Comprehensive Proteomic Analysis of the Effects of Purine Analogs

Dec 23, 2010 - The effects of CdA and fludarabine nucleoside (FdA) on the cytosolic, mitochondrial, and nuclear proteomes in human Raji lymphoma cells...
0 downloads 0 Views 3MB Size
ARTICLE pubs.acs.org/jpr

Comprehensive Proteomic Analysis of the Effects of Purine Analogs on Human Raji B-Cell Lymphoma Swetlana Mactier, Silke Henrich, Yiping Che, Philippa L. Kohnke, and Richard I. Christopherson* School of Molecular Bioscience, University of Sydney, Sydney, NSW 2006, Australia

bS Supporting Information ABSTRACT: Cladribine (CdA) and fludarabine (FdAMP) are purine analogs that induce apoptosis in chronic lymphocytic leukemia and non-Hodgkin’s lymphoma, but the mechanisms are undefined. The effects of CdA and fludarabine nucleoside (FdA) on the cytosolic, mitochondrial, and nuclear proteomes in human Raji lymphoma cells have been determined using two-dimensional fluorescence difference gel electrophoresis (DIGE) and mass spectrometry. Differentially abundant proteins have provided new insights into CdA- and FdA-induced apoptosis. Treatment with these purine analogs induced changes in proteins involved with intermediary metabolism, cell growth, signal transduction, protein metabolism, and regulation of nucleic acids. Differentially abundant mitochondrial 39S ribosomal protein L50, mTERF domain-containing protein 1, Chitinase-3 like 2 protein, and ubiquinone biosynthesis protein COQ9 have been identified in cells undergoing apoptosis. Up-regulation of several stress-associated proteins found in the endoplasmic reticulum (ER) including GRP78, ERp57, and ORP150 suggests that purine analog-induced apoptosis may result from ER stress and unfolded protein response. While mitochondria-dependent apoptosis has been associated with purine analog cytotoxicity, the likely involvement of the ER stress pathway in CdA- and FdA-induced apoptosis has been shown here for the first time. KEYWORDS: DIGE, subcellular fractionation, cladribine, fludarabine, lymphoma, apoptosis, endoplasmic reticulum stress, unfolded protein response

1. INTRODUCTION The purine analogs, cladribine (2-chloro-20 -deoxyadenosine, CdA) and fludarabine (9-β-D-arabinofuranosyl-2-fluoroadenine-50 -monophosphate, FdAMP), interfere with DNA repair in B-lymphoid cells resulting in strand breaks, and apoptosis. FdAMP is used to treat patients with chronic lymphocytic leukemia (CLL) and indolent non-Hodgkins lymphoma (NHL).1-3 CdA is effective against CLL, low-grade NHL and hairy cell leukemia (HCL).4 In contrast to the encouraging clinical outcomes for patients with FdAMP-sensitive CLL, the prognosis for patients with FdAMP-refractory CLL is poor and the treatment continues to be challenging.5 Although both drugs are used clinically for treatment of hematological malignancies, the mechanisms for induction of apoptosis remain undefined. A more complete understanding of the mechanisms of CdA- and FdAMP-induced apoptosis could lead to improved treatment of B-lymphoproliferative disorders such as CLL and NHL. FdAMP and CdA are transported into the cell via one or more nucleoside-specific membrane transporters then converted to their triphosphates, the cytotoxic forms. Fludarabine triphosphate (FdATP), is incorporated into elongating nucleic acid chains, and terminates DNA synthesis.6 FdATP primarily inhibits DNA polymerases and also other intracellular enzymes including RNA polymerases, DNA primase, DNA ligase, and ribonucleotide reductase.7,8 Cladribine triphosphate (CdATP) r 2010 American Chemical Society

acts mainly by inhibition of ribonucleotide reductase,9 resulting in an imbalance of dNTP pools and interference with the formation of NAD. It is also incorporated into DNA of dividing cells, causes DNA single-strand breaks, and inhibits DNA synthesis.10,11 FdATP and CdATP cause DNA strand breaks, resulting in accumulation of phosphorylated p53 and induction of Bax expression with translocation to mitochondria.12-15 Accumulated Bax forms pores in the outer membrane of mitochondria, releasing cytochrome c and Smac/DIABLO. The apoptosome, consisting of cytochrome c, the adaptor protein APAF-1 (apoptotic protease activating factor-1), dATP, and procaspase9 (activated to caspase-9), then activates caspases-3 and -7, with execution of the death program.16 The endoplasmic reticulum (ER) has not been associated with CdA- or FdAMP-induced apoptosis; however, the link between ER stress and cell death has recently attracted interest.17 ER stress is defined as accumulation of unfolded or misfolded proteins in the ER, which induce a coordinated adaptive program called the unfolded protein response (UPR). The UPR alleviates ER stress by suppression of protein synthesis, facilitation of protein folding via induction of ER chaperones (e.g., GRP78) Received: August 3, 2010 Published: December 23, 2010 1030

dx.doi.org/10.1021/pr100803b | J. Proteome Res. 2011, 10, 1030–1042

Journal of Proteome Research and degradation of unfolded proteins. UPR consists of three signaling pathways initiated by inositol-requiring transmembrane kinase and endonuclease 1R (Ire1-R), activation of transcription factor ATF-6 and protein kinase-like endoplasmic reticulum kinase (PERK).18-20 Unfolded proteins accumulating in the ER lumen bind to the chaperone GRP78, competitively disturbing the interactions between GRP78 and ATF-6, PERK and Ire1-R.21,22 Ire1-R and ATF-6 induce transcription of multiple genes, including chaperones (GRP78 and calreticulin), while PERK phosphorylates the transcription initiation factor eIF2-R, resulting in down-regulation of overall protein synthesis.23,24 If the ER stress is beyond the capacity of the adaptive machinery, cells undergo apoptosis.25 Apoptosis plays a major role in human diseases including cancer. The cellular responses to anticancer drugs are a complex cascade of events involving proteins, including altered protein expression, interactions, modifications, and relocalization, leading to cell cycle arrest and DNA repair or apoptosis. Protein separation by two-dimensional gel electrophoresis (2DE) with identification of proteins of interest by mass spectrometry (MS) has enabled discovery of more than 100 proteins altered during apoptosis.26,27 Proteomic analysis by 2DE and MS has also been used to identify proteins involved in clinical drug resistance.28 Identification of novel proteins involved in control of apoptosis and drug resistance may improve our understanding of drug mechanisms and lead to identification of novel drug targets. CdA and FdAMP activate apoptotic pathways. Changes such as post-translational modifications, changes in protein abundance, proteolytic processing, and alternative splicing can be monitored using 2D fluorescence difference gel electrophoresis (DIGE) and MS. A major challenge in proteomics is to separate and identify the total complement of proteins in a complex biological mixture. Low abundance proteins must be detected together with the abundant proteins, requiring a wide dynamic range. Low copy number regulatory proteins such as transcription factors, kinases and phosphatases can be detected only after additional fractionation, such as subcellular fractionation, protein and peptide affinity purification, chromatographic protein prefractionation, zoom gels of narrow pH ranges for 2DE, and preparative protein iso-electrofocusing.29,30 To maximize the identification of differentially abundant proteins during purine analog-induced apoptosis, subcellular fractionation has been used to reduce the complexity of cell extracts. We have found that treatment of the human Burkitt’s lymphoma cell line Raji, with CdA or fludarabine nucleoside (FdA) changes the levels of a number of cytosolic, mitochondrial and nuclear proteins, providing further insight regarding the mechanisms of apoptosis induced by purine analogs in B-lymphoproliferative disorders.

2. EXPERIMENTAL PROCEDURES 2.1. Growth of Raji Cells and Drug Treatment

The human Raji B-cell lymphoma cell line (American Type Culture Collection, Manassas, USA) was grown in RPMI 1640 medium (Hepes modification) supplemented with 10% fetal calf serum and 50 μg/mL gentamicin at 37 °C. Cultures of Raji cells (3  105 cells/ml, three biological replicates) were treated at IC50 levels with CdA (1 μM, 24 h) or FdA (3 μM, 24 h, SigmaAldrich, St. Louis, USA) and harvested for proteomic analysis. 2.2. Apoptosis Detection

Externalization of phosphatidylserine (PS) on cells during apoptosis was quantified by Annexin V-PE binding, while the loss

ARTICLE

of cell membrane integrity was demonstrated by binding of 7-amino-actinomycin D (7-AAD) to DNA (Annexin V-PE apoptosis detection kit 1, BD Biosciences, San Diego, CA). Briefly, cells were washed twice with PBS and resuspended at a density of 106 cells/mL in binding buffer (10 mM K.Hepes pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). An aliquot of the suspension (50 μL, 0.5  105 cells) was incubated with 2.5 μL of Annexin V-PE and 2.5 μL of 7-AAD for 15 min at room temperature. Cells were resuspended in 450 μL of binding buffer and analyzed with a FACScan flow cytometer (Becton Dickinson, San Jose, CA) using CellQuest software. 2.3. Isolation of the Cytosolic, Mitochondrial and Nuclear Fractions

