Comparative Proteomic Analysis of Chronic Myelogenous Leukemia

Comparative Proteomic Analysis of Chronic Myelogenous Leukemia Cells: Inside the Mechanism of Imatinib Resistance Germano Ferrari, Roberta Pastorelli, Francesca Buchi, Elena Spinelli, Antonella Gozzini, Alberto Bosi, and Valeria Santini* University of Florence, AUO Careggi, Department of Hematology, Florence, Italy Received July 26, 2006

Abstract: Imatinib is the first molecular targeted therapy that has shown clinical success, but imatinib acquired resistance, although a rare event, is critical during the therapy of chronic myelogenous leukaemia (CML). With the aim of better understanding the molecular mechanisms accompanying acquisition of resistance to this drug, a comparative proteomic approach was undertaken on CML cell lines LAMA 84 S (imatinib sensitive) and LAMA 84 R (imatinib resistant). Forty-four differentially expressed proteins were identified and categorized into five main functional classes: (I) heat shock proteins and chaperones; (II) nucleic acid interacting proteins (binding/ synthesis/stability); (III) structural proteins, (IV) cell signaling, and (V) metabolic enzymes. Several heat shock proteins known to complex Bcr-Abl were overexpressed in imatinib resistant cells, showing a possible involvement of these proteins in the mechanism of resistance. HnRNPs also resulted in being up-regulated in imatinib resistant cells. These proteins have been shown to be strongly and directly related to Bcr-Abl activity. To our knowledge, this is the first direct proteomic comparison of imatinib sensitive/resistant CML cell lines.

population has increased proliferation but retains the ability to differentiate. During the following accelerated phase, cell maturation is gradually lost. Finally, the acute phase, or blast crisis, is characterized by the appearance of immature cells (either lymphoid or myeloid blasts) in peripheral blood and bone marrow,8 paralleled by acquisition of further molecular abnormalities.9

Keywords: CML; imatinib resistance; proteomics

We characterized by means of a proteomic approach the phenotype of Bcr-Abl positive CML cell lines LAMA 84 S (imatinib sensitive) and LAMA 84 R (imatinib resistant). Imatinib resistance is induced in this cell line by BCR-ABL gene amplification. Total protein extracts were separated by twodimensional electrophoresis (2-DE), and 2-D gels were compared to determine differentially expressed proteins possibly directly involved in the acquisition of imatinib resistance. Matrix assisted laser desorption ionization time--of-flight mass spectrometry (MALDI-TOF) was used for protein identification.

1. Introduction Chronic myelogenous leukaemia (CML) was the first neoplasia associated with a specific chromosomal abnormality: the Philadelphia chromosome (Ph), originating from a reciprocal translocation between the long arms of chromosomes 9 and 22 [t(9;22)(q34;q11)]. This translocation produces the oncogenic fusion protein Bcr-Abl, with a constitutive tyrosine kinase activity with maintained autophosphorylation and substrate activation.1 The presence and activity of Bcr-Abl is necessary and sufficient to determine neoplastic transformation of hematopoietic multipotent cells. Transformed cells are endowed with proliferative advantages: blocking of apoptosis, genome instability, and suppression of normal hematopoiesis is induced as a consequence.2-4 Different essential pathways such as cytoskeletal organization, cell mitosis, and apoptosis are modified by the presence of the Bcr-Abl protein.5-7 Classically, CML shows a characteristic three-phase clinical history. In the initial chronic phase, the t(9;22) positive cell * Corresponding author. E-mail: [email protected]. 10.1021/pr0603708 CCC: $37.00

 2007 American Chemical Society

Specific drugs with anti-phosphotyrosine kinase activity have been synthesized to contrast the oncogenic properties of BcrAbl. Imatinib mesylate (Glivec, STI571, Novartis Inc., Basel, Switzerland) is an effective therapy for chronic phase CML. Its inhibiting activity results from the block of binding ATP to the active pocket of the tyrosine kinase, leading to selective suppression of Brc-Abl positive cell proliferation.10,11 Patients in chronic phase CML have a good response to imatinib (95% complete remission), but some may result in a refractory or loose clinical response because of innate or acquired resistance, caused by amplification of the BCR-ABL genomic locus or more frequently by point mutations within the kinase domain of BCR-ABL, which prevent imatinib binding.12,13 Modifications in cell metabolism determined by the development of imatinib resistance and the detailed molecular mechanisms involved in this event still remain unclear.

