Petrak Toman Et Al Proteomics 2009


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Petrak Toman Et Al Proteomics 2009

  1. 1. 5006 DOI 10.1002/pmic.200900335 Proteomics 2009, 9, 5006–5015 RESEARCH ARTICLE Identification of molecular targets for selective elimination of TRAIL-resistant leukemia cells. From spots to in vitro assays using TOP15 charts Jiri Petrak1,2, Ondrej Toman2, Tereza Simonova1, Petr Halada3, Radek Cmejla2, Pavel Klener1 and Jan Zivny1 1 Charles University in Prague, First Faculty of Medicine, Institute of Pathological Physiology, Prague, Czech Republic 2 Institute of Hematology and Blood Transfusion, Prague, Czech Republic 3 Institute of Microbiology, v.v.i., Academy of Sciences of the Czech Republic, Prague, Czech Republic The resistance of malignant cells to chemotherapy calls for the development of novel anti- Received: May 20, 2009 cancer drugs. TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic cytokine, Revised: July 17, 2009 which selectively induces apoptosis in malignant cells. We derived two TRAIL-resistant HL-60 Accepted: August 11, 2009 subclones, HL-60/P1 and HL-60/P2, from a TRAIL-sensitive HL-60 acute promyelocytic leukemia cell line. To identify therapeutically exploitable ‘‘weaknesses’’ of the TRAIL-resis- tant leukemia cells that could be used as molecular targets for their elimination, we performed proteomic (2-DE) analysis and compared both TRAIL-resistant subclones with the original TRAIL-sensitive HL-60 cells. We identified over 40 differentially expressed proteins. To significantly narrow the lists of candidate proteins, we excluded proteins that are known to be often differentially expressed, regardless of experiment type and tissue (the so-called ‘‘TOP15’’ proteins). Decreased expression of DNA replication and maintenance proteins MCM7 and RPA32 in HL-60/P1 cells, and the marked down-regulation of enzyme adenosine deaminase in HL-60/P2 cells, suggests increased sensitivity of these cells to DNA-interfering drugs, and adenosine and its homologues, respectively. In a series of in vitro assays, we confirmed the increased toxicity of etoposide and cisplatin to TRAIL resistant HL-60/P1 cells, and adenosine and vidarabine to HL-60/P2, compared with TRAIL-sensitive HL-60 cells. Keywords: Biomedicine / Drug resistance / HL-60 / Leukemia / TOP15 / TRAIL 1 Introduction resistance of malignant cells necessitates the development of novel therapeutic regimens, and calls for new effective Acquired or preexisting resistance to chemotherapy is a and safe drugs to target this resistant cell population. major complication in the treatment of leukemia and solid TNF-related apoptosis-inducing ligand (TRAIL) is a pro- tumors, and is often associated with therapy failure, apoptotic cytokine belonging to the tumor necrosis factor progression and/or relapse of the disease. The drug (TNF) family of death ligands [1, 2]. TRAIL induces apop- tosis in target cells by the receptor-mediated apoptotic Correspondence: Dr. Jiri Petrak, Institute of Pathological pathway [3]. While normal tissues including hematopoietic Physiology First Faculty of Medicine, Charles University U progenitor cells are resistant to TRAIL-induced apoptosis, Nemocnice, Praha 2, Czech Republic TRAIL triggers programmed death in many malignant cell E-mail: lines and primary tumor cells [4–7]. Indeed, the potential of Fax:1420-224-912-834 TRAIL as a cancer-specific therapeutic agent has been Abbreviations: ADA, adenosine deaminase; HP1a, heterochro- proposed, and several clinical trials with recombinant matin protein 1 alpha; TNF, tumor necrosis factor; TRAIL, TNF- TRAIL are under way. As with most other anti-cancer drugs, related apoptosis-inducing ligand the development of TRAIL-resistance has been reported [8], & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
