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Methylated Metabolites of Arsenic Trioxide Are More Potent Than Arsenic Trioxide as Apoptotic but not Differentiation Inducers in Leukemia and Lymphoma Cells¹

Department of Pathophysiology [G-Q. C.] and Shanghai Institute of Hematology [Z. C.], Shanghai Second Medical University, Shanghai, China; Division of Hematology/Oncology, Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029 [L. Z., Y. J., R. W., S. W.]; and Department of Pediatrics, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina [M. S., F. W.]

	ABSTRACT

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Treatment with arsenic trioxide (As₂O₃) by inducing apoptosis and partial differentiation of acute promyelocytic leukemia (APL) cells results in clinical remission in APL patients resistant to chemotherapy and all-trans-retinoic acid. As₂O₃ (iAs^III) is methylated in the liver to mono- and dimethylated metabolites, including methylarsonic acid, methylarsonous acid, dimethylarsinic acid, and dimethylarsinous acid. Methylated trivalent metabolites that are potent cytotoxins, genotoxins, and enzyme inhibitors may contribute to the in vivo therapeutic effect of iAs^III. Therefore, we compared the potency of iAs^III and trivalent metabolites using chemical precursors of methylarsonous acid and dimethylarsinous acid to induce differentiation, growth inhibition, and apoptosis. Methylarsine oxide (MAs^IIIO) and to a lesser extent iododimethylarsine were more potent growth inhibitors and apoptotic inducers than iAs^III in NB4 cells, an APL cell line. This was also observed in K562 human leukemia, lymphoma cell lines, and in primary culture of chronic lymphocytic leukemia cells, but not human bone marrow progenitor cells. Apoptosis was associated with greater hydrogen peroxide accumulation and inhibition of glutathione peroxidase activity. MAs^IIIO, in contrast to iAs^III, did not induce PML-retinoic acid receptor degradation, or restore PML nuclear bodies or differentiation in NB4 cells. In a cocultivation experiment, hepatoma-derived HepG2 cells, but not NB4 cells, methylate radiolabeled iAs^III. Methylated metabolites released from HepG2 cells are preferentially accumulated by NB4 cells. This experimental model suggests that in vivo hepatic methylation of iAs^III may contribute to As₂O₃-induced apoptosis but not differentiation of APL cells. MAs^IIIO as an apoptotic inducer should be considered in the treatment of other hematologic malignancies like lymphoma.

	INTRODUCTION

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APL⁴ is an unique subtype of acute myeloid leukemia typically carrying the specific reciprocal chromosomal translocation t(15;17) leading to the expression of the leukemia-generating fusion protein PML-RAR (1 , 2) . In 90% of de novo APL patients, ATRA treatment induces differentiation of leukemic blasts and clinical remission, and 70% have been cured by ATRA in combination with chemotherapy (3) . There is a 30% relapse, especially in APL patients presenting with high WBC. However, treatment with As₂O₃ induces complete remission in 90% in this group of chemotherapy and ATRA-resistant relapsed patients (4, 5, 6) . This suggests that As₂O₃ treatment may induce remission in APL patients by a mechanism different from ATRA or chemotherapy. Indeed, only differentiated leukemia cells have been observed in APL patients after ATRA treatment (7) , but both apoptotic and partially differentiated cells have been observed in APL patients and animal models treated with As₂O₃ (5 , 8) . This suggests that As₂O₃ may induce clinical remission in APL patients by multiple mechanisms.

