Role of NADPH Oxidase in Arsenic-Induced Reactive Oxygen Species Formation and Cytotoxicity in Myeloid Leukemia Cells

Role of NADPH oxidase in arsenic-induced reactive oxygen species formation and cytotoxicity in myeloid leukemia cells

Wen-Chien Chou*†‡, Chunfa Jie§, Andrew A. Kenedy¶, Richard J. Jonesʈ, Michael A. Trush¶, and Chi V. Dang*†¶ʈ**

*Program of Human Genetics and Molecular Biology, †Department of Medicine, §McKusick–Nathans Institute of Genetic Medicine, and ʈSidney Kimmel Comprehensive Cancer Center, School of Medicine, ¶Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, Baltimore, MD 21205

Edited by Owen N. Witte, University of California, Los Angeles, CA, and approved January 21, 2004 (received for review October 16, 2003) Arsenic has played a key medicinal role against a variety of associated and cytosolic subunits, can be stimulated by phorbol ailments for several millennia, but during the past century its myristate acetate (PMA) through protein kinase C-mediated prominence has been displaced by modern therapeutics. Recently, phosphorylation of the p47PHOX subunit (17, 18). This complex attention has been drawn to arsenic by its dramatic clinical efficacy is responsible for the production of superoxide anion (respira- against acute promyelocytic leukemia. Although toxic reactive tory burst) of professional phagocytes encountering microbial oxygen species (ROS) induced in cancer cells exposed to arsenic pathogens, and its importance in host immunity is underscored could mediate cancer cell death, how arsenic induces ROS remains by the immunocompromised congenital disease, chronic gran- undefined. Through the use of gene expression profiling, interfer- ulomatous disease (CGD), which results from mutations in one ence RNA, and genetically engineered cells, we report here that of the subunits of NADPH oxidase (19, 20). Our biochemical and NADPH oxidase, an enzyme complex required for the normal molecular biological studies reported here have uncovered a antibacterial function of white blood cells, is the main target of major role of this enzyme complex in arsenic-induced ROS arsenic-induced ROS production. Because NADPH oxidase enzyme production and cytotoxicity. We have also exploited the syner- activity can also be stimulated by phorbol myristate acetate, a gistic induction of NADPH oxidase activity and ROS production synergism between arsenic and the clinically used phorbol myris- by arsenic and PMA to provide proof-of-concept that this tate acetate analog, bryostatin 1, through enhanced ROS produc- synergy may be clinically applicable. tion can be expected. We show that this synergism exists, and that the use of very low doses of both arsenic and bryostatin 1 can Methods effectively kill leukemic cells. Our findings pinpoint the arsenic Cell Lines. NB4, U937, PLB-985, X-CGD, and HL60 cells were target of ROS production and provide a conceptual basis for an cultured in RPMI medium 1640 supplemented with 10% FBS. anticancer regimen. ML1 was maintained in RPMI medium 1640 with 7.5% FBS and 3.4 g of Hepes͞500 ml, pH 7.4. lthough arsenic has played a significant therapeutic role in Ͼ Avarious diseases for 2,000 years (1, 2), it was not used Microarray Analysis. NB4 cells were grown to a density of 105͞ml clinically for decades, until recently when clinical trials world- and were treated with 0.75 ␮M arsenic trioxide for 10 days. wide confirmed its