Quantitative Differential Proteome Analysis in an Animal Model for

Quantitative Differential Proteome Analysis in an Animal Model for Human Melanoma Katrin Lokaj,†,# Svenja Meierjohann,|,# Claudia Schu ¨ tz,† Janka Teutschbein,| Manfred Schartl,| and Albert Sickmann*,†,‡,§ Rudolf-Virchow-Center, DFG-Research Center for Experimental Biomedicine, University of Wurzburg, Versbacher Str. 9, 97078 Wurzburg, Germany, ISAS, Institute for Analytical Sciences, Bunsen-Kirchhoff-Str. 11, 44139 Dortmund, Germany, Medizinisches Proteom-Center (MPC), Ruhr-Universita¨t Bochum, Universita¨tsstrasse 150, 44801 Bochum, Germany, Department of Physiological Chemistry I, University of Wurzburg, Biocenter, 97074 Wurzburg, Germany Received July 29, 2008

In fish of the genus Xiphophorus, different grades of pigment cell lesions from nevi to melanoma can be gained by simple crossbreeding. With this model, one can easily access tissues of different malignancies from animals with highly identical genetic background. To find protein expression differences between healthy, benign and malignant tissues, we performed 2D PAGE and DIGE and found among regulated proteins antioxidant proteins that were overexpressed with increasing malignancy. Keywords: PRDX2 • PRDX6 • glutathione-S transferase • oxidative stress • tumor progression • Xmrk

Introduction Among human tumors, melanomas are one of the most aggressive cancer types with a high metastatic potential. In this context, animal models provide useful tools to examine the complex process of melanoma development. The Xiphophorus melanoma model is a well-established fish model system enabling the examination of spontaneous melanoma formation.1 Here, the oncogenic receptor tyrosine kinase (RTK) Xmrk is causative for the tumor. This EGF receptor orthologue arose from local duplication of the protooncogenic EGFR in Xiphophorus maculatus and induces many pathways also known for their relevance in human melanoma, for example, the PI3 kinase and the RAS/RAF/MAPK pathways.2 The oncogenic action of the receptor is due to two activating mutations in the extracellular domain, which lead to ligand independent dimerization and constitutive signaling, mimicking the permanent presence of a growth activating factor. This leads to cell autonomous, uncontrolled proliferation, antiapoptosis and many other features of transformed cells. In the platyfish, expression of xmrk is prevented by a repressor and no melanoma development can occur. Crossbreeding of these fish with the related species Xiphophorus hellerii, not carrying the repressor allele, results in a diminished activity of the repressor due to the heterozygous condition in fish of the F1 generation and consequently increased Xmrk* To whom correspondence should be addressed. ISAS, Institute for Analytical Sciences, Bunsen-Kirchhoff-Str. 11, 44139 Dortmund, Germany. Tel: +49 (0)231-1392105. Fax: +49 (0)231-1392310. E-mail: [email protected]. † Rudolf-Virchow-Center, DFG-Research Center for Experimental Biomedicine, University of Wurzburg. | Department of Physiological Chemistry I, University of Wurzburg. # These authors contributed equally to this work. ‡ ISAS, Institute for Analytical Sciences. § Medizinisches Proteom-Center (MPC), Ruhr-Universita¨t Bochum.

1818 Journal of Proteome Research 2009, 8, 1818–1827 Published on Web 02/27/2009

levels.2 As a result, the fish develop benign pigment cell lesions at their fins that are comparable to human nevi. Backcrossing F1 hybrids with X. hellerii leads to one-quarter of offspring in which both repressor alleles are eliminated, while the xmrk allele is maintained. This allows unrestricted xmrk expression and leads to the development of malignant melanoma (Figure 1). This tumor development is highly reproducible and permits the examination of different tumor stages by comparing healthy skin, benign lesions and malignant tissue. Furthermore, all fish have the same genetic background which is a clear advantage compared to the situation described for human melanoma samples.3 The fish used for the crossing experiments were backcrossed to the inbred X. hellerii fish more than 20 times, which guarantees a quasi-identical genetic background of the healthy fish and the fish carrying benign lesions or malignant tumors as displayed in Figure 1. In addition, the healthy fin tissue and the melanocytic lesions are from the same body compartment. A comparative protein expression analysis of the three states can enable the discrimination between events that go along with or are even causative for the development of benign lesions and malignant melanoma. To gain insight into these events, it is possible to either perform an analysis of the phosphoproteome or to monitor the overall protein amount. We have chosen to investigate the latter, as the activation of signaling proteins visualized by the phosphoproteome is often reversible and thus is only detectable in a certain time window. Compared to that, the overall protein amount is more reliable in reflecting the proteins required for the respective tumor stage. In this study, for the first time, quantitative 2DE and DIGE were applied to the Xiphophorus melanoma model. 2D PAGE separation was used for this approach because of its high 10.1021/pr800578a CCC: $40.75