2. Materials and Methods 2.1. Cell Culture. Bcr-Abl positive cell lines LAMA 84 (imatinib sensitive and resistant, referred to in this paper as LAMA 84 S and LAMA 84 R, respectively, kindly provided by Prof. Gambacorti Passerini)14 were grown in RMPI 1640 medium supplemented with 10% FCS. Cell viability was assessed by trypsin blue dye exclusion test. Cells were harvested in exponential growth phase by low-g centrifugation and washed twice in cold PBS orthovanadate and protease inhibitor cocktail (Roche Molecular Biochemicals, Indianapolis, IN). Journal of Proteome Research 2007, 6, 367-375

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Proteomic Analysis of Myelogenous Leukemia Cells

technical notes

Figure 1. CML proteome (A). Bidimensional gels of LAMA 84 S (left) and LAMA 84 R (right) total cell proteins separated by pH 3-11 nonlinear gradients. Some differentially abundant protein spots are highlighted. (B) Statistical reproducibility: intrasample scatter plots v/v of the three gels considered in this analysis for LAMA 84 S (left) and LAMA 84 R (right)

2.2. 2-D Electrophoresis. Cells were directly lysed in reswelling buffer (7 M UREA, 2% chaps, 1% DTE) and incubated for 1 h at room temperature vigorously stirring on a vortex every 10 min. Proteins were purified by ultracentrifugation (200 000g, 1 h, 4 °C), the pellet was discarded, and the protein concentration was determined by Bradford assay. Two hundred micrograms of total cell protein extracts was brought to a final volume of 125 µL with reswelling buffer and 2% v/v carrier ampholyte pH 3-11 nonlinear (Amersham Biosciences, Uppsala, Sweden), and traces of bromophenol blue were added. Proteins were adsorbed overnight onto pre-cast immobilized pH gradient strips (7 cm; pH 3-11, nonlinear Amersham Biosciences, Uppsala, Sweden), using the Immobiline Dry-Strip Reswelling Tray (Amersham Biosciences, Uppsala, Sweden). Isoelectrofocusing (IEF) was run for a total of 20 000 V h T in six discrete steps (300 V for 1 h, 500 for 1 h, 1200 for 1 h, 3200 for 1 h, 5000 V for 2 h, and 8000-20 000 V h T) on an IPG-Phor 368

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IEF apparatus (Amersham Biosciences, Uppsala, Sweden). The IPG strips were equilibrated prior to the second dimension with a two-step procedure as described elsewhere.15 SDS-PAGE was run on 12% polyacrylamide self-casted gels (7 cm × 6 cm, 1 mm thick); 2-D gels were stained by Colloidal Comassie G250 as described.16 2.3. Image Analysis and 2-D Gel Comparison. 2-D gel images were acquired by a modified UMAX Utah 1100 Image Scanner (Amersham Biosciences, Uppsala, Sweden) at 1200 dpi of resolution. Gels were analyzed and compared by the software Image Master Platinum Ed. (Amersham Biosciences, Uppsala, Sweden). Protein abundance was measured evaluating the staining volume of the spots identifying the protein. Gels were run in triplicate for each sample. Scatter plots v/v for the matching spot pools were obtained to verify the degree of reproducibility among the three replicates of each sample (Figure 1B); cor-

