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(Investigative Ophthalmology and Visual Science. 2003;44:3629-3641.)
© 2003 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.02-1225
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Comparative Proteome Analysis of Native Differentiated and Cultured Dedifferentiated Human RPE Cells

Claudia S. Alge,1,2 Sabine Suppmann,2,3 Siegfried G. Priglinger,1 Aljoscha S. Neubauer,1 Christian A. May,4 Stefanie Hauck,2,3 Ulrich Welge-Lussen,1 Marius Ueffing,2,3,5 and Anselm Kampik1,4,5

1From the Department of Ophthalmology and the 2Clinical Cooperation Group Ophthalmogenetics, Ludwig-Maximilians-University, Munich, Germany; the 3German Research Center of Environment and Health, Oberschleissheim, Germany; and the 4Department of Anatomy II, University of Erlangen-Nürnberg, Erlangen, Germany.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 Conclusion
 References
 
PURPOSE. Dedifferentiation of retinal pigment epithelial (RPE) cells is a crucial event in the pathogenesis of proliferative vitreoretinopathy (PVR). This study was designed to improve the understanding of RPE cell dedifferentiation in vitro. The protein expression pattern of native differentiated RPE cells was compared with that of cultured, thereby dedifferentiated, RPE cells.

METHODS. Differentiated native human RPE cells and monolayers of dedifferentiated cultured primary human RPE cells were processed for two-dimensional (2-D) electrophoresis. Total cellular proteins were separated by isoelectric focusing using immobilized pH gradients (IPG 3–10) and electrophoresis on 9% to 15% gradient polyacrylamide gels. Proteins were visualized by silver staining. Silver-stained gel spots were excised, digested in situ, and analyzed by matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectroscopy (MS). The resultant peptide mass fingerprints were searched against the public domain NCBInr, MSDB, and EnsemblC databases to identify the respective proteins.

RESULTS. One hundred seventy nine protein spots were analyzed and classified into functional categories. Proteins associated with highly specialized functions of the RPE, which are required for interaction with photoreceptor cells, including RPE65, cellular retinaldehyde-binding protein (CRALBP), and cellular retinol-binding protein (CRBP), were absent in dedifferentiated cultured RPE cells, whereas proteins involved in phagocytosis and exocytosis, including cathepsin D and clathrin were still present. Dedifferentiated RPE cells displayed a strong shift toward increased expression of proteins associated with cell shape, cell adhesion, and stress fiber formation, including cytokeratin 19, gelsolin, and tropomyosins, and also acquired increased expression of factors involved in translation and tumorigenic signal transduction such as annexin I and translation initiation factor (eIF)-5A.

CONCLUSIONS. Dedifferentiation of human RPE cells in vitro results in downregulation of proteins associated with highly specialized functions of the RPE and induces the differential expression of proteins related to cytoskeleton organization, cell shape, cell migration, and mediation of proliferative signal transduction. These in vitro data suggest that the dedifferentiated status of RPE cells per se may initiate PVR. Further investigation of candidate proteins may identify additional targets for treatment or prevention of diseases associated with RPE dedifferentiation.

In the adult, the retinal pigment epithelium (RPE) is a mosaic of polygonal, postmitotic cells interposed between the choroid and the neural retina and serves as the outer blood-retinal barrier regulating retinal homeostasis and visual function.1 2 The apical side of RPE cells closely associates with the outer segments of cones and rods, whereas the base of the cell attaches to Bruch’s membrane. Normally RPE cells form a quiescent monolayer, but they retain the ability to divide and do so when placed in culture2 3 or when participating in wound repair. RPE cells proliferate readily in vivo in response to a variety of stimuli such as photocoagulation and/or retinal cryotherapy.4 5 In mild injuries, involving only the RPE and photoreceptor cells, proliferating RPE cells quickly reestablish a continuous monolayer.6 7 However, after severe injury that may be associated with ocular trauma or retinal detachment, RPE cells can be detached and consequently found in the vitreous. Once in this new environment, RPE cells have been shown to dedifferentiate and exhibit a pseudometaplastic transformation into fibroblast-like, spindle-shaped cells, which become actively dividing and migratory.1 8 9 10 11 These processes are considered to be key events in the onset of proliferative vitreoretinopathy (PVR).12 13 14 15 16 17 18 19

In an animal model of PVR, Radtke et al.19 demonstrated that when RPE cells are injected into the vitreous, they cause the formation of contractile vitreal and periretinal membranes. However, this effect is more pronounced when cultured, thus dedifferentiated, RPE cells are injected than when freshly isolated native RPE cells are placed in the vitreous cavity.

Adult human RPE cells in culture escape growth arrest and fail to maintain a differentiated morphology and20 rapidly dedifferentiate at the molecular level, and markers of RPE differentiation such as RPE65 and RET-PE 10 quickly become undetectable.21 22 It has also been reported that expression of intermediate filaments is altered when the cells take on fusiform morphology23 24 and that polarity may be partially lost.25 26 27 Thus, dedifferentiated proliferative RPE cell cultures have been used to study the very early phases of PVR, both in vitro and in vivo.16 19 28 29 30 31 32

However, the molecular changes associated with dedifferentiation of RPE cells per se are not well understood, and to our knowledge, the overall cellular proteome of native differentiated RPE cells has not been characterized to date. The goal of the present study was to gain better understanding of the process of RPE cell dedifferentiation in vitro. We therefore took a proteomic approach to investigate the shift in the overall protein expression pattern between differentiated native human RPE cells and dedifferentiated cultured RPE cells. With this approach we attempted to screen for the most prominent dedifferentiation-related changes in several functional groups simultaneously. The proteomic changes illustrated in this study clearly reflect the dynamic changes in the protein expression pattern associated with dedifferentiation of the RPE. Some of the identified proteins have been described to be associated with RPE dedifferentiation or PVR, whereas other proteins may be newly linked with RPE dedifferentiation and proliferation and have not yet been described in the RPE.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 Conclusion
 References
 
