Comparative Proteome Profiling and Functional Analysis of Chronic

Comparative Proteome Profiling and Functional Analysis of Chronic Myelogenous Leukemia Cell Lines Simona Fontana⊥,† Riccardo Alessandro*,⊥,†, Marilisa Barranca,† Margherita Giordano,† Chiara Corrado,† Isabelle Zanella-Cleon,‡ Michel Becchi,‡ Elise C. Kohn,§ and Giacomo De Leo† Sezione di Biologia e Genetica, Dipartimento di Biopatologia e Metodologie Biomediche, Universita` di Palermo, Palermo, Italy, Institut de Biologie et Chimie des Proteines, UMR 5086 CNRS, IFR128, Universite´ de Lyon, Lyon, France, and Molecular Signaling Section, Medical Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, Maryland Received July 4, 2007

The aim of the present study was the molecular profiling of different Ph+ chronic myelogenous leukemia (CML) cell lines (LAMA84, K562, and KCL22) by a proteomic approach. By employing two-dimensional gel electrophoresis combined with mass spectrometry analysis, we have identified 191 protein spots corresponding to 142 different proteins. Among these, 63% were cancer-related proteins and 74% were described for the first time in leukemia cells. Multivariate analysis highlighted significant differences in the global proteomic profile of the three CML cell lines. In particular, the detailed analysis of 35 differentially expressed proteins revealed that LAMA84 cells preferentially expressed proteins associated with an invasive behavior, while K562 and KCL22 cells preferentially expressed proteins involved in drug resistance. These data demonstrate that these CML cell lines, although representing the same pathological phenotype, show characteristics in their protein expression profile that suggest different phenotypic leukemia subclasses. These data contribute a new potential characterization of the CML phenotype and may help to understand interpatient variability in the progression of disease and in the efficacy of a treatment. Keywords: chronic myelogenous leukemia cell lines; proteome profiling; tumor invasion; drug resistance.

Introduction Chronic myelogenous leukemia (CML) is caused by clonal expansion of pluripotent hematopoietic stem cells retaining their differentiation potential. The myeloid leukemia cells are characterized by t(9;22)(q34;q11) reciprocal chromosomal translocation, producing a shortened chromosome 22, the so-called Philadelphia (Ph) chromosome. This chromosomal rearrangement results in the formation of a BCR-ABL hybrid gene, the molecular hallmark of CML.1-3 BCR-ABL encodes a tyrosine kinase of 190, 210, or 230 kDa, depending on the breakpoint on the BCR gene. Several studies both in vivo and in vitro have established that the tyrosine kinase activity of BCR-ABL, affecting cell growth, adhesion, and survival,4 is responsible for the transforming and leukemogenic properties of this oncoprotein.5-7 CML is usually a triphasic disease, having a chronic, an accelerated, and a blast phase,8-10 during which progressive resistance to therapy is acquired.11-16 New evidence suggests * Corresponding author. Address: Dipartimento di Biopatologia e Metodologie Biomediche, Sezione Biologia e Genetica, Universita` di Palermo, Via Divisi 83, 90133 Palermo, Italy. Tel: +39 091 655 4608. Fax: +39 091 655 4624. E-mail: [email protected]. † Universita` di Palermo. ‡ Universite´ de Lyon. § National Cancer Institute. ⊥ These authors contributed equally to this work.

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that CML blasts arise from leukemic progenitors (rather than leukemic stem cells) that have restored self-renewal capacities.17,18 However, a more clear characterization of this cell population, responsible for CML progression, is necessary to better understand this disease and to develop new treatment strategies. The molecular fingerprinting of CML cell lines, as proposed for other tumors,19 may provide information to better define and understand the specific disease status. A key form of molecular characterization of a cytotype is the study of its protein expression profile, since proteins are molecular effectors ultimately driving a specific phenotype. Proteomic profiling is a powerful approach to investigate and to monitor the protein levels and activation state, and represents a unique method for a sensitive and simultaneous analysis of a large number of proteins. This work presents a phenotypic characterization of different Ph+ CML cell lines (LAMA84, K562, and KCL22) using a proteomic approach. We employed two-dimensional gel electrophoresis (2-DE) combined with mass spectrometry (MS) in order to define the proteomic profiles of LAMA84, K562, and KCL22 cell lines, each established from the peripheral blood of a patient with CML in blast crisis;20,21 these lines are used commonly as in vitro models of CML. A multivariate analysis demonstrated that the proteomic profiles of the three cell lines 10.1021/pr0704128 CCC: $37.00

 2007 American Chemical Society

Proteome Profiling of CML Cell Lines

are significantly different. The differences are particularly evident between LAMA84 and K562/KCL22, the latter exhibiting greater similarities to each other. We detected 35 differentially expressed proteins. LAMA84 cells preferentially expressed proteins associated with a more motile behavior, while K562 and KCL22 preferentially expressed proteins involved in drug resistance. These data demonstrate that these CML cell lines, representing the same pathological phenotype, show protein expression profile characteristics suggesting two different classes of blast crisis leukemia.

