Comparative Proteome, Transcriptome, and Genome Analysis of a Gonadal and an Extragonadal Germ Cell Tumor Cell Line Stephanie Glaesener,†,# Friedemann Honecker,*,†,# Imke M. Veltman,‡ Ad J. M. Gillis,‡ Tina Rohlfing,† Thomas Streichert,§ Benjamin Otto,§ Tim H. Brummendorf,† Leendert H. J. Looijenga,‡ Carsten Bokemeyer,† and Stefan Balabanov† Department of Oncology/Hematology/Bone Marrow Transplantation with the section of Pneumology, University Hospital Hamburg Eppendorf, Germany, Department of Pathology, Erasmus MC- University Medical Center Rotterdam, Josephine Nefkens Institute, Rotterdam, The Netherlands, and Department of Clinical Chemistry, University Hospital Hamburg Eppendorf, Germany Received March 04, 2008
Whereas clinical differences between testicular and extragonadal germ cell tumors (GCT), like reduced cisplatin sensitivity of extragonadal tumors, are well-established, little is known about underlying tumor biology. A combined approach using global proteome analysis and RT-PCR to assess mRNA levels of selected proteins on the one hand, and array comparative genomic hybridization (array-CGH), on the other hand, was used to compare two germ cell tumor (GCT) cell lines showing embryonal carcinoma histology, one of testicular, and one of extragonadal origin. Overall, the two cell lines show remarkably similar protein profiles. In total, 66 proteins were found to be differentially expressed in an at least 2-fold manner. Of these, 35 proteins (53%) could be positively identified by peptide mass fingerprinting and database search. mRNA levels of 27 differentially expressed proteins were analyzed by RT-PCR. In 17/27 genes (63%), differences in mRNA expression corresponded with differences detected on protein level, suggesting that these proteins are mainly regulated through transcription. Interestingly, no close correlation was found between proteomic and genomic analysis: 13/30 genes (43%) with higher protein levels in one cell line showed higher copy numbers of the respective gene loci in array-CGH analysis. Corresponding differences from proteome, transcriptome, and mRNA analyses were found in 9 of 27 proteins (33%). Several proteins potentially involved in cisplatin resistance were identified in the extragonadal cell line, suggesting that the cisplatin-resistant phenotype of this cell line is multifactorial. Furthermore, our data demonstrate that a combined approach of proteome, transcriptome, and genome analysis is a promising tool to gain information on gene regulation in human tumors. Keywords: extragonadal germ cell tumor • comparative proteome analysis • array-CGH • cisplatin resistance • cell lines
Introduction Male germ cell tumors (GCT) are a heterogeneous group of tumors, of which the seminomatous and nonseminomatous tumors of adolescents and young adults are clinically the most prominent (for review, see ref 1). They are the most common malignancy in men aged 20-40 years and are predominantly localized in the testis. However, a small subset of 2-5% of all GCTs occurring in adults are diagnosed at an extragonadal localization (for review, see ref 2). They * To whom correspondence should be addressed. Friedemann Honecker, M.D., Ph.D., Laboratory of Experimental Oncology, Department of Oncology/ Hematology, University Hospital Hamburg Eppendorf, Martinistr. 52, 20246 Hamburg, Germany. E-mail:
[email protected]. † Department of Oncology/Hematology/Bone Marrow Transplantation with the section of Pneumology, University Hospital Hamburg Eppendorf. # Authors contributed equally to the work. ‡ Department of Pathology, Erasmus MC-University Medical Center Rotterdam. § Department of Clinical Chemistry, University Hospital Hamburg Eppendorf.
