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Originally published In Press as doi:10.1074/jbc.M111501200 on January 4, 2002

J. Biol. Chem., Vol. 277, Issue 10, 8076-8082, March 8, 2002
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Functional Cooperation among Ras, STAT5, and Phosphatidylinositol 3-Kinase Is Required for Full Oncogenic Activities of BCR/ABL in K562 Cells*

Junko SonoyamaDagger , Itaru MatsumuraDagger , Sachiko EzoeDagger , Yusuke SatohDagger , Xian ZhangDagger , Yoshihisa KataokaDagger , Emi TakaiDagger , Masao MizukiDagger , Takashi MachiiDagger , Hiroshi Wakao§, and Yuzuru KanakuraDagger

From the Dagger  Department of Hematology/Oncology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871 and the § Helix Research Institute, 1532-3 Yana Kisarazu-shi, Chiba 292-0812, Japan

Received for publication, December 3, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

BCR/ABL tyrosine kinase generated from the chromosomal translocation t(9;22) causes chronic myelogenous leukemia and acute lymphoblastic leukemia. To examine the roles of BCR/ABL-activated individual signaling molecules and their cooperation in leukemogenesis, we inducibly expressed a dominant negative (DN) form of Ras, phosphatidylinositol 3-kinase, and STAT5 alone or in combination in p210 BCR/ABL-positive K562 cells. The inducibly expressed DN Ras (N17), STAT5 (694F), and DN phosphatidylinositol 3-kinase (Delta p85) inhibited the growth by 90, 55, and 40%, respectively. During the growth inhibition, the expression of cyclin D2 and cyclin D3 was suppressed by N17, 694F, or Delta p85; that of cyclin E by N17; and that of cyclin A by Delta p85. In addition, N17 induced apoptosis in a small proportion of K562, whereas 694F and Delta p85 were hardly effective. In contrast, coexpression of two DN mutants in any combinations induced severe apoptosis. During these cultures, the expression of Bcl-2 was suppressed by N17, 694F, or Delta p85, and that of Bcl-XL by N17. Furthermore, although K562 was resistant to interferon-alpha - and dexamethasone-induced apoptosis, disruption of one pathway by N17, 694F, or Delta p85 sensitized K562 to these reagents. These results suggested that cooperation among these molecules is required for full leukemogenic activities of BCR/ABL.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Chronic myelogenous leukemia (CML)1 is a malignant clonal disorder of hematopoietic stem/progenitor cells (as reviewed in Refs. 1-3). The diagnostic hallmark of CML is the Philadelphia chromosome, the derivative chromosome 22 resulting from the reciprocal chromosomal translocation t(9:22)(q34;q11), which is observed in over 90% of patients with CML. This translocation joins c-Abl tyrosine kinase on chromosome 9 and breakpoint cluster region (BCR) on chromosome 22, leading to the generation of the fusion gene for BCR/ABL. According to the difference in the breakpoint in BCR, three types of BCR/ABL fusion proteins, p210, p190, and p230, are generated. p210 BCR/ABL was observed in ~90% of CML patients and in a small fraction of acute lymphoblastic leukemia (ALL) patients, whereas p190 BCR/ABL and p230 BCR/ABL are associated with ALL and chronic neutrophilic leukemia, respectively. However, it still remains unknown how these three forms of BCR/ABL differ from each other in terms of their downstream signaling or induce distinct diseases. c-Abl tyrosine kinase exists in both cytoplasm and nucleus and induces apoptosis in response to DNA damage through the cooperation with the p53 homologue, p73, whereas BCR/ABL primarily localizes to cytoplasm and acts as an oncogene (for a review, see Ref. 4). This cytoplasmic localization of BCR/ABL is essential for its biologic activities because BCR/ABL entrapped into the nucleus by leptomycin B induces apoptosis with its tyrosine kinase activities (5).

As for the biologic activities of BCR/ABL in oncogenesis, a number of in vitro experiments have shown that BCR/ABL enabled primitive hematopoietic cells as well as factor-dependent hematopoietic cell lines such as Ba/F3, 32D, and FDC-P1 to proliferate under factor-deprived conditions (6-9). In addition, enforced expression of p210 or p190 BCR/ABL in Rat-1 fibroblasts caused a distinct morphologic change and conferred both tumorigenicity and capacity for anchorage-independent growth (10). Furthermore, when bone marrow cells infected with retrovirus expressing p210 BCR/ABL were transplanted into lethally irradiated mice, some of the recipients developed various types of hematologic malignancies including granulocytic hyperplasia resembling human CML, myelomonocytic leukemia, ALL, lymphomas, and erythroid leukemia (11-13). Moreover, transgenic mice expressing p210 BCR/ABL developed pre-B or T cell lymphomas, T-ALL, or myeloproliferative disorder like CML (14-16). These results indicated that BCR/ABL indeed acts as an oncogene and causes hematologic malignancies in vivo.