  2. 2. Proteomics 2009, 9, 5006–5015 5007 and TRAIL-resistant tumor and leukemia cells have been 2.2 Sample preparation for 2-DE derived by us and others [9, 10]. Specific molecular features of drug-resistant cells can be Approximately 1 Â 108 cells were harvested by centrifuga- advantageously used as targets for specific drugs. In this tion, washed twice with PBS and cell pellets were frozen at 2-DE-based proteomic study, we focused on the identifica- À801C. Samples were thawed and homogenized in lysis tion of suitable molecular targets that could be used buffer (7 M urea, 2 M thiourea, 4% CHAPS, 60 mM DTT for the selective elimination of leukemia cells resistant to and 1% ampholytes (IPG buffer pH 4–7, Amersham) TRAIL. We previously demonstrated that some proteins and containing protease inhibitor cocktail (EDTA Free, Roche protein families very often appear among differentially Diagnostics) for 20 min at room temperature. After subse- expressed proteins in published 2-DE experiments, regard- quent centrifugation at 14 000 Â g for 20 min at room less of the experiment performed or tissue studied. These temperature, supernatants were collected and protein proteins and protein families were designated as the TOP15 concentration determined by the Bradford method (Bio-Rad, [11]. Very recently, the observation of these generally CA, USA). Protein concentrations in all samples were detected proteins in comparative proteomics was confirmed equalized to 7.3 mg/mL by dilution with the lysis buffer. in different data sets from various species [12], and also backed by an automatic text analysis [13]. Since the TOP15 proteins are differentially expressed as often as in every third 2.3 2-D electrophoresis 2-DE-based study, their differential expression can hardly be deemed specific [11, 12]. If identified as differentially Isoelectric focusing was performed with a Bio-Rad Protean expressed in a 2-DE experiment, these proteins should IEF cell using 24 cm IPG strips (pH 4–7, GE, USA), using be approached with caution, and eventually excluded from rehydration loading of samples. Five replicates were run for data interpretation and the process of hypothesis formula- each cell type. Strips were rehydrated overnight in 450 mL of tion. Here, we present our approach to narrowing down the sample, representing 3.3 mg protein. Isoelectric focusing list of candidate proteins by the exclusion of these TOP15 was performed for 60 kV h, with maximum voltage not proteins. exceeding 5 kV, current limited to 50 mA per strip and By proteomic analysis of two TRAIL-resistant subclones temperature set to 181C. Focused strips were stored at and original TRAIL-sensitive HL-60 cells, we identified À801C. For SDS electrophoresis, strips were thawed, equi- proteins that were differentially expressed in two resistant librated and reduced in equilibration buffer A (6 M urea, phenotypes and the original TRAIL-sensitive HL-60 cells. 50 mM Tris pH 8.8, 30% glycerol, 2% SDS and 450 mg DTT After narrowing down the list of candidate proteins by per 50 mL of the buffer) for 15 min and then alkylated in excluding the TOP15 we identified two potential molecular equilibration buffer B (6 M urea, 50 mM Tris pH 8.8, 30% targets, and proposed and tested four chemicals selected to glycerol, 2% SDS and 1.125 mg iodacetamide per 50 mL). specifically target these processes in order to eliminate the Equilibrated strips were then placed on the top of 10% TRAIL-resistant population of leukemia cells. PAGE and secured in place by molten agarose. Electro- phoresis was performed in a tris-glycine-SDS system using a 12 gel Protean Plus Dodeca Cell apparatus (Bio-Rad) with 2 Materials and methods buffer circulation and external cooling (201C). Gels were run at constant voltage of 200 V for 6 h. Following electrophor- Unless specified otherwise, all chemicals were purchased esis, gels were washed two times for 15 min in deionized from Sigma-Aldrich (MO, USA). water to remove SDS. Washed gels were stained in CCB (Simply Blue SafeStain, Invitrogen, Carlsbad, USA) over- night, and then destained in deionized water. 2.1 Establishment and growth of TRAIL-resistant HL-60 cells 2.4 Gel image analysis The TRAIL-resistant HL-60 subclones P1 and P2 were derived from HL-60 cells (ATCC) in our previous study [9] Coomassie blue-stained gels were scanned with a GS 800 by the selective pressure of recombinant TRAIL, and stored calibrated densitometer (Bio-Rad). Image analysis was frozen under liquid nitrogen. Cells were revived and grown performed with Phoretix 2D software (Nonlinear Dynamics, in Iscove’s modified Dulbecco’s medium (Life Technologies, UK) in semi-manual mode with five gel replicates for each MD, USA) in the presence of 10% FBS in a 371C humidified cell type. Normalization of gel images was based on total atmosphere with 5% CO2. Before the current proteomic spot density, and integrated spot density values (spot analyses, the TRAIL-resistant phenotype of the revived cells volumes) were then calculated (after background subtrac- was verified by exposure to recombinant TRAIL (200 ng/mL, tion). Average spot volume values (averages from the all five Killer Trail, Apronex Biotechnologies, Czech Republic) in gels in the group) for each spot were compared between cell culture. the groups. Protein spots were considered differentially & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
