Role of the p38 Mitogen-Activated Protein Kinase Pathway in the Generation of Arsenic Trioxide–Dependent Cellular Responses

Arsenic trioxide (As 2 O 3 ) induces differentiation and apoptosis of leukemic cells in vitro and in vivo , but the precise mechanisms that mediate such effects are not known. In the present study, we provide evidence that the kinases MAPK kinase 3 (Mkk3) and Mkk6 are activated during treatment of leukemic cell lines with As 2 O 3 to regulate downstream engagement of the p38 mitogen-activated protein kinase. Using cells with targeted disruption of both the Mkk3 and Mkk6 genes, we show that As 2 O 3 -dependent activation of p38 is defective in the absence of Mkk3 and Mkk6, establishing that these kinases are essential for As 2 O 3 -dependent engagement of the p38 pathway. Pharmacologic inhibition of p38 enhances As 2 O 3 -dependent activation of the c-jun NH 2 terminal kinase (JNK) and subsequent induction of apoptosis of chronic myelogenous leukemia (CML)– or acute promyelocytic leukemia (APL)–derived cell lines. In addition, in APL blasts, inhibition of p38 enhances myeloid cell differentiation in response to As 2 O 3 , as well as suppression of Bcl-2 expression and loss of mitochondrial membrane potential. Similarly, induction of As 2 O 3 -dependent apoptosis is enhanced in mouse embryonic fibroblasts (MEF) with targeted disruption of both the Mkk3 and Mkk6 genes, establishing a key role for this pathway in the regulation of As 2 O 3 -induced apoptosis. In other studies, we show that the small-molecule p38 inhibitors SD-282 and SCIO-469 potentiate As 2 O 3 mediated suppression of myeloid leukemic progenitor growth from CML patients, indicating a critical regulatory role for p38 in the induction of antileukemic responses. Altogether, our data indicate that the Mkk3/6-p38 signaling cascade is activated in a negative regulatory feedback manner to control induction of As 2 O 3 -mediated antileukemic effects. (Cancer Res 2006; 66(13): 6763-71)


Introduction
Arsenic trioxide (As 2 O 3 ) exhibits potent antitumor effects in vitro and in vivo and is widely used in the treatment of acute promyelocytic leukemia (APL) in humans (1)(2)(3)(4). The effects of As 2 O 3 are dose dependent, with higher concentrations leading to apoptosis and lower concentrations (<0.5 Amol/L) inducing differentiation (1)(2)(3)(4). In addition to APL, arsenic is of potential therapeutic value for the treatment of other hematologic malignancies, including chronic myelogenous leukemia (CML; refs. 5,6). CML is a clonal myeloproliferative disorder of stem cells, the hallmark of which is the presence of the abnormal BCR-ABL oncogenic tyrosine kinase, which transforms the cells (7). There is evidence that As 2 O 3 can induce apoptosis of BCR-ABL-expressing cell lines (8) and inhibit the proliferation of CML leukemic blasts (9,10). These findings have suggested a potential role for this agent in the treatment of CML (5,6) but the precise mechanisms by which it induces its antileukemic effects remain to be defined.
Mitogen-activated protein kinases (MAPK) are a family of enzymes that transduce signals from the cell membrane to the nucleus in response to a wide range of stimuli, and modulate several important biological functions including gene expression, mitosis, proliferation, motility, and apoptosis (reviewed in refs. [11][12][13]. Three major groups of MAPKs exist, the extracellular signal-regulated kinase (ERK), c-jun NH 2 -terminal kinase (JNK), and p38 MAPK families (11)(12)(13). The involvement of these kinases in the generation of stress responses, as well as in signaling for various cytokines and growth factors, has been extensively studied and documented in previous studies (reviewed in refs. [11][12][13]. However, little is known on the putative roles of MAPKs in leukemogenesis or their involvement in pathways that mediate antileukemic responses (12).
