The colorectal carcinoma cell lines were kindly provided by Professor Sugiyama, Department of Gastroenterology, Graduate School of Medical and Pharmaceutical Sciences, University of Toyama, Japan and Professor Miyagi, Clinical Research Institute, Kanagawa Cancer Center, Japan. They were maintained in RPMI-1640 (Colo201, Colo205, DLD-1, HCT-15, HCT-116, HT-29, KM-12, SW480 and SW620) and DMEM (WiDr) supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin in a humidified atmosphere of 5% CO₂ at 37°C. All cells were harvested by centrifugation, rinsed with phosphate-buffered saline (PBS) and subjected to total protein extraction by sonication in RIPA lysis buffer [50 mmol/l Tris-HCl (pH 7.5), 150 mmol/l NaCl, 5 mmol/l EDTA, 0.5% Nonidet P-40, 5 mmol/l dithiothreitol, 10 mmol/l NaF and protease inhibitor cocktail (Sigma)].

Colorectal carcinomas (CRCs, n=481), adjacent adenoma (n=22), adjacent non-neoplastic mucosa (NNM, n=475), metastatic foci in lymph node metastasis (n=154) and in liver (n=24) were collected from surgical resections at the Affiliated Hospital, Kanagawa Cancer Center, Japan between 1995 and 1999. The patients with CRC included 278 men and 203 women (age range, 26–85 years; mean age, 64.1 years). Among the patients, 209 cases were accompanied by lymph node metastasis and 28 cases with liver metastasis. Thirty cases of CRCs and paired NNM were obtained from the First Affiliated Hospital of China Medical University, China, and frozen at −80°C until protein and RNA extraction by homogenization. None of the patients underwent chemotherapy, radiotherapy or adjuvant therapy prior to surgery. The patients or their relatives provided consent for the use of tumor tissue for clinical research, and our University Ethics Committee approved the research protocol. We followed up the patients by reviewing their case documents or by telephone.

All tissues were fixed in 10% neutral formalin, embedded in paraffin and cut into 4 μm sections. These sections were stained with hematoxylin and eosin (H&E) to confirm their histological characteristics. The staging for each colorectal carcinoma was evaluated according to the Union Internationale Contre le Cancer (UICC) system (14). Histological architecture of CRCs was expressed in terms of WHO classification (15). Furthermore, tumor size, depth of invasion, lymphatic and venous invasion were determined.

Representative areas of the solid tumors were identified in the H&E-stained sections of the selected tumor cases, and a 2-mm diameter tissue core per donor block was punched out and transferred to a recipient block with a maximum of 48 cores using a tissue microarrayer (Azumaya KIN-1; Azumaya, Tokyo, Japan).

Total RNA was extracted from the colorectal carcinoma cell lines or tissues using Qiagen RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. Two micrograms of total RNA was subjected to cDNA synthesis using the AMV transcriptase and random primers (Takara, Otsu, Japan). Oligonucleotide primers for PCR were 5′-AGTGCTCCGAAGACAACCC-3′ and 5′-GTTCTCCGAGACGCTGTTG-3′ for SERCA3 (134 bp, 2275–2908, NM_174953.1); and sense, 5′-CAATGACCCCTTCATTGACC-3′ and antisense, 5′-TGGAAGATGGTGATGGGATT-3′ for GAPDH (135 bp, 201–335, NM_002046.3). PCR amplification of cDNA was performed in 25-μl mixtures containing 0.125 μl Pfu (Stratagene, West Cedar Creek, USA) with 2.0 mmol MgCl₂, 2.5 μl 10X PCR buffer, 2 μl dNTP mixture, 1 μmol/l of each primer set and 100 ng of template cDNA. PCR conditions were denaturation at 95°C for 10 min, followed by 35 cycles of denaturation at 95°C for 30 sec, annealing for 30 sec and extension at 72°C for 50 sec. As a termination step, the extension time of the last cycle was increased to 7 min. The amplicons were electrophoresed in 2% agarose gel for 30 min. Real-time PCR was performed according to the protocol of the SYBR Premix Ex Taq™ II kit (Takara, Tokyo, Japan).

Amplicons were subjected to electrophoresis on 2% agarose gel and purified with the QIAquick gel extraction kit (Qiagen). After extraction, the DNA was quantified by a NanoDrop ND-1000 spectrophotometer (Laboratory & Medical Supplies, Tokyo, Japan) and then sequenced using a BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and SERCA3 primers according to the recommended protocol. The sequence data were compared with the human SERCA3 cDNA sequence (NM_174953.1) using BLAST.

