Subcellular Proteome Analysis of Camptothecin Analogue

Subcellular Proteome Analysis of Camptothecin Analogue NSC606985-Treated Acute Myeloid Leukemic Cells Yun Yu,†,| Li-Shun Wang,†,| Shao-Ming Shen,‡ Li Xia,† Lei Zhang,† Yuan-Shan Zhu,§ and Guo-Qiang Chen*,†,‡ Department of Pathophysiology, Key Laboratory of Cell Differentiation and Apoptosis of National Ministry of Education, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine (SJTU-SM, formerly Shanghai Second Medical University), Shanghai 200025, China, Institute of Health Science, Shanghai Institutes for Biological Sciences-SJTU-SM, Shanghai, China, and Department of Medicine/Endocrinology, Weill Medical College of Cornell University, New York, New York 10021 Received January 9, 2007

We reported previously that NSC606985, a camptothecin analogue, induces apoptosis of acute myeloid leukemia (AML) cells through proteolytic activation of protein kinase Cδ. Here, we analyzed protein expression profiles of fractionated nuclei, mitochondria, raw endoplasmic reticula, and cytosols of NSC606985-induced apoptotic AML cell line NB4 cells by two-dimensional electrophoresis combined with MALDI-TOF/TOF tandem mass spectrometry. In total, 90 unique deregulated proteins, including 16 compartment-compartment translocated ones, were identified. They contributed to multiple functional activities such as DNA damage repairing, chromosome assembly, mRNA processing, biosynthesis, modification, and degradation of proteins. More interestingly, several increased oxidative stress-related proteins mainly presented in mitochondria, while upregulated glycolysis proteins mainly occurred in the nuclei. With their functional analyses, the possible roles of these deregulated proteins in NSC606985-induced apoptosis were discussed. Collectively, these discoveries would shed new insights for systematically understanding the mechanisms of the camptothecin-induced apoptosis. Keywords: camptothecin • NSC606985 • leukemia • apoptosis • protein kinase C-delta • subcellular fractionation • protein profile • proteomics

Introduction Acute myeloid leukemia (AML), a heterogeneous group of hematological malignancies, frequently occurs in adults.1 In the past 20 years, substantial progresses have been made in the understanding of cytogenetic and molecular mechanisms of leukemogenesis and the improvement of survival of AML patients.2 As a typical example, cure of acute promyelocytic leukemia [APL, a unique subtype of AML which is characterized mainly by chromosome translocation t(15;17)] is now a possibility for most patients through the use of state-of-the-art treatments,3,4 including administration of all-trans retinoic acid (ATRA) and anthracycline-based chemotherapy as well as addition of arsenic trioxide.5-7 However, most AML patients still suffered from the poor prognosis.4,8 Therefore, it is imperative to develop novel agents for AML treatment. Apoptosis is arguably the most potent defense against cancer because it is the mechanism used by metazoans to eliminate deleterious cells. Over the past years, it became clear that * To whom correspondence should be addressed. No. 280, Chong-Qing South Road, Shanghai 200025, China. E-mail: [email protected] or [email protected]. † Shanghai Jiao Tong University School of Medicine. | These two authors contributed equally to this work. ‡ Institute of Health Science, Shanghai Institutes for Biological Sciences. § Weill Medical College of Cornell University.

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Journal of Proteome Research 2007, 6, 3808-3818

Published on Web 07/27/2007

anticancer drugs are able to induce apoptosis. Furthermore, apoptosis induction was found to be a common event for different classes of anticancer agents, and apoptosis induced by distinct classes of anticancer agents converges into similar downstream mechanisms, of which disruption can lead to broad drug resistance.9 On the other direction, an increasing number of cancer chemopreventive agents (e.g., certain retinoids, nonsteroidal anti-inflammatory drugs, polyphenols, and vanilloids) also stimulate apoptosis in premalignant and malignant cells, and targeting apoptosis pathways in premalignant cells may be an effective method of cancer prevention.10 Thus, the deep understanding of apoptosis would provide the basis for either chemoprevention or novel targeted therapies of cancers, the latter being capable of inducing death in cancer cells or sensitize them to established cytotoxic agents and radiation therapy.11 Over the past years, apoptosis signaling had been attracting wide interests as a hot spot in life science. It has been known that apoptosis is a complex biochemical process involving a cascade of closely coordinated factors in different cellular compartments such as nuclei, mitochondria, endoplasmic reticula (ER), and cytosols, which converge in the activation of intracellular caspases and their modification of protein substrates within the nucleus and cytoplasm.12 However, their global mechanisms remain to be further investigated. The 10.1021/pr0700100 CCC: $37.00

 2007 American Chemical Society

Subcellular Proteomics of NSC606985-Induced Apoptotic Cells

emerging proteomic technologies would bring us a chance for global analysis of apoptosis-related signaling.13-17 To date, several proteome studies of apoptotic cells were preformed with total lysates or purified compartments of different cells under various induction, which enabled the discovery of dozens of proteins altered during apoptosis and provided an alternative strategy to scan valuable proteins involved in apoptosis process.15,17-22 More recently, we reported that nanomolar concentration of NSC606985, a water-soluble camptothecin ester derivative,23 induces AML cells to undergo apoptotic cell death by mediating proteolytic activation of protein kinase Cδ (PKCδ) and caspase3.24 Herein, we performed analysis of the subcellular proteome of NSC606985-induced apoptotic APL cell line NB4 cells, and some interesting deregulated proteins were discussed.

Materials and Methods Cell Lines and Treatment. APL cell line NB4 cells25 were cultured in RPMI-1640 medium (Sigma, St Louis, MI) supplemented with 10% fetal calf serum (Gibco BRL, Gaithersburg, MD) in a 5% CO2/95% air humidified atmosphere at 37 °C. For experiments, NB4 cells were seeded at 3 × 105 cells/mL and incubated in the presence or absence of 25nM NSC606985 for 24 h. NSC606985 (kindly provided by National Cancer Institute Anticancer Drug Screen standard agent database, Bethesda, MD) was dissolved in double-distilled water as a 25µM stock solution. Flow Cytometry Analyses. To assess apoptosis, annexin-V assay was performed by the ApoAlert Annexin V kit (BD Biosciences, Palo Alto, CA) on flow cytometry (BD FACSCalibur, Palo Alto, CA). To detect intracellular level of reactive oxygen species (ROS), NSC606985-treated and untreated NB4 cells were probed with dichlorofluorescin diacetate (DCFH-DA) by the ROS assay kit (Beyotime, Shanghai, China), which is a cellpermeable probe that is de-esterified intracellularly and turns to highly fluorescent 2′,7′-dichlorofluorescin (DCF) upon oxidation by ROS. The intensity of DCF was analyzed on flow cytometry (BD FACSCalibur, Palo Alto, CA). Subcellular Fractionation and Protein Preparation. Cells were separated into four fractions according to methods described by Morand, J. P, et al.26 Briefly, NB4 cells (about 1 × 108) were harvested and rinsed with Tris-buffered sucrose (0.25 M sucrose and 10 mM Tris-HCL, pH 7.4). Then, cells were resuspended in lysis buffer (0.25 M sucrose, 10 mM Tris-HCl, pH 7.4, and 1% protease inhibitor cocktail), followed by homogenization using a glass Dounce homogenizer (Kontes, Fisher Scientific, Pittsburgh, PA) with 20 strokes at 4 °C. The homogenate was centrifuged at 1000g for 10 min at 4 °C to pellet the nucleus. The supernatant was centrifuged at 15 000g for 20 min at 4 °C to pellet the raw mitochondria. The postmitochondria supernatant was subjected to ultracentrifugation (100 000g, 50 min) at 4 °C to pellet the raw ER. From the postraw ER supernatant, the soluble cytosol proteins were precipitated with chloroform and methanol according to Klotz’s method.27 To further enrich mitochondria, the pellet of raw mitochondria was resuspended in 36% Iodixanol (Optiprep, Axis Shield /Cedarlane, Hornby, ON, CA) and overlaid with 30% and 10% Iodixanol. The gradient was ultracentrifuged (80 000g, 3 h) at 4 °C, and the enriched mitochondria were collected at the interface between two Iodixanol solutions of 10% and 30%. Then, the enriched four fractions were dissolved in lysis buffer containing 7 M urea, 2 M thiourea, 4% (m/v) CHAPS, 50 mM DTT, 40 mM Tris-Base, 0.2% Bio-lyte (pH 3-10), 10%

