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J. Biol. Chem., Vol. 281, Issue 44, 33684-33696, November 3, 2006

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Calcium-dependent Modulation of Poly(ADP-ribose) Polymerase-1 Alters Cellular Metabolism and DNA Repair^*

Melissa S. Bentle, Kathryn E. Reinicke, Erik A. Bey^¶, Douglas R. Spitz^||, and David A. Boothman^¶1

From the Departments of Pharmacology and Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106, the ^¶Department of Pharmacology, Laboratory of Molecular Stress Responses, and the Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, and the ^||Department of Radiation Oncology, Free Radical and Radiation Biology Program, Holden Comprehensive Cancer Center, University of Iowa, Iowa City, Iowa 52242

Received for publication, April 17, 2006 , and in revised form, August 17, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
After genotoxic stress poly(ADP-ribose) polymerase-1 (PARP-1) can be hyperactivated, causing (ADP-ribosyl)ation of nuclear proteins (including itself), resulting in NAD⁺ and ATP depletion and cell death. Mechanisms of PARP-1-mediated cell death and downstream proteolysis remain enigmatic. -lapachone (-lap) is the first chemotherapeutic agent to elicit a Ca²⁺-mediated cell death by PARP-1 hyperactivation at clinically relevent doses in cancer cells expressing elevated NAD(P)H:quinone oxidoreductase 1 (NQO1) levels. -lap induces the generation of NQO1-dependent reactive oxygen species (ROS), DNA breaks, and triggers Ca²⁺-dependent -H2AX formation and PARP-1 hyperactivation. Subsequent NAD⁺ and ATP losses suppress DNA repair and cause cell death. Reduction of PARP-1 activity or Ca²⁺ chelation protects cells. Interestingly, Ca²⁺ chelation abrogates hydrogen peroxide (H₂O₂), but not N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced PARP-1 hyperactivation and cell death. Thus, Ca²⁺ appears to be an important co-factor in PARP-1 hyperactivation after ROS-induced DNA damage, which alters cellular metabolism and DNA repair.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Alterations in the initiation and regulation of caspase-mediated apoptosis are associated with an array of pathological disease states, including chemotherapy resistance in cancer (1). Therefore, elucidating mechanisms that initiate non-caspase-mediated cell death are crucial for the development and use of novel anticancer agents.

A growing number of chemotherapeutic approaches focus on targeting specific DNA repair enzymes. In particular, inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1)² that sensitize cells to DNA-damaging agents are under extensive investigation (2). PARP-1 functions as a DNA damage sensor that responds to both single- and/or double-strand DNA breaks (SSBs, DSBs), facilitating DNA repair and cell survival. After binding to DNA breaks, PARP-1 converts -NAD⁺ (NAD⁺) into polymers of branched or linear poly(ADP-ribose) units (PAR) and attaches them to various nuclear acceptor proteins, including XRCC1, histones, and PARP-1 for its autoregulation (3). However, in response to extensive DNA damage, PARP-1 can be hyperactivated, eliciting rapid cellular NAD⁺ and ATP pool depletion. PARP-1-mediated NAD⁺ and ATP losses have affects on mitochondrial function by decreasing the levels of pyruvate and NADH. Loss of mitochondrial membrane potential (MMP) ensues, causing caspase-independent cell death by as yet unknown mechanisms (3). PARP-1 hyperactivation was documented in the cellular response to trauma, such as ischemia-reperfusion, myocardial infarction, and reactive oxygen species (ROS)-induced injury (3). In each case, inhibition of PARP-1 was necessary for the long-term survival of damaged cells (4).

-lapachone (-lap) elicits a unique cell death process in various human breast, lung, and prostate cancers that have elevated levels of the two-electron oxidoreductase, NAD(P)H: quinone oxidoreductase 1 (NQO1) (EC 1.6.99.2 [EC] ) (5). -lap induces an NQO1-dependent form of cell death wherein PARP-1 and p53 proteolytic cleavage fragments were noted (6), concomitant with µ-calpain activation (7). -lap-induced lethality and proteolysis were abrogated by dicoumarol (an NQO1 inhibitor), and were muted in cells deficient in NQO1 enzymatic activity (5). Restoration of NQO1 caused increases in drug sensitivity (5). In contrast to staurosporine (STS), global caspase inhibitors had little effect on -lap lethality (5). -lap-mediated cell death exhibited classical features of apoptosis (e.g. DNA condensation, trypan blue exclusion, sub-G₀-G₁ cells, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells). -lap cell death was not, however, dependent on typical apoptotic mediators, such as p53 or caspases (8). To date, the mechanisms responsible for the initiation of this unique cell death have not been delineated.

We report that -lap induces an NQO1-dependent, PARP-1-mediated cell death pathway involving changes in cellular metabolism leading to cell death. NQO1-dependent reduction of -lap results in a futile redox cycle between the parent -lap molecule and its hydroquinone form (5), wherein ROS generation causes extensive DNA damage, H2AX phosphorylation (-H2AX) and PARP-1 hyperactivation. Drastic decreases in NAD⁺ and ATP pools, in turn, inhibit DNA repair and accelerate cell death. In addition, chelation of intracellular Ca²⁺ by 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-acetoxymethyl ester (BAPTA-AM) abrogates -lap-induced: (i) -H2AX formation, (ii) PARP-1 hyperactivation, (iii) atypical PARP-1 and p53 proteolysis, and (iv) cytotoxicity without affecting NQO1-dependent ROS production. A similar Ca²⁺-sensitive cell death is observed after hydrogen peroxide (H₂O₂) exposure. Interestingly, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced PARP-1 hyperactivation is not sensitive to BAPTA-AM. These data support a critical role for Ca²⁺ as a regulator of cellular metabolism in response to ROS-induced DNA damage.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Reagents—-lap was synthesized by Dr. William G. Bornmann (MD Anderson), dissolved in dimethyl sulfoxide (Me₂SO) at 40 mM, and the concentration verified by spectrophotometric analyses (8). -lap stocks were stored at -80 °C. Hoechst 33258, 3-aminobenzamide (3-AB), dicoumarol (DIC), H₂O₂, STS, and MNNG were obtained from Sigma. BAPTA-AM was dissolved in Me₂SO and used at 5 µM unless otherwise stated. DPQ (3,4-dihydro-5[4-(1-piperindinyl)butoxy]-1(2H)-isoquinoline) was dissolved in Me₂SO and used at 20 µM. DPQ and BAPTA-AM were obtained from Calbiochem (La Jolla, CA). Z-VAD-fmk was obtained from Enzyme Systems Products (Dublin, CA), diluted in Me₂SO and used at 50 µM. DCF was dissolved in Me₂SO and used at 5 µM to monitor ROS formation. DCF was obtained from Molecular Probes (Eugene, OR).

