Proteome analysis of a human uveal melanoma primary cell culture by 2-DE and MS

REGULAR ARTICLE

Proteome analysis of a human uveal melanoma primary

cell culture by 2-DE and MS

María Pardo1, Ángel García1, Benjamin Thomas2, Antonio Piñeiro3,Alexandre Akoulitchev2, Raymond A. Dwek1 and Nicole Zitzmann1

1 Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, UK2 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK3 Instituto Galego de Oftalmoloxía (INGO), Santiago de Compostela, Spain

We present here the first proteomics analysis of uveal melanoma (UM) cells. These cells repre-sent a good model for the identification of polypeptide markers, which could be developed asdiagnostic tools. UM is the most common primary intraocular tumour in adults. In contrast toother cancers, the survival rate of patients with this malignancy has changed little over the pastfew decades; a better understanding of the molecular biology of UM oncogenesis and metastasisis needed to build the basis for the identification of novel drug targets. In the study presentedhere, proteins from a UM primary cell culture were separated by 2-DE using a pI 3–10 gradient;270 spots were analysed by LC-MS/MS, identifying 683 proteins derived from 393 differentgenes. Of those, 69 (18%) are related to cancer processes involving cell division, proliferation,invasion, metastasis, oncogenesis, drug resistance and others. To our knowledge, 96% of theproteins identified, including 16 hypothetical proteins, have never been reported in UM before.This study represents the first step towards the establishment of a UM protein database as avaluable resource for the study of this malignancy.

Received: January 17, 2005Revised: April 5, 2005

Accepted: May 10, 2005

Keywords:

2-DE / ESI-MS/MS / Uveal melanoma

4980 Proteomics 2005, 5, 4980–4993

1 Introduction

Uveal melanoma (UM) is the most common primary intra-ocular tumour in adults. Although UM and cutaneous mela-nomas are of similar embryonic origin, they differ in theirbehaviour. Local recurrence of UM is infrequent, but thiscancer often metastasises to the liver and other extraocular

sites before it is diagnosed [1–3]. Approximately 50% ofpatients with UM are thought to die within 10 years of diag-nosis, irrespective of the type of treatment [4]. Factors report-ed to be associated with an increased risk of metastatic dis-ease from UM include clinical features such as location, sizeand configuration of the tumour, as well as histological fac-tors such as cell type, mitotic activity, microcirculationarchitecture, tumour-infiltrating lymphocytes, and the pres-ence of extrascleral extension [5]. Other markers have beensuggested, involving molecular factors such as cytogeneticchanges (loss of chromosome 3) [6], deregulation of cell cycleproteins [7, 8], loss of cell adhesion proteins [9] or over-expression of apoptosis inhibitors [10]. Unfortunately,advances in eye cancer treatment have not paralleled thosemade in the treatment of other sites of cancer [11], and thesurvival rate of patients with UM has changed little duringthe past few decades [12, 13]. Thus, a better understanding of

Correspondence: Dr. María Pardo-Pérez, Oxford GlycobiologyInstitute, Department of Biochemistry, University of Oxford,South Parks Road, Oxford, OX1 3QU, UKE-mail: [email protected]: 144-1865-275216

Abbreviations: ECM, extracellular matrix; NDSB-256, non-deter-gent sulphobetain-256; Rb, retinoblastoma; UM, uveal mela-noma

2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com

DOI 10.1002/pmic.200500030

Proteomics 2005, 5, 4980–4993 Clinical Proteomics 4981

the molecular events governing UM development is neces-sary to design better and more specific drugs able to blockand eventually cure metastatic UMs.

Studies of global protein expression by proteomics tech-nology in human tumours have yielded information abouttumour heterogeneity and have led to the identification ofvarious polypeptide markers potentially useful as diagnostictools [14]. Many changes in gene expression between benignand malignant human tumours are due to post-translationalmodifications, which cannot be detected by RNA or DNAanalysis. Patients with UM can have markedly variable clin-ical courses and treatment responses, possibly due to themolecular heterogeneity found in these tumours. For thisreason, the molecular classification of UM is likely to beinformative and clinically useful as more detailed molecularanalyses of these tumours are carried out. It has been pro-posed that to be maximally effective, cancer therapy has to beindividualized, which requires a detailed understanding ofthe properties of each tumour with regard to drug sensitivityand metastasising propensity.

Primary cell cultures are better models to study the bi-ology of UM than established cell lines, as they resemblemore closely the biology of the tumour cells in the eye. Werecently published an analysis of several UM primary cellcultures, focusing on the study of alterations in cell cycleproteins [8]. One of those tumour cell cultures was char-acterised by having a non-functional hypo-phosphorylatedretinoblastoma protein (Rb), which was unable to inhibitE2F-1 and therefore was unable to stop cells from growing.We present here the analysis of this primary cell culture by 2-DE and MS. This is the first time that proteomics is appliedto analyse human UM tumour cells. We believe that thecharacterisation of the proteome of this primary cell culturerepresents an important first step towards the utilization ofproteomics technology in the study of UM and a novelapproach to search for potential markers useful as diagnostictools for this cancer.

2 Materials and methods

2.1 Materials

All cell culture materials were purchased from Invitrogen(Carlsbad, CA, USA). IPG strips (3–10 non-linear), DTT,and electrophoresis instruments for running IPG gels (Dry-Strip kit, Multiphor II, EPS 3500 XL power supply) werefrom Amersham Pharmacia Biotech (Uppsala, Sweden).Carrier ampholytes (servalytes 3–10, 2–4, 9–11) were pur-chased from BioWhittaker Ltd (Wokingham, UK), andNDSB-256 (non-detergent sulphobetain-256) from Calbio-chem-Novabiochem (Nottingham, UK). Thiourea was pur-chased from Fluka (Steinheim, Switzerland), CHAPS, ACN,tributyl phosphine, TFA and iodoacetamide from Sigma (St.Louis, MO, USA). Urea was supplied by Fischer ScientificUK Ltd (Leicester, UK), Tris by Boehringer GmbH (Mann-

heim, Germany), and trypsin (sequencing grade) by RocheDiagnostics (Hertfordshire, UK). OGT MP17 stain, theApollo II linear fluorescence scanner and software-drivenrobotic cutter were from Oxford GlycoSciences Ltd (Abing-don, UK). In-gel digestion was carried out by a DigestProworkstation from Abimed Analysen-Technik GmbH (Lan-genfeld, Germany).

2.2 Cell culture

The human UM primary cell culture was obtained from onepatient as previously described [8]. Cells were cultured inRPMI medium containing 10% inactivated foetal calf serum(FCS), 2 mM glutamine and standard antibiotics at 377C in5% CO2.

2.3 2-DE

Harvested UM cells were washed twice in PBS and disruptedby gentle sonication in sample buffer (5 M urea, 2 M thiourea,2 mM tributylphosphine, 65 mM DTT, 65 mM CHAPS, 0.15 M

NDSB-256, 1 mM sodium vanadate, 0.1 mM sodium fluoride,1 mM benzamidine). After centrifugation the supernatantwas removed, and 600 mg protein were taken up in 375 mLsample buffer. Ampholytes were added to the sample at 0.9%servalyte 3–10, 0.45% servalyte 2–4 and 9–11. IPG strips wererehydrated in the sample and IEF was carried out for 70 kVhat 177C, according to Sanchez et al. [15]. Following focussing,the IPG strips were immediately equilibrated for 10 min in4 M urea, 2 mM thiourea, 12 mM DTT, 50 mM Tris pH 6.8, 2%SDS, 30% glycerol. The IPG strips were placed on top of thesecond dimension gels and embedded with 0.5% meltedagarose. Proteins were separated in the second dimension onSDS-PAGE gradient gels (9–16% T, 2.67% C) under runningconditions of 107C, 20 mA per gel for 1 h, followed by 40 mAper gel for 4 h using an electrophoresis tank similar to thatdescribed by Amess and Tolkovsky [16]. Following electro-phoresis, the gels were fixed in 40% ethanol:10% acetic acidand stained with the fluorescent dye OGT MP17 as a basis ofreference [17]. Monochrome fluorescence images (16 bit)were obtained at 200-mm resolution by scanning gels with anApollo II linear fluorescence scanner.

