The cooperative interaction of two different signaling pathways in response to bufalin induces apoptosis in human leukemia U937 cells.

Bufalin, an active principle of Chinese medicine, chan'su, induced typical apoptosis in human leukemia U937 cells. When U937 cells were treated with 10(-8) M bufalin in the absence of serum, mitogen-activated protein (MAP) kinase activity was markedly increased 6 h after the start of treatment and elevated so for 12 h. Prior to the activation of MAP kinase, increased activities of Ras, Raf-1, and MAP kinase kinase were found, but these enzymes were transiently activated by the treatment with bufalin. These results suggest that the signal was transmitted sequentially from Ras, Raf-1, and MAP kinase kinase to MAP kinase. In association with this signal transduction, the concentration of cAMP in the cells decreased markedly, suggesting that Raf-1 was also activated by a decrease in the extent of phosphorylation by protein kinase A. In fact, pretreatment of U937 cells with forskolin and 3-isobutyl-1-methylxanthine, which are known to increase the concentration of cAMP in the cells, and subsequent treatment with bufalin resulted in a decrease in both Raf-1 activity and DNA fragmentation. To confirm the participation of MAP kinase in the apoptotic process, antisense cDNA for MAP kinase kinase 1 was expressed in U937 cells. The transformants were significantly resistant to both DNA fragmentation and cell death in response to bufalin. Our findings suggest that a pathway with the persistent activation of MAP kinase in U937 cells in response to bufalin is at least one of the signal transduction pathways involved in the induction of apoptosis.

Bufalin, an active principle of Chinese medicine, chan'su, induced typical apoptosis in human leukemia U937 cells. When U937 cells were treated with 10 ؊8 M bufalin in the absence of serum, mitogen-activated protein (MAP) kinase activity was markedly increased 6 h after the start of treatment and elevated so for 12 h. Prior to the activation of MAP kinase, increased activities of Ras, Raf-1, and MAP kinase kinase were found, but these enzymes were transiently activated by the treatment with bufalin. These results suggest that the signal was transmitted sequentially from Ras, Raf-1, and MAP kinase kinase to MAP kinase. In association with this signal transduction, the concentration of cAMP in the cells decreased markedly, suggesting that Raf-1 was also activated by a decrease in the extent of phosphorylation by protein kinase A. In fact, pretreatment of U937 cells with forskolin and 3-isobutyl-1-methylxanthine, which are known to increase the concentration of cAMP in the cells, and subsequent treatment with bufalin resulted in a decrease in both Raf-1 activity and DNA fragmentation. To confirm the participation of MAP kinase in the apoptotic process, antisense cDNA for MAP kinase kinase 1 was expressed in U937 cells. The transformants were significantly resistant to both DNA fragmentation and cell death in response to bufalin. Our findings suggest that a pathway with the persistent activation of MAP kinase in U937 cells in response to bufalin is at least one of the signal transduction pathways involved in the induction of apoptosis.
The apoptotic process can be induced by various physical and chemical stimuli and can be modulated by inhibitors of protein kinases or of phosphatases. Okadaic acid, an inhibitor of protein phosphatases, prevents radiation-induced apoptosis in human lymphoid tumor cell lines but induces apoptosis by itself in various other lines of human and rodent cells (14). Staurosporine, a relatively nonspecific inhibitor of protein kinase C, induces apoptosis very rapidly in numerous cell lines (15). These data suggest that protein kinases and their protein phosphatases might play a crucial role in triggering the apoptotic process. MAP 1 kinase is known to be involved in the early events of signal transduction, and the activation of MAP kinase signaling pathways is a key event in the proliferation and differentiation of cells. It has been reported that treatments with various reagents, such as EGF, NGF, platelet-derived growth factor, fibroblast growth factor, insulin, insulin-like growth factor I, 12-O-tetradecanoylphorbol 13-acetate (TPA), and phorbol 12,13-dibutyrate, all cause the activation of MAP kinase in quiescent fibroblastic cells (16). In the present study, we investigated the signal transduction pathway of apoptosis induced by bufalin in human promonocytic leukemia U937 cells and found that treatment of U937 cells with bufalin caused the cooperative interaction of two different signal transduction pathways, leading to abnormal activation of MAP kinase.

