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A Bcr/Abl-independent, Lyn-dependent Form of Imatinib Mesylate (STI-571) Resistance Is Associated with Altered Expression of Bcl-2

2004; Elsevier BV; Volume: 279; Issue: 33 Linguagem: Inglês

10.1074/jbc.m402290200

ISSN

1083-351X

Autores

Yun Dai, Mohamed Rahmani, Seth J. Corey, Paul Dent, Steven Grant,

Tópico(s)

Chronic Lymphocytic Leukemia Research

Resumo

The relationship between the Src kinase Lyn and Bcl-2 expression was examined in chronic myelogenous leukemia cells (K562 and LAMA84) displaying a Bcr/Abl-independent form of imatinib mesylate resistance. K562-R and LAMA-R cells that were markedly resistant to induction of mitochondrial dysfunction (e.g. loss of mitochondrial membrane potential, Bax translocation, cytochrome c, and apoptosis-inducing factor release) and apoptosis by imatinib mesylate exhibited a pronounced reduction in expression of Bcr/Abl, Bcl-xL, and STAT5 but a striking increase in levels of activated Lyn. Whereas basal expression of Bcl-2 protein was very low in parental cells, imatinib-resistant cells displayed a marked increase in Bcl-2 mRNA and/or protein levels. Treatment of LAMA-R cells with the Src kinase inhibitor PP2 significantly reduced Lyn activation as well as Bcl-2 mRNA and protein levels. Transient or stable transfection of LAMA84 or K562 cells with a constitutively active Lyn (Y508F), but not with a kinase-dead mutant (K275D), significantly increased Bcl-2 protein expression and protected cells from lethality of imatinib mesylate. Ectopic expression of Bcl-2 protected K562 and LAMA84 cells from imatinib mesylate- and PP2-mediated lethality. Conversely, interference with Bcl-2 function by co-administration of the small molecule Bcl-2 inhibitor HA14-1 or down-regulation of Bcl-2 expression by small interfering RNA or antisense strategies significantly increased mitochondrial dysfunction and apoptosis induced by imatinib mesylate and the topoisomerase inhibitor VP-16 in LAMA-R cells. In marked contrast, these interventions had little effect in parental LAMA84 cells that display low basal levels of Bcl-2. Together, these findings indicate that activation of Lyn in leukemia cells displaying a Bcr/Abl-independent form of imatinib mesylate resistance plays a functional role in Bcl-2 up-regulation and provide a theoretical basis for the development of therapeutic strategies targeting Bcl-2 in such a setting. The relationship between the Src kinase Lyn and Bcl-2 expression was examined in chronic myelogenous leukemia cells (K562 and LAMA84) displaying a Bcr/Abl-independent form of imatinib mesylate resistance. K562-R and LAMA-R cells that were markedly resistant to induction of mitochondrial dysfunction (e.g. loss of mitochondrial membrane potential, Bax translocation, cytochrome c, and apoptosis-inducing factor release) and apoptosis by imatinib mesylate exhibited a pronounced reduction in expression of Bcr/Abl, Bcl-xL, and STAT5 but a striking increase in levels of activated Lyn. Whereas basal expression of Bcl-2 protein was very low in parental cells, imatinib-resistant cells displayed a marked increase in Bcl-2 mRNA and/or protein levels. Treatment of LAMA-R cells with the Src kinase inhibitor PP2 significantly reduced Lyn activation as well as Bcl-2 mRNA and protein levels. Transient or stable transfection of LAMA84 or K562 cells with a constitutively active Lyn (Y508F), but not with a kinase-dead mutant (K275D), significantly increased Bcl-2 protein expression and protected cells from lethality of imatinib mesylate. Ectopic expression of Bcl-2 protected K562 and LAMA84 cells from imatinib mesylate- and PP2-mediated lethality. Conversely, interference with Bcl-2 function by co-administration of the small molecule Bcl-2 inhibitor HA14-1 or down-regulation of Bcl-2 expression by small interfering RNA or antisense strategies significantly increased mitochondrial dysfunction and