Dose-dependent effects of selenite (Se4+) on arsenite (As3+)-induced apoptosis and differentiation in acute promyelocytic leukemia cells

To enhance the therapeutic effects and decrease the adverse effects of arsenic on the treatment of acute promyelocytic leukemia, we investigated the co-effects of selenite (Se4+) and arsenite (As3+) on the apoptosis and differentiation of NB4 cells and primary APL cells. A 1.0-μM concentration of Se4+ prevented the cells from undergoing As3+-induced apoptosis by inhibiting As3+ uptake, eliminating As3+-generated reactive oxygen species, and repressing the mitochondria-mediated intrinsic apoptosis pathway. However, 4.0 μM Se4+ exerted synergistic effects with As3+ on cell apoptosis by promoting As3+ uptake, downregulating nuclear factor-кB, and activating caspase-3. In addition to apoptosis, 1.0 and 3.2 μM Se4+ showed contrasting effects on As3+-induced differentiation in NB4 cells and primary APL cells. The 3.2 μM Se4+ enhanced As3+-induced differentiation by promoting the degradation of promyelocytic leukemia protein–retinoic acid receptor-α (PML–RARα) oncoprotein, but 1.0 μM Se4+ did not have this effect. Based on mechanistic studies, Se4+, which is similar to As3+, might bind directly to Zn2+-binding sites of the PML RING domain, thus controlling the fate of PML–RARα oncoprotein.

Acute promyelocytic leukemia (APL) is a subtype of human acute myeloid leukemia. 1 The promyelocytic leukemia protein-retinoic acid receptor-α (PML-RARα) fusion protein, which is generated from a specific chromosome translocation t(15;17)(q22;q21), is the key driver of APL leukemogenesis. 2 Arsenic trioxide (ATO), which has been successfully used in the treatment of APL, induces the catabolism of PML-RARα oncoprotein. 3 ATO is one of the primary therapeutic agents for APL, but organ toxicity, especially for the liver and kidney, causes excessive pain for patients. 4,5 Studies on the toxicity of arsenic suggest that ATO metabolism increases its toxicity because of oxidative damage and generation of more toxic metabolites, including monomethylarsonous acid and dimethylarsinous acid. [6][7][8][9] Thus, identifying new therapeutics to decrease the adverse effects of ATO is necessary.
ATO induces both apoptosis and differentiation in human APL cells. 10 Apoptosis is an ordered cascade of enzymatic events. 11 Studies on the mechanism of ATO-induced apoptosis in APL cells suggest that ATO promotes apoptosis through the mitochondria-mediated intrinsic pathway that is induced by oxidative stress and regulated by Bcl-2 family members. 10,12,13 ATO can also induce apoptosis by inhibiting the nuclear factor-кB (NF-кB) pathway that regulates the expression of various survival proteins. 14,15 In addition to apoptosis, ATO can induce the differentiation of APL cells by degrading the PML-RARα fusion protein and activating the retinoic acid signaling pathway. 10, 16 Zhang et al. 16 reported that ATO induced the degradations of PML and PML-RARα oncoprotein by directly binding to PML. PML is a zinc-finger protein with a Cys-rich motif that contains a RING domain. The PML RING domain (PML-R) contains two Zn 2+ -binding sites (ZFs) and requires Zn 2+ for autonomous folding. 17 The conserved Cys12, Cys29, and Cys32 residues in PML-R-ZF1, and Cys24, Cys40, and Cys43 residues in PML-R-ZF2 are the binding sites for trivalent arsenic. 16 Selenium is an essential nutrient element that shows chemopreventive effect and anticancer potential. 18 Li et al. 19 suggested that high dose (5.0-20 μM) of selenite (Se 4+ ) could induce the accumulation of reactive oxygen species (ROS) and the apoptosis of NB4 cells. Subsequently, Zuo et al. 20 and Guan et al. 21 confirmed that high concentrations of Se 4+ induced the apoptosis of NB4 cells through an ROS-mediated pathway. However, the accumulation of ROS could induce adverse effects to noncancer tissues by causing oxidative damages. 22 For cancer treatment, we attempt to increase the anticancer efficacy while decreasing the adverse effects. Thus far, few studies have investigated the effects of 2.0-4.0 μM Se 4+ on the apoptosis and differentiation of human APL cells. Selenium exerts its biological functions dose-dependently. 22 In addition, selenium has chemical properties and metabolic 1 fates similar to those of arsenic. In consideration of the typical characteristics of ATO in the treatment of APL, we hypothesized that 2.0-4.0 μM Se 4+ might induce some interesting changes in APL cells, such as differentiation and the degradation of PML-RARα.