Experimental Section Cell Lines. LAMA84, K562, and KCL22 are Ph+ cell lines established from peripheral blood of patients with CML in blast crisis.20,21 The lines were kindly provided by Dr. P. Vigneri (University of Catania, Italy). Cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Cytotoxicity Assay. Methyl-thiazol-tetrazolium (MTT) cytotoxicity assays were performed on CML cell lines as described previously.22 Cells were plated in triplicate or quadruplicate at 1.5 × 104 cells per well in flat-bottomed 96-well microplates and exposed to increasing doses (0.01-10 µM) of imatinib, kindly provided by Dr. R. Bertieri (Novartis, Basel, Switzerland) for 24 h. Cell growth was plotted as a percentage of the control. Each point reported in the curve represents the mean value ( standard deviation (SD) for at least three independent experiments. Chemoinvasion and Migration Assay. LAMA84, K562, and KCL22 cell lines were assayed for their invasiveness using a modified Boyden chamber method.23 Briefly, polycarbonate filters with 8 µM pore size were coated with 6.25 µg Matrigel (Becton Dickinson, Bedford, MA). Cells (2 × 106/mL) were suspended in serum-free RPMI 1640 medium supplemented with 0.1% bovine serum albumin transferred into the upper wells. FCS (10%) was added in the bottom wells as the chemoattractant. After an 18 h incubation at 37 °C, the cells on the upper surfaces of the filters were removed, and the filters were fixed in ethanol and stained with Dif-Quick solution (Medion Diagnostics GmbH, Du¨dingen, Switzerland). Each cell line was tested in three independent experiments; the number of migrating cells in five high-power fields per well was counted at 400× magnification. Statistical differences were calculated by a two-tailed Student’s t test. Migration assays were performed using gelatin-coated filters (100 µg/mL) in otherwise identical conditions. Protein Preparations. Whole protein extracts were prepared from 1 × 107 cells. Cell pellets were washed three times with phosphate-buffered saline and then resuspended in lysis buffer (300 mM NaCl, 50 mM Tris-HCl, pH 7.6, 0.5% Triton, 1 mM phenylmethylsulphonyl fluoride, 10 µg/mL leupeptin, 10 µg/ mL aprotinin, 0.4 mM EDTA, 0.4 mM sodium orthovanadate, 10 mM sodium pyrophosphate, 10 mM NaF) on ice for 1 h. The lysates were clarified at 19000g for 15 min and, after dialysis against ultrapure distilled water, were lyophilized and stored at -80 °C until analysis. Protein concentration was determined according to the Bradford method.24 Two-Dimensional Gel Electrophoresis. Aliquots of the lyophilized cell lysate were solubilized in a buffer containing 4% CHAPS, 40 mM Tris, 65 mM 1,4-dithioerythritol (DTE), and a trace amount of bromophenol blue in 8 M urea. The first dimension separation was performed at 20 °C using com-

research articles mercial sigmoidal immobilized pH gradient (IPG) strips (18 cm long), with a pH range of 3-10 (Amersham Biosciences AB, Uppsala, Sweden,). Strips were rehydrated in 8 M urea, 2% CHAPS, 10 mM DTE, and 0.5% carrier ampholytes (IPG Buffer pH 3-10 NL, Amersham Biosciences AB). Aliquots of 60 µg (for analytical 2-DE) or 1 mg (for preparative 2-DE) were applied to the gel strip. The isoelectrofocusing was carried out by linearly increasing the voltage from 200 to 3500 V during the first 4.5 h and was continued at 8000 V for 8 h. After the run, the IPG strips were equilibrated with a solution containing 6 M urea, 30% glycerol, 2% sodium dodecyl sulfate (SDS), 0.05 M Tris-HCl, pH 6.8, and 2% DTE for 12 min, in order to resolubilize proteins and reduce disulfide bonds. The -SH groups were then blocked by substituting the DTE with 2.5% iodoacetamide in the equilibrating buffer. The focused proteins were then separated on slab vertical gels with a 9-16% polyacrylamide linear gradient (SDS polyacrylamide gel electrophoresis (SDS-PAGE)) at a constant power of 14 W/gel at 10 °C. Analytical gels were stained with ammonium silver nitrate,25 and preparative gels were stained with Coomassie Blue. Image Acquisition and Analysis. Stained gels were scanned by an ImageScanner II densitometer (Amersham Biosciences AB, Uppsala, Sweden), and the images were analyzed by the ImageMaster 2D Platinum software (Amersham Biosciences AB). A human serum internal standard was used for pI and molecular weight (MW) calibration of the 2-DE gel images.26 Individual protein spots detected in the map of each cell line were quantified and matched. Quantitative analysis was based on evaluation of the percentage volume (vol %) value. In-Gel Protein Digestion. Coomassie Blue-stained protein spots were excised from the preparative gels and cut into 1-mm pieces. In-gel digestion was performed as described by Shevchenko et al.27 with minor modifications. Destaining was obtained by successive washes with 20 mM NH4HCO3 buffer and a H2O/CH3CN (50/50) mixture. The gel was treated for proteolytic digestion with 5-20 µL of a trypsin solution (20 ng/ µL in 50 mM NH4HCO3; sequence-grade trypsin, Promega, Charbonnie`res, France) for 5 h at 37 °C. The resulting tryptic peptides were extracted from the gel by centrifugation, and the supernatant fraction was recovered. A second extraction step was performed using 10-15 µL H2O/CH3CN/HCOOH (60/36/ 4; v/v/v) for 30 min, and, finally, all extracts were pooled and used for matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) MS analysis. The extract was dried in a vacuum concentrator and resuspended in 0.1% trifluoroacetic acid (TFA) solution for liquid chromatography electrospray ionization tandem mass spectroscopy (LC-ESI/MS/MS) experiments. MALDI-TOF MS. A Voyager DE-PRO MALDI-TOF mass spectrometer (Applied Biosystems, Courtaboeuf, France) was used for peptide mass mapping in positive ion reflector mode. Spectra were acquired in the 700-5000 Da mass range. A 1 mg/200 µL solution of R-cyano 4-hydroxycinnamic acid (LaserBioLab, Sophia-Antipolis, France) in 50% CH3CN in water containing 0.1% TFA was used as the matrix. Internal calibration was done with trypsin autolysis fragments at m/z 842.5100 and 2211.1046 Da. Peptide mass fingerprinting was compared to the theoretical masses from the SwissProt sequence database using Mascot software from Matrix Science (version 2.0). The parameters for the Mascot searches were as follows: Homo sapiens as species, one missed cleavage (in some cases, better results were obtained using two missed cleavages), carboxyJournal of Proteome Research • Vol. 6, No. 11, 2007 4331