3890 Journal of Proteome Research 2008, 7, 3890–3899 Published on Web 07/22/2008
are found along the midline of the body, mainly in the retroperitoneum and the anterior superior mediastinum. It is generally believed that EGCTs follow the same principles of development as gonadal GCTs, originating from primordial germ cells.3,4 It has been hypothesized that EGCT are either the result of aberrantly migrated primordial germ cells surviving in an ectopic localization, or originate from germ cells distributed to different organs in order to provide a physiologic function.5 Alternatively, based on the genetic and cytogenetic similarities between gonadal and extragonadal tumors, some form of reverse migration of occult CIS/ ITGCNU has been suggested.6 According to this hypothesis, EGCT would also be of gonadal origin. However, besides many similarities, major differences in clinical behavior exist (Table 1). Most notably, the majority of primary mediastinal nonseminomatous GCTs exhibit an unexplained resistance to cisplatin-based chemotherapy, a biological difference that is clinically relevant.7 Recently, several studies examining the genomic constitution of GCT cell lines, including NT2 of 10.1021/pr800173g CCC: $40.75
2008 American Chemical Society
Analysis of Gonadal and Extragonadal Germ Cell Tumor Cell Line Table 1. Clinical and Biological Characteristics of Gonadal and Extragonadal Germ Cell Tumors: Similarities and Differencesa common characteristics of gonadal and extragonadal germ cell tumors
Occur in young postpubertal adults, mainly men Seminomatous and nonseminomatous histology Metastasize predominantly to the lung, liver, and bone Tumor markers AFP, β-HCG, LDH Cytogenetic abnormalities, predominantly isochromosome i(12p)
distinct characteristics of extragonadal germ cell tumors
Increased tumor bulk at presentation Predominance of nonseminomatous histology Association with Klinefelter’s syndrome Development of acute megakaryoblastic leukemia (only mediastinal nonseminomas) Higher frequency of treatment resistance (only mediastinal nonseminomas) a
Modified from ref 2.
Figure 1. Mean values of IC50 (( standard deviation) of cisplatin after 48 h of exposure to the drug (three independent experiments). The extragonadal GCT cell line NCCIT shows a significantly higher resistance to the drug than the testicular GCT cell line NT2 (p < 0.0001, Student’s t test).
gonadal and NCCIT of extragonadal origin, have been published.8,9 Interestingly, although differences between the cells lines could be detected, there were no consistent patterns allowing distinction of nonseminomas or seminomas, based on CGH data. To screen for differences that could allow a distinction between extragonadal and gonadal GCT, we used a combined approach of proteome, transcriptome, and genome analyses to compare the extragonadal cell line NCCIT with the testicular cell line NT2.
Experimental Section Cell Lines and Culture Conditions. NT2 cells, first described by Andrews et al. in 1984,10 were obtained from DSMZ (Braunschweig, Germany). NCCIT cells, first described by Teshima,11 were obtained from ATCC (Manassas, VA). NCCIT cells were cultured in DMEM F12 (1:1) medium (Gibco-BRL, Invitrogen, U.K.) containing 10% fetal calf serum (FCS) (GibcoBRL, Invitrogen, U.K.), and NT2 cells with DMEM medium supplemented with Glutamax-I (Gibco-BRL, Invitrogen, U.K.)
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containing 10% fetal calf serum (FCS) (Gibco-BRL, Invitrogen, U.K.). Cells were incubated at 37 °C in a humidified atmosphere with 5% CO2. In Vitro Cisplatin Sensitivity Assay. In vitro cytotoxicity of cisplatin was assessed using the colorimetric MTT assay. The assay was performed as previously described.12 In brief, cells were rinsed with phosphate-buffered saline (PBS), trypsinized and resuspended in 1 mL of the appropiate culture medium to count the cells in a hemacytometer chamber. In total, 4 × 103 cells/well were seeded in 96-well plates to ensure logarithmic growth. Cells were allowed to adhere overnight, and serial dilutions of the chemotherapeutic agents were added to octuplicate wells at concentrations from 0.2 to 25.6 µM. The cells were exposed to the drug