W. Y. Bode A. M., Dong Z. 2006 Cell apoptosis: Requirement of H2AX in DNA ladder formation, but not for the activation of caspase-3 Mol. Cell 23 121 132 Google Scholar Crossref Search ADS PubMed WorldCat Park G. Jeong J. W., Kim J. E. 2011 SIRT1 deficiency attenuates MPP+-induced apoptosis in dopaminergic cells FEBS Lett 585 219 224 Google Scholar Crossref Search ADS PubMed WorldCat Rinsky R. A. Young R. J., Smith A. B. 1981 Leukemia in benzene workers Am. J. Ind. Med 2 217 245 Google Scholar Crossref Search ADS PubMed WorldCat Rogakou E. P. Nieves-Neira W. Boon C. Pommier Y., Bonner W. M. 2000 Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139 J. Biol. Chem 275 9390 9395 Google Scholar Crossref Search ADS PubMed WorldCat Rogakou E. P. Pilch D. R. Orr A. H. Ivanova V. S., Bonner W. M. 1998 DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139 J. Biol. Chem 273 5858 5868 Google Scholar Crossref Search ADS PubMed WorldCat Scovassi A. I. Mariani C. Negroni M. Negri C., Bertazzoni U. 1993 ADP-ribosylation of nonhistone proteins in HeLa cells: Modification of DNA topoisomerase II Exp. Cell Res 206 177 181 Google Scholar Crossref Search ADS PubMed WorldCat Sevrioukova I. F. 2011 Apoptosis-inducing factor: Structure, function, and redox regulation Antioxid. Redox Signal 14 2545 2579 Google Scholar Crossref Search ADS PubMed WorldCat Siegel C., McCullough L. D. 2011 NAD+ depletion or PAR polymer formation: Which plays the role of executioner in ischaemic cell death? Acta Physiol. (Oxf) 203 225 234 Google Scholar Crossref Search ADS PubMed WorldCat Snyder R. Witz G., Goldstein B. D. 1993 The toxicology of benzene Environ. Health Perspect 100 293 306 Google Scholar Crossref Search ADS PubMed WorldCat Song Z. F. Ji X. P. Li X. X. Wang S. J. Wang S. H., Zhang Y. 2008 Inhibition of the activity of poly (ADP-ribose) polymerase reduces heart ischaemia/reperfusion injury via suppressing JNK-mediated AIF translocation J. Cell Mol. Med 12 1220 1228 Google Scholar Crossref Search ADS PubMed WorldCat Susin S. A. Lorenzo H. K. Zamzami N. Marzo I. Snow B. E. Brothers G. M. Mangion J. Jacotot E. Costantini P. Loeffler M. et al. 1999 Molecular characterization of mitochondrial apoptosis-inducing factor Nature 397 441 446 Google Scholar Crossref Search ADS PubMed WorldCat Virag L. 2005 Structure and function of poly(ADP-ribose) polymerase-1: Role in oxidative stress-related pathologies Curr. Vasc. Pharmacol 3 209 214 Google Scholar Crossref Search ADS PubMed WorldCat Virag L., Szabo C. 2002 The therapeutic potential of poly(ADP-ribose) polymerase inhibitors Pharmacol. Rev 54 375 429 Google Scholar Crossref Search ADS PubMed WorldCat Wang Y. Dawson V. L., Dawson T. M. 2009 Poly(ADP-ribose) signals to mitochondrial AIF: A key event in parthanatos Exp. Neurol 218 193 202 Google Scholar Crossref Search ADS PubMed WorldCat Wang Y. Kim N. S. Haince J. F. Kang H. C. David K. K. Andrabi S. A. Poirier G. G. Dawson V. L., Dawson T. M. 2011 Poly(ADP-ribose) (PAR) binding to apoptosis-inducing factor is critical for PAR polymerase-1-dependent cell death (parthanatos) Sci. Signal 4 ra20 Google Scholar PubMed OpenURL Placeholder Text WorldCat Yang M. Y. Lau S. S., Monks T. J. 2005 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ)-mediated apoptosis of human promyelocytic leukemia cells is preceded by mitochondrial cytochrome c release in the absence of a decrease in the mitochondrial membrane potential Toxicol. Sci 86 92 100 Google Scholar Crossref Search ADS PubMed WorldCat Yin S. N. Li G. L. Tain F. D. Fu Z. I. Jin C. Chen Y. J. Luo S. J. Ye P. Z. Zhang J. Z. Wang G. C. et al. 1987 Leukaemia in benzene workers: A retrospective cohort study Br. J. Ind. Med 44 124 128 Google Scholar PubMed OpenURL Placeholder Text WorldCat Yoshihara K. Itaya A. Tanaka Y. Ohashi Y. Ito K. Teraoka H. Tsukada K. Matsukage A., Kamiya T. 1985 Inhibition of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, and DNA ligase II by poly(ADP-ribosyl)ation reaction in vitro Biochem. Biophys. Res. Commun 128 61 67 Google Scholar Crossref Search ADS PubMed WorldCat Yu S. W. Andrabi S. A. Wang H. Kim N. S. Poirier G. G. Dawson T. M., Dawson V. L. 2006 Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced cell death Proc. Natl. Acad. Sci. U.S.A 103 18314 18319 Google Scholar Crossref Search ADS PubMed WorldCat Yu S. W. Wang H. Poitras M. F. Coombs C. Bowers W. J. Federoff H. J. Poirier G. G. Dawson T. M., Dawson V. L. 2002 Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor Science 297 259 263 Google Scholar Crossref Search ADS PubMed WorldCat © The Author 2012. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected] OUP Journals http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Toxicological Sciences Oxford University Press

A Dual Role for Poly(ADP-Ribose) Polymerase-1 
During Caspase-Dependent Apoptosis

Download PDF
12 pages

Loading next page...