research articles methylated cysteine as the fixed modification, oxidation of methionine as the variable modification (and in some cases with N-terminal acetylation), and a mass tolerance of ( 30 ppm for peptides. Under these conditions, a protein score greater than 55 is significant (p < 0.05). NanoLC/nanospray/tandem mass spectrometry (LC-ESI/ MS/MS). Experiments were performed on a Q-STAR XL instrument (Applied Biosystems, Courtaboeuf, France) equipped with a nanospray source using a distal coated silica-tip emitter (FS 150-20-10-D-20, New Objective, U.S.A.) set at 2300 V. Information-dependent acquisition (IDA) mode allowed peptide ions within a m/z 400-2000 survey scan mass range to be analyzed for subsequent fragmentation. MS/MS spectra were acquired in the m/z 65-2000 range for +2 to +4 charged ions. The collision energy was automatically set by the software (Analyst 1.1) and was related to the mass and the charge of the precursor ion. The MS and MS/MS data were recalibrated using internal reference ions from a trypsin autolysis peptide at m/z 842.510 [M + H]+ and m/z 421.759 [M + 2H]2+. Tryptic peptides were separated using an Ultimate-nanoLC (Dionex, Voisins Le Bretonneux, France) with a C18 PepMap micro-precolumn (5 µm; 100 Å; 300 µm × 5 mm; Dionex) and a C18 PepMap nanocolumn (3 µm; 100 Å; 75 µm × 150 mm; Dionex). The chromatographic separation was developed using a linear 60 min gradient from 0 to 50% B, where solvent A was 0.1% HCOOH in H2O/CH3CN (95/5) and solvent B was 0.08% HCOOH in H2O/CH3CN (20/80) at an approximately 200 nL/ min flow rate. Protein identification was achieved by the ProID database-searching software (Analyst, version 1.1, Applied Biosystems) using a peptide and a fragment mass tolerance of ( 0.15 and ( 0.1 Da, respectively. A ProtScore above 2.0 defined a percent confidence better than 99%. Data Analysis. Statistical comparisons between the CML cell lines were accomplished by multivariate analysis using PERMANOVA (permutational multivariate analysis of variance) on the vol % of identified spots from three independent experiments for each cell line. Data were not transformed, and the Bray-Curtis similarity was used as the distance index. For this test, p-values were calculated after 9999 permutations under the reduced models. PERMANOVA seems to be more powerful than other classical multivariate analyses allowing partitioning of variance components.28 Differences in the composition (the number of expressed protein spots) and structure (vol % of each expressed protein spot) of the proteomic profiles evaluated by PERMANOVA, were plotted by the canonical analysis of principal coordinates (CAP), this analysis was performed to visualize possible patterns in composition and structure across the different cell lines. The SIMPER (similarity percentage29) procedure was used to assess dissimilarities in the pairwise comparisons between different cell lines. All the multivariate analyses were performed with the PRIMER 6 & PERMANOVA plus software (University of Plymouth, University of Aukland). A two-tailed Student’s t test was used to assess the differences between the vol % values of identified protein spots. Statistical differences (p < 0.05) were calculated only on those protein spots exhibiting at least 3-fold changes in vol %. Morphologic Evidence of Apoptotic Cells by Acridine Orange/Ethidium Bromide Staining. LAMA84, K562, and KCL22 cells were treated with 1 and 10 µM imatinib, and apoptosis was determined by fluorescence microscopy after staining with acridine orange and ethidium bromide (AO/EB).30 After 24 h of treatment, cells were harvested by centrifugation 4332

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and AO/EB (1:1) (v/v) solution was added to the cell suspension with a final concentration of 100 µg/mL. Cells were incubated for 30 min, and their morphology was evaluated by fluorescence microscopy (Olympus BX50). At least 200 cells were counted in each experiment. Each point represent the mean value ( SD for at least three independent experiments. Western Blot Analysis. Equal amounts of cell lysate of each CML cell line after treatment with imatinib (1 and 10 µM for 24 h), were subjected to SDS-PAGE in 6 or 8% polyacrylamide gels, and transferred to nitrocellulose membrane (Protran Schleicher & Schuell, Dassel, Germany). The membrane was incubated in blocking solution (5% non-fat dry milk, 20 mM Tris, 140 mM NaCl, 0.1% Tween-20), and probed overnight at 4 °C with polyclonal antibodies against c-Abl, phospho-Abl (TYR245), and anti-actin (Cell Signaling Technology, Beverly, MA), and with monoclonal anti-phosphotyrosine (Santa Cruz Biotechnology, Santa Cruz, CA). After three washes with 20 mM Tris, 140 mM NaCl, and 0.1% Tween-20, the membrane was incubated with horseradish peroxidase-conjugated secondary antibody and proteins detected by the enhanced chemiluminescence detection system (Super Signal, Pierce, Rockford, IL).

Results and Discussion Proteome Profiling of CML Cell Lines. We created reference proteomic maps of LAMA84, K562, and KCL22 cells by applying an expression proteomics approach.31 Supplementary Figure 1 (Supporting Information) shows representative silver stained proteomic maps of CML cells (LAMA84) in which an average of 1300 independent spots of pI 4-8 and MW from 9-150 kDa were detected; Figure 1B-D shows representative 2-DE maps of each cell line (respectively LAMA84, K562, and KCL22) with spots subjected to MALDI-TOF and/or LC-ESI/MS/MS and their identification numbers; these identified spots are listed by this number in Supplementary Table 1 (Supporting Information). Altogether, we identified 191 protein spots (corresponding to 142 different proteins), of which 171 were expressed by LAMA84 cells, 180 by K562 cells, and 180 by KCL22 cells. The experimental pI and MW values of identified proteins correlated with the theoretical values reported in the SwissProt/ TrEMBL database (http://www.expasy.org/sprot/; see Supplementary Table 1, Supporting Information). As described in the literature, some observed differences between the experimental and theoretical pI/MW values and the presence of different isoforms of the same protein could reflect post-translational modifications or splice variants.19,32,33 Only spot 5, identified as beta actin (ACTB), showed an evident difference between the experimental MW (125.8 kDa) and the theoretical size (41.7 kDa). Actin isoforms with an high MW were also identified in human platelet maps deposited in the OGPWWW database (Oxford GlycoProteomics Webpage, U.K.; http://proteomewww.glycob.ox.ac.uk/2d/2d.html) federated with the WORLD-2DPAGE database (http://www.expasy.org/ ch2d/2d-index.html). The peptide mass fingerprint obtained after trypsin digestion for spots 2, 4, and 6 did not allow discrimination between ACTB and gamma actin (ACTG), since these cytoskeletal proteins differ by only four amino acids at the amino-terminal region. A similar result was obtained for spots 74 and 75, for which it was not possible to discriminate between hemoglobin subunit gamma-1 and hemoglobin subunit gamma-2 (differing by just one amino acid at position 137). Thus, we labeled these spots as ACTB/G and HBG1/HBG2 in Supplementary Table 1 (Supporting Information).


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Figure 1. Proteins (60 µg) were loaded on IPG strips (18 cm, 3.5-10 nonlinear pH gradient). The second dimension was performed on a vertical linear-gradient slab gel (9-16%). Numbers from 1 to 191 (reported in Supplementary Table 1, Supporting Information) indicate the spots identified by MALDI-TOF and/or LC-ESI/MS/MS in proteome maps of LAMA84 (A), K562 (B), and KCL22 (C) cells.

