Proteomic Analysis of MCF-7 Cell Lines Expressing the Zinc-Finger or

Dec 21, 2005 - dell'Alimentazione del CNR, Avellino, I-83100, Italy, and Dipartimento di Scienze per la Salute,. Universita` degli Studi del Molise, I...
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Proteomic Analysis of MCF-7 Cell Lines Expressing the Zinc-Finger or the Proline-Rich Domain of Retinoblastoma-Interacting-Zinc-Finger Protein Angela Chambery,† Annarita Farina,† Antimo Di Maro,† Mariangela Rossi,‡ Ciro Abbondanza,‡ Bruno Moncharmont,| Livia Malorni,§ Giuseppina Cacace,§ Gabriella Pocsfalvi,§ Antonio Malorni,§ and Augusto Parente*,† Dipartimento di Scienze della Vita, Seconda Universita` degli Studi di Napoli, I-81100 Caserta, Italy, Dipartimento di Patologia generale, Facolta` di Medicina e Chirurgia, Seconda Universita` degli Studi di Napoli, Napoli, I-80138, Italy, Centro di Spettrometria di Massa Proteomica e Biomolecolare, Istituto di Scienze dell'Alimentazione del CNR, Avellino, I-83100, Italy, and Dipartimento di Scienze per la Salute, Universita` degli Studi del Molise, I-86100 Campobasso, Italy Received December 21, 2005

To identify a growth-promoting activity related to retinoblastoma-interacting-zinc-finger (RIZ) protein, differential protein expression of MCF-7 cell lines expressing the zinc-finger or the proline-rich domain of RIZ protein was analyzed by a robust bottom-up mass-spectrometry proteomic approach. Spots corresponding to qualitative and quantitative differences in protein expression have been selected and identified. Some of these proteins have been previously reported as being associated with different types of carcinomas or involved in cell proliferation and differentiation. Knowledge of specific differentially expressed proteins by MCF-7-derived cell lines expressing RIZ different domains will provide the basis for identifying a growth-promoting activity related to RIZ gene products. Keywords: two-dimensional gel electrophoresis • mass spectrometry • proteomics • RIZ domain • peptide mass fingerprint

Introduction The retinoblastoma-interacting zinc-finger protein (RIZ) defines a series of gene products generated by the presence of alternative promoters and/or by alternative splicing of the transcripts.1 The two major gene products, RIZ1 and RIZ2, are indeed produced by alternative promoters and they only differ for the presence in the larger form (RIZ1) of an additional N-terminal 201-amino acid region, containing a 100-amino acid long PR domain (Figure 1). Previous data indicated that RIZ gene, for its RIZ1 gene product, is a putative tumor suppressor gene.2,3 In fact, forced expression of the PR-containing form of RIZ gene can induce growth arrest and apoptosis of cultured cancer cells, while deletions or inactivating mutations of the chromosomal region containing the gene (1p36) have been observed in several human cancer cell lines, as well as in solid tumors.4 Distinct functional regions have been identified in the common part of both gene products. An Rb-binding motif, sharing structural and antigenic similarities with the adenovirus * To whom correspondence should be addressed Tel: +39 0823 274583. Fax: +39 0823 274571. E-mail: [email protected]. † Dipartimento di Scienze della Vita, Seconda Universita` degli Studi di Napoli. ‡ Dipartimento di Patologia generale, Facolta` di Medicina e Chirurgia, Seconda Universita` degli Studi di Napoli. § Centro di Spettrometria di Massa Proteomica e Biomolecolare, Istituto di Scienze dell’Alimentazione del CNR. | Dipartimento di Scienze per la Salute, Universita` degli studi del Molise.

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Journal of Proteome Research 2006, 5, 1176-1185

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E1A oncoprotein, suggested a role for RIZ protein as an endogenous ligand of Rb.5 Two clusters of putative zinc-fingers, probably responsible for the initial identification of this protein as a transcription factor, are present in the central part of the molecule. A proline-rich domain, located between the two zincfinger clusters, contains a site for interaction with the estrogen receptor: a LXXLL motif identified in transcriptional coactivators interacting with nuclear receptors. RIZ protein interacts with the hormone-binding domain of estrogen receptor in a hormone-dependent manner.6 A more intriguing feature of this gene is that it is probably involved in cancer pathogenesis in a peculiar yin-yang fashion, because the shorter PR-minus gene product is always expressed in cancer cells, even in those where RIZ1 expression is disrupted. This is suggestive of a positive selection for RIZ2 expression, probably dependent on a positive effect of this gene product on cell proliferation. To identify a growth-promoting activity related to RIZ gene products, two permanently transfected MCF-7-derived cell lines were produced with cDNAs coding for a fusion protein containing the zinc-finger (MCF-7/znf, aa 359-497) or the proline-rich (MCF-7/ppp, aa 952-1052) domains of RIZ protein (Figure 1). The Zn-finger domain contains three of the eight putative Zn-finger motifs and is located in proximity of the E1Alike domain containing the Rb protein-binding motif. The proline-rich domain contains the LXXLL motif responsible for 10.1021/pr0504743 CCC: $33.50

 2006 American Chemical Society

MCF-7 Cell Lines Overexpressing RIZ Subdomains

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Figure 1. Schematic representation of RIZ gene products. Zinc-finger I (ZnF) and the proline-rich (ppp) domains of the RIZ protein are indicated with dashed lines.

the interaction with estrogen receptor. Previous studies indicated that the MCF-7/znf cells proliferated at a higher rate than the control, thus suggesting that the Zn-finger domain could be endowed with the putative oncogenic activity of RIZ2 gene product.7 Recombinant fragments of the entire protein were used in this study in order to circumvent the inability to forcedly express a complete RIZ2 protein.7 However, the existence of small RIZ gene-derived proteins could be suspected for the presence in databases of splicing variants, detected as EST. In the present study, we characterized, by a robust “bottom up” mass spectrometry proteomic approach, differential protein expression in MCF-7/znf and MCF-7/ppp cell lines compared to MCF-7/green human breast cancer cell line, used as control.