2.4 Image analysis

Scanned images were processed with a custom version ofMELANIE II (Bio-Rad, Laboratories Ltd., Hemel Hempstead,UK). Internal calibration of the 2-DE gel images with regardto pI and molecular weight was carried out on the basis of 2-DE gels separately prepared from UM primary cell culturemixed with E. coli samples, using known E. coli proteins ascalibration markers. The E. coli sample used for calibrationwas obtained from SWISS-2DSERVICE (http://us.expasy.org/ch2d/service/, courtesy of Jean-Charles Sanchez).Details of cell culture conditions can be retrieved fromhttp://www.expasy.org/ch2d/protocols/protocols.fm1.html.


4982 M. Pardo et al. Proteomics 2005, 5, 4980–4993

2.5 In-gel digestion and peptide extraction

Protein features assigned to MS analysis were excised fromthe gel by a software-driven robotic cutter. The recovered gelpieces were dried in a SpeedVac and in-gel digestion wascarried out by the automated DigestPro workstation(Abimed) according to the protocol of Shevchenko et al. [18].

2.6 MS analysis

MS analysis was carried out using a Q-TOF Micro (Micro-mass, Manchester, UK) coupled to CapLC (Waters, Milford,MA, USA). The tryptic peptides were loaded and desalted ona 300-mm id, 5-mm long C18 PepMap column (LC packings,San Francisco, CA, USA). The peptides were resolved on a75-mm id, 150-mm long C18 Atlantis NanoEase column(Waters, Milford, MA, USA). The peptide mixture was elutedwith 98% ACN containing 0.1% formic acid over 60 min at aflow rate of 250 nL/min. The gradient was as follows: 0–3 min, constant 5% ACN; 3–30 min, ACN increased to 40%;30–35 min, ACN increased to 90%; 35–40 min ACN constantat 90%; 40–45 min, ACN decreased to 5%; 45–60 min, ACNconstant at 5%. MS data were acquired and analysed byMasslynx software version 4 (Micromass) using automaticswitching between MS and MS/MS modes. The survey scan(1 s) was obtained over the mass range m/z 300–1600 in thepositive ion mode with a cone voltage of 35 V and a capillaryvoltage of 3500 V. When the signal reached a user-definedthreshold (10 counts/s), peptide precursor ions were selectedfor MS/MS (8 s total scan time) over the mass range m/z 50–2000. Fragmentation was performed using argon as the col-lision gas and with a collision energy profile (20–40 eV) opti-mised for various mass ranges of precursor ions. The select-ed precursor ions were automatically included in the exclu-sion list. The database search was performed with theMASCOT search tool (Matrix science, London, UK) screen-ing Swiss-Prot (release 45.2 of 23 Nov 2004) and TrEMBL(release 28.2 of 23 Nov 2004), and restricting the databasesearch to human taxonomy. Positive identification was onlyaccepted when the data satisfied the following criteria: (1)MS data were obtained for a full-length y-ion series of a pep-tide comprising at least eight amino acids and no missedcleavage; (2) MS data with one or two missing y-ions wereobtained for two or more different peptides comprising atleast eight amino acids and no missed cleavage.

3 Results

3.1 Proteome analysis of UM primary cell cultures by

2-DE

We have completed the first extensive investigation of the pI3–10 proteome region of a human UM primary cell culture.To do an efficient and high-resolution proteome analysis,proteins were solubilised from freshly harvested cells (pas-

sages 2–3) under reducing conditions and in the presence ofCHAPS, urea, and thiourea to minimise protein degradation(see Sect. 2.3). These experimental conditions allowed effec-tive protein separation by 2-DE applying a broad range 3–10non-linear pH-gradient for the IEF step (see Fig. 1). Thisbroad pI range, rather than zoom gels, was chosen to obtain aglobal representation of proteins on the 2-DE proteome mapwith the limited number of cells available for the study, sinceUM cells grow very slowly and are difficult to culture [8].

The pI 3–10 2-DE gels (n=4) contained 1141 6 28 (mean6 SD) protein features. On average 85% of these featureswere found to be reproducible across at least three gels as aconsequence of biological and gel-to-gel variation. The 2-DEmap represents a highly pure culture of UM cells. The purityof the culture was assessed by inspecting the cells under themicroscope and also by immunocytochemical studies thatwere performed using an antibody directed against the mel-anoma antigen gp100 (HMB-45) [8].

To obtain a fair representation of the proteins present inthe pI 3–10 UM culture proteome, 270 protein featureswere excised at random from the gels, covering a broadrange of intensities. Excised features were in-gel trypsin-digested and analysed by LC-MS/MS, resulting in the iden-tification of 683 different proteins that originated from 393different genes. Proteins identified are listed in the Supple-mentary information, which has been placed in the websupporting information for ’Proteomics’ online, with theirpI, molecular mass and descriptive functions. A short ver-sion of this table, dedicated to cancer-related proteins, isshown in Table 1.

Comparative analysis of measured molecular mass andpI versus calculated molecular mass and pI of the identifiedproteins showed a strong correlation (Fig. 2). Of the identi-fied protein features, 82% showed a difference of not morethan 6 10 kDa compared to the theoretical values calculatedon the basis of the open reading frames (ORFs) in the ge-nome (Fig. 2A). Pronounced differences between theoreticaland experimental values were rare and found predominantlyin proteins of a theoretical molecular mass of .200 kDa,which appeared in the gel as features with a significant lowermass. An example for this is the myeloid/lymphoid ormixed-lineage leukaemia protein 3 homolog, a large nuclearprotein with a theoretical molecular mass of 541,306 Dawhich appeared in the gels as truncated forms of 48,490 and47,514 Da.

The analysis of pI showed that 68% of the protein fea-tures had an experimental pI which did not deviate by morethan 6 0.5 from their theoretically calculated pI (Fig. 2B).Interestingly, several theoretically highly basic proteins werepresent in the gel as features corresponding to more acidicvariants. pI variations of this magnitude could, for example,be indicative of extensive protein phosphorylation. Examplesinclude: filamin A, 54-kDa nuclear RNA- and DNA-bindingprotein, oncogene FUS, several 40S and 60S ribosomal pro-teins, ras suppressor protein 1, and several proteins belong-ing to various histone families.