 2009 American Chemical Society

DIGE and 2DE Study of Xiphophorus Melanoma

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Figure 1. Schematic of the crossing procedure of female platyfish (X. maculatus) carrying the Tu and R allele with male swordtails (X. hellerii), which lack both alleles. The F1 generation is heterozygous for Tu and R and develops benign lesions. Repeated crossing of the female F1 generation with male swordtails leads to the following Tu and R distribution within the F2 generation: 25% carry only the Tu allele and develop malignant melanoma, 25% possess one Tu and one R allele and show benign lesions, and 50% carries no Tu and R allele or only one R allele and develop normally (each example indicated by the red box). On the right, exemplified 2D gels with regulated spot patterns (black box) are depicted.

resolution and the possibility to quantity differences between distinct disease states by supporting software. The differential analysis is based on spot intensities acquired by scanning methods of the 2DE-separated proteins that were either preelectrophoretically labeled or subsequently stained. Therefore, the study was designed as a 2DE approach based on a statistical relevant number of replicates and fluorescence staining of separated proteins. In addition, a comparative DIGE4 approach was performed. Here, the number of technical replicates is reduced due to the possibility of running a co-electrophoresis of up to three samples per gel. On the basis of these two techniques, a total of 122 differentially regulated protein spots out of approximately 1500 protein spots could be detected. Of these, 88 could be identified by nano-LC-MS/MS and Mascot searches using a FASTA whole genome sequence database of the closely related fish species medaka (Oryzias latipes).5 Since medaka proteins are not completely annotated, we identified the peptides by homology comparison of the medaka sequence to known proteins of other species, such as Danio rerio, Takifugu rubripes or Homo sapiens. Most of these proteins could be grouped into seven functional entities: sugar metabolism, amino acid metabolism, cytoskeleton-associated proteins, oxidative stress-related proteins, channels, signal transduction proteins and keratin-related proteins.

Experimental Section Reagents. CyDye DIGE fluors (minimal labeling kit), IPG buffer pH 3-10 NL, and Immobiline DryStrips pH 3-11 NL,

24 cm, were purchased from GE Healthcare, Munich, Germany. Molecular weight marker Mark12 was obtained from Invitrogen, Karlsruhe, Germany. DTT, iodoacetamide, ammonium persulfate, ethanol, agarose, acrylamide, SDS, urea, thiourea and TEMED were obtained from VWR, Darmstadt, Germany, and Complete Mini protease inhibitor cocktail was purchased from Roche (Penzberg, Germany). RuBPS was synthesized according to Rabilloud et al.6 All chemicals for liquid chromatography used in this study were obtained from VWR, Darmstadt, Germany as analytical or higher grade. Sequencing grade modified trypsin was purchased from Promega, Madison, WI. Procedures. Tissue Collection and Sample Preparation. Control or tumor tissue was obtained from fish of the following genotypes: benign pigment lesions and malignant melanoma were taken from backcross hybrids (>BC20) of female X. maculatus (WLC# 1274, Rio Jamapa strain, carrying the spotted dorsal pigment pattern) with male X. hellerii (WLC# 1373, Rio Lancetilla strain, Db-) as the recurrent parent. Whole dorsal fin was excised from X. hellerii. This served as healthy tissue control. The dorsal fin consists of the fin rays, interspersed connective tissue with some blood vessels, and is covered by normal skin. This tissue constitutes the substratum on which the benign pigment lesions grow as two-dimensional large spots, mostly covering the entire fin. For the protein analysis, the nonpigmented parts of the dorsal fin were carefully removed and only the area with the pigment lesion was processed. The malignant melanomas also grow exophytically on the dorsal fin and only secondarily invade the skin and musculature of the trunk. Only exophytic portions of the Journal of Proteome Research • Vol. 8, No. 4, 2009 1819