Growth and survival of hematopoietic cells are regulated by a number of hematopoietic growth factors. Upon the stimulation with the ligand, receptors for hematopoietic growth factors transmit mitogenic and anti-apoptotic signals through activation of their downstream molecules. To keep homeostasis of hematopoiesis, these cytokine signals are subsequently eliminated by negative feedback mechanisms including ubiquitin/proteasome-dependent protein degradation, activation of phosphatases, and induction of inhibitory molecules. By contrast, activated mutants of the upper stream signaling molecule such as TEL/platelet-derived growth factor receptor, tandem duplication of FLT3, activating point mutation of c-kit, and TEL/JAK2 cause excessive growth, survival, and consequent malignant transformation of hematopoietic cells through constitutive activation of downstream cascades. In addition to these oncogenic signaling molecules, the BCR/ABL tyrosine kinase also activates various signaling molecules including the Ras/mitogen-activated protein kinase (MAPK) pathway, the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway, and signal transducers and activators of transcription (STATs, STAT1 and STAT5), and acts as an oncogene (as reviewed in Ref. 23). As for the roles of these signaling molecules in BCR/ABL-mediated leukemogenesis, a dominant negative (DN) form of Ras inhibited the growth and survival of BCR/ABL-transformed 32D cells (17). Similarly, DN STAT5 suppressed apoptosis resistance, factor-independent proliferation, and leukemogenic potential of a CML-derived cell line, K562, and BCR/ABL-transformed 32D and Ba/F3 (18-20). In addition, a mutant form of BCR/ABL that cannot activate PI3-K did not confer leukemogenic potentials on murine bone marrow cells in vitro and in vivo, indicating that PI3-K/Akt pathway is also required for BCR/ABL-induced malignant transformation of hematopoietic cells (21). Together, these results indicated that Ras, STAT5, and PI3-K can each play essential roles in BCR/ABL-mediated leukemogenesis. However, the precise mechanisms by which each signaling molecule mediates BCR/ABL-dependent growth and survival are unknown. Additionally, the functional relationship among these signaling cascades remains to be clarified.

Therefore, in this study, we examined the functions of Ras, STAT5, and PI3-K by expressing respective DN mutant alone or in combination in p210 BCR/ABL-positive CML-derived cell line, K562. Our experiments demonstrated that Ras, STAT5, and PI3-K individually participate in BCR/ABL-dependent growth and survival of K562, whereas Ras seemed to play a central role among these molecules. Regarding this mechanism, we found that cooperation among these signaling pathways is required for maintaining the expressions of critical molecules for cell cycle progression or cell survival such as cyclin D2, cyclin D3, and Bcl-2. In addition, disruption of only one signaling pathway (Ras, STAT5, or PI3-K) made K562 cells susceptible to interferon-alpha (IFN-alpha )- or dexamethasone-induced apoptosis. These results suggested simultaneous activation of multiple signaling pathways is necessary for full leukemogenic activities of BCR/ABL and that new therapeutic strategies to abrogate at least one signaling pathway might enhance the efficacy of conventional reagents in therapy-resistant CML patients.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Reagents and Antibodies-- Recombinant human (rh) rhIFN-alpha was a gift from the Sumitomo Pharmaceutical Co. Ltd (Tokyo, Japan). Dexamethasone was purchased from Sigma and diluted to 30 mM in ethanol. An anti-pan-Ras monoclonal antibody (Ab) (OP40) was purchased from Oncogene Research Products (Cambridge, MA), an anti-STAT5b polyclonal Ab (C-17) from Santa Cruz Biotechnology (Santa Cruz, CA), and an anti-PI3-K p85alpha polyclonal Ab from MBL (Watertown, MA). An anti-phosphotyrosine Ab 4G10 was kindly provided by Dr. B. Druker (Oregon Health & Science University, Portland, OR).

Cell Lines and Cultures-- K562, a human cell line derived from a patient with CML blastic crisis, was obtained from RIKEN Cell Bank (Tsukuba, Japan) and maintained in RPMI 1640 (Nakarai Tesque, Kyoto, Japan) containing 10% fetal bovine serum (Flow, North Ryde, Australia).

Plasmid Constructs and cDNAs-- The Lac-inducible expression vectors of DN Ras (N17), DN STAT5 (694F), and DN PI3-K (Delta p85) were described previously (22, 23). Full-length cDNAs used for Northern blot analysis were kindly provided from the investigators as follows: human cyclin D1 from Dr. A. Arnold (Massachusetts General Hospital, Boston, MA); human cyclin D2 and D3 from Dr. G. Peters (Imperial Cancer Research Fund, London, United Kingdom); human cyclin A, cyclin B, cyclin E, CDK2, and CDK4 from Dr. H. Kiyokawa (University of Illinois, Cancer Center, Chicago, IL); human CDC2 from Dr. E. Harlow (Massachusetts General Hospital, Boston, MA); human Bcl-2 from Dr. Y. Tsujimoto (Osaka University, Osaka, Japan); murine Bcl-XL from Dr. T. Tsujimura (Hyogo College of Medicine, Nishinomiya, Japan).