  3. 3. 5008 J. Petrak et al. Proteomics 2009, 9, 5006–5015 expressed if they met both of the following criteria: average protein Heterochromatin protein 1 a (HP1a), cells were normalized spot volume difference 41.5-fold and statistical lysed in the lysis buffer used in the 2-DE analysis (7 M urea, significance (po0.05) of the change determined by the t-test. 2 M thiourea, 4% CHAPS, 60 mM DTT). Cleared cell lysates (14 000 Â g, 20 min) were collected and protein concentration determined by the Bradford method (Bio- 2.5 MALDI MS, protein identification Rad). Samples containing 60 mg protein were combined with SDS loading buffer containing DTT, boiled for 5 min and Differentially expressed proteins were excised from gels, cut resolved by SDS-PAGE using Novex precast 4–20% gradient into small pieces and washed several times with 50 mM gels (Invitrogen). Separated proteins were transferred onto a 4-ethylmorpholine acetate (pH 8.1) in 50% ACN (MeCN). PVDF membrane (Invitrogen) using a semi-dry blotter After complete destaining, the gel was washed with deio- (Hoeffer) for 80 min at 0.8 mA/cm2. Membranes were nized water, shrunk by dehydration in MeCN and re-swollen then blocked in PBS-T/milk (137 mM NaCl, 2.7 mM KCl, again in water. The supernatant was removed and the gel 4.3 mM Na2HPO4, 1.4 mM KH2PO4, pH 7.5, 0,1% was partially dried in a SpeedVac concentrator. Gel pieces Tween 20, 5% nonfat dry milk) overnight. Blocked were then reconstituted in a cleavage buffer containing membranes were then incubated for 3 h in the same buffer 25 mM 4-ethylmorpholine acetate, 10% MeCN and sequen- with the respective antibody, except with nonfat dry milk cing grade trypsin (5 ng/mL; Promega, WI, USA). After concentration of only 0.5%. The primary antibodies (all overnight digestion, the resulting peptides were extracted from Santa Cruz Biotechnology, CA, USA) were used in with 40% MeCN/0.5% TFA. A solution of CHCA in aqueous following dilutions: 1:30 000 for Annexin A6, 1:10 000 for 50% MeCN/0.1% TFA (5 mg/mL) was used as a MALDI PDI A3, 1:50 for HP1 alpha and 1:1000 for adenosine matrix. A sample volume of 0.5 mL was deposited on the deaminase (ADA). After four washes in PBS-T, secondary MALDI target and allowed to air-dry at room temperature. antibodies (HRP-conjugated goat anti-rabbit and donkey After complete evaporation, 0.5 mL of the matrix solution anti-goat IgG (Santa Cruz Biotechnology)) were added for was added. 1.5 h. The dilutions of secondary antibodies were 1/100 000 MALDI mass spectra were measured on an Ultraflex III for Annexin A6, 1/30 000 for PDI A3 and ADA and 1/3000 instrument (Bruker Daltonics, Bremen, Germany) equipped for HP1 a. The bound secondary antibodies were washed with a SmartbeamTM solid state laser and LIFTTM technol- twice in PBS-T, twice in PBS and then detected using an ogy for MS/MS analysis. Spectra were acquired in the mass enhanced chemiluminiscence assay (ECL, G.E, USA), range of 700–4000 Da and calibrated internally using the developed, scanned and quantified by the quantity one monoisotopic [M1H]1 ions of trypsin autoproteolytic frag- documentation system (Bio-Rad). ments (842.5 and 2211.1 Da). Peak lists in XML data format were created using the flexAnalysis 3.0 program with the SNAP peak detection 2.7 Cellular toxicity assay algorithm. No smoothing was applied, and the maximal number of assigned peaks was set to 50. After peak labeling, The toxicity of etoposide, cisplatin, adenosine and vidar- all known contaminant signals were manually removed. The abine to HL-60, HL-60/P1 and HL-60/P2 cells was measured peak lists were searched using the MASCOT search engine by a Quick Cell Proliferation Assay Kit II (BioVision, against the SwissProt 52.0 database subset of human Mountain View, USA) according to the manufacturer’s proteins