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Clinical Cancer Research Vol. 10, 2007-2014, March 2004
© 2004 American Association for Cancer Research

Molecular Oncology, Markers, Clinical Correlates

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

Takeshi Tomonaga1, Kazuyuki Matsushita2, Seiko Yamaguchi1, Masamichi Oh-Ishi3, Yoshio Kodera3, Tadakazu Maeda3, Hideaki Shimada2, Takenori Ochiai2 and Fumio Nomura1

Departments of 1 Molecular Diagnosis (F8) and 2 Academic Surgery (M9), Graduate School of Medicine, Chiba University, Chiba, Japan, and 3 Laboratory of Biomolecular Dynamics, Department of Physics, Kitasato University School of Science, Kanagawa, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Purpose: Although numerous proteome studies have been performed recently to identify cancer-related changes in protein expression, only a limited display of relatively abundant proteins has been identified. The aim of this study is to identify novel proteins as potential tumor markers in primary colorectal cancer tissues using a high-resolution two-dimensional gel electrophoresis (2-DE).

Experimental Design: 2-DE using an agarose gel for isoelectric focusing was used to compare protein profiling of 10 colorectal cancer tissues and adjacent normal mucosa. Altered expression and post-translational modification of several proteins were examined using Western blot analysis and immunohistochemistry.

Results: Ninety-seven proteins of 107 spots (90.7%) that were differentially expressed between matched normal and tumor tissues were identified by mass spectrometry. Among them, 42 unique proteins (49 spots) significantly increased or decreased in the tumors. They include eukaryotic translation initiation factor 4H, inorganic pyrophosphatase, anterior gradient 2 homologue, aldolase A, and chloride intracellular channel 1, whose elevated expression in tumor tissues was confirmed by Western blot analysis and immunohistochemistry. Interestingly, only isoform 1 of two transcript variants of eukaryotic translation initiation factor 4H was greatly up-regulated in most of the tumor tissues. Moreover, post-translational modifications of the prolyl-4-hydroxylase ß subunit and annexin A2 also were identified.

Conclusions: We identified several novel proteins with altered expression in primary colorectal cancer using agarose 2-DE. This method is a powerful technique with which to search for not only quantitative but also qualitative changes in a biological process of interest and may contribute to the deeper understanding of underlying mechanisms of human cancer.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Information at the level of the proteome is necessary to unravel the critical changes involved in disease pathogenesis. Comparative studies of protein expression in normal and disease tissues also have led to the identification of aberrantly expressed proteins that may represent new markers (1) . This disease proteomics primarily relied on a combination of two-dimensional gel electrophoresis (2-DE) to separate and visualize proteins and mass spectrometry for protein identification (1) . Development of immobilized pH gradient (IPG) gel has led to great improvements with regard to reproducibility; however, its loading capacity of proteins still is insufficient and only permits a limited display of relatively abundant proteins (2) . Low abundance proteins can be detected if the starting protein load is large, which would allow for large-scale quantitative comparisons of protein expression (3) .

Proteomic technologies also have been used to identify cancer-specific proteins that are useful for cancer diagnosis, progression, and therapeutic targets (4, 5, 6, 7, 8, 9, 10) . In addition, they have the potential to unravel important cellular events associated with cancer development, such as protein phosphorylation and degradation. Although extensive proteome analysis has identified numerous proteins overexpressed in various cancer tissues, few markers have been accepted for routine clinical use because of conflicting reports or because potential candidates have not been detected because of their low abundance (6) . Several investigators have extensively performed proteome studies of colorectal cancer; however, a limited number of proteins have been identified (11, 12, 13, 14, 15) . Thus, it is necessary to develop novel techniques that permit large-scale quantitative comparisons of protein expression between normal and cancer tissues. The agarose 2-DE method was shown previously to have a higher loading capacity than 2-DE with IPG gel for isoelectric focusing (16 , 17) . We analyzed here primary colorectal cancer tissues for protein expression using the agarose 2-DE method and identified novel proteins whose expression differs between tumor tissue and adjacent normal mucosa. Western blot and immunohistochemical analysis demonstrated that an isoform of eukaryotic translation initiation factor 4H (eIF-4H), inorganic pyrophosphatase, anterior gradient 2 homologue (hAG-2), aldolase A, and chloride intracellular channel 1 (NCC27) were overexpressed significantly in many of the primary colorectal cancer tissues. In addition, different post-translational modifications of several proteins were demonstrated between tumor and normal tissues. A tumor-specific cleavage of prolyl-4-hydroxylase ß subunit (P4HB) was observed. Conversely, annexin A2 tended to be cleaved in normal tissues. These results would be helpful not only to investigate the underlying mechanism of carcinogenesis but also to develop new biomarkers and therapeutics.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Human Tissue Samples.
Tissues from 10 patients with primary colorectal cancer were resected surgically (Table 1)Citation . Written informed consent was obtained from each patient before surgery. The excised samples were obtained within 1 h after the operation from tumor tissues and corresponding nontumor tissues that were 5–10 cm from the tumor. All of the excised tissues were placed immediately in liquid nitrogen and stored at -80°C until analysis.


