Identification of Altered Protein Expression and Post-Translational Modifications in Primary Colorectal Cancer by Using Agarose Two-Dimensional Gel Electrophoresis

Vol. 10, 2007–2014, March 15, 2004 Clinical Cancer Research 2007

Takeshi Tomonaga,1 Kazuyuki Matsushita,2 Moreover, post-translational modifications of the prolyl-4- Seiko Yamaguchi,1 Masamichi Oh-Ishi,3 hydroxylase ␤ subunit and annexin A2 also were identified. Yoshio Kodera,3 Tadakazu Maeda,3 Conclusions: We identified several novel proteins with 2 2 altered expression in primary colorectal cancer using agar- Hideaki Shimada, Takenori Ochiai, and ose 2-DE. This method is a powerful technique with which to 1 Fumio Nomura search for not only quantitative but also qualitative changes Departments of 1Molecular Diagnosis (F8) and 2Academic Surgery in a biological process of interest and may contribute to the (M9), Graduate School of Medicine, Chiba University, Chiba, Japan, 3 deeper understanding of underlying mechanisms of human and Laboratory of Biomolecular Dynamics, Department of Physics, cancer. Kitasato University School of Science, Kanagawa, Japan INTRODUCTION ABSTRACT Information at the level of the proteome is necessary to Purpose: Although numerous proteome studies have unravel the critical changes involved in disease pathogenesis. been performed recently to identify cancer-related changes Comparative studies of protein expression in normal and disease in protein expression, only a limited display of relatively tissues also have led to the identification of aberrantly expressed abundant proteins has been identified. The aim of this study proteins that may represent new markers (1). This disease pro- is to identify novel proteins as potential tumor markers in teomics primarily relied on a combination of two-dimensional primary colorectal cancer tissues using a high-resolution gel electrophoresis (2-DE) to separate and visualize proteins and two-dimensional gel electrophoresis (2-DE). mass spectrometry for protein identification (1). Development Experimental Design: 2-DE using an agarose gel for of immobilized pH gradient (IPG) gel has led to great improve- isoelectric focusing was used to compare protein profiling of ments with regard to reproducibility; however, its loading ca- 10 colorectal cancer tissues and adjacent normal mucosa. pacity of proteins still is insufficient and only permits a limited Altered expression and post-translational modification of display of relatively abundant proteins (2). Low abundance several proteins were examined using Western blot analysis proteins can be detected if the starting protein load is large, and immunohistochemistry. which would allow for large-scale quantitative comparisons of Results: Ninety-seven proteins of 107 spots (90.7%) that protein expression (3). were differentially expressed between matched normal and Proteomic technologies also have been used to identify tumor tissues were identified by mass spectrometry. Among cancer-specific proteins that are useful for cancer diagnosis, them, 42 unique proteins (49 spots) significantly increased or progression, and therapeutic targets (4–10). In addition, they decreased in the tumors. They include eukaryotic transla- have the potential to unravel important cellular events associ- tion initiation factor 4H, inorganic pyrophosphatase, ante- ated with cancer development, such as protein phosphorylation rior gradient 2 homologue, aldolase A, and chloride intra- and degradation. Although extensive proteome analysis has cellular channel 1, whose elevated expression in tumor identified numerous proteins overexpressed in various cancer tissues was confirmed by Western blot analysis and immu- tissues, few markers have been accepted for routine clinical use nohistochemistry. Interestingly, only isoform 1 of two tran- because of conflicting reports or because potential candidates script variants of eukaryotic translation initiation factor have not been detected because of their low abundance (6). 4H was greatly up-regulated in most of the tumor tissues. Several investigators have extensively performed proteome studies of colorectal cancer; however, a limited number of proteins have been identified (11–15). Thus, it is necessary to develop novel techniques that permit large-scale quantitative Received 9/30/03; revised 11/12/03; accepted 11/21/03. comparisons of protein expression between normal and cancer Grant support: Grant-in-Aid 13214016 for Priority Areas in Cancer tissues. The agarose 2-DE method was shown previously to Research to T. Tomonaga, and Grant-in-Aid for Scientific Research on have a higher loading capacity than 2-DE with IPG gel for Priority Areas in Medical Genome Science to T. Maeda from the Min- isoelectric focusing (16, 17). We analyzed here primary colo- istry of Education, Science, Sports, and Culture of Japan. The costs of publication of this article were defrayed in part by the rectal cancer tissues for protein expression using the agarose payment of page charges. This article must therefore be hereby marked 2-DE method and identified novel proteins whose expression advertisement in accordance with 18 U.S.C. Section 1734 solely to differs between tumor tissue and adjacent normal mucosa. West- indicate this fact. ern blot and immunohistochemical analysis demonstrated that Requests for reprints: Takeshi Tomonaga, Department of Molecular Diagnosis (F8), Graduate School of Medicine, Chiba University, 1-8-1 an isoform of eukaryotic translation initiation factor 4H (eIF- Inohana, Chuo-ku, Chiba 260-8670, Japan. Phone: 81-43-226-2167; 4H), inorganic pyrophosphatase, anterior gradient 2 homologue Fax: 81-43-226-2169; E-mail: [email protected]. (hAG-2), aldolase A, and chloride intracellular channel 1