technical notes relation values superior to 0.9 were considered as significant for the analysis. 2.4. In Gel Digestion and Mass Spectrometry. Spots to be identified were excised from the gels, destained by stirring twice in Ammonium Bicarbonate 0.1 M/acetonitrile ) 1:1 for 20 min, washed with acetonitrile, and allowed to dry at room temperature. Digestion buffer containing 50 mM ammonium bicarbonate and 10 ng/µL of Sequencing Grade Modified Trypsin (Promega, Madison, WI) was added on each dried spot, and in-gel digestion was performed for 2 h at 37 °C. The resulting peptides were treated by Zip-Tip C18 (Millipore), directly eluted by 2 µL of a Matrix solution (5 g/L 2,5-dihydroxybenzoic acid in 50% ACN, 0.1%TFA), onto a 384 position, 400 µm L Anchor Chip Target (Bruker Daltonics, Bremen, Germany), and allowed to dry at room temperature. Mass spectra for peptide mass fingerprinting were acquired in reflectron positive ion mode on Ultraflex MALDI-TOF/TOF mass spectrometer (matrix assisted laser desorption/ionization time-of-flight, Bruker Daltonics, Bremen, Germany), with an average of 100 laser shots/spectrum. External calibrations were performed using a 1000-4000 Da Peptide Mix (Bruker Daltonics, Bremen, Germany). Experimental peptide mass fingerprintings in the range of 600-3500 Da were compared with NCBI protein database by the software MASCOT (www.matrixscience.org). Confirmatory fragmentation analysis (MS/MS) was performed when needed. Investigation on protein function was conducted with NCBI tools (Conserved Domain Database, CDD)17 or UniProt/ TrEMBL databases. 2.5. Western Blotting. The protein concentration in the supernatants was determined, and 30 µg aliquots of each sample were boiled for 10 min, in the presence of 100 mM 2-mercaptoethanol, before being separated by SDS-PAGE in a 12.5% polyacrylamide gel and then transferred onto nitrocellulose membranes (Hybond-ECL; Amersham) by electroblotting. Specific protein expression was determined by incubating membranes in PBS containing 0.1% Tween 20 and 5% BSA (T-PBS/5% BSA; 3-5 h at room temperature) and then in a 1:1000 dilution of polyclonal anti-Hsp 90, anti-Hsp 60, antihn-RNPF/H, and anti-hnRNPK antibody (Santa Cruz Biotechnology) in T-PBS/5% BSA (16-18 h at 4 °C). To verify equal loading of samples per lane, β-tubulin expression was determined by stripping and incubating the same membranes in T-PBS/5% BSA (3-5 h at room temperature) and then in a 1:1000 dilution of a polyclonal anti-β tubulin antibody (Upstate) in T-PBS/5% BSA (16-18 h at 4 °C). Secondary antibodies, horseradish peroxidase conjugated, were anti-rabbit IgG (Sigma). Antibody coated protein bands were visualized by ECL chemiluminescence detection (Amersham, Buckinghamshire, UK).

3. Results and Discussion 3.1. Proteomic Comparison between LAMA 84 S and LAMA 84 R Cell Lines. Total cell protein extracts were separated by 2-D electrophoresis on pH 3-11 IPG strips and the bidimensional map of LAMA 84 S as compared to that of LAMA 84 R (Figures 1A and 2). After colloidal Coomassie blue staining,16 an average number of 838 and 787 spots were detected in LAMA 84 S and R, respectively. Gels were run in triplicate, and the spot staining volume parameter was used as a measure of the spot abundance. In sample reproducibility was tested by scatter plots v/v (Figure 1B), and only typical spots present in all three gels according

Ferrari et al.

to the scatter plot analysis were considered for the following steps (Figure 2). The differential proteomic pattern between the two cell lines was composed of 84 protein spots that were excised from the gel and analyzed by peptide mass fingerprinting (PMF) for protein identification. Mass spectrometry analysis allowed us to identify the characteristic protein spots as 44 unique proteins. To more easily interpret the significance of proteomic modifications between CML cell sensitive and resistant to imatinib, we thought to cluster proteins in functional classes. We thus divided differentially expressed proteins into five main functional classes reported in Table 1. 3.2. Class I: Heat Shock Proteins and Chaperones. Eleven identified proteins could be assembled into the heat shock protein and chaperone class (Table 1). Three proteins of this class were found to be more or exclusively expressed in LAMA 84 S cells: tumor rejection antigen-gp96, stress-induced phosphoprotein 1 Hsp70/Hsp90 organizing protein also called P60HOP or STI1, and a 27 kDa nuclear chloride channel. The other eight identified proteins belonged exclusively (heat shock 70 kDa protein 8 isoform 2 variant, heat shock 70 kDa protein 8 isoform 1, chaperonin 60 kDa, proteasome 26S ATPase subunit 5, chaperonin containing TCP1, and subunit 5 epsilon) or in an overexceeding amount (heat shock 90 kDa protein 1 β and VCP protein, KIAA0002) to the LAMA 84 R cell proteome. In detail, heat shock 90 kDa protein 1-βand KIAA0002 (a member of the TCP-1/cpn60 chaperonin family) proteins were upregulated more than 2-fold in LAMA R. Valosin containing protein (VCP), a protein known to bind HSP90 and play a chaperonin role beside a structural one,18,19 was also ca. 2-fold upregulated in LAMA R. VCP is a member of a family that includes putative ATP binding proteins involved in vesicle transport and fusion, 26S proteasome function, and assembly of peroxisomes. VCP, as a structural protein, is associated with clathrin and heat shock protein HSP70 to form a complex. VCP has been implicated in a number of cellular events that are regulated during mitosis, including homotypic membrane fusion, spindle pole body function, and ubiquitin-dependent protein degradation.20,21 Five chaperone proteins, as mentioned previously, were identified exclusively in LAMA R. These specific proteins are heat shock 70 kDa protein 8 isoforms 1 and 2, a 60 kDa chaperonine, a TCP1 containing chaperonine, and proteasome 26S ATPase subunit 5. It has been shown that acetylation and consequent inhibition of HSP90 function leads to increased sensitivity of CML cells to imatinib, due to ubiquitination and proteosomal degradation of Bcr-Abl protein.22-24 Bcr-Abl is by itself sufficient to induce leukemic transformation, and its mode of action is multifaceted, as it consists in provoking increased proliferation via tyrosine kinase activity but also in decreasing apoptosis and inducing chromosome instability.25 Bcr-Abl is a client of HSP 90, and the chaperonin was demonstrated to protect the oncogenic protein from degradation. Geldanamycin (specific inhibitor of Hsp90) treatment results in fact in a sensitization of Bcr-Abl positive leukemia cells to cytotoxic chemotherapy.26 The heat shock protein 90 antagonist was able to destabilize the Bcr-Abl/HSP90 chaperone complex.27 Clinical trials are ongoing with 17 aag/geldanamycin in CML refractory or resistant to imatinib.28,29 The relevance of our observations lies in the fact that not only did we find an increase in HSP 90 expression in imatinib resistant cells but also in the expression of other members of Journal of Proteome Research • Vol. 6, No. 1, 2007 369