Combination therapy is widely used in cancer treatment. The relationship between selenium and arsenic is complex. Selenium and arsenic act as metabolic and toxic antagonists. 23 Combining a low concentration of Se 4+ with ATO might decrease the toxicity and increase the curative potency of ATO in the treatment of APL. Thus, it is of great significance to evaluate the effects of combining selenium with arsenic on the apoptosis and differentiation of human APL cells.
In this study, we found dose-dependent contrasting effects of Se 4+ on arsenite (As 3+ )-induced apoptosis and differentiation in NB4 cells and primary APL cells. A 4.0-μM concentration of Se 4+ enhanced As 3+ -induced apoptosis through downregulation of NF-кB and activation of caspase-3, but 1.0 μM Se 4+ failed to elicit these effects. At 2.0-4.0 μM, Se 4+ induced cell differentiation and synergistically promoted As 3+ -induced cell differentiation. Mechanistic studies suggested that Se 4+ might bind directly to PML-R in the form of divalent selenium (Se 2+ ) to promote the degradation of PML-RARα oncoprotein.

Results
Effects of Se 4+ and As 3+ on the growth of NB4 cells. After 48 h of treatment, cells viability was determined by the Trypan blue exclusion test. 20 The viability of NB4 cells was 98%, and the viability of primary APL cells was 96%. The effects of As 3+ , Se 4+ , or their combination on the growth of NB4 cells and primary APL cells were determined by WST-1 cell proliferation assay (Figure 1). Se 4+ exerted dose-dependent effects on NB4 cell proliferation. Se 4+ at 4.0 μM significantly inhibited the growth of NB4 cells, but 1.0 μM Se 4+ did not have this effect. In addition, 1.0 μM Se 4+ markedly reduced the inhibitory effects of As 3+ on NB4 cell growth, whereas 4.0 μM Se 4+ enhanced the cell death induced by As 3+ (Figure 1a). The viability of primary APL cells (%) in response to As 3+ , Se 4+ , or their combination was also investigated. Similarly, 4.0 μM Se 4+ inhibited the proliferation of primary APL cells and enhanced As 3+ -induced cell death (Figure 1b).
Effects of Se 4+ on As 3+ -induced cell apoptosis. Concentrations of 1.0 and 4.0 μM Se 4+ were used to investigate the effects of Se 4+ on As 3+ -induced apoptosis in NB4 cells and primary APL cells. After 48 h of treatment, 2.0 μM As 3+ promoted the apoptosis of NB4 cells (Figure 2a). Compared with control, 1.0 μM Se 4+ decreased the percentage of apoptotic cells from 17.9 to 15.8%, but 4.0 μM Se 4+ increased the percentage from 17.9 to 49.0% (Figure 2a). Similar to the effects of Se 4+ on As 3+ -induced cell death, 1.0 μM Se 4+ inhibited As 3+ -induced apoptosis in NB4 cells, but 4.0 μM Se 4+ enhanced apoptosis (Figure 2a). We also investigated the effects of 1.0 and 4.0 μM Se 4+ on the apoptosis of primary APL cells (Figure 2b). The potency of Se 4+ and As 3+ in inducing primary APL cell apoptosis was low, but the characteristics were similar. At 1.0 μM, Se 4+ inhibited As 3+ -induced apoptosis from 15.9 to 9.0%, whereas 4.0 μM Se 4+ enhanced As 3+ -induced apoptosis from 15.9 to 19.5% (Figure 2b). 4+ and As 3+ on cell cycle distribution. The cell cycle is a highly regulated event that controls the growth and differentiation of cells. 24,25 Changes in cell cycle distribution might be associated with the apoptosis and differentiation of NB4 cells. Thus, we analyzed the effects of As 3+ and Se 4+ on the cell cycle distribution (   (Figures 3a-c). Similarly, 1.0 μM Se 4+ decreased the proportion of SubG 1 cells induced by As 3+ from 29.04 to 11.26%, but 4.0 μM Se 4+ increased this proportion from 29.04 to 95.06% (Figures 3d-f). Compared with the As 3+ -treated group, low concentrations (1.0 and 4.0 μM) of Se 4+ enhanced the inhibition of As 3+ in the G 1 /S transition (Figures 3d-f).