MATERIALS AND METHODS
Cell Culture-Human promonocytic leukemia U937 cells were obtained from the Japanese Cancer Resource Bank. The cells were grown in RPMI 1640 medium supplemented with 10% fetal calf serum at 37°C under 5% CO 2 in air.
Chemicals and Radioisotopes-Bufalin, myelin basic protein, and alkaline phosphatase-agarose (from bovine intestinal mucosa) were purchased from Sigma. Mouse anti-MAP kinase (ERK1ϩ2) and mouse anti-MAP kinase kinase 1 (MEK1) monoclonal antibodies were obtained from Zymed Laboratories Inc. (San Francisco, CA). Rabbit anti-Raf-1 polyclonal antibodies (C-12) and rat anti-v-Ha-Ras monoclonal antibodies (clone Y13-259) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA) and Oncogene Science (Uniondale, NY), respectively. Protein G-Sepharose 4 Fast Flow was from Pharmacia LKB Biotechnology (Uppsala, Sweden). An enzyme immunoassay system for cAMP, the ECL Western blotting detection system, and [␥-32 P]ATP (3,000 Ci/mmol) were from Amersham (United Kingdom). [ 32 P]Orthophosphoric acid in water (8,500 -9,120 Ci/mmol) was purchased from DuPont NEN. Two peptides, FLTEYVATRWYRAPEIMLN * This work was supported in part by grants-in-aid from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Treatment with Bufalin-Unless otherwise indicated, U937 cells (5 ϫ 10 6 cells) were deprived of serum for 36 h before addition of bufalin and then they were treated with 10 Ϫ8 M bufalin in serum-free RPMI 1640 medium for the indicated times. Cell viability was determined by the trypan blue-exclusion test.
Analysis of DNA Fragmentation by Agarose Gel Electrophoresis-Cells were collected by centrifugation and washed with PBS. The washed cells were lysed in a solution of 10 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0.5% (w/v) SDS, and 0.1% (w/v) RNase A, with incubation for 60 min at 50°C. The lysate was incubated for an additional 60 min at 50°C with 1 mg/ml proteinase K and then subjected to electrophoresis in a 1% (w/v) agarose gel in 40 mM Tris acetate, pH 7.5, that contained 1 mM EDTA for 60 min at 50 V. After electrophoresis, DNA was visualized by staining with ethidium bromide.
Quantification of DNA Fragmentation-The extent of DNA fragmentation was determined by the method of Wyllie (17), with slight modifications. In brief, cells were suspended in a solution of 5 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.5% (w/v) Triton X-100 and left for 20 min on ice. The suspension was centrifuged at 27,000 ϫ g for 20 min, and the fragmented DNA was recovered from the supernatant. The pellet remaining in the centrifugation tube was sonicated for 1 min at 45 W. The amount of DNA was determined by a fluorometric method using DAPI. The fluorescence intensity was measured at 454 nm with excitation at 362 nm. The percent of DNA fragmentation was defined as the ratio of the amount of fragmented DNA to the total amount of DNA.
Light Microscopy-Control and bufalin-treated U937 cells were collected by centrifugation at 250 ϫ g for 5 min and washed twice with phosphate-buffered saline (PBS). The washed cells were dried on glass slides and stained with Wright-Giemsa solution.
Preparation of Cell Lysates-U937 cells that had been treated with 10 Ϫ8 M bufalin were collected by centrifugation and washed twice with PBS. The washed cells were lysed in 0.4 ml of lysis buffer that consisted of 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.5 mM EGTA, 0.15 M NaCl, 5 g/ml aprotinin, 5 g/ml leupeptin, 5 g/ml pepstatin A, 5 g/ml antipain, 0.5 mM phenylmethylsulfonyl fluoride, 50 mM NaF, 2 mM sodium orthovanadate, 10 mM sodium pyrophosphate, and 1% (w/v) Triton X-100. The cell lysate was centrifuged at 15,000 ϫ g for 20 min. The supernatant was used for quantitation of Ras and protein kinase activities.