Cell Culture—Human MCF-7 and MDA-MB-468-NQ+ breast cancer cells were maintained and used as described (5). Human MDA-MB-231 (231) breast cancer cells that contain a 609C>T polymorphism in NQO1 (9) and are deficient in enzyme activity, were obtained from the American Type Culture Collection (Manassas, VA). Cells were stably transfected with a CMV-driven NQO1 cDNA or the pcDNA3 vector alone as described (5). All cells were grown in high glucose-containing RPMI 1640 tissue culture medium containing 5% fetal bovine serum, 2 mML-glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin at 37 °C in a 5% CO₂, 95% air humidified atmosphere (6). 231-NQO1+ (NQ+) and -NQO1 (NQ-) cells were maintained in medium containing 400 µg/ml geneticin (8), but all experiments were performed without selection. All tissue culture components were purchased from Invitrogen (Carlsbad, CA) unless otherwise stated. All cells were routinely tested and found free of myco-plasma contamination.

PARP-1 Knockdown—A puromycin-selectable-pSHAG-MAGIC2 retroviral vector containing a short hairpin small interfering RNA against PARP-1 (PARP-1-shRNA) and a non-silencing sequence (ns-shRNA) control were used to infect both NQ+ and NQ-231 cells (Open Biosystems, Huntsville, AL). Cells were then selected and grown in 0.5 µg/ml puromycin and screened for PARP-1 protein expression and NQO1 enzymatic activity.

Relative Survival Assays—Relative survival assays were performed as previously described (5). MCF-7 cells were pretreated or not with BAPTA-AM (5 µM, 30 min) then treated with or without 2-h pulses of -lap at the doses indicated, in the presence or absence of 40 µM dicoumarol. In some experiments, cells were exposed to 5 µM -lap followed by delayed (t = 0-2 h) addition of 5 µM BAPTA-AM. After drug addition, media were removed and drug-free media added. Cells were then allowed to grow for an additional 6 days and relative survival, based on DNA content (Hoechst 33258 staining), was determined (5). Prior studies using -lap showed that relative survival assays correlated directly with colony forming ability assays (5). Data were expressed as treated/control (T/C) from separate triplicate experiments (means, ± S.E.), and comparisons analyzed using a two-tailed Student's t test for paired samples.

Immunoblotting and Confocal Microscopy—Western blots were prepared as previously described (8). -PARP (sc-8007) and -p53 (DO-1) antibodies were utilized at dilutions of 1:1000 (Santa Cruz Biotechnology). The -PAR antibody was used at 1:2000 dilution (BD-Pharmingen, San Jose, CA). Antibodies to total levels of H2AX or -H2AX were used at dilutions of 1:100-1:500 and purchased from Bethyl Laboratories (Montgomery, TX) and Upstate (Charlottesville, VA), respectively. An NQO1 antibody was generated and used directly for immunoblot analyses in medium containing 10% fetal bovine serum, 1x phosphate-buffered saline, and 0.2% Tween 20 (10).

Confocal microscopy was performed as previously described (7). Cells were fixed in methanol/acetone (1:1) and incubated with -PAR (10H; Alexis, San Diego, CA) or --H2AX (Trevigen, Gaithersburg, MD) for 2 h at room temperature. Nuclei were visualized by Hoechst 33258 staining at a 1:3000 dilution. Confocal images were collected at 488 nm excitation from a krypton/argon laser using a Zeiss LSM 510 confocal microscope (Thornwood, NY). Images were representative of experiments performed at least four times. The number of PAR-positive cells and -H2AX foci/cell were quantified by counting 60 or more cells from four independent experiments (means ± S.E.).

Formation of ROS was monitored by the conversion of non-fluorescent 6-carboxy-2'7'-dichlorodihy-drofluorescin diacetate, di(acetoxymethyl ester) to fluorescent 6-carboxy-2',7'-dichlorofluorescein diacetate di(acetoxymethyl ester) (DCF) as previously described (11, 12). Briefly, MCF-7 cells were seeded at 2-3 x 10⁵ cells in 35-mm glass bottom Petri dishes (MatTek Corp., Ashland, MA) and allowed to attach overnight. Cells were loaded with 5 µM DCF in media for 30 min at 37 °C. After loading, cells were washed twice with phosphate-buffered saline, and incubated for an additional 20 min at 37 °C to allow for dye de-esterification. Confocal images of DCF fluorescence were collected using 488-nm excitation from an argon/krypton laser, 560-nm dichroic mirror, and a 500-550 nm band pass filter. Three basal images were collected before drug addition (5-8 µM -lap, +5 µM BAPTA-AM or 40 µM dicoumarol). Subsequent images were taken after the indicated treatments at 15-s intervals and similar results were found at 37 °C or rm. temp. BAPTA-AM was co-loaded with DCF where indicated. Mean pixel intensities were determined in regions of interest for at least 40 individual cells at each time point. Shown are representative traces of at least three independent experiments (means ± S.E.).