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  		Table 1 Clinical features of patients with colorectal cancer

 
Protein Extraction.
Frozen tissue samples were solubilized in lysis buffer (9.5 M urea, 2% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 1% DTT) containing protease inhibitor mixture (Complete; Roche, Mannheim, Germany) using a Polytron homogenizer (Kinematica, Littau-Luzern, Switzerland) following centrifugation (100,000 x g) for 1 h at 4°C. The supernatant was subjected either to agarose 2-DE or SDS-PAGE.

Agarose 2-DE.
Agarose gels were prepared as described previously (16 , 17) . Protein extract (500 µg) from tissues was applied to the agarose isoelectric focusing gel, and first-dimensional isoelectric focusing was conducted at 12,000 Vxhr or Vhr at 4°C, followed by fixation in 10% trichloroacetic acid and 5% sulfosalicylic acid for 1 h at room temperature. After washing with deionized water for 1 h, the agarose gel then was transferred to a 12% polyacrylamide gel, and second-dimensional SDS-PAGE was performed. The second-dimensional gel first was incubated in 30% methanol and 10% acetic acid overnight. It then was stained with PhastGel Blue R (Amersham Pharmacia Biotech, Piscataway, NJ). Each gel was scanned using Epson ES 2000 (Nagano, Japan), and NIH Image was used to measure the intensity of each spot.

Enzymatic In-Gel Digestion of Proteins.
The protein spots were excised from the gel, and in-gel tryptic digestion of proteins was performed. Briefly, the gels were cut in small pieces, destained in 50% acetonitrile/50 mM NH4HCO3, and washed with deionized water. The gel pieces were dehydrated in 100% acetonitrile for 15 min and then dried in a SpeedVac evaporator (Wakenyaku, Kyoto, Japan) for 45 min. The gel pieces were rehydrated in 10–30 µl of 25 mM Tris-Cl (pH 9)/20% acetonitrile containing 25 ng/µl trypsin (Trypsin sequence grade; Roche) for 45 min. After removal of the unabsorbed solution, the gel pieces were incubated in 10–20 µl of 50 mM Tris-Cl (pH 9)/20% acetonitrile for 20 h at 37°C. The solution containing digested fragments of proteins was transferred to a new tube, and the peptide fragments remaining in the gel also were extracted in 5% formic acid/50% acetonitrile for 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 (Perkin-Elmer, Shelton, CT), a 2.1-mm diameter and 30-mm length C8 reversed phase, and attached to the Nanospace SI-2 (Shiseido Fine Chemicals, Tokyo, Japan) as a high-performance liquid chromatography system. The flow rate of the mobile phase was 200 µl/min using buffer A (0.05% HCOOH) and buffer B (90% acetonitrile and 0.05% HCOOH). Following an initial wash with buffer A for 5 min, peptides were eluted with a linear gradient from 0–60% buffer B over an interval of either 3 min or 30 min. The purified peptides were sprayed from the high-performance liquid chromatography via a metal needle-attached AP12 (an electrospray ionization adapter) to the LCQ-Deca (ThermoQuest, San Jose, CA), which is an ion trap mass spectrometry. Data-dependent measurements of mass and tandem mass spectra of the peptides were performed according to the manufacturer’s operating specifications. SEQUEST (ThermoQuest) was used to identify proteins from mass and tandem mass data. When the SEQUEST score for the best candidate protein was <100, we inspected raw mass and tandem mass data of peptides to judge their qualities.