Detection of MAP Kinase Activity on an SDS-Polyacrylamide Gel-A separation gel containing 10% (w/v) polyacrylamide, 0.1% (w/v) SDS, and 0.5 mg/ml MBP was prepared as described previously (18) and cell lysates (40 g of protein each) were subjected to electrophoresis by the method of Laemmli (19). After electrophoresis, the gel was incubated for 1 h in 50 mM Tris-HCl, pH 8.0, that contained 20% (v/v) isopropyl alcohol to remove SDS, and then it was incubated for 1 h in buffer A containing 50 mM Tris-HCl, pH 8.0, and 5 mM 2-mercaptoethanol. Subsequently, proteins in the gel were denatured by incubation of the gel for 1 h in buffer A that contained 6 M guanidine HCl. The denatured proteins were renatured by incubating the gel for 12 h at 4°C in four changes of buffer A that contained 0.05% (w/v) Tween 20. The entire gel was then subjected to a kinase assay. After a 30-min preincubation at room temperature in buffer B (40 mM HEPES, pH 7.5, 10 mM MgCl 2 , 2 mM dithiothreitol, and 0.1 mM EGTA), the gel was incubated for 1 h in buffer B that contained 20 M [␥-32 P]ATP (25 Ci) at 30°C. The gel was washed extensively with a solution of 5% (w/v) trichloroacetic acid, 1% (w/v) sodium pyrophosphate, and then it was dried and subjected to autoradiography at Ϫ80°C on X-Omat AR 5 x-ray film (Kodak, Rochester, NY) with an intensifying screen. The incorporation of 32 P into proteins was quantified with a Bio-imaging Analyzer (BAS 2000; Fuji Photo Film Co. Ltd., Tokyo, Japan).
Immunoblotting-Cell lysates containing 20 g of protein each were subjected to SDS-polyacrylamide gel electrophoresis, and the proteins were then transferred to a polyvinylidene difluoride membrane in a wet-blotting apparatus at a constant voltage of 0.5 V/cm 2 for 3.5 h in an ice-cold bath. The membrane was then washed with 20 mM Tris-HCl buffer, pH 7.4, that contained 0.15 M NaCl (TBS), and then it was blocked by incubation with 3% (w/v) bovine serum albumin in TBS for 30 min at room temperature. The blocked membrane was subsequently probed for 1 h at room temperature with a MAP kinase-specific antibodies, diluted 1:1,000 in TBS that contained 0.1% (w/v) bovine serum albumin. After washing with TBS that contained 0.1% (w/v) Tween 20 (TTBS), the membrane was incubated for 1 h at room temperature with horseradish peroxidase-conjugated goat a antibodies against mouse IgG (H & L; American Qualex, La Mirada, CA), which had been diluted 1:2000 in TBS that contained 0.1% (w/v) bovine serum albumin. After washing with TTBS, bands of protein on the membrane were visualized with an ECL Western blotting detection system.
Isolation of RNA and Northern Blot Analysis-Total RNA was prepared by the method of Chomczynski and Sacchi (20). Fifteen g of RNA were subjected to electrophoresis on a 1% (w/v) denaturing agarose gel that contained formaldehyde and transferred to a Hybond-N membrane (Amersham). Northern blot analysis was performed as described previously (21). The ERK2 probe was prepared from 1.1-kilobase cDNA for rat ERK2.