dramatic therapeutic effects in acute promy- mRNA was isolated with the Qiagen RNeasy minikit and was elocytic leukemia (APL) (3, 4). APL is a subtype of acute subjected to Affymetrix oligonucleotide microarray analysis by myelocytic leukemia with most cases carrying the characteristic using an HG࿝U133A chip. Five replicates, including two control chromosomal translocation t(15, 17) that results in the PML- and three arsenic-treated NB4 samples, were studied. With the RAR␣ fusion protein (5). Although APL is highly responsive to ␣ expectation that only a small fraction of genes is differentially arsenic, the presence of PML-RAR fusion protein is neither expressed between samples under different treatments, the absolutely necessary nor sufficient for sensitivity to arsenic (3, 6, brightness of chips for the samples was adjusted to comparable 7). The mechanism by which arsenic is effective against APL level by normalizing the CEL file of signal values and the probe remains elusive, despite studies suggesting that arsenic can pair (perfect match and mismatch) level data of the Affymetrix promote degradation of the oncogenic PML-RAR␣ fusion expression chips, with the method of ‘‘invariant set normaliza- protein (8, 9). Paradoxically, arsenic is also an established human tion’’ (21). The normalized CEL data were then used to estimate carcinogen that can induce reactive oxygen species (ROS), the perfect match͞mismatch-model-based expression index leading to DNA damage or cell death (10–13). Some previous mechanistic studies (14, 15) were limited to (with SE) for the probe sets (22), leading to the further com- exposure of cells other than myeloid cells, or to arsenite rather putation of the fold changes and their 90% confidence intervals than arsenic trioxide for brief periods, and hence do not reflect (21). The lower bound of a 90% confidence interval, a conser- the clinical setting for cytotoxic effects of arsenic on APL cells. vative estimate of the fold change, was then used to identify To explore the molecular mechanisms of arsenic’s therapeutic differentially expressed genes. The computing was performed effects in the treatment of APL patients with daily continuous with DCHIP 1.2. infusion of arsenic trioxide, we treated a human APL cell line, NB4, for Ͼ1 week with arsenic trioxide at a dose lower than the This paper was submitted directly (Track II) to the PNAS office. plasma trough level achieved in APL patients. We reported Abbreviations: APL, acute promyelocytic leukemia; ROS, reactive oxygen species; PMA, previously that arsenic at this dose was able to down-regulate phorbol myristate acetate; DPI, diphenyleneiodonium; HRP, horseradish peroxidase; siRNA, human telomerase hTERT transcription (16). In this report, we small interference RNA; NAC, N-acetylcysteine. determined changes in gene expression profiles by using oligo- ‡Present address: Department of Internal Medicine, National Taiwan University Hospital, nucleotide microarrays, and we found that NADPH oxidase Taipei 100, Taiwan. components were dramatically up-regulated within days in my- **To whom correspondence should be addressed at: Ross Research Building, Room 1032, eloid cells treated with low-dose arsenic. NADPH oxidase, which 720 Rutland Avenue, Baltimore, MD 21205. E-mail: [email protected]. is an enzyme complex consisting of multiple membrane- © 2004 by The National Academy of Sciences of the USA