Antibodies.
Rabbit polyclonal antisera were raised against synthetic peptides corresponding to the N-terminal and COOH-terminal sequences of eIF-4H (MADFDTYDDRAY SS and GAR PRE EVV QKE QE), inorganic pyrophosphatase (MSG FST EER AAP FS and CTV PTD VDK WFH HQK N), hAG-2 (KPG AKK DTK DSR PKL and ADI TGR YSN RLY AYE), and NCC27 (TVD TKR RTE TVQ KLC and YLS NAY ARE EFA STC) and attached to keyhole limpet hemocyanin. Two peptides of each protein were immunized simultaneously to enhance the possibility of antibody production. The reactivity of the antiserum was tested by solid-phase enzyme immunoassay, and the antiserum was affinity purified by passage over a resin covalently coupled with synthetic peptides (Japan Bio Services, Saitama, Japan). Goat anti-aldolase A (C-16), rabbit anti-annexin A2 (Annexin II, H-50), and goat anti-ß-actin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-P4HB antibody was purchased from ICN Biomedicals (Aurora, OH).

Immunoblotting.
Protein extracts were separated by electrophoresis on 10–20% gradient gels (Bio-Rad, Hercules, CA). The proteins were transferred to polyvinylidene fluoride membranes (Millipore, Bedford, MA) in a tank-transfer apparatus (Bio-Rad), and the membranes were blocked with 5% skim milk in PBS. Anti-eIF-4H diluted 1:500, anti-inorganic pyrophosphatase diluted 1:100, anti-aldolase A diluted 1:100, anti-P4HB diluted 1:5000, anti-annexin A2 diluted 1:1000, and anti-ß-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 (Amersham Pharmacia Biotech).

Reverse Transcription-PCR.
Total RNA was extracted from tumor and nontumor tissues with RNeasy Mini Kit (Qiagen, 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, 5'-GGTGGCTTTGGATTCAGAAA-3' and reverse, 5'-CGAGGTTTAAGCTGGAGTCG-3'.

Immunohistochemistry.
Four-µm 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 LSAB+ 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 mounted with Malinol (Mito Pure Chemicals, Tokyo, Japan). Two pathologists evaluated immunohistochemical staining of the samples.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
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)Citation . Tumor specimens for samples largely consist of tumor cells (60–70%) and stroma (30–40%), whereas normal specimens consist of normal colon epithelial cells (60–70%) and stroma (30–40%). There are few endothelial cells and infiltrating lymphocytes either in tumor or normal specimens. Thus, there is no bias in the cellularity of the normal and tumor tissues. All of the samples were examined in duplicate or triplicate, and ~600–1000 protein spots were detected consistently in each gel. One hundred seven protein spots that were shown to increase or decrease in tumor tissues were excised, and mass spectrometry sequence was obtained from 97 spots (90.7%). Identical protein sequences were obtained from multiple spots in normal and tumor tissues, such as serum albumin and glyceraldehyde-3-phosphate dehydrogenase, and they were eliminated from additional investigation. Proteins whose expression level was not altered significantly between normal and tumor tissues (P > 0.05) also were eliminated. Forty-two proteins substantially increased or decreased in the tumors (P < 0.05; Fig. 1Citation and Table 2Citation ). These include the following proteins that were reported to have altered expression in colorectal cancer either by 2-DE or other methods: carbonic anhydrase 1, nonmetastatic cell 1 protein, two isoforms of peptidylprolyl isomerase A, manganese superoxide dismutase, keratin 18, enolase 1, calreticulin, tumor rejection antigen (gp96), and pyruvate kinase 3 (type M2; Refs. 14 , 18, 19, 20, 21 ). These results confirmed a part of our observation of the differentially expressed proteins in colorectal cancer using agarose 2-DE.



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  		Fig. 1. Coomassie-stained agarose two-dimensional gel electrophoresis (2-DE) pattern of proteins of human primary colon cancer (A) and normal adjacent mucosa (B). Total protein lysates were prepared from matched samples of tumor and adjacent normal tissue as described in "Materials and Methods." Five hundred µg 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 tumor tissue or normal mucosa, respectively.

 

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  		Table 2 Altered protein expression in primary colorectal cancer tissues

 
Among the proteins with altered expression, we focused mainly on the regulatory proteins whose expression levels have not been well studied in human primary cancers. First, immunoblot analyses of several proteins were performed to confirm the differential protein expression in tumor tissues. The most striking differences observed between tumor and adjacent normal mucosa were eIF-4H and inorganic pyrophosphatase (Fig. 2, A and B)Citation . Western blot analysis using anti-eIF-4H antibody revealed two bands in all of the tumor tissues, whereas only a faster migrating band was observed in most of the normal mucosa (Fig. 2ACitation , top, arrow and asterisk). Two transcript variants of eIF-4H have been reported recently, which include isoform 1 and 2. Isoform 2 lacks 20 amino acids of isoform 1, and this short isoform is predominant in human tissues (22) . The two bands shown in Fig. 2ACitation are likely to correspond to the two isoforms of eIF-4H. The larger isoform (isoform 1) was increased significantly in tumor tissues (0.9–79.0-fold increase; P = 0.0003). Thus, we examined the mRNA of eIF-4H by reverse transcription-PCR. The two transcript variants were found, and the expression of the larger variant was increased considerably in tumor tissues (Fig. 2E)Citation . Inorganic pyrophosphatase and aldolase A also showed significant increases in tumor tissues. Relative protein level of inorganic pyrophosphatase in tumors compared with adjacent normal tissue varied from 1.1–66.8 (P = 0.003), and that of aldolase A varied from 0.04–15.7 (P = 0.028; Fig. 2, B and CCitation ).