Previous work from our laboratory has shown that As 2 O 3 induces activation of the p38 MAPK in various neoplastic cell lines (14), suggesting a role for this pathway in the regulation of arsenicresponses in malignant cells. In the present study, we sought to define the functional relevance of activation of p38 by As 2 O 3 . Our data show that activation of p38a exhibits a negative regulatory role on the induction of apoptosis by As 2 O 3 , as evidenced by the enhanced induction of apoptosis during pharmacologic inhibition of p38 or in cells with targeted disruption of the p38a gene. In efforts to define the sequence of events that lead to arsenicdependent activation of p38, we found that the kinases MAPK kinase 3 (Mkk3) and Mkk6 are activated by As 2 O 3 and act as the upstream effectors of p38. Furthermore, apoptosis and growth inhibition promoted by As 2 O 3 were augmented in cells lacking both Mkk3 and Mkk6 genes compared with their wild-type counterparts. Finally, we show that the pharmacologic inhibitors of p38, SD-282 and/or SCIO-469, promote arsenic-mediated differentiation of APL blasts and enhance the suppressive properties of As 2 O 3 on primitive leukemic granulocyte-macrophage colony-forming unit (CFU-GM) progenitors from patients with CML. Altogether, our results suggest that the p38 MAPK signaling cascade acts as a negative feedback regulator for the generation of antileukemic responses, and its activation may account for lack of sensitivity or resistance to As 2 O 3 in different leukemia cell types.
Cell lysis, immunoprecipitations, and immunoblotting. Cells were stimulated with the indicated doses of As 2 O 3 for the indicated times and subsequently lysed in phosphorylation lysis buffer as previously described (20). Immunoprecipitations and immunoblotting using an enhanced chemiluminescence method were done as described in previous studies (20).
In vitro kinase assays. These assays were done as previously described (20,21). Briefly, cells were treated for the indicated times with the indicated doses of As 2 O 3 and lysed in phosphorylation lysis buffer. Cell lysates were then immunoprecipitated with antibodies against Mkk3 or Mkk6. The immunoprecipitates were then washed thrice with phosphorylation lysis buffer followed by two washes with kinase buffer (25 mmol/L HEPES, 25 mmol/L MgCl 2 , 25 mmol/L h-glycerophosphate, 100 Amol/L sodium orthovanadate, 2 mmol/L DTT, 20 Amol/L ATP). They were then resuspended in 30 AL of kinase buffer containing 3 Ag of glutathione S-transferase (GST)-p38a ( for the Mkk3 and Mkk6 kinase assays) and 10 ACi of [g-32 P]ATP. The reaction was subsequently incubated for 20 minutes at room temperature and terminated by addition of SDS sample buffer. Proteins were analyzed by SDS-PAGE and the phosphorylated form of p38a was detected by autoradiography.
Evaluation of apoptosis. Cells were treated with the indicated doses of As 2 O 3 for the indicated time periods. Flow cytometric assays to evaluate apoptosis by Annexin and propidium iodide staining were done essentially as previously described (23).
Mitochondrial membrane potential detection. Tetramethylrhodamine, ethyl ester, perchlorate (TMRE; Molecular Probes, Carlsbad, CA) was used to assess loss of mitochondrial membrane potential. The uptake and accumulation of TMRE in the mitochondria is driven by membrane potential (DW m ); therefore, depolarization of DW m can be represented by the loss of TMRE staining. Briefly, TMRE working solution was added directly to the medium to reach 100 nmol/L and then incubated at 37jC for 15 minutes. Cells were then washed with cold PBS once and kept on ice for immediate TMRE detection by flow cytometry.
Flow cytometric analysis. Flow cytometric studies to detect granulocytic differentiation of APL cells were done as previously described (22).
Briefly, NB-4 cells were treated with As 2 O 3 for the indicated times and cell differentiation was determined by staining with the anti-CD11b monoclonal antibody. The anti-CD11b monoclonal antibody and a matched isotype control were purchased from BD Biosciences (San Jose, CA).
Hematopoietic progenitor cell assays. Bone marrow or peripheral blood from patients with CML were collected after obtaining consent approved by the Institutional Review Board of Northwestern University. Mononuclear cells were isolated by Ficoll-Hypaque centrifugation and used for clonogenic assays in methylcellulose as previously described (23). The cells were cultured in the presence or absence of As 2 O 3 (1 Amol/L), with or without SB203580 (10 Amol/L), SD-282 (400 nmol/L), or SCIO-469 (400 nmol/L). Leukemic CFU-GM colonies were scored on day 14 of culture (23).