Single Cell Gel Electrophoresis (Alkaline Comet) Assays—DNA damage was assessed after different drug treatments by evaluating DNA "comet" tail area and migration distance (13). MCF-7 cells were pretreated with BAPTA-AM (5 µM, 30 min) or Me₂SO (1:1000 dilution), and then exposed to H₂O₂ (500 µM, 1 h), -lap (4 µM, various times), or vehicle alone, and harvested at various times. Cell suspensions (3 x 10⁵/ml cold PBS) were mixed with 1% low melting temperature agarose (1:10 (v/v)) at 37 °C and immediately transferred onto a CometSlide^TM (Trevigen). After solidifying (30 min at 4 °C), slides were submerged in prechilled lysis buffer (2.5 M NaCl, 100 mM EDTA pH 10, 10 mM Tris Base, 1% sodium lauryl sarcosinate, and 1% Triton X-100) at 4 °C for 45 min, incubated in alkaline unwinding solution (300 mM NaOH, and 1 mM EDTA) for 45 min at room temperature and washed twice (5 min) in neutral 1x TBE (89.2 mM Tris Base, 89 mM boric acid, and 2.5 mM EDTA disodium salt). Damaged and undamaged nuclear DNA was then separated by electrophoresis in 1x TBE for 10 min at 1 V/cm, fixed in 70% ethanol, and stained using SYBR-green (Trevigen). Comets were visualized using an Olympus fluorescence microscope (Melville, NY), and images captured using a digital camera. Images were analyzed using ImageJ software (14, 15) and comet tail length was calculated as the distance between the end of nuclei heads and the end of each tail. Tail moments were defined as the product of the %DNA in each tail, and the distance between the mean of the head and tail distributions in Equation 1,

(Eq.1)

where TA is the tail area, TAI is the tail area intensity, HA is the head area, and HAI is the head area intensity. Importantly, tail moment and tail area calculations yielded similar experimental results. Each datum point represented the average of 100 cells ± S.E., and data are representative of experiments performed three times.

Determination of NAD⁺ and ATP Levels—Intracellular NAD⁺ levels were measured as described (16) with modification. Briefly, cells were seeded at 1 x 10⁶ and allowed to attach overnight. Cells were pretreated for 2 h with 3-AB (25 mM), DPQ (20 µM), BAPTA-AM (5 µM), or Me₂SO and then exposed to -lap (2-20 µM) for the indicated times. Cell extracts were prepared in 0.5 M perchloric acid, neutralized (1 M KOH, 0.33 M KH₂PO₄/K₂HPO₄ (pH 7.5)), and centrifuged to remove KClO₄ precipitates. Supernatants or NAD⁺ standards were incubated 4:1 (v/v) for 20 min at 37 °C with NAD⁺ reaction mixture as described (17). Measurements from extracts were taken at an absorbance of 570 nm and intracellular NAD⁺ levels were normalized to 1 x 10⁶ cells. Data were expressed as %NAD⁺ ± S.E., for T/C samples from nine individual experiments.

ATP levels were analyzed using a luciferase-based bioluminescence assay as described (18). Data were graphed as means ± S.E. of experiments performed at least three times. Results were compared using the two-tailed Student's t test for paired samples.

NQO1 Enzyme Activity Assays—NQO1 enzymatic assays were performed as described (19) using cytochrome c (practical grade, Sigma) in Tris-HCl buffer (50 mM, pH 7.5). NADH (200 µM) was the immediate electron donor, and menadione (10 µM) the electron acceptor. Changes in absorbance were monitored using a Beckman DU 640 spectrophotometer (Beckman Coulter, Fullerton, CA). Dicoumarol (10 µM) inhibitable NQO1 levels were calculated as nmol of cytochrome c reduced/min/µg protein based on initial rate of change in absorbance at 550 nm and an extinction coefficient for cytochrome c of 21.1 nmol/liter/cm (20). Results were expressed as means ± S.E. of three or more separate experiments.

Flow Cytometry and Apoptotic Measurements—Flow cytometric analyses of TUNEL-positive cells were performed as described using APO-DIRECT^TM (BD Pharmingen) (5). Samples were analyzed in an EPICS Elite ESP flow cytometer using an air-cooled argon laser at 488 nm, 15 milliwatt (Beckman Coulter Electronics, Miami, FL) and Elite acquisition software. Experiments were performed a minimum of five times, and data expressed as means ± S.E. Statistical analyses were performed using a two-tailed Student's t test for paired samples.

Glutathione Measurements—Disulfide glutathione and total glutathione (GSH + GSSG) levels were determined using a spectrophotometric recycling assay (21, 22). Following indicated treatments, pellets were thawed and whole cell homogenates prepared as described (21, 22). Data were expressed as the %GSSG normalized to protein content, as measured using the method of Lowry et al. (23). Shown are means ± S.E. for experiments performed at least three times.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Time and Ca²⁺ Dependence of -lap-induced Cell Death—To elucidate the signaling events required for -lap-induced cell death, log-phase human MCF-7 breast cancer cells, with high levels of endogenous NQO1 activity, were tested for their sensitivities to various concentrations of -lap at various times. The purpose of these experiments was to determine the minimal time of -lap exposure required to kill the entire cell population. Cells exposed to doses of 3 µM -lap required 4hto elicit cell death, whereas 2 h exposures of -lap at 4 µM killed all MCF-7 cells (Fig. 1A).

Prior data from our laboratory demonstrated that Ca²⁺ was released within 2-5 min from endoplasmic reticulum (ER) Ca²⁺ stores after -lap treatment (24), suggesting that this was a required initiating factor in -lap-induced cell death (24). To test this, MCF-7 cells were pretreated with the intracellular Ca²⁺ chelator BAPTA-AM (5 µM, 30 min), then exposed to 4 µM -lap for various times (Fig. 1B). Under these conditions, we previously demonstrated that BAPTA-AM pretreatment was sufficient to block the rise in cytosolic Ca²⁺ caused by -lap treatment (24). BAPTA-AM abrogated -lap-induced cytotoxicity and nuclear condensation (Fig. 1B and supplemental Fig. S1A). To determine the kinetics of Ca²⁺-dependent cell death, MCF-7 cells were treated with 5 µM -lap, and 5 µM BAPTA-AM was added at various times thereafter, up to 2 h. A time-dependent decrease in survival was observed with delayed addition of BAPTA-AM (Fig. 1C), indicating that Ca²⁺ release was a necessary event, occurring before cells were committed to death. Abrogation of -lap cytotoxicity by BAPTA-AM was equivalent to that noted with NQO1 inhibition by dicoumarol (Fig. 1B). Addition of BAPTA-AM also prevented -lap-induced, atypical proteolysis (e.g. 60 kDa PARP-1 and p53 cleavage fragments), in a manner as effective as dicoumarol (supplemental Fig. S1B, lanes 3 and 4). Interestingly, Z-VAD-fmk (50 µM), a pan-caspase inhibitor, did not block atypical PARP-1 and p53 proteolysis in -lap-treated MCF-7 cells (lane 7). As expected, Z-VAD-fmk inhibited STS (1 µM)-induced caspase-mediated proteolysis (25). However, BAPTA-AM did not affect STS-induced apoptotic proteolysis (supplemental Fig. S1C). These data, in conjunction with our prior data showing -lap-induced ER Ca²⁺ release (24), support a role for Ca²⁺ in the initiation of cell death induced by this drug.

