Arsenic Trioxide Promotes Histone H3 Phosphoacetylation at the Chromatin of CASPASE-10 in Acute Promyelocytic Leukemia Cells*

Arsenic trioxide (As2O3) is highly effective for the treatment of acute promyelocytic leukemia, even in patients who are unresponsive to all-trans-retinoic acid therapy. As2O3 is believed to function primarily by promoting apoptosis, but the underlying molecular mechanisms remain largely unknown. In this report, using cDNA arrays, we have examined the changes in gene expression profiles triggered by clinically achievable doses of As2O3 in acute promyelocytic leukemia NB4 cells. CASPASE-10 expression was found to be potently induced by As2O3. Accordingly, caspase-10 activity also substantially increased in response to As2O3 treatment. A selective inhibitor of caspase-10, Z-AEVD-FMK, effectively blocked caspase-3 activation and significantly attenuated As2O3-triggered apoptosis. Interestingly, the treatment of NB4 cells with As2O3 markedly increased histone H3 phosphorylation at serine 10, an event that is associated with acetylation of the lysine 14 residue. Chromatin immunoprecipitation assays revealed that As2O3 potently enhances histone H3 phosphoacetylation at the CASPASE-10 locus. These results suggest that the effect of As2O3 on histone H3 phosphoacetylation at the CASPASE-10 gene may play an important role in the induction of apoptosis and thus contribute to its therapeutic effects on acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) 1 accounts for ϳ10 -15% of adult myeloid leukemias with 3,500 -5,500 new cases diagnosed annually (1,2). The vast majority of APL patients harbor the chromosomal translocation t(15,17)(q22;q21) involving the retinoic acid receptor ␣ (RAR␣) gene on chromosome 15 and the promyelocytic leukemia (PML) gene on chromosome 17, generally giving rise to two fusion genes, PML-RAR␣ and RAR␣-PML (1,3). Studies using transgenic mice have demonstrated that the protein product of the PML-RAR␣ fusion gene is primarily responsible for the leukemogenic property of this characteristic translocation (4,5). All-trans-retinoic acid (ATRA), a physiologically active derivative of vitamin A, can induce complete remission in most APL patients associated with an enhancement of differentiation pathways (6). Recent studies (7)(8)(9) have provided strong evidence that the induction of leukemia by the PML-RAR␣ protein relies on its ability to repress gene transcription by recruiting transcription repressor complexes. Pharmacological doses of ATRA stimulate the release of the transcription repressor complexes from PML-RAR␣, thereby activating the transcription of genes critical for normal granulocytic differentiation. However, ATRA is not curative, and resistance rapidly develops usually within 10 months of therapy (2,6). Therefore, alternative therapies are necessary.
Recently, As 2 O 3 was identified as a potent anti-leukemic agent for treating not only newly diagnosed but also relapsed APL patients (2, 10 -13), and it is remarkably effective in ATRA-refractory patients (2,12). However, the mechanisms underlying its therapeutic effects are not well understood (2,11,13). As 2 O 3 induces a drastic reorganization of the nucleus characterized by restoration of intact PML nuclear bodies followed by a progressive degradation of the PML-RAR␣ fusion protein (14,15). Although As 2 O 3 induces partial differentiation of leukemic cells (13), the apoptosis-promoting effect of As 2 O 3 is believed to play a prominent role in the remission of APL as large numbers of apoptotic myelocytes have been documented in bone marrow and peripheral blood of As 2 O 3 -treated APL patients (2,11,13). As 2 O 3 has been shown to trigger the early collapse of mitochondrial transmembrane potential (16,17), the release of cytochrome c (10), and the activation of caspases-1, -2, -3, and -8 (2,16,18).
To elucidate the key molecular events involved in mediating As 2 O 3 -induced apoptosis, we have employed the APL-derived NB4 cell line exposed to clinically relevant concentrations of As 2 O 3 and analyzed the changes in gene expression profiles using cDNA array. CASPASE-10 was among the genes upregulated with this treatment. Interestingly, treatment with As 2 O 3 also resulted in a significant increase in histone H3 phosphoacetylation, an event that is associated with altered gene transcription (19 -21). Importantly, selective inhibition of caspase-10 substantially abolished As 2 O 3 -triggered caspase-3 activation and significantly reduced As 2 O 3 -induced apoptosis. Chromatin immunoprecipitation (ChIP) assays revealed that As 2 O 3 potently enhances histone H3 phosphoacetylation at the CASPASE-10 locus. These results suggest that As 2 O 3 -induced histone H3 phosphoacetylation may be implicated in altering gene transcription profile and apoptosis and play an important role in the therapeutic effect of As 2 O 3 on APL.
