Citation
Nieuwpoort, A. F. van. (2011, March 16). Biochemical and molecular studies of atypical nevi. Retrieved from https://hdl.handle.net/1887/16632
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management of reactive oxygen species in atypical melanocytes
Frans A. van Nieuwpoort1 Arij Weerheim 1 Coby Out‐Luiting1 Paul Hensbergen2 Femke A. de Snoo3
Stan Pavel1 Wilma Bergman1 Nelleke A. Gruis1
1) Department Of Dermatology, Leiden University Medical Centre, Leiden, The Netherlands 2) Department of Parasitology, Leiden University Medical Centre, Leiden, The Neth‐
erlands 3) Centre for Human and Clinical Genetics Leiden University Medical Centre, Leiden,
The Netherlands
Submitted
Summary
A better understanding of the early stages of melanoma development might contribute to earlier intervention and a better prognosis in patients. In this study we investigated differences in protein expression between normal melanocytes and melanocytes from atypical nevi. Two‐dimensional differential in‐gel electrophoresis (2D‐DIGE) was performed on proteins derived from 18 sets of normal and atypical melanocytes from the same individual. Subsequent liquid chromatography‐tandem mass spectrometry (Maldi‐ToF‐ToF) revealed protein changes that relate to reduced management of oxidative stress in atypical melanocytes. These protein findings are in line with our earlier biochemical observations and results from gene expression analysis on the same material, providing additional evidence that a reduced management of oxidative stress, is a common feature of atypical melanocytes. Further studies are required to define how the sustained stress and accompanying oxidative DNA damage contribute to further melanoma development.
Keywords: (atypical) melanocytes, proteomics, oxidative stress
Introduction
Cutaneous melanoma is a highly malignant tumour which originates from melanocytes, the pigment producing cells of the skin. Melanoma development is a multi‐step process starting with the transition of normal melanocytes into
pigmented nevi which can further grow to form so called clinical atypical (or dysplastic) nevi. These atypical nevi can eventually progress into radial and subsequently vertical growth phase melanoma [1,2]. The complex multistage development process of melanoma is mediated via various cellular, biochemical and molecular changes [3].
Histopathological investigations show characteristic morphological changes, including proliferation and variable atypia of epidermal melanocytes, formation of irregular nests in the epidermal basal and suprabasal layers, and the interconnection of these nests and layers (bridging) [4]. Atypical melanocytes furthermore exhibit morphological alterations in melanosomes and mitochondria, similar to those observed in melanoma cells [4,5].
On the biochemical level we previously showed that atypical melanocytes in comparison with normal melanocytes show an increase in pheomelanin, iron and calcium levels resulting in elevated Reactive Oxygen Species (ROS) levels [6,7].
The ROS levels sustain in the atypical melanocytes due to lower levels of
antioxidant enzymes thereby causing chronic oxidative stress [6,7]. The latter was furthermore confirmed by gene expression profiling and subsequent gene ontology analysis that clearly demonstrated decreased expression of genes involved in the management of oxidative stress (submitted).
In the present study, the same sets of normal and atypical melanocytes used for the gene expression study, were now investigated for differences at the protein level, by using two‐dimensional differential in‐gel electrophoresis (2D‐
DIGE) [8] and liquid chromatography‐tandem mass spectrometry (Maldi‐ToF‐ToF).
In line with the lower expressed genes identified in the GO‐analysis of the gene expression study, the atypical melanocytes showed lower expression of proteins involved in the management of the oxidative status of the cell. This lowered management can lead to increased oxidative stress levels, which might play an important role in the development of oxidative DNA damage and subsequent melanoma promoting DNA mutations.