View larger version (20K): [in this window] [in a new window]   FIGURE 1. -lap-induced cell death is time- and Ca2+-dependent. A-C, cell death was examined using relative survival assays in NQO1+ MCF-7 cells. A, cells were exposed to various doses of -lap for different lengths of time to determine the minimal exposure time required for cell death. After drug exposure, media were removed and drug-free media added. Cells were then allowed to grow for an additional 6 days and relative survival, based on DNA content was determined by Hoechst 33258 staining as described under "Experimental Procedures." Student's t test for paired samples, experimental group compared with 3 µM -lap (*, p 0.001; **, p 0.005). Prior studies using -lap showed that relative survival assays directly correlated with colony forming ability. B, relative survival assays using MCF-7 cells treated with 4 µM -lap alone, or in combination with 40 µM dicoumarol or 5 µM BAPTA-AM. C, MCF-7 cells were treated with 5 µM -lap at t = 0. BAPTA-AM (5 µM) was then added to -lap-treated cells at various times afterwards as indicated.

View larger version (57K): [in this window] [in a new window]   FIGURE 2. -lap induces NQO1- and Ca2+-dependent PARP-1 hyperactivation. A, immunoblots of PAR formation as a measure of PARP-1 hyperactivation, and steady-state PARP-1 protein levels from whole cell extracts of isogenic 231-NQ+ and 231-NQ-cells treated with 6 µM -lap for 10-90 min. Relative PAR levels were calculated by densitometric analyses by NIH ImageJ using PARP loading controls wherein controls were set to 1.0. B, immunoblots of PAR formation and steady state -tubulin expression in extracts from MCF-7 cells treated with -lap or H2O2 (2 mM, control for PARP-1 hyperactivation). Other cells were pretreated with BAPTA-AM with or without -lap (5 µM). Samples were harvested at the indicated times. Relative PAR levels were calculated by densitometric analyses by NIH ImageJ using -tubulin loading controls wherein controls were set to 1.0. C, assessment of PARP-1 hyperactivation, measured by PAR formation, in MCF-7 cells treated with -lap alone or in cells pretreated with BAPTA-AM for 30 min prior to -lap exposure. PAR formation was visualized using confocal microscopy. D, quantified percentages of PAR-positive cells from confocal microscopy analyses of at least 60 cells from four independent experiments (means ± S.E.).

View larger version (26K): [in this window] [in a new window]   FIGURE 3. PARP-1-dependent NAD+ and ATP pool depletion leads to cell death after -lap exposure in MCF-7 cells. A and B, NAD+ pool depletion occurs immediately after -lap treatment in MCF-7 cells. A, MCF-7 cells were exposed to varying concentrations of -lap for 1 h, or B, 5 µM -lap alone with or without pre- and co-treatment of PARP inhibitors, (20 µM DPQ or 25 mM 3-AB) or 5 µM BAPTA-AM for various times and harvested for NAD+ content. Student's t test for paired samples, comparing experimental groups containing -lap + 3-AB or DPQ versus -lap alone are indicated (*, p 0.001; **, p 0.05, respectively). C, intracellular ATP levels were monitored using a luciferase-based bioluminescence assay in MCF-7 cells treated with -lap, or in cells pre- and co-treated with 20 µM DPQ, or 25 mM 3-AB 2 h prior to -lap addition. Differences were compared using two-tailed Student's t test. Groups having *, p 0.05 values compared with -lap alone are indicated. D, apoptotic DNA fragmentation was assessed using TUNEL assays in -lap-exposed, log-phase MCF-7 cells with or without pre- and co-treatment with DPQ or 3-AB.

To confirm that the energetic consequences of PARP-1 hyperactivation (e.g. NAD⁺ and ATP losses) were necessary and sufficient for -lap-induced cell death, the effects of 3-AB or DPQ, on apoptosis, was measured by TUNEL assay. Pretreatment with either inhibitor resulted in a reduction in apoptosis (2% and 15% total apoptosis, respectively for 3-AB and DPQ) compared with 55% in -lap-treated cells (Fig. 3D). Thus, PARP-1 inhibition by 3-AB or DPQ spared -lap-induced apoptosis in NQO1+ MCF-7 cells, consistent with the effects of these inhibitors to prevent NAD⁺ and ATP losses. Cumulatively, these data strongly suggest that Ca²⁺-dependent PARP-1 hyperactivation caused NAD⁺ and ATP loss in NQO1+ human cancer cell lines after -lap treatment.