apoptosis induced by imatinib mesylate and the topoisomerase inhibitor VP-16 in LAMA-R cells. In marked contrast, these interventions had little effect in parental LAMA84 cells that display low basal levels of Bcl-2. Together, these findings indicate that activation of Lyn in leukemia cells displaying a Bcr/Abl-independent form of imatinib mesylate resistance plays a functional role in Bcl-2 up-regulation and provide a theoretical basis for the development of therapeutic strategies targeting Bcl-2 in such a setting. Chronic myelogenous leukemia (CML) 1The abbreviations used are: CML, chronic myelogenous leukemia; RT, reverse transcriptase; FITC, fluorescein isothiocyanate; PI, propidium iodide; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; HA, hemagglutinin; PARP, poly [ADP-ribose] polymerase; bcr, breakpoint cluster region; siRNA, small interfering RNA; dsRNAi, double-stranded RNA interference; DiOC6, 3,3-dihexyloxacarbocyanine; PP2, 4-amino-5-[4-chlorophenyl]-7-[t-butyl]pyrazolo[3,4-d]pyrimidine; PP3, 4-amino-7-phenylpyrazolo[3,4-d]pyrimidine; AIF, apoptosis-inducing factor. is a hematopoietic stem cell disorder in which 90% of patients display a reciprocal translocation involving chromosomes 9 and 22, resulting in what has been designated the Philadelphia (Ph) chromosome. This translocation stems from a head-to-tail fusion of the breakpoint cluster region (bcr) at band q11 of chromosome 22 with the proto-oncogene (c-ABL) at band q34 of chromosome 9. This leads in turn to the expression in virtually all Ph+ CML patients of the chimeric fusion protein p210 Bcr/Abl, a constitutively active tyrosine kinase (1Lugo T.G. Pendergast A.M. Muller A.J. Witte O.N. Science. 1990; 247: 1079-1082Crossref PubMed Scopus (1125) Google Scholar). The etiologic role of the BCR/ABL oncogenic tyrosine kinase in the pathogenesis of CML has been well documented (2McLaughlin J. Chianese E. Witte O.N. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6558-6562Crossref PubMed Scopus (306) Google Scholar, 3Daley G.Q. Van Etten R.A. Baltimore D. Science. 1990; 247: 824-830Crossref PubMed Scopus (1929) Google Scholar). Expression of Bcr/Abl not only contributes to leukemic transformation but also represents a barrier to the successful treatment of this disorder. For example, Bcr/Abl stimulates diverse downstream survival signal transduction pathways, including those related to STAT5 (signal transducer and activator of transcription 5), Ras/Raf/mitogen-activated protein kinase kinase/extracellular signal regulated kinase and phosphatidylinositol 3-kinase/Akt that, collectively, provide affected cells with survival and proliferative advantages (4Nieborowska-Skorska M. Slupianek A. Skorski T. Oncogene. 2000; 19: 4117-4124Crossref PubMed Scopus (19) Google Scholar, 5Sonoyama J. Matsumura I. Ezoe S. Satoh Y. Zhang X. Kataoka Y. Takai E. Mizuki M. Machii T. Wakao H. Kanakura Y. J. Biol. Chem. 2002; 277: 8076-8082Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Next, Bcr/Abl confers resistance to DNA damage triggered by diverse chemotherapeutic drugs and radiation (6Slupianek A. Schmutte C. Tombline G. Nieborowska-Skorska M. Hoser G. Nowicki M.O. Pierce A.J. Fishel R. Skorski T. Mol. 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Reiffers J.J. Shea T.C. Chapuis B. Coutre S. Tura S. Morra E. Larson R.A. Saven A. Peschel C. Gratwohl A. Mandelli F. Ben-Am M. Gathmann I. Capdeville R. Paquette R.L. Druker B.J. Blood. 2002; 99: 3530-3539Crossref PubMed Scopus (1067) Google Scholar, 15Kantarjian H. Sawyers C. Hochhaus A. Guilhot F. Schiffer C. Gambacorti-Passerini C. Niederwieser D. Resta D. Capdeville R. Zoellner U. Talpaz M. Druker B. Goldman J. O'Brien S.G. Russell N. Fischer T. Ottmann O. Cony-Makhoul P. Facon T. Stone R. Miller C. Tallman M. Brown R. Schuster M. Loughran T. Gratwohl A. Mandelli F. Saglio G. Lazzarino M. Russo D. Baccarani M. Morra E. International STI571 CML Study Group N. Engl. J. Med. 2002; 346: 645-652Crossref PubMed Scopus (1835) Google Scholar). Resistance to imatinib mesylate stems from BCR/ABL gene amplification, leading to overexpression of Bcr/Abl protein (16le Coutre P. Tassi E. Varella-Garcia M. Barni R. Mologni L. Cabrita G. Marchesi E. Supino R. Gambacorti-Passerini C. 