with the following search settings: peptide toler- instructions. Five thousand cells were seeded in a 96-well ance of 50 ppm, missed cleavage site value set to two, fixed plate in 100 mL of Gibco-IMDM media (Invitrogen) with carbamidomethylation of cysteine, and variable oxidation of increasing concentrations of the chemicals tested. Cells methionine. No restrictions on protein molecular weight or were grown in 371C, 5% CO2 and humidity for 5 days pI value were applied. Proteins with a MOWSE score over with either vidarabine (adenine-9-b-Darabinofuranoside, the threshold 55 for po0.05 calculated for the used settings Fluka, Buchs, Switzerland), cisplatin (cis-diamminedi- were considered as identified. If the score was only slightly chloridoplatinum(II), Cisplan, Ebewe Pharma, Unterach, higher than the threshold value or the sequence coverage Austria) or etoposide (40 demethyl-epipodophyllotoxin too low, the identity of the protein candidate was confirmed 9-[4,6-O-(R)-ethylidene-beta-D-glucopyranoside], 40 -dihydro- by MS/MS analysis. gen phosphate, Ebewe Pharma) or for two days with adenosine monophosphate (ICN Pharmaceuticals, Costa Mesa, USA). After cultivation, 5 mL of WTS reagent (Quick 2.6 Western blotting Cell Proliferation Assay Kit II, BioVision) was added to each well and cells were incubated for 2 h under standard Cells pellets were solubilized in 300 mL of lysis buffer culture conditions. Absorbance was measured on a SunRise (50 mM Tris pH 7.4; 1% Triton X-100, Protease inhibitor microplate absorbance reader (Tecan, Switzerland) with a cocktail, 1 tablet per mL (EDTA Free, Roche Diagnostics)) 450 nm reading filter and 650 nm reference filter. The and lysed on ice for 20 min. For the detection of nuclear absorbance of free medium was used as the background & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
  4. 4. Proteomics 2009, 9, 5006–5015 5009 level. Triplicate samples were grown and measured for each group and average values were calculated. Because of the different proliferation potential of individual cell lines and subclones, the data were normalized. The measured absor- bance of control cells grown in media without any drugs added was set as (100%). 3 Results Our aim was to identify differences in protein expression between the TRAIL-resistant and TRAIL-sensitive HL-60 cells, and find potential drug targets based on specific expression features. Therefore, we looked for ‘‘an Achilles heel’’ in the TRAIL-resistant cells that could serve as a drug target for the selective elimination of these resistant cells. 3.1 TRAIL-resistant HL-60 subclones The HL-60 acute myeloid leukemia cell line is sensitive to TRAIL-induced apoptosis [6]. In our previous work, we derived two distinct TRAIL-resistant HL-60 subclones, designated as HL-60/P1 and HL-60/P2 [9]. These subclones differ from each other in the expression levels of TRAIL receptors, the CD14 myeloid marker and of several anti- apoptotic genes. The survival time of immunodeficient mice transplanted with either HL-60/P1 or HL-60/P2 also differs, P2 being the more aggressive population [9]. HL-60/P1 and HL-60/P2 TRAIL-resistant cells were stored frozen and thawed for the current study. 3.2 Proteomic analysis We performed proteomic analysis and compared expression patterns of leukemia cells HL-60 and HL-60/P1 and HL-60/ P2 subclones in total cell homogenates. Using 2-D electro- phoresis in large polyacrylamide gels, we reproducibly detected 1180 (725) spots on CCB-stained gels (Fig. 1). Compared with HL-60 cells, we found 22 and 24 protein Figure 1. 2-DE analysis of TRAIL-sensitive HL-60 and TRAIL- spots to be significantly quantitatively changed (change resistant HL-60/P1 and HL-60/P2 cells, performed on 24 cm strips 41.5-fold, po0.05) in HL-60/P1 and HL-60/P2 TRAIL- pH 4–7 and 10% SDS-PAGE. Proteins were stained with CCB. resistant subclones, respectively. Relative differences in Numbered arrows indicate differentially expressed proteins compared with HL-60. expression ranged from 1.5 to as much as almost ten-fold. Using MALDI-TOF/TOF-MS we identified differentially expressed proteins in all selected spots (Tables 1 and 2). One spot (No. 4 in HL-60/P2 cells) contained two proteins and was excluded from further data interpretation. In total, we expression profiles also differ. Decreased expression of three