Co-treatment with As 2 O 3 enhances selective cytotoxic effects of STI-571 against Bcr- Abl-positive acute leukemia cells

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1 (2001) 15, Nature Publishing Group All rights reserved /01 $ Co-treatment with As 2 O 3 enhances selective cytotoxic effects of STI-571 against Bcr- Abl-positive acute leukemia cells M Porosnicu 1, R Nimmanapalli 2, D Nguyen 1, E Worthington 1, C Perkins 1 and KN Bhalla 2 2 Interdisciplinary Oncology Program, Moffitt Cancer Center, University of South Florida, Tampa and 1 Sylvester Comprehensive Cancer Center, University of Miami School of Medicine, Miami, FL, USA By inhibiting the tyrosine kinase (TK) activity of Bcr-Abl, STI- 571 induces differentiation and apoptosis of HL-60/Bcr-Abl (with ectopic expression of p185 Bcr-Abl) and K562 (containing endogenous expression of p210 Bcr-Abl) but not of the control HL-60 cells. Treatment with arsenic trioxide (As 2 O 3 ) lowers Bcr- Abl protein levels and induces apoptosis of the Bcr-Ablpositive leukemic blasts (Blood 2000; 95: 1014). Here, we demonstrate that compared to treatment with STI-571 (0.25 to 1.0 M) oras 2 O 3 (0.5 to 2.0 M) alone, combined treatment with As 2 O 3 and STI-571 induced significantly more apoptosis of HL- 60/Bcr-Abl and K562 but not HL-60/neo cells (P 0.05). Combined treatment with As 2 O 3 and STI-571 also resulted in greater reductions in the levels of Bcl-x L, XIAP and Akt, and inhibition of Akt kinase activity. Co-treatment with As 2 O 3 inhibited STI- 571-induced hemoglobin, which was associated with the cleavage and downregulation of GATA-1 transcription factor involved in erythroid differentiation. These data demonstrate that a treatment strategy which combines an agent that lowers Bcr-Abl levels, eg As 2 O 3, with an agent that inhibits Bcr-Abl TK activity, eg STI-571, can potently induce apoptosis and differentiation of Bcr-Abl-positive human leukemic cells. (2001) 15, Keywords: STI-571; As 2 O 3 ; Bcr-Abl; Bcl-x L ; AKT Introduction The dysregulated activity of the tyrosine kinase (TK) encoded by the bcr-abl fusion gene is responsible for many of the malignant features observed in the leukemic blasts of chronic myelogenous leukemias (CML) in blast crisis, as well as ALL (acute lymphoblastic leukemia) blasts. 1 The fusion gene either encodes for the p210 or p185 TK. This has been implicated in the pathogenesis of CML or 30 to 50% of adult ALL. 1,2 Bcr- Abl expressing leukemic blasts display resistance to apoptosis, even when exposed to high doses of antileukemic drugs. 2 6 Consistent with this, the ectopic or endogenous protein expression of Bcr-Abl in HL-60/Bcr-Abl or K562 cells, respectively, was demonstrated to block the mitochondrial permeability transition ( m) and release of cytochrome c (cyt c), thereby inhibiting the activation of the executioner caspases and apoptosis. 5,6 Ectopic or endogenous Bcr-Abl protein expression upregulates several antiapoptotic mechanisms including the protein levels of Bcl-x L as well as the activities of NF B and Akt kinase. 5 9 NF B transactivates and upregulates the levels of the IAP (inhibitor of apoptosis protein) family which are known to inhibit the activity of caspases-9, -7 and ,11 Recent studies have shown that the inhibition of Bcr- Abl TK activity by a relatively specific inhibitor STI-571 induces differentiation and apoptosis as well as causes in vitro and in vivo eradication of Bcr-Abl-positive human leukemia cells Exposure to STI-571 was shown to lower TK activity Correspondence: K Bhalla, Interdisciplinary Oncology Program, Moffitt Cancer Center, University of South Florida, Magnolia Drive, MRC 3 East, Room 3056, Tampa, FL 33612, USA; Fax: Received 26 July 2000; accepted 20 January 2001 but not the levels of Bcr-Abl protein STI-571 also lowered Bcl-x L levels and Akt kinase and NF B activities as well as induced intracellular Hb and the expression of the monocytic and myeloid differentiation marker CD11b. 14 Arsenic trioxide (As 2 O 3 ) is a clinically active agent against acute promyelocytic leukemia (APL). 