Biomethylation is the major metabolic pathway for iAs in humans and many animal species (9) . In this pathway iAs undergo metabolic conversion that includes reduction of iAs^v to iAs^III with subsequent methylation yielding mono- and dimethylated metabolites (10 , 11) . The postulated scheme is as follows: iAs^v iAs^III MAs^V MAs^III DMAs^V DMAs^III MAs^III and DMAs^III have been detected in the urine of humans exposed to inorganic arsenic in drinking water (11 , 12) and in human hepatoma (HepG2) cells exposed to iAs^III (11) . Notably, chemical derivatives of MAs^III are significantly more toxic for cultured cells (13 , 14) and laboratory animals (15) than either iAs^v or iAs^III. DMAs^III derivatives are more toxic than iAs species in most cell types tested (13 , 14) . Although As₂O₃ has been found to induce remission in APL patients and induce apoptosis in cultured APL cells (4 , 5 , 8 , 16) , the metabolism of As₂O₃ and the effect of its methylated metabolites have not been tested. It is possible that methylated As₂O₃ metabolites that form in vivo may contribute to the therapeutic effect of As₂O₃ in APL.

NB4 cells, a cell line derived from t(15;17) APL (17, 18, 19, 20) were used to compare the induction of apoptosis and differentiation by iAs^III to methylated arsenicals that are chemical precursors of methylated trivalent metabolites of iAs, MAs^IIIO and DMAs^IIII. The effects of these arsenicals on H₂O₂ production, glutathione peroxidase activity, caspase activation, PML-RAR protein degradation, and PML nuclear body formation were also examined. We also studied the production and distribution of iAs^III metabolites in a cocultivation cell culture system containing HepG2, a hepatoma-derived cell line and NB4 cells. The cytotoxic effect of MAs^IIIO and iAs^III was also compared in K562 leukemia cells, lymphoma cells, primary cultures of CLL cells, and in normal human bone marrow progenitor cells. The data indicate that MAs^IIIO, and to a lesser extent DMAs^IIII, were more potent growth inhibitors and apoptotic inducers than iAs^III in leukemia and lymphoma cells but not in human bone marrow progenitor cells. H₂O₂ accumulation and GPx inhibition but not degradation of PML-RAR correlated with greater MAs^IIIO-induced apoptosis in NB4 cells. NB4 cells did not methylate iAs^III, but mono- and dimethylated metabolites formed from iAs^III in HepG2 cells released into the medium and were preferentially taken up by NB4 cells.

	MATERIALS AND METHODS

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Arsenicals and Reagents.
iAs^III and iAs^V, sodium salts (at least 99% pure), were purchased from Sigma (St. Louis, MO). MAs^V (sodium salt, 98% pure) was obtained from Chem Service (West Chester, PA) and DMAs^V (98% pure) from Strem (Newburyport, MA). MAs^IIIO (CH₃AsO) and DMAs^IIII [(CH₃)₂AsI] were synthesized by Dr. William R. Cullen (University of British Columbia, Vancouver, British Columbia, Canada) using methods described previously (21 , 22) . Identity and purity of these compounds were confirmed using ¹H NMR and mass spectrometry. In aqueous solutions, MAs^IIIO and DMAs^IIII form the respective (methylarsonous and dimethylarsenous) oxyanions. The hydrolysis of oxides and iodides of MAsIII has been documented previously by Petrick et al. (15) using ¹H NMR and mass spectrometry. Carrier-free [⁷³As]iAs^v was purchased from Los Alamos Meson Production Facility (Los Alamos, NM). [⁷³As]iAs^III was prepared from [⁷³As]iAs^v by reduction with metabisulfite/thiosulfate reagent (23) . Yields of [⁷³As]iAs^III in this reaction as determined by TLC (24) typically exceed 95%.