Table 1. UM primary cell culture proteome: cancer-related proteins

Protein namea) Swiss-Protentry

Mol. massgel (Da)

pI gel General functionb)

(A) Cell division and proliferation

Annexin A2 (1) P07355 25 59226 83228 51230 61130 91734 73435 86436 061

5.415.976.976.656.627.126.476.27

Calcium-regulated membrane-binding protein. It has arole in cell proliferation and its regulation is alteredin pancreatic cancer [59]

CENP-F kinetochore protein (2) P49454 79 521 5.76 Involved in chromosome segregation during mitosis.Interacts with retinoblastoma protein (Rb), CENP-Eand BUBR1. Breast carcinoma [27]

Ezrinc) (3) P15311 81 161 6.07 Probably involved in connections of major cytoskeletalstructures to the plasma membrane. UM [20]

GTP-binding nuclear protein RAN (4)P62826 23 76324 11124 111

4.886.307.02

Involved in chromatin condensation and control of cellcycle. Teratocarcinoma [50]

Heat shock protein 75-kDa,mitochondrial precursor c) (5)

Q12931 108 646 5.18 Control of cell proliferation and cellular aging. Binds toRb1. Belongs to the Hsp90 family. UM [21]

Mitotic checkpoint proteinBUB3 (6)

O43684 40 693 6.82 Required for kinetochore localization of BUB1. Over-expressed in gastric cancer [60]

Oncoprotein 18 (7) P16949 18 605 5.68 Regulation of the microtubule filament system. Over-expressed in acute leukaemia [61]

Proliferation-related proteinp80 (8)

P78559 149 390 5.92 Structural protein. Ovarian carcinoma [Chen et al.,submitted (FEB-2001) to EMBL/GenBank/DDBJ data-bases]

Stress-70 protein, mitochondrialprecursor c) (9)

P38646 76 338 5.48 Control of cell proliferation and cellular aging. Belongsto the Hsp70 family. Colon carcinoma [33]; UM [21]

Tumor protein D54 (10) O43399 28 66930 113

5.245.11

May play a role in calcium-mediated signal transduc-tion and cell proliferation. Human breast carcinoma[62]

(B) Heat shock and chaperones

60-kDa heat shock protein, mito-chondrial precursor c) (11)

P10809 61 60362 874

5.365.29

Chaperone (Hsp60 family). Colon carcinoma [63];breast carcinoma [64]; UM [21]

78-kDa glucose-regulated proteinprecursor (12)

P11021 30 195 6.35 Chaperone (Hsp70 family). Breast carcinoma [64];colon carcinoma [33]

Calreticulin precursor (13) P27797 56 77057 646

4.464.45

Chaperone promoting folding. Human hepatoma [65];colon carcinoma [33]

Heat shock protein HSP 90-betac)

(14)P08238 108 646 5.18 Chaperone (Hsp90 family). Colon carcinoma [33];

gastric cancer [66]; carcinogenic endometrium [67];UM [21]

Heat-shock protein beta-1c) (15) P04792 18 60522 39424 93325 21425 49725 78426 36726 86327 36827 834

5.686.946.605.996.525.345.156.065.545.87

Involved in stress resistance and actin organization.Belongs to the small Hsp20 family. Human renal cellcarcinoma, testis, breast and ovarian cancer [68]; UM[21]



Table 1. Continued


Mol. massgel (Da)


28 90428 95229 03430 832

5.295.295.884.44

Peptidyl-prolyl cis-trans isomeraseA (16)

P62937 16 40218 60519 217

7.506.776.89

Chaperone. Non-small cell lung cancer [69]

Protein disulfide isomerase A3precursor (17)

P30101 56 77057 24157 71631 501

5.766.315.655.75

Rearrangement of -S-S- bonds in proteins. Gastriccancer [70]; breast carcinoma [64]; colon carcinoma[33]

Protein disulfide isomeraseprecursor(18)

P07237 53 69453 97955 431

6.106.344.86

Disulfide isomerase family. Colon carcinoma [33]

(C) Invasion and metastasis

150-kDa oxygen-regulated proteinprecursor (19)

Q9Y4L1 150 208153 778

5.255.26

Cytoprotective cellular mechanisms. Up-regulated intumours, especially in breast tumours, and associat-ed with tumour invasiveness. Astrocytoma [71]

Alpha enolase (20) P06733 31 50135 86445 15747 64048 49049 435

5.756.476.346.856.996.40

Glycolytic pathway. Colon carcinoma [33]; colorectalcancer cell line Caco-2 [72]; head and neck cancerinvasion and metastasis [14]

Alpha-actinin 4 (21) O43707 114 648116 129117 043117 433

5.365.135.855.52

Actin-binding. May be associated with cancer metas-tasis due to enhanced cell motility [73]

Cathepsin B precursor (22) P07858 25 78433 40634 519

5.345.275.27

Protease involved in intracellular degradation andturnover of proteins. Implicated in tumour invasionand metastasis [74]; gastric carcinoma [75]; murinesquamous carcinoma cells [76]; colon carcinoma[77]

Cathepsin D precursor (23) P07339 26 82628 928

5.575.62

Acid protease active in intracellular protein and ex-tracellular matrix breakdown. Involved in breastcancer and hepatocellular carcinoma cell strains [78]

Cathepsin Z precursor (24) Q9UBR2 35 669 5.43 Peptidase activity. Related to aggressive phenotypes inskin melanoma [79]

Galectin-1 (25) P09382 13 48913 57314 23414 29015 10015 22016 12916 84523 670

5.125.277.018.136.825.125.135.476.21

Plays a role in cellular proliferation, differentiation, ad-hesion, neoplastic transformation, apoptosis, neo-plastic and extracellular matrix interaction [80].Binds CD45, CD3 and CD4

Galectin-3 (26) P17931 27 83427 991

5.877.85

Involved in cell-cell and cell-matrix interactions andcancer progression. Diagnostic/prognostic markerfor specific cancer types, such as thyroid and pros-tate [81]



Table 1. Continued


Mol. massgel (Da)


Keratin, type I cytoskeletal 16 (27) P08779 39 03341 153

5.925.89

KRT16 and KRT17 are coexpressed only in pathologicalsituations such as metaplasias and carcinomas ofthe uterine cervix. Breast cancer [57]

Keratin, type I cytoskeletal 17 (28) Q04695 41 15342 447

5.896.75

KRT16 and KRT17 are coexpressed only in pathologicalsituations such as metaplasias and carcinomas ofthe uterine cervix. Breast cancer [57]

Keratin, type I cytoskeletal 18c) (29) P05783 42 13442 48143 53844 621

5.146.055.396.61

Interconverted phenotype, related to an increased abil-ity to invade a basement membrane matrix in vitro.Breast cancer [36]; UM [19]; skin melanoma [82]

Keratin, type II cytoskeletal 7 (30) P08729 54 742 5.48 Differential diagnosis of tumours [83]. Breast cancer[57]

Keratin, type II cytoskeletal 8c) (31) P05787 42 13449 19353 26854 30154 742

5.145.426.035.595.48

Interconverted phenotype, related to an increased abil-ity to invade a basement membrane matrix in vitro.Breast cancer [36]; UM [19]; skin melanoma [82]

LIM and SH3 domain protein 1 (32) Q14847 34 83135 02435 18535 51335 86436 061

6.806.246.866.316.476.27

Focal adhesion protein necessaryfor cell migration andsurvival in response to growth factors and extracellular matrix proteins. Breast carcinoma [84]

Melanoma-associated antigenMUC18 (33)

P43121 36 816 6.06 Allows interaction with cellular elements of the vas-cular system enhancing haematogenous tumourspread in skin melanoma [39]. Increases metastasisof human prostate cancer cells [85]

Metastasis inhibition factor nm23c)

(34)P15531 19 385

19 8424.705.87

Synthesis of nucleoside triphosphates. Reducedamount in tumour cells of high metastatic potential.UM [86]

Mitogen-inducible 2 interacting pro-tein (35)

Q8WUP2 46 725 5.58 Anchoring site for cell-ECM adhesion proteins and fi-lamin-containing actin filaments. Implicated in cellshape modulation (spreading) and motility. Cervicaladenocarcinoma [87]

S100 calcium-binding protein A11(Calgizzarin)c) (36)

P31949 13 10413 524

5.155.98

UM [22]; breast carcinoma [84]