identified 20 (HL-60/P1) and 21(HL-60/P2) individual proteins (HP1a, PDI A3 and annexin A6) is common for proteins. These differentially expressed proteins are involved both HL-60/P1 and HL-60/P2 cells compared with HL-60 in various aspects of cellular metabolism, including energy cells. metabolism, cellular stress, cytoskeletal components and To confirm the results of our proteomic study by an regulatory proteins. independent method, we verified the altered expression of Since HL-60/P1 and HL-60/P2 are two phenotypically four proteins (HP1a, PDI A3, annexin A6 and ADA) by distinct cell subclones [9], it is not surprising that their Western blotting analysis (Fig. 2). & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
  5. 5. 5010 J. Petrak et al. Proteomics 2009, 9, 5006–5015 often as in every third 2-DE-based study, their differential expression can hardly be deemed specific [11–13]. If identified as differentially expressed, these proteins should be approached with caution and could even be excluded from data interpretation and the process of hypothesis formulation. Unsurprisingly, many of the 41 differentially expressed proteins identified in our current study belong among these TOP15 proteins, namely, enolase 1, HSP 27, peroxiredoxin 2, HSC71 Grp78, pyruvate kinase M1/M2 and TOP15 protein families such as tubulins, annexins, actins and protein disulfide isomerases (marked by ‘‘YES’’ in Tables 1 and 2). By exclusion of the individual TOP15 proteins and the members of TOP15 protein famil- ies, we simplified our list of candidate proteins from 20 (HL- 60/P1 cells) and 21 (HL-60/P2 cells) original candidates to only 16 and 11 individual proteins, respectively. This step focused our attention on the proteins that are more likely to be specific and relevant to the molecular features of TRAIL resistant cells. Our original aim was to identify ‘‘the Achilles heel’’ of the TRAIL-resistant cells – a protein or a pathway that could serve as a target for the selective elimination of TRAIL-resistant cells. Therefore, from the remaining 27 candidates we focused our attention on enzymes and other active proteins, which are better targets for pharmaceutical intervention than structural molecules. Three such proteins clearly stood out: the down- regulated DNA metabolism and repair proteins RPA32 and MCM7 and the down-regulated enzyme ADA, in HL-60/P1 and HL-60/P2 cells, respectively. 3.4 MCM7 and RPA32 Figure 2. Western blot analyses of protein disulfide isomerase A3 After the exclusion of TOP15 proteins only 16 candidate (PDIA3), annexin A6, HP1a and ADA. Protein samples (60 mg/well) molecules remained in the HL-60/P1 TRAIL-resistant cells. were separated on 4–20% SDS-PAGE, transferred to a PVDF We observed marked down-regulation of two proteins membrane and probed with respective primary and secondary antibodies. Densitometric quantification was performed with essential for DNA replication and DNA repair compared three independent experiments for each protein. Values for with HL-60 and HL-60/P2 cells. The DNA replication HL-60 were set as 100 percent and the HL-60/P1 and HL-60/P2 licensing factor MCM7 was down-regulated five-fold, values were calculated relative to HL-60. Average relative values whereas the replication protein A, 32 kDa subunit declined are plotted and shown with corresponding SDs. two-fold. MCM7 (minichromosomal maintenance protein 7) is a part of the MCM2–7 DNA-binding heterohexamer complex 3.3 Data interpretation and TOP15 exclusion essential for DNA replication, providing helicase activity [14, 15]. Replication protein A, 32 kDa subunit is one of The lists of all identified differentially expressed proteins three components of Replication protein A – the major found in both P1 and P2 HL-60 subclones exceeded 40 single-stranded DNA-binding protein in eukaryotes. items. We thus faced the universal dilemma of proteomic As such, the RPA complex participates in DNA replication data interpretation: How to narrow down the list of candi- and recombination, DNA damage checkpoints and date proteins? all major types of DNA repair including nucleotide In our earlier study, we demonstrated that some proteins excision, base-excision, mismatch and double-strand break and protein families often appear among the differentially repairs. [16]. expressed proteins in published 2-DE experiments, regard- Since these two proteins, critical for cell proliferation and less of the experiments performed or tissues studied. We maintenance of genome stability, are markedly down-regu- designated these ‘‘notorious’’ proteins as the TOP15 [11]. lated, we hypothesized that their DNA-related functions Since the TOP15 proteins are differentially expressed as could be partially limited in HL-60/P1 cells. Such a defect & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