for 48 h. Thereafter, the drugcontaining medium was removed and 0.2 mL of MTT solution (final concentration: 0.5 mg/mL MTT; Sigma, Germany) was added. The plates were incubated for 2 h before the medium was removed and 0.1 mL of DMSO was added. The plates were agitated for 15 min before reading optical density using a photometer (Victor2 Wallac 1420, Perkin-Elmer life sciences, Wellesley, MA) at 570 nm. All experiments were done in triplicates and were repeated at least three times. The result is expressed as drug concentration that inhibits cell growth by 50% (so-called inhibitory concentration, IC50). The difference in IC50 between the two cell lines was analyzed using the Student’s t test. Protein Preparation. For the preparation of protein extracts, culture medium was removed and adherent cells were washed once with PBS, trypsinized and pelleted by centrifugation at 1500 rpm for 5 min. Cells were lysed in a total volume of 450 µL of lysis-buffer containing 9 M urea, 4% CHAPS, 1% Pharmalyte, 1% DTT and 1% bromophenol blue at room temperature for 20 min. Subsequently, the lysate was centrifuged at 14 000 rpm for 5 min. Protein contents of the supernatant were determined by Bradford assay, using BSA as standard. Sample aliquots were stored at -80 °C until further processing. Two-Dimensional Gel Electrophoresis (2D-PAGE). 2DPAGE was performed as previously described.13 Aliquots of 450 µL of solution containing each 1.5 mg of protein extract were loaded on a linear gradient Immobiline Dry Strip (IPG Strip pH 4-7, 24 cm, Amersham Biosciences, Uppsala, Sweden). The samples were rehydrated overnight, followed by isoelectric focusing (IEF) using the Protean IEF cell (Bio-Rad, Hercules, CA). The first dimension was carried out at 10 000 V for approximately 80 kVh. After IEF, the IPG strips were equilibrated in buffer containing 6 M urea, 4% SDS and 50 mM of 1.5 M Tris-HCl plus 1% DTT for 15 min at room temperature. The IPG strips were then alkylated with 4.8% iodeacetamide in the same buffer for 15 min. The equilibrated strips were applied directly to 15% SDS-polyacrylamide gels (27 cm × 21 cm × 1.5 mm), overlaid with 0.6% agarose, and separated overnight at 120 V. The second-dimension electrophoresis was conducted in SDS-buffer containing 1.44% Glycine, 0.3% Tris-Base and 0.1% SDS at 21 °C. Gels were fixed and stained for 16 h with Coomassie Blue (0.2% Coomassie Brilliant Blue R250) and destained for 48 h with dH2O. All experiments were performed in triplicates, revealing comparable results. Image Analysis. The 2D-gels were scanned with an GS-800 Calibrated Densitometer (Bio-Rad, Hercules, CA) and subsequently digitized images were analyzed using PDQuest 7.2. software package (Bio-Rad, Hercules, CA). Following spot detection, a matchset of gels for each cell line was made. The Journal of Proteome Research • Vol. 7, No. 9, 2008 3891
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Figure 2. Representative two-dimensional (2D) gel images of proteins extracted from human NCCIT (A) and NT2 (B) cells. Samples containing 1.5 mg of total proteins were subjected to isoelectric focusing (IEF) in linear IPG strips (pH 4-7), followed by SDS-PAGE. Subsequently, the 2D-gels were stained with Coomassie blue, scanned and analyzed using PDQuest software. Differentially expressed spots are numbered and marked with arrows. Identified proteins are listed in Table 2.
individual protein spot quantity was normalized based on total density of the gel image and expressed as ppm. Quantitative analysis was performed using the Student’s t test comparing gels of the two cell lines. The confidence level was 95%. Grouping and visualization of the quantitative results were carried out by two-dimensional hierarchical clustering using the complete linkage based algorithm with Euclidian distance (R statistical packages; “stats” vers. 2.6.2, and bioconductor www.bioconductor.org, vers. 2.1). Protein Identification by Mass Spectometry. Protein spots of interest were excised from the gel using the Ettan Spot Picker, 3892