Raji cells (5  108, n = 3) were collected by centrifugation (450 g, 5 min, room temperature), washed twice in PBS, resuspended in 5 mL of lysis buffer (10 mM K.Hepes, pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 10 mM NaF, 1 mM Na3VO4, 0.2% (v/v) okadaic acid, 0.05% (v/v) NP-40), incubated on ice for 5 min and lysed with 10 gentle strokes using a Dounce glass homogenizer. The cell suspension was vortexed for 30 s and centrifuged (800 g, 5 min, 4 °C) to remove crude nuclei and cellular debris. The supernatant was centrifuged (15 000 g, 30 min, 4 °C) to isolate crude mitochondria, followed by centrifugation (100 000 g, 60 min, 4 °C) to isolate the cytosolic proteome as the supernatant depleted of the microsomal fraction. Crude mitochondria were further purified by centrifugation on a two-step sucrose gradient. Mitochondria were resuspended in 5 mL isotonic buffer (10 mM Tris-HCl, pH 7.4, 0.25 M sucrose, 1 mM EDTA) and layered onto a 1.0/1.5 M sucrose step gradient (14 mL of 10 mM Tris-HCl, pH 7.4, 1.0 M sucrose, 1 mM EDTA over 20 mL of 10 mM Tris-HCl, pH 7.4, 1.5 M sucrose, 1 mM EDTA) and centrifuged with the brake off (87 000 g, 2 h, 4 °C). The mitochondrial fraction sedimented at the 1.0/1.5 M sucrose interface and was carefully collected, diluted 2-fold in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.5 mM PMSF and centrifuged (15 000 g, 20 min, 4 °C) to pellet mitochondria. To solubilize mitochondrial proteins, pellets were resuspended in DIGE buffer (40 mM Tris base, pH 8.5, 5 M urea, 2 M thiourea, 2% (w/v) CHAPS, 2% (w/v) sulfobetaine pH 3-10). Cytosolic and mitochondrial proteins were purified using the ReadyPrep 2-D Cleanup Kit (Bio-Rad, Hercules, CA) and resuspended in DIGE buffer. The crude nuclear pellet was resuspended in 0.25 M sucrose, 10 mM MgCl2, 20 mM Tris-HCl pH 7.4 and 1 mM DTT. To further purify the nuclei, the suspension was layered onto a two-step sucrose gradient (1.3 M sucrose, 6.25 mM MgCl2, 20 mM Tris-HCl pH 7.4, 0.5 mM DTT above 2.3 M sucrose in 2.5 mM MgCl2 and 20 mM Tris-HCl, pH 7.4), then centrifuged (5000 g, 45 min, 4 °C). The purified nuclear pellet was carefully resuspended in 10 mM K. Hepes pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM DTT and 0.5 mM PMSF and centrifuged again (1000 g, 5 min, 4 °C). Nuclear pellets were resuspended in DIGE buffer. Protein concentrations were determined using the 2D Quant protein assay (GE Healthcare, Piscataway, NJ) according to the manufacturer’s protocol. 2.4. CyDye Labeling and DIGE

Protein extracts in DIGE buffer were labeled using fluorescent CyDyes (GE Healthcare, Little Chalfont, U.K.) following the manufacturer’s protocol. Protein samples from control (n = 3) or drug-treated cultures (n = 3, 50 μg protein) were labeled with 400 pmol of Cy3 or Cy5. A pooled internal standard comprising

1031

dx.doi.org/10.1021/pr100803b |J. Proteome Res. 2011, 10, 1030–1042

Journal of Proteome Research 25 μg protein from each sample was labeled with 400 pmol of Cy2. CyDyes were incubated with protein extracts on ice in the dark for 30 min and the reaction was terminated by addition of 10 nmol L-lysine followed by incubation on ice for 10 min. The Cy3- and Cy5-labeled samples were then combined with the Cy2-labeled internal standard in rehydration buffer (40 mM Trisbase, pH 8.5, 5 M urea, 2 M thiourea, 2% (w/v) CHAPS, 2 mM tributyl phosphine, 65 mM DTT, 2% (v/v) sulfobetaine pH 3-10, 1% (v/v) Bio-Lyte 3-10 ampholyte and 0.002% (w/v) Bromophenol blue). Protein samples were loaded on 17 cm pH 3-10NL (or pH 4-7) IPG gel strips and incubated overnight to allow passive in-gel rehydration. Isoelectric focusing was carried out using an IEF focusing cell (Bio-Rad Laboratories, Hercules, CA) for a total of 105 kVh. For pH 4-7 IPG strips, a paper bridge was applied at the basic end to allow the proteins with pI > 7 to escape from the IPG strip. Prior to the second dimension, the IPG strip was incubated in equilibration buffer (50 mM Tris-HCl, pH 8.8, 6 M urea, 2% (w/v) SDS, 20% (v/v) glycerol, 5 mM tributyl phosphine, 2.5% (w/v) acrylamide) for 30 min with gentle shaking. SDS-PAGE (8-18% acrylamide linear gradient) was performed using a Protean IIxi system (Bio-Rad Laboratories, Hercules, CA) at 8 °C with a two-step program; 2 mA per gel for 7 h, followed by 8 mA per gel until the end of the run. 2.5. Image Acquisition and Analysis

Proteins separated on the gel were visualized using a Molecular Imager FX scanner (Bio-Rad Laboratories, Hercules, CA). Gels were scanned between glass plates at 100 μm resolution. Cy2-labeled proteins were scanned using a 488 nm laser with a 530 nm emission filter; Cy3 was scanned using a 532 nm laser with a 605 nm emission filter; Cy5 was scanned using a 635 nm laser with a 695 nm emission filter. The Cy2 images of the internal control sample were used to normalize gel-to-gel variations and calculate standard abundances from the ratio of Cy3 and Cy5 to the Cy2 signal, within the same gel and between gels. Standard abundances were then used to calculate the protein abundance ratios for different samples. Image analysis was performed using PDQuest vr. 7.3.1 software (Bio-Rad Laboratories, Hercules, CA). 2.6. In-Gel Digestion and Mass Spectrometry

Tryptic digestion and peptide mass fingerprinting (PMF) were performed as previously described.31 Mass spectra were acquired in the mass:charge range of 800-3500 m/z on a Q-STAR XL Hybrid mass spectrometer equipped with a MALDI ion source (Applied Biosystems Inc., Foster City, CA). In some cases, protein identification was performed by MALDI-Q TOF MS/MS sequencing or liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). For MALDI-Q TOF MS/MS sequencing, peptides were subjected to N-terminal sulfonation using 4-sulfophenyl isothiocyanate (SPITC) to facilitate ionization and disintegration of peptides for MS/MS. Following tryptic digestion, 10 μL of the peptide mixture was mixed with an equal volume of 20 mg/mL SPITC in 20 mM ammonium bicarbonate and incubated (56 °C, 60 min). The reaction was stopped by addition of 1 μL of 5% (v/v) TFA and peptides were concentrated prior to MALDI-TOF MS/MS using C18 microcolumns (PerfectPure, Eppendorf, North Ryde, Australia) according to the manufacturer’s protocol. De novo peptide sequences, obtained by manual annotation of the MS/ MS spectra, were subjected to database searching against nonredundant protein sequences using the BLAST algorithm

ARTICLE

(http://www.ncbi.nlm.nih.gov/blast/Blast.cgic). For LC-MS/ MS analysis, peptides were separated on an Agilent 1100 HPLC system (20 min linear gradient, 0-60% acetonitrile with 0.1% formic acid) and analyzed using a QSTAR Elite mass spectrometer (Applied Biosystems) in an information dependent acquisition mode (IDA) using Analyst QS 1.1 software (Applied Biosystems). In IDA mode, a TOF-MS survey scan was acquired (m/z 350-1750, 0.5 s), with the three most abundant multiply charged ions (2þ to 4þ, counts >30) in the survey scan sequentially subjected to product ion analysis. Product ion spectra were accumulated for 2 s in the mass range m/z 100-1800. The MS/MS spectra were analyzed with Analyst QS 1.1 software (Applied Biosystems) and Mascot 2.0 (Matrix Science, London, U.K.) for database searching against human species (http://www.matrixscience.com). One missed cleavage per peptide, mass tolerance of 0.1 Da and variable partial oxidation for methionine were allowed. Significant (p < 0.05) Mascot scores, with at least 4 peptides matched, were considered to represent positive identification of the protein. 2.7. Western Blot Analysis

For whole cell extracts, cells were lysed in rehydration buffer. Protein samples were separated by SDS-PAGE, and transferred to a PVDF membrane (Immun-Blot PVDF Membrane, Bio-Rad Laboratories, Hercules, CA). For 2D Western blot analysis, proteins were separated on an 11 cm IPG strip pH 3-10, followed by 4-12% SDS-PAGE and then transferred to a PVDF membrane. After blocking with 5% skim milk, the membranes were incubated (4 °C, 16 h) with primary antibodies: rabbit antiERp57, rabbit anti-XBP-1, mouse anti-ORP150, mouse antiSDHA, mouse-anti RPA2, rabbit anti-PU.1, rabbit anti-calumenin, goat anti-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), mouse anti-β tubulin (BD, Franklin Lakes, NJ), mouse antiGAPDH (Millipore, North Ryde, Australia) or rabbit antiGRP78 (Cell Signaling, Danvers, MA), followed by incubation (2 h, room temperature) with a horse radish peroxidase (HRP)conjugated secondary antibody: goat-anti-mouse-HRP (Santa Cruz Biotechnology Inc.) or donkey anti-rabbit-HRP (Abcam, Cambridge, MA). Proteins were visualized using a Rapid Step ECL Reagent (Merck, Kilsyth, Australia) and ECL chemiluminescence film (GE Healthcare). Films were imaged on a Molecular Imager GS-800 densitometer (Bio-Rad) and the protein bands were quantified using Quantity One software (Bio-Rad). 2.8. Pathway Analysis