Immunoprecipitation Assays of Kinase Activity in Vitro-The activities of MAP kinase kinase and Raf-1 kinase in cell lysates were measured. Cell lysates (1 mg of protein) from U937 cells that had been treated with 10 Ϫ8 M bufalin for the indicated times were incubated for 1 h at 4°C with 20 l of a 50% (v/v) suspension of protein G-Sepharose in a washing buffer (20 mM HEPES, pH 7.4, 0.5 M NaCl, 2.5 mM MgCl 2 , 0.1% (w/v) Triton X-100, 0.005% (w/v) SDS, 2 g/ml leupeptin, 0.5 mM sodium orthovanadate, and 0.5 mM phenylmethylsulfonyl fluoride). After incubation, the supernatant was collected by centrifugation at 15,000 ϫ g for 3 min. The supernatant was allowed to react with 3 g of each antibody for 1 h at 4°C. Immune complexes were collected by centrifugation and washed 3 times with 1 ml of the washing buffer. The complexes were then washed twice with the appropriate buffer (for assays of MAP kinase kinase, 12.5 mM MOPS, pH 7.2, 12.5 mM ␤-glycerophosphate, 7.5 mM MgCl 2 , 0.5 mM EGTA, 0.05 mM NaF, 2 mM dithiothreitol, and 0.5 mM sodium orthovanadate; for assays of Raf-1 kinase, 50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 20 mM MgCl 2 , 0.1% (w/v) Triton X-100, and 1% (w/v) glycerol) in the absence of ATP and substrate proteins. Assays were initiated by addition of 20 l of the appropriate buffer that contained 50 M [␥-32 P]ATP (0.1 Ci) and 5 g of alkaline phosphatase-treated MAP kinase for assays of MAP kinase kinase, or 0.1 mg of histone V for assays of Raf-1 kinase. After a 10-min incubation at 30°C, the reaction was terminated by addition of Laemmli's loading buffer. The mixture was boiled and subjected to electrophoresis in a 10% or 15% (w/v) polyacrylamide gel. The gel was dried and autoradiographed at Ϫ80°C.
Assays of Kinase Activity in Vitro with a Synthetic Peptide-Cell lysates (40 g of protein) were prepared from U937 cells that had been transfected with the expression vector and treated with 10 Ϫ8 M bufalin. The assay of MAP kinase kinase was performed in a final volume of 100 l in an assay mixture that consisted of 50 M [␥-32 P]ATP (0.1 Ci), cell lysate (40 g of protein), 10 g of synthetic peptide (FLTEYVATRW-YRAPEIMLN), 12.5 mM MOPS, pH 7.2, 12.5 mM ␤-glycerophosphate, 7.5 mM MgCl 2 , 0.5 mM EGTA, 0.05 mM NaF, 2 mM dithiothreitol, and 0.5 mM sodium orthovanadate. After a 10-min incubation at 30°C, the reaction was terminated by the addition of 1 ml of 1 N HCl and then the mixture was boiled for 10 min. One ml of 0.1% (w/v) TFA was added to the reaction mixture, which was then applied to a Sep-pak C18 column. The column was washed with 0.1% (w/v) TFA, and the substrate peptide was eluted with 100% acetonitrile. The radioactivity incorporated into the peptide was measured in a liquid scintillation counter. The assay of MAP kinase was performed in a final volume of 30 l of an assay mixture that contained 50 M [␥-32 P]ATP (0.1 Ci), cell lysate (40 g of protein), 2 mM synthetic peptide (KRELVEPLTPAGEAPNQALLR), 25 mM HEPES, pH 7.4, 1.5 mM MgCl 2 , and 0.1 mM sodium orthovanadate. After a 10-min incubation at 30°C, the reaction was terminated by addition of 10 l of 300 mM orthophosphoric acid, and the reaction mixture was spotted onto a 1.5-cm 2 disk of P81 phosphocellulose paper (Whatman, Maidstone, UK). After the disk had been washed more than seven times by shaking for at least 5 min each in 1% phosphoric acid, the radioactivity incorporated into the substrate peptide was measured in the liquid scintillation counter.