15 Recently, we have demonstrated that As 2 O 3 treatment can induce apoptosis of multidrug-resistant human myeloid leukemic cells that express Bcr-Abl (HL-60/Bcr-Abl and K562 cells), MDR, MRP, Bcl-2 or Bcl-x L. 16 As 2 O 3 treatment had a direct effect on the mitochondria resulting in mitochondrial m and the release of cytochrome (cyt) c. This was associated with the activation of the apical caspase-9, followed by the processing and activation of the executioner caspase, such as caspase-3, cleavage of death substrates including poly (ADP-ribose) polymerase (PARP) and DNA fragmentation factor (DFF-45) and apoptosis. 16 In HL-60/Bcr-Abl and K562 cells, As 2 O 3 treatment produced a significant decline in the Bcr-Abl but not Bcl-x L protein levels. 16 In contrast to STI-571, As 2 O 3 treatment did not induce Hb but increased the expression of CD11b. 15,16 In the present studies, we have extended our observations by determining the effects of a combination of As 2 O 3 and STI- 571 on Bcr-Abl-positive leukemia cells. We demonstrate that co-treatment with As 2 O 3 significantly increases apoptosis of Bcr-Abl-positive leukemic cells by lowering Bcr-Abl and Bclx L levels and further downregulating Akt kinase activity. While As 2 O 3 co-treatment augmented STI-571-induced CD11b expression, it inhibited intracellular Hb levels, possibly by a GATA-1 mediated mechanism. Materials and methods Reagents As 2 O 3 and Ara-C were purchased from Sigma (St Louis, MO, USA). As 2 O 3 was dissolved in 1.65 mol/l sodium hydroxide (NaOH) to make a stock solution of 1.0 mm which was serially diluted in RPMI Antibodies to detect the levels of proteins including Bcl-x L, PARP, caspase-3, caspase-9 and CD11b were obtained as previously described. 16,17 Monoclonal Anti-XIAP antibody was purchased from Boehringer Mannheim (Indianapolis, IN, USA). STI-571 was kindly provided by Novartis Pharma (East Hanover, NJ, USA). Monoclonal anti-gata-1 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell culture Human leukemia HL-60/neo, and HL-60/Bcr-Abl cells and the erythroid blast crisis CML K562 cells were cultured and passaged as previously described. 6

2 Flow cytometric analysis of cell cycle status and apoptosis The flow cytometric evaluation of the cell cycle status and apoptosis was performed according to a previously described method. 16,18 The percentage of cells in the apoptotic sub-g 1 (excluding the fragments smaller than half the G 1 peak), as well as G 1, S phase, and G 2 -M phases were calculated using Multicycle software (Phoenix Flow Systems, San Diego, CA, USA). Western blot analyses of proteins Western blot analyses of Bcr-Abl (p210 and p185), Bcl-x L, Akt, XIAP, procaspase-9, caspase-3, PARP, GATA-1 and -actin were performed using specific antisera or monoclonal antibodies (see above), as described previously ,16 18 The expression of -actin was used as a loading control. Akt kinase assay In untreated and STI-571-treated cells, Akt kinase activity was determined by utilizing an immunoprecipitation kinase assay with reagents provided in a commercially available kit (New England Biolab, Beverly, MA, USA). Briefly, cell lysates were used to immunoprecipitate Akt utilizing a polyclonal Akt antibody. Immunoprecipitates were then incubated with GSK-3 fusion protein in the presence of ATP and kinase buffer, allowing immunoprecipitated Akt to phosphorylate GSK-3, which was analyzed by Western blotting using a phospho- GSK-3 / (serine 219) antibody cells. The percentage of apoptotic cells was calculated for each experiment, as described previously. 20 Statistical analysis Significant differences between values obtained in a population of leukemic cells treated with different experimental conditions were determined by paired t-test analyses. A oneway ANOVA was also applied to the results of the various treatment groups, and post hoc analysis was performed using the Bonferroni correction method. Results Combination of STI-571 and As 2 O 3 induces more apoptosis than either agent alone We determined the apoptotic effects of the various clinically achievable concentrations of As 2 O 3 and STI-571, administered alone or in combination, in HL-60/neo, HL-60/Bcr-Abl and K562 cells. 21 Figure 1 demonstrates that as compared to 773 Immunophenotyping for differentiation markers and hemoglobin production HL-60/neo, HL-60/Bcr-Abl, and K562 cells were treated with various concentrations of As 2 O 3 or STI-571 for 2 days. Cells were then washed with PBS, resuspended in 100 l FACS wash buffer containing 10 l PE-conjugated anti-human CD11b antibody (Pharmingen, San Diego, CA, USA) and analyzed by flow cytometry. 