View larger version (44K): [in this window] [in a new window]   FIGURE 4. PARP-1 plays an essential role in-lap-induced apoptotic cell death as monitored by TUNEL. A, immunoblots of steady state PARP-1 and -tubulin protein levels from whole cell extracts of 231-NQ+ ns-shRNA, 231-NQ+ PARP-1-shRNA, 231-NQ-ns-shRNA, and 231-NQ-PARP-1-shRNA cell lines. Relative PARP-1 protein levels were calculated by densitometry analyses using -tubulin loading controls by NIH ImageJ wherein controls were set to 1.0. B, immunoblots of PAR, PARP-1, and -tubulin protein levels from control and -lap (6 µM, 10-90 min)-exposed 231-NQ+ ns-shRNA or PARP-1-shRNA cells. Relative PAR levels were calculated by densitometry analyses using -tubulin loading controls by NIH ImageJ. C, PARP-1 protein knock-down does not alter NQO1 enzymatic activity. NQO1 enzyme activities were determined for 231-NQ+ ns-shRNA, 231-NQ+ PARP-1-shRNA, 231-NQ-ns-shRNA, and 231-NQ-PARP-1-shRNA cells. NQO1 enzyme activities were calculated as nmol of cytochrome c reduced/min/µg protein. D, PARP-1 is necessary for NAD+ loss after -lap exposure. 231-NQ+ ns-shRNA and 231-NQ+ PARP-1-shRNA cells were treated with 6 µM -lap for 0-120 min at which times NAD+ content was determined as previously described. E, -lap-induced ATP loss is PARP-1-dependent. ATP levels were monitored in 231-NQ+ ns-shRNA or PARP-1-shRNA cells after exposure to -lap (6 µM, 0-120 min). Student's t test for paired samples, comparing experimental groups 231-NQ+ ns-shRNA versus 231-NQ+ PARP-1-shRNA are indicated (*, p 0.05; **, p 0.001). F, knock-down of PARP-1 protein protects cells from -lap-induced cell death. 231-NQ+ ns-shRNA, and 231-NQ+ PARP-1-shRNA cells were exposed to a 2-h pulse of -lap and monitored for apoptosis 24-h later. Students's t test for paired samples, experimental group compared with vehicle control (*, p 0.001).

NQO1-dependent DNA Damage after -lap Treatment—To date, there is little direct evidence that -lap treatment causes DNA damage as assessed by alkaline or neutral filter elution, p53 induction, or covalent complex protein-DNA formation (8, 32, 33). However, many of these prior studies were performed in cells expressing little to no NQO1 (34). PARP-1 hyperactivation typically requires DNA damage, therefore, we examined NQO1-positive cells exposed to -lap for DNA breaks, by measuring -H2AX. H2AX contains a highly conserved serine residue (Ser¹³⁹), that is rapidly phosphorylated upon DNA damage (35). Significant -H2AX foci formation was observed in 30 min, similar to that seen 15 min following 5 Gy (Fig. 5A). In contrast, total H2AX and -tubulin levels remained unchanged. These results were confirmed by confocal microscopy (Fig. 5B).

Because Ca²⁺ chelation blocked both -lap-induced PARP-1 hyperactivation and cell death, we tested the effects of BAPTA-AM on -H2AX foci formation. Similar to the immunoblot analyses and the PAR formation kinetics (Fig. 2C), -lap-treated MCF-7 cells showed -H2AX foci in 10 min (17 foci/cell) of exposure, with peak levels at 60 min (40 foci/cell) (Fig. 5C). Importantly, -lap-induced -H2AX foci formation was partially abrogated by BAPTA-AM addition, with fewer -H2AX foci noted in 30-90 min (5-25 foci/cell respectively) (Fig. 5C). These results were confirmed by immunoblot analyses (Fig. 5D).

Ca²⁺ Chelation Modulates DNA Repair after -lap Treatment—We postulated that metabolism of -lap by NQO1 would generate superoxide, peroxide, and other ROS (36). We directly monitored intracellular ROS formation, using the conversion of non-fluorescent 6-carboxy-2',7'-dichlorodihydrofluorescin to fluorescent 6-carboxy-2',7'-dichlorodihydrofluorescein (DCF). Indeed, exposure of MCF-7 cells with 5 or 8 µM -lap treatment, caused an increase in fluorescence within 5 min compared with Me₂SO-treated cells (Fig. 6A, left panel). Region-of-interest analyses showed an 2000-fold increase in fluorescence with -lap alone over control cells, which could be abrogated by inhibiting NQO1 activity with dicoumarol (Fig. 6A, right panel). Because BAPTA-AM has moderate affinity for divalent cations other than Ca²⁺, we explored the possibility that BAPTA-AM may protect cells from DNA damage and subsequent cell death by interfering with Fenton chemistry. Cells pretreated with 5 µM BAPTA-AM and then exposed to 5 or 8 µM -lap exhibited no significant difference in the rate or extent of ROS formation compared with -lap alone-treated cells (Fig. 6A). These results were confirmed by examining the oxidative state of MDA-MB-468-NQ+ cells after treatment with 4 µM -lap in the presence or absence of 5 µM BAPTA-AM. -lap treatment caused an 65% rise in disulfide glutathione (GSSG) levels, that persisted during drug exposure (Fig. 6B). Addition of BAPTA-AM did not alter the kinetics or levels of GSSG formation during -lap exposure (Fig. 6B). These data suggest that the protective effects of BAPTA-AM on -lap-treated NQO1+ cells were not caused by interference with -lap-induced ROS formation. Similar results were found in 231-NQ+ cells (data not shown).

To assess the effects of Ca²⁺ on DNA damage and repair,-lap-treated MCF-7 cells were analyzed by alkaline comet assays to monitor total DNA strand breaks with or without BAPTA-AM addition. -lap-treated cells exhibited significant DNA strand breakage by 10 min, resembling the positive control (H₂O₂), and after 30 min, -lap-induced DNA damage exceeded those levels (Fig. 6C and supplemental Fig. S3). Cells pretreated with BAPTA-AM exhibited less DNA damage compared with -lap alone, and their repair of DNA damage correlated well with their ability (or lack thereof) to survive (Figs. 6C and 1B).

We then examined the kinetics of repair in MCF-7 cells following a 2-h -lap exposure with or without BAPTA-AM pretreatment. After -lap exposure, DNA damage persisted and gradually increased over time (Fig. 6C), indicative of inhibition of DNA repair and consistent with the drop in NAD⁺ and ATP levels (Fig. 3, B and C). Although cells treated with -lap and BAPTA-AM at 2 h exhibited equivalent damage to 10 min of -lap exposure alone (4.6 ± 0.2 versus 4.7 ± 0.4, p > 0.5 comet microns, respectively), BAPTA-AM pretreated cells were protected from PARP-1 hyperactivation (Fig. 2B), as well as decrements in NAD⁺ levels (Fig. 3B). BAPTA-AM-pretreated cells showed a time-dependent recovery from DNA damage (Fig. 6C and supplemental Fig. S3). In contrast, -lap-exposed cells showed extensive DNA damage with no signs of DNA repair. Collectively, these data suggest that NQO1-mediated metabolism of -lap leads to the generation of ROS and subsequent DNA damage that hyperactivates PARP-1.