S100 calcium-binding protein A6(Calcyclin)c) (37)

P06703 10 47710 542

5.195.12

Inducible by growth factors and overexpressed inacute myeloid leukaemias. Human colorectal ade-nocarcinoma tumorigenesis and invasion/metasta-sis [88]; colon carcinoma progression [89]. UM [22]

Tropomyosin 1 alpha chain (38) P09493 32 14134 51934 708

4.705.274.66

Actin-binding protein. Breast cancer [57]

Tropomyosin alpha 4 chain (39) P67936 30 15631 287

4.624.64

Actin-binding protein. Ovarian carcinoma [90]

Tropomyosin beta chain (40) P07951 30 15631 28731 54434 51934 70835 909

4.624.645.035.274.664.61

Actin-binding protein. Breast cancer [57]



Table 1. Continued


Mol. massgel (Da)


Vimentinc) (41) P08670 32 15342 13443 53844 01644 62146 61450 69454 30155 431

6.035.145.395.286.614.995.075.595.34

Interconverted phenotype, related to an increased abil-ity to invade a basement membrane matrix in vitro.Breast cancer [36]; UM [19]; skin melanoma [82];osteosarcoma [91]

(D) Oncogenes

DJ-1 protein (42) Q99497 24 496 5.86 May be a novel mitogen-dependent oncogene productinvolved in a Ras-related signal transduction path-way [42]. Prostate cancer [92]

Oncogene EMS1 (43) Q14247 83 686 5.27 Tyrosine phosphorylation in transformed cells maycontribute to cellular growth regulation and trans-formation. Hepatocellular carcinoma [93]; humanprimary oesophageal squamous cell carcinoma [94]

Oncogene FUS (44) P35637 62 874 5.98 May play a role in maintenance of genomic integrity[95]

Ras-related protein R-Ras2 (Te-ratocarcinoma oncogene) (45)

P62070 24 30324 691

5.535.98

Aberrant R-Ras function may stimulate malignanttransformation, via up-regulation of part of the Rassignal transduction pathway [50, 96, 97]

(E) Drug resistance

14–3-3 protein gamma (46) P61981 28 747 4.77 Cell signalling. Interacts with RAF1. Human lung can-cer tissues [98]. Related to chemoresistance in skinmelanoma cell lines [99]

54-kDa nuclear RNA- and DNA-binding protein (47)

Q15233 55 431 5.34 mRNA processing. Present in breast tumour cell linesand prostate cancer [100]

Annexin A1 (48) P04083 31 50132 15335 66936 15936 612

5.756.035.436.566.55

Modulates drug resistance in tumour cells [101].Upregulated in hairy cell leukaemia [102]; human pi-tuitary adenomas and a growth hormone-secretingcarcinoma [103]

Elongation factor 1-gamma (49) P26641 45 954 6.40 Implicated in translation. Pancreatic carcinoma [104];hepatocellular carcinoma [105]; oesophageal carci-noma [106]; gastric carcinoma [107]

Major vault protein(lung resistance protein)c) (50)

Q14764 124 622 5.43 May be involved in nucleo-cytoplasmic transport.Overexpressed in many multidrug-resistant cancercells and primary tumour samples [108, 109]; gan-gliogliomas [110]; glioblastoma cell lines andtumours [111]; non-small cell lung cancer [112];UM [23]

Multidrug resistance-associatedprotein MGr1-Ag (51)

P08865 34 708 4.66 Belongs to the S2P family of ribosomal proteins. Coloncarcinoma [113]

Tumor rejection antigen 1 (grp94)c)

(52)P14625 106 864

110 9166.365.01

Chaperone (Hsp90 family). Related to drug resistancein tumour cells. May serve as vaccine in human can-cer. Breast cancer [114]; UM [21]



Table 1. Continued


Mol. massgel (Da)


(F) Others

AF15q14 isoform 2 (53) Q8WXA6 25 592 5.41 Cancer/testis-associated gene [115]; acute myeloidleukaemia [116]

Arginine-rich, mutated in earlystage tumours (54)

Q86U67 16 841 8.35 Unknown

Colonic and hepatic tumour over-expressed protein (55)

Q14008 53 979 6.34 Stabilizes microtubules. Over-expressed in hepatomasand colonic tumours [117]

Elongation factor 1-delta (56) P29692 36 816 5.02 Implicated in translation. Hepatocellular carcinoma[105]; colorectal carcinoma [118]

Glutathione S-transferase P (57) P09211 24 30324 691

5.536.70

Conjugation of reduced glutathione to a wide numberof exogenous and endogenous hydrophobic elec-trophiles [119]. Colon carcinoma [33]; prostate tu-mours [120]

Glyceraldehyde 3-phosphate dehy-drogenase, liver (58)

P04406 32 12833 31533 41933 59834 73435 35335 453

7.787.005.417.047.127.946.13

Increased glycolysis in cancer cells reveals an impor-tant role of energy creating reaction in cancer cellgrowth. Lung cancer [121]

Golgi-associated plant pathogene-sis-related protein 1 (59)

Q9H4G4 13 080 8.51 Interacts with CAV1. Belongs to the CRISP family. Braintumours [122]

Myeloid/lymphoid or mixed-lineageleukaemia protein 3 homolog (60)

Q8NEZ4 47 51448 490

6.996.99

Belongs to a coactivator complex of nuclear receptors,involved in transcriptional coactivation

Nuclear protein Hcc-1 (61) P82979 32 153 6.03 May participate in important transcriptional or trans-lational control of cell growth, metabolism and car-cinogenesis. [123]. Increased in hepatocellular carci-noma and pancreatic adenocarcinoma [124]

Nucleophosmin (62) P06748 36 81643 476

6.065.93

Ribosome assembly/transport. Overexpression in can-cer cells may inactivate p53 and tumour progression[125]. Colon carcinoma [33]; skin melanoma [58]

Peroxiredoxin 2 (63) P32119 23 08223 670

5.505.27

Redox regulation of the cell. Colon carcinoma [33]

Protein C7orf24 (64) O75223 22 053 5.04 Chromosomal region 7p12, which contains GBAS, isamplified in approximately 40% of glioblastomas

RuvB-like 1 (65) Q9Y265 51 98652 314

6.086.59

May have a role in nuclear processes such as recombi-nation and transcription. Essential cofactor in the c-Myc oncogenic transformation [56]

Superoxide dismutase [Mn],mitochondrial precursor (66)

P04179 22 394 6.94 Iron/manganese superoxide dismutase family. Breastcarcinoma [64]

Transformation up-regulatednuclear protein (67)

P61978 61 60362 87466 770

5.365.295.39

Pre-mRNA processing; it is a c-myc transcription fac-tor. Breast cancer [57]

Translationally controlled tumourprotein (68)

P13693 23 42023 763

5.724.88

Found in several healthy and tumour cells includingerythroleukaemia cells, gliomas, skin melanomas,hepatoblastomas, and lymphomas. Chemoresis-tance malignant skin melanoma [126]; breast carci-noma [64]



Table 1. Continued


Mol. massgel (Da)


Triosephosphate isomerase (69) P60174 24 30324 69125 28425 58626 46526 863

5.536.705.226.956.896.06

Role in several metabolic pathways. Breast carcinoma[64]; colon carcinoma [33]; lung adenocarcinoma[127]

a) Number in brackets indicates location in Fig. 1.b) Function either known or predicted by homology using Swiss-Prot and NCBI.c) Protein identified previously in human UM.

Figure 1. Image of the pI 3–10 proteome coverage 2-DE map of the human UM primary cell culture analysed.Numbers indicate the location of cancer-related proteins from Tab. 1.