research articles isopropanol, and 12.5% water-saturated isobutanol, followed by centrifugation (35 000g, 1 h) at 4 °C. The supernatant was quantified using Bio-Rad RC DC protein assay kit (Bio-Rad, Hercules, CA) and aliquoted. The protein samples were stored at -80 °C until analysis. Two-Dimensional Electrophoresis (2-DE) and Image Analysis. After dilution in rehydration buffer containing 8 M urea, 2% (m/v) CHAPS, 25 mM DTT, 0.2% Bio-lyte (3-10, pI range), and 0.002% bromophenol blue, 160 µg of protein was applied onto 17 cm IPG strips (NL, pH 3-10, Bio-Rad). The first dimension was carried out on a Protean IEF Cell system (BioRad) as we described previously.28 After IEF, the IPG strips were equilibrated in a buffer (6 M urea, 20% glycerol, and 2% SDS in 0.05 M Tris-HCl buffer, pH 8.8) containing 2% (w/v) DTT and 2.5% (w/v) iodoacetamide, respectively. The seconddimensional separation was carried out on 12.5% SDS-polyacrylamide gels, followed by the silver staining.28 Silver-stained gels were scanned using GS-800 calibrated imaging density meter (Bio-Rad). The spots were automatically detected using PDQuest Image Analysis Software version 7.2 (Bio-Rad) and visually checked for undetected or incorrectly detected spot. In-Gel Digestion and Mass Spectrometry. The protein spots were cut out of 2-D gels using Gelpix Spot-Excision Robot (Genetix, Hampshire, U.K.) and transferred into 96-well plate and incubated in silver destaining solution (equal volume of 30 mM potassium ferricyanide and 100 mM sodium thiosulfate) at room temperature,29 followed by washing with Milli-Q water, 25 mM ammonium bicarbonate/50% ACN, and 100% ACN. When the gel pieces were dried in a vacuum, the proteins were digested overnight in 10 µL of trypsin (4 ng/µl, Trypsin Gold, mass spectrometry grade, Promega, Madison, WI) in 25 mM ammonium bicarbonate at 37 °C. The reaction was terminated with 2 µL 1% of TFA, and the peptide fragments were enriched and desalted with ZipTip pipet (Millipore, Billerica, BA) tips according to the manufacturer. Tryptic peptides were lyophilized and resuspended in 1 µL of matrix solution containing 5 mg/mL R-cyano-4-hydroxycinnamin acid prepared in 50% ACN/0.1% TFA. The samples were spotted onto the MALDI sample target plate. Peptide mass spectra were obtained on a MALDI-TOF-TOF mass spectrometer (4700 Proteomics Analyzer, Applied Biosystem, Foster City, CA). The peptide mass fingerprints were obtained in the mass range between 800 and 4000 Da with ca. 5000 laser shots. Trypsin autolytic peaks were used for internal calibration of the mass spectra. Up to 5 most intense peaks excluding the known background peaks or keratin peaks were selected for subsequent MS/MS data acquisition. Collision induced energy issued from atmosphere was adjusted to 5 × 10-7 Torr for MS/ MS spectra acquisition. Protein identification was processed and analyzed by searching the Swiss-Prot protein database using the MASCOT search engine of Matrix Science that integrated in the Global Protein Server Workstation. The mass tolerance, the most important parameter, was limited to 50 ppm. The results from both the MS and MS/MS spectra were accepted as a good identification when the GPS score confidence was higher than 95%. Western Blot. The protein lysates were loaded onto 10% SDS-PAGE, and electrophoretically transferred to NC membrane (Bio-Rad, Hercules, CA). The membranes were blotted with antibodies against R-enolase (a gift from Dr. Kato, Institute for Developmental Research, Aichi, Japan), GSTP1 (BD Biosciences, Palo Alto, CA), calnexin (Stressgen, Canada), laminB, active caspase-3, poly-ADP ribose polymerase (PARP), cytoJournal of Proteome Research • Vol. 6, No. 9, 2007 3809

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Figure 1. Subcellular expression profiles of NB4 cells with and without NSC606985 treatment. (A-C) NB4 cells were treated with or without NSC606985 (25nM) for hours as indicated. Then, the percentages of annexin-V+/PI- (low right quadrant) and annexin-V+/PI+ (upper right quadrant) cells were detected on flow cytometry (A), caspase-3 and PARP proteins in whole cell extracts were detected by Western blot with β-actin as loading control (B), and fractionated nuclei (Nuc), ER, mitochondria (Mit), and cytosols (Cyt) were monitered with Western blots (C). Panel D shows 4 pairs of representative 2-DE gel images respectively for four fractions of NB4 cells with or without NSC606985 for 24 h. For images of every fraction, down-regulated and up-regulated protein spots are labeled, respectively, on untreated and treated group with serial numbers.

chrome C (Santa Cruz, CA), and β-actin (Oncogene, San Diego, CA), followed by incubation with horseradish peroxidase (HRP)conjugated secondary antibody (Dako Cytomation, Denmark). The protein signal was detected by luminol detection reagent (Santa Cruz, CA). Statistical Analysis. The values were expressed as mean ( SD. The paired t test was used for statistical analysis between two groups. Significant level was set at p < 0.05.

Results As we described previously,24 25nM NSC606985 treatment for 24 h significantly induced the NB4 cell line to undergo apoptosis, as estimated by annexin-V assay, proteolytic activation of caspase-3, cleavage of PARP, and PKCδ protein as well as release of mitochondrial cytochrome C to cytosol (Figure 1A-C). Then, nuclei (Nuc), ER, mitochondria (Mit), and cytosols (Cyt) of NB4 cells were relatively fractionated, as verified by the corresponding subcellular resident proteins, including nuclear protein lamin B, ER resident protein calnexin, mitochondrial protein cytochrome C, and cytosolic protein β-actin (Figure 1C). Then, protein extracts were applied to 2-DE. Gels of three patches of cell cultures with the same treatment were run simultaneously to keep a good reproducibility, followed 3810

Journal of Proteome Research • Vol. 6, No. 9, 2007

by analysis of PDQuest software. The protein spots from three patches of gels were matched. The match rate was 88.5 ( 6.34%. The intensity of spots were normalized between gels by the total density in gel images. Protein spots were compared between corresponding fractions, and the significant changes (paired t test, p < 0.05) in a consistent direction (increase or decrease) among three patches were judged as deregulated ones and cut for identification. Figure 1D shows 4 pairs of representative 2-DE gel images with the deregulated spots marked, respectively, for four fractions of NSC606985-treated and untreated NB4 cells. As representative spots, Nuc92 and Mit37 were significant, which were both identified as R-enolase, up-regulated in the nuclei and mitochondria (Figure 2A). Totally, 96, 38, 38, and 41 up- or down-regulated spots were found, respectively, in nuclear, raw ER, mitochondrial, and cytosolic fractions (Table 1). Out of these 213 deregulated spots, 189 were successfully identified by analysis of MALDI-TOF/ TOF mass spectrometry with PMF and/or MS/MS followed by database searching, as shown in Figure 2 and summarized in Table 2. Totally, there were 90 unique proteins to be identified. They were classified into metabolism, DNA damage repair and chromosome assembly, transcription and mRNA processing, biosynthesis, modification and degradation of proteins, cy-