4578–4583 ͉ PNAS ͉ March 30, 2004 ͉ vol. 101 ͉ no. 13 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0306687101 Downloaded by guest on September 26, 2021 Fig. 1. Microarray analysis of NB4 cells before and after arsenic treatment. Expression profiles of the 24 genes (with the lower bound fold change of the 90% confidence intervals Ն5) were shown across the five samples in Eisen’s heat map. No As, untreated NB4 cells; As, arsenic-treated NB4 cells. Red and green colors represent high and low expression levels, respectively. The corresponding gene symbols, fold changes, and the lower bound fold change of the 90% confidence intervals are also listed. –, down-regulated gene expression. Those genes related to ROS production were marked with an asterisk. The genes are ordered from the most down-regulated genes to the highest MEDICAL SCIENCES up-regulated genes, based on the lower bound fold change.

Real-Time PCR. Detection of hTERT was described (16). Expres- sion of other genes was determined by reverse transcription followed by SYBR green real-time PCR. All primer sequences for the genes tested are available on request. cDNA was gener- ated by first heating a 15-␮l mixture containing 15 ␮g of total RNA and 1 ␮g of random primers (Promega) to 70°C for 5 min. After immediate chilling on ice, 5 ␮lof5ϫ reaction buffer, 5 ␮l of dNTP (2.5 mM each), 40 units of RNase inhibitor, and 200 Fig. 2. Up-regulation of NADPH oxidase and eosinophil peroxidase expres- units of Moloney murine leukemia virus reverse transcriptase sion by arsenic. (A) Absent immunohistochemical staining in control cells (Left) (Promega) were added, and the mixture was incubated at 37°C and intense staining of eosinophil peroxidase in arsenic-treated NB4 cells for 1 h. Ten nanograms of cDNA was subjected to SYBR green (Right). The staining in arsenic-treated cells depended on the primary anti- PHOX quantitative real-time PCR. Every tube of 20 ␮l contained 500 body (data not shown). As, arsenic. (B) Immunoblotting of p47 and p67PHOX shows dramatic increases in the protein levels in NB4 cells after arsenic nM each of primer, 200 ␮M dNTP, and PCR buffer with 1.75 ͞ ␮ treatment. ␣-Tubulin was used as a loading control. (C) Arsenic increased mM MgCl2 0.5 l of 15,000-fold diluted SYBR (Molecular ͞ mRNA of all of the NADPH oxidase subunits in NB4 cells as measured by SYBR Probes) 0.5 units of PlatinumTaq (Invitrogen). All primers were green quantitative real-time PCR. designed to cross introns and span Ͻ400 base pairs of the mRNA. All PCRs were performed at 95°C for 5 min, followed by 40 cycles of 95°C for 30 sec, 60°C for 30 sec, and 72°C for 30 123 (Sigma) for 30 min at 37°C. The ROS was determined by the sec. The signals were detected with the ABI 7700 sequence fluorescent intensity by flow cytometry with excitation at 490 nm detection system. All of the signals were normalized by the and emission at 520 nm. expression levels of large acidic ribosomal protein (RPLP0). ROS Detection by Luminol Chemiluminescence. To detect extracel- ␮ ϫ 6 Immunohistochemical Staining. The cells were prepared by cyto- lular and intracellular ROS, 10 M luminol was added to 1 10 cells in 2 ml of aerated complete PBS (PBS with 0.5 mM spin and were fixed with methanol and then acetone for 2 min ͞ ͞ MgCl2 0.7 mM CaCl2 0.1% glucose) supplemented with 10 each. The staining procedures were performed with the Vec- ␮ ͞ tastain kit (Vector Laboratories). After blocking, the cells were g ml horseradish peroxidase (HRP). The chemiluminescence was measured continuously in a Berthold LB9505 (Pforzheim, stained with 5 ␮g͞ml antieosinophil peroxidase antibody (Re- Germany) six-channel luminometer at 37°C for 30 min. search Diagnostics, Flanders, NJ), followed by washes and anti-mouse secondary antibody. The reaction was stopped by Superoxide Detection by Lucigenin-Derived Chemiluminescence. One washing with water. The slides were counterstained with hema- ͞ million arsenic-treated or control cells were suspended in 2 ml toxylin eosin. of air-aerated complete PBS. Lucigenin (5 ␮M) was added to the cells, and the chemiluminescence was detected as described Detection of ROS by Flow Cytometry. NB4 cells treated with 0.75 above. ␮M arsenic for 10 days were washed with PBS and were resuspended in complete medium with original concentration of PMA Stimulation. After 30 min of recording as described above, arsenic, followed by incubation with 0.5 ␮M dihydrorhodamine the chemiluminescence signals were than recorded for another

Chou et al. PNAS ͉ March 30, 2004 ͉ vol. 101 ͉ no. 13 ͉ 4579 Downloaded by guest on September 26, 2021 30 min after the addition of 50 nM of PMA into the reaction mixture.