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transferred to 96-well-plates, washed with Millipore-purified water and 50% acetonitrile/water using the Ettan Digester (both Amersham Biosciences, Uppsala, Sweden). In-gel protein digestion was performed as previously described with minor modifications.14 Trypsin (10 µL/sample) (sequencing grade, Promega, Mannheim, Germany) was added to each sample for 4 h at 37 °C. Digested peptide samples were mixed with the matrix solution containing R-cyano-4-hydroxycinnamic acid, spotted for MALDI-MS measurement onto target slides, and analyzed using a matrix-assisted laser desorption/ionization time-of- flight (MALDI-TOF) mass spectrometer (Amersham
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Analysis of Gonadal and Extragonadal Germ Cell Tumor Cell Line
Table 2. Differentially Expressed Proteins of NCCIT Cells Compared to NT2 Cells after Identification with MALDI-TOFa
protein name
NT2 versus NCCIT accession no. fold spot (NCBI, theor. theor. change coverage chromosomal protein RNA chromosomal no. Swiss-Prot) MW (kDa) pl protein (%) localization expression expression representation
Cell CAG33298.1 P29373 gi|999893 P62873
Signaling/Differentiation 15.7 5.3 2.1 66.4 15.7 5.5 5.5 * 26.8 6.5 13.8 6.51 38.02 5.6 2.6 32
15q24.1 1q23.1 12p13.31 1p36.33
v v V V
V T T V
V v v V
Ion Transport 27.34 5.1 2.2
6p21.33
v
V
V
Membrane and Vesicle Trafficking 6 CAI16496.1 22.74 5.4 2.5 32
9q12-q21.2
v
v
v
Cytoskeleton and Cell Movement 7 AAB88188.1 38.95 4.7 2.9 25.1 8 NP_002264.1 53.69 5.5 2.3 29.6 9 CAI16744.1 55.54 5.3 2.6 27.3
6p21.33 12q13.13 10q24.33
v v v
v V v
V T V
36.4
1p13.2
V
v
V
CRABP1 CRABP2 Triosephosphate Isomerase (Tim), (TPI1) Guanine nucleotide-binding protein G(I)/G(S)/G(T) beta subunit 1, (GNB1)
1 2 3 4
Chloride intracellular channel 1 variant, (CLIC1)
5 BAD97099.1
Annexin A1, (ANXA1) Beta Tubulin, (TUBB) Keratin 8, (KRT8) Internexin neuronal intermediate filament protein, alpha, (INA) F-Actin capping protein alpha-1 subunit variant, (CAPZA1) Fatty-acid-binding protein, epidermal (E-FABP) (Psoriasis-associated), (FABP5) Aldehyde dehydrogenase 1 family, member B1, (ALDH1B1) Chain H, The structure of Human Mitochondrial Aldehyde Dehydrogenase In Complex With The Antidipsotropic Inhibitor Daidzin Glutathione S synthetase, (GSS) Creatine kinase, (CKB) Prosome beta-subunit; HSBpros26, (PSMB4) Tryptophanyl tRNA synthetase (WARS) ER-60 protein, (ERp60) Enoyl-CoA hydratase Dihydropyrimidinase-like 3, (DPYSL3) HNRPH1 protein, (HNRPH1) RuvB-like 2 (EC 3.6.1) (48-kDa TATA box-binding protein-interacting protein), (RUVBL2) HNRPF protein, (HNRPF) Prohibitin (PHB) Enolase Tumor rejection antigen (gp96) 1 variant, (TRA1) Heat shock protein gp96 precursor Parkinson disease (autosomal recessive, early onset) 7, (PARK7) TRAP1 Human pre-mRNA splicing factor (C1QBP) Unknown Unactive progesterone receptor, 23 kDa Serine (or cysteine) proteinase inhibitor, clade B ovalbumin), member 9, (SERPINB9) Hypothetical protein LOC51031 Purine nucleoside phosphorylase, (NP) a
10 BAD96946.1
33.06
11 Q01469
Metabolism 15.37 6.84 81.6
48
8q21.1
v
v
V
12 AAP36086.1
57.68
6.4
3.7
17.6
9p13.2
v
T
v
13 Gi|49258360
54.94
5.7
3.3
14
0
v
*
*
14 CAB93423.1 15 gi|180570 16 AAB31085.1
52.54 42.87 25.95
5.7 5.34 5.7
2.3 7.4 2.4
35.2 23 46.4
20q11.22 14q32.32 1q21.3
V V V
V V T
v T T
17 AAA61298.1
53.41
5.7
3.1
21.7
14q32.2
V
*
T
18 AAC51518.1 19 CAA66808.1 20 AAH39006.1
57.16 31.81 62.36
5.9 8.9 6
3.1 2.6 3.4
32.5 26.6 17.4
15q15.3 10q26.3 5q32
V V V
V * V
V v V
DNA-Binding and Transcriptional Regulation 21 AAH01348.1 49.5 5.9 2.9 23.8 5q35.3 22 Q9Y230 51.17 5.49 2.0 16 19q13.3
v V
v V
V v
23 gi|76780063 24 AAP36079.1 25 CAA47179.1
10q11.21 17q21.23 12p13
V V V
V V *
V V v
Protein Folding and Chaperones 26 BAD92771.1 66.16 5.1 3.1 14.8
12q23.3
V
v
T
27 AAK74072.1 28 CAB52550.1
90.35 20.04
4.8 6.3
6.9 3.4
15.1 37.6
12q23.3 1p36.23
V V
* V
* V
29 AAC24722.1
70.64
6.6
4.1
28.7
45.98 29.84 49.86
5.4
2.1
44.4
5.38 5.6 5.8
2.5 2.1 2.2
26 47.4 13.3
16p13.3
V
V
v
Function Unknown/Other 30 AAA73055.1 31.29 4.7 3.1 32
17p13.2
V
v
V
31 AAY14958.1 32 gi|23308579
18.48 18.97
6.3 4.35
8.5 2.1
27.2 32
2p25.3 12q13.3
V V
* *
* *
33 gi|4758906
43
5.61
3.3
43
6p25
V
V
V
34 gi|34850074 35 AAV68044.1
33.57 32.33
5.4 6.5
2.2 7.2
38 37.7
14q11.2
V V
* V
* V
Spot numbers are equal to the spot numbers in Figure 1.