technical notes

Figure 2. Differential proteomic analysis. Spots present in both LAMA 84 S and LAMA 84 R with different staining volume are circled and labeled. Identifications are provided. Istograms show the different values of average staining volumes; error bars display the variability of the value among the replicates for the considered spots.

the same family of heat shock proteins and of several chaperone proteins (VCP protein, KIAA0002, heat shock 70 kDa protein 8 isoforms 1 and 2, a 60 kDa chaperonine, a TCP1 containing chaperonine, and proteasome 26S ATPase subunit 5). This finding is extremely important because it sheds lights onto what could be considered a new general mechanism of acquired resistance to chemotherapy. The enhanced expression of chaperone proteins could be a shared characteristic of malignancies bearing an oncogene product and could therefore constitute a target for therapy. It seems evident that not a unique heat shock protein, as indicated in previous studies, but a series of proteins with similar functions and roles must be druggable to overcome completely their cotransforming effects. Therefore, not only the HSP 90 specific inhibitor geldanamycin but a series of compounds or synthetic molecules targeting common domains of chaperones should be envisaged as future therapeutic tools. The 2-DE proteomic approach we applied was apparently extremely efficacious in pointing out molecules involved in conferring the resistant phenotype. 370


The heat shock protein complex is known to interact with Bcr-Abl proteins.30 Heat shock protein 70, recently described also as an inhibitor of apoptosis,31 was suggested to be overexpressed in Bcr-Abl positive, imatinib resistant cells, possibly also contributing to the resistant phenotype.32 It has also been shown that high ectopic expression of HSP 70 in acute leukaemia cells leads to resistance to cytarabine, etoposide, and TRAIL induced apoptosis by interference with preand post mitochondrial apoptosis.33,34 The same authors suggested HSP70 inhibition as a strategy to enhance the imatinib sensitizing effects of the HSP90 blockade.33 All these observations are consistent with our findings. Quite surprisingly, STI-1 (Hop/p60), tumor rejection antigengp96, and the 27 kDa nuclear chloride channel are expressed at higher levels in LAMA 84 S. This seems to be in contrast with the observation of the significant increase of several important Hsp proteins in LAMA 84 R and deserves some consideration. Molecular chaperones undergo multistep assembly, and Hop, which binds both Hsp70 and Hsp90, can facilitate the

technical notes

Ferrari et al.