Differentially abundant proteins were analyzed by the Ingenuity Pathway Analysis software (Ingenuity Systems, http:// analysis.ingenuity.com), which relates gene products based on their interactions and functions. The list of differentially abundant proteins (Swiss-Prot accession numbers, Table 1) was loaded into the Ingenuity software, which identified biological networks and canonical pathways. The software computes a p-score for each possible network according to the fit to the input proteins, where p-score = -log10(p-value). Scores of 1.3 or higher have at least a 95% confidence of not being generated by chance. The significance of the association between the data set and the canonical pathway was measured in 2 ways: (1) A ratio is displayed of the number of genes from the data set that map to the pathway divided by the total number of genes that map to the canonical pathway. (2) Fischer’s Exact Test was used to compare the number of proteins that occur in a given pathway relative to the total number of occurrences of those proteins in all functional annotations stored in the Ingenuity Pathways Data Base. 1032

dx.doi.org/10.1021/pr100803b |J. Proteome Res. 2011, 10, 1030–1042

Journal of Proteome Research

ARTICLE

Table 1. Differentially Abundant Mitochondrial/ER Proteins (p < 0.05) in Raji Cells Treated with CdA (1 μM, 24 h) or FdA (3 μM, 24 h) abundance ratioc SSPa accession numberb

CdA/control p-value FdA/control p-value theoretical MW/pI

protein ID

Differentially abundant cytosolic proteins 1401

P06748

Nucleophosmin

0.66

0.052

0.40

0.007

1902

Q59FC6

94 kDa glucose-regulated protein

0.32

0.009

0.20

0.003

32440/4.6 92411/5.0

2205

P61086

Ubiquitin-conjugating enzyme E2-25 kDa

0.40

0.000

0.66

0.011

22407/5.3

3502

P50453

Serpin B9, proteinase inhibitor 9

2.61

0.037

42376/5.6

4101

P16949

Stathmin

0.47

0.001

17292/5.7

5301

P30040

Endoplasmic reticulum protein ERp29

0.42

0.022

28975/6.7

5305

P61289

Proteasome activator 28 subunit γ

0.45

0.002

0.72

0.038

29561/5.6

5603 5702

Q9UQ80 P50579

Proliferation-associated protein 2G4 Methionine aminopeptidase 2

4.03 0.61

0.047 0.016

2.41 0.50

0.014 0.044

41654/7.1 52858/5.5

6301

P15927

Replication protein A2, 32 kDa

0.34

0.006

0.64

0.030

29228/5.7

6303

P06730

Eukaryotic translation initiation factor 4E

2.03

0.005

1.25

0.041

6707

Q01518

Adenylyl cyclase-associated protein 1

0.36

0.011

51510/8.1

7402

P04406

Glyceraldehyde-3-phosphate dehydrogenase

1.78

0.021

36031/8.2

7403

P04406

Glyceraldehyde-3-phosphate dehydrogenase

2.14

0.007

8604

P12268

Inosine-50 -monophosphate dehydrogenase 2

20.7d

0.000

1405

O75208

Ubiquinone biosynthesis protein COQ9

2.49

0.013

1601

Q6IAW5

Calumenin

4.18

1809

P11021

78 kDa glucose-regulated protein (GRP78)

2708

P30101

2810 3702

25082/5.7

36031/8.2 13.7d

0.039

0.017

1.50

0.037

4.26

0.008

2.35

0.029

72071/5.0

Protein disulfide-isomerase A3 (PDIA3, ERp57)

5.41

0.009

3.01

0.010

56644/6.1

P30101

Protein disulfide-isomerase A3 (PDIA3, ERp57)

10.0

0.013

4.26

0.037

P30101

Protein disulfide-isomerase A3 (PDIA3, ERp57)

9.19

0.042

2902

Q9Y4L1

150 kDa oxygen-regulated protein (ORP150)

8.42

0.025

4102 4307

Q8N5N7 O75489

Mitochondrial 39S ribosomal protein L50 NADH dehydrogenase [ubiquinone] Fe-S protein 3

2.84 4.42

0.021 0.000

4606

Q96E29

mTERF domain-containing protein 1

0.37

0.021

5102

P10606

Cytochrome c oxidase subunit Vb

3.93

0.003

8401

Q5VUV7

Chitinase 3-like 2

0.40

0.014

9207

P48047

ATP synthase subunit O, mitochondrial

55770/6.4

Differentially abundant mitochondrial proteins

0.42

0.023

3404

0.40

0.015

5101

2.25

0.005

22923/5.7 37084/4.4

56644/6.1 56644/6.1

10.8

0.043

111266/5.1

3.12

0.030

18313/7.7 30223/6.9

0.67

0.046

47941/8.6

1.75

0.044

13696/9.0 42093/7.2 23291/9.9

0.25

0.010

0.50 0.48

0.037 0.025

0.37

0.001

Differentially abundant nuclear proteins 403 1103

Q09028 Q13185

Histone-binding protein RBBP4 Heterochromatin protein 1 (HP1γ)

0.68 0.67

0.046 0.050

1302

P06753

Tropomyosin-3

5.70

0.046

1304

Q13247

Splicing factor arginine/serine-rich 6 (SFRS6)

1305

P60709

Actin, cytoplasmic 1

2504

P60709

Actin, cytoplasmic 1

3.23

0.009

41710/5.2

1606

P06748

Nucleophosmin

2.70

0.048

32555/4.6

2301

O00299

Chloride intracellular channel protein 1

2.80

0.018

26906/5.1

2302 2702

Q07955 Q9BQE3

Splicing factor, arginine/serine-rich 1 Tubulin alpha-1C chain

0.40 4.02

0.028 0.026

27728/9.1 49863/4.9

2705

P61978

Heterogeneous nuclear ribonucleoprotein K

0.32

0.029

50944/5.7

3702

P61978

Heterogeneous nuclear ribonucleoprotein K

0.44

0.023

50944/5.7

2707

Q12874

Splicing factor 3A subunit 3

0.32

0.021

58812/5.3

2710

P10809

Heat shock protein 60

2.91

0.003

61016/5.7

3302

O75934

Pre-mRNA-splicing factor SPF27

0.49

0.007

26143/5.5

3504

O60506

Heterogeneous nuclear ribonucleoprotein Q

4.69

0.013

65642/8.7

3808 4102

P38646 Q9NPD3

Stress-70 protein Exosome complex exonuclease rRNA-processing protein 41 (RRP41)

2.97

0.015

3.29

1033

47626/4.8 20798/5.2 29000/4.7

0.046

39587/11.4 41710/5.2

0.22

0.034

73635/6.0 26383/6.1

dx.doi.org/10.1021/pr100803b |J. Proteome Res. 2011, 10, 1030–1042

Journal of Proteome Research

ARTICLE

Table 1. Continued abundance ratioc SSPa accession numberb

CdA/control p-value FdA/control p-value theoretical MW/pI

protein ID

4409 4505

P50213 Q13148

Isocitrate dehydrogenase [NAD] subunit alpha TAR DNA-binding protein 43

4.14 0.39

0.031 0.002

34483/6.0 44711/5.9

4804

P11142

Heat shock cognate 71 kDa protein

2.78

0.001

70855/5.3

4901

P43243

Matrin-3

0.40

0.031

94565/5.7

5406

P31942

Heterogeneous nuclear ribonucleoprotein H3

0.50

0.012

36903/6.3

8307

P31942

Heterogeneous nuclear ribonucleoprotein H3

0.27

0.029

5411

Q7Z3B4

Nucleoporin p54 (NUP54)

5610

Q9Y265

RuvB-like 1

0.49

0.008

6409 7405

P14866 P14866

Heterogeneous nuclear ribonucleoprotein L Heterogeneous nuclear ribonucleoprotein L

0.54

0.028

7105

P84103

Splicing factor, arginine/serine-rich 3 (SFRS3)

0.25

0.043

19330/10.1

7515

P19338

Nucleolin

3.30

0.022

76614/4.6

8107

P98179

RNA-binding motif protein 3 (RBM3)

10.2

0.030

17170/8.9

36903/6.4 2.30

0.020

55435/6.0 50196/5.9

0.48 0.37

0.030 0.027

64092/6.6 64092/6.6

a

Spot numbers refer to those in Figures 2 and 3. b Accession numbers of proteins were derived from the Swiss-Prot database. c The average abundance ratio was calculated by dividing the average spot intensity of the drug-treated sample by the average spot intensity of the control. Theoretical isoelectric point and molecular weight were obtained from Mascot (http://www.matrixscience.com) or ExPASy database (http://expasy.org). d Western blotting of IMPDH2 ( CdA or ( FdA showed an acidic shift for the IMPDH2 spot consistent with phosphorylation in the presence of purine analog rather than up-regulation of the protein. Blank spaces indicate no significant change in abundance.

Figure 1. Induction of apoptosis in Raji cells by purine analogs. Samples of cells were taken after incubation with the drug or dimethylformamide (control), stained with Annexin V-PE and 7-AAD, and analyzed by flow cytometry as described in Experimental Procedures. (A) Control, (B) CdA (1 μM, 24 h) and (C) FdA (3 μM, 24 h).