Few studies of CML cell lines have addressed the characterization of changes in expressed proteins following treatment with antiproliferative/cytodifferentiative drugs or between the imatinib-sensitive and -resistant phenotype.34-37 Of the proteins identified in this report, 105 proteins, indicated with an asterisk in Supplementary Table 1 (Supporting Information; 74% of those identified), have not been reported previously to be expressed in either CML cell lines or CML patients. Approximately 63% of the identified proteins have been described in the literature as cancer-related.38-45,46 We classified these proteins according to their potential role in cancer progression into seven different groups: (i) invasion and metastasis (18 proteins), (ii) cell cycle regulation and cell proliferation (7), (iii) proteins involved in metabolic and biosynthetic processes (21), (iv) stress-related proteins and chaperones (14), (v) detoxification and drug resistance (13), (vi) mRNA processing proteins (3), and (vii) others (13 proteins with multiple roles; Table 1). Differences were seen between the three cell lines for some proteins belonging to the groups of

invasion and metastasis (CAH1, CAH2, PLSL, S10A4, S10A6, and TPM3), detoxification and drug resistance (ANXA1, GSTO1), stress-related proteins (HSP71, HSP7C, and HSPB1) and others (GDIS). Moreover, among the cancer related proteins, CAH1 and CAH2, STMN1, HMBG1, ALDOA, ROA1 and RSSA were linked to a leukemia phenotype. Stathmin 1 or oncoprotein 18 (STMN1 or Op18) is essential for cell cycle progression and has been described as a major cytosolic phosphoprotein constituent of leukemia cells.47,48 High-mobility group box protein 1 (HMGB1) is a ubiquitous nuclear protein defined by Mu ¨ ller and colleagues as a chromatin protein with a cytokine function having implications for therapeutic intervention on inflammatory diseases as well as cancer.49 This protein has been found to be overexpressed in many solid tumors and also in leukemia cell lines.49 In particular, it has been shown that HMGB1 expression is higher in myeloid than in lymphoid cells and lower in irreversibly differentiated cells.50 Fructose-bisphosphate aldolase A (ALDOA) is a glycolytic and homotetrametric enzyme predominantly distributed in muscle Journal of Proteome Research • Vol. 6, No. 11, 2007 4333

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Table 1. Cancer Related Proteins Identified in CML Cell Lines abbreviated name

AC numbera

ACTB CAH1 CAH2 COF1 ENOA

P60709 P00915 P00918 P23528 P06733

actin, cytoplasmic 1 carbonic anhydrase 1 carbonic anhydrase 2 cofilin-1 alpha-enolase

EZRI FSCN1 K1C18 K2C8 LEG1 MIF MOES PLSL S100P S10A4 S10A6 TPM3 TPM4

P15311 Q16658 P05783 P05787 P09382 P14174 P26038 P13796 P25815 P26447 P06703 P06753 P67936

ezrin fascin keratin, type I cytoskeletal 18 keratin, type II cytoskeletal 8 galectin-1 macrophage migration inhibitory factor moesin plastin-2 protein S100-P protein S100-A4 protein S100-A6 tropomyosin alpha-3 chain tropomyosin alpha-4 chain

Pa, Bra, Br, Co Ga, Pa,86 Leu,87 CoRec60 Ga, Pa,86 Leu,88 CoRec,60 ReCaC59 OSCC, Co, Pa Bra, Uv mel, Pa, Es, Lu, Br, Co, He/Ne,89 BrCaC, CoCaC90 Uv mel, HCC Bra, Es, ESCC BrCaC, Uv mel, Co Co, BrCaC, Uv mel, Es, Lu, Ga Uv mel, Pa Co,91 Ga92 Ut ce,93 Bra Ov,64 Br,64 Pr,65 PrCaC94 Pa95 Co,69 HCC-C,67 Gb96 Uv mel, Co70 HCC, BrCaC73 Co, Br, Uv mel

CLIC1 NPM PA2G4 PCBP1 PEBP PHB STMN1

O00299 P06748 Q9UQ80 Q15365 P30086 P35232 P16949

Cell Cycle Regulation and Cell Proliferation chloride intracellular channel protein 1 nucleophosmin proliferation-associated protein 2G4 poly(rC)-binding protein 1 phosphatidylethanolamine-binding protein prohibitin stathmin

HCC, Ga,97 Co Uv mel, HCC Bra, Lu BrCaC98 HCC, Br Es, Lu Bra, Uv mel, HCC, Lu, Leu,47,48 Co