Quantitation of GDP and GTP Bound to Ras-U937 cells were maintained for 34 h in phosphate-free RPMI 1640 medium in the absence of fetal calf serum. The cells were then labeled for 2 h with 0.1 mCi/ml [ 32 P]orthophosphate and treated with 10 Ϫ8 M bufalin for the indicated times. The labeled cells were washed twice with TBS and lysed in lysis buffer. The lysates were centrifuged at 15,000 ϫ g for 20 min at 4°C, and the supernatants were collected. The supernatants were incubated with 20 l of a 50% (w/v) suspension in lysis buffer of protein G-Sepharose for 1 h, and the the mixture was centrifuged at 15,000 ϫ g for 3 min. Ras-specific monoclonal antibodies that had previously been allowed to react with protein G-Sepharose were added to each supernatant, and the immune complexes were collected and washed 3 times with a solution that contained 20 mM HEPES, pH 7.4, 0.5 M NaCl, 2.5 mM MgCl 2 , 0.1% (w/v) Triton X-100, 0.005% (w/v) SDS, 2 g/ml leupeptin, 0.5 mM sodium orthovanadate, and 0.5 mM phenylmethylsulfonyl fluoride. The bound GDP and GTP were dissociated from the immune complexes by incubation at 65°C for 5 min in 20 mM Tris-HCl, pH 7.5, 1 mM GTP, 1 mM GDP, 20 mM EDTA, and 2% (w/v) SDS, and purified by thin layer chromatography on PEI-cellulose plates with 0.75 M KH 2 PO 4 , pH 3.5, as the mobile phase. The radioactivity incorporated into GDP and GTP was quantified with the Bio Imaging Analyzer.
Purification of MAP Kinases from Rat Brain-MAP kinases were purified from rat brain as described previously (22). MBP was used as the substrate protein instead of human protein 34 or MAP2. During purification, the enzymatic activity of each fraction was measured and, simultaneously, MAP kinases were detected by immunoblot analysis with MAP kinase-specific and phosphotyrosine-specific antibodies. Purified MAP kinases were dephosphorylated for 1 h at 30°C by incubation with alkaline phosphatase-agarose in 10 mM Tris-HCl, pH 9.0, 1 mM MgCl 2 , and 1 mM ZnCl 2 , and they were then submitted to the assay of MAP kinase kinase activity.
Determination of the Intracellular Concentration of cAMP-U937 cells that had been treated with 10 Ϫ8 M bufalin were collected and washed with PBS. The cells were counted, and 5 ϫ 10 6 cells were homogenized in 0.5 ml of 6% (w/v) trichloroacetic acid. The homogenate was centrifuged at 15,000 ϫ g for 10 min, and the supernatant was collected. The supernatant was then subjected to an enzyme immunoassay (Amersham) for cAMP in accordance with the manufacturer's instructions.
Cloning of cDNA for Human MAP Kinase Kinase 1 and Construction of Sense and Antisense Expression Vectors-To obtain cDNA for MAP kinase kinase 1, we used the reverse transcription-PCR technique (23). Total RNA was isolated from U937 cells and 1. Transfection of U937 Cells-Transfections were performed by electroporation (Gene Pulsor, Bio-Rad) as described previously (24). A pulse of electricity was delivered to 0.4 ml of a suspension of cells (4 ϫ 10 6 cells) that contained 10 g of linearized plasmid DNA. The cells were plated in 96-well microplates (1 ϫ 10 4 cells per well) and cultured in RPMI 1640 medium. After 24 h in culture, geneticin (G418 sulfate; Life Technologies, Inc.) was added at a final concentration of 400 g/ml, and subclones were isolated 2 to 3 weeks later.

Induction of Apoptosis in U937 Cells by Bufalin-When human leukemia U937 cells were maintained for 36 h in medium
without fetal calf serum, to circumvent any effects of growth factors in the serum, and were then treated with 10 Ϫ8 M bufalin in this medium, cell viability decreased markedly with time, as shown in Fig. 1A. We examined whether or not the cell death caused by bufalin under these conditions might have been due to apoptosis. Fig. 1B shows the results of agarose gel electrophoresis of DNA extracted from cells that had been treated with 10 Ϫ8 M bufalin for various times. A ladder pattern typical of internucleosomal fragmentation, which is considered to be an early event in programmed cell death, was detected when cells had been treated with bufalin for more than 6 h. Furthermore, the amount of fragmented DNA, quantified by a fluorometric method using DAPI, increased gradually with time, as shown in Fig. 1A. No fragmentation of DNA occurred under the same conditions in the absence of bufalin (Fig. 1C). Morphological observations of U937 cells that had been treated with 10 Ϫ8 M bufalin for 30 h under a light microscope revealed the condensation of chromatin in nuclei and the degradation of nuclei (results not shown). These results indicated that bufalin caused apoptosis in U937 cells under these conditions.