3.2 Functional classification of the UM primary

culture proteome

Successfully identified proteins were grouped according totheir functional classification into the following categories:cytoskeletal, membrane proteins, metabolic enzymes, mito-chondrial, nuclear, protein processing, signalling, vesicular,unknown and miscellaneous (see Supplementary Table).

Relevant information on protein function was retrieved fromthe Swiss-Prot and GenBank databases and relevant refer-ences therein. The distribution of proteins within thesegroups is presented for the pI 3–10 region in Fig. 3A. Thelargest class represented is the one of proteins involved inprotein processing (22%). This group includes proteinsdedicated to protein degradation, such as components of theproteasome and the ubiquitin pathways, and to protein syn-



Figure 2. Graphs showing the correlation between theoreticaland experimental molecular mass and pI values for the humanUM primary cell culture analysed. (A) Molecular mass; (B) pI.

thesis. The second most abundant group was found to com-prise cytoskeletal proteins (19%) and includes proteinsinvolved in the regulation of actin polymerisation and majorcomponents of microtubules, e.g. tubulins. Other maingroups (11% each) include nuclear (e.g. histones, hnRNPs)and signalling proteins (e.g. 14–3-3, R-Ras2).

Of the identified proteins, 26 were classified in the groupof proteins of unknown function, which also includes 16 hy-pothetical proteins. The existence of these hypothetical pro-teins had previously been predicted on the basis of ORFsidentified in the genomic sequences, and their presence wasconfirmed here by detailed analysis of the MS/MS spectra. Ofthe analysed proteins present in the pI 3–10 region, 96%have never been reported before in UM (see SupplementaryTable).

3.3 Potential novel markers in UM

Sixty-nine proteins (18%) identified in this study have beenpreviously described as cancer related. We classified theminto the following groups: cell division and proliferation,heat shock and chaperones, invasion and metastasis, onco-genes, drug resistance and others (Tab. 1; Fig. 3B). Some ofthese proteins have already been suggested as markers forUM: cytokeratins 8 and 18 (epithelial keratins present incarcinomas) together with vimentin (a mesenchymal marker

Figure 3. Pie chart representing categories of protein functionfound in the UM primary cell culture, following 2-DE separation.(A) General proteome; (B) Cancer-related proteins.

typical of melanomas), the combined presence of whichconstitutes the so-called interconverted phenotype [19]; theprotein ezrin, associated with cell migration and recognition[20]; the chaperones heat-shock protein beta-1, Hsp70 andHsp90 [21]; the calcium binding proteins S100A11 andS100A6 [22]; and the drug resistance-related proteins majorvault protein [23], and tumour-rejecting antigen 1 [21]. How-ever, most of the proteins we found have not been describedin UM before (Tab. 1) and may be useful for the developmentof diagnostic markers. This group includes proteins relatedto cell proliferation, e.g. the CENP-F kinetochore protein,oncoprotein 18 (Op18) and tumour protein D54; proteinsthat participate in cell-cell, cell-extra cellular matrix (ECM)interaction and ECM breakdown such as melanoma-asso-ciated antigen MUC18, cathepsins B, D and Z, galectins 1and 3, LIM and SH3 protein 1, and mitogen-inducible 2interacting protein; the oncogenes DJ-1 protein, oncogeneEMS1, oncogene FUS, and Ras-related protein R-Ras2; drug-resistance proteins like 14–3-3 protein gamma, elongationfactor 1-gamma, and multidrug resistance-associated proteinMGr1-Ag; and other cancer-related proteins such as colonicand hepatic tumour over-expressed protein, myeloid/lym-phoid or mixed lineage leukaemia protein 3 homolog,



nuclear protein Hcc-1, nucleophosmin, transformation up-regulated nuclear protein, and translationally controlledtumour protein (Fig. 1).

4 Discussion

4.1 The proteome of UM primary cell cultures

analysed by 2-DE

We report here the first proteome analysis of human UMcells. Bearing in mind that there may be differences betweenpatients, this study will form the basis of a UM databasewhich will be extended by similar proteome analyses of nor-mal uveal tissues and other UM primary cell cultures. A UMdatabase may be used to analyse differential protein expres-sion in different UM patients to achieve a better treatmentand survival prediction. Primary cell cultures are a bettermodel to study the biology of UM than cell lines as they moreclosely resemble the tumour cells in the eye. In a recentlypublished analysis of several UM primary cell cultures, wefocused on the study of alterations in cell cycle proteins [8].One of those continuously dividing tumour cell cultures fea-tured a non-functional hypophosphorylated Rb. An initialproteomic analysis was carried out to identify proteins boundto the non-functional Rb in those tumour cells that mightexplain the lack of functionality of Rb [8]. This study wasextended here into an extensive characterization of the pro-teome of those UM cells by a combination of 2-DE for proteinseparation and LC-MS/MS for protein analysis. The proteinidentification data reported here are based on peptidesequence tags identified by MS/MS, which provide higheridentification accuracy than MALDI-based PMF strategies[24]. The protein identifications and gel annotations can befound on our web-based database (http://www.bioch.ox.-ac.uk/glycob/ogp).

2-D gel analysis showed that only a few proteins exhib-ited large differences between experimental and theoreticalmolecular weights. Observed differences were primarily dueto the presence of truncated forms of the protein. In thesecases, the full-length proteins were large proteins(.200 kDa) that are generally poorly resolved on 2-D gels.Some of these proteins, especially cytoskeletal ones, havebeen reported to undergo in vivo fragmentation in other celltypes [25, 26]. The experimental and predicted pI of the pro-teins identified correlated well for the majority of the pro-teome, with deviations of greater than 6 0.5 in only 30% ofcases. These cases represent proteins that are extensivelypost-translationally modified with charged or charge mod-ifying groups (e.g. phosphorylation, sulphation).

4.2 Functional groups and cancer-related proteins

The proteome analysis of the UM primary cell cultureallowed us to identify many interesting proteins that mightpredict the biological behaviour of UM. An important per-

centage of the proteins identified are cancer related and havebeen classified according to their role in tumour progression(Tab. 1). Cancer-related proteins comprise proteins involvedin cell division, such as CENP-F kinetochore protein, a cellcycle-related nuclear protein with maximum expression inthe G2 and M phases of the cell cycle, which has previouslybeen shown to be associated with malignancy [27–29], andOp18, which is up-regulated in acute leukaemia and signifi-cantly correlates to a high percentage of cells in the S phaseof the cell cycle in response to external signals [30].

The expression of certain groups of heat shock proteinshas been described in the literature as a prognostic factor inmany tumours [31]. Here we identified several heat shockproteins and chaperones, three already described in UM(heat-shock protein beta-1, Hsp70, and Hsp90 [21]) as well asproteins like Hsp60, calreticulin precursor [32, 33] and pro-tein disulphide isomerases [33, 34] which have not beenreported before and might be implicated in this malignancy.

UM has a great propensity to metastasise hematogen-ously and to develop distant metastasis, in particular in theliver. It has been hypothesised that micrometastases mayalready be present at the time of primary tumour detection.Therefore, much effort is now being directed at finding anearly marker in the primary tumour that could indicate itsmetastatic potential. Here we present potential marker can-didates. Among those related to enhanced cell mobility are a-actinin 4, several keratins (K8, K18), ezrin, different trompo-myosins and vimentin. The presence of the interconvertedphenotype (vimentin and cytokeratins 8 and 18) correlateswith an increased ability to invade a basement membranematrix in vitro in breast carcinoma as well as in cutaneousmelanoma and UM [19, 35, 36]. In the group related to inva-sion and metastasis, we included proteins related to cell-celland cell-matrix interactions and different proteases thatmight play an important role in invasion by degrading theextracellular matrix. Noteworthy is the presence of the mela-noma-associated antigen MUC18 (MCAM), described herefor the first time in UM. This adhesion protein is expressedmost strongly in metastatic lesions and advanced primarytumours, and is only rarely detected in benign lesions [37,38]. The ability of this protein to interact with cellular ele-ments of the vascular system suggested its role in promotingthe haematogenous tumour spread [39] and indeed MUC18is regarded as potential target for immunotherapy in skinmelanoma [40]. A more detailed analysis about the possibleimplication of MUC18 in UM tumour growth and metastasisis currently under investigation.