research articles malignant melanomas were used here. For cell lysis and protein solubilization, 20 mg of fins was added to 300 µL of rehydration buffer (8 M urea, 2 M thiourea, 4% CHAPS, and Complete Mini (one tablet for 18 mL of buffer), and for DIGE samples, additionally 30 mM Tris-HCl, pH 8.5). Samples were homogenized using a Turrax dispersing tool (IKA, Staufen, Germany) three times for 30 s with intermediate 1 min cooling periods on ice. The protein amount of each sample was determined by Amido Black assay.7 Twenty milligrams of fin material corresponded to approximately 800 µg of protein. The reproducibility of the preparation method was checked by match of spot pattern and comparable spot intensities within the different tissue groups. DIGE, 2DE and Imaging. In all 2DE and DIGE experiments, six biological replicates per group (healthy tissue, benign lesion and malignant tumor) were used. For the DIGE experiment, a dye swapping approach was performed, meaning that each of the six biological samples were divided into two halves and either labeled with Cy3 or independently with Cy5 to reduce CyDye depending variances.8 DIGE labeling was performed according to the manufacturer using 400 pmol of fluorochrome per 50 µg of protein. An internal standard was generated by pooling equal protein amounts of each sample followed by labeling with Cy2. Half of the healthy, benign, and malignant samples were labeled with Cy3 or Cy5, respectively, following the experimental design described by Lilley et al.8 Afterward the internal standard was pooled with two different samples (malignant/healthy, healthy/ benign, or benign/malignant). Each 2DE sample was applied on an IPG strip and 2DE was performed as described in the following. All samples were passively rehydrated into 24 cm IPG strips (3-10 NL) at 20 °C for 12 h. Isoelectric focusing was performed at a total of 50 kVh (hold 150 V for 2 h, hold 300 V for 2 h, ramp to 500 V in 2 h, ramp to 1000 V in 3 h, ramp to 4000 V in 3 h, hold at 6000 V for 7 h) using an Ettan IPGphor from GE Healthcare, Munich, Germany. After IEF, IPG strips were shortly rinsed with water and incubated in equilibration buffer (6 M urea, 30% glycine, 2% SDS, and 50 mM Tris HCl, pH 8.8) supplemented with 130 mM DTT for 20 min. This step was followed by an exchange of equilibration buffer, which was now supplemented with 280 mM iodoacetamide and further incubation for 20 min. Equilibrated IPG strips were placed on top of 12.5% polyacrylamide gels that were casted using an Ettan DALT six gel gelcaster (GE Healthcare, Munich, Germany). Mark 12 (Invitrogen, Karlsruhe, Germany) was used as molecular weight marker for SDS-PAGE. The second dimension was carried out using the Ettan Dalt Six Elect, Unit 230 from GE Healthcare, Munich, Germany. First, 5 W/gel was applied for 30 min and afterward the power was raised to 17 W/gel or max. 100 W until the running front reached the end of the glass plate (approximately 4.5 h). After finishing the second dimension, the DIGE gels were scanned on a PharoxFX plus (Bio-Rad, Munich, Germany). The DIGE gels were scanned with 100 µm using excitation/emission wavelengths specific for Cy2 (488/520 nm), Cy3 (532/580 nm), and Cy5 (633/670 nm), respectively. Gels of the standard 2DE were fixed, washed and stained with RuBPS as described elsewhere.9 RuBPS stained gels were also scanned on the PharoxFX plus with 100 µm using excitation/emission wavelengths 532/605nm. The photomultiplier tube voltage was adjusted on an abundant nonregulated protein spot for all Cy dyes and gels to ensure an equal intensity for all gels close to saturation. 1820