Effects of Bufalin on the Signal Transduction Pathway That Involves MAP Kinase-Since MAP kinase is known to be important in the regulation of cell growth and differentiation (25,26), we examined whether the activity of MAP kinase in U937 cells might be altered by the treatment with 10 Ϫ8 M bufalin. As shown in Fig. 2A, the intensity of the bands of a phosphorylated protein with a molecular mass of 42 kDa, corresponding to ERK2, increased significantly within 6 -12 h after the start of treatment with bufalin. Estimation of the activity of MAP kinase from the extent of incorporation of 32 P into MBP using a Bio-Imaging analyzer indicated that the activity increased gradually for the first 3 h and then markedly 6 h after the start of treatment with bufalin. The maximum activation of the enzyme was detected at 6 h after the start of treatment with bufalin, and then it decreased slightly (Fig. 2B). It was surprising to us that the elevated activity of MAP kinase persisted for 12 h. To our knowledge, such an anomalous and continuous pattern of activation of MAP kinase has never been reported previously.
We next compared the activation of MAP kinase in U937 cells by bufalin with that by EGF. As shown in Fig. 2B, EGF induced a rapid and transient increase in the activity of MAP kinase, as demonstrated previously in other cell lines (26,30). The extent of the activation of MAP kinase by EGF was approximately 2-fold, which was almost the same extent as that by bufalin. Immunoblot analysis of a lysate of U937 cells that had been treated with bufalin showed that the level of the phosphorylated form of MAP kinase also increased significantly 6 h after the start of treatment (Fig. 2C, P-ERK2). Furthermore, Northern blot analysis of ERK2 revealed that its mRNA was expressed at almost a constant level for 12 h after the addition of bufalin to cultures (data not shown). These results indicated that the activation of MAP kinase was due to its phosphorylation by a protein kinase upstream of the MAP kinase signal pathway. Therefore, we examined the changes in activity of components upstream of the MAP kinase. As shown in Fig. 3, the activity of MAP kinase kinase in U937 cells also increased during the treatment with bufalin, reaching a maximum 3 h after the start of treatment, and then it decreased gradually to the basal level. Rather similar changes in activity were observed for Raf-1 (Fig. 3B). Ras activity, measured as the ratio of Ras-bound GTP/(GTP ϩ GDP), increased rapidly after the start of treatment with bufalin and then decreased rapidly 30 min after the addition of bufalin (Fig. 3C). Considering all the evidence, we can conclude that bufalin sequentially activates Ras, Raf-1, MAP kinase kinase, and MAP kinase in U937 cells.
Effects of Bufalin on the Intracellular Concentration of cAMP-Phosphorylation of Ser-43 of Raf-1 by a cAMP-dependent protein kinase (protein kinase A) is known to inhibit Raf-1 activity (27,28). We examined whether bufalin could change the intracellular concentration of cAMP in U937 cells and, thus, affect the activity of Raf-1. The intracellular level of cAMP in U937 cells dropped sharply just after the start of the treatment with 10 Ϫ8 M bufalin, and it decreased to approximately 50% of the control value within 6 h after the start of the treatment (Fig. 4A). By contrast, treatment of U937 cells with EGF caused a sharp and transient increase in the concentration of cAMP in the cells (Fig. 4B), as reported previously in isolated, perfused rat hearts (29). This result prompted us to examine whether agents that increase intracellular levels of cAMP, such as IBMX and forskolin, could antagonize the effect of bufalin on the activation of Raf-1. As shown in Fig. 5, the activation of Raf-1 in U937 cells by bufalin was significantly , and Ras (C) in U937 cells that had been treated for the indicated times with 10 Ϫ8 M bufalin were investigated. Protein kinase activities in A and B were measured by an immunoprecipitation in vitro kinase assay as described under "Materials and Methods." The intensity of the bands of phosphorylated proteins on autoradiograms was quantified with a Bio-Imaging analyzer. C, the amounts of GDP and GTP bound to Ras were measured as described under "Materials and Methods," and the activity is shown as the ratio of bound GTP to bound (GTP ϩ GDP).