  6. 6. Proteomics 2009, 9, 5006–5015 5011 Table 1. Differential protein expressions in HL-60/P1 cells Fold Spot Protein name SwissProt Matched Sequence Mascot Excluded change number number peptides coverage (%) Score (TOP 15) Proteins differentially expressed in P1 cells Up-regulated 10 9 Thioredoxin-like protein 1 O43396 14 52 179 3.8 17 Sjoegren syndrome/scleroderma O60232 10 59 153 autoantigen 1 3.3 15 Heat-shock protein b-1 (HSP27) P04792 9 55 141 YES 2.1 20 Cofilin-1 P23528 14 72 196 1.6 11 Splicing factor, arginine/ Q07955 17 58 256 serine-rich 1 Down-regulated À5.7 1 DNA replication licensing factor P33993 27 49 373 MCM7 À3.7 22 Lamin-A/C P02545 9 13 87 À3.4 6 Proliferation-associated protein 2G4 Q9UQ80 16 28 136 À3 8 Macrophage-capping protein P40121 10 35 121 À2.6 16 78 kDa glucose-regulated protein P11021 10 19 160 YES À2.5 18 Heterochromatin protein 1a P45973 9 43 144 À2.5 7 Protein phosphatase 1 regulatory Q15435 13 39 133 subunit 7 À2.2 2 Annexin A6 P08133 30 46 341 YES À2.2 21 Stathmin P16949 11 42 144 À2.1 12 Replication protein A 32 kDa subunit P15927 11 48 154 À2.1 13 F-actin capping protein subunit b P47756 15 48 196 À2 3 Protein disulfide-isomerase A3 P30101 12 22 135 YES precursor À2 5 Tryptophanyl-tRNA synthetase, P23381 12 33 180 cytoplasmic À1.9 10 F-actin capping protein subunit a-1 P52907 11 48 186 À1.9 14 6-phosphogluconolactonase O95336 10 40 122 À1.8 19 Stathmin P16949 8 41 96 À1.6 4 Protein disulfide-isomerase A3 P30101 26 47 344 YES precursor would make these cells highly sensitive to a treatment with responsible for severe combined immunodeficiency disease chemicals and clinically used genotoxic drugs that interfere [17, 18]. Deficiency of ADA causes increased levels of with DNA replication and the maintenance of DNA integ- intracellular adenosine. The most widely accepted mechan- rity. Typical examples of such drugs are the DNA cross- ism of ADA deficiency implicates the formation of intra- linker cis-platin and the inhibitor of topoisomerase cellular deoxyadenosine, deoxyATP and/or S-adenosyl II, etoposide. homocysteine as well as pyrimidine starvation as the direct cause of intracellular toxicity [18, 19]. Based on these facts, we hypothesized that markedly decreased expression of 3.5 ADA ADA in HL-60/P2 cells could partially reduce the detox- ification capacity of these cells and make them vulnerable to In the HL-60/P2 subclone, only 11 of the original 21 increased adenosine concentrations. candidate proteins remained after the exclusion of TOP15 ADA is also normally responsible for deamidation, and proteins and protein families. The most notable alteration of hence inactivation of synthetic purine anti-metabolite expression in HL-60/P2 cells was the down-regulation of vidarabine (adenine arabinoside, Ara-A) used as a cytostatic ADA. Based on 2-DE data, levels of ADA are decreased and anti-viral drug. Sensitivity of cells to the cytostatic action approximately sixfold, with more than a fivefold decline of vidarabine is inversely proportional to the activity of ADA confirmed by western blotting. ADA participates in purine [20]. In accordance with this, we hypothesized that the metabolism where it degrades adenosine or 20 -deoxy- observed sixfold decreased expression of ADA would result adenosine, producing inosine or 20 -deoxyinosine, respec- in the higher toxicity of vidarabine to HL-60/P2 cells tively. In humans, a congenital defect in this enzyme is compared with HL-60/P1 and HL-60 cells. & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