We also report for the first time four different oncogenesfound in UM that might be useful for the early diagnosis ofthis cancer: DJ-1 protein, oncogene EMS1, oncogene FUSand Ras-related protein R-Ras2. Le Naour and collaborators[41] found a high level of DJ-1 protein in the serum of breastcancer patients, which suggested that this protein can trans-locate from the intracellular to the extracellular environmentduring tumour development. This protein is also able totransform mouse NIH3T3 cells in cooperation with ras [42].



The oncogene EMS1, or cortactin, is an actin-binding proteininvolved in the restructuring of the cortical actin cytoskele-ton. It is redistributed into cell-matrix contact sites in humancarcinoma cells by overexpression and post-translationalmodification, implying a role in the modulation of cellularadhesive properties [43, 44]. The oncogene FUS is involved ina form of malignant myxoid liposarcoma and acute myeloidleukaemia through chromosomal translocation [45–47]. Sev-eral authors reported that there are no mutations in NRAS inUM [48, 49], but no information has been published to dateabout R-Ras. R-Ras2 or TC21 was found present in humanteratocarcinoma [50] and a mutation (Leu to Gln) was iden-tified in an ovarian tumour [51]. It seems that this proteinmediates transformation and cell survival via activation ofthe phosphatidylinositol 3-kinase/Akt and NF-kB signallingpathway [52]. The exact role of these oncogenes in UM ispresently under investigation.

Metastatic UM is known to be profoundly chemoresis-tant and consequently to have a very poor prognosis for sur-vival. The expression of P-glycoprotein, tumour rejectionantigen 1 (gp94) and major vault protein, all of which parti-cipate in drug resistance and tumour invasiveness have beenreported before in UM [21, 23, 53]. Here we show not onlythe presence of major vault protein and gp94 but also otherdrug-resistance related proteins described for the first timein UM, such as 14–3-3 protein gamma, 54-kDa nuclear RNA-and DNA- binding protein, annexin A1, elongation factor 1-gamma and multidrug resistance-associated protein MGr1-Ag.

Other interesting proteins presented in this work havebeen associated with different cancers, but their role inoncogenesis is still not well known. Among these are a cou-ple of proteins related to the c-myc oncogene, which is fre-quently overexpressed in UM [54, 55]: RuvB-like protein,which is an essential co-factor in the c-myc oncogenic trans-formation [56], and transformation up-regulated nuclearprotein, which is a transcription factor for c-myc expression[57]. Both proteins might be playing a role in c-myc activationin UM. The presence of nucleophosmin protein is alsonoteworthy since it strongly correlates with cutaneous mela-noma progression [58]. Our findings might support theimplication of nucleophosmin in ocular melanoma.

In summary, we present here the first proteome analysisof a UM primary cell culture. A significant proportion of theproteins reported have not been described in UM before.These include 16 hypothetical proteins and a number ofcancer-related proteins (see Table 1 and SupplementaryTable). This information will lead to a better understandingof the biological mechanisms that rule the transformationand dissemination of uveal melanocytes and may suggestpotential targets for drug development.

The authors thank Prof. Carmela Capeans-Tomé and Prof.Manuel Sánchez-Salorio for their continuous support. This workwas funded by the Oxford Glycobiology Institute endowment.

5 References

[1] Eskelin, S., Pyrhonen, S., Summanen, P., Hahka-Kemppinen,M., Kivela, T., Ophthalmology 2000, 10, 1443–1449.

[2] Margo, C. E., Cancer Control 2004, 11, 304–309.

[3] Diener-West, M., Earle, J. D., Fine, S. L., Hawkins, B. S. et al.,Arch. Ophthalmol. 2001, 119, 969–982.

[4] Bergman, L., Seregard, S., Nilsson, B., Lundell, G., Invest.Ophthalmol. Vis. Sci. 2003, 44, 3282–3287.

[5] Singh, A. D., Shields, C. L., Shields, J. A., Melanoma Res.2001, 11, 255–263.

[6] Horsthemke, B., Prescher, G., Bornfeld, N., Becher, R., GenesChromosomes Cancer 1992, 4, 217–221.

[7] Mouriaux, F., Maurage, C. A., Labalette, P., Sablonniere, B.,Invest. Ophthalmol. Vis. Sci. 2000, 41, 2837–2843.

[8] Pardo, M., Piñeiro, A., de la Fuente, M., Garcia, A. et al., J.Cell. Biochem. 2004, 93, 708–720.

[9] Anastassiou, G., Schilling, H., Stang, A., Djakovic, S., Oncol-ogy 2000, 58, 83–88.

[10] Loercher, A. E., Harbour, J. W., Curr. Eye Res. 2003, 27, 69–74.

[11] Jemal, A., Thomas, A., Murray, T., Thun, M., CA Cancer J.Clin. 2002, 52, 23–47.

[12] Damato, B., Eye 2001, 15, 155–158.

[13] Diener-West, M., Hawkins, B. S., Markowitz, J. A., Schachat,A. P., Arch. Ophthalmol. 1992, 110, 245–250.

[14] Wu, W., Tang, X., Hu, W., Lotan, R. et al., Clin. Exp. Metas-tasis 2002, 19, 319–326.

[15] Sanchez, J. C., Rouge, V., Pisteur, M., Ravier, F. et al., Elec-trophoresis 1997, 18, 324–327.

[16] Amess, B., Tolkovsky, A. M., Electrophoresis 1995, 16, 1255–1267.

[17] Hassner, A., Birnbaum, D., Loew, L. M., J. Org. Chem. 1984,49, 2546–2551.

[18] Shevchenko, A., Jensen, O. N., Podtelejnikow, A. V.,Sagliocco, F. et al., Proc. Natl. Acad. Sci. USA 1996, 93,14440–14445.

[19] Hendrix, M. J., Seftorm, E. A., Seftor, R. E., Gardner, L. M. etal., Lab. Invest. 1998, 78, 53–63.

[20] Makitie, T., Carpen, O., Vaheri, A., Kivela, T., Invest. Oph-thalmol. Vis. Sci. 2001, 42, 2442–2449.

[21] Missotten, G. S., Journee-de Korver, J. G., de Wolff-Rouen-daal, D., Keunen, J. E., Invest. Ophthalmol. Vis. Sci. 2003, 44,3059–3065.

[22] Van Ginkel, P. R., Gee, R. L., Walker, T. M., Hu, D. N., Biochim.Biophys. Acta 1998, 1448, 290–297.

[23] van der Pol, J. P., Blom, D. J., Flens, M. J., Luyten, G. P.,Invest. Ophthalmol. Vis. Sci. 1997, 38, 2523–2530.

[24] Mann, M., Hendrickson, R. C., Pandey, A., Annu. Rev. Bio-chem. 2001, 70, 437–473.

[25] Lee, A., Morrow, J.S., Fowler, V.M., J. Biol. Chem. 2001, 276,20735–20742.