 
/lp/oxford-university-press/a-dual-role-for-poly-adp-ribose-polymerase-1-during-caspase-dependent-GXkSn6SWyH

References (44)

Publisher
Oxford University Press
Copyright
© The Author 2012. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected]
Subject
RESEARCH ARTICLE
ISSN
1096-6080
eISSN
1096-0929
DOI
10.1093/toxsci/kfs142
pmid
22523229
Publisher site
See Article on Publisher Site

Abstract

Abstract 2,3,5-Tris(glutathion-S-yl)hydroquinone (TGHQ), a metabolite of benzene, catalyzes the generation of reactive oxygen species (ROS) and caspase-dependent apoptosis in human promyelocytic leukemia (HL-60) cells. We now report that TGHQ induces severe DNA damage, as evidenced by DNA ladder formation and H2AX phosphorylation. The subsequent activation of the DNA nick sensor enzyme, poly(ADP-ribose) polymerase-1 (PARP-1), leads to the rapid depletion of ATP and NAD and the concomitant formation of poly(ADP-ribosylated) proteins (PARs). PJ-34 (a PARP-1 inhibitor) completely prevented the formation of PARs, partially attenuated TGHQ-mediated ATP depletion, but had little effect on NAD depletion. Intriguingly, although z-vad-fmk (a pan-caspase inhibitor) attenuated TGHQ-induced apoptosis, cotreatment with PJ-34 led to a further decrease in apoptosis, suggesting that PARP-1 participates in caspase-dependent apoptosis. Indeed, PARP-1 inhibition reduced TGHQ-induced caspase-3, -7, and -9 activation, at least partially by attenuating cytochrome c translocation from mitochondria to the cytoplasm. In contrast, PJ-34 potentiated TGHQ-induced caspase-8 activation, suggesting that PARP-1 plays a dual role in regulating TGHQ-induced apoptosis via opposing effects on the intrinsic (mitochondrial) and extrinsic (death-receptor) pathways. PARP-1 knockdown in HL-60 cells confirmed that PARP-1 participates in effector caspase activation. Finally, PJ-34 also inhibited TGHQ-induced apoptosis-inducing factor (AIF) nuclear translocation, but neither c-jun NH(2)-terminal kinase nor p38 MAPK (p38 mitogen-activated protein kinase) activation was required for AIF translocation. In summary, TGHQ-induced apoptosis of HL-60 cells is accompanied by PARP-1, caspase activation, and AIF nuclear translocation. TGHQ-induced apoptosis appears to primarily occur via engagement of the mitochondrial-mediated pathway in a process amenable to PARP inhibition. Residual cell death in the presence of PJ-34 is likely mediated via the extrinsic apoptotic pathway. TGHQ, PARP-1, apoptosis, caspase, AIF, TGHQ Poly(ADP-ribose) polymerase-1 (PARP-1) is a member of a family of proteins that constitute a superfamily of at least 18 putative PARP proteins, based on protein domain homology and enzymatic function (Diefenbach and Burkle, 2005). PARP-1 
is activated by DNA strand nicks and breaks, which can be induced by a variety of environmental stimuli or by endogenous reactive oxygen species (ROS) (Andrabi et al., 2006; Virag and Szabo, 2002). In the absence of DNA damage, poly(ADP-ribosylation) is a rare event but increases over 100-fold upon DNA damage. PARP-1 catalyzes the formation of ADP-ribose polymers from nicotinamide adenine dinucleotide (NAD+) on various nuclear proteins, including histones (Adamietz and Rudolph, 1984), DNA polymerases (Yoshihara et al., 1985), topoisomerases (Scovassi et al., 1993), and PARP-1 itself (Erdelyi et al., 2005). Although PARP-1 may facilitate DNA repair and cell survival in response to mild genotoxic stimuli, it contributes to cell death in the face of severe DNA damage (Virag, 2005). Although PARP-1–dependent NAD+ consumption and energy failure are thought to contribute to cell death, the precise mechanisms by which PARP-1 regulates apoptotic cell death remain unclear. Apoptosis-inducing factor (AIF) is a flavin adenine dinucleotide–containing, NADH-dependent oxidoreductase residing in the mitochondrial intermembrane space (Sevrioukova, 2011). It is believed to play a central role in the regulation of PARP-induced cell death, because it is released into the cytoplasm after PARP-1 activation and ultimately translocates to the nucleus where it triggers chromatin condensation and large-scale DNA degradation in a caspase-independent manner (Siegel and McCullough, 2011). Moreover, AIF has emerged as a protein critical for cell survival, reflecting the complexity and multilevel regulation of AIF-mediated signal transduction (Sevrioukova, 2011). However, the relationship between AIF and PARP-1 in cell death remains unclear. Benzene is a ubiquitous chemical widely used in the manufacturing industry. However, benzene causes bone marrow suppression in rodents and is hematotoxic and leukemogenic in humans (Rinsky et al., 1981; Yin et al., 1987). Because benzene must be metabolized to mediate its harmful effects, a number of benzene metabolites have been implicated in benzene-mediated hematotoxicity (Eastmond et al., 1987; Snyder et al., 1993). 2,3,5-Tris(glutathion-S-yl)hydroquinone (TGHQ), a metabolite of benzene, causes hematotoxicity in rats and induces apoptosis in human promyelocytic leukemia (HL-60) cells in a ROS-dependent fashion (Bratton et al., 1997, 2000; Yang et al., 2005). Thus, in this study, we investigated the role of PARP-1 in TGHQ-mediated, ROS-dependent HL-60 cell apoptosis, and in particular the relationship between PARP-1 activation and caspase- and AIF-mediated cell death. Materials and Methods Reagents Caution. TGHQ is nephrotoxic and nephrocarcinogenic in rats and must therefore be handled with protective clothing and in a ventilated hood. Chemicals. Unless otherwise specified, all chemicals were purchased from Sigma Chemical Co. (St Louis, MO). All reagents were of the highest grade commercially available. TGHQ was synthesized in the laboratory according to established methodology (Lau et al., 1988). Antibodies and all other assay kits: GAPDH, c-jun NH(2)-terminal kinase (JNK), p-JNK, p38, p-p38, cytochrome c, caspase-3, -7, -8, cleaved caspase-9, β-actin, CoxIV, and AIF were all purchased from Cell Signaling Technology (San Diego, CA). The antibody for poly(ADP-ribose) was purchased from Biomol International (Plymouth Meeting, PA). The DNA fragmentation kit was purchased from Invitrogen (Carlsbad, CA). The pan-caspase inhibitor z-vad-fmk and the annexin-V/propidium iodide (PI) kit were obtained from BD Bioscience. Cell Culture Conditions The promyelocytic leukemia HL-60 cell line was obtained from the American Type Culture Collection (Manassas, VA). HL-60 cell suspensions were cultured in RPMI 1640 medium (Gibco BRL, Grand