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Among six glycolysis-related proteins identified, as shown in Table 2, five were elevated in nuclei, ER, and/or mitochondria but not in cytosols. They were triosephosphate isomerase (TPI1), D-3-phosphoglycerate dehydrogenase (PHGDH), phosphoglycerate mutase 1 (PGAM1), R-enolase (ENO1), and galactokinase (GALK1). For example, R-enolase, which mainly presented in cytosols, significantly increased in nuclei and mitochondria with no significant alterations of total R-enolase and those in ER and cytosols of NSC606985-treated NB4 cells (Figure 2A/D). Of note, we also found two down-regulated spots in the nuclei (Nuc38/39, Table 2), which were both identified as glycolysis-related phosphoglycerate kinase 1 (PGK1) but presented smaller MW than PGK1. It remains to be investigated whether they are isoforms of PGK1 and whether they are involved in glycolysis. In NSC606985-treated NB4 cells, glutathione S-transferase P (GSTP1) increased in nuclei, ER, and mitochondria (Figure 4A), which could be confirmed by Western blot (Figure 4B). However, other 6 significantly upregulated oxidative stressrelated proteins, including thioredoxin-like protein 2 (TXNL2), protein DJ-1 (PARK7), thioredoxin, and peroxiredoxin 1, 2, and 6 (PRDX1, 2 and 6) (Table 2), were identified exclusively in mitochondria. Meanwhile, 24 h NSC606985 treatment significantly increased ROS level, as estimated by the mean fluorescence of DCF (Figure 4C).

Discussion

Figure 2. NSC606985 treatment increases R-enolase protein in nuclear and mitochondrial fractions. (A) Enlarged 2-DE map of the area including R-enolase in four fractions of NB4 cells with or without NSC606985 treatment for 24 h, which is pointed by arrows. (B) Peptide mass fingerprinting of tryptic peptides from the spot Nuc92. “T” indicates trypsin autolytic peptides for internal calibration, and asterisks indicate peaks matched to R-enolase. (C) A representative fragmentation spectrum (m/z ) 1143.61 in panel B) of MALDI-TOF-TOF tandem MS is shown with the insertion of the identified sequence. (D) Western blot for R-enolase protein in subcelluar fractions of NB4 cells with NSC606985 treatment for hours as indicated, with ponceau red staining as loading control. Table 1. Numbers of Deregulated Spots and Identified Proteins of NB4 Cells after NSC606985 Treatmenta down-regulateda up-regulateda

nuclei ER mitochondria cytosols total a

42/42 13/13 10/7 23/20 88/82

54/46 25/20 28/25 18/16 125/107

totala

%a

96/88 38/33 38/32 41/36 213/189

45.07/46.56 17.84/17.46 17.84/16.93 19.25/19.05

Deregulated spot/identified protein numbers.

toskeleton and membrane trafficking, oxidative stress, signal transduction, and others according to their primary functions (Table 2 and Figure 3), out of which 16 proteins presented translocation among cell compartments during apoptosis (Table 3).

In an overall scenario, the development of malignant cancers including leukemia results from deregulated proliferation, abnormal differentiation and/or an inability of cells to undergo apoptosis. Accordingly, basic cytological mechanisms of anticancer drugs are to inhibit proliferation and/or to induce apoptosis/differentiation in sensitive tumor cells. As a camptothecin analogue, NSC606985 significantly induces AML cells to undergo apoptosis.24 To understand its mechanisms, this work identified a group of deregulated proteins by comparative subcellular proteomic analysis of non-apoptotic and apoptotic NB4 cells in the presence and absence of NSC606985 treatment. These increased or decreased proteins, which mainly presented in nuclei and mitochondria, involved various functional activities (Table 2 and Figure 3). Meanwhile, a set of proteins underwent translocations among different cellular compartments. It should be pointed out that due to the limitation of 2-DE analysis to detect hydrophobic, very acidic and very basic proteins as well as proteins of low concentrations, we could not identify all proteins modulated by NSC606985, although subcellular fractionation could enrich proteins to a degree. In addition, we could not exclude the possibility that observed changes of some spots identified on 2-DE gels was due to post-translational modifications, which remain to be further identified. It has been known that the common underlying mechanism for chemotherapeutic drugs-induced apoptosis might be a damage to DNA, lipid components of cell membranes, and cellular proteins.30,31 As topoisomerase I inhibitors, camptothecin analogues have been shown to damage DNA possibly by generating replication-mediated DNA double-strand breaks and inhibiting DNA religation.32-34 Some proteins involved in transcription, translation, and degradation of proteins were also deregulated by NSC606985 treatment. For example, far upstream element-binding protein-1 (FUBP1), one of the calibrated molecular tools to adjust gene expression,14 stimulates expression of c-myc in undifferentiated cells by activating its Journal of Proteome Research • Vol. 6, No. 9, 2007 3811

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Table 2. Functional Classifications of the Deregulated Proteins in NSC606985-Induced Apoptotic NB4 Cellsa

classification/ gene symbol

protein name

TPI1

Triosephosphate isomerase

PHGDH PGAM1

D-3-phosphoglycerate dehydrogenase Phosphoglycerate mutase 1

ENO1

Alpha-enolase

GALK1 PGK1

Galactokinase (isoform) Phosphoglycerate kinase 1

ITPA APRT

Inosine triphosphate pyrophosphatase Adenine phosphoribosyltransferase

GART

(fragment) Trifunctional purine biosynthetic protein adenosine-3 Deoxyuridine 5′-triphosphate nucleotidohydrolase

DUT

RAD23B RUVBL2 RBBP4 RBBP7 CBX3 TMPO LMNA LMNB1

BUB3 NSBP1 NFYC ACTL6A FUBP1

HNRPA1 HNRPC

HNRPK

SYNCRIP

C1QBP

3812

b

acc. no.

PMF MS/MS mean-fold pI (thero./ MW (thero./ c d e f g spot no. N/C (n ) 3) exptl. ) exptl.) Pep. Cov Sco. Pep. Sco.

1. Metabolism a. Glucolysis P60174 ER-36 Mit-32 O43175 Nuc-96 P18669 Nuc-79 Nuc-80 Mit-34 P06733 Nuc-91 Nuc-92 Nuc-93 Mit-37 P51570 Nuc-90 P00558 Nuc-38 Nuc-39 b. Nucleoside Q9BY32 Nuc-61 P07741 Nuc-64 Mit-25 P22102 Nuc-77

14.40 ( 7.21 25.60 ( 6.11 14.30 ( 16.1 appear 2.31 ( 0.39 4.62 ( 4.21 appear 5.72 ( 4.11 11.60 ( 5.40 2.89 ( 1.04 appear 0.61 ( 0.54 0.13 ( 0.17

6.5/6.9 6.5/6.9 6.3/6.8 6.7/5.6 6.7/7.3 6.7/7.3 7.0/7.0 7.0/7.0 7.0/7.2 7.0/7.2 6.0/6.1 8.3/6.4 8.3/6.4

26.8/24.2 26.8/24.2 57.2/61.6 28.8/24.9 28.8/26.0 28.8/26.0 47.3/54.2 47.3/54.3 47.3/54.3 47.3/54.3 42.7/46.0 44.9/31.3 44.9/33.7