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Lokaj et al. Quantification Analysis. Image analysis was done using the protein quantification software PDQuest Advanced 8.0.1 (BioRad, Munich, Germany) according to the manufacturer’s recommendations. Briefly, Student’s t test (95%) as part of the PDQuest software was used for statistical analysis of the data. Protein spots which show an up- or down-regulation of the expression by factor g2 compared to healthy tissue (as control) were shown in a match list. All matches were checked manually. Only spots that were found regulated by both 2DE and DIGE experiments were used for MS identification. In-Gel Digestion. Protein spots were excised by an ExQuest Spot Cutter (Bio-Rad, Munich, Germany). Sample preparation for mass spectrometry was performed according to a modified protocol by Shevchenko et al.10,11 Samples were washed twice, alternating with 50 mM ammonium hydrogencarbonate and 25 mM ammonium hydrogencarbonate buffer, 50% (v/v) acetonitrile. Gel pieces were dried in a SpeedVac (Thermo Electron, Dreieich, Germany) and rehydrated with 4 ng of trypsin in 50 mM ammoniumhydrogen carbonate buffer. Digestion was performed overnight at 37 °C. The resulting peptides were extracted by application of 15 µL of 5% formic acid for 15 min at 37 °C, which was repeated twice. Protein Identification. Proteins were analyzed by means of nano-LC-MS/MS. In detail, an Ultimate 3000 (Dionex, Idstein, Germany) coupled with an ESI-linear iontrap (LTQ XL, Thermo Electron, Dreieich, Germany) was used. The LC setup consisted of an autosampler (WPS, Dionex, Idstein, Germany) and a column compartment (FLM, Dionex, Idstein, Germany) before nano-LC separation (Ultimate 3000, Dionex). Precolumns (100 µm inner diameter × 20 mm length, Synergy Hydro-RP C18 5 µm particle size, Phenomenex, Aschaffenburg, Germany) and separation columns (75 µm inner diameter × 150 mm length, Synergy Hydro-RP C18 3 µm particle size, Phenomenex) were custom build. Samples were loaded onto the precolumn with a flow rate of 6 µL/min 0.1% TFA for 5 min. Gradient elution was performed using a linear gradient from 95% solvent A (0.1% formic acid) to 50% solvent B (84% acetonitrile, 0.1% formic acid) during a time period of 33 min. Solvent A was 0.1% formic acid in water. Separation was followed by rinsing the column with 95% solvent B for 5 min before equilibration with 5% solvent B prior to the next separation. Peptides were directly eluted into the ESI-linear iontrap (LTQ XL) using distal-coated fused silica tips (New Objectives, Woburn, MA) with spray voltage set to 1800 V. A survey scan (m/z 400-2000) was followed by five MS/MS scans fragmenting of the five most intensive peptide signals (1000 cps, 30 ms). Duplicate detection of one mass within 30 s led to dynamic exclusion for 180 s. Mass spectra obtained from LC-MS/MS analysis were used to identify the corresponding peptides by the Mascot algorithm12 (version 2.1.6). The raw data conversion was done with the LCQ-DTA.EXE as plug-in to Mascot Daemon with the following parameters: (a) minimum mass, 400; (b) maximum mass, 3000; (c) grouping tolerance, 1.4; (d) min scans/group, 1; (e) intermediate scans, 1; (f) precursor charge, auto. Searches were conducted against the Ensembl FASTA database of O. latipes (Japanese medaka) from May 25, 2007 with 25 880 sequences (ftp://ftp.ensembl.org/pub/current_fasta/oryzias_ latipes/) using the following parameters set in Mascot: (a) fixed modification, carbamidomethyl (C); (b) variable modification, oxidation (M); (c) peptide and MS/MS tolerance, (1.5 and 0.5 Da, respectively; (d) ion score cutoff, 35; (e) significance threshold, p < 0.05; (f) enzyme trypsin with miss cleavage, max

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DIGE and 2DE Study of Xiphophorus Melanoma 1; (g) instrument, ESI-QUAD_TOF. After manual validation a protein was accepted as “identified” if at least two different peptides with a score >35 were found, and the cumulative score was >100. Cell Culture. Human melanoma cell lines A375, A375M, DX3, LT5.1 were maintained in DMEM supplemented with penicillin (400 U/mL), streptomycin (50 µg/mL), L-glutamine (300 µg/mL) and 10% fetal calf serum and were kept under a humidified atmosphere of 5% CO2 at 37 °C. Western Blot. Samples were homogenized in lysis buffer (20 mM HEPES (pH 7.8), 500 mM NaCl, 5 mM MgCl2, 5 mM KCl, 0.1% deoxycholate, 0.5% Nonidet-P40, 10 µg/mL aprotinin, 10 µg/mL leupeptin, 200 µM Na3VO4, 1 mM PMSF and 100 mM NaF) using a mortar. For each sample, 50 µg of cell lysate was resolved by SDS/PAGE and transferred to nitrocellulose according to standard Western blotting protocols. Antiperoxiredoxin II antibody (H-40) and anti-β-actin antibody (C4) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and were diluted 1:500 and 1:5000, respectively. Antiperoxiredoxin VI antibody was produced in rabbits immunized with the bovine serum albumin-coupled C-terminal peptide of PRDX6, (CELPSGKKYLRYTPQP) and was a gift from S. Werner, ETH Zurich, Switzerland.

Table 1. 2DE and DIGE Analysis in Comparison and Distribution of Regulated Spots Which Were Identified within the Indicated Experiments (A) 2DE and DIGE Analysis in Comparison DIGE

max number of detected spots regulated spots (by ratio e0.5, g2, % of max detected spots) DIGE and 2DE intersection of regulated spots regulated spots exclusively found in DIGE or 2DE

2DE

1457

1117

91 (6.2%)

111 (10.0%)

80

80

11

31

(B) Distribution of Regulated Spots Which Were Identified within the Indicated Experiments

spots identified by the medaka database spots not identified by the medaka database spots containing more than one protein