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Cell. 2003; 112: 831-843Abstract Full Text Full Text PDF PubMed Scopus (561) Google Scholar). Recently, a putatively Bcr/Abl-independent form of imatinib mesylate resistance has been reported by several groups (20Nimmanapalli R. O'Bryan E. Huang M. Bali P. Burnette P.K. Loughran T. Tepperberg J. Jove R. Bhalla K. Cancer Res. 2002; 62: 5761-5769PubMed Google Scholar, 21Donato N.J. Wu J.Y. Stapley J. Gallick G. Lin H. Arlinghaus R. Talpaz M. Blood. 2003; 101: 690-698Crossref PubMed Scopus (596) Google Scholar, 22Donato N.J. Wu J.Y. Stapley J. Lin H. Arlinghaus R. Aggarwal B. Shishodin S. Albitar M. Hayes K. Kantarjian H. Talpaz M. Cancer Res. 2004; 64: 672-677Crossref PubMed Scopus (210) Google Scholar). Specifically, Bcr/Abl-positive CML cells cultured in the continuous presence of imatinib mesylate (20Nimmanapalli R. O'Bryan E. Huang M. Bali P. Burnette P.K. Loughran T. Tepperberg J. Jove R. Bhalla K. Cancer Res. 2002; 62: 5761-5769PubMed Google Scholar, 21Donato N.J. Wu J.Y. Stapley J. Gallick G. Lin H. Arlinghaus R. Talpaz M. Blood. 2003; 101: 690-698Crossref PubMed Scopus (596) Google Scholar), or obtained from certain CML patients who have progressed while receiving imatinib mesylate (22Donato N.J. Wu J.Y. Stapley J. Lin H. Arlinghaus R. Aggarwal B. Shishodin S. Albitar M. Hayes K. Kantarjian H. Talpaz M. Cancer Res. 2004; 64: 672-677Crossref PubMed Scopus (210) Google Scholar), display a decline in Bcr/Abl protein and/or mRNA levels and a corresponding increase in expression/activity of the Lyn and Hck kinase (21Donato N.J. Wu J.Y. Stapley J. Gallick G. Lin H. Arlinghaus R. Talpaz M. Blood. 2003; 101: 690-698Crossref PubMed Scopus (596) Google Scholar, 22Donato N.J. Wu J.Y. Stapley J. Lin H. Arlinghaus R. Aggarwal B. Shishodin S. Albitar M. Hayes K. Kantarjian H. Talpaz M. Cancer Res. 2004; 64: 672-677Crossref PubMed Scopus (210) Google Scholar). Lyn and Hck represent members of the Src tyrosine kinase family and have been implicated in the regulation of cell survival and proliferation, among numerous other functions (23Lionberger J.M. Wilson M.B. Smithgall T.E. J. Biol. Chem. 2000; 275: 18581-18585Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar, 24Bates R.C. Edwards N.S. Burns G.F. Fisher D.E. Cancer Res. 2001; 61: 5275-5283PubMed Google Scholar). Lyn exists in two isoforms, p56 and p53, derived from alternatively spliced mRNAs (25Stanley E. Ralph S. McEwen S. Boulet I. Holtzman D.A. Lock P. Dunn A.R. Mol. Cell. Biol. 1991; 11: 3399-3406Crossref PubMed Scopus (59) Google Scholar). The catalytic activity of Lyn is tightly regulated through tyrosine phosphorylation at the conserved Tyr-508 (26Shangary S. Lerner E.C. Zhan Q. Corey S.J. Smithgall T.E. Baskaran R. Exp. Cell Res. 2003; 289: 67-76Crossref PubMed Scopus (11) Google Scholar). Evidence derived from both murine and human model systems suggests that Lyn is a potentially important downstream target of Bcr/Abl (27Danhauser-Riedl S. Warmuth M. Druker B.J. Emmerich B. Hallek M. Cancer Res. 1996; 56: 3589-3596PubMed Google Scholar); moreover, Lyn has been shown to be activated in Bcr/Abl-positive cells obtained from CML patients in blast crisis as well as in HL-60 human leukemia cells transfected with Bcr/Abl (28Ptasznik A. Urbanowska E. Chinta S. Costa M.A. Katz B.A. Stanislaus M.A. Demir G. Linnekin D. Pan Z.K. Gewirtz A.M. J. Exp. Med. 2002; 196: 667-678Crossref PubMed Scopus (90) Google Scholar). Together, such findings raise the possibility that activation of Lyn can subserve the anti-apoptotic functions of Bcr/Abl, including conditions in which expression of Bcr/Abl is diminished, for whatever reason. Although the role that Lyn plays in protecting cells from lethal stimuli has been examined in some detail (29Grishin A.V. Azhipa O. Semenov I. Corey S.