Lac-inducible System-- To express the target cDNA, we used a LacSwitchTM II inducible expression system (Stratagene, La Jolla, CA). In short, K562 cells were initially transfected with an expression vector of Lac repressor (LacR), pCMV-LacI, by electroporation (250 V, 960 microfarads). The transfected cells were screened by culturing with 0.5 mg/ml hygromycin (Sigma). Of several hygromycin-resistant clones, one clone in which LacR was most intensely expressed was further transfected with a Lac-inducible vector pOPRSVI each containing DN Ras, DN STAT5, and DN PI3-K. The expression vector of pOPRSVI contains RSV promoter linked to the Escherichia coli lactose operon, and the expression of target cDNA is suppressed by LacR through the lactose operon. In response to the addition of isopropyl-beta -D-thiogalactopyranoside (IPTG) into the culture medium, LacR is released from lactose operon and transcription of the target cDNA is initiated. After the selection with 1 mg/ml G418 (Invitrogen), induction levels of each target protein were examined by Northern blot analyses before and after 0.5 mM IPTG treatment in several clones. To express DN mutant in combination, K562/N17 or K562/694F was further cotransfected with pOPRSVI containing 694F or Delta p85 and an expression vector puromycin, pBABE-puro. After the selection with 1 mg/ml puromycin (Sigma), the induction levels of the target proteins in each clone were examined by Western blot analyses.

Luciferase Assays-- Three tandem repeats of AP-1-binding sequence in the polyomavirus Py enhancer and three tandem repeats of STAT5-binding sequence in the beta -casein promoter were linked to the murine minimal JunB promoter (-42 to +136), and subcloned into pGL3-Basic-Luc (Promega, Madison, WI) to construct reporter genes for Ras and STAT5 activities (named 3×AP-1-Luc and 3×beta -Cas-Luc, respectively). Luciferase assays were performed by using the Dual-Luciferase reporter system (Promega), in which transfection efficiency was monitored by cotransfected pRL-CMV-Rluc, an expression vector of Renilla reniformis luciferase. In short, cultured cells (1 × 107 cells/sample) were electroporated (250 V, 960 microfarads) with 10 µg of an appropriate reporter gene together with 2 µg of pRL-CMV-Rluc. After a 12-h recovery period in the culture medium, the cells were cultured with or without IPTG for 36 h. The cells were lysed in lysis buffer supplied by the manufacturer, followed by the measurement of firefly (Photinus pyralis) and Renilla luciferase activities on luminometer LB96P (Berthold Japan, Tokyo, Japan). The relative firefly luciferase activities were calculated by normalizing transfection efficiency according to the Renilla luciferase activities.

Northern Blot Analysis-- The isolation of total cellular RNA and the method for Northern blot were described previously (24).

Western Blot Analysis-- Preparation of cell lysates, gel electrophoresis, and immunoblotting were performed according to the methods described previously (25). Immunoreactive proteins were visualized with the enhanced chemiluminescence detection system (PerkinElmer Life Sciences).

PI3-K Assays-- Total cellular lysates were obtained from 5 × 106 cells, and tyrosine-phosphorylated proteins were immunoprecipitated with 2 µg of an anti-phosphotyrosine Ab at 4 °C. The immunoprecipitates were washed twice with lysis buffer, twice with buffer containing 0.5 M LiCl and 0.2% Nonidet P-40, and finally with 10 mM HEPES (pH 7.4) and 0.15 M NaCl. Then, 30 µl of 10 mM phenyl phosphate (dissolved in 20 mM HEPES (pH 7.4)) was added. The kinase reaction was performed in 100 µl of kinase buffer containing 0.2 mg/ml phosphatidylinositol, 40 µM ATP, 30 mM MgCl2, and 10 µCi [gamma -32P]ATP at 37 °C for 10 min. Then, the reaction was stopped by adding 100 µl of 1 N HCl, and the lipid layer was extracted with 200 µl of chloroform/methanol (1:1 v/v). The extracts were washed with methanol/1 N HCl (1:1 v/v), separated by thin layer chromatography (TLC) using silica gel G60 (250 mm, Merck, Darmstadt, Germany) in chloroform/methanol/H2O/NH4OH (43:38:7:5, v/v), and subjected to autoradiography.

DNA Content Analysis-- The DNA content of cultured cells was examined by staining with propidium iodide (PI) and analyzed using a FACSort (Beckon Dickinson, Oxnard, CA) with a program Modfit LT2.0 (Beckon Dickinson) as previously described (23).