H₂O₂ Causes Ca²⁺-dependent PARP-1 Hyperactivation—To examine the universality of Ca²⁺-modulated PARP-1 function in response to other DNA damaging agents, we examined responses to H₂O₂ or MNNG. Unlike -lap, H₂O₂ treatment caused PARP-1 hyperactivation in both 231-NQ+ and 231-NQ-cells (Fig. 7A). However, expression of NQO1 required higher doses of H₂O₂ to cause PAR formation in 231 cells (Fig. 7A). These data suggest that NQO1 has a broader antioxidant role by protecting against ROS-induced damage as previously proposed (37-39). Consistent with -lap, however, was the abrogation of H₂O₂-induced PAR formation by BAPTA-AM in 231 cells independent of NQO1 activity (Fig. 7B).

H₂O₂ treatment also caused a dose-dependent increase in apoptosis in both 231-NQ- and 231-NQ+ cells that was blocked by BAPTA-AM (Fig. 7C). However, 231-NQ+ cells were much less sensitive to H₂O₂ than 231-NQ-cells. ATP loss was seen within minutes of H₂O₂ exposure in 231-NQ-, but not in NQO1-positive 231-NQ+ cells (supplemental Fig. S4). In addition, PARP-1 hyperactivation and cell death in response to equivalent doses of H₂O₂ in 231-NQ-cells was much more robust than in 231-NQ+ cells (Fig. 7, B and C). Interestingly, as noted with -lap exposure, treatment of MCF-7 cells with 200 µM H₂O₂ for 2 h resulted in formation of a 60-kDa PARP-1 and 40-kDa p53 fragments. This atypical proteolysis was effectively inhibited by BAPTA-AM pretreatment (supplemental Fig. S5).

Finally, BAPTA-AM had no effect on PARP-1 hyperactivity or cytotoxicity caused by treatment with the monofunctional DNA-alkylating agent, MNNG (Fig. 7E). Because MNNG does not cause Ca²⁺ release like -lap or H₂O₂, these data suggest that Ca²⁺ modulation of PARP-1 hyperactivation is unique to ROS-producing agents.

View larger version (55K): [in this window] [in a new window]   FIGURE 5. -lap-induced -H2AX foci formation is abrogated by BAPTA-AM pretreatment. A, immunoblot analyses of -H2AX, total H2AX, and -tubulin protein levels in whole cell extracts from MCF-7 cells exposed to -lap for various times, or IR (5 Gy) harvested after 15 min. B, visualization of -H2AX foci in MCF-7 cells at various times after treatment with 5 µM -lap or 15 min post-IR (5 Gy) by confocal microscopy. C, BAPTA-AM pretreatment followed by -lap exposure abrogates -H2AX foci formation in MCF-7 cells as visualized by confocal microscopy. The number of -H2AX foci per cell was determined from at least 60 cells for each treatment group from four independent confocal experiments (means ± S.E.). D, immunoblot of -H2AX, total H2AX, and -tubulin protein levels in whole cell extracts from MCF-7 cells exposed to -lap for various times with or without BAPTA-AM (5 µM) pretreatment, or IR (5 Gy) harvested after 15 min.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

View larger version (28K): [in this window] [in a new window]   FIGURE 6. Ca2+ modulates DNA repair in -lap-treated cells. A,-lap treatment causes the formation of ROS that is not blocked by BAPTA-AM pretreatment. MCF-7 cells were loaded with DCF as described under "Experimental Procedures." Images were collected before drug treatment (0 min) and every 15 s during treatment up to 5 min. Cells were treated with -lap (5 or 8 µM) with or without BAPTA-AM pretreatment or co-treatment with dicoumoral. Values represent the average of total cellular DCF fluorescence expressed as arbitrary units (AU) from 0-5 min (left panel). X-fold changes in fluorescence were calculated as the difference in fluorescence at 5 min minus the initial fluorescence obtained at the time of drug addition (right panel). Results are expressed as means ± S.E. from 20-30 cells per treatment group. Shown are representative data from three independent experiments. B, BAPTA-AM does not block -lap-induced ROS production in NQO1+ MDA-MB-468 cells as monitored by changes in levels of disulfide glutathione (%GSSG) following treatment with 4 µM -lap alone, or in cells pretreated for 30 min with 5 µM BAPTA-AM and then exposed to 4 µM -lap. C, DNA damage in MCF-7 cells during (left) or following (right) vehicle (Me2SO) alone, 500 µM H2O2 (), 4 µM -lap, 5 µM BAPTA-AM () or in MCF-7 cells pretreated with 5 µM BAPTA-AM prior to -lap (4 µM) exposure. DNA damage was assessed by alkaline comet assays at the indicated times. The comet tail area of 100 cells (means ± S.E.) for each time and condition were quantified with NIH ImageJ software and normalized to untreated cells. Similar results were obtained by analyzing comet tail length.

PARP-1 hyperactivation in response to -lap treatment was not cell type-specific and has been observed in all cells that express elevated NQO1 levels (Fig. 2A, and supplemental Fig. S2, A-C). As a result, cells exposed to -lap exhibited depletion of NAD⁺ and ATP, occurring 30-60 min during drug exposure. NAD⁺ and ATP losses were, in part, PARP-1-mediated since PARP inhibitors (e.g. 3-AB and DPQ) partially abrogated nucleotide loss (Fig. 3, B and C). Chemical inhibition of PARP-1, or PARP-1 protein knock-down, not only prevented NAD⁺ and ATP losses, but also abrogated -lap-induced apoptosis (Figs. 3D and 4, D-F). These data established PARP-1-mediated NAD⁺ and ATP losses as crucial upstream events in -lap-mediated cell death.

PARP-1-mediated alterations in cellular metabolism caused by -lap reported in NQO1-expressing cells in this study explain many of its purported effects in vitro and in vivo. These include, but are not limited to: (i) inhibition of NFB activation via inhibition of IKK- (41), (ii) lack of caspase activation (6) and p53 stabilization (8), and (iii) inhibition of Topoisomerase (Topo) I and Topo II- (42). Furthermore, -lap can initiate cell death independently from Bax and/or Bak activation as changes in mitochondrial outer membrane permeabilization (MOMP) can occur via PARP-1-mediated NAD⁺ loss.³ Thus, the results reported here appear to explain all prior phenomena reported in cells exposed to -lap.