Table 1 Clinical features of patients with colorectal cancer pieces, destained in 50% acetonitrile/50 mM NH4HCO3, and Sample Dukes washed with deionized water. The gel pieces were dehydrated in number Age Sex Locationa stage 100% acetonitrile for 15 min and then dried in a SpeedVac 1 6 57 Male S B evaporator (Wakenyaku, Kyoto, Japan) for 45 min. The gel 2 27 51 Male R D pieces were rehydrated in 10–30 ␮lof25mM Tris-Cl (pH 3 29 51 Male R C 9)/20% acetonitrile containing 25 ng/␮l trypsin (Trypsin se- 4 34 59 Male R C quence grade; Roche) for 45 min. After removal of the unab- 5 35 72 Female S B sorbed solution, the gel pieces were incubated in 10–20 ␮lof 6 36 67 Male T C 7 37 49 Male R C 50 mM Tris-Cl (pH 9)/20% acetonitrile for 20 h at 37°C. The 8 112 72 Male S C solution containing digested fragments of proteins was trans- 9 117 49 Male Ce D ferred to a new tube, and the peptide fragments remaining in the 10 118 85 Male S B gel also were extracted in 5% formic acid/50% acetonitrile for a S, sigmoid colon; R, rectum; T, transverse colon; Ce, cecum. 20 min at room temperature. Mass Spectrometry of Proteins. Digested peptides equivalent to the maximum of 10–20 pmol of a protein in a 2-DE spot were injected into an Aquapore RP-300 column (NCC27) were overexpressed significantly in many of the pri- (Perkin-Elmer, Shelton, CT), a 2.1-mm diameter and 30-mm mary colorectal cancer tissues. In addition, different post-trans- length C8 reversed phase, and attached to the Nanospace SI-2 lational modifications of several proteins were demonstrated (Shiseido Fine Chemicals, Tokyo, Japan) as a high-performance between tumor and normal tissues. A tumor-specific cleavage of liquid chromatography system. The flow rate of the mobile ␤ prolyl-4-hydroxylase subunit (P4HB) was observed. Con- phase was 200 ␮l/min using buffer A (0.05% HCOOH) and versely, annexin A2 tended to be cleaved in normal tissues. buffer B (90% acetonitrile and 0.05% HCOOH). Following an These results would be helpful not only to investigate the initial wash with buffer A for 5 min, peptides were eluted with underlying mechanism of carcinogenesis but also to develop a linear gradient from 0–60% buffer B over an interval of either new biomarkers and therapeutics. 3 min or 30 min. The purified peptides were sprayed from the high-performance liquid chromatography via a metal needle- MATERIALS AND METHODS attached AP12 (an electrospray ionization adapter) to the LCQ- Human Tissue Samples. Tissues from 10 patients with Deca (ThermoQuest, San Jose, CA), which is an ion trap mass primary colorectal cancer were resected surgically (Table 1). spectrometry. Data-dependent measurements of mass and tan- Written informed consent was obtained from each patient before dem mass spectra of the peptides were performed according to surgery. The excised samples were obtained within 1 h after the the manufacturer’s operating specifications. SEQUEST (Thermo operation from tumor tissues and corresponding nontumor tis- Quest) was used to identify proteins from mass and tandem sues that were 5–10 cm from the tumor. All of the excised mass data. When the SEQUEST score for the best candidate tissues were placed immediately in liquid nitrogen and stored at protein was Ͻ100, we inspected raw mass and tandem mass data Ϫ80°C until analysis. of peptides to judge their qualities. Protein Extraction. Frozen tissue samples were solubi- Antibodies. Rabbit polyclonal antisera were raised lized in lysis buffer (9.5 M urea, 2% 3-[(3-cholamidopropyl) against synthetic peptides corresponding to the N-terminal and dimethylammonio]-1-propanesulfonate, and 1% DTT) contain- COOH-terminal sequences of eIF-4H (MADFDTYDDRAY SS ing protease inhibitor mixture (Complete; Roche, Mannheim, and GAR PRE EVV QKE QE), inorganic pyrophosphatase Germany) using a Polytron homogenizer (Kinematica, Littau- (MSG FST EER AAP FS and CTV PTD VDK WFH HQK N), Luzern, Switzerland) following centrifugation (100,000 ϫ g) for hAG-2 (KPG AKK DTK DSR PKL and ADI TGR YSN RLY 1hat4°C. The supernatant was subjected either to agarose AYE), and NCC27 (TVD TKR RTE TVQ KLC and YLS NAY 2-DE or SDS-PAGE. ARE EFA STC) and attached to keyhole limpet hemocyanin. Agarose 2-DE. Agarose gels were prepared as described Two peptides of each protein were immunized simultaneously previously (16, 17). Protein extract (500 ␮g) from tissues to enhance the possibility of antibody production. The reactivity was applied to the agarose isoelectric focusing gel, and first- of the antiserum was tested by solid-phase enzyme immunoas- dimensional isoelectric focusing was conducted at 12,000 Vxhr or say, and the antiserum was affinity purified by passage over a Vhr at 4°C, followed by fixation in 10% trichloroacetic acid and resin covalently coupled with synthetic peptides (Japan Bio 5% sulfosalicylic acid for1hatroom temperature. After washing Services, Saitama, Japan). Goat anti-aldolase A (C-16), rabbit with deionized water for 1 h, the agarose gel then was transferred anti-annexin A2 (Annexin II, H-50), and goat anti-␤-actin anti- to a 12% polyacrylamide gel, and second-dimensional SDS-PAGE bodies were purchased from Santa Cruz Biotechnology (Santa was performed. The second-dimensional gel first was incubated in Cruz, CA). Mouse anti-P4HB antibody was purchased from 30% methanol and 10% acetic acid overnight. It then was stained ICN Biomedicals (Aurora, OH). with PhastGel Blue R (Amersham Pharmacia Biotech, Piscataway, Immunoblotting. Protein extracts were separated by NJ). Each gel was scanned using Epson ES 2000 (Nagano, Japan), electrophoresis on 10–20% gradient gels (Bio-Rad, Hercules, and NIH Image was used to measure the intensity of each spot. CA). The proteins were transferred to polyvinylidene fluoride Enzymatic In-Gel Digestion of Proteins. The protein membranes (Millipore, Bedford, MA) in a tank-transfer appa- spots were excised from the gel, and in-gel tryptic digestion of ratus (Bio-Rad), and the membranes were blocked with 5% skim proteins was performed. Briefly, the gels were cut in small milk in PBS. Anti-eIF-4H diluted 1:500, anti-inorganic pyro-