ALDOA ADRO

P04075 P22570

ATPB BLVRB COX5A

P06576 P30043 P20674

EF1A1 EF1B EF1G EF2 EFTU FAAA FUMH G3P IPYR KCRB LDHB PGAM1 PIMT RBBP4 RSSA TPIS

P68104 P24534 P26641 P13639 P49411 P16930 P07954 P04406 Q15181 P12277 P07195 P18669 P22061 Q09028 P08865 P60174

CALR CH60

P27797 P10809

ENPL GRP75 GRP78 HS90B HSP71 HSP7C HSPB1 PDIA1 PDIA3 PPIA STIP1 TCTP

P14625 P38646 P11021 P08238 P08107 P11142 P04792 P07237 P30101 P62937 P31948 P13693

protein name

tumor type and cancer cell linesb,38-45,46

Invasion and Metastasis

4334

Proteins Involved in Metabolic and Biosynthetic Processes fructose-bisphosphate aldolase A Leu,54 Br, Lu,51,53 Li,52 Ga,52 Co, BrCaC NADPH:adrenodoxin oxidoreductase, mitochondrial Co,99 CoCaC100 [precursor] ATP synthase beta chain, mitochondrial [precursor] Bra, HCC, Co flavin reductase Br, OSCC45 cytochrome c oxidase polypeptide Va, mitochondrial Ga [precursor] elongation factor 1-alpha 1 Es, Br,101 BrCaC101 elongation factor 1-beta Br,101 BrCaC101 elongation factor 1-gamma Br,102 BrCaC102 elongation factor 2 HCC elongation factor Tu, mitochondrial [precursor] BrCaC, Es fumarylacetoacetase HCC fumarate hydratase, mitochondrial [precursor] Bra glyceraldehyde-3-phosphate dehydrogenase Bra, Uv mel, Br, BrCaC, Es, Pa inorganic pyrophosphatase Co creatine kinase B-type Bra, Ga, Co102 L-lactate dehydrogenase B chain Bra, Lu phosphoglycerate mutase 1 HCC, Ga, Lu protein-L-isoaspartate(D-aspartate) O-methyltransferase Bra histone-binding protein RBBP4 Leu103 40S ribosomal protein SA Leu,57 HCC triosephosphate isomerase Bra, Uv mel, Br, Co, HCC, Ga, Lu, Pa Stress-Related Proteins and Chaperones calreticulin 60 kDa heat shock protein, mitochondrial [precursor] endoplasmin [precursor] Stress-70 protein, mitochondrial [precursor] 78 kDa glucose-regulated protein [precursor] heat shock protein HSP 90-beta heat shock 70 kDa protein 1 heat shock cognate 71 kDa protein heat-shock protein beta-1 protein disulfide-isomerase [precursor] protein disulfide-isomerase A3 [precursor] peptidyl-prolyl cis-trans isomerase A stress-induced-phosphoprotein 1 translationally controlled tumor protein


Bra, Br, Co, Uv mel, Pa Bra, Uv mel, BrCaC, Co, HCC Lu,104 Pa, Uv mel, Co Uv mel, HCC Uv mel, Bra, Lu,104 HCC Uv mel Bra, HCC HCC, Co OSCC, Bra, Uv mel, HCC, Ga, Lu HCC, Ga Uv mel, Co, BrCaC, HCC, Ga Uv mel, BrCaC, Co, Br, Pa Lu Uv mel

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Proteome Profiling of CML Cell Lines Table 1 (Continued) abbreviated name

AC numbera

ANXA1 EF1G GSTO1 GSTP1 LGUL PGK1 PRDX1 PRDX2 PRDX3 PRDX5 PRDX6 SODC THIO

P04083 P26641 P78417 P09211 Q04760 P00558 Q06830 P32119 P30048 P30044 P30041 P00441 P10599

Detoxification and Drug Resistance annexin A1 elongation factor 1-gamma glutathione transferase omega-1 glutathione S-transferase P lactoylglutathione lyase phosphoglycerate kinase 1 peroxiredoxin-1 peroxiredoxin-2 thioredoxin-dependent peroxide reductase peroxiredoxin-5, mitochondrial [precursor] peroxiredoxin-6 superoxide dismutase [Cu-Zn] thioredoxin

OSCC, Uv mel Uv mel OvCaC82 Bra, OSCC, Uv mel, Br, BrCaC, Co Bra, HCC Bra, Br, Co Br, Co, Es, Lu105 Uv mel, Es OSCC, Bra, HCC, Lu105 Co Bra Bra, Br BrCaC, Br, Lu105

HNRPK ROA1 ROA2

P61978 P09651 P22626

mRNA Processing Proteins heterogeneous nuclear ribonucleoprotein K heterogeneous nuclear ribonucleoprotein A1 heterogeneous nuclear ribonucleoproteins A2/B1

HCC, Lu,106 LuCaC106 Bra, Lu,107 LuCaC,107 Leu,56 Co Ga, Lu,107 LuCaC,107

GDIR GDIS HMGB1 NDKA NDKB PARK7 PROF1 PSA6 RUVB1 SH3L3 UBIQ UQCR1

P52565 P52566 P09429 P15531 P22392 Q99497 P07737 P60900 Q9Y265 Q9H299 P62988 P31930

VDAC1

P21796

tumor type and cancer cell linesb,38-45,46

protein name

Others Rho GDP-dissociation inhibitor 1 Rho GDP-dissociation inhibitor 2 high mobility group protein B1 nucleoside diphosphate kinase A nucleoside diphosphate kinase B protein DJ-1 profilin-1 proteasome subunit alpha type 6 RuvB-like 1 SH3 domain-binding glutamic acid-rich-like protein 3 ubiquitin ubiquinol-cytochrome-c reductase complex core protein I, mitochondrial [precursor] voltage-dependent anion-selective channel protein 1

Br OSCC Co,102 LeuC,49 Pa OSCC, HCC, Co, Uv mel, Co Uv mel, Bra, Pa Pa Co Uv mel Bra Br HCC Bra, Co

a

Accession number used in the SwissProt database; all tumors are of human origin. b Br: breast cancer; Bra: brain cancer; Co: colon cancer; CoRec: Colorectal cancer; Es: esophageal cancer; Ga: gastric cancer; Gb: gallbladder cancer; HCC: hepatocellular cancer; He/Ne: head and neck cancer; Leu: leukemia; Lu: lung cancer; OSCC: oral squamous cell carcinoma; Ov: ovarian cancer; Pa: pancreatic cancer; Pr: prostate cancer; Uv mel: uveal melanoma; Ut ce: uterine cervical cancer; C denotes cell lines, BrCaC: breast cancer cell lines; CoCaC: colon cancer cell lines; HCC-C: hepatocellular carcinoma cell lines; LeuC: leukemia cell lines; LuCaC: lung cancer cell lines; OvCa: ovarian cancer cell lines; PrCaC: prostate carcinoma cell lines; ReCaC: renal cancer cell lines.

tissues. This protein has been shown to be overexpressed in tissues and sera of patients affected by breast, lung, liver, gastric, and colon cancers.40,46,51-53 Recently ALDOA was also indicated as a leukemia-associated antigen in chronic myeloid leukemia.54 Heterogeneous ribonucleoprotein A1 (hnRNP A1 or ROA1) is an ubiquitously expressed hnRNP protein and has an important role in pre-mRNA and mRNA metabolism.55 Increased levels of hnRNP A1 have been observed in myeloid progenitor cells expressing the p210BCR/ABL oncoprotein, in mononuclear cells from patients affected by CML blast crisis, and during disease progression.56 Moreover, a direct role of hnRNP A1 in the regulation of normal hematopoiesis and BCR/ ABL-dependent leukemogenesis has been demonstrated.56 DNA microarray analysis revealed that hnRNP A1 and 40S ribosomal protein SA (RSSA) genes are significantly up-regulated in patients with refractory acute leukemia, suggesting that they may play a role as biomarkers of prognosis.57 Our study represents the first step toward the establishment of a CML protein database and may provide a useful platform for studying the protein profile alterations of CML cells induced by drug treatment and by drug-resistance acquisition. Comparative Proteomic Analysis of 2-DE Maps of CML Cell Lines. Statistical analysis with PERMANOVA yielded significant differences in the composition (number of expressed protein