Materials and Methods Cell cultures
After approval by the Review Board of Leiden University Medical Centre, 18 patients attending the Pigmented Lesions Clinic of the Department of Dermatology consented to have one atypical nevus excised. From the most atypical part of this atypical nevus a small biopsy was taken for review by a pathologist to confirm atypia and to exclude primary melanoma. Fat tissue was discarded and normal skin at the tips of the excision was removed for the purpose of normal melanocyte culture. Both the normal skin and remaining atypical part of the nevus were incubated with trypsin solution containing 0.25% Trypsin 250 (Difco labs, Detroit, MI, USA), 0.02 g EDTA (Mallinckrodt Baker, Deventer, The Netherlands) and 0.1 g d(+)‐Glucose‐monohydrate (Mallinckrodt Baker, Deventer, The Netherlands) for 18 hours at 4 C. The epidermis and dermis from both parts were separated,
collected and resuspended in cell culture medium (Ham’s F10 with penicillin (100 U/ml), streptomycin (100 U/ml), L‐glutamine (all obtained from Invitrogen, Breda, The Netherlands), 1% Ultroser G (Biosepra, Cipbergen inc, Fremont California, USA) including the following growth factors: Endothelin‐1 (5 ng/ml), basic‐FGF (5 ng/ml), Cholera toxin (30 µg/ml), IBMX (33 µM) and TPA (8 nM, all obtained from Sigma Aldrich, Zwijndrecht, The Netherlands). Both the normal and atypical melanocytes were cultured for a maximum of 6 passages while being in the log‐
phase to minimize differences due to cell cycle effects.
Isolation of proteins
For protein isolation, the cells were rinsed with PBS and incubated for 5 min with trypsin solution containing 0.01% Trypsin 250 (Difco labs, Detroit, MI, USA), 0.02 g EDTA (Mallinckrodt Baker, Deventer, The Netherlands) and 0.1 g d(+)‐Glucose‐
monohydrate (Mallinckrodt Baker, Deventer, The Netherlands). The reaction was terminated by adding 4 ml of cell culture medium. Cells were spun down for 5 min at 1100 rpm. The cell pellet was resolved in 1 ml of TRIzol® reagent (Invitrogen, Paisley, Scotland, United Kingdom). Total protein was obtained using TRIzol®
reagent (Invitrogen, Life Technologies, Breda, The Netherlands) according to the manufacturer’s instructions. After isolation the total protein was stored pelleted at
−20 °C. Prior to use in 2D gel electrophoresis, the air‐dried protein pellets were solubilised in 9 mol/L urea, 2% Chaps (3‐[(3‐cholamidopropyl)dimethylammonio]‐
1‐propanesulphonate), 0.5% IPG buffer (=isoelectric focusing buffer, Amersham–
Pharmacia Biotech, Roosendaal, The Netherlands), and 5 mg/mL dithiothreitol. The protein concentration was measured by the BCA‐Protein Assay Kit (colorimetric) from Pierce (Rockford, IL, USA) according to the manufacturer’s protocol.
Labelling of the proteins with cy‐dyes
Samples were labelled using the fluorescent cyanine dyes developed for 2‐D DIGE (Amersham–Pharmacia Biotech) following the manufacturer’s recommended protocols. Protein (10 ug) was labelled with 1000 pmol of amine reactive cyanine dyes, freshly dissolved in anhydrous dimethyl formamide. The labelling reaction was incubated at room temperature in the dark for 30 min. The reaction was terminated by addition of 10 nmol lysine. Equal volumes of sample buffer (7 M urea, 2 M thiourea, 2% amidosulfobetaine‐14, 20 mg/mL DTT and 2% Pharmalytes 3–10) were added to each of the labelled protein samples and the two samples were mixed.
Rehydration buffer (7 M urea, 2 M thiourea, 2% amidosulfobetaine‐14, 2 mg/mL DTT and 1% Pharmalytes 3–10) was added to make up the volume to 250 ml prior to 2D‐DIGE. To check for labelling differences between cy3 and cy5 dye swap experiments were performed.
2D‐DIGE
2D electrophoresis was performed as described previously [9]. Briefly, the protein mix was loaded on 18‐cm isoelectric focusing ready‐made IPG (Immobilised pH Gradient) strips with a non‐linear gradient of pH 3–10 (Amersham–Pharmacia Biotech). Rehydration of the IPG strips was performed for 12 h at 30 V after which proteins were focused for 65,000 V h (IPGphor, Amersham–Pharmacia Biotech).