Biosciences, Uppsala, Sweden). All measurements were performed in the positive-ion reflection mode at an accelerating
voltage of 23 kV and delayed-pulsed ion extraction. The instrument was calibrated externally. All peptide samples were Journal of Proteome Research • Vol. 7, No. 9, 2008 3893
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Figure 3. Hierarchichal clustering with Euclidean distance of 35 proteins identified by peptide mass fingerprinting and database search. The values are z-score scaled over the rows.
measured as monoisotopic masses. Database searches (NCBInr, nonredundant protein database) were performed using the MASCOT software from Matrix Science with carboxymethylation of cysteine and methionine oxidations as variable modifications (probability value p < 0.05). RNA Isolation and Reverse Transcriptase PCR. RNA was isolated from 1 × 107 NCCIT and NT2 cells using TRIzol (Invitrogen, Karlsruhe, Germany) according to the manufacturer’s protocol. cDNA was prepared by reverse transcription of 700 ng of total RNA using oligo(dT)15 primer and Superscript II reverse transcriptase (Invitrogen, Karlsruhe, Germany). cDNA was amplified by 25 cycles of PCR using REDTaq polymerase (Sigma, Germany). Twenty-five microliters of PCR mix consisted of 12.5 µL of REDTaq, cDNA µg/mL, 1 µL of primer mix (100 pmol/µL), and 14 µL of dH2O. PCR program was run at 95 °C for 4 min, 95 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min, 72 °C for 10 min, followed by 4 °C until collection of samples. Primer sequences are given in Table 3S in Supporting Information. Array-Comparative Genomic Hybridization (Array-CGH). The analyses of array-CGH of NCCIT and NT2 presented here have been published previously.9 Genomic DNA was extracted as described before.15 Briefly, each sample was treated with proteinase K followed by phenol/chloroform extraction and DNA was dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Array-CGH was performed using the 3K BAC/PAC array 3894
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containing 3600 clones, covering the full genome at on average a 1 Mb-spacing (produced at the Leiden University Medical Center), based on clone sets publicly distributed by the Welcome Trust Sanger Institute (http://www.ensembl.org). Labeling and hybridization were performed as described previously.16 Scanning was done on a ScanArray Express HT Microarray Scanner (Perkin-Elmer) and resulting images were analyzed with the GenePix Pro 5.0 software (Axon Instruments). Clone positions were based on Ensembl (v35, May 2005; www.ensembl.org). Gene positions of the differentially expressed genes were verified with Ensembl (v36, November 2005; www.ensembl.org). The baseline DNA copy number (log 2 ) 0) in the cell lines refers to triploidy.
Results Cytotoxicity of Cisplatin in NCCIT and NT2 Cells by MTT Assay. In vitro analysis of drug sensitivity to cisplatin, analyzed by colorimetric MTT assay for cytotoxicity, showed a more than 4-fold higher resistance of the extragonadal cell line NCCIT, compared to NT2 cells of testicular origin (IC50 1.7 versus 0.4 µM; p < 0.0001, Student’s t test, see Figure 1). Results were consistent in three independent experiments. 2-D Gel Electrophoresis of Protein Extracts from NCCIT versus NT2 Cells. Protein lysates of NCCIT and NT2 cells were analyzed by 2D-PAGE. On average, 2D-PAGE analysis revealed
Analysis of Gonadal and Extragonadal Germ Cell Tumor Cell Line
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Figure 4. mRNA expression, detected by RT-PCR, of differentially expressed proteins in NCCIT and NT2 cells with their corresponding spot numbers. The primer list is given in Supporting Information (Table 3S); expression of genes is normalized to the housekeeping gene RPLPO.
Figure 5. Copy number aberrations for NCCIT and NT2, determined by 3.6 K BAC/PAC array-CGH. Of note, loss of material of the Y chromosome, and gain of 12p, the cytogenetic hallmark of GCT, is present in both cell lines.