Figure 2. Constrained ordination plot obtained by CAP on the identified protein spots (vol %). Symbols represent replicated samples of the three CML cell lines. Journal of Proteome Research • Vol. 6, No. 11, 2007 4335

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Table 2. Average Bray-Curtis Dissimilarity (%) for Identified Protein Spots in Pairwise Comparisons between CML Cell Lines LAMA84-K562 LAMA84-KCL22 K562-KCL22

21.19% 20.61% 14.43%

Table 3. Qualitative Differences among LAMA84, K562, and KCL22 Cells NR spota

abbreviated name

AC numberb

exp. pI/MW (kDa)c

LAMA84

K562

KCL22

7 9 14 21 51 52 61 69 72 73 74 90 95 96 97 98 103 111 112 128 139 154 155 162 184

ADRO AL1A1 ANXA1 CAH2 FKBP3 FSCN1 G3P GSTO1 HBA1 HBAZ HBG2 HSPB1 K1C18 K2C8 K2C8 KCRB LGUL NONO NONO PLSL PRDX6 ROA2 ROA2 S10A4 TPM3

P22570 P00352 P04083 P00918 Q00688 Q16658 P04406 P78417 Q9BX83 P02008 P69892 P04792 P05783 P05787 P05787 P12277 Q04760 Q15233 Q15233 P13796 P30041 P22626 P22626 P26447 P06753

6.77/51.8 6.28/50.9 6.64/32 6.65/25.3 7.28/25.2 6.48/51.5 7.04/30.7 5.99/26.2 6.92/10.6 6.78/10.4 6.58/11 5.93/23.5 5.43/43.6 5.40/53.5 5.62/52.8 5.47/41.4 5.06/19 7.07/58 7.12/58 5.20/63.4 5.88/24.2 6.94/33.4 7.05/30.9 5.58/9.7 4.76/31

Ad A A P A A A A A A P A A A A A P A A P A P P P P

Pd P P A P P A P P P A P A P P P A P P A A A P A A

P P P A P P P P P P P P P P A P A P P A P A A A P

a Spot number reported on the 2-DE maps in Figure 1. b Accession number used in the SwissProt database. c Experimental pI and MW values calculated by ImageMaster 2D Platinum 6.0 software carried out using human serum as the internal standard. d A: absent; P: present.

spots) and relative quantity (vol % of each expressed protein spot) of LAMA84, K562, and KCL22 cells proteomic profiles (p ) 0.01). These results are displayed by the CAP plot that also showed the high reproducibility between the three replicates examined within each cell line (Figure 2). The dissimilarity among the three CML cell lines was revealed by pairwise comparisons between LAMA84 and K562 (21.2% dissimilarity) and KCL22 (20.6% dissimilarity), respectively, with these latter exhibiting lower dissimilarity with each other (14.4% dissimilarity; SIMPER procedure, Table 2). Twentyfive protein spots representing the range of qualitative differences (Table 3) and nine showing quantitative differences in

expression levels (Table 4) were highlighted. Only protein spots exhibiting at least a 3-fold change in vol % and p < 0.05 were considered in the quantitative analysis. Implied functional biological differences between LAMA84 and K562 or KCL22 based upon protein patterns suggested a different phenotypic characterization of these CML lines. Some proteins exclusively or preferentially expressed in LAMA84 cells are involved in positive regulation of invasion (CAH1, CAH2, GDIS, PLSL, S10A4, S10A6, and TPM3). K562 and KCL22 cells had low or absent expression of those proteins and were instead characterized by exclusive or preferential expression of proteins with anti-apoptotic function or those involved in drug resistance: ANX1, GSTO1, HSPB1 (one isoform), HSP71, and HSP7C (the basic isoform). The carbonic anhydrase family (CAHs) plays an important role in extracellular acidification; a possible involvement of these enzymes in tumor invasion has been proposed.58 In vitro studies in renal cancer cell lines showed the direct role of carbonic anhydrase II (CAH2) in regulating the invasive ability of these cells.59 It was reported that expression of CAH1 and 2 correlated with the biological aggressiveness of colorectal cancer and the presence of synchronous distant metastases.60 We observed specific expression of CAH1 and CAH2 in LAMA84 cells with reduction in K562 and KCL22 cells. CAH1 exhibited a 8.7- and 12.6-fold increase in LAMA84 cells vs K562 and KCL22 cells, respectively, with CAH2 expressed exclusively in LAMA84 cells (Tables 3 and 4 and Figure 3A). The Rho GDP dissociation inhibitors (RhoGDIs) are pivotal regulators of Rho GTPase function.61,62 LAMA84 cells overexpress RhoGDI-2 (GDIS), the one human RhoGDI originally found only in hematopoietic tissues and cells, more recently described in bladder, ovarian, and breast cancers.62 This protein exhibited a 5.8- and 4.5-fold increase in LAMA84 cells over K562 and KCL22 cells (Table 4 and Figure 3A). A conflict in results is found describing the role of GDIS in metastasis regulation. This protein has been shown to act as a metastasis suppressor in bladder cancers,63 while in breast cancer cells, its overexpression correlates with motile and invasive phenotype.62 Moreover, the invasive capacity of highly metastatic MDA-MB-231 breast cancer cells is specifically related to overexpression of GDIS, but not of RhoGDI-1 (GDIR).62 No differences in expression levels of GDIR were observed in the three CML cell lines. Plastin-2 or L-plastin (PLSL) is an actin-bundling protein expressed predominantly in cells of haemopoietic origin, but is also overexpressed in many malignant human solid tumors.64,65 In vitro and in vivo experiments using prostate carcinoma cell lines demonstrated that down-regulation of

Table 4. Over/Underexpressed Proteins in LAMA84 vs K562 and KCL22 Cells NR spot

abbreviated name

AC number

exp. pI/MW (kDa)

trend in LAMA84 vs K562 cellsa

p-valuesa

trend in LAMA84 vs KCL22 cellsa

12 13 20 62 64 84 88 145 163

ANXA1 ANXA1 CAH1 G6PD GDIS HSP71 HSP7C RANG S10A6

P04083 P04083 P00915 P11413 P52566 P08107 P11142 P43487 P06703

6.31/34.2 6.44/32.3 6.47/26.2 6.35/54.2 5.11/22.1 5.53/65.3 5.47/66 5.13/24.9 5.06/8.8

-4.90 -4.73 8.72 -3.34 5.82 -4.52 -4.11 -4.52 7.21

0.0027 0.0001 0.0017 0.0187 0.0021 0.0094 0.0230 0.0057 0.0119

-5.15 -4.13 12.59 NDEb 4.48 -4.10 -3.47 -4.47 6.72

p-valuesa

0.0068 0.0135 0.0029 0.0011 0.0037 0.0119 0.0011 0.0121

a Only protein spots exhibiting at least 3-fold changes in vol % and p < 0.05 (calculated by Student’s t test) were considered differentially expressed between the CML cell lines. Differences with p < 0.05 were considered significant. b NDE: no differential expression.