3. RESULTS AND DISCUSSION To quantify the proportion of apoptotic cells in Raji cultures exposed to CdA or FdA, samples were analyzed using the Annexin V-PE apoptosis detection kit. Figure 1 shows the percentages of viable, apoptotic and dead cells after treatment with a purine analog. The proportions of cells in early apoptosis after treatment with CdA (1 μM, 24 h) or FdA (3 μM, 24 h) were 36.3% (p < 0.0001) and 27.2% (p < 0.0001), respectively, compared to 8.4% in control cultures (Figure 1). Cytosolic, mitochondrial and nuclear fractions were obtained from Raji cells treated with a purine analog. The enrichment and purity of each fraction was assessed by Western blotting probing for specific cytosolic, nuclear, and mitochondrial proteins (Figure S1, Supporting Information). Cytosolic (glyceraldehyde 3-phosphate dehydrogenase, GAPDH), nuclear (PU.1) and mitochondrial (succinate dehydrogenase A, SDHA) proteins were detected in whole cell extracts and at higher abundance in their respective fractions indicating the enrichment. GAPDH, PU.1 and SDHA were exclusively detected in the cytosolic, nuclear and mitochondrial fractions, respectively, indicating the purity of these fractions. DIGE analysis of the cytosolic fraction of control and drug-treated cultures revealed 14 protein spots belonging to 13 protein species

that were differentially abundant (p < 0.05) in cells treated with CdA (1 μM, 24 h) and 10 proteins after treatment with FdA (3 μM, 24 h, Table 1). In the mitochondrial fraction, 13 protein spots (11 protein species) differed in abundance in cells treated with CdA, and 8 protein spots (7 protein species) after treatment with FdA. In the nuclear fraction, 24 protein spots belonging to 21 protein species differed in abundance after CdA treatment and 10 protein spots (9 protein species) differed in abundance in FdAtreated cells (Table 1). None of these proteins has previously been associated with CdA- or FdA-induced apoptosis. Figures 2 and3 show representative DIGE gels of cytosolic, mitochondrial and nuclear fractions, Figure 4 shows close-up sections for some proteins; the SSP numbers indicate differentially abundant proteins (p < 0.05). These proteins were identified by PMF and MS/ MS analysis (Table S1, Supporting Information). Protein spots 3404 and 5101 were of low abundance and not identified. Proteins that changed in abundance may contribute to the mechanisms underlying CdA- or FdA-induced apoptosis. The Human Protein Reference Database (http://www.hprd.org/) was used to group the differentially abundant proteins according to their biological functions (Figure 5). The potential roles of some of these proteins in apoptosis are discussed below. 1034

dx.doi.org/10.1021/pr100803b |J. Proteome Res. 2011, 10, 1030–1042

Journal of Proteome Research

ARTICLE

Figure 2. Representative DIGE images for the cytosolic and mitochondrial proteomes of Raji cells. (A) Cytosolic proteome resolved on a 17 cm pH 3-10NL IPG strip and run on an 8-18% acrylamide SDS-PAGE. Numbers indicate differentially abundant protein spots (refer to Table 1). (B) Same cytosolic sample analyzed on a 17 cm pH 4-7 IPG strip. Numbers indicate differentially abundant proteins additional to those in (A). (C) Mitochondrial proteome resolved on a 17 cm pH 3-10NL IPG strip; (D) same mitochondrial sample resolved on a 17 cm pH 4-7 IPG strip. Numbers indicate differentially abundant proteins additional to those in (C).

Figure 3. Representative DIGE image for the nuclear proteome of Raji cells. Nuclear proteins were resolved on a 17 cm pH 3-10 IPG strip and run on a 8-18% acrylamide SDS-PAGE. Numbers indicate differentially abundant protein spots (Refer to Table 1).

3.1. Intermediary Metabolism

DIGE analysis showed that inosine 50 -monophosphate dehydrogenase 2 (IMPDH2, spot 8604) increased in Raji cells treated

with CdA (20.7-fold) or FdA (13.7-fold, Figure 4, Table 1). However, Western blotting showed an acidic shift for the IMPDH2 spot consistent with phosphorylation in the presence of a purine analog, rather than up-regulation of the protein (data not shown). A recent study showed that IMPDH2 is phosphorylated at serines 159 and 160.32 Inhibition of IMPDH2 by benzamide riboside, mycophenolate, or tiazofurin sensitizes methotrexateresistant cells to methotrexate.33 We have found that FdAinduced cytotoxicity against Raji cells was significantly enhanced when cells were treated with FdA in combination with mycophenolate (10 μM, 48 h). Cell viability for single drugs was 75% for FdA (3 μM, 48 h) and 91.5% for mycophenolate (10 μM, 48 h), while viability decreased to 41.8% for these drugs in combination (Figure S2, Supporting Information). Inhibition of IMPDH2 by mycophenolate clearly enhances the cytotoxicity of FdA against Raji cells; such synergy might be useful clinically. Inhibition of IMPDH2 would lead to a decrease in GTP. G-proteins such as Ras are activated by GTP; inhibition of IMPDH2 and consequent depletion of guanine nucleotides has been associated with apoptosis.34 In addition, a recent study has shown that IMPDH2 is associated with polyribosomes, suggesting that this enzyme may also have a role in regulation of translation.35 Cytochrome c oxidase subunit Vb (Cox Vb, spot 5102) increased 3.93-fold upon treatment with CdA and 1.75-fold 1035

dx.doi.org/10.1021/pr100803b |J. Proteome Res. 2011, 10, 1030–1042

Journal of Proteome Research

ARTICLE

Ubiquinone biosynthesis protein COQ9 (spot 1405) was upregulated in mitochondria of Raji cells treated with CdA (2.49fold, Table 1). COQ9 is required for the biosynthesis of coenzyme Q,39 which prevents apoptosis by inhibiting mitochondrial depolarisation;40 lack of coenzyme Q induces apoptosis.41 Increased abundance of COQ9 in CdA-treated Raji cells could increase coenzyme Q and contribute to cell survival. 3.4. Regulation of Nucleobase, Nucleoside, Nucleotide and Nucleic Acid Metabolism

Figure 4. Close-up sections of DIGE gels showing differentially abundant proteins between (A) control, (B) CdA-, and (C) FdA-treated Raji cells. Proteins are marked with their standard spot numbers (SSPs) according to Table 1. Arrows indicate protein spots that change in abundance.

with FdA (Table 1). Cox Vb may act as an antiapoptotic protein36 and increases in Cox Vb after treatment with CdA or FdA may contribute to drug resistance. NADH dehydrogenase [ubiquinone] iron-sulfur protein 3 (NDUFS3) was up-regulated by CdA (spot 4307, 4.42-fold) and FdA (3.12-fold, Table 1). Up-regulation of NDUFS3, a subunit of the mitochondrial respiratory chain complex I, has been associated with production of toxic reactive oxygen species.37 ATP synthase subunit O was down-regulated in Raji cells treated with CdA (spot 9207, 0.42-fold, Table 1), which could lower cellular ATP. 3.2. Cell Growth, Maintenance, Communication and Signal Transduction

Proliferation-associated protein 2G4 (spot 5603), also known as ErbB3-binding protein (Ebp1), increased in Raji cells treated with CdA by 4.03-fold and with FdA by 2.41-fold (Table 1). Bose et al. demonstrated that Ebp1 binds Bcl-2 mRNA in human HL60 leukemia cells, potentially contributing to overexpression of the Bcl-2 protein, with suppression of apoptosis.38 Up-regulation of Ebp1 in Raji cells treated with CdA or FdA could be a survival response to the cytotoxity. 3.3. Other Functions

A number of proteins of unknown function were found in this study. mTERF domain-containing protein 1 (spot 4606) decreased in abundance in mitochondria of Raji cells after treatment with CdA (0.37-fold) and FdA (0.67-fold). The abundance of mitochondrial Chitinase-3 like 2 (Chi3L2, spot 8401) decreased after treatment with CdA by 0.40-fold. No association of mTERF domain-containing protein 1 or Chi3L2 with apoptosis has been reported and the roles of these proteins in CdA-induced apoptosis needs further investigation.