Experimental Section Materials. Chemical reagents and trypsin TPCK-treated were from Sigma (Milan, Italy). Immobilized pH gradient (IPG) buffers, IPG strips, and electrophoresis apparatus were purchased from Amersham-Biosciences (Milan, Italy). Electrophoresis reagents, including acrylamide, N,N-methylenebisacrylamide, N,N,N′,N′-tetramethylethylenediamine, ammonium persulfate and sodium dodecyl sulfate (SDS) were from BioRad (Milan, Italy). All other reagents were of analytical grade. Plasmids Preparation and Cell Culture. MCF-7/znf, MCF7/ppp and MCF-7/green cell lines were produced as previously reported7. Cells were grown in 75-cm2 flasks in Dulbecco’s modified Eagle’s medium (DMEM), additioned with 2 mM L-glutamine, 100 U/mL penicillin G, 0.1 mg/mL streptomycin, 10% FCS in a 5% CO2 environment at 70-80% confluence. All tissue culture reagents were from Invitrogen Corporation, Carlsbad, CA. Sample Preparation. Monolayer cultures of the three cell lines were harvested, after three washes in ice-cold PBS, by incubating them with a solution containing trypsin (0.5 g/L) and EDTA (0.2 g/L). After centrifugation at 13 000 rpm for 15 min at 4 °C in a F2402H rotor (Beckman centrifuge GS-15R), cell pellets were washed and 0.8 × 106-cell aliquots were mixed and lysed with 60 µL of lysis buffer (40 mM Tris-Cl containing

8 M urea, 4% CHAPS, 65 mM DTT and 1 mM PMSF). Total proteins were extracted by freeze-thawing several times the cell pellet in liquid nitrogen. Samples were further centrifuged at 13 000 rpm for 15 min at 4 °C, to eliminate cellular debris, and sonicated. The supernatant was collected and protein concentration determined by the Bradford method, according to manufacturer’s instructions (Biorad, Milan, Italy). Lysates were aliquoted and stored at -80 °C until use. Two-Dimensional (2-D) Gel Electrophoresis. Triplicate gels were made for each cell line, and a total amount of 1 mg of protein per gel was analyzed, according to manufacturer’s instructions. Samples to be processed by isoelectrofocusing (IEF), were diluted with rehydratation buffer (8 M urea, 0.5% CHAPS, 0.2% DTT, 0.5% immobilized pH gradient ampholytes and 0.002% bromophenol blue). The precast IPG strips (3-10 linear pH gradient, 18 cm long, Amersham Biosciences, Milan, Italy), used for the first dimension, were passively rehydrated with 340 µL of rehydratation buffer at room temperature for 12 h under low-viscosity paraffin oil. IEF was then performed in a IPGphor isoelectric focusing cell (Amersham Biosciences, Milan, Italy), according to the following protocol: 500 V for 700 Vh, 1000 V for 1400 Vh, 8000 V for 34500 Vh. Strips were then equilibrated twice for 15 min with gentle shaking in equilibration solution (6 M urea, 50 mM Tris-Cl buffer, 30% glycerol, 2% SDS, 0.002% bromophenol blue) additioned with 1% DTT in the first equilibration step and 2.5% iodoacetamide to alkylate thiols, in the second. Separation by protein mass was performed in an Ettan DALTsix Electrophoresis Unit on homogeneous polyacrylamide gels (12% T, 0.03% C). The equilibrated strips were sealed to the top of the vertical gel with agarose (agarose 0.5% and trace of bromophenol blue dissolved in SDS/Tris running buffer) and electrophoresis was carried out at constant power (2.5 W/gel for 30 min followed by 100 W for 4 h and 30 min) and temperature (20 °C), until the blue dye reached the bottom of the gel. At the end of the electrophoresis, the protein spots were visualized, after washing gels in 50% methanol/0.1% acetic acid for 2 h, by incubating gels with colloidal Coomassie blue stain (2% phosphoric acid, 10% ammonium sulfate, 20% methanol, 0.1% Coomassie brilliant blue G-250) for 4-8 h, followed by three individual 2-h washes Journal of Proteome Research • Vol. 5, No. 5, 2006 1177