Prior to the second dimension, IPG strips were equilibrated in 1% dithiothreitol (w/v) followed by 2.5% iodoacetamide (w/v), both for 15 min in 50 mM Tris–HCl, pH 8.8, 6 M urea, 30% glycerol, and 2% SDS (sodium dodecyl sulphate). After this procedure, the strips were placed on top of a polyacrylamide gel (13%
homogeneous, 2.6% cross‐linking, 0.1% SDS, and 375 mM Tris–HCl, pH 8.8) and run for 45 min at 5 W/gel followed by 4 h at 15 W/gel (Ettan Dalt, Amersham–
Pharmacia Biotech). Gels were scanned in low‐fluorescent glass plates using a Typhoon 9400 scanner (GE Health care UK ltd., Buckinghamshire, UK, equipped with DeCyder™ 2 Software v6.5 ) at 600 V with excitation wavelengths for
fluorescence Cy3 532 nm and Cy5 633 nm with a resolution of 100 micron resulting in 2 images per gel. Images were saved as 16 bit tagged image file format (TIFF) files (65,536 levels) with standard image orientation.
Data analysis of 2D‐DIGE
All TIFF images of the 2D‐DIGE gels were analysed using Delta2D v3.4 software (Dodecon, Greifswald, Germany) to detect spots. After spot detection the spots were quantified and normalised based on the total spot volume of one gel and the matched spot volumes were subsequently normalised across the whole set. The normalised spot volumes were compared between images in the set. Statistical significant differences between normalised spot volumes were determined using the Welch‐modified Student’s t‐test and step‐down Westfall and Young method as a correction for the false discovery rate resulting in 99 statistically differently expressed protein spots.
In‐gel digestion and peptide mass finger print by MALDI‐ToF‐ToF
Protein spots of interest were excised, and dehydrated in 100% acetonitrile for 10 min. After removal of the acetonitrile, gel pieces were dried in a Speed‐Vac (Savant Instruments, Holbrook, New York) and subsequently allowed to re‐swell in a trypsin solution (20 ng per mL (Promega Benelux, Leiden, the Netherlands)) in 50 mM ammonium bicarbonate (pH 7.9) for 45 min on ice. Further incubation was performed overnight at 37 °C. Peptide fragments were extracted for 20 min at RTusing 100 mL 25 mM ammonium bicarbonate, followed two times by an extraction using 60 mL 0.1% trifluoric acid/50% acetonitrile. The extraction solutions were pooled, concentrated to approximately 30 mL (Speed‐ Vac, Savant Instruments), and desalted over Poros 50R2 (Applied Biosystems, Forster City, California). For characterization by MALDI‐ToF (Matrix Assisted Laser Desorption/
Ionization‐Time of Flight) MS (Ultraflex, Bruker, Germany), peptides were directly eluted with matrix solution (a‐cyanocinnamic acid). All mass fingerprint and MS/MS data were searched against the human protein database using the Mascot program (Matrix Science, Boston, Massachusetts).
Results
Protein analysis by 2D‐DIGE
Eighteen sets of normal and atypical melanocytes were subjected to 2D‐
DIGE to determine protein expression alterations characteristic for this very early step in melanoma development. In figure 1, a representative example of a comparison of proteins of normal melanocytes (green, cy3) and its corresponding protein expression in atypical melanocytes (red, cy5) is shown. To check for labelling efficiency between cy3 and cy5 a dye swap on various samples was performed. The dye swap did not indicate large differences in the labelling efficiency of the same sample with either cy3 or cy5 (data not shown).
Each of the 2D‐DIGE‐gels contained on average 2205 spots, 90% of these spots were reproducible in at least 15 gel sets. Protein spots (n=99) that were differentially expressed between normal and atypical melanocytes in at least 15 gel sets (determined by the Welch‐modified Student’s t‐test and step‐down Westfall and Young method) were excised and subsequent Maldi‐ToF‐ToF analysis and database mining resulted in the annotation of 70 proteins (Supplementary table 1). In table 1 the 16 most differentially expressed proteins (p<0.03 and at least 2‐fold expression difference) are given. Proteins most significantly lower expressed in atypical melanocytes were ribosomal proteins S6, S7 and L9. These mitochondrial related proteins are involved in the protein translation process in mitochondria [10,11].