FMK was from Enzyme Systems (Livermore, CA). Flow cytometry and estimation of sub-G 1 apoptotic populations were performed as described previously (22). Where indicated, percentages of dead cells were assessed by direct cell counting after trypan blue staining using a hemacytometer. For caspase activity determinations, 100-g aliquots of cell lysate were prepared and used following the manufacturers' recommendations to assay caspase-2 (BioVision, Mountain View, CA), caspase-3 (Molecular Probes, Eugene, OR), or caspase-10 activity (R&D, Minneapolis, MN) by fluorometry.
Caspases-2, -3, and DNA fragmentation factor-45 were detected by Western blot analysis using monoclonal antibodies (Transduction Laboratories, Lexington, KY). Caspase-8 was detected by Western blotting using a rabbit polyclonal antibody (BD Biosciences). Active caspase-3 was detected by Western blotting using a rabbit polyclonal antibody that recognizes the active/cleaved caspase-3 (Asp-175) (Cell Signaling, Beverly, MA). Modified histone H3 was detected by Western blotting as described previously (23). Crude histone proteins were extracted as described previously (23) and separated through electrophoresis using 10% NuPAGE gels and MES buffer (Invitrogen). Phosphorylation of histone H3 at serine 10 was detected by Western blotting using an antibody specific for Ser-10-phosphorylated histone H3 (Cell Signaling). Histone H3 phosphoacetylation was detected by Western blotting using an antibody, which is specific for histone H3 phosphorylated at Ser-10 and acetylated at Lys-14 (Clontech). Histone H3 hyperacetylation was detected by Western blotting using an antibody specific for histone H3 that is acetylated at both Lys-9 and Lys-14 (Clontech).
cDNA Array-DNase-treated total RNA (5 g), prepared from either untreated or As 2 O 3 -treated cells, was used to synthesize cDNA probes using reverse transcriptase, [␣-33 P]dATP, and a specific CDS primer mix for genes represented on the Atlas human cancer 1.2 array (Clontech) containing 1,176 human cDNAs. Arrays were hybridized and washed according to the manufacturer's instructions and were visualized and quantitated using a PhosphorImager. Signal intensities were normalized to the signals of housekeeping genes in each membrane, and simple ratios (treated relative to untreated) were calculated. The array analysis was performed at least twice for each time point.
ChIP and Real-time PCR-ChIP was performed using 1.5ϫ10 8 cells as described previously (23). Ten percent of the chromatin solution was used for isolating input DNA, and the remainder was used for performing ChIP assays. ChIP procedures were carried out either using 10 l of rabbit pre-immune serum or 100 l of purified antibody specifically recognizing phosphoacetyl-histone H3 (p-Ser-10/Ac-Lys-14) (Clontech). Immunoprecipitated DNA was heated at 65°C for Ն6 h to reverse the formaldehyde cross-links, extracted with phenol/chloroform, precipitated, and dissolved in water. Real-time PCR was performed using SYBR Green PCR Master Mix and GeneAmp 5700 Sequencing Detection System (Applied Biosystems, Foster City, CA). Primers 5Ј-TG-CTTACCAGCGGCTACAC-3Ј and 5Ј-CCCTCCCTACCCCAGAAAC-3Ј were used to amplify the CASPASE-10 genomic DNA spanning a 151-bp region in the second intron of CASPASE-10 (24). Human ␤-GLOBIN was amplified using primers 5Ј-GGCAAGGTGAACGTGG-ATGAAGTTGGTG-3Ј and 5Ј-GGAGTGGACAGATCCCCAAAGGACTC-AAAG-3Ј, which span a 234-bp region from the first to the second exon of the human ␤-GLOBIN gene (25). PCR amplification was carried out using these parameters: 10 min at 95°C followed by 45 cycles of denaturation (95°C, 15 s), annealing (60°C, 30 s), and extension (72°C, 30 s). Upon the completion of the PCR reactions, PCR products were resolved on 2% agarose gels and visualized under UV light to confirm the quality of the PCR amplification.