intersection DIGE + 2DE

only found by DIGE

only found by 2DE

∑

57

8

23

88

19

3

7

29

4

-

1

5

Results and Discussion Analysis of Differentially Expressed Proteins. After 2D PAGE, the images of the four single experiments (see Supporting Information 2DE 1., 2DE 2., DIGE 1. and DIGE 2.) were separately analyzed for regulated spots by the PDQuest Advanced 8.0.1 software. Within each experiment, images of the healthy, benign and malignant samples were compared with each other and the regulation factors were calculated for malignant/healthy, benign/healthy and benign/malignant. Afterward, the 2DE and DIGE experiments were compared among the replicates (2DE 1. with 2DE 2., DIGE 1. with DIGE 2.) and within the different methods (2DE with DIGE). In the two DIGE experiments, a total of 1457 spots were found and 91 of them were reproducibly and significantly up- or downregulated (ratio 2). In the two 2DE experiments, in total, 1117 protein spots were detected and 111 protein spots (approximately 10%) were regulated. Comparing the regulated spots between the DIGE and the 2DE experiments, an intersection of 80 regulated spots was detected, corresponding to approximately 5% of the total resolved protein pattern (Table 1A). A certain amount of regulated spots was also found exclusively in one of the two types of experiments. The DIGE and the 2DE experiment showed 11 and 31 regulated spots, respectively, that did not overlap with the regulated spots from the other method. In total, 122 regulated spots were detected in the 2DE and the DIGE experiments by 2D PAGE analysis using the PDQuest Advanced 8.0.1 software (Table 1A). Altogether, the intersection of regulated protein spots was satisfactory. The usage of different label and detection techniques may account for the slight differences observed. Our data show that, besides obstacles that might come along with mass spectrometric analysis (e.g., by coelutions of peptides, quenching effects, automated choice of precursor ions or dynamic exclusion settings), the classical 2DE approach is a robust technique to gain a good overview of protein classes being regulated during melanoma development. Nevertheless, the 2DE technique suffers of its general limitations like limited dynamic range, inferior resolution of proteins with very acidic

or basic properties or those of extreme size or hydrophobicity that can be overcome using gel-free proteomics.13 For DIGE, a dye swapping approach was used by us to minimize technical variances of possible different labeling efficiencies of the CyDyes and the different fluorescence characteristics of acrylamide at the different excitation wavelengths for Cy2, -3 and -5.8 For the ruthenium-based fluorescence dye RuBPS, it should be noted that it is not as sensitive as CyDye. However, both staining methods have a high linearity over up to 5 orders of magnitude.6,8 Furthermore, it is most likely that both staining methods, the pre-electrophoretic CyDye labeling and the postelectrophoretic ruthenium-based dye, have different preferences to certain protein types. This may result in a slightly different protein pattern, thus, influencing the outcome of searches for regulated protein spots.14 Identification of Differentially Expressed Proteins. Out of the 122 regulated protein spots, 88 (72.2%) could be identified by nano-LC-MS/MS and Mascot searches using the FASTA database of the closely related medaka (O. latipes). However, 29 spots (23.8%) could not be identified by this means (Table 1B). The 88 identified proteins spots correspond to 63 different proteins (Table 2). As reported in several gel-based proteome studies, it was observed that different protein gel spots belong to the same protein, because of possible protein isoforms or modifications such as phosphorylation,15 deamidation16 or glycosylation.17 This phenomenon was found, for example, for glyceraldehyde 3-phosphate dehydrogenase which was present in two spots (62 and 63, Table 2) as depicted in the mastergel shown in Figure 2. Xiphophorus cell lines and brain tissue, as well as medaka brain tissue, were used before in classical 2D-PAGE analyses.18-20 Still, only a limited number of proteins could be identified by these approaches, which was due to the limited genome sequence information of the Xiphophorus genome and even the medaka genome at the time of publication. In medaka, DIGE was performed with brain tissue under normal and hypoxic conditions, before MALDI-TOF/TOF-MS and de novo Journal of Proteome Research • Vol. 8, No. 4, 2009 1821

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Lokaj et al. a

Table 2. Groups of Identified Regulated Protein Spots

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Table 2. Continued

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Lokaj et al.

Table 2. Continued

a Proteins were identified either according to annotations from the Ensembl database (http://www.ensembl.org/Oryzias_latipes/Info/Index) (see Supporting Information) or, if there was no medaka protein annotation, by homology search using D. rerio, T. rubripes or H. sapiens databases (where indicated; proteins presented without a species name were found as annotated proteins in the O. latipes database). The red color indicates a downregulation of gfactor 2. Green color represents an upregulation gfactor 2. n.d, not defined (ratios could not be calculated because spot intensities were not suitable for calculation); (, coefficient of variance; “*”, “+”, “#”, “°” or “∼” mark identical proteins within one group.

Table 3. Categorization of Proteins Up- And Downregulated in Benign Lesions versus Healthy Tissue and Malignant versus Healthy Tissuea group

amino acid metabolism channels cytoskeleton except keratin and keratin-related proteins keratin and keratin-related proteins ROS-associated signal transduction sugar metabolism others

proteins downregulated

proteins upregulated

5 8*

3 12*

3* 2 7

2* 6 1 6 10

a Asterisk (*) indicates two different spots from the same protein that behaved controversially (one spot being present in the group of downregulated proteins, the other in the group of upregulated proteins). Those belonged to β-actin 1 (cytoskeletal proteins) and keratin 5 (keratin and keratin-related proteins).