761 spots on each gel (Figure 2). Comparing the two cell lines by PDQuest software, 66 spots with an at least 2-fold difference were identified. In total, 47 spots were more abundant or uniquely found in NCCIT, whereas 19 spots were less abundant or absent when comparing NCCIT with NT2 (see Figure 2 and
Table 2). All 66 spots were subjected to mass spectrometry using MALDI-TOF-MS. Thirty-five (53%) proteins could be positively identified by peptide mass fingerprinting and database search. Hierarchichal clustering of identified proteins was performed with complete linkage based on Euclidean distance Journal of Proteome Research • Vol. 7, No. 9, 2008 3895
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Figure 6. Combined analysis of proteome, mRNA expression levels of identified proteins and array-CGH, comparing differences in NT2 versus NCCIT cells. v depicts relatively more abundance of protein, mRNA, or chromosomal material in NT2, compared with NCCIT; V depicts less abundance in NT2, compared with NCCIT.
(R statistical packages) for visualization of the separation between NCCIT and NT2 cells (Figure 3). Analysis of mRNA Expression of Differentially Expressed Proteins. For all identified proteins, mRNA expression analysis was performed by semiquantitative RT-PCR. Interpretable results were obtained for 27 genes (77%) (Figure 4). The analysis revealed that for 17 proteins (63%), differences in mRNA expression, normalized to the expression of the housekeeping gene RPLPO, corresponded with differences seen in protein expression. This suggests that these proteins are mainly regulated through transcription. Therefore, differences in gene translation and post-translational protein modifications are likely to be involved in a significant number of those genes where differences in protein and mRNA expression did not match. Array-CGH of NCCIT and NT2. Global results of array-CGH have been reported previously,9 and are shown in Figure 4. Overall, chromosomal gains and losses of both cell lines show a pattern characteristic for nonseminomatous GCTs.1 Namely, gain of chromosomal material of 7, 12p, 21, and X, and losses on chromosome 18 as hallmarks of germ cell tumor cells were detectable in both cell lines. Comparison of Protein Expression, mRNA Levels, and Array-CGH Data. To gain insights into mechanisms of regulation of protein expression, we looked for correlations between protein expression, mRNA levels, and representation of chromosomal material by array-CGH. No close correlation was found comparing results of proteome and genome analyses: in 13 of 30 proteins (43%) with stronger expression in one cell line, a relatively higher representation of chromosomal material was found in the same cell line by array-CGH analysis, whereas in 17 cases, (57%), no correlation or even the opposite pattern was found. Comparing results from array-CGH with mRNA analysis, corresponding results were seen in only 12 of 27 cases (44%). In total, corresponding differences from proteome, transcriptome, and genome levels were found in merely 9 of 27 proteins (33%) (Table 2, and Figures 6 and 7). Interestingly, in all but one of these genes (Annexin A1), uniformly lower levels were found in NT2 compared to NCCIT. However, whether these differences are due to net losses in NT2 cells or result from respective gains in NCCIT cannot be determined by our approach, which looked at relative, but not absolute, changes in copy numbers.