12 17 19 12 18 11 17 17 9 27 15 7 12

58 68 44 50 59 50 43 46 23 60 46 24 31

124 192 148 92 137 89 183 172 42 240 124 73 67

appear 17.30 ( 3.06 22.26 ( 1.54 appear

5.5/5.4 5.8/5.6 5.8/5.6 6.3/6.3

21.8/22.5 19.6/20.8 19.6/20.8 108.9/23.8

6 11 13 16

40 76 76 18

9.7/5.9 9.7/5.9

26.9/19.1 26.9/19.1

11 11

4.8/4.8 4.8/4.8 5.5/5.8

43.2/60.3 43.2/60.3 51.2/51.4

4.7/4.9 4.7/4.9 4.9/5.0 5.2/5.2 7.8/7.4 6.6/6.2 5.1/5.0 5.1/5.0

Nuc-36 0.11 ( 0.15 Cyt-15 0.15 ( 0.08 2. DNA Damage Repair and Chromosome Assembly a. DNA Damage Repair UV excision repair protein RAD23 homolog B P54727 Nuc-2 0.30 ( 0.27 Cyt-6 0.06 ( 0.02 RuvB-like 2 Q9Y230 Nuc-14 disappear b. Chromosome Assembly Chromatin assembly factor 1 subunit C Q09028 Nuc-7 0.02 ( 0.01 Nuc-26 0.03 ( 0.02 Histone acetyltransferase type B subunit 2 Q16576 Cyt-7 0.26 ( 0.04 Chromobox protein homologue 3 Q13185 Nuc-35 0.29 ( 0.30 (fragment) Thymopoietin isoform alpha P42166 Nuc-81 appear (fragment) Lamin A/C P02545 Nuc-78 appear (fragment) Lamin B1 P20700 Nuc-60 appear ER-32 appear 3. Transcription and mRNA Processing a. Transcription Mitotic checkpoint protein BUB3 O43684 Nuc-88 appear Nuc-89 appear Nucleosomal binding protein 1 P82970 Nuc-6 0.16 ( 0.09 Nuclear transcription factor Y subunit gamma Q13952 Nuc-15 0.26 ( 0.23 (fragment) Actin-like protein 6A O96019 Cyt-41 appear Far upstream element-binding protein 1 Q96AE4 Nuc-41 disappear Nuc-42 disappear ER-11 0.18 ( 0.08 ER-12 0.09 ( 0.03 ER-13 0.07 ( 0.04 Cyt-21 0.09 ( 0.02 Cyt-22 0.12 ( 0.05 Cyt-23 0.06 ( 0.01 b. mRNA Processing (fragment) Heterogeneous nuclear ribonucleoprotein P09651 Nuc-69 appear A1 Heterogeneous nuclear ribonucleoproteins C1/C2 P07910 Nuc-55 appear Nuc-56 appear ER-7 0.11 ( 0.06 ER-8 0.08 ( 0.01 ER-21 appear ER-22 appear Cyt-26 appear Cyt-27 appear Heterogeneous nuclear ribonucleoprotein K P61978 Nuc-1 0.34 ( 0.08 Cyt-1 0.16 ( 0.02 Cyt-8 disappear (fragment) Heterogeneous nuclear ribonucleoprotein Q O60506 Nuc-52 appear Nuc-53 appear Nuc-85 6.03 ( 1.74 Mit-16 appear Mit-17 appear Cyt-25 appear Complement component 1, Q subcomponent binding Q07021 Nuc-34 0.14 ( 0.19 protein Nuc-58 appear


P33316

4 3

148 173

3 3 3 4

65 96 67 141

4

211

77 165 141 77

1 2 2 2

46 51 49 38

44 44

76 76

3 4

33 135

12 17 21

25 41 48

76 153 137

3 1

133 18

47.8/51.1 47.8/26.5 48.1/50.7 20.9/20.1 75.9/25.7 74.4/24.9 66.5/24.5 66.5/24.5

14 11 14 9 10 19 12 12

35 18 33 36 21 22 20 24

109 84 93 56 71 87 91 87

2 2 2 2

95 10 59 137

3

53

6.4/6.8 6.4/6.9 4.5/4.6 5.8/4.7 5.9/7.1 7.2/7.3 7.2/7.5 7.2/7.3 7.2/7.4 7.2/7.5 7.2/7.5 7.2/7.3 7.2/7.4

37.6/41.2 37.6/41.2 31.5/50.4 50.6/37.6 47.9/43.1 67.6/80.1 67.6/80.1 67.6/80.1 67.6/80.1 67.6/80.1 67.6/73.4 67.6/80.1 67.6/80.1

23 16

49 37

192 97

12 19 25 14 12 28 25 20 21

27 34 40 29 29 46 45 31 25

50 114 146 100 88 229 176 171 161

2 1 2

47 39 49

4 2 3 2 3 3

63 40 76 67 46 121

9.3/5.7

38.8/18.2

10

27

94

3

73

5.0/5.1 5.0/5.1 5.0/5.1 5.0/5.1 5.0/5.4 5.0/5.4 5.0/5.4 5.0/5.4 5.4/5.2 5.4/5.2 5.4/5.4 8.7/5.8 8.7/5.8 8.7/7.1 8.7/5.8 8.7/5.8 8.7/5.8 4.7/4.6

33.7/39.6 33.7/37.9 33.7/39.6 33.7/37.9 33.7/37.8 33.7/36.8 33.7/37.8 33.7/36.8 51.2/69.3 51.2/69.3 51.2/52.6 69.8/46.6 69.8/46.6 69.8/42.2 69.8/46.6 69.8/46.6 69.8/46.6 31.7/22.1

13 11 10 11 7 9

29 24 24 28 20 20

67 56 46 55 28 38

22 16 10 18 27 15 19 16 22 7

44 38 27 32 40 28 31 29 36 37

161 105 58 179 198 81 119 111 156 64

3 3 3 2 2 3 3 1 4 4 3 3

45 51 184 90 29 145 109 40 99 243 93 114

4 3 3 2

72 39 56 96

4.7/4.4

31.7/29.0

10

50

85

3

70

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Subcellular Proteomics of NSC606985-Induced Apoptotic Cells Table 2. (Continued)


NCL RPLP2 EEF1B2

EEF1G EIF5A

PSMC3

PSMA1 PSMA2 PSMA6 COPS8

TUBB TUBB2C none CFL1 TPT1 GSN DNM2 LASP1 CCT5 ACTB

CPNE1 AHSA1

GSTP1

TXN TXNL2 PARK7 PRDX1 PRDX2 PRDX6 ARHGDIA ARHGDIB

RANBP1 RAN PEBP1 YWHAE G3BP

protein name

b

acc. no.

PMF MS/MS mean-fold pI (thero./ MW (thero./ c d e f g spot no. N/C (n ) 3) exptl. ) exptl.) Pep. Cov Sco. Pep. Sco.