Figure 2. Differentially expressed and identified protein spots (marked with numbers) present on an inverted overlay of healthy (red) and benign (green) DIGE gel images. The contrast of each gel was transformed to 2000 high/400 low counts. Within the white square, the contrast was 3000 high/500 low counts. High, small counts make weak signals darker; low, bigger counts reduce the image background.

protein sequencing were performed.20 Yamashita and colleagues compared the protein expression of Xiphophorusderived EHS fibroblast cells maintained at 28 °C or heatshocked at 37 °C.21 Here, 2D-PAGE was not followed by protein identification, so the upregulation of heatshock proteins was only suggested based on the protein size. Oehlers et al. used Xiphophorus brain extract to establish an optimized MALDITOF method that enabled good quality but still time-consuming de novo peptide sequencing.19 In contrast to that, we could show that the utilization of the recently published medaka FASTA database was very useful for identifying large numbers of Xiphophorus proteins as homologues of closely related species. Thus, it is a time-saving and efficient alternative to de novo protein sequencing in this organism. Most of the proteins identified by us belong to seven different functional groups, namely, amino acid metabolism, channels, cytoskeleton proteins, keratin-related proteins, reactive oxygen species associated proteins, signal transduction and sugar metabolism (Table 3 and Figure 3). Within the groups, the protein gel spots could be subdivided into 54 upregulated 1824


(61.4%) and 34 downregulated protein gel spots (38.6%), which are illustrated in Figure 3. In two cases, we found that different gel spots of the same protein behaved controversially in terms of the direction of regulation (Table 3). These proteins were β-actin 1 and keratin 5. Both are prone to numerous protein modifications. It is likely that the different spots constitute different modifications and that a certain type of modification goes along with the healthy or malignant state. However, in all other cases, all spots belonging to one protein behaved largely similarly. Remarkably, the entire groups of both the channel proteins and the sugar metabolism proteins were upregulated. A drastic increase of glucose-metabolizing enzymes is a common feature of fast-dividing tumor cells.22 Because of their uncontrolled proliferation and the accompanying energy requirement, the mitochondrium-dependent ATP supply may not be efficient enough anymore, and the cells may become more and more dependent on ATP supply by glycolysis. Upregulation of certain enzymes (such as betaine aldehyde dehydrogenase, as in this case) could indicate an increasing contribution of anaerobic metabolism to energy supply.22 In contrast, all five proteins of the amino acid and homocysteine catabolism were downregulated in the tumor lesions, suggesting that the energy supply from amino acid degradation does not play a role for this melanoma. The largest group of regulated proteins were cytoskeletal proteins that encompassed 19 members corresponding to 26


Figure 3. Functional classification of 88 spots regulated between healthy tissue and benign lesions or healthy and melanoma tissue. Functional subdivision of 54 upregulated gel spots (A) and 34 downregulated gel spots (B).

protein gel spots (15 up- and 11 downregulated). For example, cytoplasmic actin 1 was upregulated and destrin was downregulated (Table 2). The strong regulation of cytoskeletal proteins can most likely be explained by the different cellular shape and the high mobility of melanoma cells compared to healthy cells. In benign lesions, seven cytoskeletal-associated proteins were upregulated, while 11 were upregulated in malignant tissue. An overrepresentation of cytoskeleton-associated proteins is a common observation during tumor development and was also observed in human epithelialovarian cancer and breast cancer.23,24 The second largest group of regulated proteins were those involved in the prevention of reactive oxygen species (ROS) accumulation. A majority of these proteins were upregulated in the malignant tumor, suggesting the effort to counteract cellular stress. Peroxiredoxin-2 and -6, and glutathione S-transferase Mu3 were the most strongly upregulated proteins. Peroxiredoxin-2 was upregulated 2.8 times in malignant tumor compared to benign lesion. Peroxiredoxin-6 and glutathione S-transferase Mu3 were found in more than one spot (Table 2). The more acidic proteins spots (in Table 2, spots 91 and 42) were only highly regulated in the benign lesions compared to healthy samples, while the regulation between benign and malignant samples was not significant. In contrast, the protein spots in the less acidic regions were upregulated in correlation to melanoma progression. For example, glutathione S-transferase Mu3 (in Table 2, spot 69) showed a regulation factor(benign/healthy) of 3 and a regulation factor(malignant/benign) of approximately 6. A group with a high content of regulated protein spots consisted of keratin and keratin-related proteins, such as keratins 4 and 5, cytokeratin 8 and keratin 8-like protein. Keratin 4 and 5 were present in more than one spot, indicating various protein modifications. In addition, keratin 5 was apparently modified in multiple ways, as it was detected in eight regulated protein spots. Here, the downregulated proteins spots were found on the more acidic pH range (pH ap-