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  		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 ß-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, ß-actin as a loading control (42 kDa). The expressions of eIF-4H, inorganic pyrophosphatase, and aldolase A were increased significantly in tumor tissues (P < 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.

 
Although there is no bias in the cellularity of the normal and tumor tissues, whole tissue sections include nonepithelial components, and the altered protein expression in our 2-DE analysis may emanate from such nonepithelial components. Thus, the differential protein expression in colorectal cancer also was validated by immunohistochemical analysis to demonstrate the cellular origin and distribution of the identified proteins. Frozen tissue sections of colorectal cancers and adjacent normal tissues were stained with several antibodies. As shown in Fig. 3Citation , immunohistochemistry with antibodies to eIF-4H and hAG-2 showed strong nuclear staining, whereas those to aldolase A and NCC27 showed relatively mild nuclear staining. Examination of the several tissue sections showed similar staining patterns, indicating that these proteins actually are expressed in the tumor cells and not in the surrounding mesenchymal cells.



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  		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 x400.

 
During the course of the 2-DE analysis, we encountered several interesting observations. For example, P4HB (Fig. 1ACitation , spot T23) migrated slightly faster than what was expected, considering the actual molecular mass of the protein (57 kDa). Annexin A2, calreticulin, triosephosphate isomerase 1, and vimentin were found in tumor and normal tissues with different molecular mass and isoelectric point values (Fig. 1Citation ; annexin, spots T21 and N5–1, 2; calreticulin, spots T1 and N7; triosephosphate isomerase 1, spots T10 and N4; and vimentin, spots T36 and N8). All of the differences are probably caused by the post-translational modifications, and it also was examined with Western blot analysis. A faster migrating band of P4HB in most cancer tissues but not in normal mucosa was clearly shown (Fig. 4ACitation , arrow), which indicates that P4HB tends to be cleaved in tumors. Conversely, a faster migrating band of annexin A2 in normal mucosa was scarcely observed in tumor tissues (Fig. 4BCitation , arrow). The peptide sequence of the protein (spot N5–1) as revealed by mass spectrometry contained only the N-terminal half of annexin A2 (data not shown). These results suggest that the COOH-terminus of annexin A2 is cleaved in normal colon mucosa, whereas it is resistant to such proteolytic cleavage in colorectal cancer. These post-translational modifications may be important mechanisms for the development of colorectal cancer.



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

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study we showed that agarose 2-DE is a powerful technique to detect quantitative changes or modifications of proteins compared with the conventional 2-DE method. The most prominent advantage of the technique is the high loading capacity of the agarose gel used for isoelectric focusing. Oh-ishi et al. (16) carefully compared previously the Coomassie Blue-stained 2-DE patterns of agarose 2-DE with conventional IPGs using the same amount of crude extracts and found that protein spot densities were much higher in agarose 2-DE than in IPGs. In this study, we were able to detect 600-1000 protein spots and 107 differentially expressed proteins with Coomassie Blue staining. These numbers appear to be less than that of previous reports. It is because most studies visualized protein spots with silver staining, which is 10–100 times more sensitive than Coomassie Blue staining. However, the absolute intensity of spots visualized with silver staining is controlled by the length of reaction and is variable for different proteins. Moreover, silver-stained protein spots tend to be difficult to identify proteins by mass spectrometry because each spot often contains small amount of protein and the proteins also are modified by formaldehyde used as a fixative. For example, a previous proteomic study of primary colorectal cancer using IPGs and silver staining identified only 18 of 57 (32%) proteins, whereas we were able to identify 97 of 107 (90.7%) proteins in this study (14) . Although recent progress of mass spectrometry makes it possible to detect small amount of proteins, it is safe to start with a large amount of protein to identify proteins efficiently. There are several steps between excision of protein spots from gel, in gel digestion, and protein identification by mass spectrometry, and a large portion of the proteins are likely to be lost during the steps. Thus, the agarose 2-DE with its high loading capacity is recommended to evaluate the quantitative difference of altered protein expression levels and to identify the proteins with higher efficiency. Using this technique, we were able to identify several novel proteins of altered expression in colorectal cancer. Furthermore, agarose 2-DE facilitates the discovery of unique disease-related post-translational modifications.