phosphatase diluted 1:100, anti-aldolase A diluted 1:100, anti- P4HB diluted 1:5000, anti-annexin A2 diluted 1:1000, and anti-␤-actin diluted 1:500 in blocking buffer were used as primary antibodies. Goat antirabbit IgG horseradish peroxidase, goat antimouse IgG horseradish peroxidase (Bio-Rad) diluted 1:3000, and rabbit antigoat IgG horseradish peroxidase (Cappel, West Chester, PA) diluted 1:500 in blocking buffer were used as secondary antibodies. Antigens on the membrane were detected with enhanced chemiluminescence detection reagents (Amer- sham Pharmacia Biotech). Reverse Transcription-PCR. Total RNA was extracted from tumor and nontumor tissues with RNeasy Mini Kit (Qia- gen, Tokyo, Japan). cDNA was synthesized from total RNA with the first-strand cDNA synthesis kit for reverse transcription-PCR (Roche). Using the cDNA as a template, eIF-4H cDNA was amplified with suitable primers: forward, 5Ј-GGT- GGCTTTGGATTCAGAAA-3Ј and reverse, 5Ј-CGAGGTTTA- AGCTGGAGTCG-3Ј. Immunohistochemistry. Four-␮m sections of frozen tissue were fixed on slide glasses with acetone for 10 min at 4°C, followed by treatment with 0.3% H2O2 in methanol for 15 min at room temperature. After washing three times with PBS, nonspecific binding of antibodies was blocked with blocking buffer (1% swine serum/PBS) for 1 h. Tissues then were incubated for 1 h with anti-eIF-4H diluted 1:100, anti-hAG diluted 1:10, anti-aldolase A diluted 1:50, and anti-NCC27 diluted 1:200 in 1% BSA/PBS. After washing with PBS, DAKO LSABϩ Kit (DAKO Japan, Kyoto, Japan) was used to visualize tissue antigens according to manufacturer’s instructions. Tissue sections were counterstained with hematoxylin for 30 s and dehydrated with 100% ethanol and xylene, and coverslips were Fig. 1 Coomassie-stained agarose two-dimensional gel electrophoresis (2-DE) pattern of proteins of human primary colon cancer (A) and mounted with Malinol (Mito Pure Chemicals, Tokyo, Japan). normal adjacent mucosa (B). Total protein lysates were prepared from Two pathologists evaluated immunohistochemical staining of matched samples of tumor and adjacent normal tissue as described in the samples. “Materials and Methods.” Five hundred ␮g of protein then were subjected to agarose 2-DE, followed by staining with PhastGel Blue R. Spots T1-T37 and N1-N9 are proteins whose expression is elevated in RESULTS tumor tissue or normal mucosa, respectively. Whole cell lysates from 10 matched samples of tumor or adjacent normal mucosa were separated by agarose 2-DE, and proteins were visualized by Coomassie Blue staining (Fig. 1). have altered expression in colorectal cancer either by 2-DE or Tumor specimens for samples largely consist of tumor cells other methods: carbonic anhydrase 1, nonmetastatic cell 1 pro- (60–70%) and stroma (30–40%), whereas normal specimens tein, two isoforms of peptidylprolyl isomerase A, manganese consist of normal colon epithelial cells (60–70%) and stroma superoxide dismutase, keratin 18, enolase 1, calreticulin, tumor (30–40%). There are few endothelial cells and infiltrating lym- rejection antigen (gp96), and pyruvate kinase 3 (type M2; Refs. phocytes either in tumor or normal specimens. Thus, there is no 14, 18–21). These results confirmed a part of our observation of bias in the cellularity of the normal and tumor tissues. All of the the differentially expressed proteins in colorectal cancer using samples were examined in duplicate or triplicate, and ϳ600– agarose 2-DE. 1000 protein spots were detected consistently in each gel. One Among the proteins with altered expression, we focused hundred seven protein spots that were shown to increase or mainly on the regulatory proteins whose expression levels have decrease in tumor tissues were excised, and mass spectrometry not been well studied in human primary cancers. First, immu- sequence was obtained from 97 spots (90.7%). Identical protein noblot analyses of several proteins were performed to confirm sequences were obtained from multiple spots in normal and the differential protein expression in tumor tissues. The most tumor tissues, such as serum albumin and glyceraldehyde-3- striking differences observed between tumor and adjacent phosphate dehydrogenase, and they were eliminated from addi- normal mucosa were eIF-4H and inorganic pyrophosphatase tional investigation. Proteins whose expression level was not (Fig. 2, A and B). Western blot analysis using anti-eIF-4H altered significantly between normal and tumor tissues (P Ͼ antibody revealed two bands in all of the tumor tissues, whereas 0.05) also were eliminated. Forty-two proteins substantially only a faster migrating band was observed in most of the normal increased or decreased in the tumors (P Ͻ 0.05; Fig. 1 and Table mucosa (Fig. 2A, top, arrow and asterisk). Two transcript vari- 2). These include the following proteins that were reported to ants of eIF-4H have been reported recently, which include