Island, NY) supplemented with 20% fetal bovine serum. No antibiotics were used in our experiments, and cells were incubated in a humidified atmosphere with 5% CO2 in air at 37°C. Exponentially growing cells were seeded at a cell density of 1.0 × 106 cells/ml. DNA Fragmentation Assay Apoptotic DNA fragmentation in HL-60 cells was determined using the Quick Apoptotic DNA Ladder Detection Kit (Invitrogen). Briefly, cells were treated with different concentrations of TGHQ for the indicated time periods. After the incubation, cells were harvested by centrifugation and washed with phosphate-buffered saline (PBS). DNA fragments were extracted according to the manufacturer’s instructions. Finally, DNA fragments were visualized following electrophoresis on a 1% agarose gel. Histone Extraction HL-60 cells were washed with 3 ml PBS and lysed in 1 ml ice-cold hypotonic lysis buffer (10mM Tris-HCl, pH 8.0, 1mM KCl, 1.5mM MgCl2, and 1mM DTT). Histones were extracted with 0.4N H2SO4 overnight at 4°C and then precipitated in trichloroacetic acid on ice for 0.5 h. The resulting pellet was washed with ice-cold acetone. Pellets were dried in air and subjected to Western blot analysis. Western Blot Analysis After specified treatments, HL-60 cells were washed twice with PBS and lysed in ice-cold RIPA (Radio-Immunoprecipitation Assay) lysis buffer. For nuclear protein extraction, the Nuclear Extraction Kit from Panomics, Inc. was used. After incubation for 15 min on a rotator at 4°C, cell lysates were centrifuged to remove cell debris. Protein concentrations were determined with detergent-compatible reagent (Bio-Rad Laboratories, Hercules, CA). Samples were incubated with sample buffer and boiled for 5 min at 100°C, and resolved in SDS polyacrylamide gels. After electrophoresis, proteins were transferred to a PVDF membrane (Millipore, Billerica, MA). After blocking with 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (TBST), membranes were incubated with appropriate primary antibodies (1:1000 dilution) overnight at 4°C. After washing in TBST, membranes were incubated with appropriate horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology). After thoroughly washing in TBST, bound antibodies were visualized using standard chemiluminescence on autoradiographic film. Preparation of Cytosolic and Mitochondrial Extracts After treatment of cells with TGHQ, cytosolic and mitochondrial extracts were prepared as described (Holden and Horton, 2009). Cells were harvested and centrifuged at 100 RCF at 4°C to pellet the cells. The supernatant was aspirated, and the cells were washed by gentle pipetting into 1 ml of ice-cold PBS. The cell suspension was again centrifuged at 4°C to pellet the cells, the supernatant aspirated and the cell pellet resuspended by gentle pipetting into 400 µl of ice-cold buffer 1 (150mM NaCl, 50mM HEPES, pH 7.4, 25 µg/ml digitonin, and 1mM PMSF). The cell suspension was gently mixed at 4°C for 10 min and then centrifuged at 2000 RCF to pellet the cells. The supernatants (cytosolic fraction) were saved. Cell pellets were resuspended by vortexing in 400 µl of ice-cold buffer 2 (150mM NaCl, 50mM HEPES, pH 7.4, and 1% NP40), incubated on ice for 30 min,and then centrifuged at 7000 RCF to pellet nuclei and cell debris. The supernatant, containing the mitochondrial proteins, was then collected for further analyses. Annexin-V/PI Staining Annexin-V/PI staining was used to detect early apoptotic cells during apoptotic progression. After treatment with TGHQ for 4 h alone or in combination with specific inhibitors, cells were collected and washed with cold PBS twice and then resuspended with 100 µl of binding buffer (10mM HEPES, 140mM NaCl, and 2.5mM CaCl2, pH 7.4) at a concentration of 1 × 106 cells/ml. The cell suspension (100 µl) was mixed with 5 µl of FITC-conjugated annexin-V and 5 µl of PI and incubated for 15 min at room temperature in the dark. Thereafter, cells were immediately added to 400 µl of binding buffer and analyzed by flow cytometry (Becton Dickinson, San Jose, CA). The dual-parametric dot plots were used for calculation of the percentage of nonapoptotic live cells in the lower left quadrant (annexin-V–/PI–), early apoptotic cells in the lower right quadrant (annexin-V+/PI–), late apoptotic cells in the upper right quadrant (annexin-V+/PI+), and necrotic cells in the upper left quadrant (annexin-V–/PI+). Cellular ATP Assay HL-60 cells were treated with test compounds. After the indicated time, cells were collected, resuspended in RPMI 1640, and seeded in opaque-walled 96-well plates. ATP content per well was determined using the CellTiter-Glo luminescent assay (Promega, Madison, WI) according to the manufacturer’s instructions. Determination of Total NAD Content Cells were seeded in 6-well plates at 2 × 106 cells/well and treated with 200µM TGHQ with or without PJ-34 (10µM) cotreatment for varying periods of time. NAD was extracted by addition of 1.0M HClO4 to dishes of cells on ice. Cell extracts were neutralized to pH 7.0 by addition of 2M KOH/0.66M KH2PO4. NAD concentrations were determined by enzymatic cycling assays as described (Jacobson and Jacobson, 1997). Small Interfering RNA Transfection Knockdown of PARP-1 was performed in HL-60 cells using predesigned ON-TARGETplus siRNA SMART pool purchased from Dharmacon. The transfections were performed by the electroporation method (Lonza, Nucleofector Program T-019), and the same numbers of the transfected cells were seeded in 6-well plates for treatment. ON-TARGETplus nontargeting siRNA pool was used as a control for nonsequence-specific effects. Statistical Analyses Data are expressed as mean ± SE. Statistical differences between the treated and control groups were determined by Student’s paired t-test. Differences between groups were assessed by one-way ANOVA using the SPSS software package for Windows. Differences between means were considered significant if p < 0.05. Results PARP-1 Is Activated in Response to TGHQ-Mediated DNA Damage TGHQ induced typical oligonucleosomal DNA ladder formation, confirming cell death in HL-60 cells is apoptotic in nature (Fig. 1A). To further confirm the DNA-damaging effect of TGHQ, cell lysates were analyzed for the presence of the phosphorylated form of the histone variant H2AX (γ-H2AX), an indicator of the presence of DNA double-strand breaks (Rogakou et al., 1998). TGHQ significantly induced the formation of γ-H2AX in HL-60 cells (Fig. 1B). PAR formation also increased in HL-60 cells following treatment with TGHQ and reached a peak 3 h after