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with deionized water. Proteins were classified as being differentially expressed between the three cell lines when differences in spot intensity were consistently observed in two or more experiments. Gel Scanning and Image Analysis. Resulting 2-D gels were imaged and analyzed for comparison, to provide data on matching spots. Gels were scanned using a Molecular Dynamics densitometer, model 375-A (Amersham Biosciences, Milan, Italy). Gel image analysis was processed for spot detection, background subtraction and matching using ImageMaster 2D Elite 3.1 software (Amersham Biosciences). Intensity of each spot was quantified by calculation of spot volume after normalization of the image using the total spot volume normalization method.8 Relative intensity (RI ) vi/vt) of each spot was calculated by dividing volume of the spot by the total volume of the detected spots on the gels, multiplied by the total area of all the spots. Spots were described as showing qualitative variation when they were either present or absent in comparison with the reference gel, and quantitatively different to corresponding control when their normalized total volume values differed significantly at P < 0.05, based on ANOVA analysis. Protein Identification by MALDI-TOF Mass Spectrometry. Selected differential protein spots were excised from gels and destained by washing twice with 100 µL aliquots of water and performing a further washing step with 50% acetonitrile. The gel pieces were then dried in a SpeedVac Vacuum (Savant Instruments, Holbrook, NY) and rehydrated with 10 µL of 50 mM ammonium bicarbonate, followed by the addition of 5 µL of a 70 ng/mL TPCK porcine trypsin solution. Digestion was performed by incubation at 37 °C for 3 h. Further amounts of buffer solution without trypsin was added when necessary to keep the gel pieces wet during the digestion. Peptides were extracted in two steps by sequential addition of 1% trifluoroacetic acid (TFA) and then of 2% TFA/50% acetonitrile for 5 min in a sonication bath. The combined supernatants were concentrated in the SpeedVac Vacuum for mass spectrometry analysis. When necessary, the tryptic peptide mixture was extracted and purified with Millipore ZIPTIP C18 column (Milan, Italy). After in situ tryptic digestion proteins were identified by peptide mass fingerprint (PMF) based on matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), as follows. Tryptic peptides were mixed with an equal volume of saturated R-cyano-4-4-hydroxycinnamic acid matrix solution [10 mg/mL in ethanol:water (1: 1; v:v), containing 0.1% TFA] and spotted onto a MALDI-TOF target plate. The droplet was dried at room temperature. Once the liquid was completely evaporated, the sample was loaded into the mass spectrometer and analyzed. Peptide spectra were collected on a MALDI LR mass spectrometer (Waters Corporation, Milford, MA) in the positive ion reflectron mode. The instrument was externally calibrated using a tryptic alcohol dehydrogenase digest (Waters, Milford, MA) as standard. The protonated monoisotopic mass of ACTH peptide (m/z 2465.199) was used as internal lock mass to further improve the peptide mass accuracy to within 50 ppm. All spectra were processed and analyzed using the MassLynx 4.0 software (Waters, Milford, MA). The obtained spectra were used to identify proteins in the SWISSPROT protein sequence database by using Protein Lynx Global Server 2.0 software. The following searching parameters were used: mass tolerance 50 ppm; allowed number of missed cleavage sites up to 1; cysteine residue 1178

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Figure 2. Representative 2DE image obtained from MCF-7/green (A), MCF-7/ppp (B) and MCF-7/znf (C) cell lines. 2DE were performed using a pH linear range of 3-10 in the first dimension and SDS-PAGE in the second. Circles visualize qualitatively and quantitatively changing spots among the three Coomassie blue stained electropherograms. Spots number correspond to identified proteins reported in Table 1.

modified as carbamidomethyl-cys; minimum number of matched-peptides 3; the isotope masses were used. Immunoblotting. For Western blot analysis aliquots corresponding to equal amounts (20 µg of proteins) from MCF/ green, MCF-7/znf and MCF-7/ppp cell lysates were resolved by 12% SDS-PAGE and transferred onto nitrocellulose membrane (Sartorius, Go¨ttingen, Germany) with an electroblot apparatus (Bio-Rad, Milano, Italy), according to the manufac-

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Figure 3. Peptide mass fingerprinting of protein spot P8 in MCF-7/ppp cell line 2DE map.

turer’s instructions. The membrane was blocked with 5% BSA in PBS for 1 h at 37 °C and washed with PBTS (0.1% Tween-20 in PBS, w/v). Subsequently, it was incubated with anti-enolase (H-300, sc-15343, Santa Cruz Biotechnology, CA) diluted 1:1000 in PBTS for 1 h at 37 °C and then, after washing with PBTS, with alkaline phosphatase-conjugated secondary antibody (goat anti-rabbit IgG; Sigma, Milan, Italy) diluted 1:1000 in PBTS for 1 h at 37 °C. Immunoreactive protein bands were visualized by color development with 5-bromo-4-chloro-3-indolyl-phosphate and nitro blue tetrazolium (Fast BCIP/NTB buffered substrate tablet, Sigma).

Results 2-D gel Analysis of Differentially Expressed Proteins. Total cell proteins from two permanently transfected MCF-7-derived cell lines coding for a fusion protein containing the zinc-finger (MCF-7/znf) or the proline-rich (MCF-7/ppp) domains of RIZ protein were compared by 2-D gel electrophoresis (2DE) to the MCF-7/green human breast cancer cell line, used as a control. For a reliable analysis of protein expression, 2-D gel maps of each cell line were performed and analyzed in triplicate. The pattern of moderately to darkly stained proteins on a 2DE gel, after colloidal Coomassie blue staining, was reproducible between several runs. Representative 2DE gel maps for MCF7/green, MCF-7/ppp and MCF-7/znf cell lines are displayed in Figure 2A, B, and C, respectively. The MCF-7/ppp and MCF7/znf maps were compared with the control-2DE MCF-7/green map for spot matching. A total of 29 unmatched spots between MCF-7/znf or MCF-7/ppp and MCF-7/green cell lines was detected by 2DE gel image analysis software. MALDI-TOF-MS PMF Analysis of the Differential Protein Spots. The selected proteins were identified for their peptide mass fingerprint (PMF) by MALDI-TOF mass spectrometry. To determine the accuracy of the matched result, two matched