Superoxide Detection by Cytochrome c Reduction. The procedures were performed by adding 1.5 mg͞ml cytochrome c to 1 ϫ 106 cells with or without 50 nM PMA or 300 units͞ml superoxide dismutase. The mixture was shaken at 37°Cfor1h.The supernatant was measured by spectrophotometry at 550 nm. The amount of reduced cytochrome c was determined by converting the absorbance with extinction coefficient of 28 per mM. For inhibitor studies, 10 ␮M diphenyleneiodonium (DPI) was added to cells in complete PBS and was incubated for 5 min at 37°C before measurement of chemiluminescence or cytochrome c reduction.

RNA Interference. Three million NB4 cells growing in log phase were washed twice and were resuspended in 500 ␮l of electro- poration buffer (21 mM Hepes͞137 mM NaCl͞5mMKCl͞0.7 ͞ mM Na2HPO4 6 mM glucose, pH 7.15) containing either 0.5 nmol of scrambled small interference RNA (siRNA) (5Ј- CACGCUCGGUCAAAAGGUUdTdT-3Ј)orp47PHOX siRNA (5Ј-GAGUACCGCGACAGACAUCdTdT-3Ј, Dharmacon, Lafayette, CO) in a 4-mm gap cuvette (BTX, Holliston, MA). The mixture was then electroporated with 1,500 ␮F and 200 volts by using Gene Pulser II (Bio-Rad). After 48 h, the cells were treated with 1.5 ␮M arsenic for another 48 h. The cells were harvested for ROS detection and immunoblotting.

Determination of Viability. NB4 cells (105 per ml) without or with arsenic (0.75 ␮M) treatment for 10 days were then exposed to PMA (0.2 nM) or bryostatin 1 (0.75 or 1 nM) with or without concomitant presence of 10 mM N-acetylcysteine (NAC), and the cell viability was followed up for another 6 days by using the Trypan blue exclusion method. At the fourth day of viability assay, control or arsenic-treated cells without or with NAC coincubation were evaluated by luminol plus HRP chemiluminescence. Bryostatin 1 (1 nM) was added in the middle of the assay to detect the induction of ROS production. Results Arsenic Effects on Gene Expression Profiles of NB4 Cells. To explore the molecular mechanisms of arsenic’s therapeutic effects in the treatment of APL patients with daily continuous infusion of arsenic trioxide, we treated a human APL cell line, NB4, for 10 days with 0.75 ␮M arsenic trioxide, a dose slightly lower than the plasma trough levels achieved in APL patients (23). We reported previously that arsenic at this dose was able to down-regulate human telomerase hTERT transcription (16). Multiple replicate experiments were analyzed by microarray hybridizations, including three microarrays for arsenic-treated and two microarrays for control NB4 cells. The effect of arsenic treatment verified by real-time PCR showed the Ͼ99% down-regulation of hTERT expression, which is too low for reliable microarray analysis (data not shown). With arsenic exposure, NB4 cells continued to proliferate although at a slower rate compared with control (data not shown). Gene expression index was estimated for the samples with the perfect match͞mismatch multiplicative statistical (22). The high correlation of gene expression index between samples

Fig. 3. Induction of ROS formation from NADPH oxidase by arsenic. Chemi- luminescence measured with luminol plus HRP (A), lucigenin (B), and ﬂow cytometry (C) by using dihydrorhodamine 123 showed signiﬁcant ROS induction in NB4 cells treated with arsenic. (D and E) Luminol plus HRP (D) and cytochrome c reduction (E) showed that induction of ROS was further dramatically enhanced by the addition of 50 nM PMA. Incubation with DPI completely blunted the baseline, arsenic-induced, or PMA-stimulated chemiluminescence and cytochrome c reduction. SOD, superoxide dismutase.