RNA-binding motif protein 3 (RBM3) was up-regulated 10.2fold by FdA (spot 8107, Table 1). Martinez-Arribas et al. correlated the expression of the RBM genes RBMX, RBM10 and RBM3 with the expression of genes involved in apoptosis.42 Expression of all three RBM genes was associated with expression of the Bax gene, a central member of the caspase apoptotic pathway. RBM3 expression was also associated with expression of wild-type p53. These results seem to support the hypothesis by Sutherland et al. that RBM proteins play a role in the regulation of apoptosis.43 An isoform of nucleolin (pI 7.5-8.0, Mr ≈110 kDa) was up-regulated 3.3-fold in FdA-treated Raji cells (spot 7515, Table 1). In another Burkitt’s lymphoma cell line, BL60, nucleolin was identified as an apoptosis-associated protein involved in anti-immunoglobulin M antibody-mediated apoptosis.44 Replication protein A (RPA) is a heterotrimeric complex with subunits of approximately 70 kDa (RPA1), 32 kDa (RPA2) and 14 kDa (RPA3).45 DIGE analysis revealed that in the cytosolic fraction of Raji cells, subunit RPA2 (spot 6301) is downregulated upon treatment with CdA (0.34-fold) and FdA (0.64fold). RPA facilitates the DNA unwinding process during replication initiation and elongation and participates in the DNA damage recognition step of the nucleotide excision repair pathway.46,47 Previous studies have shown that RPA2 becomes phosphorylated in response to DNA damage48 and this phosphorylation stimulates DNA repair.49 Western blot analysis confirmed down-regulation of RPA2 in cytosolic fractions with concurrent appearance of higher molecular weight bands after treatment with FdA or CdA (Figure 6A). Higher molecular weight bands for RPA2 following DNA damage have been previously described and identified as phosphorylated forms of RPA2.50 RPA is a nuclear protein but in cell fractionation experiments, including the present work, RPA is found in the cytosolic fraction.45,51 Szuts et al. reported the importance of cytosolic RPA in regulating human DNA replication.52 A recent study shows that decreases of RPA1 and RPA2 in the soluble cell fraction are associated with parallel increases of these proteins in the chromatin fraction in response to 5-aza-20 -deoxycytidine, and that association of RPA with chromatin may be required for phosphorylation of p53 by ATR.53 Western blot analysis revealed the accumulation of RPA2 in nuclear fractions following treatment with purine analogs and the concurrent appearance of higher molecular weight bands (Figure 6B). Western blot (2D) analysis of nuclear fractions showed an acidic shift in RPA2, indicating phosphorylation of this protein upon treatment with purine analogs (Figure 6C). 3.5. Protein Metabolism

Identified proteins associated with protein metabolism include: 94 kDa glucose-regulated protein (spot 1902), proteasome activator 28 subunit γ (PA28γ, spot 5305), eukaryotic translation initiation factor 4E (eIF4E, spot 6303), methionine 1036

dx.doi.org/10.1021/pr100803b |J. Proteome Res. 2011, 10, 1030–1042

Journal of Proteome Research

ARTICLE

Figure 5. Biological functions of differentially abundant proteins in Raji cells treated with (A) FdA or (B) CdA. The Human Protein Reference Database (http://www.hprd.org/) was used to group the proteins according to their functions.

Figure 6. Western blots of replication protein A2 (RPA2) in (A) cytosolic and (B) nuclear extracts of Raji cells treated with FdA or CdA. Tubulin and total protein stain were used as loading controls. (C) 2D Western blot analysis of nuclear RPA2. Abbreviations: C, control; FdA, fludarabine nucleoside; CdA, cladribine; RPA2-P, phosphorylated RPA2. The Figure is representative of three independent experiments. Statistical significance (* p < 0.05, ** p < 0.01) between control and drug-treated samples refers to densitometry measurements on RPA-2 bands.

aminopeptidase 2 (MetAP2, spot 5702), ubiquitin-conjugating enzyme E2 25 kDa (E2-25K, spot 2205), and mitochondrial 39S ribosomal protein L50 (MRP-L50, spot 4102, Table 1). CdA increased the levels of MRP-L50 by 2.84-fold. All mitochondrial ribosomal proteins (MRPs) are encoded by nuclear genes and in addition to their role in protein synthesis, some are involved in apoptotic signaling.54,55 Endoplasmic reticulum protein ERp29 (spot 5301, Table 1) may function as a molecular chaperone facilitating folding of proteins56 and is involved in drug-induced UPR.57 3.6. Cladribine and Fludarabine Induce Accumulation of ER Chaperones in Mitochondria

Calumenin (spot 1601), 150 kDa oxygen-regulated protein (ORP150, spot 2902), 78 kDa glucose-regulated protein (GRP78, spot 1809) and protein disulfide-isomerase A3 (PDIA3, spots 2708, 3702 and 2810) are ER chaperones that increase in the mitochondrial fraction in response to CdA or FdA (Table 1). Studies using electron and fluorescence microscopy have revealed that the ER is in close proximity to mitochondria.58,59

This intimate association is consistent with the reported mechanism of transport of certain lipids into mitochondria60 and Ca2þ signaling.61 Hence, it is likely that some level of ER proteins will remain even in a pure mitochondrial fraction. In fact, protein mapping of the mitochondrial fraction on a 2D gel revealed that 9% of all identified proteins are ER-associated proteins (Wong and Christopherson, unpublished data). The increase in calumenin in Raji cells treated with a purine analog was shown by Western blotting (Figure 7). Accumulation of calumenin in the mitochondria of Raji cells treated with CdA (4.18-fold) or FdA (1.50-fold) could decrease the concentration of free mitochondrial Ca2þ and inhibit apoptosis. ORP150, a member of the heat shock protein family, increased 8.42-fold in mitochondria with CdA, and 10.8-fold with FdA. Increased abundance of ORP150 in purine analog-treated Raji cells has been confirmed by Western blotting (Figure 7). Expression of ORP150 inhibits apoptosis induced by hypoxia and celecoxib,62,63 suggesting that this chaperone may be antiapoptotic. Although upregulation of ORP150 during apoptosis and its involvement in ER 1037

dx.doi.org/10.1021/pr100803b |J. Proteome Res. 2011, 10, 1030–1042

Journal of Proteome Research

Figure 7. Western blot analysis of whole cell extracts of some differentially abundant proteins in Raji cells treated with FdA or CdA. Actin and succinate dehydrogenase A (SDHA) were used as loading controls. Abbreviations: C, control; FdA, fludarabine nucleoside; CdA, cladribine. The Figure is representative of three independent experiments. Statistical significance (* p < 0.05, ** p < 0.01) between control and drugtreated samples refers to densitometry measurements.

stress have been reported,63,64 this study provides evidence that CdA and FdA trigger accumulation of OPR150 in mitochondria. DIGE analysis revealed increased levels of GRP78 in mitochondria of Raji cells treated with CdA (4.26-fold) or FdA (2.35-fold). GRP78 serves as a master modulator for the UPR network by binding to the ER stress sensors ATF6, PERK and Ire1; one characteristic of the UPR is induction of GRP78. Increased abundance of GRP78 in purine anolog-treated Raji cells was confirmed by Western blotting (Figure 7). Unfolded proteins accumulating in the ER lumen bind to GRP78, thereby competitively disturbing the interaction between GRP78 and Ire1, which upon dissociation of GRP78 oligomerizes and autophosphorylates.22 Activated Ire1 catalyzes the splicing of the X-boxbinding protein 1 (XBP1) mRNA to produce an active transcription factor. Activated XBP1 leads to expression of target genes including ER chaperones (GRP78, PDI).22 Increased expression of XBP1 in CdA- and FdA-treated Raji cells has been demonstrated by Western blotting (Figure 7). Our results indicate that CdA and FdA induce ER stress and increased expression of ER chaperones through the activation of XBP1. GRP78 is antiapoptotic, stress induction of GRP78 represents an important

ARTICLE

pro-survival arm of the UPR, with implications for cancer progression and drug-resistance.65 GRP78 prevents apoptosis caused by disturbance of ER homeostasis;65 reduction of GRP78 has been associated with UPR leading to apoptosis.66 Interestingly, Sun et al. observed the localization of GRP78 to mitochondria in cells undergoing the UPR.67 Another protein often associated with ER stress and UPR is protein disulfide-isomerase A3 (PDIA3) or ERp57.68,69 Three isoforms of ERp57 were up-regulated in the mitochondrial proteome with CdA (spot 2708, 5.41-fold; spot 3702, 9.19-fold and spot 2810, 10.0-fold) and two isoforms with FdA (spot 2708, 3.01-fold; and spot 2810, 4.26-fold, Table 1). The increased abundance of ERp57 in CdA- and FdA-treated Raji cells was confirmed by Western blotting (Figure 7). ERp57 is mainly localized in the ER, but has been found in other cellular compartments.70,71 Although an increase in ERp57 during apoptosis has been reported,68,69 the present study shows an increase in ERp57 in mitochondria of cells treated with a purine analog. CdA and FdA induce accumulation of ERp57 in mitochondria, similar to GRP78, ORP150 and calumenin. The localization of these proteins on or in mitochondria, and their functions, remain to be investigated. Knock-down of ERp57 increased the apoptotic response,69 suggesting that down-regulating ER stress responses may enhance apoptosis induced by oxidative stress-inducing drugs acting through the ER stress pathway. Thus, ERp57 together with GRP78, ORP150 and calumenin are likely to promote cell survival and are potential targets for cancer therapy. While CdA and FdA are structurally similar, there are some differences in their effects. Overall CdA seems to have a much stronger effect on subcellular proteomes of Raji cells than FdA. More proteins in all three subcellular compartments differed in abundance in Raji cells treated with CdA. However, CdA induced higher rates of apoptosis than FdA (Figure 1), and the stronger effect of CdA on protein abundance may be due to increased apoptosis. For the cytosolic and mitochondrial fractions, the majority of FdA-induced changes were also observed with CdA, but the two drugs had different effects on protein abundance in the nuclear fraction. CdA induced accumulation of several stressassociated proteins like heat shock protein 60, stress-70 protein and heat shock cognate 71 kDa protein in the nucleus of Raji cells. The profiles of proteins up-regulated by CdA and FdA (Table 1) may provide the basis for a screening test for drug sensitivity with CLL samples from patients. While a number of proteins have been identified that change in abundance in Raji cells upon treatment with FdA or CdA, no known targets of FdAMP or CdA have been identified in this study. One possible explanation is that FdAMP and CdA act mainly by inhibiting DNA polymerases and ribonucleotide reductase, respectively, rather than changing the abundance of these proteins (see Introduction). In addition, 2D gels may not resolve low abundance proteins due to a low dynamic range. 3.7. Network and Canonical Pathway Analysis