4. Protein biosynthesis, Modification and Degradation a. Protein Biosynthesis and Modification (fragment) Nucleolin P19338 Nuc-66 appear 4.6/6.2 60S acidic ribosomal protein P2 P05387 ER-10 0.16 ( 0.02 4.4/4.5 (fragment) 60S acidic ribosomal protein P2 P05387 ER-34 appear 4.4/4.6 Elongation factor 1-beta P24534 Mit-4 0.37 ( 0.09 4.5/4.6 Mit-6 0.11 ( 0.09 4.5/4.6 Mit-7 0.01 ( 0.003 4.5/4.6 Nuc-21 0.14 ( 0.03 4.5/4.6 Nuc-22 0.39 ( 0.11 4.5/4.6 Nuc-23 0.31 ( 0.16 4.5/4.6 Nuc-24 0.17 ( 0.05 4.5/4.6 (Fragment) Elongation factor 1-gamma P26641 Cyt-32 disappear 6.7/5.9 Eukaryotic translation initiation factor 5A P63241 Nuc-65 0.32 ( 0.03 5.1/5.1 Cyt-16 0.27 ( 0.02 5.1/5.1 (Fragment) Eukaryotic translation initiation factor 5A Nuc-68 appear 5.1/6.1 b. Protein Degradation 26S protease regulatory subunit 6A P17980 Nuc-51 appear 5.1/5.2 ER-20 appear 5.1/5.2 Mit-15 appear 5.1/5.2 Cyt-24 appear 5.1/5.2 Proteasome subunit alpha type 1 P25786 Nuc-82 4.02 ( 1.77 6.2/6.7 Proteasome subunit alpha type 2 P25787 Nuc-75 5.02 ( 0.42 7.1/7.4 Mit-30 appear 7.1/7.4 Proteasome subunit alpha type 6 P60900 Mit-33 appear 6.3/6.7 Signalosome subunit 8 Q99627 Nuc-63 appear 5.3/5.5 5. Cytoskeleton and Membrane Trafficking a. Cytoskeleton Tubulin beta-2 chain P07437 Nuc-47 3.53 ( 1.58 4.8/4.8 Tubulin beta-2C chain P68371 Mit-14 3.97 ( 1.57 4.8/4.8 Tubulin alpha-ubiquitous chain P68363 Nuc-48 appear 4.9/5.3 Cofilin-1 P23528 Nuc-70 8.14 ( 0.99 8.3/8.3 Translationally-controlled tumor protein P13693 Mit-21 3.58 ( 1.83 4.8/4.9 (fragment) Gelsolin precursor P06396 Cyt-40 appear 5.9/7.2 (fragment) Dynamin-2 P50570 Nuc-86 appear 7.0/7.2 Cyt-39 appear 7.0/7.2 LIM and SH3 domain protein 1 Q14847 Nuc-40 0.17 ( 0.15 6.6/7.1 T-complex protein 1, epsilon subunit P48643 Cyt-5 0.29 ( 0.07 5.5/5.5 Beta-actin P60709 Nuc-28 disappear 5.3/4.5 Cyt-29 3.90 ( 0.43 5.3/5.0 Cyt-30 5.07 ( 0.63 5.3/5.0 Cyt-31 7.49 ( 1.38 5.3/5.1 b. Membrane Trafficking Copine-1 Q99829 Cyt-2 0.31 ( 0.12 5.5/5.3 Activator of 90 kDa heat shock protein ATPase O95433 Cyt-9 0.46 ( 0.12 5.4/5.2 homologue 1 6. Oxidative Stress Glutathione S-transferase P P09211 Nuc-76 8.26 ( 2.05 5.4/5.7 ER-33 6.63 ( 1.05 5.4/5.7 Mit-22 20.1 ( 3.66 5.4/5.7 Thioredoxin P10599 Mit-26 9.73 ( 3.46 4.8/4.7 Thioredoxin-like protein 2 O76003 Mit-18 14.51 ( 6.17 5.3/5.2 Protein DJ-1 Q99497 Mit-23 10.74 ( 4.52 6.3/5.9 Peroxiredoxin 1 Q06830 Mit-29 appear 8.3/7.4 Peroxiredoxin 2 P32119 Mit-24 27.29 ( 16.08 5.7/5.7 Peroxiredoxin 6 P30041 Mit-31 appear 6.0/6.6 7. Signal Transduction Rho GDP-dissociation inhibitor 1 P52565 Nuc-31 0.16 ( 0.16 5.0/5.0 Rho GDP-dissociation inhibitor 2 P52566 Nuc-32 0.13 ( 0.07 5.1/5.1 Nuc-71 appear 5.1/6.8 Nuc-72 appear 5.1/6.8 Nuc-73 8.45 ( 2.30 5.1/7.2 Mit-28 appear 5.1/6.8 Cyt-14 Appear 5.1/5.1 Cyt-36 appear 5.1/6.8 Cyt-37 appear 5.1/6.8 Ran binding protein 1 P43487 Nuc-27 0.57 ( 0.38 5.2/5.2 GTP-binding nuclear protein Ran P62826 ER-37 6.65 ( 1.50 7.0/7.2 Phosphatidylethanolamine-binding protein Nuc-37 0.30 ( 0.16 7.4/7.8 14-3-3 protein epsilon P62258 Cyt-12 0.49 ( 0.08 4.6/4.6 GAP SH3-domain binding protein 1 Q13283 ER-1 0.07 ( 0.04 5.4/5.4 ER-2 0.23 ( 0.03 5.4/5.5 Cyt-3 0.12 ( 0.08 5.4/5.5 Cyt-4 0.15 ( 0.06 5.4/5.5

76.2/15.9 11.6/16.8 11.6/15.4 24.8/27.3 24.8/26.6 24.8/26.1 24.8/27.3 24.8/26.9 24.8/26.6 24.8/26.1 50.7/19.1 16.9/16.8 16.9/16.8 16.9/15.9

13

19

60

2 1 3 2 3 2

26 86 242 143 143 75

5 11 8 5 9 9 12 12

76 41 35 27 47 47 50 52

53 92 68 34 101 117 98 99

2 3 3 2 2 2 2

97 96 73 35 164 146 154

6 7 5

35 50 39

62 62 63

49.4/44.3 49.4/44.3 49.4/44.3 49.4/44.3 29.8/27.8 25.9/23.7 25.9/23.7 27.8/24.7 23.3/20.3

30 10 19 25 12 13 8 11 5

46 27 48 51 38 67 41 50 31

184 48 125 174 107 164 61 96 45

3 2 2 3 3 4 2 3

84 28 18 86 41 174 89 77

50.1/58.7 50.0/58.7 50.8/53.7 18.6/17.8 19.7/22.4 86.0/40.2 98.3/43.9 98.3/43.9 30.1/37.4 60.1/62.3 42.1/26.8 42.1/24.6 42.1/24.3 42.1/23.8

29 31 17 10 17

53 55 50 67 50

229 266 131 66 115

3 4 1 3 4 2

50 138 31 83 100 128

12 17 11 18 6 12 14 11

22 20 29 33 24 36 40 30

62 77 60 96 51 65 76 67

3 2 2 2 1

67 40 229 224 100

59.6/62.3 38.4/39.5

13 20

23 60

969 164

3 3

84 114

23.4/22.7 23.4/22.7 23.4/22.7 11.9/13.1 37.7/39.6 20.1/22.6 22.3/24.2 21.9/21.6 25.0/28.5

12 8 15 8 9 7 12 14 7

65 54 71 44 27 33 51 41 36

115 80 148 51 53 46 97 146 54

2 3 3 2 1 3 2 2

113 210 76 15 12 57 84 24

23.2/24.3 23.0/23.7 23.0/21.2 23.0/21.7 23.0/21.7 23.0/21.8 23.0/23.7 23.0/21.8 23.0/21.2 23.5/25.3 24.6/24.2 21.0/21.6 29.3/29.2 52.2/58.6 52.2/58.6 52.2/58.6 52.2/58.6

11 9 6 11 15 6 11 10 9 12 9 11 10 21 26 21 24

38 53 47 58 65 23 57 61 53 47 37 66 34 52 60 46 57

71 103 68 158 128 46 70 71 59 79 81 109 65 157 213 131 197

2 2

72 18

2 3 3 2

25 113 73 17

4 3 4 3 3 3 4

138 114 97 30 110 54 172

Journal of Proteome Research • Vol. 6, No. 9, 2007 3813

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Table 2. (Continued) PMF


ANP32A ANP32B

SUGT1 PCNA TOMM40

HSPD1

HBLD2 IMMT

HTRA2 COX5A PDHX ATP5B HSPA9B

TXNDC5 NACA

CALR

PDIA3 HSPA5

DHFR CLIC1 AKR1A1 ESD GRWD1

NPM37 CLEC11A RPSA

protein name

acc. no.b

MS/MS

mean-fold pI (thero./ MW (thero./ exptl.) Pep.e Covf Sco.g Pep. Sco. spot no. N/Cc (n ) 3) exptl.d)