Isolation of Human RPE Cells
Eyes from eight human donors were obtained from the Munich University Hospital Eye Bank and processed within 4 to 16 hours after death. The average donor age was 46 ± 6 years. None of the donors had a known history of eye disease. Methods for securing human tissue were humane, included proper consent and approval; complied with the Declaration of Helsinki, and were approved by the local ethics committee. Human retinal pigment epithelium (RPE) cells were harvested according to a procedure described previously.33 In brief, whole eyes were thoroughly cleansed in 0.9% NaCl solution, immersed in 5% poly(1-vinyl-2-pyrrolidone)-iodine (Jodobac; Bode-Chemie, Hamburg, Germany), and rinsed again in the sodium-chloride solution. The anterior segment from each donor eye was removed, and the posterior poles were examined with the aid of a binocular stereomicroscope to confirm the absence of gross retinal disease. Next, the neural retinas were carefully peeled away from the RPE-choroid-sclera with fine forceps. The eyecup was rinsed with Ca2+ and Mg2+-free Hanks’ balanced salt solution and filled with 0.25% trypsin (Invitrogen/Gibco, Karlsruhe, Germany) for 30 minutes at 37°C. The trypsin was carefully aspirated and replaced with Dulbecco’s modified Eagle’s medium (DMEM; Biochrom, Berlin, Germany) supplemented with 10% fetal calf serum (FCS; Biochrom). The medium was gently agitated with a pipette, releasing the RPE into the medium by avoiding damage to Bruch’s membrane. The suspended RPE cells were then transferred to a 35-mm2 Petri dish and checked by microscope for cross contamination.

Human RPE Cell Culture
The RPE cell suspension was transferred to a 50-mL flask (Falcon, Wiesbaden, Germany) containing 20 mL of DMEM (Biochrom) supplemented with 20% FCS (Biochrom) and maintained at 37°C and 5% CO2. Epithelial origin was confirmed by immunohistochemical staining for cytokeratin (CK) using a pan-CK antibody (Sigma-Aldrich, Deisenhofen, Germany).34 The cells were tested and found free of contaminating macrophages (anti-CD11; Sigma) and endothelial cells (anti-von Willebrand factor, Sigma-Aldrich; data not shown). After reaching confluence, primary RPE cells were subcultured to passages 2 to 3 and maintained in DMEM (Biochrom) supplemented with 10% FCS (Biochrom) at 37°C and 5% CO2. Cells were grown on plastic 10-cm tissue culture dishes until they had reached no more than 80% to 90% confluence, to assure that they are still in a proliferative, presumably dedifferentiated, state, and were then maintained under serum-free conditions for 48 hours to reduce the influence of serum stimulation.

Sample Preparation
For preparation of protein lysates from native RPE cell preparations, suspensions of freshly isolated human RPE cells were transferred to a 2.0-mL microcentrifuge tube and washed twice in Ca2+-Mg2+-free 1x phosphate-buffered saline (PBS; pH 7.4), followed by centrifugation at 800 rpm for 5 minutes. Cell pellets were then resuspended in nanopure water containing a protease inhibitor cocktail (Complete Mini; Roche Diagnostics, Inc., Mannheim, Germany) and snap frozen in liquid nitrogen. Cells were then disrupted by grinding with a Teflon glass homogenizer (Braun Biotech International, Melsungen, Germany), lyophilized, and stored at -80°C for future use. Proteins were solubilized in denaturing lysis buffer containing 9 M urea (Merck, Darmstadt, Germany), 2 M thiourea (Merck), 4% 3-([3-cholamidopropyl]dimethylammonio-2-hydroxy-1-propanesulfonate (CHAPS; Sigma-Aldrich), 1% dithioerytherol (Merck), 2.5 µM EGTA (Sigma-Aldrich), 2.5 µM EDTA (Sigma-Aldrich), and protease inhibitors for 4 hours at room temperature (RT). To minimize interindividual differences in native human RPE, protein lysates from five age-matched donor eyes were pooled.

For isolation of whole cellular protein extracts from RPE cultures, cells were washed once with serum-free medium, followed by a wash in 1x PBS (pH 7.4) and a third wash in 0.5x PBS to reduce contamination with salts. Subsequently, cells were collected and lysed in denaturing lysis buffer, as described earlier. Lysates were then cleared by centrifugation at 22,000g for 45 minutes at RT. Protein concentrations were determined by the Bradford protein assay reagent (Bio-Rad, Munich, Germany). Freshly prepared lysate containing 125 µg total protein was loaded onto each gel.

Two-Dimensional Electrophoresis
First-dimension isoelectric focusing (IEF) was performed with precast 18-cm immobilized pH gradient (IPG) strips (18 cm Immobiline DryStrip 3-10 NL; Amersham Pharmacia Biotech, Braunschweig, Germany). IPG strips were rehydrated overnight with 125 µg protein diluted to 350 µL with reswelling solution (9 M urea, 2 M thiourea, 4% CHAPS, 2.5 µM EGTA, 2.5 µM EDTA, 1% DTE, 4 mM Tris, 0.25% (wt/vol) bromophenol blue (BPB), and 0.7% (vol/vol) IEF ampholytic carriers (Pharmalytes pH 3–10; Amersham Pharmacia Biotech). IEF was performed at 20°C with a commercial system (Multiphore II; Amersham Pharmacia Biotech), with the initial voltage limited to 50 V (2 hours) and then increased stepwise to 8000 V and held until 101 kV/h was reached. Immediately after IEF, the IPG strips were equilibrated for 10 minutes in buffer containing 6 M urea, 2% SDS, 50 mM Tris-HCl (pH 6.8), 30% glycerol, and a trace of BPB under reducing conditions (65 mM DTE added), followed by another 10-minute incubation in the same buffer under alkylating conditions (135 mM iodoacetamide added). Equilibrated IPG strips were then placed on top of 9% to 15% linear gradient polyacrylamide gels 20 cm x 18 cm x 0.75 mm in size and embedded in 0.5% agarose (Serva, Heidelberg, Germany) in running buffer (384 mM glycin, 50 mM Tris, 0.2% SDS) and electrophoresed at a constant current of 8 mA/gel at 10°C, until the dye front had reached the bottom of the gel (approximately 16 hours). All gels were silver stained35 and dried between cellophane sheets.