4580 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0306687101 Chou et al. Downloaded by guest on September 26, 2021 MEDICAL SCIENCES

Fig. 4. NADPH oxidase is the main source of arsenic-induced ROS. (A) p47 PHOX protein was induced by arsenic in cells transfected with scrambled (Scram.) siRNA, but this induction is diminished in cells transfected with p47PHOX siRNA (Inset). The lucigenin chemiluminescence induced by arsenic in p47 PHOX siRNA-transfected cells was signiﬁcantly diminished compared with those transfected with scrambled siRNA. As, arsenic. (B) Lucigenin chemiluminescence was absent in X-CGD cells before and after arsenic treatment. In contrast, the baseline chemiluminescence of the parental cells, PLB-985, was enhanced by arsenic treatment. (C) Cytochrome c reduction assay showed PMA-enhanced superoxide production in arsenic-treated parental cells PLB-985 but not in X-CGD cells. (D) mRNAs of most NADPH oxidase subunits in PLB-985 or X-CGD cells were increased by arsenic, as determined by real-time PCR.

ranges from 0.970 to 0.991, suggesting only a small fraction of oxidation stress-related protein (eosinophil peroxidase, EPX) genes is differentially expressed between the samples with and (refs. 26 and 27 and Fig. 1, asterisk). These findings strongly without arsenic exposure (data not shown). To identify the up- suggest that ROS production and NADPH oxidase activity are and down-regulated genes, a 90% confidence interval was induced by arsenic. Further inspection of the raw microarray computed for the fold change of the averaged expression index data revealed up-regulation (Ͼ2-fold) of another scaffolding of each gene between the samples with and without arsenic protein, S100A9 (24), and all of the other NADPH oxidase treatment. The lower bound of a 90% confidence interval, a components, including p67PHOX, CYBA (p22 subunit of cyto- conservative estimate of the fold change, served as the practical chrome b558), CYBB (gp91PHOX), p40PHOX, and rac2, which is a way of identifying differentially expressed genes (21). Of 22,000 small G protein important in activating the NADPH oxidase genes on the array, 15 and 9 were up- and down-regulated, complex (data not shown). To confirm the increased levels of respectively, by arsenic with a lower bound fold change Ն5 (Fig. these ROS-related proteins, we performed immunohistochem- 1). Real-time PCR confirmed the microarray results for all 14 ical staining for eosinophil peroxidase (Fig. 2A) and immuno- genes randomly selected for verification (data not shown). blotting for p47PHOX and p67PHOX (Fig. 2B). We also used real-time PCR and confirmed that mRNAs for all of the Up-Regulation of NADPH Oxidase Components by Arsenic. Among NADPH oxidase components were significantly up-regulated in those 14 selected were genes involved in oxidant production such NB4 cells after arsenic treatment (Fig. 2C). In these experi- as NADPH oxidase components p47PHOX (NCF1) (17), NADPH ments, NB4 cellular morphology or expression of CD11b and oxidase assembly scaffolding protein (S100A8) (24), arachido- CD15 were unaltered (data not shown), suggesting that differ- nate lipoxygenase activating protein (ALOX5AP) (25), and entiation does not play a major role, although induction of

Chou et al. PNAS ͉ March 30, 2004 ͉ vol. 101 ͉ no. 13 ͉ 4581 Downloaded by guest on September 26, 2021 dismutase, confirming the ROS produced in arsenic-treated NB4 cells was superoxide anion (Fig. 3E). The absence of signals of cytochrome c reduction in arsenic-treated cells without challenge with PMA might reflect the lower sensitivity of this assay compared with chemiluminescence. We also tested arsenic induction of ROS and NADPH oxidase activation in other myeloid cell lines, including ML1 (monocytic), PLB-985 (mono- blastic leukemia), HL60 (promyelocytic leukemia), and U937 (monocytic leukemia). We found that all but U937 produced higher amount of ROS as measured by chemiluminescence after arsenic treatment, with the corresponding elevation of mRNAs of both p47 PHOX and p67PHOX (data not shown).