research articles proximately 4), while the upregulated spots were shifted to the less acidic side (pH approximately 5). Still, most of the spots belonging to this group were downregulated in benign and malignant tissue, which might be due to the fact that the relative amount of keratin in healthy skin is larger compared to benign and malignant tissue where the neoplastic cells take over a large percentage of the tissue. The smallest consistent group of proteins belonged to the signal transduction proteins. Here, a G0 protein was upregulated in benign and malignant tissue compared to the healthy control. The transducin β chain and the GDP dissociation inhibitor 2, the latter being responsible for limiting G protein signaling. were downregulated in both benign lesions and malignant tumor tissue when compared to healthy tissue. The remainder of 17 other identified proteins, which did not belong to one of the functional classes mentioned above, were a mixture of predicted (hypothetical) proteins (e.g., downregulated hypothetical protein LOC436665), proteins which appeared only as single regulated protein (e.g., apolipoprotein A-I.) or proteins whose function was not clearly described in literature (e.g., Annexin A11).25 However, it was apparent that serum proteins like apolipoprotein A-I and serumtransferrin precursor were most highly expressed in the malignant tissue. This might probably reflect a higher blood vessel density in the melanoma compared to the other tissue and raises a point that requires future confirmation. Peroxiredoxin Expression in Xiphophorus Tissue and Human Melanoma. To confirm the upregulation of oxidative stress-related enzymes in an independent assay, we chose the well-conserved proteins PRDX2 and PRDX6 and performed Western blot analysis of healthy tissue, benign lesions and malignant melanoma tissue. For each tissue type, samples of two different fish were used. For both PRDX2 and PRDX6, no signal was observed in healthy tissue, while expression of the respective protein increased with malignancy from benign tissue to melanoma. Thus, the Western blot data of both proteins corresponded to the situation seen on the 2D gels (Figure 4). In our previous work, the Xiphophorus system has proven very useful in identifying proteins that play a role in human melanoma, but were not ascribed a role for melanomagenesis previously. To gain information about the status of PRDX2 and PRDX6 in human melanoma, we chose two pairs of cell lines of the same origin which are known to comprise different metastatis potential. A375 M was described to behave more metastatic than its sister cell line A375, while LT5.1 is more metastatic than DX.3.26,27 Western blot analysis showed that PRDX2 was visibly expressed in all cell lines, similar to PRDX6. However, cells with a stronger metastatic potential expressed a higher amount of PRDX6 compared to their respective counterparts (Figure 5). It is generally known that oxidative stress is enhanced in tumor tissue, including melanoma (e.g., reviewed in Toyokuni et al.).28 In comparison to normal human melanocytes, melanoma cell lines contain higher levels of superoxide.29 Enhanced ROS levels can have different cellular effects. If only slightly increased, they can act as second messengers and thereby induce and maintain the tumorigenic phenotype of cancer cells. Many proteins are regulated via their oxidation state and contain well-accessible redox-sensitive cysteine residues, for example, various kinases, phosphatases, transcription factors and cell cycle regulators.30-32 In addition, DNA is prone to oxidative lesions caused by ROS, such as 8-hydroxyguanine, which can undergo base pairing with adenine and thereby leads Journal of Proteome Research • Vol. 8, No. 4, 2009 1825

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Lokaj et al. PRDX6 activity is presumably involved in carcinogen-induced lung tumor development in mice.44 A possible link between malignancy and PRDX expression in melanoma will be investigated in the future.

Conclusion

Figure 4. Visualization of PRDX2 and PRDX6 by 2DE and Western blot. (A) Enlarged 2D gel details of healthy (H), benign (B), and malignant (M) samples. Protein spot no. 71 was identified as PRDX6, while spot no. 72 corresponded to PRDX2 (Table 2). (B) Western Blot analysis of two samples each of healthy skin, benign lesions, and malignant lesions, probed against PRDX2 and PRDX6. β-Actin was used as loading control.