Image Analysis
Of the 12 silver-stained gels per cell type (native RPE; dedifferentiated cultured RPE), the 3 best focusing results were selected for calculating a virtual average gel on computer (Z3 software package; Compugen, Haifa, Israel). The computational calculation of virtual average gels reduced spot variations in individual gels from one sample resulting from limitations in two-dimensional electrophoresis (2-DE) reproducibility. Because spot integration of more than three gels from a given sample resulted in reduced quality of the virtual average gel, no more than three 2-DE gels per cell type were included in the virtual average gel. To create the virtual average gel, silver-stained 2-DE gels were scanned at 12 bit/300 dpi. Scans were then matched for each of the two cell types, using 42 to 56 common spots as landmarks, and processed on computer (in the Z3 Raw Master Gel [RMG]mode; Compugen). Next, spot detection was performed on each RMG by image analysis software (PD-Quest; Bio-Rad). RMGs from a given experimental condition (differentiated native versus dedifferentiated cultured RPE) were analyzed manually to determine quantitative and qualitative differences in protein expression, because computer-assisted software comparison failed to integrate the rather diverse protein patterns stemming from differentiated native versus dedifferentiated cultured RPE.

In-Gel Digestion and MALDI-TOF Analysis
Central areas (2 x 2 mm) of silver stained spots were excised from dried 2-DE gels, transferred to 96-well plates (Nunc 22-260; Nunc, Wiesbaden, Germany), rehydrated in 100 µL nanopure water for a minimum of 30 minutes, destained with 30 mM potassium ferricyanide (Sigma-Aldrich) and 100 mM sodium thiosulfate (Merck),36 and washed three times in 100 µL 40% acetonitrile (15 minutes each) to extract residual water. Acetonitrile was then removed, and a 10-µL digestion solution containing 0.01 µg/µL trypsin (Sequencing Grade Modified Trypsin; Promega, Mannheim, Germany) in 1 mM Tris-HCl (pH 7.5) was added for enzymatic cleavage.

After incubation under humid conditions at 37°C for 12 hours, 0.5 µL aliquots of the tryptic digests were mixed with 0.5 µL matrix consisting of 2,5-dihydroxybenzoic acid (Sigma-Aldrich; 20 mg/mL in 20% acetonitrile and 0.1% trifluoroacetic acid [TFA]) and 2-hydroxy-5-methoxybenzoic acid (Fluka, Buchs, Switzerland; 20 mg/mL in 20% acetonitrile and 0.1% TFA) at a 9:1 ratio (vol/vol) and spotted onto a 400-µm anchor steel target (Bruker-Daltronik, Bremen, Germany). MALDI-TOF peptide mass fingerprints were obtained on a mass spectrometer (Reflex III; Bruker-Daltronik) equipped with an ion source. Mass analyses obtained in the positive ion reflector mode were run automatically. For calibration, angiotensin-2-acetate (Mr 1046.54), substance P (Mr 1347.74), bombesin (Mr 1619.82), and ACTH 18-3 (Mr 2465.20) were recorded. Internal calibration using peptides resulting from autodigestion of trypsin was also performed.

Database Research
Trypsin specifically cleaves proteins at the C terminus of lysine and arginine residues, thereby generating a fingerprint of peptide masses that can be searched in databases. Database searches were performed with the assistance of commercial software (Mascot software; Matrix Science, London, UK).37 Parameter settings were 150 ppm mass accuracy with one miscleavage allowed, and the search was performed in all available mammalian and, in assorted cases, all eukaryotic sequences. Peptide mass fingerprints (PMFs) were searched for matches with the virtually generated tryptic protein masses of the protein databases NCBInr (http://ncbi.nih.gov/ National Center for Biotechnical Information, Bethesda, MD), MSDB (csc-fserve.hh.med.ic.ac.uk/msdb.html/ Proteonomics Department, Hammersmith Campus, Imperial College, London, UK), and EnsemblC (http://www.ensembl.org/ Sanger Centre, Hinxton, UK). A protein was regarded as identified, if the following four criteria were fulfilled: (1) the MOWSE score38 (http:www.hgmp.mrc.ac.uk/ Molecular Weight Search, Human Genome Mapping Project Resource Centre, Sanger Centre) was above the 5% significance threshold for the respective database, (2) the matched peptide masses were abundant in the spectrum, (3) the theoretical isoelectric point (pI) and the molecular weight (Mr) of the search result could be correlated with the 2-DE position of the corresponding spot, and (4) the matched sequence did not contain more than 20% uncleaved peptides. Functional classification was based on the classification provided in the TrEMBL (http://embl-heidelberg.de/ European Molecular Biology Laboratory, Heidelberg, Germany) and SwissProt protein knowledge databases (http://www.expasy.org/ Swiss Institute of Bioinformatics, Geneva, Switzerland). All databases are provided in the public domain by the host institutions.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 Conclusion
 References
 
2-DE and Image Analysis
It was the main objective of this study to identify those proteins that are differentially expressed as a result of RPE dedifferentiation in vitro. To probe this proteomic shift, 2-DE gels of differentiated native and dedifferentiated cultured human RPE cells were analyzed. Figures 1 and 2 show the computationally estimated average RMG images of differentiated native RPE cells (Fig. 1) and of dedifferentiated cultured RPE cells (Fig. 2) . Spot identification by the image analysis software (PD-Quest; Bio-Rad) allowed for the resolution of 1985 to 2014 spots in the RMGs of both cell types. Although 2-DE gels from differentiated native and dedifferentiated cultured RPE cells exhibited certain identical protein expression profiles, the images were too different to apply a computational comparative image analysis. For this reason, RMGs were manually compared, and the most distinct differentially expressed proteins were chosen for protein identification. Furthermore, randomly selected high-abundance protein spots found in both cell types were excised to serve as landmark proteins.



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  		FIGURE 1. Silver-stained 2-DE image of differentiated native human RPE cells. Differentiated native human RPE cells were freshly isolated from human donor eyes and processed as described in Materials and Methods. Approximately 125 µg of protein was separated on an 18-cm, pH 3 to 10 nonlinear IPG strip, followed by 9% to 15% SDS-PAGE. The images are virtual average images calculated from three real gel images. Numbered spots were identified by MALDI-TOF MS and are listed in Table 1 .

 


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  		FIGURE 2. Silver-stained 2-DE image of dedifferentiated cultured primary human RPE cells of passage 3. RPE cells were grown on tissue culture plastic plates until they reached 80% to 90% confluence. Cells were then depleted of serum for 48 hours and harvested. Processing was as in Figure 1 . Numbered spots are identified in Table 2 .

 
Protein Identification
Proteins were identified from 71 of 88 gel spots excised from 2-DE gels of native RPE and from 62 of 91 gel spots cut from the cultured RPE cell samples, for an average spot identification yield of 74%. For 20% of the remainder, the MS data were of insufficient quality, and protein identification was not possible. Another 3.1% could not be identified because of contamination of the tryptic digests with keratins, and no identity could be assigned to a further 2.9% despite good MS data, most likely because there was none of the respective sequences in the databases used.