  7. 7. 5012 J. Petrak et al. Proteomics 2009, 9, 5006–5015 Table 2. Differential protein expressions in HL-60/P2 cells. Identification of proteins with Mascot Score. Fold Spot Protein name SwissProt Matched Sequence Mascot Excluded change number number peptides coverage Score (TOP 15) (%) Proteins differentially expressed in P2 cells Up-regulated 7.3 6 Septin-11 Q9NVA2 10 29 121 4 22 Peroxiredoxin-2 P32119 8 43 125 YES 2.6 23 a-Enolase P06733 10 24 113 YES 2.4 14 S-formylglutathione hydrolase P10768 9 42 111 2.1 19 GrpE protein homolog 1, mitochondrial Q9HAV7 9 47 124 precursor 1.8 20 Heat shock cognate 71 kDa protein P11142 9 19 119 YES Down-regulated À6.4 10 Adenosine deaminase P00813 17 43 205 À4.8 7 Pyruvate kinase isozymes M1/M2 P14618 15 37 72 YES À4 9 Heat shock cognate 71 kDa protein P11142 7 14 63Ã YES À3.6 2 Nucleolin (Protein C23) P19338 14 23 163 À2.5 12 Tubulin b chain P07437 15 29 165 YES À2.4 8 Ubiquinol-cytochrome-c reductase P31930 15 43 132 complex core protein 1 À2.4 21 Heterochromatin protein 1 a P45973 4 24 59Ã À2.3 15 Proteasome activator complex subunit 2 Q9UL46 10 44 135 À2.2 1 Annexin A6 P08133 14 26 126 YES À2.2 11 Pyruvate kinase isozymes M1/M2 P14618 6 14 172 YES À2.1 4 Hydroxymethylglutaryl-CoA synthase, Q01581 15 31 149 cytoplasmic 4 Actin, cytoplasmic 1 P60709 5 21 39Ã À2 17 Heat-shock protein b-1 (HSP27) P04792 6 27 65Ã YES À1.9 13 Tubulin b chain P07437 14 31 150 YES À1.7 16 Proteasome activator complex subunit 1 Q06323 17 59 198 À1.6 18 NADH dehydrogenase [ubiquinone] O75489 15 54 216 iron–sulfur protein 3 À1.6 24 Cofilin-1 P23528 12 62 182 À1.6 5 Protein disulfide-isomerase A3 precursor P30101 23 44 270 YES À1.5 3 Protein disulfide-isomerase precursor P07237 22 50 321 YES Ã MS/MS confirmation of identification Spot Protein Peptide number name 4 Actin cytoplasmic 1 SYELPDGQVITIGNER 17 Heat-shock protein LATQSNEITIPVTFESR, b-1(HSP27) LFDQAFGLPR 21 Heterochromatin CPQIVIAFYEER protein 1 a 9 Heat shock cognate ARFEELNADLFR 71 kDa protein 3.6 Cell assays We tested the toxic effect of increasing doses of etoposide (inhibitor of topoisomerase II), cis-platin, adenosine and To test our hypotheses that HL-60/P1 cells are sensitive vidarabine on the growth and survival of HL-60, HL-60/P1 to a treatment interfering with DNA-replication and and HL-60/P2 cells in culture. The toxic effect of these integrity, and that P2 cells are vulnerable to adenosine respective treatments was considered as the percentage of and sensitive to vidarabine, we set up series of in vitro surviving cells, as measured by mitochondrial activity cell assays. (Fig 3). In accordance with our hypotheses, etoposide and & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
  8. 8. Proteomics 2009, 9, 5006–5015 5013 cis-platin were both significantly more toxic to HL-60/P1 vidarabine. Unlike the effect of DNA-interfering agents and cells than to HL-60 and HL-60/P2. These DNA-interfering anti-metabolites, adenosine toxicity in ADA-deficient cells is drugs eliminated 60% of the HL-60/P1 population at high independent of DNA replication. Therefore, adenosine nM or low mM concentrations in five days, while reducing toxicity to HL-60/P2 cells was demonstrated in an assay with the HL-60 and HL-60/P2 cell population only by 0–20%. a shorter incubation period (2 days only). As hypothesized, Similarly, both adenosine and vidarbine were markedly adenosine monophosphate added to the media reduced the more toxic to HL-60/P2 cells than to HL-60/P1 or HL-60 P2 cell population by more than 60% at the 1000 mM cells. Vidarabine completely eliminated the P2 population at concentration, while in contrast stimulated the proliferation a concentration of 100 mM, while only marginally reducing of HL-60 and HL-60/P1 cells. the viability of HL-60. The complete elimination of P1 would To summarize, our two hypotheses based on proteomic require a more than two-fold higher concentration of data were tested and confirmed in a series of in vitro cellular Figure 3. Cellular toxicity assay. Cells were seeded and grown with increasing concentrations of either cis-platin, etoposide, adenosine monophosphate or vidarabine (single dose at the time of seeding) and cultivated for 5 days, except for cells exposed to AMP which were grown for only 2 days. Decreased mitochondrial activity measured by a Quick Cell Proliferation Assay Kit reflects the number of surviving cells and hence the toxicity of individual tested chemicals. Average values from triplicate experiments are shown. & 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