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10172-10177Crossref PubMed Scopus (65) Google Scholar), the functional relationship that exists between this Src kinase and Bcr/Abl remains to be fully elucidated. Moreover, no information exists concerning interactions hyperactivated Lyn might have with Bcl-2 family members. To address these issues, we have examined the molecular profile of human CML cells that have become resistant to imatinib mesylate in association with the loss of Bcr/Abl and activation of the Lyn kinase. Here we report that in such cells activation of Lyn is associated with a pronounced increase in the levels of the anti-apoptotic protein Bcl-2 and that disruption of Lyn activation through either pharmacologic or genetic strategies has a significant functional impact on Bcl-2 expression and resistance to mitochondria-dependent apoptosis. In addition, pharmacological or genetic disruption of Bcl-2 function increases the susceptibility of these resistant cells to lethality of imatinib mesylate or the topoisomerase II inhibitor VP-16. These findings may have implications for the development of new therapeutic strategies directed against leukemia cells exhibiting novel forms of imatinib mesylate resistance. Cells and Reagents—LAMA84 were purchased from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). Imatinib-resistant LAMA84 cells (LAMA-R) were generated by culturing cells in gradually increasing concentrations of imatinib mesylate (beginning at 0.1 μm and increasing in stepwise increments of 0.1 μm) until a level of 1 μm (30Yu C. Krystal G. Dent P. Grant S. Clin. Cancer Res. 2002; 8: 2976-2984PubMed Google Scholar). Parental K562 and imatinib-resistant K562-R cells (generated as described above for LAMA-R cells) were kindly provided by Dr. L. Varticovski (Tufts University School of Medicine, Boston). Cells were cultured in 10% fetal bovine serum/RPMI 1640 medium as described previously (30Yu C. Krystal G. Dent P. Grant S. Clin. Cancer Res. 2002; 8: 2976-2984PubMed Google Scholar). K562-R and LAMA-R were maintained in the medium containing 1 μm imatinib mesylate and were washed free of drug before all experimental procedures. All experiments were performed utilizing logarithmically growing cells (4–6 × 105 cells/ml). Imatinib mesylate (Gleevec, STI-571), provided by Dr. Elizabeth Buchdunger (Novartis Pharmaceuticals, Basel, Switzerland), was dissolved in Me2SO at a stock concentration of 50 mm, stored at –20 °C, and subsequently diluted with serum-free RPMI medium prior to use. 4-Amino-5-[4-chlorophenyl]-7-[t-butyl]pyrazolo[3,4-d]pyrimidine (PP2), a selective inhibitor of the Src family tyrosine kinases (31Wilson M.B. Schreiner S.J. Choi H.J. Kamens J. Smithgall T.E. Oncogene. 2002; 21: 8075-8088Crossref PubMed Scopus (120) Google Scholar), 4-amino-7-phenylpyrazolo[3,4-d]pyrimidine (PP3), a negative control for PP2, and ethyl-2-amino-6-bromo-4-[1-cyano-2-ethoxy-2-oxoethyl]-4H-chromene-3-carboxylate (HA14-1), a cell-permeable and low molecular weight Bcl-2 inhibitory ligand (32Wang J.L. Liu D. Zhang Z.J. Shan S. Han X. Srinivasula S.M. Croce C.M. Alnemri E.S. Huang Z. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7124-7129Crossref PubMed Scopus (1184) Google Scholar, 33An J. Chen Y. Huang Z. J. Biol. Chem. 2004; 279: 19133-19140Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar), were purchased from Calbiochem and Biomol (Plymouth Meeting, PA). cpm-1285 (a cell-permeable Bcl-2-binding peptide derived from the BH3 domain encompassing residues 140–165) and its negative control peptide cpm-1285mt (a mutant in which Leu-151 is replaced by Ala) (34Wang J.L. Zhang Z.J. Choksi S. Shan S. Lu Z. Croce C.M. Alnemri E.S. Korngold R. Huang Z. Cancer Res. 2000; 60: 1498-1502PubMed Google Scholar) were provided by Calbiochem. Etoposide (VP-16), a topoisomerase II inhibitor, was obtained from Sigma. These agents were dissolved in Me2SO as a stock solution and stored at –80 °C. In all experiments, the final concentration of Me2SO did not exceed 0.1%. Transient and Stable Transfection with cDNAs—cDNAs encoding full-length, kinase-defective (K275D, in which Asp is substituted for Lys-275 in the ATP-binding pocket), and constitutively active (Y508F, in which Tyr-508 is substituted in the conserved tail of the C terminus with Phe) Lyn were subcloned into pcDNA3 containing an HA tag (26Shangary S. Lerner E.C. Zhan Q. Corey S.J. Smithgall T.E. Baskaran R. Exp. Cell Res. 2003; 289: 67-76Crossref PubMed Scopus (11) Google Scholar). Bcl-2 cDNA (wild type) in pUSEamp was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). LAMA84 and K562 cells (1 × 106 per condition) were transiently transfected with three forms of Lyn cDNA (i.e. wild type, K275D, and Y508F) and Bcl-2 cDNA, respectively, using the Amaxa Nucleofector Device (program T-16) with Cell Line-specific Nucleofector Kit V (Amaxa GmbH, Cologne, Germany). Alternatively, K562 and LAMA84 cells were transfected with K275D and Y508F constructs as well as their empty vector counterparts (pcDNA3), respectively, and stably transfected clones were selected by limited dilution using G418. Bcl-2 RNA Interference and Antisense Oligonucleotides—1 × 106 LAMA84 and LAMA-R cells were transfected with 2 μg of Bcl-2-annealed dsRNAi oligonucleotide (5′-GUACAUCCAUUAUAAGCUGTT-3′/3′-TTCAUGUAGGUAAUAUUCGAC-5′, Orbigen, San Diego, CA) and SignalSilence Control siRNA (Cell Signaling, Beverly, MA), respectively, using the Amaxa Nucleofector Device (program T-16, Kit V). Alternatively, LAMA84 and LAMA-R cells were transfected with 5 μgof a Bcl-2 antisense oligonucleotide (5′-TCTCCCAGCGTGCGCCAT-3′, G3139, Calbiochem) or a scrambled control oligonucleotide (5′-TACCGCGTGCGACCCTCT-3′, G3622, Calbiochem) as described for transfection with dsRNAi. Transfection efficiency (>80% at 24 h post-transfection) was monitored by using fluorescein-labeled dsRNAi or antisense oligonucleotide and flow cytometric analysis. Bcl-2 protein levels were determined by Western blot analysis. Apoptosis and Viability—The extent of apoptosis was evaluated by annexin V-FITC staining and flow cytometry. Briefly, 1 × 106 cells were stained with annexin V-FITC (Pharmingen) and 5 μg/ml propidium iodide (PI, Sigma) in 1× binding buffer (10 mm Hepes/NaOH, pH 7.4, 140 mm NaOH, 2.5 mm CaCl2) for 15 min at room temperature in the dark. The samples were analyzed by flow cytometry within 1 h to determine the percentage of cells displaying annexin V+ (early apoptosis) or annexin V+/PI+ staining (late apoptosis). The ViaCount assay was performed to evaluate cell viability in the experiments involving transient transfection. 1 × 105 cells were stained with Guava ViaCount Reagent, and the percentage of viable cells was determined using a Guava Personal Cytometer (Gauva Technologies, Hayward, CA) as per the manufacturer's instructions. The ViaCount assay distinguishes between viable and nonviable cells, based on the differential permeability of DNA-binding dyes in the ViaCount Reagent, and was found to correlate closely with the results of annexin/PI staining. Mitochondrial Membrane Potential (Δψm)—2 × 105 cells were stained with 40 nm 3,3-dihexyloxacarbocyanine (DiOC6; Molecular Probes Inc., Eugene, OR) in phosphate-buffered saline at 37 °C for 20 min and then analyzed by flow cytometry. The percentage of cells exhibiting decreased level of DiOC6 uptake, which reflects loss of Δψm, was determined using FACScan from BD Biosciences. Cell Growth and Survival (MTT Assay)—5 × 104 (in 100 μl volume)/well cells were seeded into 96-well plates and incubated with 20 μl of CellTiter 96® AQueous One Solution (Promega, Madison, WI) as per the