Cell Culture and Treatment.
NB4 human APL cells (kindly provided by Dr. M. Lanotte, Hopítal Saint-Louis, Paris, France; Ref. 17 ) and the NB4-derived ATRA-resistant R4 line (kindly provided by W. Miller, Jr., Lady Davis Institute for Medical Research, Montreal, Canada; Ref. 25 ) and K562 cells (obtained from ATCC) were cultured in RPMI 1640 supplemented with 10% FCS (JRH BioScience, Lenexa, KS) in a humidified atmosphere of 95% air and 5% CO₂ at 37°C. HepG2 cells (obtained from ATCC) were cultured in MEM supplemented with 10% FCS. Jurkat and Namalwa human lymphoma cells (obtained from ATCC) were cultured in RPMI 1640 adjusted to contain 1.5 g/liter of sodium bicarbonate, 4.5 g/liter of glucose, 10 mM HEPES, 1.0 mM sodium pyruvate and 10% FCS. CLL cells were obtained from blood from two patients with consent according to Institutional Review Board regulations. B-cell populations were isolated using RosetteSep B Cell Enrichment mixture (StemCell Technologies, Vancouver, British Columbia, Canada), which used antibodies against CD2, CD3, CD16, CD36, and CD56. This negative selection system enabled the isolation of B cells that were of >95% purity by FACS analysis without activating the cells through any surface proteins. Whole blood was incubated with RosetteSep (50 µl of RosetteSep mixture/ml whole blood) at room temperature for 20 minutes. Samples were then diluted 1:2 with PBS +2% FCS and centrifuged over Ficoll-Hypaque density medium (Amersham Biosciences, Piscataway, NJ). B cells were collected from the Ficoll:plasma interface, washed in PBS, and ready for use. For treatment with arsenicals, cells were seeded at 2 x 10⁵ cells/ml and cultured in the above-mentioned medium with or without the indicated doses of compounds for the indicated time. Cell viability was estimated by trypan blue dye exclusion, and cell numbers were counted by hemacytometer.

Annexin-V Assay.
Apoptotic cells were detected by Annexin-V assay. Generally 5–10 x 10⁵ cells after treatments were washed twice with PBS. Then, cells were labeled by Annexin-V-FITC and PI in binding buffer according to the instruction in the Annexin V-FITC Apoptosis Detection kit provided by the manufacturer (Oncogene, Cambridge, MA). Fluorescent signals of FITC and PI were detected, respectively, by FL1 (FITC detector) at 518 nm and FL2 at 620 nm on FACScan (Becton Dickson, San Jose, CA). The log of Annexin-V-FITC fluorescence was displayed on the X axis and the log of PI fluorescence on the Y axis, and data were analyzed using the CELLQuest (Becton Dickson) program. For each analysis, 10,000 events were recorded.

Measure of Intracellular Hydrogen Peroxide Production.
The intracellular hydrogen peroxide level was detected as we reported before by using 5,6-carboxy-2',7'-dichloroflurescein-diacetate (Molecular Probe, Eugene, OR; Ref. 20 ). Briefly, 2 h before ending the indicated treatment, 10 µM 5,6-carboxy-2',7'-dichloroflurescein-diacetate was added into the medium and continued incubation for 2 h at 37°C, and the fluorescence intensity was measured by FACScan (Becton Dickinson).

Glutathione Peroxidase Activity Assays.
Cells (5 x 10⁷) were washed twice with PBS, resuspended in PBS, sonicated for 10 s, centrifuged at 14,000 rpm for 10 min, and the supernatants subjected to enzyme assays. Glutathione peroxidase activity was determined using commercial kits (Calbiochem, San Diego, CA). One milliunit of enzyme activity was defined as 1 nmol of NADPH oxidized to NADP per milligram of protein per min.

Immunofluorescence and Confocal Laser Microscopy Analysis.
Cells were smeared onto slides and kept on -80°C. For immunofluorescence staining of PML, which was described previously (26) , cells were fixed in 20% methanol and antihuman PML monoclonal antibody 5E10 and then with FITC-conjugated secondary antibodies. Fluorescence was observed using a Leica confocal laser microscope with excitation at 488 nm (green). Images were overlaid in PhotoShop.