Discussion The biology underlying the development of extragonadal germ cell tumors (EGCT) is poorly understood. It has been 3896
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suggested that the precursor cells of these tumors show an apoptotic defect, enabling cells to survive in an aberrant localization and without the support of their physiologic gonadal microenvironment. In nontransformed mouse germ cells, aberrant migration to an extragonadal localization invariably leads to initiation of apoptosis.17 Clinical data demonstrate that especially mediastinal nonseminomas show decreased sensitivity to cisplatin-based chemotherapy.7 Interestingly, in the present study, drug sensitivity assay showed a more than 4-fold higher resistance to cisplatin of the extragonadal GCT cell line NCCIT when compared to the cell line NT2 of testicular origin, in line with a previous analysis.18 Besides this difference, however, the two cell lines share a number of important characteristics, most importantly their histology, which has been described as embryonal carcinoma. Furthermore, both cell lines show the capacitiy to differentiate upon external stimuli like exposure to all-trans retinoic acid.10 Recently, we and others have shown that proteomic analyses can be used to identify differentially expressed proteins in primary tumor material from renal clear cell carcinoma and GCTs of the seminomatous type.19,20 To our knowledge, no data on global protein expression in GCT cell lines has been reported to date. We therefore undertook a proteomic screening analysis to compare these two GCT cell lines, giving special attention to factors potentially involved in differentiation, cellular response to chemical stimuli and stress, and cell death. Despite the different localization of the primary tumors, both the global proteome and genome analyses were found to show more similarities than differences between the two cell lines. To investigate mechanisms of gene regulation, we also analyzed mRNA levels of proteins that were found to be differentially expressed by our proteomics approach. Comparing proteome and transcriptome, we found corresponding differences in 63% of the respective genes. We therefore postulate that the majority of genes in this analysis are regulated on the transcriptional level. Furthermore, we compared array-CGH patterns with the results of the proteome and the mRNA analysis. Even though the disadvantages of cell lines are well-recognized, for example, acquisition of secondary alterations in vitro, a recent analysis looking at genomic copy number and expression patterns in GCT concluded that the majority of altered regions in primary tumor material was also found in the cell lines.8 Recently, a comparison of the profiles of gene expression profiling and array-CGH of two cell lines, one of which has also been analyzed in this study (NT2), has been published.21 However, a study comparing gene copy numbers and expression in testicular germ cell tumors recently
Analysis of Gonadal and Extragonadal Germ Cell Tumor Cell Line
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failed to identify unique patterns that could distinguish between the two major groups of GCT, seminomas and nonseminomas.8 In our analysis, a uniform pattern of over- or underrepresentation was seen in only 33% of genes. In the vast majority, gene copy numbers and expression levels were higher in NCCIT than in NT2. Combining the results from genome, transcriptome, and proteome profiling demonstrates the complexity of gene regulation in cancer and underlines the necessity to look for proteins as effector molecules rather than exclusively relying on data from gene arrays or array-CGH.
Figure 7. Images of three representative Coomassie blue-stained 2D-gel protein spots showing differences between NCCIT and NT2 cells. These proteins have been described in drug resistance and in DNA repair, and could be involved in cisplatin resistance seen in the extragonadal cell line NCCIT. (A) Corresponding histograms show quantified expression levels of each spot. Gray bars in a histogram represent protein of NCCIT, and black bars in a histogram represent protein of NT2 cells. Quantitative differences in protein expression levels are shown in parts per million (ppm), as defined by the PDQuest software (B). The results of respective RT-PCR analyses are shown in comparison to the housekeeping gene RPLPO (C). Areas showing chromosomal localization of the proteins and the difference in chromosomal representation in NCCIT and NT2 are given in (D). The localization of the gene on the chromosome is marked with a dotted line.
Resistance to cisplatin-based chemotherapy is a major problem in nonseminomatous germ cell tumors found in the mediastinum.7 We therefore gave particular attention to factors potentially involved in drug resistance showing more abundant expression in NCCIT, a cell line derived from a mediastinal mixed germ cell tumor. A number of chromosomal loci have previously been reported to be involved in cisplatin resistance in primary GCTs.22 Furthermore, in GCT cell lines with acquired cisplatin resistance, region 16q22-23 was found to be consistently overrepresented.22 Of this total set of chromosomal regions, only 15q23-24 was overrepresented in NCCIT compared to NT2, whereas regions 1q31-32, 9q22, 9q32-34, and 16q22-23 were in fact overrepresented in NT2. Other regions did not show differences between the two cell lines. Therefore, it does not seem likely that differences in these loci can solely explain the higher resistance to cisplatin found in NCCIT compared to NT2. On the other hand, by our approach, a number