manufacturer's instructions, and the absorbance at 490 nm was recorded using a 96-well plate reader (Molecular Devices, Sunnyvale, CA). Western Blot—Whole-cell pellets were lysed in SDS sample buffer, and 30 μg of protein for each condition was subjected to Western blot analysis following the procedures described in detail previously (35Dai Y. Yu C. Singh V. Tang L. Wang Z. McInistry R. Dent P. Grant S. Cancer Res. 2001; 61: 5106-5115PubMed Google Scholar). Where indicated, the blots were reprobed with antibodies against β-actin (rabbit polyclonal, Transduction Laboratories, Lexington, KY) or α-tubulin (mouse monoclonal, Calbiochem) to ensure equal loading and transfer of proteins. The following antibodies were used as primary antibodies: phospho-Bcr (Tyr-177) antibody (rabbit polyclonal, Cell Signaling), c-Abl antibody (mouse monoclonal, Santa Cruz Biotechnology, Santa Cruz, CA), STAT5 antibody (rabbit polyclonal, Santa Cruz Biotechnology), Bcl-xL antibody (rabbit polyclonal, Cell Signaling), anti-PARP (poly [ADP-ribose] polymerase, mouse monoclonal, Biomol), phospho-Lyn (Tyr-507) antibody (rabbit polyclonal, Cell Signaling), Lyn antibody (rabbit polyclonal, Cell Signaling), anti-human Bcl-2 oncoprotein (mouse monoclonal, Dako, Carpinteria, CA), Bax antibody (rabbit polyclonal, Santa Cruz Biotechnology), XIAP antibody (mouse monoclonal, Transduction Laboratories), phospho-CrkL (Tyr-207) antibody (rabbit polyclonal, Cell Signaling), CrkL (32H4) antibody (mouse monoclonal, Cell Signaling), phospho-Hck (Tyr-411) antibody (rabbit polyclonal, Santa Cruz Biotechnology), anti-Hck antibody (rabbit polyclonal, Upstate Biotechnology, Inc.), and HA probe (rabbit polyclonal, Santa Cruz Biotechnology). In some cases, the density of blots was quantified using FluoChem 8800 Imaging System (Alpha Innotech, San Leandro, CA) and VideoTesT-Master software (VideoTesT, Ltd., St. Petersburg, Russia). Translocation of Bax, Cytochrome c, and AIF—4 × 106 cells were washed in phosphate-buffered saline and lysed by incubating for 30 s in lysis buffer (75 mm NaCl, 8 mm Na2HPO4, 1 mm NaH2PO4, 1 mm EDTA, and 350 μg/ml digitonin). After centrifuged at 12,000 × g for 1 min, the supernatant (cytosolic fraction) was collected in an equal volume of 2× sample buffer, and the pellet (mitochondria-rich fraction) was lysed by sonication in 1× SDS sample buffer. For both cytosolic and pellet fractions, the proteins were quantified, and 30 μg of protein per condition was separated by 15% SDS-PAGE and subjected to Western blot, as described above, using Bax antibody, cytochrome c antibody (mouse monoclonal, Santa Cruz Biotechnology), and AIF antibody (mouse monoclonal, Santa Cruz Biotechnology) as primary antibody. RT-PCR—Total RNA was isolated from 1 × 106 cells using RNeasy mini kit (Qiagen, Valencia, CA) with QIAshredder spin column (Qiagen) as per the manufacturer's instructions. 1 μg per condition of total RNA was subjected to RT-PCR using One-step RT-PCR kit (Qiagen) and PTC-200 Peltier Thermal cycler (MJ Research, Reno, NV). The primers (forward, 5′-CGACTTCGCCGAGATGTCCAGGCAG-3′; reverse, 5′-GACCCACGGATAGACCCGGTGTTCA-3′) were used for Bcl-2. RT-PCR was performed under the conditions as follows: reverse transcription at 50 °C for 30 min, initial PCR activation step at 95 °C for 15 min, three-step cycling (denaturing at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 1 min) for 30 cycles, and final extension at 72 °C for 10 min. The reactions were run, in parallel, for actin as endogenous control. PCR products of Bcl-2 (388 bp) were analyzed in 2% agarose gel with ethidium bromide. Real Time Quantitative RT-PCR—Total RNA was prepared as described above. The real time RT-PCR was performed on the ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA) using the TaqMan One-step PCR Master Mix Reagents kit (P/N, 4309169). All the samples were tested in triplicate under the conditions recommended by the fabricant. The