FIG. 2. Activation of MAP kinase in response to bufalin. MAP kinase activity was detected on an SDS-polyacrylamide gel that contained MBP as described under "Materials and Methods." Lanes 1-7 correspond to cells treated with 10 Ϫ8 M bufalin for 0, 0.5 h, 1 h, 3 h, 6 h, 9 h, and 12 h, respectively. MAP kinase activity was detected in an SDS-polyacrylamide gel that contained MBP as described under "Materials and Methods." B shows the quantitation of the results in A and results obtained with EGF (10 Ϫ8 M bufalin (E), 30 nM EGF (q)). C shows the results of immunoblot analysis of cells treated with bufalin under the same conditions as described in A. Lanes 1-6 correspond to lysates (20 g of protein) of U937 cells that had been treated with bufalin for 0, 1, 3, 6, 9, and 12 h. Immunoblotting was performed with MAP kinase-specific antibodies.
inhibited by pretreatment of cells with IBMX or forskolin. The results suggest that a marked decrease in the concentration of cAMP in U937 cells upon treatment with bufalin results in inactivation of protein kinase A, with subsequent activation of Raf-1. The above results raise the possibility that IBMX and forskolin might inhibit the fragmentation of DNA in U937 cells that is induced by bufalin by inhibiting Raf-1 via its phosphorylation. Indeed, the induction of fragmentation of DNA by bufalin was inhibited by pretreatment of cells with either IBMX or forskolin (Fig. 6). The inhibitory effect on bufalininduced fragmentation of DNA in U937 cells became evident 6 to 12 h after the start of treatment with bufalin. Thus, Raf-1 was activated by two mechanisms, namely, as a consequence of the increased activity of Ras, as described above, and as a result of a decrease in levels of cAMP. It seems likely that together these factors caused the persistent activation of MAP kinase.
Effect of Expression of Antisense cDNA for MAP Kinase Kinase on the Induction of Apoptosis-To verify the contribution of the MAP kinase signaling pathway to apoptosis caused by bufalin, sense and antisense cDNAs for MAP kinase kinase were inserted into the cloning site of the mammalian expression vector pMAM-neo. Transcription of the insert in this vector is under the control of the Rous sarcoma virus promoter and a dexamethasone-inducible long terminal repeat of a mouse mammary tumor virus (30). The linked selectable marker (neo), which confers resistance to G418, is controlled by the SV40 promoter. The expression plasmids were used to transfect U937 cells, and G418-resistant colonies were subjected to assays of protein kinase activity (Fig. 7). When cells transfected with pMAM-neo (no insert), with pMAM-sense MAP kinase kinase-neo, and with pMAM-antisense MAP kinase kinase-neo were treated with bufalin without pretreatment with dexamethasone, approximately 1.5-fold activation of MAP kinase kinase was observed in each case (Fig. 7A), indicating that the three lines of transfected cells retained the responsiveness to bufalin. The activities of MAP kinase kinase in cells that had been transfected with pMAM-neo or pMAM-sense MAP kinase kinase-neo and pretreated with 1 M dexamethasone for 20 h were slightly increased by treatment with 10 Ϫ8 M bufalin for 3 h. By comparison, the activation of MAP kinase kinase by bufalin in cells that had been transfected with pMAM-antisense MAP kinase kinase-neo was markedly decreased by the pretreatment with dexamethasone. The activities of MAP kinase in the transfected cells responded similarly to dexamethasone and bufalin, respectively. As shown in Fig. 7B, activation of MAP kinase by bufalin in U937 cells that had been transfected with pMAM-antisense MAP kinase kinase-neo was also significantly suppressed by the pretreatment with dexamethasone (p Ͻ 0.001).