Table 2 Altered protein expression in primary colorectal cancer tissues Protein Database SEQ Coverage Fold increase P spot no.a Protein identification accession no.b M.W. scorec (%)d (average Ϯ SD)e (t test) T1 calreticulin ref͉NP_004334.1ʈ 48142 118.8 23.7 2.55 Ϯ 1.80 0.0186 T2 similar to tumor rejection antigen (gp96) 1 gb͉AAH09195.1͉ 35450 189.1 28.3 5.60 Ϯ 4.84 0.0101 T3 protein disulfide isomerase pirʈS55507 56679 207.0 26.3 13.57 Ϯ 13.02 0.0072 T4 pyruvate dehydrogenase beta ref͉NP_000916.1͉ 39249 125.1 24.2 4.59 Ϯ 2.38 0.0081 T5 pyruvate kinase-3 ref͉NP_002645.1͉ 57914 301.8 36.9 5.77 Ϯ 2.64 0.0032 T6 inorganic pyrophosphatase ref͉NP_066952.1͉ 32660 117.0 25.6 3.11 Ϯ 1.37 0.0135 T7 chloride intracellular channel 1 (NCC27) gb͉AAC25675.1͉ 26924 166.0 30.7 1.98 Ϯ 0.77 0.0257 T8 stathmin 1/oncoprotein 18 ref͉NP_005554.1͉ 17303 41.7 21.5 N.D.f T9 non-metastatic cells 1 protein (NM23-H1) ref͉NP_000260.1͉ 17149 122.8 38.2 N.D.f T10 triosephosphate isomerase 1 ref͉NP_000356.1͉ 26669 347.6 55.4 4.62 Ϯ 4.38 0.0098 T11 aldolase A pdb͉1ALD͉ 39289 100.0 11.8 13.02 Ϯ 13.87 0.0047 T12 phosphoglycerate kinase 1 sp͉P00558͉ 44728 100.0 10.3 18.35 Ϯ 22.85 0.0005 T13 malate dehydrogenase 2 ref͉NP_005909.1͉ 35531 444.3 51.8 7.22 Ϯ 6.44 0.0017 T14 heterogeneous nuclear ribonucleoproteins A2/B1 sp͉P22626͉ 37430 43.4 17.3 8.81 Ϯ 2.72 0.0131 T15 porin 31HM/voltage-dependent anion channel 1 (VDAC1) gb͉AAB20246.1͉ 30641 120.0 19.9 6.87 Ϯ 3.61 0.0006 T16 peroxiredoxin 1/natural killer-enhancing factor A pirʈI54533 22127 144.0 27.6 1.85 Ϯ 0.52 0.0118 T17-1 peptidylprolyl isomerase A sp͉P05092͉ 17881 32.5 16.5 11.41 Ϯ 3.79 0.0075 T17-2 peptidylprolyl isomerase A pdb͉1AWR͉A 17881 70.0 42.1 14.08 Ϯ 8.25 0.0117 T18 cofilin 1 (non-muscle) ref͉NP_005498.1͉ 18502 145.7 44.6 6.18 Ϯ 1.16 0.0038 T19 non-metastatic cells 2 protein (NM23B, NM23-H2) ref͉NP_002503.1͉ 17298 134.5 49.3 8.71 Ϯ 2.33 0.0216 T20 heterogeneous nuclear ribonucleoprotein C (C1/C2) gb͉AAH03394.1͉ 33598 195.1 27.8 8.57 Ϯ 4.81 0.0077 T21 annexin A2 gb͉AAH09564.1͉ 38618 436.6 60.2 6.32 Ϯ 4.00 0.0106 T22 KIAA0038/eukaryotic translation initiation factor 4H dbj͉BAA05063.1͉ 25186 102.5 32.5 12.33 Ϯ 3.69 0.0317 T23 prolyl-4-hydroxylase, beta subunit ref͉NP_000909.2͉ 57098 411.6 49.2 9.16 Ϯ 7.65 0.0196 T24-1 enolase 1 (alpha) ref͉NP_001419.1͉ 47169 61.5 15.9 9.84 Ϯ 5.19 0.0220 T24-2 enolase 1 (alpha) gb͉AAB88178.1͉ 36308 224.8 33.0 3.82 Ϯ 2.06 0.0135 T25 voltage-dependent anion channel 2 (VDAC2) gb͉AAH00165.1͉ 30412 214.5 41.3 3.18 Ϯ 1.31 0.0005 T26 heat shock 60kD protein 1 (chaperonin) gb͉AAH10112.1͉ 59812 441.7 40.9 2.24 Ϯ 0.85 0.0138 T27 peroxiredoxin 5 ref͉NP_036226.1͉ 22026 190.5 37.9 4.39 Ϯ 1.17 0.0258 T28 adenylyl cyclase-associated protein ref͉XP_029844.6͉ 51813 240.9 32.4 6.11 Ϯ 4.74 0.0122 T29 manganese superoxide dismutase (MnSOD) pdb͉1JA8͉A 22089 146.0 66.2 2.07 Ϯ 0.71 0.0007 T30 keratin 18 ref͉NP_000215.1ʈ 48058 579.1 52.8 1.43 Ϯ 0.36 0.0072 T31 anterior gradient 2 homolog (hAG-2) ref͉NP_006399.1ʈ 19979 70.8 25.7 21.76 Ϯ 18.77 0.0224 T32 L-lactate dehydrogenase M chain pdb͉1I10͉A 36558 327.3 46.5 2.18 Ϯ 0.62 0.0358 T33 transgelin 2 ref͉NP_003555.1ʈ 22391 167.8 48.7 9.34 Ϯ 9.96 0.0158 T34 heat shock 70kD protein 8 ref͉NP_006597.1ʈ 70898 200.0 17.6 7.95 Ϯ 2.98 0.0353 T35 tropomyosin 1 (alpha) ref͉NP_000357.2ʈ 32876 110.0 30.3 3.99 Ϯ 2.61 0.0278 T36 vimentin ref͉XP_167414.1ʈ 53652 482.0 53.2 7.54 Ϯ 3.10 0.0288 T37 ATP synthase (ATPSA1) ref͉NP_004037.1ʈ 59751 506.9 54.2 5.10 Ϯ 1.99 0.0392