Table 1. Differentially Expressed Proteins in LAMA 84 S and LAMA 84 R id

1 2 3 4 5 6 7 8 9 10 11

gi|61656607 gi|15215418 gi|48257098 gi|5803181 gi|4588526 gi|62896815 gi|5729877 gi|40789055 gi|49522865 gi|24497435 gi|24307939

1 2 3 4 5 6 7 8

gi|4504865 gi|52627149 gi|76780063 gi|48145673 gi|37078490 gi|55958544 gi|460789 gi|3041664

9 10

gi|54696064 gi|4503571

1 2 3 4 5 6

gi|48257098 gi|32015 gi|37852 gi|34234 gi|54696574 gi|15277503

1 2 3 4 5

gi|48255891 gi|67464424 gi|4099506 gi|58530845 gi|33875631

1 2 3 4 5

gi|35505 gi|41350401 gi|20070125 gi|31645 gi|51479152

6 7 8 9 10 11

gi|62896585 gi|860986 gi|4503571 gi|55925942 gi|56081766 gi|24159117

a

name

Class I: Chaperones/Heat Shock Proteins tumor rejection antigen (gp96) 1 heat shock 90 kDa protein 1β valosin containing protein stress-induced phosphoprotein 1 nuclear chloride channel heat shock 70 kDa protein 8 isoform 2 variant heat shock 70 kDa protein 8 isoform 1 KIAA0002 chaperonin proteasome 26S ATPase subunit 5 chaperonin containing TCP1, subunit 5 (epsilon) Class II: Nucleic acid binding/Synthesis/Stability KH-type splicing regulatory protein TRF2 interacting telomeric RAP1 protein heterogeneous nuclear ribonucleoprotein F heterogeneous nuclear ribonucleoprotein H1 far upstream element binding protein 1 heterogeneous nuclear ribonucleoprotein K transformation upregulated nuclear protein deoxyuridine 5′-triphosphate nucleotidohydrolase, mitochondrial precursor (dUTPase) (dUTP pyrophosphatase) eukaryotic translation initiation factor 3, subunit 2β, 36 kDa enolase 1 Class III: Structural Proteins valosin containing protein R-tubulin vimentin laminin binding protein actin, γ1 ACTB protein Class IV: Cell Signaling protein kinase C substrate 80K-H isoform 2 chain A, 14-3-3 protein epsilon (human) complexed to peptide erbB3 binding protein EBP1 zyxin ANP32A protein Class V: Metabolic Enzymes pyruvate kinase migration-inducing gene 10 protein prolyl 4-hydroxylase, β subunit glyceraldehyde-3-phosphate dehydrogenase ATP synthase, H+ transporting, mitochondrial F0 complex, subunit d, isoform b adenylyl cyclase-associated protein variant protein disulfide isomerase enolase 1 purine nucleoside phosphorylase phosphoglycerate mutase 1 chain A, site specific mutant (H64a) of human carbonic anhydrase II at high resolution

MW

pI

sequence coverage (%)

vol S/Ra

92637 83638 71660 63381 27333 53609 71138 61108 61229 48510 60201

4.77 4.97 4.94 6.40 5.02 5.62 5.37 5.54 5.70 7.11 5.45

46 36 33 31 19 46 54 44 45 43 41

2.492 0.494 0.514 S S R R 0.456 R R R

73542 44446 46069 49455 67645 47812 51396 27045

6.84 4.64 5.38 5.79 7.18 5.46 5.13 9.65

44 21 27 37 32 52 48 40

S 1.223 0.572 0.366 R R R R

36976 47566

5.38 7.01

60 47

0.842 5.909

71660 50685 53724 31916 42192 40620

4.94 4.95 5.06 4.84 5.31 5.55

49 47 35 38 51 29

0.514 1.621 3.193 3.348 1.514 R

60348 26954 38391 62730 24195

4.34 4.92 7.15 6.22 4.75

32 39 62 28 37

1.530 1.763 0.593 R 0.315

58551 44614 57578 36244 15834

7.58 8.30 4.76 8.26 6.60

64 61 27 53 60

2.533 1.206 1.229 1.905 S

51969 57141 47566 32381 28928 29233

8.07 6.10 7.01 6.45 6.67 6.84

44 35 47 30 72 48

0.884 0.852 5.909 R R 0.311

S ) Identified only in LAMA 84 S. R ) Identified only in LAMA 84 R.

progression through the intermediate stages of assembly. Through its ability to simultaneously bind both Hsp70 and Hsp90, Hop serves as an adaptor to coordinate the recruitment of both proteins.35-37 Hop can also inhibit Hsp90 clientstimulated ATPase activity. Hop does not possess independent chaperone activity, so there could be an additional Hop activity other than passive binding to Hsp70 and Hsp90, as has been hypothesized from evidence gathered studying glucocorticoid receptor function in vivo. Post-translational modification of Hop could also alter the equilibrium of its activity, modulating Hsp 70 and Hsp 90 function. On the basis of these recent findings, one could speculate over a regulatory role of Hop in Hsp complex interaction with Bcr-Abl, but indeed, deeper investigations are required to interpret the loss of expression of Hop demonstrated in LAMA 84 R cells.