NADPH Oxidase Is the Main Source of Arsenic-Induced ROS Production. Although arsenic induced both NADPH oxidase and ROS production, whether the ROS came from NADPH oxidase remains to be determined. The dramatic PMA stimulation of ROS production by arsenic-treated NB4 cells strongly implicates NADPH oxidase as the source, because PMA is known to activate latent NADPH oxidase (17). To address this issue further, we first used DPI, a flavoprotein inhibitor of NADPH oxidase. We found that arsenic-treated cells did not exhibit any ROS production after the addition of DPI, even after PMA stimulation (Fig. 3 D and E). Although DPI is regarded as an NADPH oxidase inhibitor, it is not specific (29). Hence, we inhibited P47PHOX expression by siRNA in NB4 cells. ROS production as measured by chemiluminescence of lucigenin or luminol plus HRP after arsenic treatment and PMA stimulation was dramatically reduced after suppression of p47PHOX induction by arsenic (Fig. 4A and data not shown). We also used the myeloid cell line PLB-985 and its gp91PHOX-knockout derivative, X-CGD (30), as another genetic approach to test our hypothesis that NADPH oxidase is the main source of arsenic-induced ROS production. We found that X-CGD exhibited no baseline, arsenic-induced, or PMA-stimulated ROS as measured by luminol plus HRP (data not shown) and lucigenin chemiluminescence (Fig. 4B and data not shown). In contrast, the parental cells Fig. 5. Synergistic cytotoxicity between arsenic and either PMA or bryostatin PLB-985 responded to arsenic with enhanced lucigenin signals 1 (Bryo). (A) PMA or byrostatin 1 at the doses used did not prevent cell (Fig. 4B) and luminol plus HRP (data not shown). PMA proliferation. Without arsenic, the cells continued proliferation in the pres- stimulation further augmented the chemiluminescence as mea- ence of PMA or bryostatin 1 in absence (Left) or presence (Right) of NAC. NAC sured by using luminol plus HRP and lucigenin in the parental had mild toxicity to NB4 cells. (B Left) Synergistic toxicity is evident in cells cells (data not shown). Cytochrome c reduction also showed pretreated with arsenic and then exposed to PMA or byrostatin 1, as compared increased superoxide production in PLB-985 after arsenic treat- with cells pretreated with arsenic only. (Right) NAC blocks the synergistic ment and PMA stimulation, but not in X-CGD (Fig. 4C). Most toxicity. (C) NAC decreased arsenic-induced ROS signals. After the addition of of the NADPH oxidase components were also up-regulated by 1 nM bryostatin, the arsenic-treated cells showed signiﬁcant induction of arsenic in PLB-985 and X-CGD (Fig. 4D), but due to the lack of chemiluminescence of luminol plus HRP than those cells coincubated with PHOX NAC. the functional gp91 component in X-CGD cells (30), there was no functional NADPH oxidase enzyme complex. These results indicate that NADPH oxidase is the main source of myeloid differentiation by arsenic (28) could trigger NADPH arsenic-induced ROS production. oxidase expression. These data indicated that arsenic potently induces components of NADPH oxidase. Synergism of Cytotoxicity Between Arsenic and PMA or Bryostatin 1. Because oxidants can exert cytotoxicity (12, 31–33), and PMA Induction of ROS by Arsenic in Myeloid Cells. Corresponding to the dramatically enhances oxidant production in cells pretreated activation of NADPH oxidase, ROS was greatly enhanced by with arsenic, we tested whether arsenic and PMA are synergis- arsenic treatment as verified by several approaches for detection tically cytotoxic. First, we identified a dose of PMA (0.2 nM) that did not significantly reduce cell viability (Fig. 5A), but could of ROS such as chemiluminescence of luminol with or without enhance ROS production in arsenic-treated NB4 cells (data not HRP, lucigenin, and flow cytometry by using dihydrorhodamine shown). At this dose, PMA potently synergized with arsenic in 123 as a probe (Fig. 3 A–C). The enhancement of chemilumi- cytotoxicity (Fig. 5B). We extended our finding to a clinically nescence of either luminol or lucigenin was dramatically ampli- used PMA analog, bryostatin 1, a macrocyclic lactone isolated fied by the addition of 50 nM PMA (Fig. 3D and data not shown) from a marine bryozoan Bugula neritina (34). Its antileukemic that is known to stimulate NADPH oxidase activity (17). These effect was suggested by in vitro studies (35, 36) and preliminary data indicate that arsenic dramatically induces the latent form of clinical trials (37, 38). We identified doses of bryostatin 1 (0.5–1 NADPH oxidase. We also verified the production of superoxide nM) that still maintained cellular proliferation (Fig. 5A) and through its ability to reduce cytochrome c (Fig. 3E). The greatly enhanced ROS production (data not shown). Bryostatin reduction of cytochrome c in arsenic-treated cells challenged 1 also synergized with arsenic in killing the cells (Fig. 5B). The with PMA was completely rescued by addition of superoxide synergism between arsenic and either PMA or bryostatin 1