N1 smooth muscle myosin heavy chain 11 isoform SM1 ref͉NP_002465.1͉ 227339 728.9 28.3 2.59 Ϯ 0.54 0.0015 N2 tropomyosin 2 (beta) sp͉PO7951͉ 32851 118.0 21.8 1.62 Ϯ 0.33 0.0064 N3 carbonic anhydrase I ref͉NP_001729.1ʈ 28870 102.4 39.1 2.41 Ϯ 0.83 0.0083 N4 triosephosphate isomerase 1 ref͉NP_000356.1ʈ 26669 64.4 21.7 2.10 Ϯ 0.67 0.0268 N5-1 annexin A2 gb͉AAH09564.1͉ 38618 275.9 33.6 2.45 Ϯ 0.85 0.0126 N5-2 annexin A2 gb͉AAH09564.1͉ 38618 242.7 35.4 1.80 Ϯ 0.46 0.0260 N6 selenium binding protein 1 ref͉NP_003935.1ʈ 52313 161.9 34.3 2.38 Ϯ 0.20 0.0068 N7 calreticulin ref͉NP_004334.1ʈ 48142 287.0 40.3 4.84 Ϯ 3.16 0.0035 N8 vimentin ref͉XP_167414.1ʈ 53652 317.3 45.2 2.63 Ϯ 1.31 0.0327 N9 alpha 3 type VI collagen, isoform 2 precursor ref͉NP_476505.1ʈ 325177 198.3 5.7 14.52 Ϯ 14.88 0.0073 a Protein spots increased in tumor tissue (Fig. 1A;T1–37) or in adjacent normal mucosa (Fig. 1B;N1–9). b Reference for the protein identification. c SEQUEST score of the candidate proteins. The quality of the candidates was carefully judged as described in “Materials and Methods.” d Sequence coverage of MS/MS analysis of protein. e Intensity of the spots in 2-DE gel of ten matched samples (tumor vs normal) was measured with NIH image software. The differences for all proteins were significant (P Ͻ 0.05). f The intensity of these spots was unable to be quantitated due to the background noise near the dye front.