Effects of Se
Effects of Se 4+ on arsenic uptake. Inductively coupled plasma-mass spectrometry (ICP-MS) was used to detect arsenic in NB4 cells. 26,27 As shown in Figure 4, 1.0 and 4.0 μM Se 4+ inhibited and promoted the uptake of As 3+ respectively, and this result can explain the dose-dependent contrasting effects of these concentrations of Se 4+ on As 3+induced apoptosis.  (Figure 5a). However, As 3+ significantly increased cellular ROS after 36 h of treatment. Adding Se 4+ eliminated the ROS generated by As 3+ (Figure 5b). The expression of heme oxygenase-1 (HMOX1), a key oxidative stress response enzyme that is upregulated in the presence of elevated ROS, was analyzed by RT-PCR ( Figure 5c). 29,30 As 3+ upregulated the expression of HMOX1, but Se 4+ alone had no significant effect. Furthermore, Se 4+ inhibited the upregulation of HMOX1 induced by As 3+ (Figure 5d). 4+ and As 3+ on the expression of apoptotic factors. We analyzed the expression of key apoptotic factors by RT-PCR and western blot ( Figure 6). As 3+ upregulated the Bax pro-apoptotic factor and downregulated the Bcl-2 antiapoptotic factor at both gene and protein levels (Figures 6a and b). Se 4+ (1.0 and 4.0 μM) did not obviously regulate the expression of Bcl-2, but it downregulated the expression of Bax. Moreover, Se 4+ (1.0 and 4.0 μM) showed antagonistic effects with As 3+ on the regulation of Bax and Bcl-2 ( Figures 6c and d). The results suggested that Se 4+ inhibited the mitochondria-mediated apoptosis. 13 To clarify the mechanism of Se 4+ -induced apoptosis, we studied the effects of Se 4+ on the expression of NF-кB and caspase-3 ( Figure 6). Se 4+ (1.0 and 4.0 μM) obviously downregulated the expression of NF-кB. Se 4+ at 4.0 μM induced the activation of caspase-3, but 1.0 μM Se 4+ did not have this effect. Moreover, we found that 4.0 μM Se 3+ enhanced the regulation of As 3+ in the expression of NF-кB and caspase-3 ( Figure 6).    Interactions between Se 4+ and PML-R. To investigate whether Se 4+ promotes the catabolism of PML-RARα oncoprotein by directly interacting with PML-R, we analyzed the interactions between Se 4+ and PML-R. The intrinsic ultraviolet-visible (UV-vis) absorption peak of PML-R at 280 nm is primarily caused by Trp47, and the intensity of this peak can indicate perturbation of the microenvironment around Trp47. 17,31 After incubation with Se 4+ for 15 min, the intensity of the 280 nm peak was increased. Compared with Zn 2+ and As 3+ , Se 4+ increased the intensity at 280 nm more obviously (Figure 8a). The conformational changes of PML-R were also detected by circular dichroism (CD). 27 The conformation of the PML-R zinc-finger domain was disordered. 17 Zn 2+ induced PML-R folding to a stable structure (Figure 8b). Similarly, Se 4+ and As 3+ promoted the folding of PML-R ( Figure 8b). Compared with Zn 2+ and As 3+ , Se 4+ evidently increased the β-pleated sheet of PML-R (Figures 8b and c). As evidenced by the hydrodynamic radius (R H ) of PML-R analyzed by dynamic light scattering (DLS), PML-R was in an unfolded state. Se 4+ induced the folding of PML-R, 32 because R H was decreased from 9.1 to 4.3 nm (Figure 8d). Changes in the maximum emission wavelength and intensity of the synchronous fluorescence spectrum (Δλ = 60 nm) can reflect the microenvironment around Trp47 for PML-R. 17,33 Se 4+ dramatically decreased the synchronous fluorescence intensity of PML-R at 285 nm with an increasing mole ratio, but Zn 2+ and As 3+ slightly decreased this fluorescence (Figure 8e). Spectrographic analysis suggested that Se 4+ and As 3+ promoted the folding of the PML-R zinc-finger domain. Differently, Se 4+ affected the conformation of PML-R more remarkably. Zhang et al. 16 reported that Cys residues in PML-R-ZFs were involved in the binding of As 3+ . To determine the mechanism of Se 4+ binding, the effects of Se 4+ on thiol groups of PML-R were analyzed by the method of Ellman. 