Results
We first determined whether As 2 O 3 treatment induces phosphorylation/activation of p38 MAPK in the CML-derived KT-1 cell line. Cells were incubated in the absence or presence of As 2 O 3 (2 Amol/L) for various times and total cell lysates were resolved by SDS-PAGE and immunoblotted with an antibody against the phosphorylated/activated form of p38. Treatment of KT-1 cells with As 2 O 3 induced strong phosphorylation/activation of p38, which was detectable after 30 minutes of treatment and persisted up to 120 minutes of incubation of the cells with As 2 O 3 (Fig. 1A). On the other hand, the levels of p38 protein expression were similar before and after As 2 O 3 treatment of the cells (Fig. 1A). Thus, the p38 MAPK is phosphorylated/activated in an As 2 O 3 -inducible manner in BCR-ABL-expressing cells, suggesting that its function may be important for the induction of the antileukemic effects of In subsequent studies, we sought to identify upstream kinases that regulate phosphorylation and activation of the p38 MAPK in response to As 2 O 3 . As MAPK kinases 3 and 6 (Mkk3 and Mkk6) have previously been shown to phosphorylate p38 in response to stress (15,24,25), we examined their involvement in the regulation of the arsenic-activated p38 MAPK pathway. Treatment with As 2 O 3 resulted in phosphorylation of Mkk3/Mkk6 in both KT-1 cells (1B) and the APL-derived NB-4 cell line (1C), in which p38 is also activated in response to As 2 O 3 or retinoids (14,22). Consistent with this, in experiments in which immunocomplex kinase assays were done in anti-Mkk3 and anti-Mkk6 immunoprecipitates (Fig. 1D), there was detectable As 2 O 3 -dependent Mkk kinase activity. Thus, the kinases Mkk3 and Mkk6 are activated in an As 2 O 3 -dependent manner in sensitive cells, suggesting that they may play regulatory roles in the activation of the p38 pathway by As 2 O 3 .
To directly define the roles that Mkk3 and Mkk6 may play in the activation of p38 by As 2 O 3 , we examined the phosphorylation/ activation of p38 in double-knockout MEFs for both the Mkk3 and Mkk6 genes (15). Mkk3 +/+ Mkk6 +/+ and Mkk3 À/À Mkk6 À/À MEFs were incubated in the presence or absence of As 2 O 3 . The cells were subsequently lysed and cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-phospho-p38 antibody. As 2 O 3 treatment resulted in phosphorylation/activation of p38 in Mkk3/6 +/+ cells whereas such phosphorylation was defective in the MEFs with targeted deletion of both the Mkk3 and Mkk6 genes (Fig. 1E), establishing that activation of the p38 MAPK cascade by As 2 O 3 requires the upstream functions of Mkk3 and Mkk6.
We subsequently sought to determine the functional relevance of activation of the Mkk3/6-p38 MAPK pathway during treatment of BCR-ABL-expressing cells with As 2 O 3 . We first examined the effects of a p38 inhibitor, the pyridinyl imidazole compound SB203580, on the induction of apoptosis by As 2 O 3 . Treatment of KT-1 cells with As 2 O 3 resulted in induction of apoptosis, as determined by Annexin V/propidium iodide staining, whereas the SB203580 inhibitor alone did not induce apoptosis of the cells ( Fig. 2A). However, concomitant addition of the p38 inhibitor together with As 2 O 3 in the cultures strongly enhanced the induction of apoptosis as compared with cells treated with As 2 O 3 alone ( Fig. 2A). We also did similar studies using the SD-282 and SCIO-469 specific inhibitors of p38. The activity of these inhibitors against the p38 MAPK has been extensively docu-mented previously (18,19,26). As in the case of the SB203580 inhibitor, both SD-282 and SCIO-469 potentiated the induction of apoptosis by As 2 O 3 (Fig. 2B). We did experiments in which the expression of p38a was blocked in CML-derived cells using the