isoform 1 and 2. Isoform 2 lacks 20 amino acids of isoform 1, reverse transcription-PCR. The two transcript variants were and this short isoform is predominant in human tissues (22). The found, and the expression of the larger variant was increased two bands shown in Fig. 2A are likely to correspond to the two considerably in tumor tissues (Fig. 2E). Inorganic pyrophos- isoforms of eIF-4H. The larger isoform (isoform 1) was in- phatase and aldolase A also showed significant increases in creased significantly in tumor tissues (0.9–79.0-fold increase; tumor tissues. Relative protein level of inorganic pyrophos- P ϭ 0.0003). Thus, we examined the mRNA of eIF-4H by phatase in tumors compared with adjacent normal tissue varied

Fig. 2 Proteins up-regulated in primary colorectal cancers. Total protein lysates prepared from 10 matched samples of tumor (T) and adjacent normal tissue (N) were resolved on 10–20% gradient polyacrylamide gel and immunoblotted with several antibodies. Intensity of each band was measured using NIH Image, and the relative protein levels between tumor and normal tissue normalized with ␤-actin were calculated. A, eukaryotic translation initiation factor 4H (eIF-4H). The arrow and asterisk likely correspond to isoform 1 (27 kDa) and 2 (25 kDa) of eIF-4H, respectively. Isoform 1 was observed only in tumor tissues. B, inorganic pyrophosphatase (32 kDa). C, aldolase A (39 kDa). D, ␤-actin as a loading control (42 kDa). The expressions of eIF-4H, inorganic pyrophosphatase, and aldolase A were increased significantly in tumor tissues (P Ͻ 0.05). E, total RNAs were prepared from matched samples of tumor (T) and adjacent normal tissue (N), and reverse transcription-PCR was performed to examine the eIF-4H transcript. The faster and slower migrating bands correspond to the two transcript variants of eIF-4H, and expression of the isoform (isoform 1) was increased significantly in tumor tissues.