spots (G4-G5, P13-P14, and Z13-Z14 in MCF-7/green, MCF-7/ ppp and MCF-7/znf cell lines, respectively) were identified with MALDI-TOF-MS. The results showed that the selected matched spots on the three maps contained the same protein, identified as the heat-shock protein (HSP 27) involved in stress resistance and actin organization (SWISS-PROT accession no. P04792). The 29 differential protein spots, detected comparing MCF-7/ ppp and MCF-7/znf cells to the control cell line, were excised from the Coomassie blue-stained gels, in situ digested with trypsin and analyzed by MALDI-TOF-MS, as described in the Methods section. Figure 3 shows an example of the PMF map of the protein spot P8. Data of the proteins identified in the SWISS-PROT protein sequence database are reported in Table 1, where the main properties of the proteins are summarized (theoretical pI, Mr, number of matched peptides, sequence coverage and matching probability). Several differential protein spots were identified in both MCF-7/znf and MCF-7/ppp cell lines compared to MCF-7/green. Some of these proteins were previously reported as being associated with different types of carcinomas or involved in cell proliferation and differentiation. Energy Metabolism. We identified one differentially expressed glycolytic enzyme involved in energy metabolism. In both MCF-7/znf and MCF-7/ppp cell lines we found increased amounts of alpha-enolase isoforms (Figure 4A; SWISS-PROT accession number: P06733, spots P8 and P9 in MCF-7/ppp and Z8 and Z9 in MCF-7/znf) compared to MCF-7/green (spots G2 and G3). Western blotting experiment using an immunospecific antibody confirmed the overexpression of enolase (Figure 4B), which catalyses the dehydration of 2-phosphoglycerate to phosphoenolpyruvate. RNA Metabolism. Two isoforms of a protein implicated in the regulation of RNA metabolism were upregulated in MCF7/znf and MCF-7/ppp cell lines. The overexpression of a splicing factor (SF2), arginine/serine rich 1 (Figure 4B; SWISSJournal of Proteome Research • Vol. 5, No. 5, 2006 1179

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Figure 4. Example of differential expression of protein involved in energy metabolism (A), RNA maturation and protease (B) and detoxification of cytotoxic product (C, D). Representative subsections of 2DE images are shown for R-enolase (A), SF2 and cathepsin D (C), Hsp70 (D) and PDI (E). Spots ID is numbered according to Table 1. (B) Representative Western blot for enolase in MCF-7/green (1), MCF-7/ppp (2), and MCF-7/znf (3). Table 1. Identification of Differentially Expressed Proteins in MCF-7/Green (G series), MCF-7/ppp (P series) and MCF-7/Znf (Z series) Cell Lines spot ID

G1 G2 G3 G4 G5 G6 G7 P1 P2 P3 P5 P6 P7 P8 P9 P10 P11 P12 P13 P14 P15 P16 Z1 Z2 Z3 Z7 Z8 Z9 Z10 Z11 Z12 Z13 Z14 Z15 Z16

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protein identified

accession no.

coverage

% Pr

peptide matches

theoretical Mr/pI

Alpha enolase Alpha enolase Alpha enolase Heat-shock protein beta-1 (HSP27) Heat-shock protein beta-1 (HSP27) Desmuslin Heat-shock protein beta-1 (HSP27) Stress-70 protein, mitochondrial Stress-70 protein, mitochondrial Protein disulfide-isomerase Protein disulfide-isomerase A3 Protein disulfide-isomerase A3 Keratin, type II cytoskeletal 8 Alpha enolase Alpha enolase Splicing factor, arginine/serine-rich 1 Splicing factor, arginine/serine-rich 1 Cathepsin D Heat-shock protein beta-1 (HSP27) Heat-shock protein beta-1 (HSP27) Keratin, type II cytoskeletal 1 Nucleoside diphosphate kinase A Stress-70 protein, mitochondrial Stress-70 protein, mitochondrial Protein disulfide-isomerase Keratin, type II cytoskeletal 8 Alpha enolase Alpha enolase Splicing factor, arginine/serine-rich 1 Splicing factor, arginine/serine-rich 1 Cathepsin D Heat-shock protein beta-1 (HSP27) Heat-shock protein beta-1 (HSP27) Keratin, type II cytoskeletal 1 Nucleoside diphosphate kinase A

P06733 P06733 P06733 P04792 P04792 O15061 P04792 P38646 P38646 P07237 P30101 P30101 P05787 P06733 P06733 Q07955 Q07955 P07339 P04792 P04792 P04264 P15531 P38646 P38646 P07237 P05787 P05787 P05787 Q07955 Q07955 P07339 P04792 P04792 P04264 P15531

48.0 47.3 64.4 44.9 49.7 22.2 35.1 57.0 57.9 58.3 52.1 55.8 52.7 54.3 65.8 57.1 70.0 47.8 53.2 40.5 33.0 44.1 54.3 50.5 60.4 59.1 37.4 54.7 59.9 64.0 35.3 40.0 53.2 31.1 44.1

98.5 96.9 100 100 100 69.7 100 98 98 100 100 100 85.7 100 99.9 100 99.9 99.5 100 100 82.6 64.4 77.4 94.1 100 100 7.9 100 100 100 100 100 100 99.9 64.4

24 22 31 9 9 36 7 47 46 37 30 42 40 25 30 20 20 23 10 8 21 7 39 38 37 37 15 29 21 23 16 9 11 18 7