small interfering RNA (siRNA) method and the induction of apoptosis by As 2 O 3 in KT-1 cells was compared in the presence or absence of p38a. The p38a siRNA inhibited expression of p38a ( Fig. 2C) whereas the induction of apoptosis was enhanced in cells transfected with p38a siRNA as compared with cells Figure 1. As 2 O 3 -dependent phosphorylation/activation of the p38 MAPK and its upstream effectors Mkk3 and Mkk6. A, KT-1 cells were incubated in the absence or presence As 2 O 3 (2 Amol/L) for the indicated times. Equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with an anti-phospho-p38 antibody (left ). The same blot was stripped and reprobed with anti-p38 antibody to control for equal loading (right ). B, KT-1 cells were incubated in the absence or presence As 2 O 3 (2 Amol/L) for the indicated times. Equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with an anti-phospho-Mkk3/6 antibody (left). The same blot was stripped and reprobed with anti-Mkk3 antibody to control for equal loading (right ). C, NB-4 cells were incubated in the absence or presence of As 2 O 3 (2 Amol/L) for the indicated times. Equal amounts of total cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-phospho-Mkk3/6 antibody (left). The same blot was stripped and reprobed with an anti-Mkk3 antibody to control for protein loading (right ). D, KT-1 cells were incubated for the designated times in the absence or presence of As 2 O 3 (2 Amol/L). Cell lysates were immunoprecipitated with an anti-Mkk3 antibody or control nonimmune rabbit immunoglobulin (RIgG ), and immunoprecipitated proteins were subjected to in vitro kinase assays using GST-p38a as an exogenous substrate. Proteins were resolved by SDS-PAGE and phosphorylated proteins were detected by autoradiography (left ). NB-4 cells were incubated for the indicated times in the absence or presence of As 2 O 3 (2 Amol/L). Cell lysates were immunoprecipitated with an anti-Mkk6 antibody or control nonimmune goat immunoglobulin (GIgG ), and immunoprecipitated proteins were subjected to in vitro kinase assays using GST-p38a as an exogenous substrate. Proteins were then resolved by SDS-PAGE and phosphorylated proteins were detected by autoradiography (right ). E, Mkk3/6 +/+ and Mkk3/6 À/À MEFs were incubated in the absence or presence of As 2 O 3 (2 Amol/L) for 120 minutes. The cells were lysed and equal amounts of total cell lysates were analyzed by SDS-PAGE and immunoblotted with anti-phospho-p38 antibody (left ). The same blot was stripped and reprobed with an anti-p38 antibody to control for protein loading (right ).
transfected with control siRNA (Fig. 2D). Taken altogether, these findings strongly suggested a putative role for p38, acting as a negative regulator of As 2 O 3 -induced apoptosis in BCR-ABLexpressing cells.
To further establish the role of the p38 MAPK pathway on the regulation of As 2 O 3 -induced apoptosis, studies using immortalized MEFs with targeted deletion of gene-components of the p38 MAPK pathway were subsequently done. Mkk3 +/+ Mkk6 +/+ and Mkk3 À/À Mkk6 À/À MEFs were treated with As 2 O 3 for 48 hours and the percentage of apoptotic cells was determined by flow cytometry. Treatment of Mkk3 À/À Mkk6 À/À cells with As 2 O 3 resulted in significantly enhanced apoptosis as compared with Mkk3 +/+ Mkk6 +/+ cells (Fig. 3A). Similarly, when the antiproliferative effects of As 2 O 3 were compared in the wild-type MEFs and the double Mkk3 À/À Mkk6 À/À knockout MEFs, there was a substantial augmentation of the inhibitory effects of different concentrations of As 2 O 3 in the absence of Mkk3 and Mkk6 (Fig. 3B). Altogether, these studies provided firm evidence implicating Mkk3 and Mkk6 as negative regulators of As 2 O 3 -induced apoptosis and growth