from 1.1–66.8 (P ϭ 0.003), and that of aldolase A varied from During the course of the 2-DE analysis, we encountered 0.04–15.7 (P ϭ 0.028; Fig. 2, B and C). several interesting observations. For example, P4HB (Fig. Although there is no bias in the cellularity of the normal 1A, spot T23) migrated slightly faster than what was ex- and tumor tissues, whole tissue sections include nonepithelial pected, considering the actual molecular mass of the protein components, and the altered protein expression in our 2-DE (57 kDa). Annexin A2, calreticulin, triosephosphate isomer- analysis may emanate from such nonepithelial components. ase 1, and vimentin were found in tumor and normal tissues Thus, the differential protein expression in colorectal cancer with different molecular mass and isoelectric point values also was validated by immunohistochemical analysis to demon- (Fig. 1; annexin, spots T21 and N5–1, 2; calreticulin, spots strate the cellular origin and distribution of the identified pro- T1 and N7; triosephosphate isomerase 1, spots T10 and N4; teins. Frozen tissue sections of colorectal cancers and adjacent and vimentin, spots T36 and N8). All of the differences are normal tissues were stained with several antibodies. As shown probably caused by the post-translational modifications, and in Fig. 3, immunohistochemistry with antibodies to eIF-4H and it also was examined with Western blot analysis. A faster hAG-2 showed strong nuclear staining, whereas those to aldol- migrating band of P4HB in most cancer tissues but not in ase A and NCC27 showed relatively mild nuclear staining. normal mucosa was clearly shown (Fig. 4A, arrow), which Examination of the several tissue sections showed similar indicates that P4HB tends to be cleaved in tumors. Con- staining patterns, indicating that these proteins actually are versely, a faster migrating band of annexin A2 in normal expressed in the tumor cells and not in the surrounding mucosa was scarcely observed in tumor tissues (Fig. 4B, mesenchymal cells. arrow). The peptide sequence of the protein (spot N5–1) as

Fig. 3 Immunohistochemical analysis of eukaryotic translation initiation factor 4H (eIF-4H; A and B), anterior gradient 2 homologue (hAG-2; C and D), aldolase A (E and F), and chloride intracellular channel 1 (NCC27; G and H). Frozen sections of normal colon epithelium (A, C, E, and G) and colon cancer tissue (B, D, F, and H) were stained with antibodies as described in “Materials and Methods.” Arrowheads show normal colon epithelial glands, and arrows show areas of stained tumor cells. Strong nuclear staining was observed for eIF-4H and hAG-2, whereas relatively mild staining, mainly in the nucleus, was observed for aldolase A and NCC27. All of the magnification is ϫ400.

revealed by mass spectrometry contained only the N-terminal DISCUSSION half of annexin A2 (data not shown). These results suggest that the COOH-terminus of annexin A2 is cleaved in normal In this study we showed that agarose 2-DE is a powerful colon mucosa, whereas it is resistant to such proteolytic technique to detect quantitative changes or modifications of cleavage in colorectal cancer. These post-translational mod- proteins compared with the conventional 2-DE method. The ifications may be important mechanisms for the development most prominent advantage of the technique is the high loading of colorectal cancer. capacity of the agarose gel used for isoelectric focusing. Oh-ishi

Fig. 4 Post-translational modifications of the prolyl-4-hydroxylase ␤ subunit (P4HB) and annexin A2 in colorectal cancers. Western blot analyses of P4HB (A) and annexin A2 (B) were performed as in Fig. 2. -Indicates the full-length proء tein; the arrow indicates its cleaved product. Several bands between the asterisk and arrow in (B) also may be cleavage products of annexin A2.

et al. (16) carefully compared previously the Coomassie Blue- human homologue of the secreted Xenopus laevis protein stained 2-DE patterns of agarose 2-DE with conventional IPGs XAG-2, which is expressed in the cement gland, an ectodermal using the same amount of crude extracts and found that protein organ in the head associated with anteroposterior fate determi- spot densities were much higher in agarose 2-DE than in IPGs. nation during early development. This protein was reported In this study, we were able to detect 600-1000 protein spots and recently to express in estrogen receptor-positive breast tumors