47.0/7.5 47.0/7.5 47.0/7.5 22.7/6.3 22.7/6.3 172.7/5.1 22.7/6.3 73.7/6.2 73.7/6.2 57.1/4.8 56.7/6.3 56.7/6.3 53.5/5.6 47.0/7.5 47.0/7.5 27.6/10.5 27.6/10.5 26.6/5.6 22.7/6.3 22.7/6.3 65.8/8.4 17.1/6.1 73.7/6.2 73.7/6.2 57.1/4.8 53.5/5.6 47.0/7.5 47.0/7.5 27.6/10.5 27.6/10.5 26.6/5.6 22.7/6.3 22.7/6.3 65.8/8.4 17.1/6.1

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Figure 5. Magnification of protein differentially expressed in MCF-7/green, MCF-7/ppp and MCF-7/Znf cell lines. Representative subsections of 2DE images are shown for PDI A3 (A), keratin K8 (B), and NDPKA (C). Spots ID is numbered according to Table 1.

PROT accession no. Q07955, spot P10 and P11 in MCF-7/ppp and Z10 and Z11 in MCF-7/znf), identified as a pre-mRNA splicing factor,9 could be related to RNA metabolism. Protease. The acid protease cathepsin D (SWISS-PROT accession no. P07339), a secreted, 46-kDa estrogen-induced glycoprotein was overexpressed both in MCF-7/ppp and MCF7/znf cell lines compared to MCF-7/green (Figure 4B, spot P12 in MCF-7/ppp and Z12 in MCF-7/znf). Detoxification of Cytotoxic Products. Evidence of tumorassociated protein misfolding was provided by the selective upregulations of various molecular chaperones, including the heat-shock protein Hsp70 (SWISS-PROT accession no. P38646; Figure 4C, spot P1 and P2 in MCF-7/ppp and Z1 and Z2 in MCF-7/znf). A further alteration related to the stress-response protein family involved the up-regulation of different protein disulfide isomerases, including PDI (SWISS-PROT accession no. P07237; Figure 4D, spots P3 and Z3 in MCF-7/ppp and MCF-7/znf, respectively) and PDIA3 (SWISS-PROT accession no. P30101; Figure 5A; spots P5 and P6 in MCF-7/ppp). Cytoskeleton. A differential feature of MCF-7/znf and MCF7/ppp cell lines was found to be related to the differential expression of cytoskeleton proteins such as keratins. Both in the MCF-7/znf and MCF-7/ppp 2-D maps, levels of keratin K8 were increased (Figure 5B, spot P7 and Z7). Miscellaneous. Spots P16 and Z16 in MCF-7/ppp and MCF7/znf, respectively, were identified as nucleoside diphosphate kinase A, the A subunit of the nucleoside diphosphate kinase (NDPK) enzyme (SWISS-PROT accession no. P15531; Figure 5C). The A subunit is encoded by the nm23.h1 gene, whereas the nm23.h2 gene encodes the B subunit. Both genes are 88% identical. Isoforms and Degradation Products of Proteins. In most cases, the experimental pI and Mr of proteins from 2DE were in agreement with their theoretical values, determined with the EXPASY tool. For some proteins, however, the presence of several isoforms with different pI and Mr suggested that posttranslational modifications (phosphorylation, glycosylation, limited proteolysis), degradation or mRNA alternative splicing had occurred. We observed some discrepancies for spot G7 (Figure 6A), which was identified as the heat-shock protein beta-1, also found in spots G4 and G5, and for the intermediate filament protein desmin (SWISS-PROT accession no.: O15061;

Figure 6. Magnification of degradation product detected in MCF7/green (A and B), MCF-7/ppp and MCF-7/Znf (C) cell lines. Representative subsections of 2DE images are shown for desmin (A), Hsp 27 (B), and keratin K1 (C). Spots ID is numbered according to Table 1.

Figure 6B, spot G6), which was undetactable in the MCF-7/ znf and MCF-7/ppp 2-D maps. Putative degradation products were also found for protein spots P15 and Z15 in MCF-7/ppp and MCF-7/znf, respectively, identified as the cytoskeletal type II keratin K1 (SWISS-PROT accession no. P04264; Figure 6C).

Discussion To investigate a growth-promoting activity related to RIZ2 gene products, two permanently transfected MCF-7-derived cell lines were produced with the cDNA coding for a fusion protein containing the zinc-finger or the proline-rich domains of RIZ protein.7 This strategy was chosen to bypass a suspected lethal effect of the forcedly expressed RIZ2 form, because several attempts to transfect HeLa or MCF-7 cells with cDNA coding for a RIZ2 gene product never produced any viable clone expressing the insert.7 Furthermore, evidence provided a valid background supporting our approach. Chromatin immunoprecipitation experiments indicated that recombinant fragments were acting on the same regulatory elements where the presence of RIZ protein was demonstrated confer an optimal estrogen response10,10bis (see Supporting Information). It was not possible to define if the recombinant proteins had a direct dominant effect or a dominant negative effect, but the specificity of the recombinant fragment effect was confirmed by the comparison with the GFP transfected cells. Furthermore, fluorescence observation indicated a nuclear localization of Znf-recombinant fragment predominantly concentrated in the nuclei, with a positive nucleolar pattern analogous to the wildtype protein, whereas prolin-rich fragment was present both in nuclear and cytoplasm compartments.7 In the present study we characterize, by a well-suited bottom-up mass spectrometry-based proteomic approach, the differential protein expression of MCF-7/znf and MCF-7/ppp cell lines compared to MCF-7/green human breast cancer cell line, utilized as control. As zinc-finger domain expression bestowed a higher proliferation rate to MCF-7 cell line, consistent with small differences in expression of proteins relevant to cell cycle control (such as cyclin A and D1),7 we would expect to reveal specific proteins involved in cell cycle regulation. Anyway, differential expression of cyclins was modest, probably also as a consequence of the use on nonsynchronized cells; therefore, it is reasonable to hypothesize that such variations, if present, were not detectable by 2D gel analysis. Journal of Proteome Research • Vol. 5, No. 5, 2006 1181