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Figure 3. The 2-DE panels are representative of three independent experiments for each cell line and show differences of spot intensity among LAMA84, K562, and KCL22 cells: (A) proteins involved in positive regulation of motile and invasive behaviour; (B) proteins associated with drug resistance and anti-apoptotic activity. For each protein spot included in the group of the quantitative differences (Table 4), the level of expression in the three CML cell lines was indicated as vol % in the correspondent histogram. The values reported in the graphs are the mean of three independent experiments ( SD. Statistic significance of the data was calculated by the Student’s t test: *p < 0.05; **p < 0.01; ***p < 0.001.

L-plastin induced a decrease of invasive behavior of these cells.65 Furthermore, the positive effect of L-plastin overexpression on parameters associated with metastasis, including proliferation, migration, and invasion was demonstrated in colon cancer cell lines.66 L-plastin was expressed only by

LAMA84 cells, those with the invasion proteomic functional profile (Table 3 and Figure 3A). S10A4 (p9Ka, CAPL, or calvasculin) and S10A6 (calcyclin) are two members of the S100 calcium-binding proteins family, associated with cancer invasion and metastasis. Studies on Journal of Proteome Research • Vol. 6, No. 11, 2007 4337

research articles hepatocellular cancer (HCC) cell lines have shown that the S10A4 overexpression is associated with high metastatic capacity and a prognostic significance of this protein was also proposed for colorectal cancer.67-69 A high level of S10A6 was also observed in human colorectal adenocarcinoma tissue where association with tumorigenesis and invasion/metastasis was suggested.70 As reported in Tables 3 and 4, and shown in Figure 3A, S10A4 was exclusively expressed in LAMA84 cells, and S10A6 protein exhibited a higher level in LAMA84 cells in comparison with K562 (7.21-fold increase) and KCL22 cells (6.72-fold increase). Tropomyosins (TPMs) are a family of cytoskeletal proteins present in virtually all eukaryotic cells, where they bind actin filaments and stabilize their structure.71 Previous studies have demonstrated that TPMs (TPM1, TPM2, TPM3, and TPM4) are expressed more in highly metastatic cell lines than in cells with a low metastatic potential.72,73 Moreover, overexpression of specific isoforms of TPMs, such as TPM1 and TPM30nm and TPM4, was also correlated with the presence of lymph node metastasis and with the HER2-positive status of breast cancer.72-74 In LAMA84 cells, we have identified two isoforms of the tropomyosin alpha-3 chain (TPM3), showing the same pI value (4.76), but different MW values (kDa 33.1 and kDa 31). In K562 and KCL22 cells, only the isoform with higher MW (33.1 kDa) was expressed (Table 3 and Figure 3A). Annexin A1 (ANX1) belongs to a large family of Ca2+dependent phospholipid binding proteins with several functions including intracellular membrane vesicular trafficking and exocytosis.75 Involvement of this protein in the regulation of drug resistance has been proposed, and ANX1 has been described as a stress protein, with cytoprotective activity for cells exposed to stress signals and cytotoxic agents.76 Constitutively high expression of endogenous ANX1 has been shown to contribute to the resistance of leukemic blasts to tumor necrosis factor and possibly to immune-mediated killing.77 Finally, it has been observed that tumor cells derived from ovarian and breast cancer patients with clinically resistant tumors had high levels of ANX1 and an absence of detectable levels of P-glycoprotein (P-gp1), the multidrug resistance protein 1 (MRP1), or of the breast cancer resistance protein (BCRP).78 In the present study we have identified three different isoforms of ANX1 (Supplementary Table 1, Supporting Information). As shown in Figure 3B, two of these isoforms (pI 6.31/ MW 34.2 kDa and pI 6.44/MW 32.3 kDa) were found to be overexpressed in K562 and KCL22 cells in comparison with LAMA84 cells, while the more basic isoform (pI 6.64/MW 34 kDa) was completely absent in LAMA84 cells (Table 3). In particular, the ANXI isoform with pI 6.31 and MW 34.2 kDa exhibited a 4.9- and 5.15-fold decrease in LAMA84 cells vs K562 and KCL22 cells, respectively; the isoform with pI 6.44 and MW 32.3 kDa exhibited a 4.73- and 4.13-fold decrease in LAMA84 cells vs K562 and KCL22 cells, respectively (Table 4). Glutathione S-transferases (GSTs) are a family of multifunctional detoxification enzymes, present in all living organisms. Their main function is the detoxification of electrophilic compounds, and they are divided into two distinct super-family members: the membrane-bound microsomal and cytosolic family members. The mammalian cytosolic GSTs form seven distinct classes termed alpha, mu, pi, sigma, theta, zeta, and a new class of human GSTs designated omega (GSTO1).79,80 GSTs have been implicated in the development of resistance against chemotherapy agents, likely through direct detoxification or acting as an inhibitor of the MAP kinase pathway.79 Recently, 4338


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Figure 4. Motile and invasive properties of LAMA84, K562, and KCL22 cells. The histograms show the results of migration and invasion assays of CML cell lines, tested in a modified Boyden chamber as described in the Materials and Methods section. The motile and invasive properties of CML cells was determined by the average number of migrating cells/well obtained from at least 40 wells (( SD). LAMA84 cells exhibited an approximate 3-fold increase in migration compared to K562 cells and an approximately 2.5-fold increase compared to KCL22 cells. LAMA84 cells were more invasive than K562 (2.5-fold increase) and KCL22 cells (2.3-fold increase). Statistic significance was by the Student’s t test: ***p < 0.001.