inhibition. We also did experiments using MEFs with targeted disruption of the p38a gene (16). As shown in Fig. 3C, treatment of wild-type p38a +/+ MEFs with As 2 O 3 resulted in phosphorylation/ activation of p38a whereas p38a protein expression was absent in cells with targeted deletion of the p38a gene (p38a À/À ; Fig. 3C). In experiments in which the induction of apoptosis by As 2 O 3 was compared between p38a +/+ and p38a À/À MEFs, the numbers of cells undergoing apoptosis after arsenic treatment were higher in p38a À/À cells as compared with parental p38a +/+ cells (Fig. 3D). However, as the increase in As 2 O 3 -mediated apoptosis was not as pronounced in the p38a cells when compared with the Mkk3/6 À/À MEFs, we speculated that compensatory activities of other p38 isoforms might account for such differences in the MEFs. Consistent with such a hypothesis, pretreatment of p38a À/À MEFs with the p38 inhibitor SB203580 further potentiated As 2 O 3 -induced apoptosis (Fig. 3D). On the other hand, when similar studies were done in double-knockout MEFs for both Jnk1 and Jnk2 (17), there was no enhancement of As 2 O 3 -dependent apoptosis (Fig. 3E). On the contrary, there was decreased apoptosis in the Jnk1 À/À Jnk2 À/À MEFs as compared with Jnk1 +/+ Jnk2 +/+ MEFs (Fig. 3E), confirming and expanding on the results of previous studies in which it was shown that pharmacologic inhibition of Jnk reverses As 2 O 3dependent apoptosis (27). As 2 O 3 -induced apoptosis in malignant cells has previously been shown to involve opening of the mitochondrial permeability transition pore, which is dependent on a balance between the prosurvival and proapoptotic family members of the Bcl-2 family (28). It is also well established that Bcl-2 plays an important role in protecting against As 2 O 3 -induced apoptosis in certain malignant cell types (29)(30)(31)(32). We examined the effects of pharmacologic inhibition of the p38 on the suppression of Bcl-2 expression by As 2 O 3 treatment in the NB-4 APL cell line. Cells were exposed to As 2 O 3 for 24 hours, in the absence or presence of SB203580, and the expression of Bcl-2 protein was determined. As expected, treatment of cells with As 2 O 3 resulted in down-regulation of Bcl-2 (Fig. 4A). However, such As 2 O 3 -dependent down-regulation of Bcl-2 was further enhanced by the addition of SB203580 in the cultures as compared with treatment with As 2 O 3 alone (Fig. 4A). Consistent with the enhanced suppression of Bcl-2 expression, inhibition of p38 with either SB203580 or SCIO-469 potentiated arsenic-induced loss of mitochondrial membrane potential (DW m ; Fig. 4B). Altogether, these findings suggest a putative mechanism by which p38 inhibition enhances arsenic-dependent apoptosis in cells of promyelocytic origin, involving Bcl-2 down-regulation and enhanced loss of mitochondrial membrane potential.
To further understand the mechanisms by which p38 inhibition may promote As 2 O 3 -induced apoptosis of target cells, we examined the effects of p38 inhibitors on other signaling pathways of which the function is essential for the induction of apoptosis by As 2 O 3 . It is now well established by the work of others (27), and confirmed by our experiments using Jnk1/2 knockout MEFs (Fig. 3E), that activation of the Mkk4/JNK pathway is essential for the induction of As 2 O 3 -dependent apoptosis. When either NB-4 (Fig. 4C) or KT-1 (Fig. 4D) cells were concomitantly treated with SB203580, SCIO-469, or SD-282, we noticed a strong enhancement of Jnk1/Jnk2 phosphorylation in response to As 2 O 3 . Thus, pharmacologic inhibition of the p38 MAPK pathway seems to enhance activation of Jnk by As 2 O 3 , and such events may also contribute to the induction of apoptosis on target cells.