27,31 A total of 4.76 (thiol group/mol of protein) thiol groups were detected in PML-R (Figure 8f). Unlike Zn 2+ and As 3+ , Se 4+ dramatically eliminated the thiol groups in PML-R, indicating stronger coordination with Cys residues (Figure 8f). Subsequently, the Cys residues involved in selenium binding were detected by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF)-MS. After being incubated with Zn 2+ , As 3+ , or Se 4+ and then alkylated with iodoacetamide (IA), PML-R was digested by trypsin for analysis. The peaks corresponding to Cys9 (IA-modified) and Cys12 (IA-modified) were detected (Figure 8g). Compared with PML-R, adding Zn 2+ , As 3+ , or Se 4+ decreased the intensities of these two peaks (Figures 8g-j).

Discussion
The chemical properties and metabolic fates of selenium are similar to those of arsenic. 23 Uniquely, selenium is an essential nutrient element that shows lower genotoxicity, cytotoxicity, and oxidative toxicity. 18 As ATO has been successfully used in the treatment of APL, combination therapy has been advocated. 34 Arsenic generates ROS and induces oxidative damage that limits its application in the treatment of diseases, including cancers. 6,35 However, low concentrations of selenium can eliminate ROS and protect organisms. 18 In consideration of the complicated interaction between selenium and arsenic, 23 the co-effects of low doses of Se 4+ and As 3+ on the apoptosis and differentiation of APL cells were evaluated.
Bcl-2, Bax, NF-кB, and caspase-3, which play key roles in ATO-induced cell apoptosis, 10,14,15 were selected to study the mechanism for the apoptosis of NB4 cells induced by As 3+ (2.0 μM), Se 4+ (1.0 and 4.0 μM), or a combination of As 3+ and Se 4+ . As 3+ arrested the G 1 /S transition, increased the cellular ROS, upregulated the Bax pro-apoptotic factor, downregulated the Bcl-2 and NF-кB anti-apoptotic factors, and activated caspase-3. These changes of NB4 cells suggested that As 3+ induced the apoptosis of NB4 cells by promoting the mitochondria-mediated intrinsic pathway and inhibiting the NF-кB pathway. 10,[13][14][15] Concentrations of Se 4+ greater than 2.0 μM inhibit the growth of NB4 cells. 19 However, when the concentration of selenium increased to a certain extent, it will induce the accumulation of ROS in vivo and lead to adverse effects. 22 In this work, 1.0-4.0 μM Se 4+ inhibited the generation of cellular ROS and blocked mitochondria-mediated apoptosis by downregulating the expression of Bax and upregulating the expression of Bcl-2. However, Se 4+ arrested the cell cycle at G 0 /G 1 , downregulated the expression of NF-кB, and induced the activation of caspase-3 to promote NB4 cell apoptosis. Because the promoting effects exceeded the inhibitory effects, 4.0 μM Se 4+ induced the apoptosis of NB4 cells. At 1.0 μM, Se 4+ inhibited As 3+ uptake, eliminated As 3+ -generated ROS, and prevented the cells from undergoing As 3+ -induced apoptosis by increasing the expression of Bcl-2. Contrarily, 4.0 μM Se 4+ enhanced As 3+ -induced apoptosis and arrested the cell cycle at the G 0 /G 1 phase. On one hand, 4.0 μM Se 4+ promoted the uptake of As 3+ and enhanced the downregulation of NF-кB and the activation of caspase-3 that were induced by As 3+ . On the other hand, 4.0 μM Se 4+ inhibited the apoptosis of NB4 cells by eliminating As 3+ -generated ROS and inhibiting the regulatory effects of As 3+ toward the Bax pro-apoptotic factor and the Bcl-2 antiapoptotic factor. In general, As 3+ -induced apoptosis was more prone to promotion by 4.0 μM Se 4+ than to inhibition.