The effective differential expression of some of the previously mentioned proteins (namely, Hsp 90 and Hsp60) was checked by SDS-PAGE and Western blotting with specific antibodies. The pattern of expression demonstrated confirmed the data of 2-D gels (Figure 3). 3.3. Class II: Nucleic Acid Binding/Synthesis/Stability. Ten proteins of this class were differentially expressed in the bidimensional maps of the two cell lines (Table 1). Enolase 1 is a glycolitic enzyme that has been shown also to bind to an element acting on gene control and located in the c-myc promoter.38 The transcriptional regulation activity of this protein, more than its function in glycolysis, seems to be most relevant in the acquisition of imatinib resistance. This protein was more than 5-fold overexpressed in the LAMA S cell line, and the fact that enolase1 has been shown to have growth and Journal of Proteome Research • Vol. 6, No. 1, 2007 371


Figure 3. Different abundance of several identified proteins confirmed by Western blot. Hsp 90 ) heat shock protein 90 kDa; hsp 60 ) heat shock protein 60 kDa; hnRNP ) heterogeneous nuclear ribonucleoproteins; and βtub ) tubulin β used as control.

tumor suppressor activity39 suggests that its loss or its significant decrease may lead to the acquisition of a more malignant phenotype, as is apparent in LAMA R cells. TRF2 interacting telomeric RAP1 protein was up-regulated (about only 1.2-fold more expressed) in LAMA 84 S and shows transcriptional regulating activity. Far upstream element (FUSE) binding protein 2 was identified only in LAMA S. This protein (named also KSRP), modifies RNA splicing and interacts with c-src in a differentiation depending way.40 Three nucleic acid interacting proteins were up-regulated in LAMA R. These are: eukaryotic translation initiation factor 3 subunit 2 β and heterogeneous nuclear ribonucleoprotein F and H1 (hnRNPF and hnRNPH1) expressed 1.19-, 1.75-, and 2.73-fold, respectively. Only the latter can thus be considered significant. Heterogeneous nuclear ribonucleoprotein K (hnRNPK) and transformation up-regulated nuclear protein were identified only in LAMA R. In the nucleus, a nascent RNA transcript associated with various proteins, forming heterogeneous ribonucleoproteins (hnRNPs). RNPs that contain fully processed mRNAs are called messenger RNPs and are exported to the cytosol through nuclear pores. Heterogeneous nuclear ribonucleoproteins are RNA binding proteins complexing with heterogeneous nuclear RNA (hnRNA). Their association with pre-mRNAs in the nucleus influence pre-mRNA, metabolism, and transport.41-43 372