Yan and colleagues suggested a role of GSTO1 as a marker for platinum resistance in human ovarian cancer cells.81 In the three CML cell lines analyzed in the present study, GSTO1 expression was exclusive of K562 and KCL22 cells (Table 3 and Figure 3B). Stress or heat shock proteins (HSPs) represent a set of highly conserved proteins that accumulate in cells in response to temperature increase and to different kinds of stress. HSPs function mainly as molecular chaperones and have strong cytoprotective effects. Mammalian HSPs have been classified into four major families according to their molecular size: HSP90, HSP70, HSP60, and the small HSPs (such as HSP27 and HSP10).82 Recently, HSPs were also described as regulators of apoptosis. Among the four families of HSPs, proteins with antiapoptotic (HSP27 and HSP70) and proapoptotic (HSP60 and HSP10) functions have been distinguished.82 Interestingly, the HSPB1 (HSP27) isoform showing pI 5.93 and MW 23.5 kDa was completely absent in LAMA84 cells (Table 3 and Figure 3B). Similarly, HSP71 and the HCP7C isoform with pI 5.47 and MW 66 kDs exhibited lower quantity in LAMA84 cells than in K562 and KCL22 cell lines (HSP71: 4.52- and 4.10-fold decrease in LAMA84 cells vs K562 and KCL22 cells, respectively; HCP7C isoform: 4.11- and 3.47-fold decrease in LAMA84 cells vs K562 and KCL22 cells, respectively; Table 4 and Figure 3B). Overexpression of HSP70 was recently reported as being associated with imatinib resistance in chronic myeloid leukemia, consistent with our finding.83 Functional Implication of Proteins Differentially Expressed Between LAMA84 and K562/KCL22 Cells. In order to investigate whether the observed differences in protein expression profiles of LAMA84, K562, and KCL22 cells could affect cell behavior, we performed migration, chemoinvasion, and cytotoxicity assays. As shown in Figure 4, LAMA84 cells had higher motile and invasive ability than K562 and KCL22 cells (p < 0.001). Significant differences were not observed between K562 and KCL22 cells in both migration and invasion assays. These data paralleled the results highlighted by comparative proteomic analysis revealing overexpression in LAMA84 cells of proteins associated with an invasive and transmigrating phenotype.

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Figure 5. The imatinib dose-response curves for LAMA84, K562, and KCL22 cells. Cell growth was measured by MTT assay after 24 h of treatment with imatinib and was plotted as a percentage of control (Ctrl) growth. Each point represents the mean ( SD for three independent experiments.

Figure 7. Effect of imatinib treatment on BCR-ABL phosphorylation (A) and on the general tyrosine phosphorylation pattern (B) in the three CML cell lines. Whole cell lysates were prepared from CML cells treated with 1 and 10 µM of imatinib for 24 h and subjected to western blot analysis with antibodies against p-BCR-ABL, BCR-ABL, and phosphorylated tyrosine as described in the Materials and Methods section. Protein loading was controlled by probing the stripped membrane with a polyclonal anti-ACTB antibody (lower panels). These results were confirmed by three independent experiments for each cell line. Ctrl: untreated cells.

treatment, likely related to the expression of several proteins implicated in drug resistance and anti-apoptotic activity.

Conclusion Figure 6. Apoptosis after incubation of LAMA84, K562, and KCL22 with 1 and 10 µM of imatinib for 24 h. Apoptosis was measured by fluorescence microscopy. Results are expressed as the mean of three independent experiments ( SD. Statistic significance was calculated between LAMA84 and K562 or KCL22 cells by the Student’s t test: * p < 0.05; ** p < 0.01. CTRL: untreated cells.

Cytotoxicity assays revealed a different responsiveness of the three cell lines to imatinib, the major therapeutic agent for the treatment of patients with CML.84,85 The graph in Figure 5 shows that after 24 h of treatment, K562 and KCL22 cells were less sensitive than LAMA84 cells to imatinib. AO/EB staining showed that both 1 and 10 µM of imatinib caused a significant increase of apoptosis in LAMA84 cells in comparison with K562 and KCL22 cells (p < 0.05; Figure 6). This reduced sensitivity of K562 and KCL22 cells to imatinib was also confirmed by evaluating BCR-ABL phosphorylation as well as the overall the pattern phospho-tyrosine proteins. The western blots in Figure 7 show that 24 h of imatinib-treatment induced a marked decrease of BCR-ABL phosphorylation already with 1 µM imatinib, and a complete inhibition with 10 µM. In contrast, 1 µM imatinib only slightly inhibited the BCR-ABL phosphorylation in K562 and KCL22 cells, while a 10 µM dose caused a significant inhibition only in K562 cells. The general pattern of tyrosine phosphorylated proteins indicated a greater downregulation of kinase activity in imatinib-treated LAMA84 cells compared to K562 and KCL22 cells. These data suggest an intrinsic resistance of K562 and KCL22 cells to imatinib

In conclusion, these data present a reference proteomic map for LAMA84, K562, and KCL22 Ph+ CML cell lines, suggesting a new subset description as a function of their proteomic phenotype. Comparative proteome analysis of these three cell lines, commonly considered representative of the same clinical phenotype, revealed intrinsic differences in protein expression that translated to cellular behavior. Our study demonstrated that cells of blast crisis can display different and selective properties, such as an marked invasive capacity or an intrinsic propensity to drug resistance. The differences observed in this study may be applied to clinical samples to further dissect interpatient variability in the progression of disease and in the efficacy of treatment. These data may contribute to a further biological characterization of late-phase CML blasts, leading to optimization of the treatment strategies.

Acknowledgment. M.B. is a fellow of the Universita` of Palermo, M.G. is a Ph.D. student in Immunopharmacology at the Universita` of Palermo, and C.C. is a fellow of Italian Association for Cancer Research (AIRC). This work was supported by the Italian Association for Cancer Research (AIRC) to G.D.L. and R.A., and MURST to R.A. and to G.D.L. We thank Dr. Marco Milazzo and Dr. Chiara Romano for multivariate statistical analysis. Supporting Information Available: Supplementary Figure 1; Supplementary Table 1 reporting all the protein spots identified by MALDI-TOF and/or LC-ESI/MS/MS and numJournal of Proteome Research • Vol. 6, No. 11, 2007 4339

research articles bered in the maps in Figure 1. This information is available free of charge via the Internet at http://pubs.acs.org.

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