As 2 O 3 Stimulates Apoptosis and Induces CASPASE-10 Expression in NB4
Cells-Treatment of NB4 cells with As 2 O 3 led to a time-dependent cleavage of caspase-2 and caspase-3 as indicated by the progressive decrease in the unprocessed caspases. However, it did not affect the expression of another apoptosis-related protein, DNA fragmentation factor-45 (Fig.  1A). Confirming the previous report that As 2 O 3 activates caspase-3 in APL cells in vivo (12), we observed a substantial increase in caspase-3 activity after 24 h in As 2 O 3 -treated NB4 cells. The activity of caspase-3 reached its maximal levels at 48 -72 h (Fig. 1B). In addition, treatment with As 2 O 3 caused the appearance of an apoptotic sub-G 1 population (Fig. 1C) and a marked increase in cell death (Fig. 1D). It has been demonstrated that that As 2 O 3 at concentrations above 0.5 M induces apoptosis in APL cells (10). Consistent with this report, we also observed a substantial increase in caspase-3 activity in cells treated with 0.8 and 1.6 M As 2 O 3 but not in cells treated with lower doses of As 2 O 3 (0.2 and 0.4 M). The increase was detected by both Western blotting using an antibody specific for the active/cleaved caspase-3 ( Fig. 2A) and fluorometric assays (Fig. 2B). Similarly, a significant increase in caspase-2 activity was detected by fluorometric assays in cells treated with 0.8 and 1.6 M As 2 O 3 but not in cells treated with lower doses (0.2 and 0.4 M) (Fig. 2C). The substantial increases in caspase-2 and caspase-3 activity in cells treated with 0.8 and 1.6 M As 2 O 3 were reflected in a significant increase in trypan bluepositive cells (Fig. 2D). It has been reported that in patients receiving daily intravenous As 2 O 3 infusion, the plasma As 2 O 3 concentration stays within a range between 0.5 and 3 M for the most time with a peak level between 4.2 and 6.7 M shortly after infusion (10). Based on this report, we chose a highly effective and clinically relevant dose of 1.6 M for most of the subsequent analyses.
To understand the molecular basis for the As 2 O 3 -induced apoptosis, we performed a large-scale gene expression analysis to identify genes regulated by As 2 O 3 . Total RNA was isolated from either untreated or As 2 O 3 -treated cells, and radiolabeled reverse transcripts were used to hybridize cDNA arrays containing 1,176 human gene transcripts closely related to a variety of cancers (Atlas Human Cancer Arrays, Clontech). As 2 O 3 consistently caused a Ն2-fold change in the expression of 25 genes on the DNA array, representing ϳ2% of genes analyzed. Among the most interesting genes up-regulated by As 2 O 3 was CASPASE-10 ( Fig. 3A), which has been shown to play an important role in apoptosis (26,27).
To further characterize the effect of As 2 O 3 on CASPASE-10 expression, Northern blot analysis was performed. Consistent with the cDNA array results, CASPASE-10 mRNA levels increased markedly with the As 2 O 3 treatment, reaching its highest expression (ϳ7-fold) after 12 h of treatment and gradually declining thereafter (Fig. 3B).
The effect of As 2 O 3 on caspase-10 activity was examined using fluorometric assays. As 2 O 3 treatment resulted in a timedependent increase in caspase-10 activity, which peaked between 48 and 72 h after the addition of As 2 O 3 to the medium, and decreased abruptly thereafter, possibly because of protein degradation during the late stages of apoptosis (Fig. 4A). The activation of caspase-10 by As 2 O 3 was dose-dependent. Significant activation of caspase-10 only occurred in cells treated with 0.8 and 1.6 M As 2 O 3 (Fig. 4B). Importantly, treatment with the caspase-10 inhibitor Z-AEVD-FMK led to a significant reduction of the apoptosis triggered by As 2 O 3 as indicated by the considerable decrease in cell death (Fig. 4C). These observations demonstrate the important role of caspase-10 in As 2 O 3mediated cytotoxicity.