dosing (Fig. 2). FIG. 1. Open in new tabDownload slide TGHQ induces DNA damage in HL-60 cells. Cells were treated with various concentrations of TGHQ for the indicated periods of time. (A) Fragmented genomic DNA was extracted and resolved on 1% agarose gel. Apoptotic DNA was visualized by ethidium bromide staining. (B) Western blot analysis of H2AX and γ-H2AX levels. Cells were treated with TGHQ (200µM) for various periods of time and histones extracted and subjected to Western blot analysis. FIG. 1. Open in new tabDownload slide TGHQ induces DNA damage in HL-60 cells. Cells were treated with various concentrations of TGHQ for the indicated periods of time. (A) Fragmented genomic DNA was extracted and resolved on 1% agarose gel. Apoptotic DNA was visualized by ethidium bromide staining. (B) Western blot analysis of H2AX and γ-H2AX levels. Cells were treated with TGHQ (200µM) for various periods of time and histones extracted and subjected to Western blot analysis. FIG. 2. Open in new tabDownload slide TGHQ induces PARP-1 activation. HL-60 cells were treated with 200µM TGHQ for various periods of time, and PAR accumulation was determined by Western blot analysis. Equal loading was confirmed by Ponceau S staining. For densitometric analysis of Western blots, values are expressed as fold change relative to the appropriate controls. Values represent the mean ± SE (n = 3). Significantly different from controls at †p < 0.05; *p < 0.01. FIG. 2. Open in new tabDownload slide TGHQ induces PARP-1 activation. HL-60 cells were treated with 200µM TGHQ for various periods of time, and PAR accumulation was determined by Western blot analysis. Equal loading was confirmed by Ponceau S staining. For densitometric analysis of Western blots, values are expressed as fold change relative to the appropriate controls. Values represent the mean ± SE (n = 3). Significantly different from controls at †p < 0.05; *p < 0.01. TGHQ Induces ATP and NAD Depletion in HL-60 Cells The remarkable PARP activation led us to subsequently examine the effect of TGHQ on ATP and NAD+ concentrations in HL-60 cells. TGHQ caused a rapid depletion of both cellular ATP (Fig. 3A) and NAD+ (Fig. 3B) in HL-60 cells within 6 h of exposure, indicating the overactivation of PARP-1 in response to TGHQ-induced DNA damage. FIG. 3. Open in new tabDownload slide TGHQ causes ATP and NAD depletion in HL-60 cells. Cells were treated for up to 6 h with TGHQ in the presence or absence of a PARP inhibitor, PJ-34 (10µM). (A) ATP content was determined using the ATP luminescent assay. (B) NAD concentration was determined with the method described in Materials and Methods. Data represent the mean ± SE (n ± 3). •, control; ○, PJ-34; ■, TGHQ, and □, (TGHQ+PJ-34). Significantly different from controls at *p < 0.001. FIG. 3. Open in new tabDownload slide TGHQ causes ATP and NAD depletion in HL-60 cells. Cells were treated for up to 6 h with TGHQ in the presence or absence of a PARP inhibitor, PJ-34 (10µM). (A) ATP content was determined using the ATP luminescent assay. (B) NAD concentration was determined with the method described in Materials and Methods. Data represent the mean ± SE (n ± 3). •, control; ○, PJ-34; ■, TGHQ, and □, (TGHQ+PJ-34). Significantly different from controls at *p < 0.001. PARP-1 Modulates TGHQ-Induced HL-60 Cell Apoptosis To gain an insight into whether the activation of the pro apoptotic PARP pathway is involved in TGHQ-induced HL-60 cell apoptosis, we further determined by flow cytometry whether PJ-34, a specific PARP inhibitor, could ameliorate apoptosis. PJ-34 itself did not lead to any apoptosis in HL-60 cells (Fig. 4). Interestingly, however, apoptosis induced by TGHQ (200µM) was decreased from 25.7 to 18.9% (an ~30% reduction) in the presence of PJ-34, indicating that PARP-1 regulates TGHQ-induced apoptosis (with the caveat that PJ-34 is behaving exclusively as a PARP-1 inhibitor). To test the efficiency of PJ-34 as a PARP inhibitor, the effect of PJ-34 on PAR-modified proteins, as well as ATP and NAD concentrations in TGHQ-treated cells, was analyzed. Protein-bound PAR was evident in TGHQ-treated HL-60 cells (Fig. 5A) but was completely absent in PJ-34–treated cells. It is noteworthy that PAR formation in response to TGHQ-induced DNA damage decreases with increasing concentrations of TGHQ. Whether this occurs subsequent to the activation of caspase-3, and the concomitant cleavage (and inactivation) of PARP-1 (Kaufmann et al., 1993), remains to be determined. However, consistent with this view, the cleaved form of PARP-1 increased in parallel with increases in caspase-3 activation (Fig. 5B). Similarly, PJ-34 markedly attenuated TGHQ-mediated ATP depletion (Fig. 3A). In contrast, PJ-34 had little effect on TGHQ-mediated decreases in NAD concentrations (Fig. 3B). FIG. 4. Open in new tabDownload slide PJ-34 protects against TGHQ-induced HL-60 cell apoptosis. Cells were treated with various concentrations of TGHQ for 4 h, followed by costaining with PI and FITC-conjugated annexin-V, which specifically detects the translocation of phosphatidylserine to the cell surface. Cells were then analyzed by flow cytometry. FIG. 4. Open in new tabDownload slide PJ-34 protects against TGHQ-induced HL-60 cell apoptosis. Cells were treated with various concentrations of TGHQ for 4 h, followed by costaining with PI and FITC-conjugated annexin-V, which specifically detects the translocation of phosphatidylserine to the cell surface. Cells were then analyzed by flow cytometry. FIG. 5. Open in new tabDownload slide PJ-34 inhibits TGHQ-induced PARP-1 activation. Cells were treated with various concentrations of TGHQ in the presence or absence of PJ-34 for 3 h. (A) PAR accumulation was determined with a PAR antibody, SA-216. Equal loading was confirmed by Ponceau S staining. (B) Effect of TGHQ on PARP-1 cleavage and caspase-3 activation. For the quantitative analysis of Western blots by densitometry, values are expressed as fold changes relative to the appropriate controls. Values are given as mean ± SE (n = 3). †p < 0.05; *p < 0.001 compared with control. FIG. 5. Open in new tabDownload slide PJ-34 inhibits TGHQ-induced PARP-1 activation. Cells were treated with various concentrations of TGHQ in the presence or absence of PJ-34 for 3 h. (A) PAR accumulation was determined with a PAR antibody, SA-216. Equal loading was confirmed by Ponceau S staining. (B) Effect of TGHQ on PARP-1 cleavage and caspase-3 activation. For the quantitative analysis of Western blots by densitometry, values are expressed