In addition to apoptosis, 2.0-4.0 μM Se 4+ induced the differentiation of NB4 cells and primary APL cells. The combination of 3.2 μM Se 4+ and 0.4 μM As 3+ enhanced the differentiation of NB4 cells. The PML-RARα fusion protein is the key driver of APL leukemogenesis and the target of ATO. 2 The differentiation of human APL cells induced by ATO is related to the degradation of PML-RARα fusion protein. 16 In consideration of the similarity between arsenic and selenium, we hypothesized that Se 4+ -induced differentiation of NB4 cells and primary APL cells might be related to the degradation of PML-RARα fusion protein. The results of western blot confirmed the hypothesis that Se 4+ caused the decomposition of PML-RARα oncoprotein in both NB4 cells and primary APL cells.
The Cys-rich zinc-finger domain of PML-R is the binding domain of As 3+ . 16 Similar to As 3+ , Se 4+ was readily bound to thiol groups in vitro. 36 To investigate whether Se 4+ directly binds to PML-R, the interactions between Se 4+ and PML-R were studied. The zinc-finger domain of PML-R contains two PML-R-ZFs. 17 The spectroscopic data herein showed that Zn 2+ , As 3+ , and Se 4+ induced the folding of PML-R and exposed the residues of Cys24, His26, Cys40, and Cys43 near Trp47 in PML-R-ZF2. Compared with Zn 2+ and As 3+ , Se 4+ induced conformational changes of PML-R evidently and uniquely. In addition, more thiol groups in PML-R were involved in the binding of Se 4+ . GSSeSG is another selenium substrate for proteins that formed by the reduction of Se 4+ with glutathione. 36,37 Shi et al. 35 reported that Se 2+ was the terminal form that bound to Cys-rich proteins. In the binding reaction, one Se 4+ needed four Cys residues. Two Cys residues were oxidized to a Cys-Cys pair in the process of reducing Se 4+ , whereas the other two bound to the reduced Se 2+ in the form of RSSeSR. 35 The in vitro experiments on the interaction between Se 4+ and PML-R suggested that Se 4+ might be reduced to Se 2+ that then bound PML-R. The large conformational changes of PML-R might be ascribed to the formation of disulfide bonds. Moreover, MALDI-TOF-MS spectra showed that Cys9 and Cys12 at PLM-R-ZR1 were involved in the binding of Se 4+ . Therefore, Se 2+ might be the form of selenium that promoted the in vivo degradation of PML-RARα fusion protein by directly binding to PML-R-ZFs.
