Fludarabine and Cladribine Induce Changes in Surface Proteins on

Article pubs.acs.org/jpr

Fludarabine and Cladribine Induce Changes in Surface Proteins on Human B‑Lymphoid Cell Lines Involved with Apoptosis, Cell Survival, and Antitumor Immunity Philippa L. Kohnke, Swetlana Mactier, Juhura G. Almazi, Ben Crossett, and Richard I. Christopherson* School of Molecular Bioscience, University of Sydney, Sydney, NSW 2006, Australia S Supporting Information *

ABSTRACT: Fludarabine and cladribine are purine analogues used to treat hematological malignancies. Alone or in combination with therapeutic antibodies, they are effective in treating patients with chronic lymphocytic leukemia and nonHodgkin's lymphoma. However, the mechanisms of action of these drugs are not well understood. Plasma membrane proteins perform a variety of essential functions that can be affected by malignancy and perturbed by chemotherapy. Analysis of surface proteins may contribute to an understanding of the mechanisms of action of purine analogues and identify biomarkers for targeted therapy. The surface of human cells is rich in N-linked glycoproteins, enabling use of a hydrazide-coupling technique to enrich for glycoproteins, with iTRAQ labeling for quantitative comparison. A number of plasma membrane proteins on human leukemia and lymphoma cells were affected by treatment with a purine analogue, including decreases in CD22 (an adhesion and signaling molecule) and increases in CD205 (a “damaged cell marker”) and CD80 and CD50 (T-cell interaction molecules). Purine analogues may affect B-cell receptor (BCR) signaling and costimulatory molecules, leading to multiple signals for apoptosis and cell clearance. Fludarabine and cladribine induce differential effects, with some cell survival proteins (ECE-1 and CD100) more abundant after fludarabine treatment. Cell surface proteins induced by fludarabine and cladribine may be targets for therapeutic antibodies. KEYWORDS: plasma membrane, N-linked glycoproteins, hydrazide coupling, cladribine, fludarabine, lymphoma, leukemia, iTRAQ

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INTRODUCTION

2-CdA are active against indolent lymphomas refractory to single agent therapy.8 However, some patients respond poorly or develop resistance to purine analogues, especially those with progressive CLL that has a poor prognosis. 2-FaraA and 2-CdA induce strand breaks in DNA, with incorporation of the analogue into DNA, leading to chain termination.9,10 Accumulation of strand breaks results in phosphorylation of p53 that accumulates and activates the intrinsic apoptotic pathway.11−13 The triphosphate derivatives of 2-FaraA and 2-CdA inhibit DNA and RNA polymerases and ribonucleotide reductase, arrest the cell cycle,14,15 affect apoptosis or signal transduction pathways, and induce apoptosis.16,17 2-FaraA and 2-CdA have multiple mechanisms

Hematological malignancies may be treated with the purine analogues fludarabine (9-β-D-arabinofuranosyl-2-fluoroadenine5′-monophosphate, 2-FaraA) and cladribine (2-chloro-2′deoxyadenosine, 2-CdA). 2-FaraA is administered as the nucleoside 5′-monophosphate (2-FaraAMP) derivative to improve solubility.1 2-FaraAMP, alone or in combination with other drugs, is effective in the treatment of B-cell chronic lymphocytic leukemia (CLL) and low-grade non-Hodgkin's lymphoma (NHL).2,3 2-CdA is used to treat CLL, low-grade NHL, and hairy cell leukemia (HCL).4,5 The combination of a purine analogue with a therapeutic antibody such as rituximab (anti-CD20) is effective against CLL and NHL.6 2-FaraAMP, cyclophosphamide, and rituximab (FCR) are used to treat CLL and produce a high proportion of complete remissions and prolonged survival.6,7 Rituximab and © 2012 American Chemical Society

Received: January 25, 2012 Published: July 30, 2012 4436

dx.doi.org/10.1021/pr300079c | J. Proteome Res. 2012, 11, 4436−4448

Journal of Proteome Research

Article

of 2-FaraA (100 μM, 24 h) or 2-CdA (1 μM, 24 h).35 Control cultures were treated with the corresponding amount of dimethylformamide as a vehicle control.

of action with differences in mechanisms and specificity for leukemia and lymphoma subtypes. A more complete understanding of these mechanisms could lead to improved treatment of CLL and NHL. Analysis of the effects of 2-CdA and 2-FaraA on surface proteins may lead to identification of up-regulated targets for therapeutic antibodies. Human Raji (B-cell Burkitt's lymphoma) and MEC1 (B-cell CLL in prolymphocytoid transformation) cell lines are suitable models to study the effects of purine analogues on Blymphoproliferative disorders.18,19 We have already determined the effects of 2-CdA and 2-FaraA on the nuclear, mitochondrial, and cytosolic proteomes of Raji cells.20,21 These drugs induced differential abundance of proteins involved in signaling ER stress and activation of the unfolded protein response, a novel mechanism for purine analogue-induced apoptosis.20 We have also determined the effects of purine analogues on a limited number of cluster of differentiation antigen (CD antigens) using DotScan antibody microarrays,22 but global effects on the cell surface proteome of Raji cells are unknown. Cell surface proteins mediate communication between the cancer cell and its microenvironment23 with cellular functions that can be transformed by disease or affected by drugs.24−27 Although cell surface proteins are normally difficult to analyze,28−30 we have exploited the fact that many are Nlinked glycoproteins.31 A shot-gun hydrazide-coupling method has been used, where the polysaccharide residues were oxidized to dialdehydes that were coupled to hydrazide beads for fractionation.32,33 Deglycosylated peptides were selectively released for analysis by treatment with peptide-N-glycosidase F (PNGaseF) that cleaves N-linked glycans from peptides. Isobaric tags for relative and absolute quantitation (iTRAQ) labeling of these released peptides and analysis by liquid chromatography coupled to tandem mass spectrometry (LCMS/MS) enabled quantitative comparison between control and drug-treated human Raji cells. Using hydrazide coupling, we have identified more than 230 N-linked glycoproteins on the surface of Raji cells and analyzed their responses to 2-FaraA and 2-CdA. We have confirmed that the proteomics results reflect consistent responses between different human B-cell lines, suggesting specific mechanisms of action and global effects for purine analogues.