cycling conditions are as follows: 48 °C/30 min; 95 °C/10 min; and 40 cycles of 95 °C/15 s and 60 °C/1 min. The cycle threshold was determined to provide the optimal standard curve values (0.98 to 1.0). The probes (5′-CCTGGTGGACAACATCGCCCTGT-3′) and primers for Bcl-2 (forward, 5′-CCTGGTGGACAACATCGCCCTGT-3′; reverse, 5′-GCCGGTTCAGGTACTCAGTCAT-3′) were designed using the Primer Express 2.0 version. The probes were labeled at the 5′ end with 6-carboxyfluorescein and at the 3′ end with 6-carboxytetramethylrhodamine. Ribosomal RNA (18 S rRNA) from the Pre-developed TaqMan Assay Reagents (P/N, 4310893E) was used as endogenous control. Statistical Analysis—For analysis of apoptosis, Δψm, MTT, viability, and real time quantitative RT-PCR, values represent the means ± S.D. for at least three separate experiments performed in triplicate. The significance of differences between experimental variables was determined by using the Student's t test. Imatinib-resistant CML Cells Exhibit Loss of Bcr/Abl and Down-regulation of Bcr/Abl Downstream Targets—The response of K562 and LAMA84 cells and their resistant counterparts (K562-R and LAMA-R) to imatinib mesylate was compared. Following an exposure to 0.5–10.0 μm imatinib, K562 (48 h) and LAMA84 cells (24 h) displayed progressive increases in annexin V/PI positivity, whereas their resistant counterparts were minimally affected (Fig. 1, A and B). Thus, both the K562-R and LAMA-R cell lines displayed marked resistance to imatinib mesylate-mediated lethality. Attempts were then made to establish the basis for imatinib mesylate resistance in the two resistant cell lines. Parental K562 and LAMA84 cells, as well as their resistant counterparts, were exposed to 1 μm imatinib mesylate for 48 (K562 and K562-R) and 24 h (LAMA84 and LAMA-R), after which Western blot analysis was performed. As shown in Fig. 1C, both K562-R and LAMA-R cells displayed a dramatic reduction in levels of total and phosphorylated Bcr/Abl, as well as markedly diminished expression of Stat5 and Bcl-xL, well described Bcr/Abl downstream targets (36Horita M. Andreu E.J. Benito A. Arbona C. Sanz C. Benet I. Prosper F. Fernandez-Luna J.L. J. Exp. Med. 2000; 191: 977-984Crossref PubMed Scopus (325) Google Scholar), compared with parental cells. Treatment of both sensitive and resistant cells with imatinib mesylate resulted in reductions in Bcl-xL levels, whereas expression of the inhibitor of apoptosis protein XIAP was only diminished following imatinib mesylate exposure in sensitive cells. Consistent with the results shown in Fig. 1, A and B, imatinib mesylate-induced PARP cleavage was largely abrogated in K562-R and LAMA-R cells. Thus, both K562-R and LAMA-R cells appeared to exhibit a Bcr/Abl-independent form of imatinib mesylate resistance, analogous to that previously described by Donato et al. (21Donato N.J. Wu J.Y. Stapley J. Gallick G. Lin H. Arlinghaus R. Talpaz M. Blood. 2003; 101: 690-698Crossref PubMed Scopus (596) Google Scholar, 22Donato N.J. Wu J.Y. Stapley J. Lin H. Arlinghaus R. Aggarwal B. Shishodin S. Albitar M. Hayes K. Kantarjian H. Talpaz M. Cancer Res. 2004; 64: 672-677Crossref PubMed Scopus (210) Google Scholar) in K562 cells and patient samples. Bcr/Abl-independent Imatinib Resistance Is Associated with Marked Activation of Lyn and Increased Expression of Bcl-2— Expression of phosphorylated Bcr/Abl and related proteins in resistant cells was examined next. As shown in Fig. 2A, levels of phospho-Bcr/Abl (activated) were dramatically reduced in K562-R and LAMA-R compared with their parental counterparts. Furthermore, consistent with earlier results involving K562 cells (21Donato N.J. Wu J.Y. Stapley J. Gallick G. Lin H. Arlinghaus R. Talpaz M. Blood. 2003; 101: 690-698Crossref PubMed Scopus (596) Google Scholar), phosphorylation of the Src kinase Lyn was markedly increased in both resistant cell lines

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