The most remarkable changes in colorectal cancer were eIF-4H and hAG-2. eIF-4H is one of the translation initiation factors, and its gene is known to be deleted in Williams syndrome, a multisystem development disorder caused by the deletion of contiguous genes at 7q11.23. Alternative splicing of this gene generates two splice variants, isoform 1 and 2. The smaller variant is far more abundant than the larger one in humans, which is consistent with our results in normal colon epithelium (22) . In most of the tumor tissues, however, expression of the larger isoform was increased significantly compared with normal tissues, which might indicate the aberrant regulation of translational initiation in tumor cells. The specific function of the two isoforms remains to be elucidated. hAG-2 is the human homologue of the secreted Xenopus laevis protein XAG-2, which is expressed in the cement gland, an ectodermal organ in the head associated with anteroposterior fate determination during early development. This protein was reported recently to express in estrogen receptor-positive breast tumors (23) . Inorganic pyrophosphatase catalyzes the hydrolysis of PPi to inorganic phosphate, which is important for the phosphate metabolism of cells. The expression of this protein also is up-regulated in lung adenocarcinoma, which may relate to the increased requirement for energy in rapidly growing tumors (9) . NCC27 is a nuclear chloride ion channel protein that regulates ionic traffic between nucleus and cytoplasm. It also was known to be involved in regulation of the cell cycle (24) . Increased protein expression of NCC27 has been reported recently in human breast ductal carcinoma in situ (10) . Aldolase A is involved in the glycolytic pathway and has been shown to be overexpressed in lung, liver, and stomach cancers (25 , 26) . Serum aldolase A levels were elevated, and autologous antibodies to the protein were produced in the serum of lung cancer patients (27 , 28) . Thus, the proteins identified to be up-regulated in our study also could be good candidates for a tumor marker of colorectal cancer. We were unable to identify some proteins that were identified by the previous 2-DE study of colorectal cancer, such as elongation factor 2, dimethylaminohydrolase, annexin IV, lysophospholipase, and prohibitin (14) . It is probably because of the difference of 2-DE and staining methods.

Prolyl-4-hydroxylase is a key post-translational modifying enzyme in collagen synthesis, and it also participates in antioxidation or detoxification reactions. Chen et al. (9) previously showed that one isoform of P4HB was overexpressed significantly in lung adenocarcinoma and suggested that it may help clear the toxic byproducts resulting from increased metabolism in the tumor. Our results by immunoblot analysis showed only a faster migrating band of P4HB was overexpressed in most of the tumor tissues, which is likely to be the cleavage product of P4HB rather than an isoform. Detection of such a cleavage product of the protein could be useful for early diagnosis of colorectal cancer. In contrast to P4HB, annexin A2 was resistant to cleavage in tumor tissues. Annexins are a family of proteins that bind phospholipids in a calcium-dependent manner. They have been proposed recently to regulate intracellular vesicular transport, calcium sensors, and signal transduction (29 , 30) . In addition, annexins contain a KFERQ-like motif, which has been implicated in the targeting of mammalian proteins for degradation in lysosomes (31) . The resistance of annexin A2 to the proteolysis in colorectal cancer suggests a failure to halt transducing intracellular signals such as growth stimuli, which may cause deregulated cell proliferation in cancer cells. The significance and precise mechanism of the proteolysis of these proteins remain to be investigated. Uncovering the mechanisms responsible for the altered levels or post-translational modification of proteins observed in our study will be important to understand carcinogenesis and may be critical for cancer control and prevention.


   ACKNOWLEDGMENTS
 
We thank Kaori Dobashi and Mamoru Satoh for their technical assistance.


   FOOTNOTES
 
Grant support: Grant-in-Aid 13214016 for Priority Areas in Cancer Research to T. Tomonaga, and Grant-in-Aid for Scientific Research on Priority Areas in Medical Genome Science to T. Maeda from the Ministry of Education, Science, Sports, and Culture of Japan.

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

Requests for reprints: Takeshi Tomonaga, Department of Molecular Diagnosis (F8), Graduate School of Medicine, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. Phone: 81-43-226-2167; Fax: 81-43-226-2169; E-mail: tomonaga{at}faculty.chiba-u.jp

Received 9/30/03; revised 11/12/03; accepted 11/21/03.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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