technical notes All the proteins included in this family are located in the nucleus and have typical nucleic acid binding properties, but there is evidence that some of them are shuttle proteins between nucleus and cytoplasm.44 High levels of hnRNPs have been associated with tumor progression and resistance to chemotherapy induced apoptosis in several neoplasms.44-48 Consistently with our findings, it has recently been reported that p210BCR/ABL increases expression and activity of hnRNPK in transfected cell lines and primary CML CD34+ cells, in a dose- and kinase-dependent manner through the activation of the MAPKERK1/2 pathway.49 Notari et al. have described that hnRNPK up-regulates MYC protein expression upon binding to MYC IRES. Moreover, hnRNPK down-regulation and interference with its translational activity impairs proliferation, clonogenic potential, and leukemogenesis of BCR/ABL-transformed cells and/or primary CD34+ CML-BC cells through inhibition of the IRES-dependent translation of MYC mRNA.49 The impossibility of identifying this protein in LAMA 84 S cells that are Bcr-Abl positive could be attributed to expression levels below the threshold of detection of the staining procedure. FUSE binding protein 1 was also detected exclusively in the LAMA R 2-D map. FBP-1 regulates myc expression binding to a single stranded far upstream element (FUSE), located 5′ to the myc promoter, acting both as activator or as repressor of transcription.50 Translation initiation factors are known to contain the WD40 domain that covers a variety of functions, including adaptor/ regulatory modules in signal transduction, pre-mRNA processing, and cytoskeleton assembly.51 Another protein, deoxyuridine 5′-triphosphate nucleotidohydrolase, a mitochondrial precursor (dUTPase, one enzyme of the nucleotide metabolism located in the mitochondrion that catalyzes the production of dUMP), was exclusively expressed in LAM84R. Mono-dimensional SDS-PAGE electrophoresis and Western blotting with anti-hnRNPF/H and anti-hnRNPK were consistent with the variation in expression indicated by 2-D and MS (Figure 3). 3.4. Class III: Structural Proteins. Four of the six identified proteins belonging to this class were overexpressed in LAMA 84 S: actin γ-1, Rtubulin, vimentin, and laminin binding protein (Table 1). These proteins play a role in cytoskeleton assembly and cell integrity. The identified Laminin bindin protein originally thought to be a laminin receptor whose mRNA were overexpressed in several human carcinomas52,53 is now recognized as a member of the ribosomal protein S2P family.54 In addition to his chaperon function, the already mentioned Valosin containing protein (VCP), overexpressed in LAMA R cells, exerts a structural role binding to heat shock protein 90 kDa.18,19 3.5. Class IV: Cell Signaling. Five proteins can be assigned to this functional class (Table 1). Two of them, protein kinase C substrate 80K-H isoform-2 and chain A-14--3-3 protein epsilon were overexpressed in LAMA S (staining volume S/R ) 1.530 and 1.763, respectively). While the first one is involved in protein kinase cascade and in GLUT4 vesicle trafficking,55 14--3-3 proteins mediate signal transduction by binding to phosphoserine containing proteins. 14--3-3 protein homologues are involved also in growth factor signaling via MEK kinases.56,57 Two proteins of this class were found to be more expressed in LAMA R: erb-B3 binding protein EBP1 and ANP32A protein,

technical notes

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Figure 4. Diagram of a tentative model of proteome modifications in imatinib resistant CML cells. Red circles indicate proteins overexpressed in LAMA 84 R; green circles indicate proteins overexpressed in LAMA 84 S. Gray circles indicate proteins not identified in this experiment.

both involved in intracellular signal cascade and nucleocytoplasmic transport. Finally, zyxin, a zinc binding phosphoprotein that concentrates at adhesion foci and along the actin cytoskeleton, was identified only in LAMA R. Zyxin has an N-terminal prolinerich domain and three LIM domains in its C-terminal half. The proline-rich domain may interact with SH3 domains of proteins involved in signal transduction pathways, while the LIM domains are involved in protein-protein interaction.58 Zyxin has a role of messenger in the signal transduction pathway that promotes adhesion-stimulated changes in gene expression and may regulate the cytoskeletal organization of actin bundles. 3.6. Class V: Metabolic Enzymes. This is the broader class, including enzymes involved in several metabolic pathways. Eleven proteins were associated with this class (Table 1). Six of them were up-regulated in LAMA S: migration-inducing gene 10 protein, related to phosphoglycerate kinase and glycolytic metabolism, and the subunit β of prolyl-4-hydroxylase was only slightly overexpressed (S/R vol ) ca. 1.2). Three glycolytic enzymes (glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase, and enolase) were expressed from 1.9- to 5.9-fold more in LAMA S. Enolase-1, previously associated to class II for its DNA binding properties,38 was included also in this class for its well-characterized phosphopyruvate hydratase activity. Recent works showed alternative functions for this protein: Ejeskar et al. evidenced a tumor suppressor activity of enolase-

1, and high expression levels of this enzyme correlated with growth inhibitory effects and cell death in neuroblastoma cells.39 Moreover, there is recent evidence on the capability of enolase to elicit the immune response in several pathologies like autoimmune retinopathy and Behcet’s disease.59,60 The subunit d of isoform B of the F0 complex-ATP synthase, involved in ATP synthesis coupled to proton transport, was identified exclusively in LAMA S. Considering the five proteins of this class typical of LAMA 84 R cells, adenylyl ciclase associated protein and protein disulfide isomerase showed a vol S/R next to 0.85, while chain A of carbonic anydrase was significantly overexpressed (vol S/R ) 0.31). Purine nucleoside phosphorylase and the glycolytic enzyme phosphoglycerate mutase 1 were identified only in LAMA R. In Figure 4, we elaborated a tentative scheme of the pathways involved in imatinib resitance in LAMA 84 cells and their cross-talks.