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Sample Preparation

Extracts of Raji cells (5 × 108, n = 3) were fractionated by differential centrifugation to separate the microsomal fraction from other cellular compartments,36 and this fraction also contained plasma membranes, with Golgi and ER membranes and some contamination from soluble proteins of the cytoplasm. The microsomal pellet was resuspended in 8 M urea and 1% (w/v) SDS in 50 mM Tris-HCl, pH 7.8. Protein concentrations were determined using the 2D Quant protein assay (GE Healthcare, Piscataway, NY) according to the manufacturer's protocol. Equal amounts of protein for each biological replicate (5−10 mg) were taken for glycopeptide enrichment. Proteins were reduced with 10 mM TCEP at 37 °C for 1 h and alkylated with 50 mM iodoacetamide at room temperature for 30 min in the dark. Samples were diluted with 50 mM Tris-HCl, pH 8.0, to concentrations of urea < 1 M and SDS < 0.1% (w/v). Trypsin (Promega, Sydney Australia) was added at a ratio of 1:100 (enzyme:protein) and incubated overnight at 37 °C. N-Linked Glycopeptide Capture

The complex tryptic peptide mixture was acidified to pH < 2 with trifluoroacetic acid, before desalting and concentration by solid-phase extraction (SPE) using an activated Oasis HLB SPE column (Waters Corporation, Milford, MA). Samples were dried by vacuum centrifugation (Concentrator Plus, Eppendorf, North Ryde, NSW, Australia) and then resuspended in coupling buffer (100 mM sodium acetate, 150 mM sodium chloride, pH 5.5). The samples were oxidized with 15 mM sodium periodate (fresh) for 1 h in the dark at room temperature with rotation, before quenching with 25 mM sodium sulfite for 10 min. Quenched samples were coupled to washed Bio-Rad Affi-Prep HZ resin (100 μL packed resin) overnight at room temperature. The resin was washed sequentially with PBS, 1.5 M NaCl, 50% ACN (w/v), 100% methanol, and 100 mM triethylammonium bicarbonate (four times each) to remove nonglycosylated peptides. N-linked glycopeptides were released from the resin with 1 μL of PNGaseF (15000 IU, glycerol-free) incubated overnight at 37 °C. The deglycosylated peptides were collected by washing the resin with 3 × 50% (v/v) acetonitrile, and the combined eluate was dried by vacuum centrifugation.

MATERIALS AND METHODS

Reagents

2-CdA was a kind gift from the National Cancer Institute (Bethesda, MD). Gentamicin, L-glutamine, and fetal calf serum were from Invitrogen (Mulgrave, VIC, Australia). Hydrazideresin was from Bio-Rad Laboratories (Hercules, CA). PNGaseF was from New England Biolabs (Beverly, MA). All other chemicals were from Sigma-Aldrich (St. Louis, MO), unless otherwise stated.

iTRAQ Labeling

Dried peptides enriched from glycopeptide capture were iTRAQ-labeled (4-plex) according to the manufacturer's protocol (Applied Biosystems, Foster City, CA). Labeling of the samples in the biological sets 1 and 2 used 114 as the control; in the biological set 3, 117 was the control to remove label choice bias. Each mixed sample was then desalted by SPE and dried by vacuum centrifugation.

Cell Lines and Culture Conditions

Human Raji (B-cell Burkitt's lymphoma) and MEC1 (B-CLL) cells from the American Type Culture Collection (Manassas, VA34) were grown in HEPES-buffered RPMI 1640 medium with 10% fetal calf serum, 50 μg/mL gentamicin, and 2 mM Lglutamine at 37 °C in a nonhumidified atmosphere without CO2. Raji cultures (three biological replicates) at a density of 3 × 105 cells/mL were treated with IC50 levels of 2-FaraA (3 μM), 2-CdA (1 μM), or methotrexate (MTX) (1 μM) for 24 h before harvesting for analysis.20,22,35 For confirmation studies using flow cytometry, MEC1 cells were treated with IC50 levels

Two-Dimensional Liquid Chromatography Tandem Mass Spectrometry

For the 2D LC-MS/MS, each set of desalted, iTRAQ-labeled samples was separated using an online strong cation exchange (SCX) column coupled to a reverse phase (RP) column, with step-gradient elution using an Agilent 1100 Capillary and Nano LC system (Agilent, Palo Alto, CA). The 2D LC-MS/MS conditions are described in the Supporting Information. 4437