Metabolic Studies in a Cocultivation Culture System.
HepG2 cells were cultured in monolayers in 12-well culture plates with MEM-supplemented 10% serum, and cells were cultured at 37°C in a humidified incubator in an atmosphere of 95% air and 5% CO₂ to 90% confluence (1.6 x 10⁶ cells/well). Before exposure to iAs^III, MEM was replaced with a supplemented Williams’ medium E (27) with 10% fetal bovine serum (1.5 ml/well), and cultures were left to equilibrate for 1 h. The supplemented Williams’ medium E has been shown previously to provide an optimal support for methylation of arsenite in hepatic cells (27) . Carrier-free [⁷³As]iAs^III was added into the equilibrated HepG2 cultures (1 µCi/well) along with desirable amounts of iAs^III carrier, and plates were returned to a culture incubator for additional 30 min to allow for uptake of iAs^III. Suspension of NB4 cells was introduced into the HepG2 cultures in inserts (Becton Dickinson, Franklin Lake, NJ) with 0.4-µm pore size (1 x 10⁶ cells in 0.5 ml Williams’ medium E per insert). Individual cultures of HepG2 in monolayers and NB4 cells in inserts were used in parallel with the combined cultures. The final concentrations of iAs^III in the combined cultures reached 0.1 or 1 µM. The [⁷³As]iAs^III-treated individual and combined cultures of HepG2 and NB4 cells were kept in a culture incubator for 48 h. Radiolabeled arsenic metabolites were analyzed separately in each type of cells and in a combined medium (medium in inserts + medium in well) using techniques described previously (24) . To release protein-bound metabolites and to facilitate analysis of chemical species, medium and cells were treated with acidic CuCl solution (pH 1.0) at 100°C (28) , and oxidized with hydrogen peroxide. Oxidized CuCl extracts were separated by TLC (24) . The distribution of the radioactivity on the developed TLC plates was analyzed with an AMBIS 4000 imaging detector (Ambis, San Diego, CA). Because of the CuCl treatment and subsequent oxidation of samples used for TLC, this procedure cannot determine original oxidation states of arsenic in metabolites. Thus, arsenic metabolites in this series of experiments are generically referred to as iAs, MAs, and DMAs.

Colony-forming Assays of Human Bone Marrow Progenitor Cells.
Following informed consent according to Mount Sinai School of Medicine Institutional Review Board regulations, bone marrow from three normal adults were collected into syringes containing 50 units of Heparin. To obtain MNCs, bone marrow was diluted 1:1 with medium, layered onto an equal volume of Ficoll-Paque, and centrifuged at 450 x g for 30 min at 18°C. For methylcellulose cultures, MNCs were cultured according to methods described previously. Each ml of culture contained 300,000 MNCs, 0.8% methylcellulose in medium, 30% fetal bovine serum, 1% BSA (COHN fraction IV), 0.1 mmol/liter 2-mercaptoethanol, 0.1 units of penicillin, and 0.1 µg/ml streptomycin. Erythroid cultures contained erythropoietin (2 units/ml). Myeloid cultures contained GM colony-stimulating factor (2 ng/ml) and interleukin 3 (50 units/ml). Cultures were performed in triplicate in 0.3-ml volume in Nunc four-well dishes (Intermed, Roskilde, Denmark) and incubated at 37°C in 4% CO₂ with or without As₂O₃ or MAs^IIIO. Colonies derived from CFU-E were counted on day 7 and those from BFU-E and CFU-GM on day 13 using a Stereozoom dissecting microscope (Bausch & Lomb, Rochester, NY).

	RESULTS

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Methylation of iAs^III Increases Cytotoxity in NB4 Cells.
We compared growth inhibition and cell death by treatment with iAs^III, iAs^V, and their monomethylated and dimethylated derivatives in NB4 cells at concentrations of 0.5, 1, and 2 µM for 1–3 days. MAs^IIIO treatment was more growth inhibitory (IC₅₀ = 0.3 µM) than iAs^III, whereas DMAs^IIII was similar to iAs^III (IC₅₀ 1.0 µM) at 3-day treatment (Fig. 1A) . There was minimal loss of viability after treatment with 0.5 µM of iAs^III, DMAs^IIII, and MAs^IIIO. However, 1 µM of MAs^IIIO caused 90% loss of viability of NB4 cells, whereas iAs^III and DMAs^IIII remained at 15% (Fig. 1B) . Treatment with up to 2 µM of pentavalent methylated arsenicals (Mas^V and DMAs^V) did not significantly inhibit growth or decrease viability of NB4 cells (data not shown).