Proteins whose abundances were altered during apoptosis induced by a purine analog (Table 1) were up-loaded with their accession numbers into the Ingenuity Pathway Analysis software. The top scoring network for proteins differentially abundant in Raji cells treated with FdA was protein degradation and cellular function and maintenance (score = 35). For proteins that differ in abundance after treatment with CdA, the top scoring network (score = 65) was cell death, protein degradation and posttranslational modification (Figure S3, Supporting Information). 1038

dx.doi.org/10.1021/pr100803b |J. Proteome Res. 2011, 10, 1030–1042

Journal of Proteome Research

ARTICLE

Figure 8. Signaling and metabolic pathways analysis for proteins that differ in abundance in Raji cells treated with (A) FdA or (B) CdA. Significance threshold was set at -log(0.05). Pathways associated with purine analog-induced apoptosis display -log(p-value) > 1.3 (bars). Ratios (dots) were calculated by dividing the number of proteins identified in this study by the total number of proteins involved in those pathways. Proteins are indicated by their gene names. COX5B, cytochrome c oxidase subunit Vb; ERP29, endoplasmic reticulum protein ERp29; HSPA5, 78 kDa glucose-regulated protein; IMPDH2, inosine-50 -monophosphate dehydrogenase 2; NDUFS3, NADH dehydrogenase [ubiquinone] Fe-S protein 3; RPA2, replication protein A2; UBE2K, ubiquitin-conjugating enzyme E2; RBBP4, histone-binding protein RBBP4.

Analysis of signaling and metabolic pathways revealed that several pathways were significantly (-log(p-value) > 1.3) in-

volved in FdA- and CdA-induced apoptosis (Figure 8). Mitochondrial dysfunction, oxidative phosphorylation, ER stress, 1039

dx.doi.org/10.1021/pr100803b |J. Proteome Res. 2011, 10, 1030–1042

Journal of Proteome Research DNA methylation and transcriptional repression signaling pathways were correlated with differentially abundant proteins in Raji cells treated with FdA or CdA. The ER stress pathway displayed the highest ratio (the number of proteins identified in this study by the total number of proteins involved in ER stress pathways) for both analysis sets indicating the likely involvement of this pathway in purine analog-induced apoptosis. Mitochondrial dysfunction usually leads to an inadequate supply of energy, alterations in cellular ion homeostasis, and induces apoptotic and nonapoptotic cell death.72 While mitochondria-dependent apoptosis has been associated with purine analog cytotoxicity, the likely involvement of the ER stress pathway in FdA- and CdAinduced apoptosis has been shown here for the first time.

4. CONCLUSIONS This study has provided additional insights into the cellular mechanisms activated during apoptosis induced by CdA or FdA. Four mitochondrial proteins, mTERF domain-containing protein 1, mitochondrial 39S ribosomal protein L50, Chitinase-3 like 2, and ubiquinone biosynthesis protein COQ9, have been identified for the first time as associated with apoptosis. Increased apoptosis of Raji cells when FdA was used in combination with mycophenolate, an inhibitor of IMPDH, suggests an important and unexpected role for this purine-pathway enzyme in purine analog-induced apoptosis. Differentially abundant proteins identified in purine analogtreated Raji cells include those classically involved in the ER stress pathway and UPR, for example, GRP78, ORP150 and ERp57. The UPR in cells undergoing ER stress is characterized by up-regulation of ER chaperones, which can improve cell survival by facilitating correct folding or assembly of ER proteins, preventing their aggregation. Inhibitors of calumenin, ORP150, ERp57 and GRP78, may reduce survival responses offering clinical benefit as drugs. In addition, identification of several UPR-associated proteins as well as the induction of the UPR transcription factor XBP1, provides evidence that ER stress and UPR are also involved in purine analogue-induced apoptosis. A number of recent studies have described the involvement of both ER- and mitochondria-dependent pathways in apoptosis.73-75 The accumulation of GRP78 in mitochondria during ER stress has been described; our study shows that other chaperones (calumenin, ORP150 and ERp57) also accumulate in mitochondria. The results presented here strongly suggest that, in cells treated with CdA or FdA, apoptosis is driven by mitochondrial and ER stress-mediated pathways. ’ ASSOCIATED CONTENT

bS

Supporting Information Figure S1 showing Western blot analysis of cytosolic, nuclear and mitochondrial fractions. Table S1 summarizing the MS data for identified proteins. Figure S2 showing effects of FdA and mycophenolate alone and in combination on the viability of Raji cells. Figure S3 showing network analysis for proteins identified in Raji cells treated with FdA or CdA. Figure S4 showing MS/MS spectrum for cytochrome c oxidase subunit Vb (single peptidebased protein identification). This material is available free of charge via the Internet at http://pubs.acs.org.

’ AUTHOR INFORMATION Corresponding Author

*Richard I. Christopherson Ph.D., School of Molecular Bioscience, University of Sydney, Sydney, NSW 2006, Australia.

ARTICLE

Tel: 61-2-9351-6031. Fax: 61-2-9351-4726. E-mail: richard. [email protected].

’ ACKNOWLEDGMENT This research was supported by a grant from the CLL Australian Research Consortium and the CLL Global Research Foundation. 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. ’ REFERENCES (1) Tam, C. S.; Wolf, M.; Prince, H. M.; Januszewicz, E. H.; Westerman, D.; Lin, K. I.; Carney, D.; Seymour, J. F. Fludarabine, cyclophosphamide, and rituximab for the treatment of patients with chronic lymphocytic leukemia or indolent non-Hodgkin lymphoma. Cancer 2006, 106 (11), 2412–20. (2) Nabhan, C.; Gartenhaus, R. B.; Tallman, M. S. Purine nucleoside analogues and combination therapies in B-cell chronic lymphocytic leukemia: dawn of a new era. Leuk. Res. 2004, 28 (5), 429–42. (3) Hagenbeek, A.; Eghbali, H.; Monfardini, S.; Vitolo, U.; Hoskin, P. J.; de Wolf-Peeters, C.; MacLennan, K.; Staab-Renner, E.; Kalmus, J.; Schott, A.; Teodorovic, I.; Negrouk, A.; van Glabbeke, M.; Marcus, R. Phase III intergroup study of fludarabine phosphate compared with cyclophosphamide, vincristine, and prednisone chemotherapy in newly diagnosed patients with stage III and IV low-grade malignant NonHodgkin’s lymphoma. J. Clin. Oncol. 2006, 24 (10), 1590–6. (4) Blum, K. A.; Johnson, J. L.; Niedzwiecki, D.; Piro, L. D.; Saven, A.; Peterson, B. A.; Byrd, J. C.; Cheson, B. D. Prolonged follow-up after initial therapy with 2-chlorodeoxyadenosine in patients with indolent non-Hodgkin lymphoma: results of Cancer and Leukemia Group B Study 9153. Cancer 2006, 107 (12), 2817–25. (5) Tsimberidou, A. M.; Keating, M. J. Treatment of fludarabinerefractory chronic lymphocytic leukemia. Cancer 2009, 115 (13), 2824–36. (6) Huang, P.; Chubb, S.; Plunkett, W. Termination of DNA synthesis by 9-beta-D-arabinofuranosyl-2-fluoroadenine. A mechanism for cytotoxicity. J. Biol. Chem. 1990, 265 (27), 16617–25. (7) Plunkett, W.; Huang, P.; Gandhi, V. Metabolism and action of fludarabine phosphate. Semin. Oncol. 1990, 17 (5 Suppl 8), 3–17. (8) Gandhi, V.; Plunkett, W. Cellular and clinical pharmacology of fludarabine. Clin. Pharmacokinet. 2002, 41 (2), 93–103. (9) Parker, W. B.; Bapat, A. R.; Shen, J. X.; Townsend, A. J.; Cheng, Y. C. Interaction of 2-halogenated dATP analogs (F, Cl, and Br) with human DNA polymerases, DNA primase, and ribonucleotide reductase. Mol. Pharmacol. 1988, 34 (4), 485–91. (10) Griffig, J.; Koob, R.; Blakley, R. L. Mechanisms of inhibition of DNA synthesis by 2-chlorodeoxyadenosine in human lymphoblastic cells. Cancer Res. 1989, 49 (24 Pt 1), 6923–8. (11) Hirota, Y.; Yoshioka, A.; Tanaka, S.; Watanabe, K.; Otani, T.; Minowada, J.; Matsuda, A.; Ueda, T.; Wataya, Y. Imbalance of deoxyribonucleoside triphosphates, DNA double-strand breaks, and cell death caused by 2-chlorodeoxyadenosine in mouse FM3A cells. Cancer Res. 1989, 49 (4), 915–9. (12) Rao, V. A.; Plunkett, W. Activation of a p53-mediated apoptotic pathway in quiescent lymphocytes after the inhibition of DNA repair by fludarabine. Clin. Cancer Res. 2003, 9 (8), 3204–12. (13) Genini, D.; Adachi, S.; Chao, Q.; Rose, D. W.; Carrera, C. J.; Cottam, H. B.; Carson, D. A.; Leoni, L. M. Deoxyadenosine analogs induce programmed cell death in chronic lymphocytic leukemia cells by damaging the DNA and by directly affecting the mitochondria. Blood 2000, 96 (10), 3537–43. (14) Marzo, I.; Perez-Galan, P.; Giraldo, P.; Rubio-Felix, D.; Anel, A.; Naval, J. Cladribine induces apoptosis in human leukaemia cells by 1040