16 Alternatively, untreated or As 2 O 3 - treated cells were washed in PBS and intracellular Hb levels were determined by a previously described method. 5 Apoptosis assessment by Annexin-V staining After drug treatment, cells were washed in PBS and resuspended in 100 l of staining solution (containing Annexin-V, fluorescein and PI in a HEPES buffer; Annexin-V-FLUOS Staining Kit, Boehringer Mannheim). Following incubation at room temperature for 15 min, cells were analyzed by flow cytometry. 16,17 Morphology of apoptotic cells After treatment with or without As 2 O 3, cells were washed and resuspended in PBS. Cytospin preparations of the cell suspensions were fixed and stained with Wright stain. Cell morphology was determined by light microscopy. In all, five different fields were randomly selected for counting of at least Figure 1 Combined treatment with As 2 O 3 plus STI-571 induces more apoptosis than treatment with either of the agents alone. HL- 60/Bcr-Abl (a) or K562 (b) were exposed to either As 2 O 3 (0.5 or 1.0 M) or STI-571 (0.25 or 1.0 M) alone or in combinations for 48 h. Following this, cells were harvested and the percentage of apoptotic cells was determined by Annexin V staining followed by flow cytometry (see text). The values shown as the bar graphs represent a mean of three experiments ± s.d. (error bars).

3 774 Table 1 As 2 O 3 and/or STI-571-induced apoptosis of HL-60/neo, HL-60 Bcr-Abl and K562 cells HL-60/neo HL-60/Bcr-Abl K562 AV a PI b AV a PI b AV a PI b Control 3.9 ± ± ± STI-571, 0.5 M 5.7 ± ± ± As 2 O 3, 2.0 M 6.6 ± ± ± As 2 O 3 + STI ± ± 1.3 c ± 2.0 c 39.4 Combined treatment with As 2 O 3 and STI-571 induced more apoptosis than either agent alone of HL-60/Bcr-Abl and K562 but not HL- 60/neo cells. Cells were treated with the indicated concentrations of the drugs for 48 h. Following this, the percentages of apoptotic cells were determined by either flow cytometry after Annexin V (AV) staining, or PI (propidium iodide) staining and DNA content analysis to estimate hypodiploid cells (sub-g1 fraction). a Values (AV only) represent the mean ± s.e. of three separate experiments. b Values are mean of two experiments performed in duplicate. c Values are significantly different (P 0.01) from those observed in cells treated with STI-571 or As 2 O 3 alone. Figure 2 Co-treatment with STI-571 and As 2 O 3 induces cytosolic accumulation of cyt c, processing of caspase-9 and -3 as well as the cleavage of PARP in Bcr-Abl-positive cells. Cells were treated with 0.5 M STI-571 and/or 2.0 M As 2 O 3 for 48 h. Following this, either the S100 fractions and cell lysates from the treated cells were utilized for Western analysis of cytosolic cyt c or of caspase-9 (panel a), -3 or PARP (panel b), respectively (see text). treatment with either STI-571 (0.25 and 1.0 M) oras 2 O 3 (0.5 and 1.0 M) alone for 48 h, combined treatment with these agents resulted in significantly more apoptosis (P 0.05), as determined by Annexin V staining followed by flow cytometry to detect the cell surface expression of phosphatidyl serine. 16,17 Table 1 demonstrates that this effect of As 2 O 3 on STI- 571-induced apoptosis was also seen with higher concentrations of As 2 O 3 (2.0 M). Similar findings were observed when the percentages of apoptotic cells were estimated by light microscopic assessment of morphologic features of apoptosis (data not shown). We investigated whether the increased apoptosis observed with the combined treatment with As 2 O 3 and STI-571 was due to the sensitizing effect of STI-571 on As 2 O 3 -induced cytosolic accumulation of cyt c, resulting in the increased processing and activity of caspase-9 and -3. Immunoblot analyses in Figure 2a demonstrates that, as compared to treatment with either agent alone, the combined treatment with STI-571 and