Terminal Deoxynucleotidyltransferase-mediated Biotin-dUTP Nick End Labeling (TUNEL) Assays-- TUNEL assays were performed with an In Site cell death detection kit (Roche Molecular Biochemicals). Briefly, cells were fixed with 4% paraformaldehyde in PBS for 30 min, transferred into permeabilization solution (0.1% Triton X-100 in 0.1% sodium citrate), and incubated on ice for 2 min. After washing with PBS, the cells were resuspended in TUNEL reaction mixture containing terminal deoxynucleotidyltransferase enzyme and digoxigenin-nucleotide. Incorporation of nucleotides into 3'-DNA fragmented ends was detected by flow cytometry.

Annexin-V Staining-- Cells were washed with RPMI 1640 twice and resuspended in 100 µl of labeling solution containing avidin-annexin-V conjugates at room temperature for 30 min. The cells were rinsed and developed with fluorescein-conjugated avidin (Becton Dickinson) at 4 °C for 30 min. The stained cells were analyzed by flow cytometry.

Assays for Caspase-3 Activities-- Caspase-3 activities were measured with a PhiPhiLux-G1D2 kit (OncoImmunin, College Park, MD). Briefly, cells were washed with PBS and resuspended in 50 µl of substrate solution supplied by the manufacturer, which contains the caspase-3-specific substrate. After 60 min of incubation in a 5% CO2 incubator at 37 °C, the cells were suspended in 500 µl of dilution buffer supplied by the manufacturer and subjected to flow cytometry. In this system, caspase-3 activities are measured by fluorescence that is derived from the cleaved substrate specific for caspase-3.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Inducible Expression of DN-Ras, DN-STAT5, and DN-PI3-K in K562-- BCR/ABL has been reported to activate various signaling molecules such as Ras/MAPK pathways, PI3-K/Akt pathways, and STATs (as reviewed in Refs. 2 and 3). To clarify the roles of each signaling molecule in BCR/ABL-mediated cell growth and survival, we inducibly expressed a DN form of Ras (N17), STAT5 (694F), and PI3-K (Delta p85) by using a Lac-inducible system, in which expression of the target protein was induced by IPTG treatment; each transfected clone was designated as K562/N17, K562/694F, and K562/Delta p85, respectively. As shown in Fig. 1A, Western blot analysis on the whole cell lysates revealed that addition of IPTG induced expression of N17, 694F, and Delta p85 after 4 h, and their expression reached a maximum at 24 h and was retained until 48 h. Moreover, IPTG-induced DN mutant proteins were far more abundant than their respective endogenous proteins detected in K562/Mock (a clone transfected with an empty Lac-inducible vector).


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  		Fig. 1.   Inducible expression of N17, 694F, and Delta p85 and their effects on respective signaling pathways. A, each clone was cultured with 0.5 mM IPTG, and whole cell lysates were prepared at the times indicated. Equal amounts of whole cell lysates were subjected to SDS-PAGE followed by Western blotting with anti-Ras, anti-STAT5b, and anti-PI-3K p85alpha antibodies, respectively. IB, immunoblotting. B, effects of IPTG-induced N17 and 694F on respective signaling pathways. K562/N17, K562/694F, and K562/Mock were transfected with 10 µg of a reporter gene indicated and 2 µg of pRL-CMV-Rluc by electroporation. After 12 h, the cells were cultured with or without IPTG for 36 h, and then subjected to luciferase assays. The relative firefly luciferase activities were calculated by normalizing transfection efficiency according to the Renilla luciferase activities. The experiments were performed in triplicate, and similar results were obtained from at least three independent experiments. The results are shown as the mean ± S.D. of triplicate experiments. C, effects of IPTG-induced Delta p85 on BCR/ABL-dependent PI3-K activities. K562/Delta p85 and K562/Mock were cultured with or without 0.5 mM IPTG for 48 h, or with 100 nM wortmannin for 2 h. Equal amounts of cell lysates were immunoprecipitated with an anti-phosphotyrosine Ab, and PI3-K activities were assayed in the immunoprecipitates using [gamma -32P]ATP and phosphatidylinositol as a substrate. The extracted lipids were separated by TLC and visualized by autoradiography. The position of migrated phosphatidylinositol 3-phosphate (PI3P) is indicated. Endo., endogenous; Exo., exogenous.