as fold changes relative to the appropriate controls. Values are given as mean ± SE (n = 3). †p < 0.05; *p < 0.001 compared with control. Caspase-Dependent HL-60 Cell Death Is Regulated by PARP-1 The broad-spectrum caspase inhibitor, z-vad-fmk, significantly inhibited TGHQ-induced HL-60 cell apoptosis (Fig. 6A), completely abrogating apoptosis at a concentration of 80µM. TGHQ-induced apoptosis in HL-60 cells is, thus, caspase dependent. Interestingly, the residual apoptosis occurring in HL-60 cells exposed to lower concentrations (< 80µM) of z-vad-fmk was further reduced when PJ-34 was added to the cell cultures, suggesting that PARP-1 can influence caspase(s) activation. To further verify this possibility, the PARP-1 gene was knocked down in HL-60 cells and caspase-3 (the effector caspase) activation in the presence of TGHQ was determined. Caspase-3 activation was greatly attenuated in PARP-1 siRNA-treated cells (Fig. 6B). FIG. 6. Open in new tabDownload slide Genetic or pharmacological inhibition of PARP-1 prevents TGHQ-mediated caspase activation and attenuates apoptosis. (A) Cells were treated with PJ-34 and/or different concentrations of z-vad-fmk in the presence or absence of 200µM of TGHQ for 4 h, followed by costaining with PI and FITC-conjugated annexin-V, which specifically detects the translocation of phosphatidylserine to the cell surface. Cells were then analyzed by flow cytometry. (B) Expression of PARP-1 was knocked down in HL-60 cells by using siRNA with Nucleofector Kits according to the manufacturer’s protocol. Western blot analysis was performed to determine PARP-1 and β-actin expression or caspase-3 activation. Data are representative of three independent experiments. For the densitometric analysis of the Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at †p < 0.01 and *p < 0.001 via one-way ANOVA. FIG. 6. Open in new tabDownload slide Genetic or pharmacological inhibition of PARP-1 prevents TGHQ-mediated caspase activation and attenuates apoptosis. (A) Cells were treated with PJ-34 and/or different concentrations of z-vad-fmk in the presence or absence of 200µM of TGHQ for 4 h, followed by costaining with PI and FITC-conjugated annexin-V, which specifically detects the translocation of phosphatidylserine to the cell surface. Cells were then analyzed by flow cytometry. (B) Expression of PARP-1 was knocked down in HL-60 cells by using siRNA with Nucleofector Kits according to the manufacturer’s protocol. Western blot analysis was performed to determine PARP-1 and β-actin expression or caspase-3 activation. Data are representative of three independent experiments. For the densitometric analysis of the Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at †p < 0.01 and *p < 0.001 via one-way ANOVA. PARP-1 Plays a Dual Role in TGHQ-Induced Caspase Activation To further clarify the role of PARP-1 in TGHQ-induced apoptosis, the activation of a series of caspases was investigated. Caspases-3, -7, -8, and -9 were all markedly activated in response to TGHQ (Fig. 7A,C) as revealed by the cleaved caspase(s) band(s) detection, suggesting that both the intrinsic and death-receptor pathways participate in TGHQ-induced apoptosis. Strikingly, PJ-34 significantly attenuated TGHQ-induced caspase-3, -7, and -9 cleavage, confirming that PARP-1 is involved in TGHQ-induced caspase activation. Furthermore, TGHQ-mediated cytochrome c translocation from mitochondria to the cytoplasm was attenuated following the inhibition of PARP-1 (Fig. 7B), suggesting that PARP-1 supports TGHQ-induced caspase-3, -7, and -9 activation by assisting in mitochondrial cytochrome c release. In contrast, inhibition of PARP-1 potentiated caspase-8 cleavage (Fig. 7C). These findings were replicated in experiments using an alternative PARP inhibitor, 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone (data not shown). Thus, PARP-1 plays a dual role in regulating TGHQ-induced apoptosis via opposing effects on the intrinsic (mitochondrial) and extrinsic (death-receptor) pathways. FIG. 7. Open in new tabDownload slide PARP-1 plays a dual role in TGHQ-induced caspase activation. HL-60 cells were treated with various concentrations of TGHQ in the presence or absence of PJ-34 for 6 h. (A) Western blot analysis determined caspase-3, cleaved caspase-7, cleaved caspase-9, and β-actin expression. Data shown are representative of three independent experiments. (B) Western blot analysis determined cytochrome c, GAPDH, and CoxIV expression. (C) Western blot analysis determined caspase-8 and β-actin expression. For the densitometric analysis of the Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at #p < 0.05; †p < 0.01 and *p < 0.001 via one-way ANOVA. FIG. 7. Open in new tabDownload slide PARP-1 plays a dual role in TGHQ-induced caspase activation. HL-60 cells were treated with various concentrations of TGHQ in the presence or absence of PJ-34 for 6 h. (A) Western blot analysis determined caspase-3, cleaved caspase-7, cleaved caspase-9, and β-actin expression. Data shown are representative of three independent experiments. (B) Western blot analysis determined cytochrome c, GAPDH, and CoxIV expression. (C) Western blot analysis determined caspase-8 and β-actin expression. For the densitometric analysis of the Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at #p < 0.05; †p < 0.01 and *p < 0.001 via one-way ANOVA. PARP-1 Also Plays a Dual Role in Curcumin-Induced 
Caspase Activation Curcumin induces apoptosis in HL-60 cells by activating caspase-8 and caspase-3 (Anto et al., 2002). We, therefore, examined whether PARP-1 plays a similar dual role during curcumin-induced apoptosis of HL-60 cells. Indeed, inhibition of PARP-1 with PJ-34 attenuated curcumin-induced caspase-3 activation, while simultaneously potentiating caspase-8 activation, confirming that PARP-1 plays a dual role in regulating caspase activation in HL-60 cells (Fig. 8). FIG. 8. Open in new tabDownload slide PARP-1 plays a dual role in curcumin-induced caspase activation. HL-60 cells were treated with various concentrations of curcumin in the presence or absence of PJ-34 for 24 h. Western blot analysis determined caspase-3, caspase-8, and β-actin expression. For the densitometric analysis of the Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at *p < 0.001 via one-way ANOVA. FIG. 8. Open in new tabDownload slide PARP-1 plays a dual role in curcumin-induced caspase activation. HL-60 cells were treated with various concentrations of curcumin in the presence or absence of PJ-34 for 24 