To further address the role of caspase-10 in the apoptosis cascade, the effect of the selective caspase-10 inhibitor Z-AEVD-FMK on caspase-3 activation was examined (Fig. 5). In cells treated with As 2 O 3 , an ϳ20-fold increase in caspase-3 activity was detected after 48 h using fluorometric assay. Importantly, the caspase-10 inhibitor Z-AEVD-FMK at a very low concentration of 2 M substantially attenuated the As 2 O 3 -induced caspase-3 activation, decreasing caspase-3 activity by 75% at the 48-h time point (Fig. 5A). The partial inhibition of caspase-3 by Z-AEVD-FMK was confirmed by Western blot analysis using an antibody that specifically recognizes the active/cleaved caspase-3 (Fig. 5B). It is worth noting that although higher concentrations of Z-AEVD-FMK further decreased caspase-3 activity, even at a high concentration of 50 M, the caspase-10 inhibitor was unable to completely abolish the As 2 O 3 -induced caspase-3 activation (Fig. 5C). Likewise, the high concentrations of Z-AEVD-FMK (50 M) only partially blocked the apoptotic process triggered by As 2 O 3 (Fig. 5D). These observations suggest that in addition to caspase-10, other early caspase(s) may also contribute to the activation of caspase-3 by As 2 O 3 . Indeed, a cleaved form of caspase-8 was detected by Western blotting in cells treated with As 2 O 3 (Fig.  5B), confirming the earlier report by Kitamura et al. (18) that caspase-8 is rapidly activated in response to As 2 O 3 treatment and plays a significant role in As 2 O 3 -induced apoptosis.
As 2 O 3 Induced Phosphorylation of Serine 10 and Acetylation of Lysine 14 on Histone H3-We have previously shown that arsenite-stimulated MAPK phosphatase-1 induction is associated with an increase of phosphoacetyl-histone H3 on its chromatin (23). To assess whether similar chromatin changes were implicated in the As 2 O 3 -mediated induction of apoptosis, we examined histone H3 modification in NB4 cells treated with different doses of As 2 O 3 using antibodies specific for various modified histone H3 molecules (Fig. 6A). As shown in Fig. 5A, lower doses of As 2 O 3 (0.2 and 0.4 M) did not significantly alter the level of either phospho-histone H3 (p-Ser-10) or phosphoacetyl-histone H3 (p-Ser-10/Ac-Lys-14). However, at higher doses (0.8 and 1.6 M), As 2 O 3 induced a significant increase in both the phospho-histone H3 (p-Ser10) and phosphoacetyl-histone H3 (p-Ser10/Ac-Lys14) levels. The effect of As 2 O 3 on histone H3 phosphoacetylation did not appear to be caused by a nonspecific inhibition of the histone deacetylase activity, because As 2 O 3 treatment did not result in a significant increase in either Lys-14-acetylated histone H3 or hyperacetylated histone H3 (Ac-Lys-9/Ac-Lys-14) (Fig. 6A). Histone H3 phosphorylation at Ser-10 and dual modification (phosphorylation at Ser-10 and acetylation at Lys-14) occurred with similar kinetics in As 2 O 3 -treated cells (Fig. 6B), supporting the notion that acetylation at the Lys-14 residue and phosphorylation at the Ser-10 residue are coupled events (19). Comparable loading of the crude histone proteins among the various samples was verified by Western blotting using a monoclonal antibody recognizing total histone H3.