The proteins glutathione‐S‐transferase Omega, ferritin heavy chain and cytochrome b‐c1 complex subunit Rieske, HSP27, ATP synthase and tyrosine 3‐
monooxygenase activation protein were also found to be expressed at much lower levels in the atypical melanocyte. These proteins are involved in the management of oxidative stress [12‐15], the energy production of the cell [16] and eumelanin synthesis [17]. Other proteins in atypical melanocytes we found to be expressed at lower levels were vimentin, annexin A2, which are respectively part of the actin filament and the intermediate filament system of the melanocyte [18,19].
Proteins found to be higher expressed in atypical melanocytes were eukaryotic translation initiation factor 3, GTP‐binding rho‐like protein cdc42, prolyl 4‐hydroxylase, platelet‐activating factor acetylhydrolase and DnaJ homolog subfamily B. These proteins play a role in protein synthesis and folding [20‐24].
Discussion
In order to define changes in protein expression in the earliest stage of melanoma development, we compared protein profiles within 18 sets of normal melanocytes and melanocytes obtained from atypical nevi. The protein profile comparison and subsequent peptide analysis resulted in the identification of 70 differentially expressed proteins.
Melanocytes from atypical nevi showed much lower expression of mitochondrial related proteins (MRPs): ribosomal proteins S6, S7 and L9. These MRPs are involved in the synthesis of the proteins involved in the electron
transport chain located within the mitochondrion. The low expression of the MRPs in atypical nevi could lead to a malfunctioning of the electron transport chain leading to accumulation of electrons which leak into the cytosol of the cell giving rise to reactive oxygen species (ROS) [25]. In addition, we found lower expression of cytochrome bc1 complex Rieske, which plays a role in maintaining the structure of the mitochondrial membrane [26]. The lower expression of this protein
increases leakage of electrons out of the mitochondria resulting again in increasing ROS levels [26] in atypical melanocytes.
In contrast to the lowered expression of MRPs in atypical nevi in our study, Simon et al. observed over‐expression of the ribosomal proteins MRPL3, MRPS11 and MRPS10 in late stage metastatic cell lines [27]. They suggest that the up‐regulation of these ribosomal proteins could reflect the higher level of
metabolic activity in melanoma cells as compared to normal melanocytes.
Therefore, more detailed studies on MRPs are needed to define the precise role and functions of the MRPs in relation to melanoma development and progression.
Previous biochemical studies by our group already suggested that atypical melanocytes have a reduced capacity to reduce oxidative stress levels, which can be partly explained by down‐regulation of the antioxidant enzyme glutathione [6,7]. The results in the current study indicate that other antioxidant enzymes such as glutathione‐S‐transferase, ferritin and HSP27 are down‐regulated in the atypical melanocyte as well. Especially the diminished protein expression of glutathione‐S‐
transferase is of interest, since this enzyme catalyses the interaction of glutathione with a wide variety of compounds thereby being the first step in the ROS reducing process [28,29]. It is anticipated that the lowered protein expression of
glutathione‐S‐transferase, besides the already reduced level of glutathione itself, could underlie the elevated levels of ROS in atypical melanocytes [6,7].
Expressed at lower levels were the cytoskeletal proteins vimentin and annexin in atypical melanocytes, which are respectively part of the actin filament or the intermediate filament system. The actin filaments in the cell provide structural support and allow the cell to move [30]. Vimentin is regarded as a cytoskeletal safeguard, which protects the cell against various stresses such as heat shock and oxidative stress [31‐35]. A change in cell morphology is known to be one of the consequences of oxidative stress injury [36‐39]. Therefore lowered expression of both the vimentin and the actin filaments could explain the morphological atypia of melanocytes in atypical nevi [4,6,7].
The group of Mirabelli et al. showed that actin not only becomes oxidised by ROS but also by quinones [36,40]. Especially in the atypical melanocyte the oxidation of actin by quinones can be of importance since these melanocytes display elevated levels of pheomelanin intermediates (which are quinones) as a result of increased pheomelanin synthesis. We also observed a lower level of ferritin, the iron store of the cell. Decreased levels of ferritin increase ROS
formation by releasing free iron that acts as a catalyst for hydroxyl radicals [41‐43].
Melanin precursors can also liberate iron from ferritin, which can subsequently increase the level of ROS via the Fenton reaction [6,44].