h. Western blot analysis determined caspase-3, caspase-8, and β-actin expression. For the densitometric analysis of the Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at *p < 0.001 via one-way ANOVA. Nuclear AIF Translocation Is PARP-1 Dependent In normal cells, AIF is confined to the mitochondrial intermembrane space. However, under certain conditions of stress, the outer mitochondrial membrane becomes permeable, and AIF can translocate to the nucleus, causing DNA condensation and fragmentation, and cell death. Because evidence s uggests that PARP-1 initiates a nuclear signal that propagates to mitochondria and triggers the release of AIF leading to cell death (Huber et al., 2004), we next investigated whether AIF nuclear translocation contributes to TGHQ-induced apoptosis of HL-60 cells. TGHQ induced marked nuclear AIF accumulation, which was significantly abrogated following PARP-1 inhibition (Fig. 9), suggesting that the DNA damage–induced PARP-1 activation plays an important role in mediating nuclear AIF translocation. FIG. 9. Open in new tabDownload slide PARP-1 activation mediates nuclear AIF translocation. HL-60 cells were treated with increasing concentrations of TGHQ in the presence or absence of PJ-34 for 5 h. Nuclear protein was extracted with the Nuclear Extraction Kit. Western blot analysis was performed to determine AIF, Cox IV, H3, and MEK1/2 levels in nucleus. Cox IV is used as the marker for mitochondrial protein, H3 is the marker for nuclear protein, and MEK1/2 is the marker for cytoplasmic protein. The membrane was stained by Ponceau S. For the densitometric analysis of Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at †p < 0.05; *p < 0.01. FIG. 9. Open in new tabDownload slide PARP-1 activation mediates nuclear AIF translocation. HL-60 cells were treated with increasing concentrations of TGHQ in the presence or absence of PJ-34 for 5 h. Nuclear protein was extracted with the Nuclear Extraction Kit. Western blot analysis was performed to determine AIF, Cox IV, H3, and MEK1/2 levels in nucleus. Cox IV is used as the marker for mitochondrial protein, H3 is the marker for nuclear protein, and MEK1/2 is the marker for cytoplasmic protein. The membrane was stained by Ponceau S. For the densitometric analysis of Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at †p < 0.05; *p < 0.01. PARP-1 Inhibition Potentiates JNK and p38 MAPK Activation It has been suggested that during ischemia/reperfusion injury, JNK and/or p38 MAPK lie downstream of PARP activation, and may be required for PARP-mediated AIF translocation (Song et al., 2008). We, therefore, examined the response of JNK and p38 MAPK in HL-60 cells triggered to undergo apoptosis following treatment with TGHQ. Exposure of HL-60 cells to TGHQ enhanced both JNK and p38 phosphorylation (Fig. 10). However, although inhibition of PARP-1 reduced TGHQ-induced AIF translocation (Fig. 9), this effect occurred in the context of potentiated JNK1/2 and p38 MAPK activation (Fig. 10). Thus, activation of JNK and p38 MAPK is not sufficient for PARP-1–mediated nuclear AIF translocation. FIG. 10. Open in new tabDownload slide PJ-34 potentiates TGHQ-mediated activation of JNK and p38 MAPKs. HL-60 cells were treated with increasing concentrations of TGHQ in the presence or absence of PJ-34 for 6 h. Western blot analysis was performed to determine JNK, p38, phosphorylated JNK, or phosphorylated p38 expression. Data are representative of three independent experiments. For the densitometric analysis of the Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at #p < 0.05; †p < 0.01 and *p < 0.001 via one-way ANOVA. FIG. 10. Open in new tabDownload slide PJ-34 potentiates TGHQ-mediated activation of JNK and p38 MAPKs. HL-60 cells were treated with increasing concentrations of TGHQ in the presence or absence of PJ-34 for 6 h. Western blot analysis was performed to determine JNK, p38, phosphorylated JNK, or phosphorylated p38 expression. Data are representative of three independent experiments. For the densitometric analysis of the Western blots, values are expressed as fold change relative to the appropriate controls and represent the mean ± SE (n = 3). Significantly different from controls at #p < 0.05; †p < 0.01 and *p < 0.001 via one-way ANOVA. Discussion In this study, we have shown that PARP-1 has the ability to regulate the nuclear translocation of AIF, and perhaps more importantly, to differentially regulate caspase activation during ROS-induced apoptosis of HL-60 cells. Despite the nuclear translocation of AIF during TGHQ-mediated apoptosis of HL-60 cells, DNA fragmentation is oligonucleosomal in nature (Fig. 1A) rather than the characteristic high-molecular-weight (MW) fragments associated with nuclear AIF (Siegel and McCullough, 2011). The ability of nuclear AIF to initiate DNA fragmentation has recently been linked to its ability to form a complex with the histone γ-H2AX and the nuclease cyclophilin A (Baritaud et al., 2010). Levels of γ-H2AX increase significantly in our model of HL-60 cell death, which is consistent with γ-H2AX being implicated in the caspase-3/caspase–activated DNAase pathway of apoptotic cell death (Lu et al., 2006; Rogakou et al., 2000). Perhaps additional posttranslational modifications of γ-H2AX might determine its relative affinity for either cyclophilin A or caspase-activated DNAase during cell death. Consistent with the oligonucleosomal nature of DNA fragmentation (Fig. 1), caspases are activated during TGHQ- induced HL-60 cell apoptosis (Fig. 7), and PARP-1 also modulates the caspase response. Thus, although PAR proteins are detected in TGHQ-treated cells and AIF is released from mitochondria and migrates to the nucleus, TGHQ-induced apoptosis in HL-60 cells is caspase dependent (Fig. 6A). Caspase- dependent apoptosis can be activated via two main pathways: the extrinsic pathway, which originates from the activation of cell-surface death-receptors, such as Fas and tumor necrosis factor- receptor 1, culminating in caspase-8 activation; and the intrinsic pathway, which originates from the mitochondrial release of cytochrome c and culminates in the activation of caspase-9 through the cytochrome c/apoptotic protease-activating factor-1 (Apaf-1)/procaspase-9 heptamer. Both the intrinsic (caspases-3, -7, and -9) and extrinsic (caspase-8) pathways are activated during TGHQ-induced apoptosis (Fig. 7). However, whereas inhibition of PARP-1 attenuates the activation of caspases-3, -7, and -9, it actually potentiates caspase-8 activation. Consistent with