  9. 9. 5014 J. Petrak et al. Proteomics 2009, 9, 5006–5015 assays, and two cellular processes were proposed as potential the best potential drug targets-MCM7 and RPA2 in HL-60/P1 therapeutic targets. Four proposed drugs aimed at these cells, and ADA in HL-60/P2 cells. We subsequently hypo- targets were tested and shown to be effective for the elimin- thesized that the observed down-regulation of MCM7 and ation of HL-60/P1 and HL-60/P2 TRAIL-resistant cells RPA2 proteins in TRAIL-resistant P1 leukemia cells makes in vitro. these cells vulnerable to clinically used DNA-interfering agents such as etoposide and cis-platin. Successful in vitro testing in cell cultures demonstrated that HL-60/P1 cells are 4 Discussion markedly more sensitive to etoposide than TRAIL-sensitive HL-60 and resistant HL-60/P2 cells. Similarly, we proposed Resistance to anti-cancer drugs is the major cause of and experimentally verified that markedly decreased expres- chemotherapy failure, and necessitates the application of sion of ADA makes HL-60/P2 cells vulnerable to the drug combined drug regimens and the development of new anti- vidarabine and to high concentrations of the nucleoside cancer molecules. The new and very promising apoptosis- adenosine. inducing molecule – recombinant death ligand TRAIL – is Based on 2-DE analysis of TRAIL-resistant leukemia undergoing several clinical trials with promising preli- cells, we revealed specific molecular targets that can be used minary results. However, as with all other anti-cancer drugs, (at least in vitro) for the selective elimination of two TRAIL- TRAIL-based therapies are likely to be complicated by resistant subclones derived from established HL-60 leuke- TRAIL-resistant cancer cells. We used expression proteo- mia cells. If TRAIL is approved for clinical use, questions on mics as a tool for the identification of potential molecular how to target TRAIL-resistant tumors will become immi- targets of TRAIL-resistant leukemia cells. We looked for nent. Therefore, any information on the specific molecular proteins or pathways that could be used as targets for the features of resistant cancer cells and especially their ‘‘weak selective elimination of TRAIL-resistant cells. The discovery spots’’ will become invaluable. We are fully aware that the of such target molecules could be invaluable for the isolation and characterization of drug-resistant cancer cells improvement of future TRAIL-based therapies. The last from individual patients in order to optimize therapy is, decade has witnessed a massive expansion of proteomic indeed, technically challenging and incomparably more approaches into molecular oncology and cell physiology. complex. Nevertheless, we believe that our work has However, proteomic analyses only seldom produce a direct, demonstrated a basic ‘‘proof of concept’’ for the possible experimentally verified, insight into cell physiology or implementation of proteomics in the development of pathology. The interpretation of data derived from expres- patient-tailored anti-cancer therapy. sion proteomics studies and verification of the hypotheses generated remain among the most challenging tasks of This research was supported by grants from the Czech Science current proteomics. A typical expression proteomics Foundation (GACR) 305/09/1390 and 204/07/0830, from the experiment produces lists of quantitatively or qualitatively Ministry of Health of the Czech Republic (MZCR) MZCR/ changed proteins. The question remains how to transform UHKT No. 023736, IGA MZ NR8317-4, NR8930-4, NS10300- the resulting list of differentially expressed proteins into 3 and the Ministry of Education, Youth and Sports of the Czech plausible and verifiable hypothesis addressing the molecular Republic (MSMTCR) projects LC06044, MSM 0021620806, mechanism. What candidate proteins are central or specific MSM 0021620808 and the Institutional Research Concept for the process studied? How do we narrow down the list of AV0Z50200510 (IMIC). Special thanks to Mrtva Ryba differentially expressed candidate proteins? Here, we iden- tified 41 differentially expressed proteins in TRAIL-resistant The authors have declared no conflict of interest. HL-60/P1 and HL-60/P2 subclones compared with the original HL-60 TRAIL-sensitive cells. Which one of these 41 proteins could serve as a molecular target for the selective 5 References elimination of resistant cells? 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