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Figure 1. Workflow for analysis of the effects of 2-FaraA and 2-CdA on plasma membrane proteins. Raji cells were grown in culture as a control, 2FaraA-treated (3 μM, 24 h), 2-CdA-treated (1 μM, 24 h), or MTX-treated (1 μM, 24 h), and then were lysed, fractionated, and analyzed. The membrane fraction was collected by centrifugation (100000g, 60 min, 4 °C), digested with trypsin, and oxidized with sodium periodate, and N-linked glycopeptides were coupled to hydrazide-resin. Other peptides were removed by extensive washing, and N-linked glycopeptides were released by PNGaseF. The formerly glycosylated peptides were labeled with iTRAQ tags and analyzed by 2D LC-MS/MS, and the data were searched using ProteinPilot.

allowed if the site of N-linked glycosylation was present and deamidated by the action of PNGaseF, increasing the confidence of the assignment.38 Only peptides with deamidation sites at the consensus N-linked glycosylation sequon were chosen to contribute to the quantitative ratio for relative comparison. All database peptide sequences, iTRAQ area MS/ MS fragmentation, and putative glycosylation sites were confirmed by manual inspection of the MS/MS spectra to determine an accurate sequence assignment, iTRAQ ratio reporting, and correctly deamidated glycosylation site. Drug-treated to control ratios were normalized to ensure that mixing and experimental discrepancies of the ratios were corrected. Normalization was applied to glycoprotein matches (with the sequence Asn-X-Ser/Thr) so that the median iTRAQ ratio was 1:1 control to drug-treated, as most cell surface proteins within the sample were not predicted to change based on previous results.22 Gene Ontology information was gathered from the Human Protein Reference Database (www.hprd. org),39 and trans-membrane prediction was gathered from the TMHMM algorithm (www.cbs.dtu.dk/services/TMHMM).40

The LC eluate was subjected to positive ion nanoflow electrospray analysis using a QSTAR XL MS/MS system (Applied Biosystems/MDS SCIEX) in information-dependent acquisition mode (IDA). In IDA mode, a TOF-MS survey scan was acquired (m/z 400−1800, 1 s) with the three most intense multiply charged ions (counts >60) in the survey scan sequentially subjected to product ion analysis. Product ion spectra were accumulated for 2 s in the mass range m/z 100− 1600 with a modified Enhance all mode Q2 transition setting favoring low mass ions so that the iTRAQ ion intensities were enhanced for quantification. Dynamic exclusion was used with a 45 s and 200 ppm window. Data Analysis

MS/MS data were analyzed using ProteinPilot v3.0 (Applied Biosystems) that uses the Paragon algorithm37 to perform database matching for protein identification, protein grouping to remove redundant hits, and comparative quantitation. The protein database used for all searches was a combination of UniProtKB, Swiss-Prot, and Trembl (accessed December, 2008, and March, 2012), IPI human (version 3.58), and a file of common proteomics contaminants (included in the ProteinPilot 3.0 software), with a total of 833880 sequences. ProteinPilot parameters used an iTRAQ (4-plex) method, with enzyme:trypsin and thorough identification with a focus on biological modifications and amino acid substitutions, with special factors of urea denaturation and hydrazide enrichment, which increased the potential assignment of the following variable modifications: carbamidomethylated cysteine (+57), oxidized methionine (+16), and deamidation of asparagine (+1). We report peptide identifications with confidence >95% and protein identifications with ProtScore 1.3, which represents >95% statistical confidence in ProteinPilot. The false discovery rate was chosen as 5%, although the spectra of matching peptide sequences were manually inspected to reduce false positives. Proteins confirmed as nonglycoprotein contaminants were selected based on two peptides identified with 95% confidence. Glycoproteins identified with only one peptide sequence were

Flow Cytometry

To confirm proteins identified as differentially abundant by iTRAQ analysis, selected glycoproteins were analyzed using a FACScalibur flow cytometer (Becton Dickinson, San Jose, CA) with a 488 nm argon laser, running CellQuest Pro software version 5.2 (Becton Dickinson). FITC- or PE-conjugated monoclonal antibodies against CD100, CD205, CD79a, CD79b, and isotype controls were obtained from Biolegend (San Diego, CA), antibodies against CD50 and CD80 and their isotype controls were obtained from Beckman Coulter (Gladesville, NSW, Australia). Raji and MEC1 cells were treated with purine analogues as described above and labeled with fluorescent antibodies using standard procedures. Fluorescence detection used logarithmic amplification; data are the averages of three biological replicates. 4438



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Figure 2. MS/MS spectrum of N-linked glycopeptide AN#LTVVLLR (572.36 m/z, 2+) originating from CD54 (ICAM1). The symbol # represents the N-glycosylation site, and the mass difference of 115 between y7 and y8 confirms the deamidation of asparagine in the peptide resulting from PNGaseF treatment.

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proteins was low, ∼20% of the total proteins (Figure 3B). The identifications include 55 CD antigens, commonly used for leukemia classification. Furthermore, the majority of N-linked glycoproteins had at least one transmembrane domain, with 149 having 1−2 putative or known transmembrane regions, and a further 48 were multipass transmembrane proteins (Figure 3B).