4 As 2 O 3 induces significantly more accumulation of cyt c in K562 cells. This was not evident in HL-60/Bcr-Abl cells. The combined treatment yielded significant processing of procaspase-9 and -3. The PARP cleavage activity of caspase-3 also appeared to be maximal when the cells were treated with STI- 571 plus As 2 O 3. These events were not observed in HL-60/neo cells treated with identical doses and exposure intervals to STI-571 and/or As 2 O 3 (Figure 2a and b). The immunoblots presented in Figure 2 are representative of the findings of three experiments. Combined treatment with STI-571 and As 2 O 3 reduces the levels of antiapoptotic Bcl-x L, Akt, XIAP and Akt kinase activity in Bcr-Abl-positive leukemic cells Since As 2 O 3 and STI-571 have been shown to downregulate Bcr-Abl levels and its TK activity, respectively, 14,16 we investigated the effect of the combination of STI-571 plus As 2 O 3 on Bcl-x L, Bcr-Abl and Akt levels and the Akt kinase activity. As shown in Figure 3a, treatment with As 2 O 3, but not STI-571, inhibited Akt levels in HL-60/Bcr-Abl and slightly in K562 cells. However, co-treatment with STI-571 enhanced As 2 O 3 - induced downregulation of Akt levels resulting in the proportional lowering of its kinase (GSK-3 phosphorylation) Figure 3 Effects of As 2 O 3 and/or STI-571 on the levels of Bcr-Abl, Bcl-x L, Akt and XIAP, as well as on the activity of Akt kinase. HL- 60/neo, HL-60/Bcr-Abl and K562 cells were treated with 0.5 M STI- 571 and/or 2.0 M As 2 O 3 for 48 h. Following this, cell lysates were utilized to determine Akt kinase activity (see text) or Western blot analysis was performed to determine the levels of XIAP, Bcl-x L and AKT (panel a) as well as Bcr-Abl levels (panel b). Evaluation of actin expression was used to control for equal protein loading. activity. While treatment with As 2 O 3 lowered Bcr-Abl levels, which we have also previously reported, 16 STI-571 did not affect Bcr-Abl levels (Figure 3b). As compared to treatment with As 2 O 3 alone, a combined treatment with STI-571 and As 2 O 3 produced a greater decline in Bcr-Abl levels in HL- 60/Bcr-Abl, but not in K562 cells (Figure 3b). As also previously reported, 16 while treatment with As 2 O 3 alone had little effect on Bcl-x L levels, the combined treatment with STI-571 and As 2 O 3 abrogated Bcl-x L levels both in K562 and HL- 60/Bcr-Abl cells (Figure 3a). Since it has been previously reported that during apoptosis XIAP is processed and downregulated by caspase-3 activity, 22 we examined the combined effect of STI-571 and As 2 O 3 on XIAP levels in these cell types. As shown in Figure 3a, marked inhibition of XIAP levels was observed when HL-60/Bcr-Abl and K562 cells were exposed to a combination of STI-571 and As 2 O 3. XIAP levels were minimally affected in HL-60/neo cells. A combination of lower concentrations of STI-571 (0.25 M) and As 2 O 3 (1.0 M) produced an attenuated effect on Bcl-x L, Akt and XIAP levels (data not shown). Co-treatment with As 2 O 3 enhances STI-571-induced CD11b expression but not intracellular Hb The effect of STI-571 and/or As 2 O 3 on the expression of the monocytic and myeloid marker CD11b was determined by flow cytometry. 23 Figure 4 demonstrates that exposure to STI- 571 induced CD11b expression in HL-60/Bcr-Abl and K562 but not in HL-60/neo cells. In contrast, treatment with As 2 O 3 induced CD11b in all cell types. Combined treatment with STI-571 and As 2 O 3 induced more CD11b in all cell types, but the effect was pronounced in K562 and HL-60/Bcr-Abl cells. The data in Figure 4 are representative of three experiments. Since previous studies had shown that STI-571 but not As 2 O 3 induces Hb, in Bcr-Abl-positive leukemic cells, 14,16 we determined the combined effect of STI-571 and As 2 O 3 on these cells. Figure 5 demonstrates that STI-571 increased intracellular Hb in HL-60/Bcr-Abl and K562 but not in HL-60/neo cells. Contrary to the effect on CD11b expression, cotreatment with As 2 O 3 inhibited STI-571-induced Hb in both HL-60/Bcr-Abl and K562 cells. It is noteworthy that the untreated HL-60/Bcr-Abl as well as K562 cells displayed higher levels of intracellular Hb as compared to HL-60/neo cells, which had barely detectable Hb levels. Results presented in Figure 5 are representative of three experiments. Recent studies have demonstrated that GATA-1 transcription factor is required for the terminal erythroid differentiation, 24 and the erythroid progenitors undergo developmental arrest and apoptosis when GATA-1 is either absent or cleaved by active caspases during apoptosis. 25,26 Therefore, we investigated the processing of GATA-1 in STI-571- and/or As 2 O 3 - treated HL-60/Bcr-Abl and K562 vs HL-60/neo cells. As shown in Figure 6, while the untreated HL-60/Bcr-Abl and K562 cells express GATA-1, it is barely detectable in HL- 60/neo cells. In addition, in HL-60/Bcr-Abl and K562 cells, co-treatment with As 2 O 3 and STI-571 resulted in greater processing of GATA-1 than treatment with either of the agents alone. Discussion Several strategies have been developed to target either the mrna or protein encoded by the bcr-abl fusion gene, which 775