IPTG-induced N17, 694F, and Delta p85 Inhibit Their Corresponding Signaling Pathways Almost Completely-- At first, we evaluated the efficacy of DN mutants on their respective signaling pathways. The effectiveness of N17 and 694F was measured by luciferase assays with reporter plasmids for Ras (3×AP-1-Luc) and STAT5 (3×beta -Cas-Luc), respectively. With reference to basal activities (activities of JunB-MP-Luc, a backbone reporter plasmid without an element), 3×AP-1-Luc was activated by ~6-fold in both K562/Mock and IPTG-untreated K562/N17, suggesting that the Ras/MAPK pathway is activated by BCR/ABL in K562 (Fig. 1B). Although IPTG treatment hardly affected 3×AP-1-Luc activities in K562/Mock, IPTG-induced N17 reduced its activities to the basal level in K562/N17. Similarly, IPTG treatment almost completely inhibited BCR/ABL-induced 3×beta -Cas-Luc activities (~7-fold induction) in K562/694F but not in K562/Mock (Fig. 1C). We also assessed the efficacy of IPTG-induced Delta p85 by PI3-K assays. As shown in Fig. 2C, PI3-K was activated in K562/Mock and IPTG-untreated K562/Delta p85, whereas IPTG treatment inhibited its activities in K562/Delta p85 as efficiently as a PI3-K inhibitor, wortmannin. These results suggested that IPTG-induced N17, 694F, and Delta p85 could inhibit their corresponding signaling pathways almost completely. Because Ras has been reported to affect activities of PI3-K and STAT5 in other cell types (26, 27), we investigated the functional cross-talk among these molecules by examining the effects of N17, 694F, and Delta p85 on the other signaling pathways. Except that STAT5 activities were slightly augmented by N17, none of these molecules affected the activities of the other two signaling pathways (data not shown).


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  		Fig. 2.   Effects of N17, 694F, and Delta p85 on the growth of K562. A, the cells of indicated clones were seeded at a cell density of 50/µl, cultured with or without IPTG, and total number of viable cells were counted by trypan blue dye exclusion method. The results are shown as the mean ± S.D. of triplicate cultures. B, the cells were cultured with or without IPTG for 5 days. DNA content of the cultured cells was examined by PI staining and analyzed on FACSort.

Effects of N17, 694F, and Delta p85 on Proliferation and Survival of K562-- Next, we evaluated the effects of each DN mutant on the growth of K562. Under the culture without IPTG, K562/N17, K562/694F, and K562/Delta p85 showed growth curves similar to K562/Mock (Fig. 2A). Although IPTG treatment did not affect the growth of K562/Mock, IPTG-induced N17, 694F, and Delta p85 inhibited the growth (evaluated at day 5) by 90, 55, and 40%, respectively (Fig. 2A). We also performed DNA content analysis before and after 5-day culture with IPTG in these clones. As shown in Fig. 2B, IPTG-induced N17, 694F, and Delta p85 suppressed the proportion of proliferating cells (percentage of the cells in S or G2/M phase at 120 h: K562/N17, 10%; K562/694F, 21%; K562/Delta p85, 28%). In addition to the growth suppression, N17 was found to induce apoptosis, which was detected as a subdiploid fraction, in a small proportion (12%) of the cultured cells (Fig. 2B). By contrast, neither 694F nor Delta p85 induced apoptosis.

Effects of N17, 694F, and Delta p85 on the Expressions of Cell Cycle Regulatory Molecules-- To elucidate the mechanisms by which Ras, STAT5, and PI3-K mediate BCR/ABL-dependent growth of K562, we examined changes in expressions of cell cycle regulatory molecules in K562/N17, K562/694F, and K562/Delta p85 by Northern blot analysis during the 120-h IPTG treatment. As shown in Fig. 3, cyclin D2 expression was suppressed in all clones as early as 24 h and remained suppressed during the test period. Cyclin D3 expression was reduced by N17 after 24 h, and by 694F and Delta p85 after 48 h. The expression of cyclin E was suppressed by N17 but not by 694F or Delta p85, whereas that of cyclin A was reduced by Delta p85 but not by N17 or 694F. We did not detect cyclin D1 expression or significant changes in the expression levels of cyclin B, CDK2, CDK4, or CDC2 in these clones.


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  		Fig. 3.   Effects of N17, 694F, and Delta p85 on the expressions of cell cycle regulatory molecules. The cells were cultured with IPTG, and total cellular RNA was isolated at the time indicated. Northern blot analysis was performed with 32P-labeled probes indicated. The expression of beta -actin mRNA was examined as a loading control.

Cooperation among Ras, STAT5, and PI3-K Is Required for BCR/ABL-dependent Cell Growth and Survival-- To investigate whether these signaling molecules could support BCR/ABL-dependent cell growth and survival cooperatively, we inducibly expressed two of N17, 694F, and Delta p85 in combination; these clones were designated as K562/N17+694F, K562/N17+Delta p85, and K562/694F+Delta p85, respectively. In these clones, IPTG-induced DN mutants inhibited their respective signaling pathways efficiently (data not shown). As shown in Fig. 4A, IPTG treatment suppressed the growth of K562/N17+694F, K562/N17+Delta p85, and K562/694F+Delta p85 completely. Consistent with these data, the proportion of proliferating cells severely decreased after 5-day IPTG treatment in K562/N17+694F (percentage of the cells in S or G2/M phase at 120 h, 5%), K562/N17+Delta p85 (5%), and K562/694F+Delta p85 (14%). In addition, IPTG treatment evoked severe apoptosis in these clones (percentage of apoptotic cells: K562/N17+694F, 65%; K562/N17+Delta p85, 68%; K562/694F+Delta p85, 59%).