It is well established that, in contrast to high doses, low doses of As 2 O 3 promote differentiation of APL blasts to granulocytes (1-4). There is also evidence from our previous work that pharmacologic inhibition of p38 enhances the induction of differentiation of APL cells in response to all-trans-retinoic acid (22). We examined the effects of p38 inhibitors on the induction of As 2 O 3 -induced differentiation of NB-4 cells. As 2 O 3 treatment of NB-4 cells resulted in granulocytic differentiation of a fraction of cells, as determined by the induction of CD11b expression on the surface of the cells (Fig. 5A). Cotreatment of cells with either SD-282 or SCIO-469 strongly enhanced the percentage of CD11b-expressing cells ( Fig. 5A and B). On the other hand, cotreatment of cells with the MAPK/ERK kinase (MEK)/ERK inhibitor PD98059 reversed the induction of differentiation, suggesting that the MEK/ERK pathway promotes arsenic-mediated NB-4-cell differentiation (Fig. 5B) in a manner similar to what is seen in the case of retinoid-dependent differentiation of APL cells (22). As As 2 O 3 is also known to inhibit the growth of NB-4 cells (27), we cannot absolutely exclude the possibility that the enhanced differentiation by the p38 inhibitors is not due in part to selective killing of undifferentiated cells by As 2 O 3 . However, this is unlikely as it has also previously been shown that arsenic does not induce significant apoptosis at the low concentrations used here (27). In addition, in further analysis in which the proportion of differentiated cells among 1 Â 10 4 live cells analyzed by flow cytometry was compared with the fraction of differentiated cells present in the total population of cells (live and dead) counted, similar patterns of CD11b induction were observed in response to Figure 3. Targeted disruption of the Mkk3 and Mkk6, or p38a, genes potentiates As 2 O 3 -induced apoptosis and growth inhibition. A, Mkk3 +/+ 6 +/+ and Mkk3 À/À 6 À/À MEFs were incubated in the absence or presence of As 2 O 3 (2 Amol/L) for 48 hours. The total percentage of apoptotic cells was determined by Annexin V/propidium iodide staining, followed by flow cytometry. Columns, mean of three experiments; bars, SE. B, Mkk3 +/+ 6 +/+ and Mkk3 À/À 6 À/À MEFs were incubated in the absence or presence of the indicated final concentrations of As 2 O 3 for 3 days. Cell proliferation was assessed by MTT assay. Points, means of three experiments; bars, SE. C, p38a +/+ and p38a À/À MEFs were incubated in the absence or presence of As 2 O 3 (2 Amol/L) for the indicated times. The cells were then lysed and total cell lysates were resolved by SDS-PAGE and immunoblotted with an anti-phospho-p38 MAPK antibody (top left ). The same blot was subsequently stripped and reprobed with an anti-p38 MAPK antibody (top right ). The same blot was subsequently stripped and reprobed with an antitubulin antibody to control for protein loading (bottom ). D, p38a+/+ and p38a À/À MEFs were incubated for 48 hours in the absence or presence of As 2 O 3 (2 Amol/L) for 48 hours, in the presence or absence of SB203580. The percentage of apoptotic cells was calculated by Annexin V/propidium iodide staining using flow cytometric analysis. Columns, mean of three experiments; bars, SE. E, Jnk1 +/+ 2 +/+ and Jnk1 À/À Jnk2 À/À MEFs were incubated for 48 hours in the absence or presence of As 2 O 3 (2 Amol/L) for 48 hours. Induction of apoptosis was evaluated by flow cytometry after Annexin V/propidium iodide staining. Columns, mean of three experiments; bars, SE. p38 and Antileukemic Responses www.aacrjournals.org treatment combinations of arsenic with the p38 inhibitors (data not shown). This makes it unlikely that that the combination therapy selectively killed undifferentiated cells. Thus, pharmacologic inhibition of p38 enhances arsenic-induced differentiation of APL blasts to granulocytes, suggesting that the p38 pathway negatively regulates leukemic cell differentiation.
To obtain information on the role of p38 in the generation of As 2 O 3 -dependent responses in a more physiologically relevant system, studies were done with primary leukemic progenitors from patients with CML. Bone marrow or peripheral blood mononuclear cells from different patients with CML were treated them with As 2 O 3 in the absence or presence of different pharmacologic inhibitors of p38, including SB203580, SD-282, or SCIO-469, and subsequently assayed for leukemic CFU-GM progenitor colony formation. Concomitant treatment with As 2 O 3 and the pyridinyl imidazole compound SB203580 enhanced suppression of leukemic CFU-GM progenitors ( Fig. 6A; ref. 14). Importantly, the p38 inhibitors SD-282 and SCIO-469 also augmented the suppressive effects of As 2 O 3 on leukemic CFU-GM colony formation (Fig. 6B). Using paired t-test statistical analysis, the effects of either SD-282 (two-tailed P = 0.0061) or SCIO-469 (two-tailed P = 0.0044) were found to be statistically significant. Thus, different classes of pharmacologic inhibitors of p38 enhance the antileukemic properties of As 2 O 3 in vitro, suggesting that such effects may also occur during concomitant administration of p38 inhibitors and As 2 O 3 to leukemia patients in vivo.