research articles Overexpression of keratin K8, was clearly detected both in both MCF-7/znf and MCF-7/ppp cell lines when compared to MCF-7/green. Trask et al.11 demonstrated that normal cells produce K5, K6, K7, and K17 keratins, whereas tumor cells produce mainly K8, K18, and K19 keratins, usually not expressed in normal breast epithelial cells. This distribution was confirmed in tumor samples,12 and cytokeratin immunodetection is routinely used in histopathology to highlight epithelial cells and to help discriminate benign from malignant cells in tumor biopsies. Furthermore, several proteins that we found up-regulated in MCF-7/znf and MCF-7/ppp cell lines compared to MCF-7/green, including heat shock protein and PDI, were also identified in the first large-scale proteomic analyses of other carcinomas, such as human lung cancers.13,14 Interestingly, stable transfection of MCF-7 cell lines with znf or ppp RIZ domains increased the expression levels of the glycolytic enzyme R-enolase, confirmed also by Western blot analysis. Mammalian enolase is composed of three isozyme subunits (alpha, beta, and gamma), which can form cell type- and development-specific homodimers or heterodimers. In the glycolytic pathway, enolase catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate, which in turn is dephosphorylated into pyruvate to yield ATP. Indeed, R-enolase is a multifunctional enzyme that, beside glycolysis, plays a role in various processes such as growth control, tolerance to hypoxia, and allergic responses.15 R-enolase constitute a clear example of proteins for which alternative functions have been identified, also named “moonlighting proteins.”16 It was also reported that enolase may function in the intravascular and pericellular fibrinolytic system, due to its ability to bind and activate plasminogen on the surface of several cell-types, such as leukocytes and neurons.15 Other groups have resolved different R-enolase protein spots in 2-D gel from human cell samples,17 demonstrating that R-enolase was expressed at relatively high levels in proliferating human cells. Antibodies against R-enolase are present in serum of patients with cancerassociated retinopathy syndrome (CAR), a progressive blinding disease occurring in the presence of disseminated tumor growth, primary lung small-cell carcinoma and other malignancies.16 Two recent papers described an alternative 38-kDa R-enolase gene translation product identical to the myc-binding protein-1 (MBP-1).18 The N-terminal region of MBP-1 binds to the c-myc protooncogene and negatively regulates its expression. The N-terminal end of full-length R-enolase also binds the c-myc promoter, at least in vitro.19 Down-regulation of c-myc both at the transcriptional and translational level is a prerequisite for differentiation in many cell types. MBP-1 cDNA shares 97% similarity with the cDNA encoding the glycolytic enzyme R-enolase and both genes have been mapped to the same region of human chromosome 1 (corresponding also to chromosome region coding for RIZ gene), suggesting that the MBP-1 and R-enolase might be alternative translation products of a full-length R-enolase mRNA. This shorter form of R-enolase is able to bind the MBP-1 consensus sequence and to downregulate the expression of a luciferase reporter gene under the control of the c-myc P2 promoter.15 The finding that a transcriptional repressor of the c-myc oncogene is an alternatively translated product of the ENO1 gene, which maps to a region of human chromosome 1 frequently deleted in human cancers, makes ENO1 a potential candidate as tumor suppressor. Variations in glycolysis rate may affect substrate availability for the tricarboxylic acid cycle and subsequent oxidative phosphorylation, influencing in turn ATP levels, thus affecting ATP1182

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dependent reactions including creatine and nucleoside diphosphate phosphorylation. It was also reported that the commonly observed overexpression of glycolytic enzymes in tumor cells compared to normal tissue20 may be an effect of enhanced proliferation rather than be a specific feature of the neoplastic phenotype.21 In our biological system, we demonstrate that forced expression of both ppp and Znf RIZ domains increase the expression of PDI, one of the most abundant proteins in endoplasmic reticulum (ER). PDI is a multidomain and multi-functional member of the thioredoxin superfamily, with two catalytically active thioredoxine domains, and catalyses thiol-disulfide oxidation, reduction, and isomerization, the last of which occurs directly through intramolecular disulfide rearrangement or through cycles of reduction and oxidation.22-24 During the past years, 17 human PDI-family members in the ER have been reported,25 with a wide range of domain architectures and active-site chemistries. Whereas PDI interacts directly with, and also folds, non-native proteins, PDIA3 (also known as ERp57) is known to act in vitro and in vivo on glycosylated substrates interacting with the ER-resident lectins calnexin and calreticulin.26,27 Although the family name implies that all members have a role in protein disulfide isomerization, only a subset of them are able to efficiently catalyze this reaction, whereas others are probably not directly involved in native disulfide bond formation. Despite the large body of in vitro and in vivo experimental data on the PDI-family member activities, there are still many unanswered questions regarding the physiological functions of individual proteins and their mechanisms of action, especially within the complex ER environment. In humans, there are nearly 20 members of the PDI family, implying a complexity of the system that we are far from understanding. In particular, it will be necessary to determine whether the different enzymes have overlapping or separate and distinct substrate specificities. Other obvious questions to address include a systematic evaluation of the transcriptional regulation of all PDI-family members, the cellular mechanisms for regulation of their redox state and the physiological relevance of their unusual locations that have been reported.28 There is also a need to understand how PDI-family members interact with each other and with other ER-resident folding catalysts and chaperones. For some PDI, it was suggested a cooperation with ER chaperones of the heat-shock protein 70 (Hsp70) family,29,30 that we found to be overexpressed in MCF7/znf and MCF-7/ppp cell lines. There is already evidence for chaperone organization within the ER, and PDIs have been reported to form a complex with several ER chaperones and folding factors.31 However, much more work is needed to determine the dynamics of such complexes, how they are formed, and what their effects are on the folding process. Other interaction partners that act as modulators also need to be identified. The cellular content of various PDI isoforms is regulated by glucose deprivation,32 oxidative stress,33 differentiation,34 and neoplastic phenotype.35,36 In light of its multiple cellular roles, manipulation of PDI expression may produce pleiotropic effects. Changes both in Hsp70 and PDI levels may play an important role in protein folding and stress response. Forced-expression of ppp or Znf RIZ domains influences the expression of the acid protease cathepsin D, an aspartyl protease that participate in tumor development.37,38 Several studies in cancer cells reported overexpression (described both at mRNA and protein levels) and copious secretion of cathepsin