Therefore the increased pheomelanin production in the atypical melanocytes could be regarded as a plausible cause underlying several of the observed protein differences.
Proteins that were higher expressed in atypical melanocytes compared to normal melanocytes were eukaryotic translation initiation factor 3, GTP‐binding rho‐like protein cdc42, prolyl 4‐hydroxylase, platelet‐activating factor
acetylhydrolase and DnaJ homolog subfamily B. These proteins are involved in protein synthesis and folding [20‐24]. In Alzheimer’s disease and Parkinson’s disease it is known that due to high levels of ROS proteins show problems with correct protein folding and this could be anticipated in atypical melanocytes as well [45‐49]. Further research is needed to determine the exact role of protein misfolding in melanoma development and progression.
In addition to our study, only a few proteomic studies have been published focussing mainly on the late melanoma development stages [50‐54].
Bernard et al. compared 12 melanoma cell lines representing radial growth phase melanoma, vertical growth phase melanoma and metastatic tumours, with two melanocyte cell cultures to identify candidate molecular markers for melanoma progression [53]. In total only 8 proteins were identified as candidate markers of melanoma progression. Proteins up‐regulated in melanoma in comparison to normal melanocytes were hepatoma derived growth factor (HDGF) and
nucleophosmin/B23. Down‐regulated proteins in melanoma were cathepsin D and glutathione‐S‐transferase. HDGF has DNA‐binding activity and may play a role in cellular proliferation and differentiation [55]. Nucleophosmin/B23 is implicated in multiple functions, including ribosomal protein assembly and transport [56].
Cathepsin D is located in the lysosomes and melanosomes and is involved in protein breakdown [57]. Lowered glutathione‐S‐transferase was the only protein in common with our analysis, suggesting that most proteins found in the study from Bernard et al. are associated with late stage melanoma progression.
Also Carta et al. studied protein expression differences between short‐
term cultured normal melanocytes and primary and metastatic melanoma cell lines albeit from one patient to investigate the presence of pathogenic mechanisms playing a role in melanocytic transformation and melanoma
development [54]. Thirty‐seven differentially expressed proteins were identified in this study. The authors classified the differential proteins into three groups:
structural proteins, stress proteins and enzymes in ATP production. Most of the proteins in the stress protein group corresponded to heat shock proteins (HSP).
HSPs function as a chaperone of proteins, protecting them from environmental stress and HSPs play a role in transporting proteins to their target organelles [58,59].
With respect to the stress protein group, Carta et al. observed an up‐regulation of HSP 27, 60 and 70 whereas the antioxidant enzymes glutathione transferase omega, thiodoxin‐dependent peroxide reductase were down‐regulated in all melanoma cells [54]. Over expression of HSP60 and HSP70 has been described in various cancer types, including malignant melanoma [60]. In our study we
observed a lower expression of HSP27 in the atypical melanocytes, which contrasts to the results of Carta et al. A role for HSP27 to prevent actin depolymerisation has been described in various studies as an adaptive response to oxidative stress [61‐
63]. As mentioned above, actin filaments are a key role player in providing a cell structural support. The lowered HSP27 level that we observed in atypical
melanocytes in comparison with normal melanocytes is another factor that could contribute to the observed morphological atypia of melanocytes in atypical nevi [4,6,7].
In contrast to the studies of Bernard et al. and Carta et al. who
concentrated on the late melanoma progression stages, our study focused on the early stage of melanoma progression. Most of our differentially expressed proteins show that atypical melanocytes are insufficient in the management of oxidative stress in comparison with their normal counterpart.
These findings are consistent with the results of previous biochemical and gene expression studies of our group [6,7], and further support the view that oxidative stress may play an important role in the early stages of melanoma development [64‐67]. There is supportive evidence that sustained oxidative stress is related to oxidative DNA damage [68]. Whether oxidative DNA damage furthermore introduces genetic alterations leading to malignant transformation is not yet clear and needs to be the subject of further study.