A unique feature of PARP-1-mediated cell death stimulated by -lap was that administration of BAPTA-AM abrogated PARP-1 hyperactivation (Fig. 2B, supplemental Fig. S2D), nucleotide pool loss (Fig. 3B), atypical proteolyses (assessed by p53 and 60 kDa PARP-1 cleavage) (supplemental Fig. S1B), and cell death (Fig. 1, B and C). When BAPTA-AM was added >20 min after -lap treatment, cells were not protected from cell death (Fig. 1C), suggesting that events occurring within the first 20 min of drug exposure committed cells to death. BAPTA (free acid form) did not alter NQO1 activity in vitro (24). This appears to be confirmed by the inability of BAPTA-AM to prevent ROS generation, which arises from NQO1-mediated metabolism of -lap. Instead, our data suggest that the ability of BAPTA-AM to prevent -lap-induced lethality in NQO1+ cancer cells was caused by the specific prevention of PARP-1 hyperactivation. The observed differences in the amount of DNA damage and -H2AX foci formation between -lap alone and that of -lap co-administered with BAPTA-AM suggest that preventing PARP-1 hyperactivation and subsequent changes in cellular metabolism can allow for cell recovery, noted by more rapid and extensive DNA damage repair (Figs. 5C and 6C). Recent data suggest that protein phosphatase 2A (PP2A) dephosphorylates -H2AX and is required for DSB repair (43). It is possible that Ca²⁺ chelation not only prevents PARP-1 hyperactivation, but also augments -H2AX dephosphorylation through PP2A activity. ROS-induced ER Ca²⁺ release may poison PP2A. However, we favor the theory that NQO1-mediated -lap-induced ER Ca²⁺ release has its predominant affect on PARP-1 hyperactivation, thereby inhibiting DNA repair and cell recovery.

View larger version (59K): [in this window] [in a new window]   FIGURE 7. H2O2 causes Ca2+-dependent PARP-1 hyperactivation and cell death. A, PARP-1 hyperactivation after H2O2 exposure occurs regardless of NQO1 status. Immunoblot analyses of PAR, NQO1, and -tubulin protein levels in whole cell extracts from 231-NQ- and 231-NQ+ cells after exposure to varying doses of H2O2 for 15 min. B, PARP-1 hyperactivation after H2O2 treatment is Ca2+-dependent. 231-NQ-(left) and 231-NQ+ cells (right) were pretreated with BAPTA-AM or vehicle alone for 30 min and then treated with varying doses of H2O2 and harvested after 15 min. Immunoblots of PAR, and -tubulin protein levels from whole cell extracts were then analyzed. C, Ca2+ chelation protects 231-NQ- and 231-NQ+ cells from H2O2-induced apoptosis. TUNEL assays were performed in H2O2-exposed, log-phase 231-NQ- and 231-NQ+ cells, with or without pretreatment with 5 µM BAPTA-AM. D, MNNG-induced PARP-1 hyperactivation is not blocked by Ca2+ chelation. Immunoblots of PAR and -tubulin protein levels from whole cell extracts of MCF-7 cells treated with MNNG or in cells pretreated for 30 min with BAPTA-AM and then exposed to MNNG are shown. Cells were treated for the indicated times and immediately harvested.

PARP-1 hyperactivation was also observed following high doses of MNNG, however, this response was not affected by BAPTA-AM (Fig. 7D). These data highlight two separate PARP-1 regulatory mechanisms. First, ROS-induced, PARP-1-mediated cell death appears to require Ca²⁺ as a cofactor, whereas alkylation-mediated PARP-1-induced cell death does not. We propose that Ca²⁺ release following ROS-induced stress directly influences PARP-1 and PARG function. Both Mg²⁺ and Ca²⁺ exert significant (3-fold increases) allosteric activation of PARP-1 auto(ADP-ribosyl)ation in vitro that is inhibited by EDTA addition (45). We, therefore, speculate that Ca²⁺ chelation modulates PAR synthesis by dampening PARP-1 auto(poly-ADP)-ribosylation. Furthermore, since increases in [Ca²⁺] can inhibit PARG function by up to 50% in vitro, maintenance of homeostatic Ca²⁺ levels after drug treatment would, thereby, restore the normal turnover of PAR by PARG, lifting PARP-1 self-inhibition (46). Our data are consistent with the hypothesis that both PAR synthesis and degradation can be modulated by BAPTA-AM to spare the cell from metabolic catastrophe via Ca²⁺-mediated NAD⁺ and ATP losses (Figs. 2B, and 3, B and C) (47). The remaining PARP-1 activity would be necessary for DNA break repair, ultimately providing a survival advantage to damaged cells (Figs. 1B, 5C, 6C and 7C). We are currently exploring the mechanism by which Ca²⁺ modulates PARP-1 hyperactivation and subsequent DNA repair after H₂O₂ or -lap treatments versus MNNG.

There appears to be some disagreement as to the role of Ca²⁺ in PARP-1-dependent cell death. Ca²⁺ can hyperactivate PARP-1 in the absence of DNA breaks (48). In neuronal cells, glutamate caused Ca²⁺-mediated ROS production through mitochondrial dysfunction, leading to DNA damage, PARP-1 hyperactivation, and cell death. Furthermore, Ca²⁺ chelators, such as BAPTA-AM, EGTA-AM, and Quin-2-AM, protected against other oxidative stress-induced apoptotic and necrotic cell death mechanisms (49, 50). In these studies, Ca²⁺ chelation did not directly inhibit PARP-1 activity, but rather prevented DNA damage by inhibiting ROS. Contrary to these observations, in our system BAPTA-AM did not alter the direct production of ROS or oxidative stress in H₂O₂-or -lap-exposed cells (Fig. 6A). Therefore, there does not appear to be an interference with transition metal-mediated oxidant production by BAPTA-AM (e.g. Fenton reaction) as previously suggested after H₂O₂ treatment (51, 52). In fact, our data demonstrate that -lap caused equivalent ROS production in both -lap alone and -lap + BAPTA-AM treated cells. In contrast to -lap alone-treated cells, cells pretreated with BAPTA-AM did not exhibit notable PARP-1 hyperactivation, associated NAD⁺ and ATP losses, and showed a decrease in DNA damage over time (supplemental Table S1). While these data are suggestive of ongoing DNA repair in the presence of Ca²⁺ chelators, we cannot discount that these observations could also be the result of a decrease in the initial amount of DNA damage created in NQO1+ cells in response to -lap. Initial DNA damage and active DNA repair would be indistinguishable in these experiments. Thus, although we believe it is unlikely, the Ca²⁺-dependence of PARP-1 hyperactivation could be an indirect consequence of a BAPTA-AM-mediated decrease (i.e. protection) in the initial amount of DNA lesions created in response to -lap. Future studies will address this issue by utilizing DNA repair-compromised NQO1+ cells.