research articles

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D.39-42 A significant increase in the cytosol of neoplastic cells was shown in carcinomas of uterus, ovary, lung, intestines, colon, and many other organs.43-48 Cathepsin D endopeptidase activity degrades many intracellular and endocytosed proteins, as well as extracellular matrix proteins and proteins of the basal epithelium. Human cathepsin D can stimulate tumor growth by acting directly or indirectly as a mitogenic factor for cancer cells, independently of its catalytic activity and it can also promote tumor angiogenesis. On the other hand, it has also been described as a pro-apoptotic factor.49 Furthermore, a marked expression of cathepsin D, detected in human breast cancer cell lines by Westley and Rochefort,50,51 has been associated with a poor prognosis.39,52,53 Production of an inhibitor of this protease (i.e., antichymotrypsin) in breast cancer cells was demonstrated using 2DE,54 indicating the importance of the extracellular protease/antiprotease balance for the development of breast cancer.

crotubule mitotic spindle polymerization, and protein synthesis. A decrease in NDKA levels was observed in Caco-2 cells upon differentiation.67 Thus, NDKA expression, was higher in cells which just ceased to proliferate, as compared to fully differentiated cells. These data are in agreement with other reports supporting a role of NDKA in cell proliferation.68,69 In addition, the present data agree with decreased NDKA levels measured in differentiated cell systems.70 The nm23.h2 gene product (NDKB) is identical to PuF, a c-myc transcription factor. Its transcriptional activity is independent from the NDKP activity. The role of c-myc in proliferation and differentiation is well established. It is possible that a change in NDKA levels compared to NDKB causes a shift in subunit arrangement within the active enzyme hexamer. This may implicate a direct biological role of NM23 proteins in c-myc-dependent mechanisms involved in cell proliferation and differentiation.

An arginine/serine-rich splicing factor 1 (SF2) involved in RNA metabolism9 was upregulated in our transgenic cells. SF2 has a role in preventing exon skipping, ensuring splicing accuracy and regulating alternative splicing. It belongs to a conserved protein family recognized by a monoclonal antibody binding the active transcription site of RNA polymerase II. This family, named SR for the serine- and arginine-rich carboxyterminal domains of its members, consists of at least five different proteins with molecular masses of 20, 30, 40, 55, and 75 kDa, whose sequence revealed that they are related, but not identical. While the functions of SR proteins in splicing have been well documented in vitro, elucidation of their roles in vivo is not yet established. There is growing evidence that modulation of gene expression at the pre-mRNA level is an important regulatory mechanism in tumorigenesis.55 Pre-mRNA processing proteins mediate many aspects of preparing a final mRNA transcript for polysomal translation, including alternative splicing, stability, and transport of pre-mRNA,56,57 and thus can influence the translation into proteins of numerous mRNAs, including those leading to carcinogenesis. Computational analysis of mRNAs derived from tumor or normal samples identified 845 alternatively spliced isoforms significantly associated with human cancer.58 Accordingly, expression of several SR proteins, including ASF/SF2, increases during mouse mammary gland tumorigenesis,59 in benign tumors and in nonneoplastic proliferating lung compared to normal lung tissue.60 Furthermore, several studies suggested that changes in SR protein phosphorylation may influence alternative splicing of pre-mRNAs encoding apoptotic regulators.61-64 Recently, it has been demonstrated that cell motility, an activity important for embryogenesis, tissue formation and tumor metastasis, is controlled by SF2/ASF through alternative splicing of the Ron protooncogene.65 However, although gene targeting experiments showed that SR proteins are essential for cell viability and/or animal development,66 the specific role(s) that individual SR proteins play in general cell physiology is largely unknown.