Acknowledgements
Prof. W. Mooi (Department of Pathology, Free University Medical Center, Amsterdam, The Netherlands) is acknowledged for histopathological analysis of the excised nevi. F.v.N. is supported by the Netherlands Organization for Scientific Research (grant number 015.001.060) and by the Dutch Cancer Society (grant number RUL2003‐2805). N.G. is a recipient of an Aspasia fellowship of the Netherlands Organization for Scientific Research.
Table I: Top 16 differentially expressed proteins in normal and atypical melanocytes determined by 2D-DIGE.
Swiss‐
prot id Protein name
Mol. Mass calculated (Da)1
pI obs.
2
pI calc.3
% Pept.
Coverage4 Ratio (AM
/ NM)5 P‐value General function
Q9Y2R9 Ribosomal protein S7 22113 10.09 10 43 0.1310 0.010946 Translational elongation
P32969 Ribosomal protein L9 21964 9.96 9.96 29 0.22334 0.016167 Translational elongation
P78417
Glutathione S‐transferase omega 1‐
1 27833 6.23 6.24 21 0.23627 0.013815
Exhibits glutathione‐dependent thiol transferase and dehydroascorbate reductase activities
P62753 Ribosomal protein S6 28842 10.9 10.85 23 0.31890 0.012139
Controlling cell growth and proliferation through the selective translation of particular classes of mRNA
O75947
ATP synthase, H+ transporting, mitochondrial F0 complex, subunit
d 18537 5.21 5.21 41 0.32135 0.027965
Mitochondrial membrane ATP synthase that produces ATP from ADP
P04792 Heat shock protein 27 22427 7.83 5.98 57 0.36622 0.023047 Involved in stress resistance and actin organization.
P02794 Ferritin heavy chain 21252 5.3 5.08 46 0.40067 0.006873
Stores iron in a soluble, non‐toxic, readily available form. Important for iron homeostasis.
Q59EQ2
Tyrosine 3‐monooxy‐
genase/tryptophan 5‐
monooxygenase activation protein 28179 4.76 4.73 26 0.41836 0.012436 oxido‐reductase
P08670 Vimentin 53710 5.06 5.06 43 0.4478 0.023374 Vimentins are class‐III intermediate filaments
P07355 Annexin A2 36631 8.32 7.56 53 0.47142 0.00642 May be involved in heat‐stress response
Swiss‐
prot id Protein name
Mol. Mass calculated
pI obs.
2
pI calc.3
% Pept.
Coverage4
P47985
Cytochrome b‐c1 complex subunit
Rieske 29934 8.55 6.3 5 0.47725 0.008578
Component of the ubiquinol‐cytochrome c reductase complex
Q5U0F4
Eukaryotic translation initiation
factor 3 36878 5.38 5.38 32 3.2155 0.018194 protein synthesis
O94844 GTP‐binding rho‐like protein cdc42 20123 5.25 4.83 55 2.88699 0.017896
The protein may play a role in small GTPase‐mediated signal transduction and the organization of the actin filament system.
P07237 Prolyl 4‐hydroxylase 57480 4.76 4.69 46 2.79651 0.009987
This multifunctional protein catalyzes the formation, breakage and rearrangement of disulfide bonds.
Q6IBR6
Platelet‐activating factor
acetylhydrolase 25569 5.57 5.57 2.46963 0.008975 Lipid metabolic process
Q9UBS4 DnaJ (Hsp40) homolog, subfamily B 40774 5.81 5.81 30 2.33737 0.011076
Binds directly to both unfolded proteins that are substrates for ERAD and nascent unfolded peptide chains.
1) Calculated molecular mass of the protein in Dalton (Da) 2) Isoelectric point (pI) observed in this study
3) pI calculated according to the Mascot database 4) Percentage peptide coverage
5) Protein ratio atypical melanocyte (AM) / normal melanocyte (NM)
Figure 1: representable 2D‐DIGE image of a comparison of normal melanocytes (labelled with cy3 (green)) compared to the atypical melanocytes (labelled with cy5 (red)) obtained from the same individual. A yellow colour means equal expression off a protein in both normal and atypical melanocyte, red is over expression in atypical melanocytes while green depicts over expression of a protein in the normal melanocyte. The labelled arrows show spots that were excised and identified on the mass spectrometer. To help identification of spots throughout the gel set also not differentially expressed proteins were excised.
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