8. Cell Proliferation, Differentiation, and Apoptosis Acidic leucine-rich nuclear phosphoprotein 32 family P39687 Cyt-28 2.93 ( 0.19 4.0/3.9 member A (fragment) Acidic leucine-rich nuclear phosphoprotein Q92688 Cyt-33 appear 3.9/4.3 32 family member B Cyt-34 appear 3.9/4.4 Suppressor of G2 allele of SKP1 homologue Q9Y2Z0 Cyt-10 0.42 ( 0.06 5.1/5.1 Proliferating cell nuclear antigen P12004 ER-25 10.85 ( 3.87 4.6/4.5 9. Resident Proteins in Mitochondria Probable mitochondrial import receptor subunit O96008 Mit-36 6.28 ( 1.20 6.8/6.8 TOM40 homologue Nuc-87 appear 6.8/6.8 60 kDa heat shock protein P10809 Nuc-10 0.21 ( 0.17 5.7/5.6 Nuc-11 disappear 5.7/5.7 Nuc-12 disappear 5.7/5.7 Nuc-13 disappear 5.7/5.7 Nuc-25 0.12 ( 0.15 5.7/4.6 Nuc-29 0.07 ( 0.08 5.7/4.6 ER-15 5.74 ( 0.67 5.7/5.6 ER-16 3.26 ( 0.47 5.7/5.7 Mit-13 5.28 ( 2.97 5.7/5.6 HESB-like domain containing protein 2 Q9BUE6 Mit-27 appear 9.2/7.0 Mitochondrial inner membrane protein Q16891 Nuc-43 appear 6.1/5.9 Nuc-44 appear 6.1/6.0 Mit-11 appear 6.1/5.9 Mit-12 appear 6.1/6.0 Serine protease HTRA2 O43464 Mit-9 0.05 ( 0.05 10.1/5.9 Cytochrome c oxidase polypeptide Va P20674 Mit-8 0.33 ( 0.03 6.3/4.7 Pyruvate dehydrogenase protein X component O00330 Mit-10 0.28 ( 0.11 8.8/6.2 ATP synthase beta chain P06576 Nuc-49 5.23 ( 1.79 5.3/5.0 ER-3 0.41 ( 0.08 5.3/5.0 75 kDa glucose regulated protein P38646 ER-14 2.86 ( 0.70 5.9/5.9 Nuc-8 disappear 5.9/5.2 Nuc-9 disappear 5.9/5.1 10. Resident Proteins in Endoplasmic Reticulum Thioredoxin domain-containing protein 5 precursor Q8NBS9 ER-4 0.23 ( 0.05 5.6/5.3 ER-5 0.13 ( 0.03 5.6/5.4 Nascent polypeptide-associated complex alpha Q13765 ER-9 0.18 ( 0.12 4.5/4.3 subunit Cyt-11 0.17 ( 0.06 4.5/4.3 ER-30 appear 4.5/4.5 Calregulin P27797 ER-6 0.27 ( 0.08 4.3/4.3 Nuc-4 0.14 ( 0.06 4.3/4.4 Nuc-5 disappear 4.3/4.3 Nuc-16 0.16 ( 0.13 4.3/4.5 Nuc-17 0.07 ( 0.07 4.3/4.6 Nuc-20 disappear 4.3/4.5 Nuc-45 appear 4.3/4.5 Protein disulfide-isomerase A3 precursor P30101 Nuc-95 0.05 ( 0.07 6.0/6.0 78 kDa glucose-regulated protein precursor P11021 Nuc-30 0.25 ( 0.29 5.1/4.5 Nuc-33 0.04 ( 0.02 5.1/4.7 11. Others Dihydrofolate reductase P00374 Nuc-74 appear 7.0/7.6 Cyt-17 disappear 7.0/7.6 Chloride intracellular channel protein 1 O00299 Nuc-59 7.52 ( 1.04 5.1/5.2 Alcohol dehydrogenase P14550 Cyt-18 0.06 ( 0.03 6.3/6.9 Esterase D P10768 Mit-35 9.81 ( 4.56 6.5/6.9 Glutamate-rich WD repeat-containing protein 1 Q9BQ67 Nuc-3 disappear 4.8/4.8 Nuc-46 appear 4.8/4.8 ER-17 4.36 ( 1.23 4.8/4.8 ER-18 2.47 ( 0.30 4.8/4.7 Nucleoplasmin-3 O7560 Nuc-62 appear 4.6/4.6 C-type lectin domain family 11 member A precursor Q9Y240 ER-19 4.60 ( 2.17 5.1/5.1 40S ribosomal protein SA P08865 Nuc-50 appear 4.8/4.7 ER-26 appear 4.8/4.7 ER-27 appear 4.8/4.8 ER-28 appear 4.8/4.9 ER-29 appear 4.8/5.4 Mit-1 0.27 ( 0.10 4.8/4.7

28.7/28.2

8

31

63

28.9/17.2

3

14

1

46

28.9/17.2 41.1/41.2 29.1/33.0

21 13

60 51

207 97

2 2 5

114 246 129

38.2/41.2

6

19

49

2

58

38.2/41.2 61.2/61.2 61.2/61.0 61.2/59.0 61.2/57.2 61.2/25.7 61.2/24.9 61.2/66.8 61.2/66.8 61.2/66.8 14.3/18.6 84.0/72.5 84.0/72.5 84.0/72.5 84.0/72.5 48.8/38.7 16.9/15.8 54.3/56.4 56.5/55.1 56.5/55.1 73.9/73.2 73.9/57.6 73.9/51.3

9 13 10 6 14 9 8 8 20 12 5 30 13 16 9 12 6 13 14 18 27 16 18

27 35 24 15 30 19 19 23 38 24 24 35 24 24 16 27 31 30 30 43 39 21 22

70 83 59 45 74 65 53 38 139 52 60 138 70 98 60 96 44 86 102 142 177 117 132

2

27

3 1 3 3 1

94 34 33 155 32

2 3

85 92

3 3 4 4 4

34 26 137 95 77

48.3/48.7 48.3/48.7 23.4/33.2

18 18 7

41 42 27

154 154 47

3 3 4

110 132 289

23.4/33.2 23.3/24.3 48.3/45.9 48.3/51.5 48.3/45.9 48.3/39.2 48.3/34.7 48.3/28.4 48.3/60.6 57.1/57.2 72.4/26.9 72.4/25.8

7 6 8 17 11 9 9 14 11 18 15 19

33 27 25 50 38 32 24 34 41 33 22 25

49 47 54 211 115 62 91 110 125 109 126 113

3 4 2 4 3

290 309 79 150 133

4 3 3 3 4 3

141 125 194 48 159 81

21.4/21.9 21.4/21.9 27.1/27.1 36.8/37.1 32.0/33.1 49.8/59.2 49.8/59.2 49.8/59.2 49.8/55.8 19.5/20.4 36.0/36.8 32.8/40.3 32.8/32.9 32.8/32.9 32.8/32.9 32.8/32.7 32.8/40.3

6 9 16 12 12 15 16 23 23 4 10 7 6 5 8 10 8

44 53 62 42 42 44 41 54 54 36 32 35 28 25 36 44 35

61 68 256 83 83 111 166 213 213 45 79 84 43 35 62 86 61

2 3 3 2 2

35 84 85 95 95

4 4 4 2

126 104 104 11

2

32

4 3 4

32 53 73

a NB4 cells were treated with or without 25nM NSC606985 for 24 h, and nucleus (Nuc), endoplasmic reticulum (ER), mitochondrion (Mit), and cytosol (Cyt) were fractionated. Then, these subcellular fractions were run on 2-DE gels, and the deregulated spots were numbered, respectively, according to their locations on the subcellular 2-DE maps of the corresponding fractions. They are classified into different sections according to their primary functions. A software-aided spot intensity ratio of the treated vs the untreated, as well as the experimental isoelectric point and molecular weight are provided for each identified protein. Theoretical isoelectric points and molecular weights are derived from the amino acid sequences in Swiss-Prot. b Accession number in Swiss-Prot database. c NSC606985 treatment vs untreatment. d Theoretical vs experimental. e Peptide counts matched in MS analysis. f Cov, coverage by the matched peptides. g The scores of identified proteins by MASCOT analysis.