Identified proteins, accession numbers, and SwissProt entry names are listed in Tables 1 and 2 . Theoretical Mr, pI, percentage of sequence coverage, probability based MOWSE score, and functional category are also included. In addition, the spot numbers are assigned on the individual RMG images in Figures 1 and 2 .


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  		TABLE 1. Proteins Identified in Differentiated Native Human RPE Cells

 

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  		TABLE 1A. (continued). Proteins Identified in Differentiated Native Human RPE Cells

 

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  		TABLE 2. Proteins Identified in Dedifferentiated Cultured Human RPE Cells

 

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  		TABLE 2A. (continued). Proteins Identified in Dedifferentiated Cultured Human RPE Cells

 

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  		TABLE 2B. (continued). Proteins Identified in Dedifferentiated Cultured Human RPE Cells

 
As seen before from 2-DE analysis, 25 proteins of a total of 90 different proteins identified were found in multiple spots on several positions on the gel. For example, two spots were found at the same Mr but separated by approximately 0.5 pH units (N62,N63 cathepsin D; N31,N35 F1-ATPase-ß chain), and one protein was found at different Mrs but with the same pI (C4,C18; glucosidase II). Finally, four proteins were identified at slightly different Mrs and pIs (N58,N61 CRALBP; N5,N7 mitofilin; C45,C46 14-3-3 protein; C41,C42,C43 tropomyosin alpha, tropomyosin 4, tropomyosin alpha 3 chain.), suggesting the presence of different isoforms. These patterns clearly differed from a more frequently observed configuration, where spots were aligned like strings of pearls. This was observed for 15 proteins, which are represented with a series of two to three different spots, close together in a row (e.g., N14,N15,N19 annexin VI; N27,N28 S-arrestin; N22,N23 RPE65; C29,C30 {alpha}-enolase; C32,C33 CK 18). Such a series of spots is probably due to glutamine deamidation, chemically induced during the 2-DE procedure and resulting in differentially charged proteins rather than different protein isoforms.39 We also mapped one protein (N1 cathepsin D) at a position on the gel that indicated a greater Mr than predicted and another two spots (C11 LIM-motif containing protein kinase; C31 eIF-4AI) appeared at a pI different from the predicted one. In addition, retinal pigment epithelial membrane receptor (N34) was annotated at a position that differed in Mr and pI from the predicted values. Computer assisted sequence analysis identified this protein as a bovine isoform of RPE65, in this case, probably representing a partially processed stable fragment with different Mr and pI.

Comparison of the Differentiated Native RPE Proteome with That of Dedifferentiated Cultured RPE Cells
Overall the proteome of native and cultured RPE cells displayed a high degree of similarity, but at the same time striking distinguishing marks were apparent. Proteins were defined as differentially expressed if they could not be located to the corresponding position on the 2-DE gel of the other cell type. Expression below or above the detection limit of 2-DE technology was referred to as down- or upregulation of the respective proteins.

Expression Unchanged
In a first step, randomly selected high-abundance proteins that were located in identical positions and expression levels on gels of both groups were excised to serve as landmark proteins (Tables 1 and 2) . Most of these proteins were found to be involved in metabolism (e.g., N71,C35 GADPH; N40,C55 triosephosphate isomerase). A high degree of similarity was also observed for molecular chaperones (e.g., N12,C12 heat shock 71 kDa protein) and antioxidant enzymes (e.g., N45,C49 glutathione S-transferase; N56,C50 peroxiredoxin 2). Good matching was found for the Ca2+-binding proteins annexin V (N59,C44) and annexin VI (N19,C8) and for several proteins involved in signal transduction (e.g., N56,C38; GTP-binding regulatory protein; N52,C45 14-3-3 protein epsilon). A good correlation between differentiated native and dedifferentiated cultured RPE was also observed regarding f1-ATPase (N31) and ATP synthase (N48), two mitochondrial enzymes contributing to oxidative phosphorylation, as well as for CK 8 (N53,C24), an intermediate filament protein characteristic of cells of epithelial origin. Furthermore, the lysosomal enzyme cathepsin D (N62,N63,C58) was identified in both cell types, although it appeared to be expressed at a lower level in dedifferentiated, cultured RPE.

Proteins Downregulated or Absent in Dedifferentiated Cultured RPE
Although most of the metabolic enzymes identified could be found in both, differentiated and dedifferentiated RPE, a considerable group of metabolic enzymes expressed in the native RPE proteome could not be located in the corresponding positions on 2-DE gels of dedifferentiated RPE (Table 3) . These included functions in glycolysis (N29 fructose-bisphosphate aldolase; N32 neuronal gamma enolase), in the tricarboxylic acid cycle (N54 succinyl-CoA ligase), and in protein metabolism (N26; prenylcysteine lyase). High-abundance proteins exclusively identified in differentiated RPE included interphotoreceptor retinoid-binding protein (N3; IRBP), an all-trans retinol and 11-cis retinal-binding protein located in the interphotoreceptor matrix, cellular retinol-binding protein (N51, CRBP), RPE65 (N22,N23) and cellular retinaldehyde-binding protein (N58,N61; CRALBP), all of which are involved in retinoid metabolism. Furthermore, we could not map the visual cycle proteins cone arrestin (N33,N36), S-arrestin (N27,N28), recoverin (N49), and phosducin (N60), as well as the mitochondrial motor protein mitofilin (N5,N6,N7) in 2-DE gels of cultured RPE. A significant difference was also observed in expression levels of mitochondrial proteins involved in electron transfer (N9,N10,N11 NADH-ubiquinone oxidoreductase; N37 ubiquinol-cytochrome-c reductase core protein I).


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  		TABLE 3. Proteins Downregulated or Absent in Dedifferentiated Cultured Human RPE Cells

 
Proteins Upregulated in Dedifferentiated Cultured RPE
Marked upregulation in dedifferentiated RPE was found for proteins involved in cell shape, adhesion, exocytosis, and cytoskeleton formation. A summary of proteins upregulated in dedifferentiated RPE is provided in Table 4 . Specific functions are also included.