RESULTS AND DISCUSSION Raji cells are sensitive to 2-FaraA (3 μM) and 2-CdA (1 μM), concentrations comparable to those achieved in the blood during clinical treatment. We have previously shown that onethird of Raji cells are apoptotic 24 h after drug treatment, as compared to less than 1/10 in control cultures.20 We have determined the effects of 2-FaraA and 2-CdA on the cell surface proteome of Raji cells using a proteomics approach that enriches for N-linked glycoproteins (Figure 1). MTX, an anticancer drug that produces apoptosis with a similar IC50 against Raji cells by a different mechanism, was used as an additional control. N-linked glycoproteins whose levels changed after MTX treatment were excluded from the list of proteins affected by purine analogue treatment.

Glycoproteins Affected by Purine Analogue Treatment

Normalized ratios for glycoproteins from drug-treated to control samples were selected for those identified in at least two of the three replicates and not affected by MTX treatment (Table 1). A total of 16 glycoproteins were up-regulated by one or both purine analogues, and 13 were known or predicted cell surface proteins. In addition, 17 proteins were down-regulated, with 12 known cell surface proteins. Many membrane glycoproteins affected by drug treatment are involved in cell communication, signal transduction, and the immune response (Figure 4), consistent with their functions as receptors and adhesion molecules, suggesting that the plasma membrane may mediate signaling and interactions involved in response to drug treatment. Other glycoproteins changed by drug treatment map to the endoplasmic reticulum, Golgi apparatus, and lysosomes, and their functions include protein metabolism or energy pathways. Some of these proteins correlate with previous results from our laboratory relating to protein changes in the nucleus, mitochondria, and cytoplasm, where ER stress and mitochondrial dysfunction were found to be novel pathways disrupted by purine analogues.20

Enrichment of N-Linked Glycoproteins

Quantitative analysis of three biological replicates yielded 373 putative N-linked glycopeptides from which 232 unique glycoproteins were identified, with an average of 1.6 glycopeptides per glycoprotein (Table S1 in the Supporting Information). A representative spectrum of a peptide with deamidation of asparagine at the N-linked glycosylation consensus sequence is presented in Figure 2. More than 50% of N-linked glycoproteins were annotated as plasma membrane proteins, and another 19% were annotated as integral membrane proteins (Figure 3A). Many other N-linked glycoproteins annotated as ER, Golgi apparatus, or lysosomes may shuttle to the cell surface,41 increasing the percentage of plasma membrane proteins. Contamination by nonglycosylated 4439



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Figure 3. Enrichment of N-linked glycoproteins from the plasma membrane of Raji cells. (A) The Gene Ontology subcellular locations were assigned from the Human Protein Reference Database (hprd.org), with 123 N-linked glycoproteins annotated as “plasma membrane” and 44 annotated as “integral to membrane”. (B) Data gathered from the Human Protein Reference Database and TMHMM algorithm v2.0 (www.cbs.dtu. dk/services/TMHMM) showed that the identified proteins have a single-pass (142) or multipass (48) trans-membrane (TM) domain.

Validation of Glycoproteins

hydrazide affinity protocol (Table 1) were similar in most cases to the cell surface protein abundance ratios measured by flow cytometry (Figure 5 and Table 2) or to published flow cytometry data.22

iTRAQ labeling of the glycopeptide-enriched microsomal fractions enabled quantitative comparison of drug-treated to control samples. However, the actual abundance ratios measured could represent changes in the protein level, including expression and degradation or, alternatively, variations of glycosite occupation and glycosylation abundance. Others have observed that most glycoproteins showing differential abundance in glycocapture analysis correlate with confirmatory assays that reflect a protein-level change, such as Western blots.42,43 Differentially abundant glycoproteins on Raji cells involved in apoptosis, cell survival, and antitumor immunity were selected for validation. Furthermore, MEC1 cells, as a cell line resembling CLL,44 were chosen to validate the effects of purine analogues. The identities and quantification of the plasma membrane glycoproteins CD50, CD79a, CD79b, CD80, CD100, and CD205 (n = 3 for each condition) on Raji and MEC1 cells were validated using flow cytometry (Figure 5), a technique that measures protein abundance on the cell membrane using fluorescently labeled antibodies. The glycosylation abundance ratios determined by the iTRAQ-

Up-regulation of “Damaged Cell Marker”

CD205 was up-regulated >2-fold by both 2-FaraA and 2-CdA with multiple glycopeptides identified and quantified (Table 1). Flow cytometry confirmed increases of CD205 upon 2-FaraA and 2-CdA treatment of Raji and MEC1 cells (Figure 5 and Table 2). B-cells show moderate expression of CD205 with higher levels on dendritic cells.45,46 CD205 is a recycling endocytic receptor on dendritic cells that binds extracellular antigens through lectin domains and directs them to the antigen processing machinery for presentation to T-cells.47 Antigens recognized are endocytosed, processed, and presented on major histocompatibility complex class I and class II molecules with high efficiency.48 For B-cells, CD205 has antiproliferative effects and can up-regulate costimulatory molecules.49 CD205 may also be an apoptotic or necrotic cell recognition receptor that senses dying cells, and on dendritic 4440