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Effects of trivalent arsenicals on the growth of NB4 cells. A, NB4 cells were treated with 0.5–2.0 µM iAsIII (top), MAsIIIO (middle), and DMAsIIII (bottom) for the indicated days. Total cell number was counted. Each point is the mean of triplicates with <5% variation (data not shown). B, effects of trivalent arsenicals on viability of NB4 cells. Viable cell numbers were counted by trypan-blue exclusion. All data represent an independent experiment from two repeated tests with similar results. Each point is the mean of triplicates; bars, ±SD.

View larger version (28K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Apoptosis induced by arsenicals in NB4 cells. A, NB4 cells were treated with 1 µM iAsIII, MAsIIIO, and DMAsIIII, respectively, for 24 and 48 h. Annexin-V and PI staining were measured by flow cytometry. X axis, the fluorescent intensity of Annexin-V. Y axis, the fluorescent intensity of PI. Each panel shows the percentages of Annexin-V+, PI-, and both positive cells. B, Western blot analysis of PARP cleavage. Antibody to PARP was used to detect the PARP cleavage product.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Inhibition of GPx activity. NB4 cells were treated with various concentrations of iAsIII and MAsIIIO for 8 and 24 h. GPx activity was measured as described in "Materials and Methods."

MAs^IIIO-induced Apoptosis Is Independent of PML-RAR Protein Degradation.

View larger version (61K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Effect of arsenicals on PML-RAR degradation and nuclear body formation. A, NB4 cells were treated with 1 µM MAsIIIO or 1 µM iAsIII for 24 h, and PML RAR protein was measured by Western blot analysis. B, PML nuclear bodies were assayed by confocal microscopy using PML antibody after NB4 cells were treated with 1 µM iAsIII and 0.25 µM MAsIIIO for 24 h. C, methylated arsenicals do not induce differentiation of NB4 cells. NB4 cells were treated with 0.25 µM iAsIII or MAsIIIO for 7–10 days, and differentiation was measured by expression of CD11b as measured by FACS.

MAs^IIIO Is a More Potent Cytotoxic Agent in Human Lymphoma Cells.
Some lymphoma cells are also sensitive to low concentration of As₂O₃-induced apoptosis (30) . The ability of MAs^III to induce growth inhibition and cell death was studied in two lymphoma cell lines: Jurkat cells, which are insensitive, and Namalwa cells, which are sensitive to As₂O₃-induced apoptosis. As shown in Fig. 5A , MAs^IIIO was more growth inhibitory than iAs^III in both Jurkat and Namalwa cells, because treatment with 1 µM iAs^III resulted in <10%, whereas MAs^IIIO resulted in >50% growth inhibition of both lymphoma cell lines after 3 days of treatment. This was reflected by a greater loss of cell viability after MAs^IIIO treatment (Fig. 5B) .

View larger version (34K): [in this window] [in a new window] [Download PPT slide]   Fig. 5. Effects of iAsIII and MAsIIIO on human lymphoma cell lines. A, cell growth inhibition. Jurkat and Namalwa cells were treated with 0.5–1.0 µM iAsIII and MAsIIIO for the indicated days. Total cell number was counted. B, cell viability. Viable cell numbers were counted by trypan-blue exclusion. Each point is the mean of triplicates; bars, ±SD.

View larger version (35K): [in this window] [in a new window] [Download PPT slide]   Fig. 6. Effect of arsenicals in normal human hematopoietic progenitor cells. Human bone marrow MNCs were treated with varying concentrations of iAsIII (A) and MAsIIIO (B), and assayed for CFU-E, BFU-E, and CFU-GM colonies as described in "Materials and Methods."