As 2 O 3 Stimulates the Phosphoacetylation of Histone H3 at the Chromatin of the CASPASE-10
Locus-To examine whether histone H3 modification occurs in vivo at the chromatins of CASPASE-10, ChIP assays were performed using an antibody specifically against dually modified histone H3 (23). To ascertain that equal amounts of starting chromatin material were used, DNA amplifications were carried out by realtime PCR using input chromatin DNA (not subject to immunoprecipitation) from both untreated and As 2 O 3 -treated cells and a pair of primers specific for CASPASE-10 (Fig. 7A, left panel). The very similar CASPASE-10 amplification curves and C T values, which represent the number of PCR cycles required to reach a threshold set arbitrarily at 0.5, from both the untreated and As 2 O 3 -treated input DNA indicated that the starting chromatin materials used in the subsequent ChIP assays were comparable. To detect histone H3 phosphoacetylation, chromatin solutions were incubated with a rabbit polyclonal antibody specifically recognizing phosphoacetyl-histone H3 (phospho- Ser-10/Ac-Lys-14) and protein A. Genomic DNA present in the immunoprecipitates was extracted and analyzed by real-time PCR using primers specific for CASPASE-10 (Fig. 7A, right  panel). A substantial enhancement in histone H3 phosphoacetylation at the CASPASE-10 gene was detected after As 2 O 3 treatment. The C T value was reduced from 32.6 (untreated) to 28.7 (As 2 O 3 -treated), corresponding to a 15-fold increase in chromatin-bound phosphoacetyl-histone H3. The specificity of the PCR reaction was indicated by the presence of a single band of the anticipated size on agarose gel after separating the final PCR products (Fig. 7B, upper panel). To test the specificity of the immunoprecipitation assay procedures, mock ChIP assays were carried out using rabbit pre-immune serum. The CASPASE-10 sequence was virtually undetectable in the mock immunoprecipitates (Fig. 7B, upper panel). Importantly, As 2 O 3 appeared to stimulate histone H3 phosphoacetylation only at the chromatin of a small subset of genes 2 including CASPASE-10 (Fig. 7). Phosphoacetyl-histone H3 was absent at the transcriptionally inactive ␤-GLOBIN chromatin in both untreated and As 2 O 3 -treated cells (Fig. 7B, lower pan-el), further illustrating the specificity of such modification in As 2 O 3 -stimulated cells. DISCUSSION As 2 O 3 has been shown to constitute effective therapy for APL patients relapsing from or refractory to ATRA treatment (10, 13). As 2 O 3 and ATRA appear to exert their anti-leukemic effects through partially overlapping mechanisms including those leading to the restoration of granulocyte differentiation (6,10,12,13,28). However, As 2 O 3 and ATRA therapies exhibit important differences. For example, As 2 O 3 can induce complete remission in t(15,17)-positive APL patients, regardless of their sensitivity to ATRA. In addition, As 2 O 3 can induce a high rate of molecular conversion from positive to negative for t (15,17) translocation. This is indicative of the elimination of the neoplastic progenitors that is unusual after treatment with ATRA alone (10,12,13). Recent studies (2,12,13,29) have provided considerable support for the notion that the anti-leukemic effect of As 2 O 3 lies primarily in its ability to induce apoptosis. Both in vivo and in vitro, As 2 O 3 has been found to induce apoptosis of APL cells at clinically achievable concentrations (10 -13). Here, we have confirmed that As 2 O 3 -induced apoptosis is associated with activation of the late caspases includ- ing caspases-2 and -3 (Figs. 1, 2, and 5). Through the use of large-scale gene expression analysis, we have shown that As 2 O 3 treatment of NB4 cells rapidly induced the expression of caspase-10 (Fig. 3). The marked As 2 O 3 -triggered elevation in caspase-10 activity (Fig. 4) appeared to be critical for the activation of caspase-3 (Fig. 5). This is evidenced by the observation that the caspase-10-selective inhibitor Z-AEVD-FMK at a very low concentration (2 M) markedly attenuated As 2 O 3induced caspase-3 activation. Nevertheless, it is important to note that caspase-10 is probably only partially responsible for executing As 2 O 3 -induced apoptosis, because Z-AEVD-FMK even at a high concentration of 50 M only partially inhibited the apoptotic process (Figs. 4B and 5D). This observation is consistent with our results that Z-AEVD-FMK did not totally abolish caspase-3 activation, although it significantly delayed caspase-3 activation (Fig. 5). Indeed, it has been reported that caspase-8 is activated in APL cells in response to As 2 O 3 treatment and plays a role in As 2 O 3 -induced apoptosis (18). It is possible that both caspase-10 and the structurally related caspase-8 contribute to the activation of caspase-3. In the presence of Z-AEVD-FMK, caspase-10 activity was inhibited, whereas caspase-8 was still active, resulting in attenuated caspase-3 activation (Fig. 5, A-C) and decreased apoptosis (Figs. 4C and 5D).