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Table 1. N-Linked Glycoproteins Affected by 2-FaraA (3 μM, 24 h) and 2-CdA (1 μM, 24 h) on Raji Cells Identified by 2D LCMS/MS Analysisa accession no.b O60449 P13688 Q6P4Q7 P33681 Q96JJ7 Q9HDC9 Q9Y4L1 P11912 P32942 Q01151 Q67AD6 P42892 Q16880 P11166 Q6PIU2 Q8NFQ8 P01903 Q7LGA3 P08236 P01130 Q96RQ1 P20273 Q92854 O15321 Q9BVK6 P07339 Q9NU53 P40259 Q9HD45 Q7Z7H5 P21854 Q02809 Q9Y487

protein name

(2-FaraA/ Control) meanc

2-FaraA p valued

(2-CdA/ Control) meanc

glycoproteins up-regulated by 2-FaraA or 2-CdA CD205, lymphocyte antigen 75 2.25 ± 0.28 0.001 2.19 CD66a, CEACAM1 2.27 metal transporter CNNM4 2.15 4 × 10−13 1.53 CD80, T-lymphocyte activation antigen 1.99 protein disulfide-isomerase TXNDC10 1.75 ± 0.23 0.004 adipocyte plasma membrane-associated protein 1.74 ± 0.15 4 × 10−4 hypoxia up-regulated protein 1 1.72 ± 0.21 0.001 CD79a, B-cell antigen receptor complex1.72 ± 0.37 0.019 associated protein CD50, intercellular adhesion molecule 3 1.73 ± 0.54 0.044 1.52 CD83 antigen 1.65 MHC class II antigen - Homo sapiens 1.52 ± 0.33 0.035 1.63 endothelin-converting enzyme 1 1.52 ± 0.35 0.045 2-hydroxyacylsphingosine 1-β1.53 ± 0.43 0.082 1.31 galactosyltransferase solute carrier family 2, facilitated glucose 1.44 transporter 1 arylacetamide deacetylase-like 1 1.35 ± 0.6 0.001 torsin-1A-interacting protein 2 1.35 ± 0.13 0.009 glycoproteins down-regulated by 2-FaraA or 2-CdA HLA class II histocompatibility antigen, DRα 0.37 ± 0.14 0.015 0.36 chain heparan sulfate 2-O-sulfotransferase 1 0.45 β-glucuronidase 0.48 ± 0.15 0.020 low-density lipoprotein receptor 0.53 ± 0.21 0.035 endoplasmic reticulum-Golgi intermediate 0.54 compartment protein 2 CD22, B-cell receptor 0.59 ± 0.17 0.030 0.58 CD100, semaphorin-4D 0.6 transmembrane 9 superfamily member 1 0.61 ± 0.15 0.029 transmembrane emp24 domain-containing 0.63 protein 9 cathepsin D 0.66 uncharacterized protein C6orf72 CD79b, B-cell antigen receptor complexassociated protein transmembrane 9 superfamily member 3 transmembrane emp24 domain-containing protein 4 CD72, B-cell differentiation antigen procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 V-type proton ATPase 116 kDa subunit a isoform 2

0.67 ± 0.13 0.70 ± 0.12 0.75 ± 0.1

2-CdA p valued

gene ontology functione

0.15 0.79 0.15 0.66

2 × 10−4 0.024 0.003 0.035

receptor function unknown function unknown receptor oxidoreductase function unknown chaperone B-cell receptor

± 0.37 ± 0.09 ± 0.41

0.045 4 × 10−4 0.029

± 0.16

0.018

cell adhesion function unknown MHC complex metallopeptidase transferase

± 0.2

0.013

auxiliary transport

± ± ± ±

function unknown function unknown ± 0.01

1 × 10−5

MHC class I receptor

± 0.005

7 × 10−7

± 0.03

6 × 10−5

sulfotransferase hydrolase receptor function unknown

± 0.12 ± 0.15

0.009 0.029

± 0.15

0.031

± 0.15

0.037

0.66 ± 0.09

0.009

aspartic-type signal peptidase function unknown B-cell receptor

0.73 ± 0.05

0.002

auxiliary transport function unknown

0.025

0.026

0.022 0.75 ± 0.006 0.76 ± 0.08

7 × 10−6 0.009

cell adhesion receptor binding receptor function unknown

receptor catalytic activity ATPase

a Blank spaces indicate no significant change in abundance. bAccession numbers were acquired from UniProt (http://expasy.org/). cThe drug to control mean ratio was calculated from a normalized iTRAQ ratio output from ProteinPilot. dThe p value was calculated using Student's t test (n = 3) using log2 iTRAQ values. eGene Ontology functions are listed from the Human Protein Reference Database (www.hprd.org).

cells, it mediates the uptake of apoptotic cell-derived selfantigen.50 Thus, the up-regulation of CD205 by 2-FaraA or 2CdA may facilitate the in vivo removal of cells damaged by drug treatment.