In summary, the mechanism for the effects of Se 4+ on As 3+ -induced apoptosis and differentiation in NB4 cells and primary APL cells was postulated. As shown in Figure 9, Se 4+ at low concentrations (1.0 and 4.0 μM) showed contrasting effects on As 3+ -induced apoptosis in NB4 cells and primary APL cells. On one hand, Se 4+ (1.0 and 4.0 μM) inhibited the mitochondria-mediated intrinsic apoptosis by eliminating As 3+ -generated ROS. On the other hand, Se 4+ (4.0 μM) promoted As 3+ -induced apoptosis by facilitating the downregulation of NF-ĸB and the activation of caspase-3. The effects of Se 4+ on As 3+ -induced differentiation in NB4 cells and primary APL cells were caused by the degradation of PML-RARα oncoprotein. Thus, Se 4+ , which is similar to As 3+ , might directly bind to PML-R in the form of Se 2+ to control the fate of PML-RARα fusion protein. In the meantime, Cys9 and Cys12 in PML-R-ZF1 are involved in the binding reaction.
Cell culture and cell viability assay. Human NB4 leukemia cells used in the experiments were purchased from SXBIO Biotech, Shanghai, China). NB4 cells were cultured in RPMI-1640 (KeyGEN Biotech, Nanjing, China) with 10% fetal bovine serum (FBS) at 37°C under a 5% CO 2 atmosphere. After informed consent, the bone marrow of two primary APL patients was acquired from DrumTower Hospital (Nanjing, China). Human primary APL cells were separated from the bone marrow by traditional Ficoll-Hypaque density centrifugation. 38 The fresh primary APL cells were cultured in RPMI-1640 (KeyGEN Biotech) with 15% FBS at 37°C under a 5% CO 2 atmosphere. The viability of NB4 cells and primary APL cells were determined by the Trypan blue exclusion method. 20 After culturing for 24, 48, and 96 h, cells were collected and mixed with equal volume of PBS containing 0.4% Trypan blue dye. Cell viability was calculated as the number of viable cells divided by the total number of cells with the grids on the hemacytometer. The effects of Se 4+ and As 3+ on cell growth were measured with the WST-1 cell proliferation assay kit according to the manufacturer's protocols (KeyGEN Biotech). 39 The cells were seeded at 4 × 10 4 cells/ml in a 96-well culture plate and then exposed to various concentrations of As 3+ , Se 4+ , or their combination for 48 h. Untreated cells served as controls.
Preparation of PML-R. The gene for PML-R was synthesized from Invitrogen Life Technologies (Carlsbad, CA, USA) and cloned into the NcoI-BamHI restriction sites of the pET-28a vector (Novagen, San Diego, CA, USA). The expression plasmid pET-28a-PML-R was confirmed by DNA sequencing. E. coli BL21 (DE3) pLysS was transformed by the ligated plasmid by heat shock at 42°C for 30 s. After being selected on standard kanamycin-containing agar plates, the colonies were expressed at 22°C for 10 h. The separation and purification of PML-R was similar to our previous work. 40 The concentration of PML-R was determined by a Bradford assay based on a BSA standard curve. 31 Analysis of apoptotic cells by flow cytometry. NB4 cells and primary APL cells were treated with As 3+ (2.0 μM), Se 4+ (1.0 or 4.0 μM), or their combination for 48 h. After treatment, the cells were collected, washed twice with Ca 2+ -free PBS, and stained with Annexin V-FITC and propidium iodide (PI). After double staining, the cells were recorded on a BD LSRL Fortessa flow cytometer (Franklin Lakes, NJ, USA). 39 The percentages of apoptotic cells were calculated by BD FACSDiva software (Franklin Lakes, NJ, USA). RT-PCR analysis. Total RNA was extracted from NB4 cells by RNAiso Plus (Takara, Dalian, China). Isolated total RNA (2 μg) was used to perform the reverse transcription with the PrimeScript RT reagent kit (Takara) according to the manufacturer's protocols. 39 The transcribed cDNA (2 μl) was used for PCR amplification with specific primers for HMOX1, caspase-3, Bax, Bcl-2, NF-кB, and β-Actin genes. Thirty cycles were performed under the following conditions: 30 s denaturation at 94°C, 30 s annealing at 52°C (β-Actin), 57°C (HMOX1, Bax, Bcl-2, NF-кB), or 51°C (caspase-3), and 30 s extension at 72°C. The PCR products were separated on 1% agarose gels containing ethidium bromide. The separated bands were photographed on a Gel Doc XR System (Bio-Rad, Hercules, CA, USA). Primer sequences are shown in Table 1.