dx.doi.org/10.1021/pr100803b |J. Proteome Res. 2011, 10, 1030–1042

Journal of Proteome Research caspase-dependent and -independent pathways acting on mitochondria. Biochem. J. 2001, 359 (Pt 3), 537–46. (15) Robak, T.; Lech-Maranda, E.; Korycka, A.; Robak, E. Purine nucleoside analogs as immunosuppressive and antineoplastic agents: mechanism of action and clinical activity. Curr. Med. Chem. 2006, 13 (26), 3165–89. (16) Sampath, D.; Rao, V. A.; Plunkett, W. Mechanisms of apoptosis induction by nucleoside analogs. Oncogene 2003, 22 (56), 9063–74. (17) Kim, I.; Xu, W.; Reed, J. C. Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities. Nat. Rev. Drug Discovery 2008, 7 (12), 1013–30. (18) Wang, X. Z.; Harding, H. P.; Zhang, Y.; Jolicoeur, E. M.; Kuroda, M.; Ron, D. Cloning of mammalian Ire1 reveals diversity in the ER stress responses. Embo J. 1998, 17 (19), 5708–17. (19) Yoshida, H.; Haze, K.; Yanagi, H.; Yura, T.; Mori, K. Identification of the cis-acting endoplasmic reticulum stress response element responsible for transcriptional induction of mammalian glucose-regulated proteins. Involvement of basic leucine zipper transcription factors. J. Biol. Chem. 1998, 273 (50), 33741–9. (20) Harding, H. P.; Zhang, Y.; Bertolotti, A.; Zeng, H.; Ron, D. Perk is essential for translational regulation and cell survival during the unfolded protein response. Mol. Cell 2000, 5 (5), 897–904. (21) Katayama, T.; Imaizumi, K.; Sato, N.; Miyoshi, K.; Kudo, T.; Hitomi, J.; Morihara, T.; Yoneda, T.; Gomi, F.; Mori, Y.; Nakano, Y.; Takeda, J.; Tsuda, T.; Itoyama, Y.; Murayama, O.; Takashima, A., St; George-Hyslop, P.; Takeda, M.; Tohyama, M. Presenilin-1 mutations downregulate the signalling pathway of the unfolded-protein response. Nat. Cell Biol. 1999, 1 (8), 479–85. (22) Schroder, M.; Kaufman, R. J. ER stress and the unfolded protein response. Mutat. Res. 2005, 569 (1-2), 29–63. (23) Lai, E.; Teodoro, T.; Volchuk, A. Endoplasmic reticulum stress: signaling the unfolded protein response. Physiology (Bethesda) 2007, 22, 193–201. (24) McCullough, K. D.; Martindale, J. L.; Klotz, L. O.; Aw, T. Y.; Holbrook, N. J. Gadd153 sensitizes cells to endoplasmic reticulum stress by down-regulating Bcl2 and perturbing the cellular redox state. Mol. Cell. Biol. 2001, 21 (4), 1249–59. (25) Rutkowski, D. T.; Kaufman, R. J. A trip to the ER: coping with stress. Trends Cell Biol. 2004, 14 (1), 20–8. (26) Thiede, B.; Rudel, T. Proteome analysis of apoptotic cells. Mass Spectrom. Rev. 2004, 23 (5), 333–49. (27) Neo, J. C.; Rose, P.; Ong, C. N.; Chung, M. C. beta-Phenylethyl isothiocyanate mediated apoptosis: a proteomic investigation of early apoptotic protein changes. Proteomics 2005, 5 (4), 1075–82. (28) Verrills, N. M.; Liem, N. L.; Liaw, T. Y.; Hood, B. D.; Lock, R. B.; Kavallaris, M. Proteomic analysis reveals a novel role for the actin cytoskeleton in vincristine resistant childhood leukemia--an in vivo study. Proteomics 2006, 6 (5), 1681–94. (29) Gorg, A.; Weiss, W.; Dunn, M. J. Current two-dimensional electrophoresis technology for proteomics. Proteomics 2004, 4 (12), 3665–85. (30) Stasyk, T.; Huber, L. A. Zooming in: fractionation strategies in proteomics. Proteomics 2004, 4 (12), 3704–16. (31) Gez, S.; Crossett, B.; Christopherson, R. I. Differentially expressed cytosolic proteins in human leukemia and lymphoma cell lines correlate with lineages and functions. Biochim. Biophys. Acta 2007, 1774 (9), 1173–83. (32) Olsen, J. V.; Blagoev, B.; Gnad, F.; Macek, B.; Kumar, C.; Mortensen, P.; Mann, M. Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 2006, 127 (3), 635–48. (33) Penuelas, S.; Noe, V.; Morales, R.; Ciudad, C. J. Sensitization of human erythroleukemia K562 cells resistant to methotrexate by inhibiting IMPDH. Med. Sci. Monit. 2005, 11 (1), BR6–12. (34) Huang, M.; Ji, Y.; Itahana, K.; Zhang, Y.; Mitchell, B. Guanine nucleotide depletion inhibits pre-ribosomal RNA synthesis and causes nucleolar disruption. Leuk. Res. 2008, 32 (1), 131–41. (35) Mortimer, S. E.; Xu, D.; McGrew, D.; Hamaguchi, N.; Lim, H. C.; Bowne, S. J.; Daiger, S. P.; Hedstrom, L. IMP dehydrogenase type

ARTICLE

1 associates with polyribosomes translating rhodopsin mRNA. J. Biol. Chem. 2008, 283 (52), 36354–60. (36) Wu, H.; Owlia, A.; Singh, P. Precursor peptide progastrin(1-80) reduces apoptosis of intestinal epithelial cells and upregulates cytochrome c oxidase Vb levels and synthesis of ATP. Am. J. Physiol. Gastrointest. Liver Physiol. 2003, 285 (6), G1097–110. (37) Huang, G.; Chen, Y.; Lu, H.; Cao, X. Coupling mitochondrial respiratory chain to cell death: an essential role of mitochondrial complex I in the interferon-beta and retinoic acid-induced cancer cell death. Cell Death Differ. 2007, 14 (2), 327–37. (38) Bose, S. K.; Sengupta, T. K.; Bandyopadhyay, S.; Spicer, E. K. Identification of Ebp1 as a component of cytoplasmic bcl-2 mRNP (messenger ribonucleoprotein particle) complexes. Biochem. J. 2006, 396 (1), 99–107. (39) Johnson, A.; Gin, P.; Marbois, B. N.; Hsieh, E. J.; Wu, M.; Barros, M. H.; Clarke, C. F.; Tzagoloff, A. COQ9, a new gene required for the biosynthesis of coenzyme Q in Saccharomyces cerevisiae. J. Biol. Chem. 2005, 280 (36), 31397–404. (40) Papucci, L.; Schiavone, N.; Witort, E.; Donnini, M.; Lapucci, A.; Tempestini, A.; Formigli, L.; Zecchi-Orlandini, S.; Orlandini, G.; Carella, G.; Brancato, R.; Capaccioli, S. Coenzyme q10 prevents apoptosis by inhibiting mitochondrial depolarization independently of its free radical scavenging property. J. Biol. Chem. 2003, 278 (30), 28220–8. (41) Takahashi, M.; Shimizu, T.; Moriizumi, E.; Shirasawa, T. Clk-1 deficiency induces apoptosis associated with mitochondrial dysfunction in mouse embryos. Mech. Ageing Dev. 2008, 129 (5), 291–8. (42) Martinez-Arribas, F.; Agudo, D.; Pollan, M.; Gomez-Esquer, F.; Diaz-Gil, G.; Lucas, R.; Schneider, J. Positive correlation between the expression of X-chromosome RBM genes (RBMX, RBM3, RBM10) and the proapoptotic Bax gene in human breast cancer. J. Cell Biochem. 2006, 97 (6), 1275–82. (43) Sutherland, L. C.; Rintala-Maki, N. D.; White, R. D.; Morin, C. D. RNA binding motif (RBM) proteins: a novel family of apoptosis modulators?. J. Cell Biochem. 2005, 94 (1), 5–24. (44) Brockstedt, E.; Rickers, A.; Kostka, S.; Laubersheimer, A.; Dorken, B.; Wittmann-Liebold, B.; Bommert, K.; Otto, A. Identification of apoptosis-associated proteins in a human Burkitt lymphoma cell line. Cleavage of heterogeneous nuclear ribonucleoprotein A1 by caspase 3. J. Biol. Chem. 1998, 273 (43), 28057–64. (45) Wold, M. S. Replication protein A: a heterotrimeric, singlestranded DNA-binding protein required for eukaryotic DNA metabolism. Annu. Rev. Biochem. 1997, 66, 61–92. (46) He, Z.; Henricksen, L. A.; Wold, M. S.; Ingles, C. J. RPA involvement in the damage-recognition and incision steps of nucleotide excision repair. Nature 1995, 374 (6522), 566–9. (47) Matsuda, T.; Saijo, M.; Kuraoka, I.; Kobayashi, T.; Nakatsu, Y.; Nagai, A.; Enjoji, T.; Masutani, C.; Sugasawa, K.; Hanaoka, F.; et al. DNA repair protein XPA binds replication protein A (RPA). J. Biol. Chem. 1995, 270 (8), 4152–7. (48) Zernik-Kobak, M.; Vasunia, K.; Connelly, M.; Anderson, C. W.; Dixon, K. Sites of UV-induced phosphorylation of the p34 subunit of replication protein A from HeLa cells. J. Biol. Chem. 1997, 272 (38), 23896–904. (49) Anantha, R. W.; Vassin, V. M.; Borowiec, J. A. Sequential and synergistic modification of human RPA stimulates chromosomal DNA repair. J. Biol. Chem. 2007, 282 (49), 35910–23. (50) Olson, E.; Nievera, C. J.; Klimovich, V.; Fanning, E.; Wu, X. RPA2 is a direct downstream target for ATR to regulate the S-phase checkpoint. J. Biol. Chem. 2006, 281 (51), 39517–33. (51) Braet, C.; Stephan, H.; Dobbie, I. M.; Togashi, D. M.; Ryder, A. G.; Foldes-Papp, Z.; Lowndes, N.; Nasheuer, H. P. Mobility and distribution of replication protein A in living cells using fluorescence correlation spectroscopy. Exp. Mol. Pathol. 2007, 82 (2), 156–62. (52) Szuts, D.; Kitching, L.; Christov, C.; Budd, A.; Peak-Chew, S.; Krude, T. RPA is an initiation factor for human chromosomal DNA replication. Nucleic Acids Res. 2003, 31 (6), 1725–34. 1041