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Figure 3. Functional classification of 90 unique deregulated proteins during NSC606985-induced apoptotic NB4 cells. Table 3. Translocated Proteins Identified in NSC606985-Induced Apoptotic NB4 Cells thero

gene symbol ATP5B IMMT TOMM40 HNRPC PCNA SYNCRIP

LMNB1 ARHGDIB TPI1 PHGDH PGAM1

ENO1

GALK1 APRT TUBB CFL1

spot no.

protein name

untreated cells

treated cells

acc. no.

pI

MW

pI

MW

localization

pI

MW

localization

Nuc-49 Nuc-43 Nuc-44 Nuc-87 Cyt-26 Cyt-27 ER-25 Mit-16 Mit-17 Cyt-26 ER-32 Mit-28

ATP synthase beta chain Mitochondrial inner membrane protein

P06576 Q16891

57.1

O96008 P07910

Proliferating cell nuclear antigen Heterogeneous nuclear ribonucleoprotein Q

P12004 O60506

6.8 5.1 5.1 4.5

41.2 39.6 37.9 33.0

Lamin B1 Rho GDP-dissociation inhibitor 2

P20700 P52566

56.5 84.0 84.0 38.2 33.7 33.7 29.1 69.8 69.8 69.8 66.5 23.0

5.0

Probable mitochondrial import receptor subunit TOM40 homologue Heterogeneous nuclear ribonucleoproteins C1/C2

5.3 6.1 6.1 6.8 5.0 5.0 4.6 8.7 8.7 8.7 5.1 5.1

55.2 72.5 75.5 41.2 37.8 36.8 32.4 46.6 46.6 46.6 24.5 21.8

Mit, Nuc Mit, Nuc Mit, Nuc Mit, Nuc Nuc, Cyt Nuc, Cyt Nuc, Mem Nuc, Mit Nuc, Mit Nuc, Cyt Nuc, Mem Cyt, Nuc, Mit

Triosephosphate isomerase

P60174

D-3-phosphoglycerate dehydrogenase Phosphoglycerate mutase 1

O43175 P18669

Alpha-enolase

P06733

Galactokinase Adenine phosphoribosyltransferase

P51570 P07741

6.5 6.5 6.3 6.7 6.7 6.7 7.0 7.0 7.0 7.0 6.0 5.8

26.8 26.8 57.2 28.8 28.8 28.8 47.3 47.3 47.3 47.3 42.7 19.6

22.0 23.7 24.0 24.0 61.6 25.0 26.0 26.0 54.3 54.3 54.3 54.3

5.1 5.9 6.0 6.8 5.4 5.4 4.6 5.8 5.9 5.8 5.1 6.8

ER-36 Mit-32 Nuc-96 Nuc-79 Nuc-80 Mit-34 Nuc-91 Nuc-92 Nuc-93 Mit-37 Nuc-90 Nuc-64 Mit-25 Nuc-47 Nuc-70

7.3 5.1 6.9 6.9 6.8 5.5 7.2 7.2 7.0 7.0 7.2 7.2

Tubulin beta-2 chain Cofilin-1

P07437 P23528

4.8 8.3

50.1 18.6

5.6 5.6 4.8 8.0

20.8 20.8 58.6 18.8

Mit Mit Mit Mit Nuc Nuc Nuc Nuc Nuc Nuc Nuc Nuc Cyt Cyt Cyt Cyt Cyt Cyt Cyt Cyt Cyt Cyt Cyt Cyt Cyt Cyt Cyt Cyt

6.9 6.8 6.8 5.6 7.3 7.3 7.0 7.0 7.2 7.2 6.1 5.6 5.7 4.8 8.2

24.2 23.9 61.6 24.9 26.0 26.0 54.2 54.3 54.3 54.3 46.0 20.8 21.2 58.7 18.8

Cyt, Mem Cyt, Mit Cyt, Nuc Cyt, Nuc Cyt, Nuc Cyt, Mit Cyt, Nuc Cyt, Nuc Cyt, Nuc Cyt, Mit Nuc Cyt, Nuc Cyt, Mit Cyt, Nuc Cyt, Nuc

far upstream element.35,36 When NB4 cells were treated with NSC606985 for 24 h, FUBP1 were significantly down-regulated. Meanwhile, several proteins responsible for mRNA processing were also reduced [for heterogeneous nuclear ribonucleoprotein A1 (HNRPA1), heterogeneous nuclear ribonucleoprotein K (HNRPK), and heterogeneous nuclear ribonucleoprotein Q (SYNCRIP)] or modified with shift of pI and MW on gels [for heterogeneous nuclear ribonucleoproteins C1/C2 (HNRPC)], the latter being consistent with a previous report.37 Moreover, ribosome biogenesis related proteins nucleolin (NCL) and 60s acidic ribosomal protein P2 (RPLP2), and proteins responsible for protein translation including elongation factor 1-beta (EEF1B2), elongation factor 1-gamma (EEF1G), and eukaryotic translation initiation factor 5A (E1F5A), were also downregulated. All these alterations would attenuate transcription, mRNA processing and translation. Furthermore, some protein degradation-contributing proteins were upregulated, such as 26S protease regulatory subunit 6A (PSMC3), and proteasome subunit alpha type (PSMA)-1, 2, 6, which might accelerate degradation of some proteins. NSC606985-induced damage to DNA and cellular proteins would cause an imbalance of the cellular homeostasis commonly designated as cellular stress.

As reviewed by Herr and Debatin,30 this cellular stress in turn initiates a complex cascade of stress-inducible signaling molecules in an attempt to return the cell to its previous equilibrium. The type and dose of stress within the cellular context appears to dictate the outcome of the cellular response, which is intimately converted to complex pathways mediating cellcycle control or cell death. Indeed, NSC606985 was shown to induce AML cell growth arrest and apoptosis as well, which possibly suggested that cell division was stopped to strive for DNA damage repairing at the very beginning after treatment. The kinetochore, a protein complex assembled at each centromere, serves as the attachment site for spindle microtubules. Unattached kinetochores are also the signal generators for the mitotic checkpoint, which arrests mitosis until all kinetochores have correctly attached to spindle microtubules.38 To our greatest understanding, the potential effect of camptothecins on kinetochores was not reported previously. As documented,39 mitotic checkpoint protein BUB3 (BUB3), a WD repeatcontaining mitotic checkpoint protein, localizes to kinetochores before chromosome alignment, and activates the checkpoint in response to unattached kinetochores. Of note, recent work also proposed that it acts as transcriptional repressor during Journal of Proteome Research • Vol. 6, No. 9, 2007 3815

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Figure 4. Up-regulation of glutathione S-transferase P in nuclei, ER, and mitochondria in NB4 cells with NSC606985 treatment for 24 h. (A) Enlarged 2-DE map of the area including GSTP1 in four fractions of NB4 cells with or without NSC606985 treatment for 24 h, which is pointed by arrows. (B) Western blot analysis for GSTP1 protein with ponceau red staining as loading control. (C) NB4 cells were treated with NSC606985 for 0, 6, 12, and 24 h, and levels of ROS were calculated by the mean fluorescence of DCF. In the top panel, y-axis represents fluorescent intensities of DCF, and the thin lines and bold lines represent, respectively, untreated and treated cells. The relative ROS levels (means with bar as SD of triplicates) are shown in the bottom. * P < 0.05 vs control. All experiments were repeated 3 times with similar results.