CdA (2.0-fold), while 2-FaraA had no effect (Table 1). Flow cytometry analysis of CD80 showed similar increases in abundance on Raji and MEC1 cells upon treatment with 2FaraA and 2-CdA (Table 2). The increased response of CD80 to 2-FaraA by flow cytometry as compared with iTRAQ/LCMS/MS may reflect higher sensitivity when using fluorescently labeled antibodies. CD80 is part of the T-cell costimulation pathway of CD28/ CTLA-4/CD80/CD86, where B-cell presentation of CD80/ CD86 interacts with T-cell CD28 and CTLA-4 to induce activation or inhibition of T-cells, respectively.51 This interaction is the major costimulatory signal provided by B-

Up-regulation of Cell Adhesion and Interaction Molecules

Cell−cell interactions mediate important immune system events, including the development, differentiation, and activation of immune cells. 2-FaraA and 2-CdA up-regulated a number of cell−cell interactions and costimulatory molecules, including CD50, CD80, and ECE-1. As assessed by iTRAQ/ LC-MS/MS analysis, CD80 was up-regulated on Raji cells by 24441



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Figure 4. Biological functions of differentially abundant N-linked glycoproteins. Plasma membrane glycoproteins affected by drug treatment are involved in processes that include cell communication and immune response. Other glycoproteins map to the endoplasmic reticulum, Golgi apparatus, and lysosomes and are involved in metabolism functions, such as protein metabolism or energy pathways.

cells to amplify T-cell activation.52 CD80 signaling in Raji Bcells can induce growth arrest53 and is linked to reduced proliferation, up-regulation of pro-apoptotic proteins, and down-regulation of antiapoptotic proteins, indicating the promotion of both intrinsic and extrinsic apoptosis.54 This change in the balance of apoptotic factors may initiate apoptosis and, combined with activation of cytotoxic T-cells, could have negative effects on B-cells. Cancers may escape immune surveillance by cytotoxic T-cells, although they express T-cell ligands, through mechanisms including down-regulation of costimulatory molecules such as CD80.55 Increased expression of CD80 on some cancers can initiate targeting by cytotoxic T-cells.56,57 Thus, up-regulation of costimulatory molecules such as CD80 by purine analogues may assist B-cell death through targeting for cell lysis by cytotoxic T-cells and natural killer cells. CD50 was up-regulated >1.5-fold by 2-FaraA and 2-CdA (Table 1). Flow cytometry confirmed the increase of CD50 on Raji and MEC1 cells after exposure to purine analogues, although Raji show two populations of cells expressing CD50, each increasing after treatment (Figure 5). CD50 is involved in intercellular adhesion of leukocytes and forms part of the initial adhesive contact between T-cells and antigen presenting cells, facilitating activation.58 CD50 also enables phagocytosis of apoptotic cells by a conformational change on apoptotic leukocytes that allows recognition by macrophages.59 However, elevated CD50 may enhance cancer cell proliferation60 and lead to cell migration and invasion.61 Thus, CD50 may act in concert with CD80 to assist in immune-regulated removal of apoptotic cells, or an increase in CD50 may initiate pro-survival signaling. CD66a (CEACAM1) was up-regulated by 2-CdA (2.3-fold), while 2-FaraA had no effect. CD66a is an intercellular adhesion molecule and may be an inhibitory coreceptor of the B-cell receptor (BCR).62 Soluble CEACAM1 has a pro-angiogenic function and can stimulate cell proliferation.63 Membranebound CD66a may act as a tumor suppressor, with a negative correlation between CEACAM1 expression and tumor progression that may promote apoptosis.63 Other studies have reported increased CEACAM1 induced by cytotoxic drugs.64

BCR Activation

A number of CD antigens participating in B-cell signaling through the BCR were affected by 2-FaraA or 2-CdA on Raji cells (Table 1), including decreases in CD22, CD72, CD79b, and CD100 and an increase in CD79a. B-cells depend on BCRinduced transmembrane signals for development, activation, proliferation, and effector function.65 CD79a (Ig-α) and CD79b (Ig-β) form a covalent link on the cell surface and interact with membrane IgM to form the BCR.66 CD79a was up-regulated by purine analogues on Raji and MEC1 cells, and CD79b was down-regulated by 2-CdA on Raji cells (Tables 1 and 2 and Figure 5). The CD79 heterodimer is responsible for transducing the BCR signal upon IgM binding antigen, as well as promoting endocytosis of the bound antigen.67 CD22 and CD72 are inhibitory coreceptors that directly associate with the BCR.68 The decreases in CD22 of 0.6-fold for both purine analogues were observed by CD antibody microarray and flow cytometric analysis.22 CD22 enables recruitment of proteins that inhibit BCR signaling,69 and reduced CD22 may lower the threshold for BCR activation and cause B-cells to undergo apoptosis after BCR engagement, enhancing the negative role of BCR stimulation.65,70 A decrease in CD22 reduces normal and malignant cell survival.71 CD22 is also a cell−cell interactionmediating adhesion lectin that recognizes sialic acid-containing glycoproteins from a broad range of cells, including B- and Tcells.72 Repression of CD22 on drug-treated B-cells may increase pro-apoptotic signals. CD72 also inhibits the BCR to affect B-cell signaling, expression correlates with reduced proliferation.73 CD72 is blocked by CD100,74,75 which releases the inhibitory function of CD72 signaling.68 CD100 was down-regulated 0.6-fold by 2CdA by LC-MS/MS analysis, and flow cytometry confirmed that CD100 decreased with 2-CdA (Figure 5 and Table 2) on Raji cells. Reduction of CD100 on B-cells treated by 2-CdA may release the inhibitory effect on CD72, affecting BCR-based signaling.75 The BCR balances B-cell fate, from apoptosis in immature B-cells to survival and activation in mature cells, according to the signals received.76,77 BCR signaling, in the absence of specific ligand or survival signals from T-cells, produces anergy or cell death due to activation of signaling cascades culminating in apoptosis via the intrinsic pathway.78 Our previous experiments showed that purine analogues induce 4442