Among all of the biologically relevant chemical forms of arsenic, MAs^III derivatives are the most potent enzyme inhibitor, as shown here for GPx (Fig. 3) , and cytotoxin (reviewed in Ref. 32 ). In addition, MAs^III and DMAs^III derivatives are considerably more effective than arsenite in nicking naked DNA in an in vitro system or damaging DNA in intact cells (33) . These methylated trivalent arsenicals are also potent inducers of c-Jun phosphorylation and activator protein DNA binding in human cells (14 , 34) . The results of this study suggest that leukemia cells in vivo may accumulate MAs^III produced and released from the liver of patients treated with As₂O₃. These observations suggest that methylated metabolites of As₂O₃ may contribute to therapeutic and toxic effects in APL patients. To test this possibility, we compared the extent of growth inhibition and apoptosis induced in NB4 cells and K562 cells by treatment with iAs^III, and MAs^IIIO and DMAs^IIII chemical precursors of methylated trivalent metabolites of iAs^III (Figs. 1 and 2 ). Our data indicate that the apoptosis-induction ability is MAs^IIIO>DMAs^IIII>iAs^III. MAs^IIIO treatment also inhibits growth and induces apoptosis to a greater extent than iAs^III. This suggests that the amount of MAs^IIIO in cells may mediate the sensitivity to As₂O₃-induced apoptosis in malignant cells and normal tissues such as bone marrow and liver. The unexplained and unusual severe liver toxicity in three Chinese patients treated for APL with As₂O₃ may relate to aberrant hepatocyte methylation of As₂O₃ (8) . Studies to characterize hepatic arsenic methyltransferase polymorphism, isoforms, and nutritional cofactors are required to better understand As₂O₃ as a therapeutic agent.

We compared the uptake of ⁷³As-arsenite and methylated arsenicals metabolites in NB4, which are sensitive to As₂O₃-induced apoptosis, and U937 cells, which are insensitive to As₂O₃-induced apoptosis (20) . NB4 and U937 cells have similar accumulation of ⁷³iAs, do not methylate iAs^III, and take up MAs^III and DMAs^III to similar amounts in cocultivation with HepG2 cells (Tables 3 and 4 ; data not shown). Thus, other intracellular factors contribute to the higher sensitivity of NB4 cells to MAs^IIIO-induced apoptosis. The intracellular content of glutathione, and GPx, glutathione S-transferase , and catalase activities also play an important role in catabolism of H₂O₂. In NB4, cells with low levels As2O3 treatment induces H₂O₂ accumulation and apoptosis, and when high, there is less apoptosis in leukemic cells such as U937 cells (20) . MAs^IIIO is a more potent H₂O₂ inducer than iAs^III (Table 1) and is known to have a stronger binding affinity to thiol-enzymes (21) . GPx, which has a thiol-like group required for its activation (35) , is more inhibited by MAs^IIIO (Fig. 3) , which may contribute to the greater H₂O₂ accumulation (Table 1) and apoptosis.

It has been suggested that degradation of PML-RAR and PML protein is required for As₂O₃-induced apoptosis in APL cells (18 , 19) . This would predict a greater ability of MAs^IIIO than iAs^III to degrade PML-RAR or PML. However, PML-RAR protein degradation follows iAs^III but not MAs^IIIO treatment in NB4 cells (Fig. 4A) . Moreover, iAs^III decreased abnormal PML nuclear body distribution and number as reported before (Fig. 4B) , whereas neither MAs^IIIO nor DMAs^IIII (data not shown) change the abnormal distribution of nuclear bodies in NB4 cells. Therefore, As₂O₃ triggers apoptosis by a pathway independent of PML or PML-RAR degradation, thus not limiting this therapeutic effect to APL.