A critical role for histone deacetylase-dependent transcriptional repression by PML-RAR␣ has recently been established in the pathogenesis of APL (7)(8)(9)30). Indeed, the stimulation of histone modification is emerging as a common property shared by many effective APL therapeutic regimens including ATRA and various histone deacetylase inhibitors (11,(31)(32)(33). Here, we have examined the effect of As 2 O 3 on histone H3 modification. Clinically relevant doses of As 2 O 3 were found to potently enhance histone H3 phosphorylation and phosphoacetylation (Fig. 6), modifications that are similar to those reported to occur following treatment with mitogens and stress agents (20,23,34). The role that histone H3 phosphorylation and acetylation plays in activating gene transcription has recently been documented (20,34,35). Here, we show that As 2 O 3 strongly increases histone H3 phosphoacetylation at the CASPASE-10 chromatin (Fig. 6). Thus, enhanced histone phosphoacetylation at the CASPASE-10 chromatin is likely to contribute to the observed increase in CASPASE-10 expression, caspase-10 activation, and subsequent apoptosis. As 2 O 3 may promote apoptosis through multiple mechanisms. Caspase-10 activation is probably only part of the apoptotic execution machinery, because As 2 O 3 causes Ն2-fold change in the expression of only ϳ2% of genes on the DNA array (25 genes of 1,176). In this regard, As 2 O 3 enhances gene expression in a relatively selective manner. This finding is consistent with our observation that neither As 2 O 3 significantly altered the levels of hyperacetylated (Ac-Lys-9/Ac-Lys-14) at the global levels (Fig. 5), nor did it induce histone H3 acetylation at the ␤-GLOBIN locus (Fig. 6B). Although it is unclear how the relative specificity of As 2 O 3 is achieved, we can speculate that it may involve the degradation of the PML-RAR␣ and a protein kinase that can phosphorylate histone H3 at Ser-10. Interestingly, it has been shown that As 2 O 3 induces the phosphorylation of the SMRT protein, the dissociation of SMRT from its nuclear receptor partner including PML-RAR␣, and the trans-FIG. 6. Histone H3 phosphorylation and acetylation induced by As 2 O 3 . NB4 cells were treated with As 2 O 3 . Crude histone proteins were extracted and separated on NuPAGE gel using MES buffer. Histone H3 modification was examined via Western blotting using antibodies specific for the modified histone H3. A, dose-response of histone H3 modification at 24 h. B, time course of histone H3 phosphorylation and phosphoacetylation (p-Ser-10/Ac-Lys-14) in NB4 cells treated with 1.6 M As 2 O 3 . Total histone H3 was detected using a histone H3 monoclonal antibody.
FIG. 7. Analysis of histone H3 phosphoacetylation at the chromatin of the CASPASE-10 gene. NB4 cells were treated with As 2 O 3 (1.6 M) for 48 h or left untreated, whereupon chromatin was prepared after cross-linking using formaldehyde. ChIP assays were performed using either rabbit pre-immune serum or an antibody specific for phosphoacetyl-histone H3. A, real-time PCR amplification of CASPASE-10 from template DNA isolated from the input chromatins (left graph) or from template DNA isolated from immunoprecipitated chromatins using an antibody against phosphoacetyl-histone H3 (p-Ser-10/Ac-Lys-14) (right graph). C T values representing the cycle number required to reach the threshold 0.5 are indicated in the graphs. B, final products of CASPASE-10 (upper panel) and ␤-GLOBIN (lower panel) obtained after the completion of the real-time PCR (45 cycles) using either Input DNA, ChIP DNA, or Mock ChIP DNA (no antibody) as templates. port of SMRT from the nucleus into the cytoplasm through a MAPK-mediated mechanism (36). Moreover, it has been reported that As 2 O 3 -induced apoptosis of NB4 cells is partially blocked by a pharmacological inhibitor of MAPK kinase; thus, further supporting a significant role of MAPK in the process (36). MAPK can activate two downstream kinases, MSK1 and p90 Rsk2 (37,38), which are shown to directly phosphorylate histone H3 at Ser-10. It has been shown that both epidermal growth factor and stress stimulate histone H3 phosphoacetylation through a MAPK-mediated pathway (19,23). Thus, one possible mechanism that As 2 O 3 induces apoptosis in APL cells is by promoting histone H3 phosphoacetylation and degradation of PML-RAR␣ at the promoters of ATRA-regulated genes through a process regulated at least in part by MAPKs. Further characterization of the mechanisms involved in this process may aid in the development of more effective less toxic therapeutic regimens for APL.