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  		TABLE 4. Proteins Upregulated in Dedifferentiated Cultured Human RPE Cells

 
These include the F-actin binding proteins {alpha}-actinin-1 (C3) and -4 (C57), which anchor F-actin to a variety of intracellular structures, as well as coactosin (C60). Further, an enhanced expression of vinculin (C19), a protein involved in cell adhesion, was observed. The expression of intermediate filament proteins was also altered. Both, differentiated and dedifferentiated RPE expressed CK 8 (N53,C24) and 18 (C32,C33), whereas dedifferentiated RPE cells also expressed CK-7 (C25) and -19 (C36). Furthermore, dedifferentiated cultured RPE cells expressed three isoforms of tropomyosin (C41, C42, and C43), a protein associated with stress fiber formation. Cultured cells also displayed a high abundance of ubiquitin-protein ligase (C17), annexin I (C47), annexin II (C48), translation initiation factor (eIF)-5A (C59), and eIF-4AI (C31), which are expressed at lower levels or are missing in native RPE.


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
Conclusion
 References
 
Proteomics refers to the study of the proteome—that is, the total protein complement of a genome. Unlike the genome, which is essentially the same in all somatic cells of an organism, the proteome is a dynamic entity that is not only different in different cell types, but also changes with the physiological state of a cell. Ultimately, proteins expressed in dynamic compositions determine the phenotypic expression of genomic information. An important advantage of global protein expression profiling compared with individual gene regulation studies is the ability to monitor changes in several functional groups simultaneously. The objective of this study was to further elucidate differences in the protein expression profile of differentiated native compared with dedifferentiated cultured RPE cells and to use this information to clarify further the general understanding of ocular disease associated with RPE dedifferentiation. The data compiled in Tables 3 and 4 suggest that several biological processes were affected in the present study, and a few of these processes are discussed in this section.

Proteins Downregulated in Dedifferentiated Cultured Human RPE Cells
Phototransduction and Vitamin A Metabolism.
In accordance with previous studies,21 22 40 41 we observed a downregulation of proteins associated with retinoid metabolism (CRALBP, CRBP, RPE65, and IRBP) and the visual cycle (arrestin, recoverin) in dedifferentiated cultured RPE cells (for review, see Ref. 42 ). Whereas CRALBP, CRBP, and RPE65 are biochemical markers of RPE differentiation and rapidly become undetectable in culture,21 40 41 identification of the latter ones in native RPE 2-DE gels may be due to photoreceptor cross-contamination of our RPE cell preparations. Despite extensive rinsing with PBS and repeated centrifugation at low speed, electron microscopy of consecutive sections revealed the presence of rod outer segment discs either in an intracellular location or bound to the RPE cell surface in our native RPE cell preparations (data not shown). The absence of IRBP, phosducin, brain-type fructose-bisphosphate aldolase, neuronal (gamma) enolase, and a retinal Ca2+-binding transporter in 2-DE gels from dedifferentiated RPE further substantiate this consideration.

Energy Metabolism.
Differentiated native RPE also exhibited a high abundance of the respiratory chain components NADH-ubiqinone oxidoreductase and ubiqinol-cytochrome-c reductase,43 both of which appeared to be downregulated in dedifferentiated cultured RPE cells. This may reflect the reduced energy requirement profile under cell culture conditions as opposed to the high-energy requirements in native RPE.44

Proteins Upregulated in Dedifferentiated Cultured Human RPE Cells
As expected, increased protein expression levels in dedifferentiated RPE cells can be attributed roughly to two functional groups: cytoskeleton remodeling and mediation of proliferative signal transduction.

Cell Shape, Migration, and Adhesion.
Proteins associated with the keratin cytoskeleton, actin function, and maintenance of cell shape and motility appeared to be highly abundant in dedifferentiated cultured RPE cells. In agreement with previous studies we observed an altered expression of intermediate filament proteins in dedifferentiated cultured RPE cells.23 45 CK 7, CK 8, CK 18, and CK 19 were highly abundant in dedifferentiated RPE, whereas cells isolated directly from the eye appeared to have no CK 7 and CK 19. CK 19 has been observed in proliferating RPE cells of patients with PVR23 and has also been associated with migration in cultured human RPE cells.45 46 In premalignant and malignant epithelial lesions,47 CK 19 was correlated with infiltrating characteristics and invasive ability.

In addition to alterations in intermediate filament expression, we observed an increased expression of cytoskeleton-organizing proteins in dedifferentiated RPE cells. These included {alpha}-actinin, which can bundle actin filaments into parallel arrays48 ; gelsolin, an actin-binding protein that mediates rapid remodeling of actin filaments49 ; tropomyosins, which are a main component of stress fibers in mesenchymal cells; and coactosin, a recently identified human F-actin-binding protein that has also been co-localized with stress fibers.50 In cultured fibroblasts {alpha}-actinin, gelsolin, and tropomyosin have been shown to be heavily enriched in stress fibers, which mediate cell contraction and migration.49 Furthermore, dedifferentiated RPE cells displayed an upregulation of vinculin, a protein involved in cell adhesion.51 52 Upregulation of these components may simply reflect a reorganization of the cellular cytoskeleton that is necessary for the cells to adopt to the new environment. Taken together, however, these changes may very well be coincidental with morphologic transformation and acquisition of migratory characteristics.

Cell Proliferation.
A distinct observation from this study was the upregulation of proteins that can be attributed to cell proliferation. Annexins are a family of Ca2+-dependent phospholipid-binding proteins that seem to be important regulatory proteins with pluripotent and pleiotropic roles. All annexins share the property of binding to acidic phospholipids of cellular membranes, suggesting a role in membrane-related events, particularly in membrane organization, exocytosis, and endocytosis.53 We observed a strong upregulation of annexin I and II in dedifferentiated RPE cells, whereas annexin V and VI expression levels remained unchanged. In contrast to the latter ones, annexin I is a substrate of protein kinases involved in the control of cell growth and is postulated to be involved in mitogenic signal transduction.54 Annexin II is also phosphorylated by tyrosin kinases and by protein kinase C.55 Both annexin I and II are significantly overexpressed in various human cancers,53 56 57 during liver regeneration and transformation,58 and in proliferating cultured cells.54

Translation initiation factor (eIF)-4AI and eIF-5A were expressed at detectable levels in dedifferentiated RPE cells exclusively. eIF-4AI and eIF-5A belong to a group of proteins that control the initiation phase of eukaryotic protein synthesis. A series of observations suggest that these factors may also play major roles in the regulation of cell proliferation59 and transformation (for review, see Ref. 60 ) Protein biosynthesis is one of the last steps in the transmission of genetic information on the basis of which proteins are produced to maintain the specific biological function of a cell. Currently, an increasing body of data is emerging that shows that intervention in this pathway may be an additional target for antiproliferative strategies.61 62

Neither annexin I and II nor eIF-4AI and eIF-5A have been described to play a role in RPE proliferation or dedifferentiation. Clearly, further studies are needed to evaluate the significance of these factors in control of RPE proliferation in vitro and in the situation found in PVR.