our findings, activation of caspases-3, -6, and -9 is also reduced in PARP-1–/– cells following H2O2 treatment (Blenn et al., 2011). The precise mechanisms by which PARP-1 differentially regulates the various caspases remain to be elucidated. The caspase-independent cell death pathway is associated with the activation of PARP-1 (Yu et al., 2002), and it has been proposed that either NAD+ depletion or PAR polymers themselves trigger AIF release (Alano et al., 2010; Siegel and McCullough, 2011; Yu et al., 2006). Pharmacological inhibition of PARP-1 significantly inhibited nuclear AIF translocation (Fig. 9), decreased PAR-positive proteins in TGHQ-treated HL-60 cells (Fig. 5A), but had only a nominal effect in restoring cellular NAD+ concentrations (Fig. 3B), whereas the nuclear translocation of AIF was completely abolished (Fig. 9). Thus, in HL-60 cells, the PARP-1–dependent mitochondrial release of AIF is more likely coupled to the PAR of mitochondrial proteins (Wang et al., 2009) than to NAD+ depletion. PARP-1 might, therefore, participate in regulating mitochondrial permeability by targeting mitochondrial proteins for poly(ADP-ribosylation). Direct binding of PAR to AIF may also be required for PARP-mediated cell death (Wang et al., 2011). Moreover, the restoration of cellular ATP concentrations following PARP inhibition, but not of NAD+ concentrations (Fig. 3), suggests that at least in HL-60 cells NAD+ is being consumed by mechanisms other than PARP. For example, SIRT1 consumes NAD+ during the deacetylation of SIRT1-target proteins, and inhibition of PARP-1 stimulates mitochondrial metabolism via the activation of SIRT1 (Bai et al., 2011). In addition, inhibition of SIRT1 induces apoptosis of breast cancer cells (Kalle et al., 2010) and SIRT1 deficiency attenuates MPP+-induced apoptosis of dopaminergic cells (Park et al., 2011). Maintaining SIRT1 function by preventing the complete depletion of NAD+ should, therefore, be expected to promote cell survival, consistent with the ability of PJ-34 to prevent HL-60 cell apoptosis (Fig. 4). That AIF undergoes nuclear translocation in concert with caspase activation in this model of apoptosis is unusual because AIF translocation is typically associated with caspase- independent cell death. Although this latter pathway is well recognized, mechanisms that trigger the release of AIF from the mitochondria and its translocation to the nucleus, where it initiates chromatin condensation and a characteristic high-MW (50 kbp) DNA fragmentation, and ultimately caspase-independent cell death are still unclear (Andrabi et al., 2006; Hong et al., 2004; Susin et al., 1999; Yu et al., 2002, 2006). In summary, TGHQ-induced apoptotic cell death of HL-60 cells is accompanied by PARP-1 and caspase activation and AIF nuclear translocation. The TGHQ-induced apoptosis appears to primarily occur via engagement of the mitochondrial-mediated pathway in a process amenable to PARP inhibition. The residual cell death in the presence of PJ-34 is likely mediated via the extrinsic apoptotic pathway. Unraveling the complex role of PARP-1 in cell death signaling pathways should help guide the therapeutic use of PARP inhibitors and assist in identifying novel targets for therapeutic intervention for diseases and conditions in which ROS-induced DNA damage play a critical role. Funding National Institute of Environmental Health Sciences to the Southwest Environmental Health Sciences Center (P30ES006694). REFERENCES Adamietz P., Rudolph A. 1984 ADP-ribosylation of nuclear proteins in vivo. Identification of histone H2B as a major acceptor for mono- and poly(ADP-ribose) in dimethyl sulfate-treated hepatoma AH 7974 cells J. Biol. Chem 259 6841 6846 Google Scholar PubMed OpenURL Placeholder Text WorldCat Alano C. C. Garnier P. Ying W. Higashi Y. Kauppinen T. M., Swanson R. A. 2010 NAD+ depletion is necessary and sufficient for poly(ADP-ribose) polymerase-1-mediated neuronal death J. Neurosci 30 2967 2978 Google Scholar Crossref Search ADS PubMed WorldCat Andrabi S. A. Kim N. S. Yu S. W. Wang H. Koh D. W. Sasaki M. Klaus J. A. Otsuka T. Zhang Z. Koehler R. C. et al. 2006 Poly(ADP-ribose) (PAR) polymer is a death signal Proc. Natl. Acad. Sci. U.S.A 103 18308 18313 Google Scholar Crossref Search ADS PubMed WorldCat Anto R. J. Mukhopadhyay A. Denning K., Aggarwal B. B. 2002 Curcumin (diferuloylmethane) induces apoptosis through activation of caspase-8, BID cleavage and cytochrome c release: Its suppression by ectopic expression of Bcl-2 and Bcl-xl Carcinogenesis 23 143 150 Google Scholar Crossref Search ADS PubMed WorldCat Bai P. Canto C. Oudart H. Brunyanszki A. Cen Y. Thomas C. Yamamoto H. Huber A. Kiss B. Houtkooper R. H. et al. 2011 PARP-1 inhibition increases mitochondrial metabolism through SIRT1 activation Cell Metab 13 461 468 Google Scholar Crossref Search ADS PubMed WorldCat Baritaud M. Boujrad H. Lorenzo H. K. Krantic S., Susin S. A. 2010 Histone H2AX: The missing link in AIF-mediated caspase-independent programmed necrosis Cell Cycle 9 3166 3173 Google Scholar Crossref Search ADS PubMed WorldCat Blenn C. Wyrsch P. Bader J. Bollhalder M., Althaus F. R. 2011 Poly(ADP-ribose)glycohydrolase is an upstream regulator of Ca2+ fluxes in oxidative cell death Cell Mol. Life Sci 68 1455 1466 Google Scholar Crossref Search ADS PubMed WorldCat Bratton S. B. Lau S. S., Monks T. J. 1997 Identification of quinol thioethers in bone marrow of hydroquinone/phenol-treated rats and mice and their potential role in benzene-mediated hematotoxicity Chem. Res. Toxicol 10 859 865 Google Scholar Crossref Search ADS PubMed WorldCat Bratton S. B. Lau S. S., Monks T. J. 2000 The putative benzene metabolite 2,3,5-tris(glutathion-S-yl)hydroquinone depletes glutathione, stimulates sphingomyelin turnover, and induces apoptosis in HL-60 cells Chem. Res. Toxicol 13 550 556 Google Scholar Crossref Search ADS PubMed WorldCat Diefenbach J., Burkle A. 2005 Introduction to poly(ADP-ribose) metabolism Cell Mol. Life Sci 62 721 730 Google Scholar Crossref Search ADS PubMed WorldCat Eastmond D. A. Smith M. T., Irons R. D. 1987 An interaction of benzene metabolites reproduces the myelotoxicity observed with benzene exposure Toxicol. Appl. Pharmacol 91 85 95 Google Scholar Crossref Search ADS PubMed WorldCat Erdelyi K. Bakondi E. Gergely P. Szabo C., Virag L. 2005 Pathophysiologic role of oxidative stress-induced poly(ADP-ribose) polymerase-1 activation: Focus on cell death and transcriptional regulation Cell Mol. Life Sci 62 751 759 Google Scholar Crossref Search