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Figure 5. Flow cytometry of N-linked glycoproteins affected by a purine analogue. The results of iTRAQ/LC-MS/MS analysis were confirmed for selected glycoproteins by flow cytometric analysis of 2-FaraA- (3 μM, 24 h) and 2-CdA- (1 μM, 24 h) treated Raji cells and 2-FaraA- (100 μM, 24 h) and 2-CdA- (1 μM, 24 h) treated MEC1 cells. The gray area represents untreated cells, the black outline is 2-FaraA-treated cells, and the dotted outline is 2-CdA-treated cells. The figures are representative of three technical replicates. A shift in median fluorescence intensity indicates up- or down-regulation of a surface glycoprotein. 4443



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Table 2. Differentially Abundant CD Antigens on Raji and MEC1 Cells after Treatment with 2-FaraA and 2-CdA Assessed by Flow Cytometry Raji + 2-FaraA CD antigen CD50 CD100 CD205 CD79a CD79b CD80

fold changea c

1.8 1.4 2.0 2.2 no change 1.5

Raji + 2-CdA

p valueb 1.5 2.1 1.5 1.6

× × × ×

−3

10 10−2 10−3 10−4

8.4 × 10−5

fold changea 1.3 0.7 1.3 1.4 0.7 1.3

c

MEC1 + 2-FaraA

p valueb 4.7 1.8 9.5 1.6 2 6

× × × × × ×

−2

10 10−2 10−4 10−4 10−4 10−4

fold changea 1.5 1.2 1.6 1.5 no change 1.7

MEC1 + 2-CdA

p valueb 2.3 5 5 3.6

× × × ×

−6

10 10−4 10−4 10−3

2.6 × 10−4

fold changea 1.6 2.1 2.0 1.5 no change 1.6

p valueb 3.7 8.1 5.6 2.1

× × × ×

10−5 10−6 10−5 10−3

8.3 × 10−4

Fold-change calculated by dividing the median fluorescence intensity for the drug-treated sample by that for the control (Figure 5). The p value was calculated using Student's t test (n = 3). cRaji cells show two populations with cells expressing lower and higher levels of CD50 (Figure 5). Both populations increase CD50 levels after 2-FaraA and 2-CdA treatment. The fold change was calculated by dividing the sum of median fluorescence intensity of cells expressing high (M1) and low (M2) levels of CD50 (Figure 5) for treated culture by that for the control. a

b

Figure 6. N-linked glycoproteins differentially affected by 2-FaraA or 2-CdA. β-Glucuronidase and cathepsin D are intracellular glycoproteins, and CD antigens are cell surface glycoproteins. Some N-linked glycoproteins show different responses to 2-FaraA or 2-CdA, indicating differences in the mechanisms of action of these purine analogues. Glycoproteins that are significantly differentially abundant in Raji cells treated with a purine analogue are summarized in Table 1.

intrinsic apoptotic signals from ER stress and mitochondria,20 concurrent with BCR-activated apoptosis.

tumor progression83 and correlates with poor prognosis in some cancers.84 Thus, up-regulation of CD100 on 2-FaraAtreated cells may increase pro-survival signaling and antiapoptotic mechanisms to decrease cell death after drug treatment. Endothelin-converting enzyme (ECE-1), a zinc-chelating metalloprotease on the cell surface, was up-regulated by 2FaraA (1.5-fold), while 2-CdA had no effect (Table 1). ECE-1 converts big endothelin to mitogenic endothelin-1 peptide that stimulates tumor cells, modifies the actions of tumor-infiltrating lymphocytes, and modulates matrix metalloprotease function.85 Endothelin-1 peptide may also inhibit apoptosis.86 ECE-1 is elevated in metastatic cancer and promotes cell invasion and migration.87 ECE-1 expression on tumor cells may provide cross-talk to surrounding stromal cells and increase cell growth.88 Thus, up-regulation of ECE-1 in response to a purine analogue may have a protective effect on Raji cells. The

Prosurvival Signaling

CD100, a receptor for CD72, is also the “leukocyte semaphorin 4D” expressed on T- and B-cells.79 By flow cytometry, we have found that 2-FaraA promotes CD100 expression on Raji and MEC1 cells (Figure 5). Opposite to the result with Raji cells, MEC1 cells increase CD100 expression after 2-CdA treatment (Figure 5). This difference in CD100 expression after purine analogue treatment indicates that divergent processes are involved in responses to 2-FaraA or 2-CdA. CD100 can provide proliferative signals to T-cells and can induce B-cells to aggregate and improve their survival in vitro.80 For B-CLL, CD100 may promote cell growth and survival through antiapoptotic signaling.81,82 Interaction of CD100 with plexinB1 may promote angiogenesis, invasive cell proliferation, and 4444



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survival pathways, like cell−cell adhesion networks, could increase pro-apoptotic signals to the cell, and therapeutic antibodies targeting CD50, CD100, and ECE-1, survivalpromoting molecules, may enhance drug response.