4582 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0306687101 Chou et al. Downloaded by guest on September 26, 2021 diminished after the addition of the radical scavenger NAC (Fig. NADPH oxidase complex in the APL cell line NB4, as well as 5B), with corresponding blunting of the chemiluminescence other leukemic cell lines. The protection of cells by the oxidant induced by arsenic with or without 1 nM bryostatin 1 (Fig. 5C). scavenger NAC against arsenic plus either PMA or bryostatin 1 We conclude that arsenic and either PMA or bryostatin 1 further supports the role of ROS in the cytotoxicity of leukemic synergize to enhance ROS production and tumoricidal activity. cells. Phosphorylation of the p47PHOX subunit by PMA in protein Discussion kinase C-dependent mechanisms triggers the assembly and Although arsenic has played a significant role in human medic- activation of a functional NADPH oxidase complex (17, 18). The inal history, the mechanisms underlying arsenic’s antileukemic abundant NADPH oxidase subunits induced by arsenic should activity remain enigmatic. Its ability to induce ROS production facilitate the activation of NADPH oxidase by PMA or its has been reported but the source of the ROS remained unknown. clinically used analog, bryostatin 1. We exploited this molecular Here, we provide evidence that NADPH oxidase induced by regulatory mechanism and used the synergism between arsenic arsenic is central to the mechanism of arsenic-mediated ROS and bryostatin 1 to kill leukemic cells. The concentrations of production. Not only were NADPH oxidase components in- arsenic or bryostatin 1 were much lower than experimentally or duced by arsenic concordant with ROS production in different clinically used, yet the synergistic tumoricidal effect is remark- leukemic cells, leukemic cells with p47PHOX levels diminished by able at these concentrations when the two are used together. Our RNA interference were minimally responsive to arsenic. More- biochemical and genetic studies reported here have uncovered a over, cells depleted of the gp91PHOX subunit of NADPH oxidase major role of NADPH oxidase in arsenic-induced ROS produc- by homologous recombination were totally unresponsive to tion and cytotoxicity, and also provided a conceptual basis for arsenic as compared with the wild-type parental cells in ROS the development of clinical protocols for the treatment of production. leukemias, in particular APL, through the synergism between The role of NADPH oxidase in apoptosis is implicated by the arsenic and bryostatin 1. findings that zinc, vanadium, and brain-derived neurotropic We thank M. Dinauer for PLB-985 and X-CGD cell lines and F. Racke factor could all induce NADPH oxidase activity and ROS for assistance with immunohistochemical staining and flow cytometry. production, leading to the death of nonmyeloid cells (12, 31–33). This work was supported by National Institutes of Health Grants In our studies, which began with gene expression analysis, we CA51497 (to C.V.D.) and ES03760 and ES03819 (to A.A.K. and observed that arsenic significantly induces components of the M.A.T.). MEDICAL SCIENCES

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