dx.doi.org/10.1021/pr100803b |J. Proteome Res. 2011, 10, 1030–1042

Journal of Proteome Research (53) Wang, H.; Zhao, Y.; Li, L.; McNutt, M. A.; Wu, L.; Lu, S.; Yu, Y.; Zhou, W.; Feng, J.; Chai, G.; Yang, Y.; Zhu, W. G. An ATM- and Rad3related (ATR) signaling pathway and a phosphorylation-acetylation cascade are involved in activation of p53/p21Waf1/Cip1 in response to 5-aza-20 -deoxycytidine treatment. J. Biol. Chem. 2008, 283 (5), 2564–74. (54) Chintharlapalli, S. R.; Jasti, M.; Malladi, S.; Parsa, K. V.; Ballestero, R. P.; Gonzalez-Garcia, M. BMRP is a Bcl-2 binding protein that induces apoptosis. J. Cell Biochem. 2005, 94 (3), 611–26. (55) Kim, H. R.; Chae, H. J.; Thomas, M.; Miyazaki, T.; Monosov, A.; Monosov, E.; Krajewska, M.; Krajewski, S.; Reed, J. C. Mammalian dap3 is an essential gene required for mitochondrial homeostasis in vivo and contributing to the extrinsic pathway for apoptosis. Faseb J. 2007, 21 (1), 188–96. (56) Magnuson, B.; Rainey, E. K.; Benjamin, T.; Baryshev, M.; Mkrtchian, S.; Tsai, B. ERp29 triggers a conformational change in polyomavirus to stimulate membrane binding. Mol. Cell 2005, 20 (2), 289–300. (57) Mkrtchian, S.; Sandalova, T. ERp29, an unusual redox-inactive member of the thioredoxin family. Antioxid. Redox Signal. 2006, 8 (3-4), 325–37. (58) Perkins, G.; Renken, C.; Martone, M. E.; Young, S. J.; Ellisman, M.; Frey, T. Electron tomography of neuronal mitochondria: threedimensional structure and organization of cristae and membrane contacts. J. Struct. Biol. 1997, 119 (3), 260–72. (59) Johnson, P. R.; Dolman, N. J.; Pope, M.; Vaillant, C.; Petersen, O. H.; Tepikin, A. V.; Erdemli, G. Non-uniform distribution of mitochondria in pancreatic acinar cells. Cell Tissue Res. 2003, 313 (1), 37–45. (60) Vance, J. E.; Shiao, Y. J. Intracellular trafficking of phospholipids: import of phosphatidylserine into mitochondria. Anticancer Res. 1996, 16 (3B), 1333–9. (61) Wang, H. J.; Guay, G.; Pogan, L.; Sauve, R.; Nabi, I. R. Calcium regulates the association between mitochondria and a smooth subdomain of the endoplasmic reticulum. J. Cell Biol. 2000, 150 (6), 1489–98. (62) Ozawa, K.; Kuwabara, K.; Tamatani, M.; Takatsuji, K.; Tsukamoto, Y.; Kaneda, S.; Yanagi, H.; Stern, D. M.; Eguchi, Y.; Tsujimoto, Y.; Ogawa, S.; Tohyama, M. 150-kDa oxygen-regulated protein (ORP150) suppresses hypoxia-induced apoptotic cell death. J. Biol. Chem. 1999, 274 (10), 6397–404. (63) Namba, T.; Hoshino, T.; Tanaka, K.; Tsutsumi, S.; Ishihara, T.; Mima, S.; Suzuki, K.; Ogawa, S.; Mizushima, T. Up-regulation of 150kDa oxygen-regulated protein by celecoxib in human gastric carcinoma cells. Mol. Pharmacol. 2007, 71 (3), 860–70. (64) Lai, M. Y.; Hour, M. J.; Wing-Cheung Leung, H.; Yang, W. H.; Lee, H. Z. Chaperones are the target in aloe-emodin-induced human lung nonsmall carcinoma H460 cell apoptosis. Eur. J. Pharmacol. 2007, 573 (1-3), 1–10. (65) Reddy, R. K.; Mao, C.; Baumeister, P.; Austin, R. C.; Kaufman, R. J.; Lee, A. S. Endoplasmic reticulum chaperone protein GRP78 protects cells from apoptosis induced by topoisomerase inhibitors: role of ATP binding site in suppression of caspase-7 activation. J. Biol. Chem. 2003, 278 (23), 20915–24. (66) Suzuki, T.; Lu, J.; Zahed, M.; Kita, K.; Suzuki, N. Reduction of GRP78 expression with siRNA activates unfolded protein response leading to apoptosis in HeLa cells. Arch. Biochem. Biophys. 2007, 468 (1), 1–14. (67) Sun, F. C.; Wei, S.; Li, C. W.; Chang, Y. S.; Chao, C. C.; Lai, Y. K. Localization of GRP78 to mitochondria under the unfolded protein response. Biochem. J. 2006, 396 (1), 31–9. (68) Short, D. M.; Heron, I. D.; Birse-Archbold, J. L.; Kerr, L. E.; Sharkey, J.; McCulloch, J. Apoptosis induced by staurosporine alters chaperone and endoplasmic reticulum proteins: Identification by quantitative proteomics. Proteomics 2007, 7 (17), 3085–96. (69) Corazzari, M.; Lovat, P. E.; Armstrong, J. L.; Fimia, G. M.; Hill, D. S.; Birch-Machin, M.; Redfern, C. P.; Piacentini, M. Targeting homeostatic mechanisms of endoplasmic reticulum stress to increase susceptibility of cancer cells to fenretinide-induced apoptosis: the role of stress proteins ERdj5 and ERp57. Br. J. Cancer 2007, 96 (7), 1062–71.

ARTICLE

(70) Coppari, S.; Altieri, F.; Ferraro, A.; Chichiarelli, S.; Eufemi, M.; Turano, C. Nuclear localization and DNA interaction of protein disulfide isomerase ERp57 in mammalian cells. J. Cell Biochem. 2002, 85 (2), 325–33. (71) Kimura, T.; Horibe, T.; Sakamoto, C.; Shitara, Y.; Fujiwara, F.; Komiya, T.; Yamamoto, A.; Hayano, T.; Takahashi, N.; Kikuchi, M. Evidence for Mitochondrial Localization of P5, a Member of the Protein Disulfide Isomerase Family. J. Biochem. 2008, 144 (2), 187–96. (72) Treem, W. R.; Sokol, R. J. Disorders of the mitochondria. Semin. Liver Dis. 1998, 18 (3), 237–53. (73) Kitamura, Y.; Miyamura, A.; Takata, K.; Inden, M.; Tsuchiya, D.; Nakamura, K.; Taniguchi, T. Possible involvement of both endoplasmic reticulum-and mitochondria-dependent pathways in thapsigargin-induced apoptosis in human neuroblastoma SH-SY5Y cells. J. Pharmacol. Sci. 2003, 92 (3), 228–36. (74) Li, J.; Xia, X.; Ke, Y.; Nie, H.; Smith, M. A.; Zhu, X. Trichosanthin induced apoptosis in HL-60 cells via mitochondrial and endoplasmic reticulum stress signaling pathways. Biochim. Biophys. Acta 2007, 1770 (8), 1169–80. (75) Grebenova, D.; Kuzelova, K.; Smetana, K.; Pluskalova, M.; Cajthamlova, H.; Marinov, I.; Fuchs, O.; Soucek, J.; Jarolim, P.; Hrkal, Z. Mitochondrial and endoplasmic reticulum stress-induced apoptotic pathways are activated by 5-aminolevulinic acid-based photodynamic therapy in HL60 leukemia cells. J. Photochem. Photobiol. B 2003, 69 (2), 71–85.

1042

dx.doi.org/10.1021/pr100803b |J. Proteome Res. 2011, 10, 1030–1042