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  		Fig. 4.   Cooperative effects of two DN mutants on BCR/ABL- dependent cell growth and survival. A, the cells of the indicated clones were seeded at a cell density of 50/µl, cultured with or without IPTG, and total number of viable cells was counted by trypan blue dye exclusion method. The results are shown as the mean ± S.D. of triplicate cultures. B, the cells were cultured with or without IPTG for 5 days. DNA content of the cultured cells was examined by PI staining and analyzed on FACSort.

We also performed annexin-V staining and TUNEL assays with flow cytometry after 3-day IPTG treatment (Fig. 5, A and B). Annexin-V staining detects externalization of membrane phosphatidylserine, and TUNEL assay detects DNA fragmentation, both of which are useful methods for detecting cells undergoing apoptosis (23). After 3-day culture with IPTG, N17 slightly yielded annexin-V- and TUNEL-positive populations (percentage of annexin-V- and TUNEL-positive cells: 5 and 7%, respectively), whereas 694F and Delta p85 showed little effect. In contrast, when these DN mutants were expressed simultaneously, the majority of the cultured cells became positive for these staining (percentage of annexin-V- and TUNEL-positive cells in K562/N17+694F, 95 and 93%, respectively; K562/N17+Delta p85, 91 and 78%; K562/694F+Delta p85, 72 and 69%).


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  		Fig. 5.   Characterization of DN mutant-induced apoptosis. A, the cells of the indicated clones were cultured with or without IPTG for 3 days, subjected to TUNEL assay and annexin-V staining, and analyzed on FACSort. B, the cells were cultured with or without IPTG for indicated times and subjected to FACS analysis to measure caspase-3 activities. Activities of caspase-3 were measured by a fluorescence intensity that derived from the caspase-3-cleaved substrate. Height of the right fluorescent peak indicates the degree of caspase-3 activation.

Because apoptosis is triggered by activation of caspases, we examined whether caspase-3, one of key molecules in caspase cascades, was activated during these DN mutant-induced apoptosis. In this assay we utilized here, caspase-3 activities are detected as a fluorescence peak on the right with flow cytometry. As shown in Fig. 5C, neither 694F nor Delta p85 activated caspase-3 during the test period. Additionally, only a limited but detectable degree of caspase-3 activation was induced by IPTG treatment in K562/N17. In contrast, IPTG treatment led to marked caspase-3 activation in K562/N17+694F, K562/N17+Delta p85, and K562/694F+Delta p85. These results suggested that overlapping anti-apoptotic signals were required for BCR/ABL-dependent cell survival and that these DN mutants cooperatively induced apoptosis via caspase-3 activation.

Effects of N17, 694F, and Delta p85 on the Expressions of Bcl-2 and Bcl-XL-- Because Bcl-2 family proteins play central roles in growth factor- or oncogene-induced cell survival, we examined the effects of each DN mutant on the expressions of Bcl-2 and Bcl-XL by Northern blot analysis. As shown in Fig. 6, the expression of Bcl-2 mRNA was suppressed by IPTG treatment in K562/N17, K562/694F, and K562/Delta p85. Additionally, IPTG-induced N17 reduced the expression of Bcl-XL mRNA, whereas 694F and Delta p85 showed little effect on its expression.


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  		Fig. 6.   Effects of N17, 694F, and Delta p85 on the expressions of Bcl-2 and Bcl-XL. The cells were cultured with IPTG, and total cellular RNA was isolated at the time indicated. Northern blot analysis was performed with 32P-labeled probes for Bcl-2 and Bcl-XL. The expression of beta -actin mRNA was examined as a loading control.

N17, 694F, and Delta p85 Sensitize K562 to IFN-alpha - or Dexamethasone-induced Apoptosis-- Next, we examined the effects of each DN mutant on apoptosis induced by therapeutic reagents such as IFN-alpha and dexamethasone. As shown in Fig. 7, the treatment with IFN-alpha was not able to induce apoptosis in K562/Mock regardless of IPTG treatment. In addition, K562/N17, K562/694F, and K562/Delta p85 showed resistance to IFN-alpha under the culture without IPTG. In contrast, when these clones were treated with IPTG, a considerable proportion of the cultured cells underwent apoptosis in response to IFN-alpha (percentage of apoptotic cells: K562/N17, 76%; K562/694F, 45%; K562/Delta p85, 69%). As was the case with IFN-alpha , dexamethasone-induced apoptosis was potentiated by IPTG treatment in these clones but not in K562/Mock (percentage of apoptotic cells: K562/Mock, 5%; K562/N17, 78%; K562/694F, 64%; K562/Delta p85, 69%), suggesting that three independent anti-apoptotic signals from Ras, STAT5, and PI3-K are all required to prevent K562 from IFN-alpha - or dexamethasone-induced apoptosis.