Collectively, our data suggest that PARP-1 is necessary for the initiation of cell death caused by -lap. To date, however, the endonuclease responsible for the execution of cell death in response to -lap treatment remains unknown. We therefore propose, that PARP-1-mediated NAD⁺ and ATP losses, in addition to PARG-liberated ADP-ribose, causes an influx of Ca²⁺ from extracellular and intracellular sources. Impairment of ATP-dependent membrane/organelle transporters (e.g. plasma membrane Ca²⁺ ATPases (PMCA) and sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPases (SERCA)) by ATP loss, and activation of plasma membrane cation channels (e.g. transient receptor potential-melastatin-like (TRMP)) by ADP-ribose, leads to high intracellular Ca²⁺ levels (53) sufficient to activate the Ca²⁺-dependent protease µ-calpain and commit the cell to death. Previous studies from our laboratory have demonstrated that -lap causes the downstream activation of µ-calpain resulting in its translocation to the nucleus concomitant with nuclear proteolytic cleavage of p53 and PARP-1 (7). Studies from our laboratory indicate that -lap treatment causes apoptosis-inducing factor (AIF) translocation from the mitochondria to the nucleus, leading to nuclear condensation following µ-calpain activation.⁴ To date, the mechanism responsible for PARP-1-mediated AIF release remains unclear. We speculate that AIF release under conditions of DNA damage may be mediated through a concerted effort of both µ-calpain and PARP-1. Disruption of the mitochondrial membrane potential through PARP-1-dependent NAD⁺ and ATP losses, in conjunction with µ-calpain-mediated cleavage of Bid or of AIF itself, may mediate its release from the mitochondria (54, 55).

In conclusion, our studies offer new insights into the signal transduction pathways necessary for PARP-1-mediated cell death, providing a connection between PARP-1 hyperactivation and cell death via fluctuations in Ca²⁺ homeostasis. Knowledge of this pathway may be used to understand, and effectively treat, a large number of human pathologies (e.g. ischemia-reperfusion during heart attacks and stroke, and diabetes), as well as to enhance current cancer chemotherapeutic agents through modulation of PARP-1 hyperactivation.

	FOOTNOTES

 
^* This work was supported in part by National Institutes of Health/NCI Grant 1R01CA10279201 (to D. A. B.), 1R01CA100045 (to D. R. S.), CWRU Core Grants P30CA43703 and P30CA43703-12, as well as Department of Defense Breast Cancer Program Predoctoral Fellowships, W81XWH-04-1-0301 and W81XWH-05-1-0248 (to M. S. B. and K. E. R.), respectively. This is manuscript CSCN 001 from the Cell Stress and Cancer Nanomedicine program in the Simmons Comprehensive Cancer Center at the University of Texas Southwestern Medical Center at Dallas. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental experimental procedures, Figs. S1-S5, and Table S1.

¹ To whom correspondence should be addressed: Dept. of Pharmacology, Laboratory of Molecular Stress Responses, and the Simmons Comprehensive Cancer Center, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390. Tel.: 214-648-9255; Fax: 214-648-0264; E-mail: David.Boothman{at}UTSouthwestern.edu.

² The abbreviations used are: PARP-1, poly(ADP-ribose) polymerase-1; -lap, -lapachone (3,4-dihydro-2,2-dimethyl-2H-naphthol[1,2b]pyran-5,6-dione); ROS, reactive oxygen species; H₂O₂, hydrogen peroxide; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; SSBs/DSBs, single/double-stranded DNA breaks; PAR, poly(ADP-ribose); MMP, mitochondrial membrane potential; NQO1, NAD(P)H:quinone oxidoreductase 1; STS, staurosporine; TUNEL, terminal deoxynucleotidyl transferease-mediated dUTP nick-end labeling; BAPTA-AM, (1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl ester)); Me₂SO, dimethyl sulfoxide; DIC, dicoumarol; 3-AB, 3-aminobenzamide; DPQ, (3,4-dihydro-5[4-(1-piperindinyl)butoxy]-1(2H)-isoquinoline); DCF, 6-carboxy-2'7'-dichlorodihydrofluorescin diacetate, di(acetoxymethyl ester); 231, MDA-MB-231; ns-shRNA, non-silencing shRNA; ER, endoplasmic reticulum; 231-NQ-, MDA-MB-231-NQO1-negative; 231-NQ+, MDA-MB-231-NQO1-positive; PARG, poly(ADP-ribose) glycohydrolase; IR, ionizing radiation; Topo, topoisomerase; MOMP, mitochondrial outer membrane permeabilization; PP2A, protein phosphatase 2A; PMCA, plasma membrane Ca²⁺-ATPase; SERCA, sarcoplasmic/endoplasmic reticulum ATPase; TRMP, transient receptor potential-melastatin-like; AIF, apoptosis-inducing factor.

³ W. X., Zong, E. A. Bey, and D. A. Boothman, unpublished data.

⁴ E. A. Bey and D. A. Boothman, unpublished observations.

	ACKNOWLEDGMENTS

 
We thank Drs. John J. Pink (Case Western Reserve University), Craig Thompson (U. of Pennsylvania), Wei-Xing Zong (State University of New York), and Ying Dong (University of Texas Southwestern Medical Center) for helpful discussions.

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