Figure 9
Mechanism for the effects of Se 4+ (1.0 and 4.0 μM) on As 3+ -induced apoptosis and differentiation in NB4 cells and primary APL cells. Se 4+ promotes the degradation of PML-RARα fusion protein by directly binding to the PML-R-ZFs. The decomposition of PML-RARα oncoprotein contributes to the differentiation of NB4 cells and primary APL cells. On one hand, Se 4+ (4.0 μM) prevents the cells from undergoing As 3+ -induced apoptosis by eliminating ROS, downregulating the Bax proapoptotic factor, and upregulating the Bcl-2 anti-apoptotic factor. On the other hand, Se 4+ enhances As 3+ -induced apoptosis by downregulating NF-ĸB and activating caspase-3 Western blot analysis. Total protein in NB4 cells and primary APL cells was extracted by ice-cold RIPA cell lysis buffer (Beyotime). The concentration of proteins was determined by a BCA protein quantification kit (Beyotime). Total protein (30 μg) from each sample was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane (Millipore, Billerica, MA, USA). After blocking in 5% skim milk at room temperature for 1 h, the membrane was sequentially incubated with primary and secondary antibodies. Proteins on the PVDF membrane were visualized using chemiluminescent HRP substrate (Millipore). 39 The intensities of the bands were normalized to that of β-Actin. All the experiments were repeated three or more times.
Spectrographic analysis of the interactions between Se 4+ /As 3+ /Zn 2+ and PML-R. Zn 2+ , As 3+ , and Se 4+ were each incubated with PML-R at room temperature for 15 min before spectrographic analysis. UV-vis spectra of PML-R were recorded on a Perkin Elmer Lambda-35 spectrophotometer (Waltham, MA, USA). 31 CD spectra were measured by a JASCO-J810 spectropolarimeter (Jasco Co., Tokyo, Japan). 27 The molar ratio of metal ion to PML-R was 2 : 1. Synchronous fluorescence spectra (Δλ = 60 nm) were recorded on a 48 000 DSCF time-resolved fluorescence spectrometer (SLM Co., Sunnyvale, CA, USA) equipped with 1.0 cm quartz cell. 27,33 The molar ratios of ion to PML-R were increased from 0 : 1 to 10 : 1. All spectra were obtained at room temperature.
Cysteine residues in PML-R analyzed by MALDI-TOF-MS. Before analysis, the mixtures (0.5 ml) containing 20 μM purified PML-R and 200 μM ion (Zn 2+ , As 3+ , or Se 4+ ) were incubated at 37°C for 2 h. Then, Zn 2+ -, As 3+ -, or Se 4+ -containing solutions of PML-R were mixed with 50 mM IA in the dark at room temperature for 1 h. The residual IA and ions were washed out by NH 4 HCO 3 (50 mM) using a centrifugal filter (3 kDa). The concentration of deionized PML-R was determined by the method of Bradford. The deionized PML-R was incubated with 5% trypsin overnight at 37°C. Digested peptide fragments were dissolved in 60% acetonitrile and 0.1% (v/v) trifluoroacetic acid, lyophilized, and stored at − 80°C for analysis. 27,41 Before MS analysis, the lyophilized sample was dissolved in 10 μl H 2 O and mixed with 10 μl α-cyano-4-hydroxycinnamic acid matrix solution. The mixture was dried by spotting on a polished steel target plate. The masses of peptide fragments ware recorded on an AUTOFLEX II MALDI-TOF mass spectrometer (Bruker, Madison, WI, USA) with a 20-kV acceleration voltage. 40,41 Statistical analysis. Two-tailed Student's t-tests were performed for comparisons of two groups. A P-value of o0.05 was considered statistically significant.