Discussion
As 2 O 3 is a heavy metal derivative that has potent antileukemic properties. This agent induces dramatic remissions in patients suffering from APL (1-4), whereas there is strong interest in its clinical development for other leukemias, and in particular, CML (5,6). The important antitumor properties of As 2 O 3 in vitro and in vivo have ignited extensive studies aimed to determine the mechanisms by which it induces apoptosis and antileukemic responses. It is now well established that arsenic exposure of target cells results in elevation of reactive oxygen species, loss of mitochondrial membrane potential, and release of cytochrome c, followed by activation of the caspase cascade and programmed cell death (32)(33)(34)(35). There is also evidence that generation of reactive oxygen species, in particular H 2 O 2 , depends on cellular glutathione stores and that reduced cellular glutathione (GSH) is an inhibitor of As 2 O 3 -dependent cell death by conjugating arsenic in the form of AS(GS)3 complexes and/or sequestering reactive oxygen species (36). Interestingly, GSH depletion by pretreatment of arsenicresistant APL cells with buthionine sulfoximine has been shown to restore sensitivity to As 2 O 3 -induced apoptosis (37).
Despite the significant advances on the mechanisms of arsenicinduced apoptosis and the contribution of reactive oxygen species, very little is known on the roles that other signaling pathways play in the generation of As 2 O 3 -mediated biological responses. There is some recent evidence that members of distinct MAPK families play differential roles in the generation of As 2 O 3 -dependent effects. As 2 O 3 has been shown to result in sequential activation of Mkk4 and Jnk (27,38) whereas pharmacologic inhibition of Jnk was shown to inhibit arsenicdependent apoptosis (27). Moreover, such apoptosis is defective in cells lacking Mkk4 (27), strongly suggesting that sequential activation of Mkk4 and Jnk is required for the induction of As 2 O 3 -mediated apoptosis. On the other hand, ERK1/2 are also phosphorylated/activated in response to treatment of cells with As 2 O 3 but inhibition of such activation with MEK1 inhibitors or siRNA-mediated blocking of MEK1 enhances arsenic-dependent apoptosis (39). Thus, the MEK/ERK pathway seems to exhibit an opposing role to the JNK pathway in the regulation of arsenicdependent apoptosis (39). The p38 MAPK pathway has also been shown to be activated in response to As 2 O 3 (14) and the small GTPase Rac1 was identified as an upstream regulator of this pathway (14). However, the identity of the MAPK kinases that regulate p38 activation in response to As 2 O 3 downstream of Rac1 has been unknown and the overall functional relevance of activation of this pathway by As 2 O 3 has been unclear.
In the present study, we provide the first evidence that the protein kinases Mkk3 and Mkk6 are phosphorylated and activated in an As 2 O 3 -inducible manner in human leukemic cell lines. The activation of these kinases seems to be critical for As 2 O 3dependent activation of the p38 pathway as evidenced by the lack of phosphorylation/activation of p38 in double-knockout cells for both kinases (Mkk3 À/À Mkk6 À/À ). Our data also show that arsenicmediated induction of apoptosis and growth inhibition are strongly enhanced in Mkk3 À/À Mkk6 À/À cells as compared with parental cells. Moreover, As 2 O 3 -dependent apoptosis is enhanced in cells with targeted disruption of the p38a gene. Taken p38 and Antileukemic Responses www.aacrjournals.org altogether, these data provide direct and definitive evidence that the p38 MAPK pathway acts as a negative feedback regulator to counteract induction of As 2 O 3 -induced cell death. On the other hand, our studies show that the induction of As 2 O 3 -dependent apoptosis is defective in Jnk1/Jnk2 double-knockout MEFs, directly establishing that the function of the Jnk pathway is necessary for the generation of the effects of arsenic on target cells. In other studies, we show that, in addition to SB203580 (14), different pharmacologic inhibitors of p38 (SD282 and SCIO469) potentiate the antileukemic properties of As 2 O 3 on primitive leukemic precursors derived from the peripheral blood or bone marrows of patients with CML, suggesting that activation of p38 is a physiologically relevant mechanism of resistance to the antileukemic properties of As 2 O 3 .
Our studies also show that pharmacologic inhibition of p38 enhances the induction of cell differentiation of APL blasts at the low concentration of As 2 O 3 (0.5 Amol/L) known to induce cell differentiation. Interestingly, in previous work, we had found that the p38 MAPK pathway is activated in response to treatment of NB-4 cells with all-trans-retinoic acid and negatively regulates alltrans-retinoic acid-induced cell differentiation (22). In those studies, we had found that all-trans-retinoic acid-dependent expression of CD11b was enhanced by pharmacologic inhibitors of p38 in a manner similar to the effects of p38 inhibitors on arsenic-induced differentiation of NB-4 cells. Thus, it seems that the p38 MAPK pathway is a common negative regulator of cell differentiation of APL cells in response to either As 2 O 3 or retinoids.