Figure 5. Western Blot analysis of the cell lines A375, A375M, DX-3 and LT5.1, probed against PRDX2 and PRDX6. β-Actin was used as control.

to mutations.33 These lesions also occur in response to UVirradiation. While low ROS levels can result in slightly increased mutation levels that might enhance tumorigenesis, high ROS levels can have deleterious effects on the cell, leading either to premature senescence or even apoptosis.34,35 The reduction of UV-induced oxidative stress either by chemicals such as N-acetylcysteine or by paracrine melanocyte factors such as endothelin-1 and R-melanocortin generally reduce DNA damage and thereby prevent apoptosis.36,37 However, there are contradictory reports regarding the antioxidative capacity of melanoma cells. On the one hand, in a 2D-PAGE analysis where murine melanocytes were compared to melanoma cells derived from the same origin, the latter expressed clearly lower amounts of antioxidative proteins such as GST, thioredoxin and PRDX-2 and -6.38 Accordingly, the ROS levels were higher in these cells. The PRDX-2 promoter was described to be silenced in some human melanoma cell lines.39 On the other hand, PRDX2 can endow cancer cells with a certain resistance to H2O2 and is therefore considered to act protumorigenically.40 The murine melanocyte cell line Mel AB contains a 5-fold lower amount of glutathione and displays reduced GST activity compared to RAS-transformed and metastatic cells from the same origin, and mu-type GSTs are also expressed in an enhanced manner in human melanoma.41,42 PRDX6 expression was directly linked to metastatic potential of human breast cancer cells,43 and the chemical inhibition of 1826


Here, we show that a successful proteomic analysis by 2D PAGE and DIGE shows good results for a species whose genome sequence is not entirely available, by using LC-MS data for searching the database of a related species. The upregulation of antioxidant proteins in malignant melanoma of Xiphophorus fish suggests that the malignant tissue suffers from a pronounced level of oxidative stress that is counteracted by increased levels of PRDX2, PRDX6 and GST mu3. Fittingly, PRDX6 levels were also higher in more malignant melanoma cell lines compared to their less malignant counterparts. It is most likely that the high expression of the antioxidant proteins serves both antiapoptotic purposes and alters the intracellular signaling by affecting enzymes with redox-susceptible cysteines. The exact consequences of the observed overexpression are subject to future studies. Abbreviations: 2DE, two-dimensional electrophoresis; DIGE, difference gel electrophoresis; DTT, dithiothreitol; DMF, dimethylformamide; CHAPS, 3-[(3-Cholamiddopropyl) dimethylammonio] propanesulfonic acid; HEPES, (4-(2-hydroxyethyl)1-piperazineethanesulfonic acid); IEF, isoelectric focusing; IPG, immobilizied pH gradient; RuBPS, ruthenium II tris (bathophenanthroline sulfonate); PMSF, phenylmethanesulphonylfluoride; TFA, triflouroacetic acid; LC, liquid chromatography; MS, mass spectrometry; MW, molecular weight; pI, isoelectric point.

Acknowledgment. We thank Dr. Sabine Werner for kindly providing the anti-PRDX6 antibody. This work was supported by the Deutsche Forschungsgesellschaft, Transregio 17 (“Ras-dependent pathways in human cancer”) and FZT 82. We thank the Gottfried Wilhelm Leibniz society (WGL) for financial support Supporting Information Available: Examples of 2DE and DIGE gels; variance of regulated protein spots within the 2DE and DIGE experiments; groups of identified regulated protein spots. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Meierjohann, S.; Schartl, M.; Volff, J. N. Genetic, biochemical and evolutionary facets of Xmrk-induced melanoma formation in the fish Xiphophorus. Comp. Biochem. Physiol., Part C: Toxicol. Pharmacol. 2004, 138 (3), 281–9. (2) Meierjohann, S.; Schartl, M. From Mendelian to molecular genetics: the Xiphophorus melanoma model. Trends Genet. 2006, 22 (12), 654–61. (3) Puisieux, I.; Even, J.; Pannetier, C.; Jotereau, F.; Favrot, M.; Kourilsky, P. Oligoclonality of tumor-infiltrating lymphocytes from human melanomas. J. Immunol. 1994, 153 (6), 2807–18. (4) Marouga, R.; David, S.; Hawkins, E. The development of the DIGE system: 2D fluorescence difference gel analysis technology. Anal. Bioanal. Chem. 2005, 382 (3), 669–78. (5) Wittbrodt, J.; Shima, A.; Schartl, M. Medakasa model organism from the far East. Nat. Rev. Genet. 2002, 3 (1), 53–64. (6) Rabilloud, T.; Strub, J. M.; Luche, S.; Girardet, J. L.; van Dorsselaer, A.; Lunardi, J. Ruthenium II tris (bathophenanthroline disulfonate), a powerful fluorescent stain for detection of proteins in gel with minimal interference in subsequent mass spectrometry analysis. Proteome 2000, 1, 1–14. (7) Dieckmann-Schuppert, A.; Schnittler, H. J. A simple assay for quantification of protein in tissue sections, cell cultures, and cell

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PR800578A

Journal of Proteome Research • Vol. 8, No. 4, 2009 1827

Quantitative Differential Proteome Analysis in an Animal Model for

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