of differentially expressed proteins involved in differentiation, cellular response to stress and chemical stimuli, as well as cell death, were identified. These factors, like beta tubulin, prohibitin, enolase, tumor necrosis factor-associated protein 1 (TRAP1), glutathione S synthetase, guanine nucleotide-binding protein G(I)/G(S)/G(T) beta subunit 1, and RuvBlike 2 protein are among potential candidates for increased cisplatin resistance in extragonadal GCT, calling for a multifactorial reason behind the drug resistent phenotype. In the following, three selected factors will be discussed in more detail. Glutathione S synthetase (GSS; spot 14 in Figure 1) catalyzes the second step of glutathione biosynthesis and influences the redox regulation and oxidative defense of cells. Glutathione is needed for the detoxification of methylglyoxal, a toxin produced as a byproduct of metabolism. Additionally, it can act as an antioxidant. GSS has been found to be more highly expressed in cancer cells showing drug resistance.23 Comparison shows a higher expression of GSS in NCCIT than in NT2 cells, suggesting more effective detoxification of cisplatin. Data from our combined proteome, transcriptome, and genome profiling suggest that gene expression is regulated via transcriptional mechanisms. The guanine nucleotide-binding protein G(I)/G(S)/G(T) beta subunit 1 (GNB1; spot 4 in Figure 1) belongs to the heterotrimeric GTP-binding proteins (also known as G-proteins) that anchor to the cytoplasmic cell membrane. These proteins are mediators for many cellular processes, for example, signal transduction and growth regulation. GNB1 has been found to be differentially expressed after cisplatin treatment in ovarian cancer cell lines.24 In the cell, cisplatin binds to the DNA with a preference for guanine,25 possibly indicating a role of this protein in cisplatin-induced apoptosis. In relation to NT2, expression level of the guanine nucleotide binding protein was found to be higher in NCCIT cells. These differences could be Journal of Proteome Research • Vol. 7, No. 9, 2008 3897
research articles due to loss of chromosomal material in NT2 cells coding for the respective gene on chromosome 1, as detected by array CGH. The RuvB-like 2 protein (RUVBL2; spot 22 in Figure 1) is a nuclear cofactor with a functional role in Myc-mediated oncogenesis.26 It is a component of the NuA4 histone acetyltransferase complex, which is involved in activation of diverse transcriptional programs, for example, apoptosis and DNA repair. Interestingly, high expression is found in mature and immature germ cells.27 This finding strongly argues for an immature germ cell as the common precursor cell of all GCTs, irrespective of their gonadal or extragonadal localization. From a systems biology point of view, this protein is a good example that loss of chromosomal material (as seen in NCCIT cells) does not necessarily lead to lower protein expression. As mRNA levels showed little difference between the two cell lines, higher protein expression in NCCIT cells could be the result of translational regulation (e.g., through microRNA) or posttranslational modifications. In summary, our investigations demonstrate that combining proteomic, mRNA expression and genomic analyses is valuable to characterize differences between testicular and extragonadal GCTs. With the use of proteomics as a screening approach, a number of candidates potentially involved in treatment resistance of EGCTs have been identified. In our view, these factors warrant further investigation in primary tumor material and in functional studies. Finally, to our knowledge, this study reports for the first time the proteome maps of two GCT cell lines, providing a baseline for future analyses like comparisons of GCT cell lines of different histology, or showing different response to cytotoxic treatment. A refined understanding of the mechanisms underlying the specific biology in different cancer subtypes will help to develop targeted treatment strategies and overcome treatment resistance in the future. Abbreviations: 2D-PAGE, 2-dimensional polyacrylamide gel electrophoresis; CGH, comparative genomic hybridization; EGCT; extragonadal germ cell tumor; GCT; germ cell tumor; GNB1; guanine nucleotide-binding protein G(I)/G(S)/G(T) beta subunit 1; GSS; glutathione S synthetase; ITGCNU; intratubular germ cell neoplasia unclassified; MTT; 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide; RPLPO; ribosomal protein, large, PO; RUVBL2; RuvB-like 2 protein.
Acknowledgment. This work was supported by Wilhelm Sander-Stiftung (T.R., F.H., and C.B.) and Werner Otto Stiftung (S.G., F.H., T.H.B., S.B., and C.B.). Supporting Information Available: Primer lists for RTPCRs performed are given in Table 3 (Table 3S). Raw data from array-CGH experiments for NCCIT and NT2 are given in Tables 4S and 5S, respectively. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Oosterhuis, J.; Looijenga, L. Testicular germ-cell tumours in a broader perspective. Nat. Rev. Cancer 2005, 5 (3), 210–22. (2) Schmoll, H. Extragonadal germ cell tumors. Ann. Oncol. 2002, 13 (Suppl. 4), 265–72. (3) Skakkebaek, N.; Berthelsen, J.; Giwercman, A.; Mu ¨ller, J. Carcinomain-situ of the testis: possible origin from gonocytes and precursor of all types of germ cell tumours except spermatocytoma. Int. J. Androl. 1987, 10 (1), 19–28. (4) Honecker, F.; Stoop, H.; de Krijger, R.; Chris Lau, Y.; Bokemeyer, C.; Looijenga, L. Pathobiological implications of the expression of markers of testicular carcinoma in situ by fetal germ cells. J. Pathol. 2004, 203 (3), 849–57.
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