   Conclusion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Conclusion
References
 
In the recent years, several studies have been attempted to analyze differential gene expression using cDNA-microarray technology. However, a cell’s physiological state is ultimately dictated at the protein level, and comparison of cDNA expression levels to protein levels often reveals discrepancies between these two global expression approaches, demonstrating that transcript levels cannot predict protein levels.63 64 Certainly, the inability to detect low-abundance proteins is a major drawback of the 2-DE technology. Despite this limitation, we were able to detect striking differences between differentiated and dedifferentiated RPE cells.

In the present study, we attempted to analyze dedifferentiated cultured RPE cells independent of ECM or serum stimulation in comparison with native differentiated RPE, and to draw conclusions about the potential role of RPE dedifferentiation in the onset of PVR. We are aware that the proper interpretation and clinical relevance of this study is limited by possible differences between cultured RPE cells and pseudometaplastic RPE cells in vivo, which are exposed to complex interactions that occur in the intraocular environment.

However, in summary, our data suggest a series of functional changes in dedifferentiated RPE cells in vitro, associated with the loss of RPE-specific functions, which add up to adaptive changes in cell phenotype toward a mesenchymal, migratory morphology, together with a high capacity to proliferate.

The results of this article focus attention onto a group of new proteins involved in cytoskeleton remodeling and cell proliferation, which may be involved in the initiation phase of PVR disease. Closer examination of these factors may lead to the definition of additional targets for treatment or prevention of ocular diseases associated with dedifferentiation and proliferation of RPE cells.


   Acknowledgements
 
The authors thank Katja Obholzer for excellent technical assistance and Ursula Olazabal for a critical reading of the manuscript.


   Footnotes
 
5 Contributed equally to the work and therefore should be considered equivalent senior authors. Back

Supported by the German Federal Ministry for Education and Research Grants FKZ:031U108A (MU), PRORET QLK6-CT-2000-00569 (MU), PRO AGE-RET QLK-CT-2001-00385 (MU); German Research Foundation Grant WE 2577/2-1; and German Ophthalmological Society Research Support (UW-L).

Submitted for publication November 29, 2002; revised February 12, 2003; accepted March 3, 2003.

Disclosure: C.S. Alge, None; S. Suppmann, None; S.G. Priglinger, None; A.S. Neubauer, None; C.A. May, None; S. Hauck, None; U. Welge-Lussen, None; M. Ueffing, None; A. Kampik, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Anselm Kampik, Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany; anselm.kampik{at}ak-i.med.uni-muenchen.de.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 Conclusion
 References
 