5 776 Figure 4 Co-treatment with STI-571 and As 2 O 3 induces more CD11b expression than treatment with either of the agents alone. HL-60/neo, HL-60/Bcr-Abl and K562 cells were treated with 0.5 M STI-571 and/or 2.0 M As 2 O 3 for 48 h and the percentage of cells expressing CD11b on cell surface was measured by flow cytometry. M1 represents the fraction of CD11b-positive cells. The arrow points to the control isotype antibody. Figure 6 Co-treatment with As 2 O 3 and STI-571 induces processing of GATA-1 in Bcr-Abl-positive cells. HL-60/neo, HL-60/Bcr-Abl and K562 cells were treated with 0.5 M STI-571 and/or 2.0 M As 2 O 3 for 48 h. Cell lysates were utilized for immunoblot analysis of GATA-1. Figure 5 Effect of STI-571 and/or As 2 O 3 on the intracellular hemoglobin levels. HL-60/neo, HL-60/Bcr-Abl and K562 cells were treated with 0.5 M STI-571 and/or 2.0 M As 2 O 3 for 48 h and the intracellular Hb levels were determined spectrophotometrically (see text). is pathogenetically responsible for the malignant phenotype of CML and Bcr-Abl-positive adult ALL. 27,28 However, the relatively specific inhibitors of Bcr-Abl TK have undergone more comprehensive in vitro and in vivo evaluation ,29 Of these, STI-571 has already been shown not only to induce apoptosis of Bcr-Abl-positive leukemic cells but also displays promising clinical activity against CML and adult ALL. 14,30 Results of the present studies clearly demonstrate that cotreatment with As 2 O 3, which inhibits Bcr-Abl levels, enhances

6 STI-571-induced apoptosis of Bcr-Abl-positive leukemic cells. Consistent with the previous findings that Bcr-Abl expression confers resistance against apoptosis by inhibiting the mitochondrial release and cytosolic accumulation of cyt c, 6 combined treatment with As 2 O 3 and STI-571, which lowers both the levels and activity of Bcr-Abl, induces greater cytosolic accumulation of cyt c as compared to treatment with either of the agents alone. In the presence of datp, the cytosolic cyt c is known to induce oligomerization of APAF-1, which by recruiting and oligomerizing caspase-9 and -3 induces their processing and activities. 31,32 This has been designated as the intrinsic or mitochondrial pathway to apoptosis. 33,34 Therefore, by inducing more cytosolic accumulation of cyt c, combined treatment with As 2 O 3 and STI-571 also causes greater processing of caspase-9 and -3, PARP cleavage activity of caspase-3, and apoptosis. 33 There are several molecular mechanisms that have been implicated as being involved in Bcr-Abl-mediated resistance to the intrinsic or mitochondrial pathway to apoptosis. 7 9,35 39 Increased Bcl-x L levels and Akt kinase activity, observed in Bcr-Abl-transformed cells, may contribute toward the inhibition of either the cytosolic accumulation of cyt c or the activities of the effector caspases which may mediate the resistance against the intrinsic pathway to apoptosis Binding of Bcr-Abl to the p85 regulatory subunit of PI-3 kinase (PI-3K) and an intact SH2 domain of Bcr-Abl is necessary for the activation of PI-3K as well as its downstream effector Akt kinase. 9 While not all the potential mechanisms by which Akt kinase activity suppresses apoptosis have been elucidated, 37 Akt kinase has been shown to phosphorylate Bad on serine residues S112 and S136, respectively. 37 This converts the proapoptotic, BH3 domain-only containing Bad protein into an inactive moiety sequestered in the cytosol bound to protein. 37,38 Since only the active, nonphosphorylated Bad is bound to Bcl-x L or Bcl-2, the phosphorylation of Bad frees up Bcl-x L and Bcl-2 to promote survival. 