interphase.40 Here, we identified that BUB3 protein was upregulated significantly after NSC606985 treatment. Further, a suppressor of the G2 allele of SKP1 (SUGT1), was significantly down-regulated. Recent reports showed that SUGT1 protein was required for kinetochore function during the G1/S and G2/M transitions in yeast.34 Its overexpression restores the localization of specific kinetochore proteins and chromosome alignment in HeLa cells treated with 17-allylaminogeldanamycin.41 Therefore, the altered BUB3 and SUGT1 would result in anomaly of kinetochore, which was also possibly associated with NSC606985-induced growth arrest. Apoptosis seems to be induced if DNA damage exceeds the capacity of repair mechanisms. Indeed, NSC606985 treatment significantly reduced DNA damage repairing-associated proteins UV excision repair protein RAD23 homologue B (RAD23B) and ruvB-like 2 (RUVBL2), and chromosome assembly proteins chromatin assembly factor 1 subunit C (RBBP4), histone acetyltransferase type B subunit 2 (RBBP7), and chromobox protein homologue 3 (CBX3), which were unfavorable to DNA damage repairing. In DNA damage-induced apoptosis, the activated PKCδ isozyme, which can act both upstream and 3816


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downstream of caspases, may translocate to distinct cellular compartments (mitochondria and the nuclei) as shown in Figure 1C, to reach their targets.42 Previously, we reported that the activated PKCδ, which caused the loss of mitochondrial transmembrane potential, release of cytochrome C and caspase-3 activation, exerts a critical role in NSC606985-induced apoptosis.24 However, the mechanisms underlying DNA damageinduced PKCδ activation are not clear. Cellular oxidative stress could induce DNA and protein damage and widely contributes to chemotherapeutic drugsinduced apoptosis in cancer cells.43 It was also suggested that oxidative stress activates the PKCδ kinase, as reviewed by Kanthasamy et al.44 Here, we did show that NSC606985-treated NB4 cells presented increased ROS level. However, increased ROS occurred after PKCδ activation induced by NSC606985, indicating that oxidative stress might play a role in the late stage of the agent-induced apoptosis. Regardless, several oxidative stress-related proteins were up-regulated, which mainly presented in mitochondria in NSC606985-treated NB4 cells, possibly indicating the importance of mitochondrial oxidative stress in NSC606985-induced apoptosis.45 On the other hand, some energy metabolism-related proteins were also deregulated. We showed that cytochrome c oxidase polypeptide Va (COX5A), one subunit of cytochrome oxidase complex that transfers the electrons from cytochrome c to H2O, and pyruvate dehydrogenase protein X component (PDHX), a subunit of PDH complex in citric acid cycle, were reduced during NSC606985induced apoptosis. These alterations would restrain ATP production and cause the ROS formation.46 In addition, NSC606985 treatment up-regulated some glycolysis proteins such as R-enolase, TPI1, PHGDH, and others. More interestingly, these alterations mainly occurred in the nuclei fraction. As a support, the nulear translocation and localization of PHGDH47-49 and R-enolase50,51 have also been revealed by others. suggesting that it is important to maintain enough ATP in this organelle for apoptosis development. Additionally, two putative HLA-DR-associated proteins, acidic leucine-rich nuclear phosphoprotein 32 family member A (ANP32A) and acidic leucine-rich nuclear phosphoprotein 32 family member B (ANP32B) that share 81% common amino acid residues without a variable C-terminus, were increased and cleaved, respectively. In line with these changes, ANP32A was reported to promote caspase-9 activation after apoptosome formation and thus promote apoptosis,52 while ANP32B, a homologous gene of rat PAL31, was regarded as an inhibitor of caspase-3.53 As a consequence, these two proteins might play a role in NSC606985-induced apoptosis, although it remains to be investigated whether NSC606985-induced changes of ANP32A/B are associated with activated PKCδ and whether ANP32A/B act as substrates of active caspase-3. As widely accepted, activation of caspases, which are specific cysteine proteases that either autoactivate themselves or activate other family members in a well-defined cascade depending on the type of the apoptotic stimuli, exerts a central role in apoptosis triggered by most insults. Active caspases cleave many specific intracellular proteins involved in many different types of functions, which results in an irreversible commitment to cell death. So far, a series of caspase substrates have been found, as reviewed by Fischer et al.54 Rho GDPdissociation inhibitor 2 (ARHGDIB), a known target of caspase3,55-58 was cleaved, and its fragments could be seen in nuclei, cytosols, and mitochondria in NSC606985-induced apoptotic NB4 cells. Here, we also found that full-length Rho GDP-

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dissociation inhibitor 1 (ARHGDIA) was significantly downregulated. ARHGDIA/B is known to regulate the GDP/GTP exchange exerted by the Rho proteins through inhibiting the dissociation of GDP from Rho proteins and subsequent GTPbinding. After the cleavage or down-regulation of ARHGDIA/ B, Rho proteins were apt to be activated via GTP binding, which was an important way for fragmentation of apoptotic cells into multiple apoptotic bodies as well as the subsequent phagocytosis.59 Besides lamin B1 (LMNB1) and gelsolin (GSN) which are substrates of caspase-3, respectively, in nuclei and cytosols,60-62 fragments of other two major components of nuclear matrix lamin A/C (LMNA) and thymopoietin isoform alpha (TMPO) and actin modulator Dynamin-2 (DNM2) were also detected. In addition, other two actin modulators, LIM and SH3 domain protein 1 (LASP1) and T-complex protein 1, epsilon subunit (CCT5), were reduced possibly due to degradation or cleavage. In total, these nuclear matrix and cytoskeleton proteins could be novel substrates of caspases, and their destruction would accelerate the process of apoptosis. As stated above, anticancer drugs-induced apoptosis signaling converges in the activation of intracellular caspases and their modification of protein substrates, as well as protein translocation, within the nucleus and cytoplasm.12 Prior to and after activation of caspases, translocations between subcellular compartments of some critical proteins, such as release of cytochrome c, apoptosis-inducing factor, Smac/DIABLO from mitochondria to cytosols and/or nuclei, play important and even key roles in the initiation, deveopment, and regulation of this process.63 Here, we also found several proteins that translocated among cell compartments during NSC606985induced apoptosis. In addition to three known translocating proteins, that is, PHGDH, ARHGDIB, and HNRNPC,17,47-49 we also found some new members in which translocations took place during apoptosis. These proteins included metabolism related proteins, cytoskeleton proteins, and mitochondria resident proteins (For detail, see Table 2). Notably, ran binding protein 1(RANBP1), which works as a negative regulator of regulator of chromosome condensation (RCC1) and is critical for nuclear assembly and nuclear transportation,64 was identified to be down-regulated, which might contribute to the nuclear translocation of some proteins. Totally, whether these translocations contribute to apoptosis is worthwhile to be further investigated.

Conclusions On the basis of our previous report that nanomolar concentration of camptothecin analogue NSC606985 induces AML cells to undergo apoptotic cell death, this work used 2-DE combined with MALDI-TOF/TOF to perform subcellular proteomic analysis of apoptotic AML cells. As a result, we could explore a series of deregulated proteins including translocated ones during apoptosis, most of which had not been reported previously. Further investigations on these deregulated prtoeins would shed new insights to understand the mechanisms of the camptothecin-induced apoptosis.

Acknowledgment. Supported in part by grants from Ministry of Science and Technology (NO2002CB512806, No.2006CB910104), National Natural Science Foundation (30500216; 30630034) of China, Chinese Academy of Sciences and Shanghai Science and Technology Commission (05JC14032). Dr. G. Q. Chen is a Chang Jiang Scholar of Ministry of Education of China, and is supported by Shanghai Ling-Jun Talent Program.

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Subcellular Proteome Analysis of Camptothecin Analogue

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