  1. Grierson, I, Hiscott, P, Hogg, P, Robey, H, Mazure, A, Larkin, G. (1994) Development, repair and regeneration of the retinal pigment epithelium Eye 8,255-262
  2. Zhao, S, Rizzolo, LJ, Barnstable, CJ. (1997) Differentiation and transdifferentiation of the retinal pigment epithelium Int Rev Cytol 171,225-266[Medline][Order article via Infotrieve]
  3. Campochiaro, PA, Hackett, SF. (1993) Corneal endothelial cell matrix promotes expression of differentiated features of retinal pigmented epithelial cells: implication of laminin and basic fibroblast growth factor as active components Exp Eye Res 57,539-547[CrossRef][Medline][Order article via Infotrieve]
  4. L’Esperance, FA. (1969) The ocular histopathologic effect of krypton and argon laser radiation Am J Ophthalmol 68,263-269[Medline][Order article via Infotrieve]
  5. Lincoff, H, Kreissig, I, Jakobiec, F, Iwamoto, T. (1981) Remodeling of the cryosurgical adhesion Arch Ophthalmol 99,1845-1849[Abstract]
  6. Tso, MO, Fine, BS. (1979) Repair and late degeneration of the primate foveola after injury by argon laser Invest Ophthalmol Vis Sci 18,447-461[Abstract/Free Full Text]
  7. Korte, GE, Perlman, JI, Pollack, A. (1994) Regeneration of mammalian retinal pigment epithelium Int Rev Cytol 152,223-263[Medline][Order article via Infotrieve]
  8. Machemer, R, van Horn, D, Aaberg, TM. (1978) Pigment epithelial proliferation in human retinal detachment with massive periretinal proliferation Am J Ophthalmol 85,181-191[Medline][Order article via Infotrieve]
  9. Machemer, R, Laqua, H. (1975) Pigment epithelium proliferation in retinal detachment (massive periretinal proliferation) Am J Ophthalmol 80,1-23[Medline][Order article via Infotrieve]
  10. Mueller-Jensen, K, Machemer, R, Roobik, A. (1975) Autotransplantation of retinal pigment epithelium in intravitreal diffusion chamber Am J Ophthalmol 80,530-537[Medline][Order article via Infotrieve]
  11. Mandelcorn, MS, Machemer, R, Fineberg, E, Hersch, SB. (1975) Proliferation and metaplasia of intravitreal retinal pigment epithelium cell autotransplants Am J Ophthalmol 80,227-237[Medline][Order article via Infotrieve]
  12. Kampik, A, Kenyon, KR, Michels, RG, Green, WR, de la Cruz, ZC. (1981) Epiretinal and vitreous membranes: comparative study of 56 cases Arch Ophthalmol 99,1445-1454[Abstract]
  13. Machemer, R. (1988) Proliferative vitreoretinopathy (PVR): a personal account of its pathogenesis and treatment Invest Ophthalmol Vis Sci 29,1771-1783[Free Full Text]
  14. Glaser, BM, Lemor, M. (1994) Pathobiology of proliferative vitreoretinopathy Ryan, SJ eds. Retina ,2249-2263 Mosby-Year Book Press St. Louis.
  15. Campochiaro, PA. (1997) Pathogenic mechanisms in proliferative vitreoretinopathy Arch Ophthalmol 115,237-241[Abstract]
  16. Hiscott, P, Sheridan, C, Magee, RM, Grierson, I. (1999) Matrix and the retinal pigment epithelium in proliferative retinal disease Prog Retinal Eye Res 18,167-190[CrossRef][Medline][Order article via Infotrieve]
  17. Newsome, DA, Rodrigues, MM, Machemer, R. (1981) Human massive periretinal proliferation: in vitro characteristics of cellular components Arch Ophthalmol 99,873-880[Abstract]
  18. Glaser, BM, Cardin, A, Biscoe, B. (1987) Proliferative vitreoretinopathy: the mechanism of development of vitreoretinal traction Ophthalmology 94,327-332[Medline][Order article via Infotrieve]
  19. Radtke, ND, Tano, Y, Chandler, D, Machemer, R. (1981) Simulation of massive periretinal proliferation by autotransplantation of retinal pigment epithelial cells in rabbits Am J Ophthalmol 91,76-87[Medline][Order article via Infotrieve]
  20. Campochiaro, PA, Hackett, SF, Conway, BP. (1991) Retinoic acid promotes density-dependent growth arrest in human retinal pigment epithelial cells Invest Ophthalmol Vis Sci 32,65-72[Abstract/Free Full Text]
  21. Hamel, CP, Tsilou, E, Pfeffer, BA, Hooks, JJ, Detrick, B, Redmond, TM. (1993) Molecular cloning and expression of RPE65, a novel retinal pigment epithelium-specific microsomal protein that is post-transcriptionally regulated in vitro J Biol Chem 268,15751-15757[Abstract/Free Full Text]
  22. Neill, JM, Thornquist, SC, Raymond, MC, Thompson, JT, Barnstable, CJ. (1993) RET-PE10: a 61 kD polypeptide epitope expressed late during vertebrate RPE maturation Invest Ophthalmol Vis Sci 34,453-462[Abstract/Free Full Text]
  23. Hunt, RC, Davis, AA. (1990) Altered expression of keratin and vimentin in human retinal pigment epithelial cells in vivo and in vitro J Cell Physiol 145,187-199[CrossRef][Medline][Order article via Infotrieve]
  24. Casaroli-Marano, RP, Pagan, R, Vilaro, S. (1999) Epithelial-mesenchymal transition in proliferative vitreoretinopathy: intermediate filament protein expression in retinal pigment epithelial cells Invest Ophthalmol Vis Sci 40,2062-2072[Abstract/Free Full Text]
  25. Turksen, K, Opas, M, Aubin, JE, Kalnins, VI. (1983) Microtubules, microfilaments and adhesion patterns in differentiating chick retinal pigment epithelial (RPE) cells in vitro Exp Cell Res 147,379-391[CrossRef][Medline][Order article via Infotrieve]
  26. Rizzolo, LJ. (1990) The distribution of Na+, K(+)-ATPase in the retinal pigmented epithelium from chicken embryo is polarized in vivo but not in primary cell culture Exp Eye Res 51,435-446[CrossRef][Medline][Order article via Infotrieve]
  27. Huotari, V, Sormunen, R, Lehto, VP, Eskelinen, S. (1995) The polarity of the membrane skeleton in retinal pigment epithelial cells of developing chicken embryos and in primary culture Differentiation 58,205-215[CrossRef][Medline][Order article via Infotrieve]
  28. Grisanti, S, Guidry, C. (1995) Transdifferentiation of retinal pigment epithelial cells from epithelial to mesenchymal phenotype Invest Ophthalmol Vis Sci 36,391-405[Abstract/Free Full Text]
  29. Vinores, SA, Derevjanik, NL, Mahlow, J, et al (1995) Class III beta-tubulin in human retinal pigment epithelial cells in culture and in epiretinal membranes Exp Eye Res 60,385-400[CrossRef][Medline][Order article via Infotrieve]
  30. Campochiaro, PA, Jerdan, JA, Glaser, BM. (1984) Serum contains chemoattractants for human retinal pigment epithelial cells Arch Ophthalmol 102,1830-1833[Abstract]
  31. Lee, SC, Kwon, OW, Seong, GJ, Kim, SH, Ahn, JE, Kay, ED. (2001) Epitheliomesenchymal transdifferentiation of cultured RPE cells Ophthalmic Res 33,80-86[CrossRef][Medline][Order article via Infotrieve]
  32. Ando, A, Ueda, M, Uyama, M, Masu, Y, Ito, S. (2000) Enhancement of dedifferentiation and myoid differentiation of retinal pigment epithelial cells by platelet derived growth factor Br J Ophthalmol 84,1306-1311[Abstract/Free Full Text]
  33. Alge, CS, Priglinger, SG, Neubauer, AS, et al (2002) Retinal pigment epithelium is protected against apoptosis by {alpha}B-crystallin Invest Ophthalmol Vis Sci 43,3575-3582[Abstract/Free Full Text]