Conclusions

Finally, we detect the overexpression, both in MCF-7/znf and MCF-7/ppp cell lines, of nucleoside diphosphate kinase A (NDPKA). NDPK catalyses the ATP-dependent phosphorylation of nucleoside disphosphates toward nucleoside triphosphates (NTPs). The NDPK isoenzymes consist of hexamers containing A (NDKA) and B (NDKB) subunits in different relative amounts. Therefore, NDPK may have a role in NTP-dependent cellular reactions, like DNA and RNA synthesis, GTP-dependent processes including G-protein-mediated signal transduction, mi-

Proteomic analysis is an unbiased mean of examining the molecular basis of cellular proliferation generally related to neoplasia. Several lines of investigation suggest that the RIZ locus on 1p36 plays an important role in human cancers. The RIZ1 product of this locus is a strong candidate tumor suppressor while, previous studies indicated that the Zn-finger domain could be endowed with the putative oncogenic activity of RIZ2 gene product. This work represents the first study correlating, by a proteomic mass spectrometry-based approach, the forced expression of RIZ gene Zn-finger or prolin-rich domains to differential protein expression in MCF-7 cells. Some proteins identified in this study were previously reported, in other experimental systems, as being associated with different types of carcinomas or involved in cell proliferation and differentiation. These alterations may contribute to the higher proliferation rate observed in the MCF-7/znf cell line compared to the control. Knowledge of specific proteins differentially expressed by MCF-7-derived cell lines following the introduction of RIZ protein different domains, will provide a basis for further studies aiming to identify mechanisms of growth-promoting activity activated by RIZ gene products.

Acknowledgment. This work was supported by grants from the Italian “Ministero dell’Istruzione, dell’Universita` e della Ricerca”, Fondo per gli Investimenti della Ricerca di base, Project RBNE0157EH and PRIN Project 2003060385_002. Supporting Information Available: Chromatin immunoprecipitation (ChIP) performed on MCF-derived cell lines expressing GFP fused to the proline-rich domain (MCF-7/ppp) or to the first cluster of zinc-finger motifs (MCF-7znf) of RIZ protein. This material is available free at http://pubs.acs.org. References (1) Liu, L.; Shao, G.; Steele-Perkins, G.; Huang, S. The retinoblastoma interacting zinc finger gene RIZ produces a PR domain-lacking product through an internal promoter. J. Biol. Chem. 1997, 272, 2984-2991. (2) Chadwick, R. B.; Jiang, G. L.; Bennington, G. A.; Yuan, B.; Johnson, C. K.; Stevens, M. W.; et al. Candidate tumor suppressor RIZ is frequently involved in colorectal carcinogenesis. Proc. Nat’l Acad. Sci., U.S.A. 2000, 97, 2662-2667. (3) Piao, Z.; Fang, W.; Malkhosyan, S.; Kim, H.; Horii, A.; Perucho, M.; Huang, S. Frequent frameshift mutations of RIZ in sporadic gastrointestinal and endometrial carcinomas with microsatellite instability. Cancer Res. 2000, 60, 4701-4704.

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(61) Cao, W.; Jamison, S. F.; Garcia-Blanco, M. A. Both phosphorylation and dephosphorylation of ASF/SF2 are required for premRNA splicing in vitro. RNA 1997, 3, 1456-1467. (62) Xiao, S. H.; Manley, J. L. Phosphorylation-dephosphorylation differentially affects activities of splicing factor ASF/SF2. EMBO J. 1998, 17, 6359-6367. (63) Prasad, J.; Colwill, K.; Pawson, T.; Manley, J. L. The protein kinase Clk/Sty directly modulates SR protein activity: Both hyper- and hypophosphorylation inhibit splicing. Mol. Cell. Biol. 1999, 19, 6991-7000. (64) Utz, P. J.; Hottelet, M.; van Venrooij, W. J.; Anderson, P. Association of phosphorylated serine/arginine (SR) splicing factors with the U1-small ribonucleoprotein (snRNP) autoantigen complex accompanies apoptotic cell death. J. Exp. Med. 1998, 187, 547560. (65) Ghigna, C.; Giordano, S.; Shen, H.; Benvenuto, F.; Castiglioni, F.; Comoglio, P. M.; Green, M. R.; Riva, S.; Biamonti, G. Cell motility is controlled by SF2/ASF through alternative splicing of the Ron protooncogene. Mol. Cell. 2005, 20 (6), 881-890. (66) Longman, D.; Johnstone, I. L.; Caceres, J. F. Functional characterization of SR and SR-related genes in Caenorhabditis elegans. EMBO J. 2000, 19, 1625-1637. (67) Stierum, R.; Gaspari, M.; Dommels, Y.; Ouatas, T.; Pluk, H.; Jespersen, S.; Vogels, J.; Verhoeckx, K.; Groten, J.; van Ommen, B. Proteome analysis reveals novel proteins associated with proliferation and differentiation of the colorectal cancer cell line Caco-2. Biochim. Biophys. Acta. 2003, 1650 (1-2), 73-91. (68) Cipollini, G.; Berti, A.; Fiore, L.; Rainaldi, G.; Basolo, F.; Merlo, G. et al. Down-regulation of the nm23.h1 gene inhibits cell proliferation. Int. J. Cancer 1997, 73 (2), 297-302. (69) Caligo, M. A.; Cipollini, G.; Petrini, M.; Valentini, P.; Bevilacqua, G. Down regulation of NM23.H1, NM23.H2 and c-myc genes during differentiation induced by 1, 25 dihydroxyvitamin D3. Leuk. Res. 1996, 20 (2), 161-167. (70) Olsen, E.; Rasmussen, H. H.; Celis, J. E. Identification of proteins that are abnormally regulated in differentiated cultured human keratinocytes. Electrophoresis 1995, 16 (12), 2241-2248.

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