The clinical response of APL patients to As₂O₃ treatment that is associated with differentiation induction is probably related to iAs^III (8) . Unlike iAs^III, MAs^IIIO treatment of NB4 cells does not induce differentiation in NB4 cells (Fig. 4C) and does not cooperate with ATRA to induce differentiation in R4 ATRA-resistant NB4 cells (Ref. 25 ; data not shown). Therefore, the in vivo therapeutic contribution of MAs^IIIO would be the induction of apoptosis, not differentiation. The contribution of hepatic derived MAs^III from iAs^III to therapeutic outcome remains to be determined. MAs^IIIO released from hepatocytes after metabolism of iAs^III is avidly taken up by APL cells (Table 3 ; Ref. 4 ) and may contribute more than iAs^III to the induction of apoptosis, particularly in APL cells, which have a biochemical phenotype that renders them sensitive to H₂O₂-mediated apoptosis (18) .

The finding of significant growth inhibition and apoptosis by treatment with As₂O₃ (16 , 18, 19, 20) , and to a greater extent by MAs^IIIO in lymphoma cells (Fig. 5) and in CLL cells in primary culture (Table 2) supports the evaluation of As₂O₃ and MAs^IIIO in additional clinical trials. MAs^IIIO, up to a concentration of 1 µM, although more potent than iAs^III to induce apoptosis in NB4 and death in lymphoma cells, does not show greater toxicity than iAs^III in human bone marrow as measured by colony assays (Fig. 6) . We reported previously that the combination of ascorbic acid and As₂O₃, which increases H₂O₂ accumulation and apoptosis in NB4 and lymphoma cell lines, does not enhance As₂O₃ toxicity in human bone marrow as measured by colony assays (31) . This correlates with a greater therapeutic effect of As₂O₃ in combination with ascorbic acid without increasing toxicity in a mouse lymphoma model (31) . This may be related to the observation that human bone marrow progenitor cells have high catalase levels (36) , which may render these cells more resistant to H₂O₂ accumulation after As₂O₃ treatment. The uptake and conversion of iAs^III to MAs^IIIO by HepG2 cells support the use of iAs^III or MAs^IIIO in the treatment of patients with hepatoma. There may be an increased potential for toxicity, particularly in liver with MAs^IIIO treatment. Thus, animal studies are required to evaluate this derivative before future clinical trials.

	ACKNOWLEDGMENTS

 
We thank Dr. George Acs for critical reading of this manuscript.

	FOOTNOTES

 
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.

¹ Supported in part by NIH 5R01CA85478 and Samuel Waxman Cancer Research Foundation (to S. W. and Y. J.), "one hundred-talent" programs of Chinese Academy of Sciences, and Key projects of Shanghai Committee of Science and Technology of China (to G-Q. C.), and NIH Grants ES09941 and Clinical Nutrition Research Center Grant DK 56350 (to M. S. and F. W.).

² These authors should be considered equal first authors.

³ To whom requests for reprints should be addressed, at Department of Medicine, Division of Hematology/Oncology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Phone: (212) 241-7995; Fax: (212) 996-5787; E-mail: samuel.waxman{at}mssm.edu

⁴ The abbreviations used are: APL, acute promyelocytic leukemia; RAR, retinoic acid receptor; As₂O₃, arsenic trioxide; iA, inorganic arsenical; iAs^v, pentavalent arsenate; iAs^III, trivalent arsenite; MAs^V, methylarsonic acid; MAs^III, methylarsonous acid; DMAs^V, dimethylarsine acid; DMAs^III, dimethylarsinous acid; MAs^IIIO, methylarsine oxide; DMAs^IIII, iododimethylarsine; CLL, chronic lymphocytic leukemia; ATCC, American Type Culture Collection; FACS, fluorescence-activated cell sorter; PI, propidium iodide; MA, methylarsenic; DMA, dimethylarsenic; MNC, mononuclear cell; CFU, colony-forming unit; E, erythroid; BFU, burst-forming unit; GM, granulocyte-macrophage; PARP, poly(ADP-ribose) polymerase; PML, promyelocytic leukemia; PML-RAR, promyelocytic leukemia-retinoic acid receptor alpha.

Received 11/ 1/02. Accepted 2/21/03.

	REFERENCES

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