We also examined the effect of bufalin on the induction of apoptosis in these transformants. As can be seen from Fig. 8, the fragmentation of DNA induced by bufalin in cells that had been transfected with pMAM-antisense MAP kinase kinaseneo was significantly inhibited by the pretreatment with dexamethasone. The results together suggest that the prolonged activation by bufalin of MAP kinase, a result of the cooperative interaction of two signaling pathways, induces apoptosis in U937 cells. DISCUSSION In the absence of serum, 10 Ϫ8 M bufalin induces apoptosis in human leukemia U937 cells. With the rapid increase in the fragmentation of DNA in U937 cells treated with bufalin, the activity of MAP kinase also increased markedly with time. MAP kinase activity has been reported to be stimulated by exposure of fibroblastic or PC12 cells to growth factors, such as EGF (26,31) or NGF (26). Upon treatment of these cells with EGF, maximal activation of MAP kinase occurs within 1 min after the addition of EGF, and the activity then returns rapidly to the basal level (26). Upon treatment of PC12 cells with NGF, maximal activation of MAP kinase is observed 5 min after the addition of NGF, and the activity then diminishes gradually (26). In contrast to the effects of EGF or NGF on MAP kinase activity, bufalin induced a gradual but anomalous increase in MAP kinase activity in leukemia U937 cells. The maximal activity of MAP kinase, which was about 2.4 times the basal level, was attained 6 h after the addition of bufalin to the U937 cells, and near-maximal activity was still retained 12 h after the start of treatment with bufalin. Simultaneous with the activation of MAP kinase, transient increases in the activities of enzymes in the MAP kinase cascade, such as Ras, Raf-1, and MAP kinase kinase, were found. These results indicate that the bufalin signal was transduced through MAP kinase signaling pathways.
It is well known that intracellular concentrations of cAMP are increased by certain hormones. EGF increases the intracellular concentration of cAMP in rat heart (29), mammary gland (32), and in several lines of epithelial cells when they are treated with IBMX and forskolin (33). The present study demonstrated that levels of cAMP in U937 cells were markedly decreased by bufalin. Recently, Wu et al. (27) demonstrated that an increased concentration of cAMP inhibited the activity of MAP kinase by phosphorylating Ser-43 in the regulatory domain of Raf-1. It was, therefore, expected that the extent of phosphorylation of Raf-1 would be decreased by bufalin, with subsequent activation of Raf-1. Indeed, we observed that MAP kinase was activated both by the activation of Ras and by the decrease in the concentration of cAMP. Moreover, expression of antisense DNA for MAP kinase kinase in U937 cells inhibited both the activation of MAP kinase kinase, as well as that of MAP kinase, and the induction of apoptosis by bufalin. These findings suggest that the abnormal and continuous activation of MAP kinase is at least one of the signal transduction pathways involved in the induction of apoptosis in U937 cells by bufalin.
Recently, Cowley et al. (34) and Mansour et al. (35) demonstrated that constitutive activation of MAP kinase kinase is necessary and sufficient for differentiation of PC12 cells (34) and for transformation of NIH3T3 cells (34,35). Our findings suggest that the activation of MAP kinase kinase, at least in some cases, in human leukemia cells induces cell death through apoptosis.
The activity of MAP kinase is also regulated by phosphatases. Therefore, there is a possibility that the persistent and anomalous activation of MAP kinase by bufalin might be due to the inhibition of phosphatases such as PAC1 (36) and CL100 (37). We cannot exclude this possibility at the present stage of our investigations.
A possible candidate for the receptor for bufalin in leukemia cells is Na ϩ ,K ϩ -ATPase (3) since the activity of this enzyme in various tumor cells is strongly inhibited by bufalin. Although the signal transduction pathway from Na ϩ ,K ϩ -ATPase to Ras is unknown at present, we have provided evidence in this report for the interaction of two different signaling pathways in U937 cells. The signal transduction pathway from Na ϩ ,K ϩ -ATPase on the surface of U937 cells to Ras and the possible participation of MAP kinase phosphatase in the signal pathway induced by bufalin merit further study.