The p38 MAPK pathway has previously been shown to mediate a wide array of biological responses, including regulation of apoptosis, cell survival, and differentiation (11)(12)(13)40). Interestingly, p38 has previously been shown to mediate both proapoptotic/ growth inhibitory (41)(42)(43)(44) and antiapoptotic/progrowth (45)(46)(47) signals in different systems, apparently depending on the stimulus and cell type involved. Although the precise mechanisms that account for the generation of such differential responses are unknown, one possibility is that they depend on the variable use of distinct isoforms, as there is evidence that different p38 isotypes have common and opposing effects (47). Our data also establish that in different leukemia cell types, inhibition of p38 activation results in enhanced As 2 O 3 -dependent JNK kinase activity. Interestingly, previous studies had shown that phosphorylation/activation of JNK by stress-inducing stimuli, such as lipopolysaccharide and sorbitol, is also enhanced by the p38a/h inhibitor SB203580 (48). Taken together with other studies that have shown that activation of the Mkk4/JNK cascade is essential for the generation of As 2 O 3dependent apoptosis (27), our findings suggest that a major mechanism by which p38 may impede induction of apoptosis in response to As 2 O 3 may involve direct or indirect inhibition of JNK activation.
Our findings also show that pharmacologic inhibition of p38 enhances arsenic-induced Bcl-2 down-regulation and loss of mitochondrial membrane potential in NB-4 APL cells, suggesting that, in this system, p38 up-regulates Bcl-2 expression and generation of antiapoptotic responses. Interestingly, previous studies have shown that sequential activation of Mkk6/p38h/ MAPK-activated protein kinase 3 regulates insulin-like growth factor I-dependent transcriptional induction of Bcl-2 by the nuclear transcription factor cyclic AMP response element-binding protein (45). Other studies have also shown that antisense oligodeoxynucleotides to Bcl-2 increase sensitivity of leukemic cells to As 2 O 3 (49). Moreover, arsenic-inducible voltage-dependent anion channel homodimerization, which controls mitochondria membrane potential reduction and cytochrome c release, is blocked by Bcl-2 (50). Thus, it is conceivable that the enhanced As 2 O 3 -dependent apoptosis seen by pharmacologic inhibition of p38, or in Mkk3/6 and p38a knockout cells, reflects regulatory effects on Bcl-2 expression, but other mechanisms may be involved as well. Independently of the precise mechanisms involved, our findings have potential therapeutic implications. A major obstacle in the use of As 2 O 3 as a therapeutic agent in the treatment of leukemias and solid tumors is the relative resistance of malignant cells to its proapoptotic effects. Our data raise the possibility that combinations of As 2 O 3 with pharmacologic inhibitors of p38 may be one approach to overcome As 2 O 3 resistance and raise the prospect of clinical-translational development of such combinations for the treatment of leukemias. Figure 6. Pharmacologic inhibition of p38 enhances the suppressive effects of As 2 O 3 on leukemic CFU-GM progenitors from patients with CML. A, peripheral blood mononuclear cells from three CML patients were plated in a methylcellulose culture assay system with As 2 O 3 (1 Amol/L) in the presence or absence of SB203580 (10 Amol/L). Leukemic CFU-GM progenitor colonies were scored on day 14 of culture. Columns, mean percent control of CFU-GM colony numbers for untreated cells from three experiments using different patient samples; bars, SE values. B, bone marrow or peripheral blood mononuclear cells from four different patients with CML were plated in a methylcellulose culture assay system with As 2 O 3 (1 Amol/L) and SD-282 (400 nmol/L) or SCIO-469 (400 nmol/L) as indicated. Columns, mean percent control of CFU-GM colony numbers for untreated cells from four experiments using different patient samples; bars, SE. Paired t -test analysis of the combinations of As 2 O 3 + SD282 or As 2 O 3 + SCIO-469, as compared with As 2 O 3 alone (columns marked with asterisks), showed P values of 0.0061 and 0.0044, respectively.