  34. Leschey, KH, Hackett, SF, Singer, JH, Campochiaro, PA. (1990) Growth factor responsiveness of human retinal pigment epithelial cells Invest Ophthalmol Vis Sci 31,839-346[Abstract/Free Full Text]
  35. Blum, H, Beier, H, Gross, HJ. (1987) Improved silver staining of plant proteins, RNA, and DNA in polyacrylamide gels Electrophoresis 8,93-99[CrossRef]
  36. Gharahdaghi, F, Weinberg, CR, Meagher, DA, Imai, BS, Mische, SM. (1999) Mass spectrometric identification of proteins from silver-stained polyacrylamide gel: a method for the removal of silver ions to enhance sensitivity Electrophoresis 20,601-605[CrossRef][Medline][Order article via Infotrieve]
  37. Pappin, DJ. (1997) Peptide mass fingerprinting using MALDI-TOF mass spectrometry Methods Mol Biol 64,165-173[Medline][Order article via Infotrieve]
  38. Perkins, DN, Pappin, DJ, Creasy, DM, Cottrell, JS. (1999) Probability-based protein identification by searching sequence databases using mass spectrometry data Electrophoresis 20,3551-3567[CrossRef][Medline][Order article via Infotrieve]
  39. Schmid, DG, von der Mulbe, FD, Fleckenstein, B, Weinschenk, T, Jung, G. (2001) Broadband detection electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry to reveal enzymatically and chemically induced deamidation reactions within peptides Anal Chem 73,6008-6013[Medline][Order article via Infotrieve]
  40. Nicoletti, A, Wong, DJ, Kawase, K, et al (1995) Molecular characterization of the human gene encoding an abundant 61 kDa protein specific to the retinal pigment epithelium Hum Mol Genet 4,641-649[Abstract/Free Full Text]
  41. Crabb, JW, Goldflam, S, Harris, SE, Saari, JC. (1988) Cloning of the cDNAs encoding the cellular retinaldehyde-binding protein from bovine and human retina and comparison of the protein structures J Biol Chem 263,18688-18692[Abstract/Free Full Text]
  42. McBee, JK, Palczewski, C, Baehr, W, Pepperberg, DR. (2001) Confronting Complexity: the interlink of phototransduction and retinoid metabolism in the vertebrate retina Prog Retinal Eye Res 20,469-529[CrossRef][Medline][Order article via Infotrieve]
  43. Nohl, H. (1994) Generation of superoxide radicals as byproduct of cellular respiration Ann Biol Clin (Paris) 52,199-204
  44. Cai, J, Nelson, KC, Wu, M, Sternberg, P, Jr, Jones, DP. (2000) Oxidative damage and protection of the RPE Prog Retinal Eye Res 19,205-221[CrossRef][Medline][Order article via Infotrieve]
  45. Robey, HL, Hiscott, PS, Grierson, I. (1992) Cytokeratins and retinal epithelial cell behaviour J Cell Sci 102,329-340[Abstract/Free Full Text]
  46. Fuchs, U, Kivela, T, Tarkkanen, A. (1991) Cytoskeleton in normal and reactive human retinal pigment epithelial cells Invest Ophthalmol Vis Sci 32,3178-3186[Abstract/Free Full Text]
  47. Nagle, RB, Brawer, MK, Kittelson, J, Clark, V. (1991) Phenotypic relationships of prostatic intraepithelial neoplasia to invasive prostatic carcinoma Am J Pathol 138,119-128[Abstract]
  48. Pollard, TD, Cooper, JA. (1986) Actin and actin-binding proteins: a critical evaluation of mechanisms and functions Annu Rev Biochem 55,987-1035[CrossRef][Medline][Order article via Infotrieve]
  49. Arora, PD, Janmey, PA, McCulloch, CA. (1999) A role for gelsolin in stress fiber-dependent cell contraction Exp Cell Res 250,155-167[CrossRef][Medline][Order article via Infotrieve]
  50. Provost, P, Doucet, J, Stock, A, Gerisch, G, Samuelsson, B, Radmark, O. (2001) Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75 Biochem J 359,255-263[CrossRef][Medline][Order article via Infotrieve]
  51. Opas, M, Turksen, K, Kalnins, VI. (1985) Adhesiveness and distribution of vinculin and spectrin in retinal pigmented epithelial cells during growth and differentiation in vitro Dev Biol 107,269-280[CrossRef][Medline][Order article via Infotrieve]
  52. McKay, BS, Irving, PE, Skumatz, CM, Burke, JM. (1997) Cell-cell adhesion molecules and the development of an epithelial phenotype in cultured human retinal pigment epithelial cells Exp Eye Res 65,661-671[CrossRef][Medline][Order article via Infotrieve]
  53. Della Gaspera, B, Braut-Boucher, F, Bomsel, M, et al (2001) Annexin expressions are temporally and spatially regulated during rat hepatocyte differentiation Dev Dyn 222,206-217[CrossRef][Medline][Order article via Infotrieve]
  54. Schlaepfer, DD, Haigler, HT. (1990) Expression of annexins as a function of cellular growth state J Cell Biol 111,229-238[Abstract/Free Full Text]
  55. Huang, KS, Wallner, BP, Mattaliano, RJ, et al (1986) Two human 35 kd inhibitors of phospholipase A2 are related to substrates of pp60v-src and of the epidermal growth factor receptor/kinase Cell 46,191-199[CrossRef][Medline][Order article via Infotrieve]
  56. Masaki, T, Tokuda, M, Ohnishi, M, et al (1996) Enhanced expression of the protein kinase substrate annexin in human hepatocellular carcinoma Hepatology 24,72-81[Medline][Order article via Infotrieve]
  57. Wu, W, Tang, X, Hu, W, Lotan, R, Hong, WK, Mao, L. (2002) Identification and validation of metastasis-associated proteins in head and neck cancer cell lines by two-dimensional electrophoresis and mass spectrometry Clin Exp Metastasis 19,319-326[CrossRef][Medline][Order article via Infotrieve]
  58. de Coupade, C, Gillet, R, Bennoun, M, Briand, P, Russo-Marie, F, Solito, E. (2000) Annexin 1 expression and phosphorylation are upregulated during liver regeneration and transformation in antithrombin III SV40 T large antigen transgenic mice Hepatology 31,371-380[CrossRef][Medline][Order article via Infotrieve]
  59. Williams-Hill, DM, Duncan, RF, Nielsen, PJ, Tahara, SM. (1997) Differential expression of the murine eukaryotic translation initiation factor isogenes eiF4-AI and eIF4-AII is dependent upon cellular growth status Arch Biochem Biophys 333,111-120
  60. Caraglia, M, Budillon, A, Vitale, G, Lupoli, G, Tagliaferri, P, Abbruzzese, A. (2000) Modulation of molecular mechanisms involved in protein synthesis machinery as a new tool for the control of cell proliferation Eur J Biochem 267,3919-3936[Medline][Order article via Infotrieve]
  61. Caraglia, M, Marra, M, Giuberti, G, et al (2001) The role of eukaryotic initiation factor 5A in the control of cell proliferation and apoptosis Amino Acids 20,91-104[CrossRef][Medline][Order article via Infotrieve]
  62. Nishimura, K, Ohki, Y, Fukuchi-Shimogori, T, et al (2002) Inhibition of cell growth through inactivation of eukaryotic translation initiation factor 5A (eIF5A) by deoxyspergualin Biochem J 363,761-768[CrossRef][Medline][Order article via Infotrieve]
  63. Anderson, L, Seilhamer, J. (1997) A comparison of selected mRNA and protein abundances in human liver Electrophoresis 18,533-537[CrossRef][Medline][Order article via Infotrieve]
  64. Gygi, SP, Rochon, Y, Franza, BR, Aebersold, R. (1999) Correlation between protein and mRNA abundance in yeast Mol Cell Biol 19,1720-1730[Abstract/Free Full Text]



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