ADS PubMed WorldCat Holden P., Horton W. A. 2009 Crude subcellular fractionation of cultured mammalian cell lines BMC Res. Notes 2 243 Google Scholar Crossref Search ADS PubMed WorldCat Hong S. J. Dawson T. M., Dawson V. L. 2004 Nuclear and mitochondrial conversations in cell death: PARP-1 and AIF signaling Trends Pharmacol. Sci 25 259 264 Google Scholar Crossref Search ADS PubMed WorldCat Huber A. Bai P. de Murcia J. M., de Murcia G. 2004 PARP-1, PARP-2 and ATM in the DNA damage response: Functional synergy in mouse development DNA Repair (Amst) 3 1103 1108 Google Scholar Crossref Search ADS PubMed WorldCat Jacobson E. L., Jacobson M. K. 1997 Tissue NAD as a biochemical measure of niacin status in humans Methods Enzymol 280 221 230 Google Scholar PubMed OpenURL Placeholder Text WorldCat Kalle A. M. Mallika A. Badiger J. Alinakhi Talukdar P., Sachchidanand 2010 Inhibition of SIRT1 by a small molecule induces apoptosis in breast cancer cells Biochem. Biophys. Res. Commun 401 13 19 Google Scholar Crossref Search ADS PubMed WorldCat Kaufmann S. H. Desnoyers S. Ottaviano Y. Davidson N. E., Poirier G. G. 1993 Specific proteolytic cleavage of poly(ADP-ribose) polymerase: An early marker of chemotherapy-induced apoptosis Cancer Res 53 3976 3985 Google Scholar PubMed OpenURL Placeholder Text WorldCat Lau S. S. Hill B. A. Highet R. J., Monks T. J. 1988 Sequential oxidation and glutathione addition to 1,4-benzoquinone: Correlation of toxicity with increased glutathione substitution Mol. Pharmacol 34 829 836 Google Scholar PubMed OpenURL Placeholder Text WorldCat Lu C. Zhu F. Cho Y. Y. Tang F. Zykova T. Ma W. Y. Bode A. M., Dong Z. 2006 Cell apoptosis: Requirement of H2AX in DNA ladder formation, but not for the activation of caspase-3 Mol. Cell 23 121 132 Google Scholar Crossref Search ADS PubMed WorldCat Park G. Jeong J. W., Kim J. E. 2011 SIRT1 deficiency attenuates MPP+-induced apoptosis in dopaminergic cells FEBS Lett 585 219 224 Google Scholar Crossref Search ADS PubMed WorldCat Rinsky R. A. Young R. J., Smith A. B. 1981 Leukemia in benzene workers Am. J. Ind. Med 2 217 245 Google Scholar Crossref Search ADS PubMed WorldCat Rogakou E. P. Nieves-Neira W. Boon C. Pommier Y., Bonner W. M. 2000 Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139 J. Biol. Chem 275 9390 9395 Google Scholar Crossref Search ADS PubMed WorldCat Rogakou E. P. Pilch D. R. Orr A. H. Ivanova V. S., Bonner W. M. 1998 DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139 J. Biol. Chem 273 5858 5868 Google Scholar Crossref Search ADS PubMed WorldCat Scovassi A. I. Mariani C. Negroni M. Negri C., Bertazzoni U. 1993 ADP-ribosylation of nonhistone proteins in HeLa cells: Modification of DNA topoisomerase II Exp. Cell Res 206 177 181 Google Scholar Crossref Search ADS PubMed WorldCat Sevrioukova I. F. 2011 Apoptosis-inducing factor: Structure, function, and redox regulation Antioxid. Redox Signal 14 2545 2579 Google Scholar Crossref Search ADS PubMed WorldCat Siegel C., McCullough L. D. 2011 NAD+ depletion or PAR polymer formation: Which plays the role of executioner in ischaemic cell death? Acta Physiol. (Oxf) 203 225 234 Google Scholar Crossref Search ADS PubMed WorldCat Snyder R. Witz G., Goldstein B. D. 1993 The toxicology of benzene Environ. Health Perspect 100 293 306 Google Scholar Crossref Search ADS PubMed WorldCat Song Z. F. Ji X. P. Li X. X. Wang S. J. Wang S. H., Zhang Y. 2008 Inhibition of the activity of poly (ADP-ribose) polymerase reduces heart ischaemia/reperfusion injury via suppressing JNK-mediated AIF translocation J. Cell Mol. Med 12 1220 1228 Google Scholar Crossref Search ADS PubMed WorldCat Susin S. A. Lorenzo H. K. Zamzami N. Marzo I. Snow B. E. Brothers G. M. Mangion J. Jacotot E. Costantini P. Loeffler M. et al. 1999 Molecular characterization of mitochondrial apoptosis-inducing factor Nature 397 441 446 Google Scholar Crossref Search ADS PubMed WorldCat Virag L. 2005 Structure and function of poly(ADP-ribose) polymerase-1: Role in oxidative stress-related pathologies Curr. Vasc. Pharmacol 3 209 214 Google Scholar Crossref Search ADS PubMed WorldCat Virag L., Szabo C. 2002 The therapeutic potential of poly(ADP-ribose) polymerase inhibitors Pharmacol. Rev 54 375 429 Google Scholar Crossref Search ADS PubMed WorldCat Wang Y. Dawson V. L., Dawson T. M. 2009 Poly(ADP-ribose) signals to mitochondrial AIF: A key event in parthanatos Exp. Neurol 218 193 202 Google Scholar Crossref Search ADS PubMed WorldCat Wang Y. Kim N. S. Haince J. F. Kang H. C. David K. K. Andrabi S. A. Poirier G. G. Dawson V. L., Dawson T. M. 2011 Poly(ADP-ribose) (PAR) binding to apoptosis-inducing factor is critical for PAR polymerase-1-dependent cell death (parthanatos) Sci. Signal 4 ra20 Google Scholar PubMed OpenURL Placeholder Text WorldCat Yang M. Y. Lau S. S., Monks T. J. 2005 2,3,5-tris(glutathion-S-yl)hydroquinone (TGHQ)-mediated apoptosis of human promyelocytic leukemia cells is preceded by mitochondrial cytochrome c release in the absence of a decrease in the mitochondrial membrane potential Toxicol. Sci 86 92 100 Google Scholar Crossref Search ADS PubMed WorldCat Yin S. N. Li G. L. Tain F. D. Fu Z. I. Jin C. Chen Y. J. Luo S. J. Ye P. Z. Zhang J. Z. Wang G. C. et al. 1987 Leukaemia in benzene workers: A retrospective cohort study Br. J. Ind. Med 44 124 128 Google Scholar PubMed OpenURL Placeholder Text WorldCat Yoshihara K. Itaya A. Tanaka Y. Ohashi Y. Ito K. Teraoka H. Tsukada K. Matsukage A., Kamiya T. 1985 Inhibition of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, and DNA ligase II by poly(ADP-ribosyl)ation reaction in vitro Biochem. Biophys. Res. Commun 128 61 67 Google Scholar Crossref Search ADS PubMed WorldCat Yu S. W. Andrabi S. A. Wang H. Kim N. S. Poirier G. G. Dawson T. M., Dawson V. L. 2006 Apoptosis-inducing factor mediates poly(ADP-ribose) (PAR) polymer-induced cell death Proc. Natl. Acad. Sci. U.S.A 103 18314 18319 Google Scholar Crossref Search ADS PubMed WorldCat Yu S. W. Wang H. Poitras M. F. Coombs C. Bowers W. J. Federoff H. J. Poirier G. G. Dawson T. M., Dawson V. L. 2002 Mediation of poly(ADP-ribose) polymerase-1-dependent cell death by apoptosis-inducing factor Science 297 259 263 Google Scholar Crossref Search ADS PubMed WorldCat © The Author 2012. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please email: [email protected] OUP Journals

Journal

Toxicological Sciences – Oxford University Press

Published: Jul 1, 2012

Keywords: Key Words: TGHQ PARP-1 apoptosis caspase AIF TGHQ

There are no references for this article.