[26] Umeda, T., Kouchi, Z., Kawahara, H., Tomioka, S. et al., J.Biochem. (Tokyo) 2001, 130, 535–542.

[27] Liao, H., Winkfein, R. J., Mack, G., Rattner, J. B., Yen, T. J., J.Cell Biol. 1995, 130, 507–518.

[28] Landberg, G., Erlanson, M., Roos, G., Tan, E. M., Casiano, C.A., Cytometry 1996, 25, 90–98.



[29] de la Guardia, C., Casiano, C. A., Trinidad-Pinedo, J., Baez,A., Head Neck 2001, 23, 104–112.

[30] Melhem, R. F., Strahler, J. R., Hailat, N., Zhu, X. X., Hanash,S. M., Biochem. Biophys. Res. Commun. 1991, 179, 1649–1655.

[31] Sarto, C., Binz, P. A., Mocarelli, P., Electrophoresis 2000, 21,1218–1226.

[32] Yu, L. R., Zeng, R., Shao, X. X., Wang, N., Electrophoresis2000, 21, 3058–3068.

[33] Ji, H., Reid, G. E., Moritz, R. L., Eddes, J. S., Electrophoresis1997, 18, 605–613.

[34] Rasmussen, R. K., Ji, H., Eddes, J. S., Moritz, R. L., Electro-phoresis 1997, 18, 588–598.

[35] Hendrix, M. J., Seftor, E. A., Chu, Y. W., Trevor, K. T., Seftor, R.E., Cancer Metastasis Rev. 1996, 15, 507–525.

[36] Hendrix, M. J., Seftor, E. A., Seftor, R. E, Trevor, K. T., Am. J.Pathol. 1997, 150, 483–495.

[37] Lehmann, J. M., Holzmann, B., Breitbart, E. W., Schmiege-low, P., Cancer Res. 1987, 47, 841–845.

[38] Wu, G-J., Wu, M-W. H, Wang, S-W., Liu, Z. et al., Gene 2001,279, 17–31.

[39] Lehmann, J. M., Riethmuller, G., Johnson, J. P., Proc. Natl.Acad. Sci. USA 1989, 86, 9891–9895.

[40] Mills, L., Tellez, C., Huang, S., Baker, C., Cancer Res. 2002, 62,5106–5114.

[41] Le Naour, F., Misek, D. E., Krause, M. C., Deneux, L., Clin.Cancer Res. 2001, 7, 3328–3335.

[42] Nagakubo, D., Taira, T., Kitaura, H., Ikeda, M., Biochem. Bio-phys. Res. Commun. 1997, 231, 509–513.

[43] van Damme, H., Brok, H., Schuuring-Scholtes, E., Schuur-ing, E., J. Biol. Chem. 1997, 272, 7374–7380.

[44] Schuuring, E., Verhoeven, E., Mooi, W. J., Michalides, R. J.,Oncogene 1992, 7, 355–361.

[45] Crozat, A., Aman, P., Mandahl, N., Ron, D., Nature 1993, 363,640–644.

[46] Bertrand, P., Akhmedov, A. T., Delacote, F., Durrbach, A.,Lopez, B. S., Oncogene 1999, 18, 4515–4521.

[47] Ichikawa, H., Shimizu, K., Hayashi, Y., Ohki, M., Cancer Res.1994, 54, 2865–2868.

[48] Mooy, C. M., Van der Helm, M. J., Van der Kwast, T. H., DeJong, P. T. et al., Br. J. Cancer 1991, 64, 411–413.

[49] Cruz, F. 3rd., Rubin, B. P., Wilson, D., Town, A. et al., CancerRes. 2003, 63, 5761–5766.

[50] Drivas, G. T., Shih, A., Coutavas, E., Rush, M. G., D’Eusta-chio, P., Mol. Cell. Biol. 1990, 10, 1793–1798.

[51] Chan, A. M., Miki, T., Meyers, K. A., Aaronson, S. A., Proc.Natl. Acad. Sci. USA 1994, 91, 7558–7562.

[52] Rong, R., He, Q., Liu, Y., Sheikh, M. S., Huang, Y., Oncogene2002, 21, 1062–1070.

[53] Dunne, B. M., McNamara, M., Clynes, M., Shering, S. G. etal., Hum. Pathol. 1998, 29, 594–598.

[54] Royds, J. A., Sharrard, R, M., Parsons, M. A., Lawry, J. et al.,Graefes Arch. Clin. Exp. Ophthalmol. 1992, 230, 366–371.

[55] Tulley, P. N., Neale, M., Jackson, D., Chana, J. S. et al., Br. J.Ophthalmol. 2004, 88, 1563–1567.

[56] Wood, M. A., McMahon, S. B., Cole, M. D., Mol. Cell. 2000, 5,321–330.

[57] Harris, R. A., Yang, A., Stein, R. C., Lucy, K. et al., Proteomics2002, 2, 212–223.

[58] Bernard, K., Litman, E., Fitzpatrick, J. L., Shellman, Y. G. etal., Cancer Res. 2003, 63, 6716–6725.

[59] Vishwanatha, J. K., Chiang, Y., Kumble, K. D., Hollingsworth,M. A., Pour, P. M., Carcinogenesis 1993, 14, 2575–2579.

[60] Grabsch, H., Takeno, S., Parsons, W. J., Pomjanski, N. et al.,J. Pathol. 2003, 200, 16–22.

[61] Zhu, X. X., Kozarsky, K., Strahler, J. R., Eckerskorn, C. et al., J.Biol. Chem. 1989, 264, 14556–14560.

[62] Nourse, C. R., Mattei, M. G., Gunning, P., Byrne, J. A., Bio-chim. Biophys. Acta 1998, 1443, 155–168.

[63] Ward, L. D., Hong, J., Whitehead, R. H, Simpson, R. J., Elec-trophoresis 1990, 11, 883–891.

[64] Rasmussen, R. K, Ji, H., Eddes, J. S., Moritz, R. L. et al.,Electrophoresis 1997, 18, 588–598.

[65] Yu, L. R., Zeng, R., Shao, X. X., Wang, N. et al., Electropho-resis 2000, 21, 3058–3068.

[66] Liu, X., Ye, L., Wang, J., Fan, D., Chin. Med. J. (Engl.) 1999,112, 1133–1137.

[67] Wataba, K., Saito, T., Fukunaka, K., Ashihara, K. et al., Int. J.Cancer 2001, 91, 448–456.

[68] Sarto, C., Valsecchi, C., Magni, F., Tremolada, L., Proteomics2004, 4, 2252–2260.

[69] Campa, M. J., Wang, M. Z., Howard, B., Fitzgerald, M. C.,Patz, E. F. Jr., Cancer Res. 2003, 63, 1652–1656.

[70] Ryu, J. W., Kim, H. J., Lee, Y. S., Myong, N. H. et al., J. KoreanMed. Sci. 2003, 18, 505–509.

[71] Ikeda, J., Kaneda, S., Kuwabara, K., Ogawa, S. et al., Bio-chem. Biophys. Res. Commun. 1997, 230, 94–99.

[72] Stierum, R., Gaspari, M., Dommels, Y., Ouatas, T. et al., Bio-chim. Biophys. Acta 2003, 1650, 73–91.

[73] Honda, K., Yamada, T., Endo, R., Ino, Y. et al., J. Cell. Biol.1998, 140, 1383–1393.

[74] Podgorski, I., Sloane, B. F., Biochem. Soc. Symp. 2003, 70,263–276.

[75] Cao, L., Taggart, R. T., Berquin, I. M., Moin, K. et al., Gene1994, 139, 163–169.