38 Akt kinase has also been recently shown to phosphorylate and inactivate caspase- 9, 39 thereby preventing the cleavage and activities of the executioner caspases and apoptosis. 34,39 Our findings demonstrate that STI-571 inhibits Bcl-x L and Akt kinase activity in the HL-60/Bcr-Abl and K562 but not HL-60/neo cells. Whether STI-571 inhibits the antiapoptotic mechanism of Akt kinase, mediated through Bad and Caspase-9, was not determined in the present studies. Nonetheless, the present studies do demonstrate that the combination of As 2 O 3 and STI-571 produces greater inhibition of Akt levels and activity and abrogates Bcl-x L, and this may be responsible for increased cytosolic accumulation of cyt c and apoptosis of HL-60/Bcr-Abl and K562 cells. It is noteworthy that NF B, which confers resistance to apoptosis, 8 has also been shown to be increased in Bcr-Abl-positive leukemic cells. 7,8 However, NF B activity is not a necessary component of the antiapoptotic effect exerted by Bcr-Abl. 8,41 A recent report has indicated that Bcr-Abl tyrosine kinase can support erythroid development and maturation, even in those progenitor cells that lack erythropoietin receptors. 42 This may explain the detectable Hb levels in HL-60/Bcr-Abl and K562 cells. Previous reports had also indicated that treatment of K562 cells with STI-571 could markedly increase the percentage of benzidine-positive cells. 43 Our data corroborate these findings, showing that exposure to STI-571 induced Hb in HL-60/Bcr-Abl and K562 cells. Furthermore, STI-571 also induced CD11b expression in the Bcr-Abl-positive cells. The differentiating effects of STI-571 appeared to be more related to the inhibition of Bcr-Abl TK. Co-treatment with As 2 O 3 sig- nificantly increased the induction of CD11b by STI-571, but blunted its effect on the intracellular Hb levels. The transcription factor GATA-1 is essential for erythroid maturation and differentiation, 24,25 and it has been shown to inhibit apoptosis or erythroid progenitor cells by inducing Bclx L expression. 44 During apoptosis caspase-mediated degradation of GATA-1 was shown to result in an impaired erythroid differentiation. 26 Our findings demonstrate that coincidental with the detectable levels of Hb, GATA-1 expression was also detected only in the Bcr-Abl-positive HL- 60/Bcr-Abl and K562 cells. Furthermore, the combined treatment with As 2 O 3 plus STI-571, which induced more caspase activity and GATA-1 degradation, was also associated with the blunting of Hb induction due to treatment with STI-571. However, a mechanistic link between more GATA-1 degradation and reduced Hb induction has not been definitively established. Despite promising clinical activity of STI-571 in Bcr-Ablpositive leukemias, relapses are common with its continuous use as a single agent in the blast crisis phase of this malignancy. 30,45 Recent reports have indicated that a continuous in vitro exposure of Bcr-Abl-positive human leukemic cells to STI-571 induced bcr-abl gene amplification and increased its mrna. 46,47 These cells demonstrated increased Bcr-Abl protein levels and resistance to STI-571-induced apoptosis. In a previous report, we had demonstrated that treatment with As 2 O 3 alone markedly inhibits Bcr-Abl protein levels, 16 a finding also noted when HL-60/Bcr-Abl and K562 cells were treated with a combination of As 2 O 3 plus STI-571. This suggests that a regimen consisting of As 2 O 3 and STI-571 is not only more effective in inducing cell death (associated with downregulation of antiapoptotic Bcl-x L and XIAP levels as well as inhibition of Akt kinase activity), but may also have the potential to retard the development of STI-571 resistance by abrogating Bcr-Abl levels. Therefore, the effect of As 2 O 3 on the development of resistance to STI-571 needs to be examined. Collectively, the in vitro findings presented here support the rationale to investigate the in vivo effects of the combination of As 2 O 3 plus STI-571 against Bcr-Abl-positive human leukemias that are either sensitive or resistant to STI References 1 Melo JV. The diversity of BCR-ABL fusion proteins and their relationship to leukemia phenotype. Blood 1996; 88: Faderl S, Talpaz M, Estrov Z, Kantarjian HM. Chronic myelogenous leukemia: biology and therapy. 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