documented pro-survival mechanisms triggered by elevated CD100 and ECE-1 may provide a basis for resistance to 2FaraA or 2-CdA in vivo. Differential Drug Effects

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Levels of some glycoproteins changed for only one purine analogue, or the response was variable. 2-FaraA and 2-CdA have similarities and differences in their mechanisms of action that should be reflected in these results. Proteins that are differentially modulated by 2-CdA or 2-FaraA are shown in Figure 6. The plasma membrane proteins, CD83 and CD100, are involved in cell−cell communication and immune response, while β-glucuronidase and cathepsin D are involved in energy and protein metabolism, respectively. This suggests that the two purine analogues have different effects on cellular communication and metabolism. After validation by flow cytometry, 2-FaraA up-regulated the pro-survival proteins CD50, CD100, and ECE1, whereas 2-CdA induced higher up-regulation of CD80 and a decrease in CD100 that may reduce cell survival. This comparison suggests that 2-CdA may more directly induce cell death, while 2-FaraA may induce increases of both pro- and antiapoptotic signals. The initiation of parallel survival and apoptotic pathways by 2-FaraA has been shown recently,35 and our results also indicate that drug responses involve a balance of factors that determine cell fate (Figure 7).

CONCLUSIONS Quantitative analysis of N-linked plasma membrane glycoproteins has provided additional information on the mechanisms of action of purine analogues against human Raji lymphoma cells and MEC1 leukemia cells. The two cell lines representing different B-lymphoproliferative disorders showed a high degree of correlation, indicating that the mechanisms of action and putative effects of purine analogues are significant and may translate to patients. Down-regulation of the BCR inhibitor CD22 and decreases of the CD100/CD72 complex may increase BCR activation and signaling potential, concomitant with changes to the integral BCR proteins CD79a and CD79b. Activation of BCR signaling in cells damaged by 2-FaraA or 2-CdA may induce predominant signals for apoptosis via the intrinsic pathway, with up-regulation of membrane-bound CD66a. In vivo, the up-regulation of adhesion molecules such as CD80 and CD50, combined with a concurrent apoptotic cell signal provided by up-regulated CD205, could target drug-damaged cells for lysis by cytotoxic T-cells. Thus, purine analogues may initiate multiple pathways for intrinsic apoptosis and external signals for cell clearance, explaining their potent cytotoxicity. The increases observed in CD79a and CD80 suggest that they may be suitable targets for combination treatment with synergistic therapeutic antibodies. Potential pro-survival effects induced by 2-FaraA include increased CD100 and ECE-1, which may decrease responsiveness to treatment. Further investigation may use ECE-1 and CD100 knock-downs to increase the selective cytotoxicity of 2FaraA.

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ASSOCIATED CONTENT

S Supporting Information *

Figure 7. Possible mechanisms of action of purine analogues. 2-FaraA and 2-CdA have multiple effects on levels of cell surface proteins that may contribute to survival or apoptosis. BCR-associated proteins may induce BCR signaling that can initiate apoptosis. Costimulatory proteins may facilitate engagement of cytotoxic T-cells for damaged cell removal by the cell-mediated antitumor response. Other plasma membrane proteins may induce or inhibit cell survival and affect cell fate.

Two-dimensional LC-MS/MS conditions and quantitative data and all glycoproteins identified (Table S1). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*To whom correspondence should be addressed: Prof. Richard I. Christopherson Ph.D., School of Molecular Bioscience, University of Sydney, Sydney, NSW 2006, Australia Tel: 61-29351-6031, Fax: 61-2-9351-4726, E-mail: richard. [email protected].

Putative Therapeutic Targets

CD80, up-regulated by 2-FaraA and 2-CdA (Tables 1 and 2), is a suitable target for therapeutic antibodies against Raji cells, with expression of CD80 on activated lymphocytes, dendritic cells, and macrophages. Many NHLs constitutively express CD80; galiximab, a therapeutic antibody against CD80, may be useful in treatment.57 CD205, up-regulated by 2-FaraA and 2CdA by more than 2-fold, could also be a suitable target for synergistic treatment by a purine analogue and an antibody. 2FaraA and 2-CdA up-regulated CD79a, making it a suitable antibody target, because it is B-cell specific. Therapeutic antibodies against CD79a have proved effective against NHL in vitro.89 Cytotoxic drugs induce changes that promote competing signals for cell death and survival, and the balance determines cell fate; 2-FaraA or 2-CdA affect plasma membrane proteins that may stimulate both processes. For 2-FaraA, disrupting pro-

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by a grant from the Chronic Lymphocytic Leukemia Australian Research Consortium. Helpful discussions with A/Prof. Stephen Mulligan and Dr. Giles Best are gratefully acknowledged. The analyses were facilitated by access to the Sydney University Proteome Research Unit established under the Australian Government's Major National Research Facilities program and supported by the University of Sydney. 4445



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ABBREVIATIONS BCR, B-cell receptor; CD antigen, cluster of differentiation antigen; 2-CdA, cladribine; CLL, chronic lymphocytic leukemia; 2-FaraA, fludarabine nucleoside; iTRAQ, isobaric tags for relative and absolute quantitation; LC-MS/MS, liquid chromatography coupled to tandem mass spectrometry; MTX, methotrexate; NHL, non-Hodgkin's lymphoma; PNGaseF, peptide-N-glycosidase F; SPE, solid-phase extraction

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