[76] Coulibaly, S., Schwihla, H., Abrahamson, M., Albini, A. et al.,Int. J. Cancer 1999, 83, 526–531.

[77] Keppler, D., Sloane, B. F., Enzyme Protein 1996, 49, 94–105.

[78] Ding, S. J., Li, Y., Shao, X. X., Zhou, H. et al., Proteomics2004, 4, 982–994.

[79] Rumpler, G., Becker, B., Hafner, C., McClelland, M. et al., Exp.Dermatol. 2003, 12, 761–771.

[80] van den Brule, F., Califice, S., Castronovo, V., Glycoconj. J.2004, 19, 537–542.

[81] Califice, S., Castronovo, V., Van Den Brule, F., Int. J. Oncol.2004, 25, 983–992.

[82] Chu, Y. W., Seftor, E. A., Romer, L. H., Hendrix, M. J., Am. J.Pathol. 1996, 148, 63–69.

[83] Vojtesek, B., Staskova, Z., Nenutil, R., Bartkova, J. et al.,Neoplasma 1990, 37, 333–342.

[84] Tomasetto, C., Moog-Lutz, C., Regnier, C. H., Schreiber, V. etal., FEBS Lett. 1995, 373, 245–249.

[85] Wu, G. J., Peng, Q., Fu, P., Wang, S. W. et al., Gene 2004, 327,201–213.



[86] Ma, D., Luyten, G. P., Luider, T. M., Jager, M. J., Niederkorn,J. Y., Invest. Ophthalmol. Vis. Sci. 1996, 37, 2293–2301.

[87] Tu, Y., Wu, S., Shi, X., Chen, K., Wu, C., Cell 2003, 113, 37–47.

[88] Komatsu, K., Murata, K., Kameyama, M., Ayaki, M. et al.,Oncology 2002, 63, 192–200.

[89] Stulik, J., Osterreicher, J., Koupilova, K., Knizek, J. et al.,Eur. J. Cancer 2000, 36, 1050–1059.

[90] Ota, T., Suzuki, Y., Nishikawa, T., Otsuki, T. et al., Nat. Genet.2004, 36, 40–45.

[91] Gupta, A. K., Aubin, J. E., Waye, M. M., Gene 1990, 86, 303–304.

[92] Hod, Y., J. Cell. Biochem. 2004, 92, 1221–1233.

[93] Chuma, M., Sakamoto, M., Yasuda, J., Fujii, G. et al., J.Hepatol. 2004, 41, 629–636.

[94] Arai, H., Ueno, T., Tangoku, A., Yoshino, S. et al., CancerGenet. Cytogenet. 2003, 146, 16–21.

[95] Aman, P., Panagopoulos, I., Lassen, C., Fioretos, T. et al.,Genomics 1996, 37, 1–8.

[96] Keely, P. J., Rusyn, E. V., Cox, A. D., Parise, L. V., J. Cell. Biol.1999 145, 1077–1088.

[97] Cox, A. D., Brtva, T. R., Lowe, D. G., Der, C. J., Oncogene1994, 9, 3281–3288.

[98] Qi, W., Liu, X., Qiao, D., Martinez, J. D., Int. J. Cancer 2005,113, 359–363.

[99] Sinha, P., Kohl, S., Fischer, J., Hutter, G. et al., Electropho-resis 2000, 21, 3048–3057.

[100] Ishiguro, H., Uemura, H., Fujinami, K., Ikeda, N. et al., Int. J.Cancer 2003, 105, 26–32.

[101] Wang, Y., Serfass, L., Roy, M. O., Wong, J. et al., Biochem.Biophys. Res. Commun. 2004, 314, 565–570.

[102] Falini, B., Tiacci, E., Liso, A., Basso, K. et al., Lancet 2004,363, 1869–1870.

[103] Mulla, A., Christian, H. C., Solito, E., Mendoza, N. et al.,Clin. Endocrinol. (Oxf) 2004, 60, 107–119.

[104] Lew, Y., Jones, D. V., Mars, W. M., Evans, D. et al., Pancreas1992, 7, 144–152.

[105] Shuda, M., Kondoh, N., Tanaka, K., Ryo, A. et al., AnticancerRes. 2000, 20, 2489–2494.

[106] Mimori, K., Mori, M., Inoue, H., Ueo, H. et al., Gut 1996, 38,66–70.

[107] Mimori, K., Mori, M., Tanaka, S., Akiyoshi, T., Sugimachi,K., Cancer 1995, 75, 1446–1449.

[108] Scheffer, G. L., Wijngaard, P. L., Flens, M. J., Izquierdo, M.A. et al., Nat. Med. 1995, 1, 578–582.

[109] Scheffer, G. L., Schroeijers, A. B., Izquierdo, M. A., Wiemer,E. A., Scheper, R. J., Curr. Opin. Oncol. 2000, 12, 550–556.

[110] Aronica, E., Gorter, J. A., van Vliet, E. A., Spliet, W. G. et al.,Epilepsia 2003, 44, 1166–1175.

[111] Zhang, R., Tremblay, T. L., McDermid, A., Thibault, P., Sta-nimirovic, D., Glia 2003, 42, 194–208.

[112] Volm, M., Koomagi, R., Mattern, J., Efferth, T., Br. J. Cancer2002, 87, 251–257.

[113] Yow, H. K., Wong, J. M., Chen, H. S., Lee, C. G. et al., Proc.Natl. Acad. Sci. USA 1988, 85, 6394–6398.

[114] Haverty, A. A., Harmey, J. H., Redmond, H. P., Bouchier-Hayes, D. J., J. Surg. Res. 1997, 69, 145–149.

[115] Takimoto, M., Wei, G., Dosaka-Akita, H., Mao, P. et al., Br. J.Cancer 2002, 86, 1757–1762.

[116] Hayette, S., Tigaud, I., Vanier, A., Martel, S. et al., Oncogene2000, 19, 4446–4450.

[117] Charrasse, S., Mazel, M., Taviaux, S., Berta, P. et al., Eur. J.Biochem. 1995, 234, 406–413.

[118] Scanlan, M. J., Chen, Y. T., Williamson, B., Gure, A. O. et al.,Int. J. Cancer 1998, 76, 652–658.

[119] Moscow, J. A., Fairchild, C. R., Madden, M. J., Ransom, D.T. et al., Cancer Res. 1989, 49, 1422–1428.

[120] Alaiya, A. A., Oppermann, M., Langridge, J., Roblick, U. etal., Cell. Mol. Life Sci. 2001, 58, 307–311.

[121] Tokunaga, K., Nakamura, Y., Sakata, K., Fujimori, K. et al.,Cancer Res. 1987, 47, 5616–5619.

[122] Murphy, E. V., Zhang, Y., Zhu, W., Biggs, J., Gene 1995, 159,131–135.

[123] Fukuda, S., Wu, D. W., Stark, K., Pelus, L. M., Biochem.Biophys. Res. Commun. 2002, 292, 593–600.

[124] Choong, M. L., Tan, L. K., Lo, S. L., Ren, E. C. et al., FEBSLett. 2001, 496, 109–116.

[125] Maiguel, D. A., Jones, L., Chakravarty, D., Yang, C., Carrier,F., Mol. Cell. Biol. 2004, 24, 3703–3711.

[126] Sinha, P., Kohl, S., Fischer, J., Hutter, G. et al., Electropho-resis 2000, 21, 3048–3057.

[127] Chen, G., Gharib, T. G., Huang, C. C., Thomas, D. G. et al.,Clin. Cancer Res. 2002, 8, 2298–2305.


Documents

Proteome analysis of a human uveal melanoma primary cell culture by 2-DE and MS