4. Conclusion Imatinib has been a milestone in the treatment of cancer. Its incredible clinical efficacy has indeed contributed to a conceptual revolution in cancer research. Molecular targeted therapy has in fact been demonstrated to be achievable and successful. Nevertheless, we face the fact that CML cells find their way out and acquire resistance to imatinib. Among the frequent point mutations of the Abl kinase domain, at least one (T315I) has until present not been sensitive to any of the Journal of Proteome Research • Vol. 6, No. 1, 2007 373


numerous new compounds synthesized to inhibit mutated BcrAbl forms. To overcome this problem, imatinib has been combined with other drugs endowed with some activity against CML cells (i.e., HDAC inhibitors, bortezomib, interferon, and arsenic trioxide), and some of these combinations were successful in restoring imatinib sensitivity. To ameliorate the rate of success of imatinib/drug combinations, one should identify beforehand the possible and more significant molecular targets in resistant CML cells. The molecular modifications accompanying Bcr-Abl point mutations or its gene amplifications have never been analyzed in depth. Our work has clarified proteome alterations in imatinib resistant versus sensitive CML cells. We believe that the identification of a substantial number of proteins belonging to the heat shock family, and evidently playing a pivotal role in the maintenance of the resistant phenotype, can be a sound background for development of targeted therapeutic agents, not only in CML but possibly in other neoplasms. Although some authors26,30,31 had observed an involvement of HSP 90 and indirectly of HSP70 in imatinib resistance, we showed a significant increase of eight Hsp proteins, whose activity is also connected to inhibition of apoptosis. Broad involvement of Hsp proteins could in fact represent a common shared mechanism in tumor progression, and we think that our findings should prompt further investigation. The same speculations held for hnRNPs, whose importance and significance in stabilizing and shuttling the message of the Bcr-Abl oncoprotein seems fundamental to determine imatinib resistant phenotype. We showed the increased expression of three hnRNPs in resistant CML cells. Consistent with our observations, hnRNPK expression and subcellular localization were recently shown to be directly related to Bcr/Abl presence and activity via a MAPK pathway.49 The significant increase in expression of the hnRNPK protein we observed could be a direct consequence of the enhanced activity and amount of Bcr-Abl fusion protein in LAMA 84 R cells, in a sort of auto-maintaining loop. At the same time, it contributes in a fundamental manner to the hyperactivity of the oncogenic tyrosine kinase, characterizing imatinib resistant CML cells. Proteomic studies of CML cells are not numerous at present. Our study has clearly and decisively identified specific proteins and a family of proteins associated with imatinib resistance. Because of the high interest in detecting biomarkers of imatinib and of other tyrosine kinase inhibitor resistances, we believe that further investigations of the protein expression signature of CML cells are warranted to allow the development of additional molecular targeted therapeutic agents.

Acknowledgment. We thank Prof. Rossi Ferrini for continuous support, Prof. Moneti (CISM), Ente Cassa di Risparmio (ECR) for fellowship and grant support (G.F. and R.P.), MIUR, and Novartis for a fellowship to A.G. References (1) Liu, J.; Campbell, M.; Guo, J. Q.; Lu, D.; Xian, Y. M.; Andersson, B. S.; Arlinghaus, R. B. Oncogene 1993, 8 (1), 101-9. (2) Elefanty, A. G.; Hariharan, I. K.; Cory, S. EMBO J. 1990, 9 (4), 1069-78. (3) Sattler, M.; Griffin, J. D. Int. J. Hematol. 2001, 73 (3), 278-91. (4) Sattler, M.; Griffin, J. D. Semin Hematol. 2003, 40, (2 Suppl. 2), 4-10. (5) Salgia, R.; Li, J. L.; Ewaniuk, D. S.; Pear, W.; Pisick, E.; Burky, S. A.; Ernst, T.; Sattler, M.; Chen, L. B.; Griffin, J. D. J. Clin. Invest. 1997, 100 (1), 46-57. (6) Cambier, N.; Chopra, R.; Strasser, A.; Metcalf, D.; Elefanty, A. G. Oncogene 1998, 16 (3), 335-48.

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Comparative Proteomic Analysis of Chronic Myelogenous Leukemia

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