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  		Fig. 7.   Potentiation of IFN-alpha - or Dexamethasone-induced apoptosis by N17, 694F, and Delta p85. The cells were treated with 3000 units/ml IFN-alpha or 100 µM dexamethasone in the presence or presence of IPTG for 4 days. The DNA content of the cultured cells was examined by PI staining and analyzed on FACSort.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

During the last decade, a number of studies have been made to clarify the mechanisms how signaling molecules activated by cytokines or oncogenes regulate the functions and expressions of cell cycle regulatory molecules. Ras was shown to up-regulate the expressions of cyclin D1 and c-myc, to down-regulate the protein expression level of cyclin-dependent kinase (CDK) inhibitor p27Kip1, and to activate cdc25 phosphatases, thereby inducing cell cycle progression from G1 to S phase (for a review, see Ref. 28). In addition, Ras activities influence cell cycle machinery at several phases. Meanwhile, STAT5 was reported to mediate the growth of hematopoietic cells through inducing cyclin D1 and pim-1 (24, 29). PI3-K/Akt was also assumed to promote cell cycle progression through induction of cyclin D3, stabilization of cyclin D1, degradation of p27Kip1 and phosphorylation of p21WAF1 (30-33). These studies indicated that Ras, STAT5, and PI3-K can individually promote cell cycle progression from G1 to S phase. In accord with these results, we found here that Ras, STAT5, and PI3-K each contribute to BCR/ABL-dependent cell growth, although Ras seemed to play the most important role among these molecules. Although Ras and STAT5 have been reported to induce cyclin D1 expression (24, 28, 34), we could not detect any cyclin D1 expression in K562. Therefore, cyclin D2, cyclin D3, and cyclin E were supposed to regulate CDK activities required for G1/S progression (i.e. activities of CDK4, CDK6, and CDK2) instead of cyclin D1 in K562. Under these circumstances, the expressions of cyclin D2 and cyclin D3 were inhibited by either N17, 694F, or Delta p85. These results suggested that the functional role of each signal is not redundant but indispensable for the expressions of cyclin D2 and cyclin D3. Furthermore, as the expression of cyclin E and that of cyclin A, which regulates G2/M phase, were specifically inhibited by N17 and Delta p85, respectively, each signaling molecule was supposed to individually contribute to BCR/ABL-dependent growth through the induction of their unique target gene(s).

Excessive cell cycle progression lacking anti-apoptotic signals has been shown to result in aggressive cell death (as reviewed in Refs. 35 and 36). Therefore, most oncogenic signaling molecules transmit both anti-apoptotic signals and mitogenic signals to their downstream cascades simultaneously. Among these molecules, Ras mediates anti-apoptotic signals, at least in part, by activation of PI3-K (26), although we did not detect this relationship in K562. In addition, Ras induces the expressions of anti-apoptotic molecules, Bcl-2 and Bcl-XL (37). Similarly, STAT5 induces these expressions (38, 39). Meanwhile, PI3-K/Akt exerts anti-apoptotic effects by inhibiting the function of pro-apoptotic molecules, BAD, FKHRL1 (by phosphorylation), and caspases (by degradation) (as reviewed in Ref. 40). In agreement with these anti-apoptotic roles of signaling molecules, BCR/ABL was found to induce expressions of Bcl-2 and Bcl-XL and phosphorylation of Bad in host cells probably through the activation of Ras, STAT5, and PI3-K (34, 41-44). In the present study, BCR/ABL-mediated Bcl-2 expression was disrupted by each of N17, 694F, and Delta p85, implying that cooperation among three signaling molecules was required for maintaining its expression. In addition, when two DN mutants were coexpressed, K562 underwent severe apoptosis, suggesting that the remaining one pathway, i.e. Ras, STAT5, or PI3-K alone, was not able to support the growth or survival of K562. However, because both oncogenic Ras (Ha-RasG12V) and constitutively active STAT5 (1*6-STAT5) were shown to induce Bcl-2 expression and enabled factor-dependent Ba/F3 cells to proliferate under factor-deprived conditions (23, 29), it was possible that BCR/ABL may not activate Ras, STAT5, or PI3-K to the full extent in K562 and possibly in CML cells, and that, for this reason, the simultaneous activation of multiple signaling cascades was necessary for leukemogenic activities of BCR/ABL.