107 differentially expressed proteins with Coomassie Blue (23). Inorganic pyrophosphatase catalyzes the hydrolysis of PPi staining. These numbers appear to be less than that of previous to inorganic phosphate, which is important for the phosphate reports. It is because most studies visualized protein spots with metabolism of cells. The expression of this protein also is silver staining, which is 10–100 times more sensitive than up-regulated in lung adenocarcinoma, which may relate to the Coomassie Blue staining. However, the absolute intensity of increased requirement for energy in rapidly growing tumors (9). spots visualized with silver staining is controlled by the length NCC27 is a nuclear chloride ion channel protein that regulates of reaction and is variable for different proteins. Moreover, ionic traffic between nucleus and cytoplasm. It also was known silver-stained protein spots tend to be difficult to identify pro- to be involved in regulation of the cell cycle (24). Increased teins by mass spectrometry because each spot often contains protein expression of NCC27 has been reported recently in small amount of protein and the proteins also are modified by human breast ductal carcinoma in situ (10). Aldolase A is formaldehyde used as a fixative. For example, a previous pro- involved in the glycolytic pathway and has been shown to be teomic study of primary colorectal cancer using IPGs and silver overexpressed in lung, liver, and stomach cancers (25, 26). staining identified only 18 of 57 (32%) proteins, whereas we Serum aldolase A levels were elevated, and autologous antibod- were able to identify 97 of 107 (90.7%) proteins in this study ies to the protein were produced in the serum of lung cancer (14). Although recent progress of mass spectrometry makes it patients (27, 28). Thus, the proteins identified to be up-regulated possible to detect small amount of proteins, it is safe to start with in our study also could be good candidates for a tumor marker a large amount of protein to identify proteins efficiently. There of colorectal cancer. We were unable to identify some proteins are several steps between excision of protein spots from gel, in that were identified by the previous 2-DE study of colorectal gel digestion, and protein identification by mass spectrometry, cancer, such as elongation factor 2, dimethylaminohydrolase, and a large portion of the proteins are likely to be lost during the annexin IV, lysophospholipase, and prohibitin (14). It is prob- steps. Thus, the agarose 2-DE with its high loading capacity is ably because of the difference of 2-DE and staining methods. recommended to evaluate the quantitative difference of altered Prolyl-4-hydroxylase is a key post-translational modifying protein expression levels and to identify the proteins with higher enzyme in collagen synthesis, and it also participates in antioxi- efficiency. Using this technique, we were able to identify sev- dation or detoxification reactions. Chen et al. (9) previously eral novel proteins of altered expression in colorectal cancer. showed that one isoform of P4HB was overexpressed signifi- Furthermore, agarose 2-DE facilitates the discovery of unique cantly in lung adenocarcinoma and suggested that it may help disease-related post-translational modifications. clear the toxic byproducts resulting from increased metabolism The most remarkable changes in colorectal cancer were in the tumor. Our results by immunoblot analysis showed only eIF-4H and hAG-2. eIF-4H is one of the translation initiation a faster migrating band of P4HB was overexpressed in most of factors, and its gene is known to be deleted in Williams syn- the tumor tissues, which is likely to be the cleavage product of drome, a multisystem development disorder caused by the de- P4HB rather than an isoform. Detection of such a cleavage letion of contiguous genes at 7q11.23. Alternative splicing of product of the protein could be useful for early diagnosis of this gene generates two splice variants, isoform 1 and 2. The colorectal cancer. In contrast to P4HB, annexin A2 was resistant smaller variant is far more abundant than the larger one in to cleavage in tumor tissues. Annexins are a family of proteins humans, which is consistent with our results in normal colon that bind phospholipids in a calcium-dependent manner. They epithelium (22). In most of the tumor tissues, however, expres- have been proposed recently to regulate intracellular vesicular sion of the larger isoform was increased significantly compared transport, calcium sensors, and signal transduction (29, 30). In with normal tissues, which might indicate the aberrant regula- addition, annexins contain a KFERQ-like motif, which has been tion of translational initiation in tumor cells. The specific func- implicated in the targeting of mammalian proteins for degradation of the two isoforms remains to be elucidated. hAG-2 is the tion in lysosomes (31). The resistance of annexin A2 to the

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Takeshi Tomonaga, Kazuyuki Matsushita, Seiko Yamaguchi, et al.

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