Although the induced expression of N17 alone was sufficient for suppressing Bcl-2 and Bcl-XL expressions, N17 was less effective in inducing apoptosis than N17+694F or N17+Delta p85. In addition, 694F+Delta p85 induced apoptosis as efficiently as N17+694F or N17+Delta p85, whereas Bcl-XL expression was maintained in this clone (data not shown). Together, these data raised a possibility that an additional anti-apoptotic molecule(s) other than Bcl-2 and Bcl-XL, which would be regulated by Ras, STAT5, and/or PI3-K, might also control BCR/ABL- dependent cell survival. Among several candidate molecules, NF-kappa B seemed to be most likely because NF-kappa B was reported to be activated by BCR/ABL and to be involved in the resistance to drug-induced apoptosis in K562 (45, 46).

K562 was shown to be resistant to apoptosis induced by actinomycin D, camptothecin, etoposide, and cycloheximide as well as Fas-induced apoptosis (47, 48). Regarding this mechanism, it has been reported that BCR/ABL-mediated expression of Bcl-XL, activities of protein kinase Ciota, and VLA-5-mediated cell adhesion were involved in these drug-resistance (45, 49-51). In addition, Sluplanek et al. (52) recently demonstrated that STAT5-induced RAD51, a mammalian homologue of the E. coli RecA protein was essentially important for resistance to cisplatin and mitomycin C. In addition, we found here that disruption of one signaling cascade by N17, 694F, or Delta p85 equally sensitized K562 to IFN-alpha - or dexamethasone-induced apoptosis. Because Bcl-2 expression was down-regulated in these clones, Bcl-2 was also supposed to play some role in BCR/ABL-mediated drug-resistance.

In summary, we showed here that Ras, STAT5, and PI3-K pathways cooperatively contribute to BCR/ABL-dependent cell growth and survival in K562 cells. Although STI571 has been shown to be practically effective in a considerable proportion of CML patients, many patients in advanced stage become refractory to this drug as a result of reactivation of BCR/ABL signal transduction (53, 54). Thus, further studies to elucidate the functional network among these signaling molecules would provide more useful information to design new therapeutic strategies that target these molecules and to overcome the drug resistance.

    ACKNOWLEDGEMENTS

We thank Drs. A. Arnold, G. Peters, H. Kiyokawa, E. Harlow, Y. Tsujimoto, and T. Tsujimura for providing the plasmids.

    FOOTNOTES

* This work was supported by grants from the Ministry of Education, Science and Culture of Japan; the Mochida Foundation; the Ichiro Kanehara Foundation; the Uehara Memorial Foundation; the Naito Foundation; and the Japan Medical Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 81-6-6879-3871; Fax: 81-6-6879-3879; E-mail: matumura@bldon.med.osaka-u. ac.jp.

Published, JBC Papers in Press, January 4, 2002, DOI 10.1074/jbc.M111501200

    ABBREVIATIONS

The abbreviations used are: CML, chronic myelogenous leukemia; MAPK, mitogen-activated protein kinase; STAT, signal transducers and activators of transcription; PI3-K, phosphatidylinositol 3-kinase; LacR, Lac repressor; Ab, antibody; IPTG, isopropyl-beta -D-thiogalactopyranoside; TUNEL, terminal deoxynucleotidyltransferase-mediated biotin-dUTP nick end labeling; BCR, breakpoint cluster region; ALL, acute lymphoblastic leukemia; IFN, interferon; DN, dominant negative; PI, propidium iodide; NF-kappa B, nuclear factor kappa B; PBS, phosphate-buffered saline; CDK, cyclin-dependent kinase.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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C. Selleri, J. P. Maciejewski, N. Montuori, P. Ricci, V. Visconte, B. Serio, L. Luciano, and B. Rotoli
Involvement of nitric oxide in farnesyltransferase inhibitor-mediated apoptosis in chronic myeloid leukemia cells
Blood, August 15, 2003; 102(4): 1490 - 1498.
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M. J. Lindemann, M. Benczik, and S. L. Gaffen
Anti-apoptotic Signaling by the Interleukin-2 Receptor Reveals a Function for Cytoplasmic Tyrosine Residues within the Common gamma (gamma c) Receptor Subunit
J. Biol. Chem., March 14, 2003; 278(12): 10239 - 10249.
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J. M. Golas, K. Arndt, C. Etienne, J. Lucas, D. Nardin, J. Gibbons, P. Frost, F. Ye, D. H. Boschelli, and F. Boschelli
SKI-606, a 4-Anilino-3-quinolinecarbonitrile Dual Inhibitor of Src and Abl Kinases, Is a Potent Antiproliferative Agent against Chronic Myelogenous Leukemia Cells in Culture and Causes Regression of K562 Xenografts in Nude Mice
Cancer Res., January 15, 2003; 63(2): 375 - 381.
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N. J. Donato, J. Y. Wu, J. Stapley, G. Gallick, H. Lin, R. Arlinghaus, and M. Talpaz
BCR-ABL independence and LYN kinase overexpression in chronic myelogenous leukemia cells selected for resistance to STI571
Blood, January 15, 2003; 101(2): 690 - 698.
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