Denatured protein was separated on an SDS-polyacrylamide gel (10% acrylamide) and transferred to an Amersham Hybond membrane (Amersham, Germany), which was then blocked overnight in 5% skim milk in TBST (10 mmol/l Tris-HCl, 150 mmol/l NaCl, 0.1% Tween-20). For immunoblotting, the membrane was incubated for 15 min with the mouse antibody against SERCA3 (1:200) (XL-6, sc-101265; Santa Cruz Biotechnology, Santa Cruz, CA, USA). Then, it was rinsed with TBST and incubated with anti-mouse IgG conjugated to horseradish peroxidase (1:1,000) (Dako, Carpinteria, CA, USA) for 15 min. All of the incubations were performed in a microwave oven to allow intermittent irradiation as recommended by Li et al(16). Bands were visualized on X-ray film (Fujifilm, Tokyo, Japan) using ECL-Plus detection reagents (Santa Cruz Biotechnology). Finally, the membrane was washed with Western Blot Stripping Solution (pH 2.0–3.0; Nacalai, Tokyo, Japan) for 1 h and treated as previously described except for the anti-GAPDH antibody (1:10,000) (Sigma) as the internal control.

Cells were grown on glass coverslips, washed twice with PBS, fixed with 4% formaldehyde for 10 min at room temperature and permeabilized with 0.2% Triton X-100 in PBS for 10 min at room temperature. After washing with PBS, cells were incubated overnight at 4°C with the mouse antibody against SERCA3 (1:50) (Santa Cruz Biotechnology). The cells were then washed with PBS and incubated with anti-mouse Alexa Fluor 594 IgG (1:2,000) (Invitrogen). Nuclei were stained using 1 μg/ml DAPI (Sigma) for 30 min at 37°C. Finally, coverslips were mounted with SlowFade^® Gold Antifade reagent (Invitrogen) and were observed under a laser confocal scanning microscope (Leica, Wetzlar, Germany).

Consecutive sections were deparaffinized with xylene, rehydrated with alcohol, and subjected to antigen retrieval by irradiation in target retrieval solution (TRS; Dako) for 15 min with a microwave oven (Oriental Rotor Co., Ltd., Tokyo, Japan). The sections were quenched with 3% hydrogen peroxide in absolute methanol for 2 min to block endogenous peroxidase activity. Bovine serum albumin (5%) was then applied for 5 min to prevent non-specific binding. The sections were incubated with the anti-SERCA3 antibody (1:50) (XL-6, sc-101265; Santa Cruz Biotechnology) for 15 min, and were then treated with the anti-mouse conjugated to horseradish peroxidase (Dako) antibody for 15 min. All of the incubations were performed in a microwave oven to allow intermittent irradiation as previously described (17). After each treatment, the slides were washed with TBST three times for 1 min. Binding sites were visualized with 3,3′-diaminobenzidine. After counterstaining with Mayer’s haematoxylin, the sections were dehydrated, cleared and mounted. Omission of the primary antibody was used as a negative control.

As indicated in Fig. 1, SERCA3 protein was positively localized in the cytoplasm. One hundred cells were randomly selected and counted from 5 representative fields of each section in a blinded manner by two independent observers (W.-F.G. and H.-C.Z.). Inconsistent data were assessed by both observers until a final consensus was reached. The positive percentage of counted cells was graded semi-quantitatively according to a 4-tier scoring system: negative (−), 0–5%; weakly positive (+), 6–25%; moderately positive (++), 26–50%; and strongly positive (+++), >50%.

To perform RNA-DNA in situ hybridization (ISH) for SERCA3, a digoxygenin-labeled SERCA3 probe was constructed in 35-cycle PCR using the above-mentioned primers and 50 ng template DNA of 30-cycle products from the DLD-1 cDNA using Pfu polymerase (Stratagene, La Jolla, CA, USA). Sections (4-μm) were deparaffinized and digested with 20 μg/ml proteinase K in 50 mmol/l Tris-HCl at 37°C for 10 min. Subsequently, 20 μl of a 1:20 probe dilution in hybridization buffer (22 mmol/l Tris-HCl, pH 7.4, 2.75 mmol/l ethylenediaminetetraacetic acid, 660 mmol/l NaCl, 1X Denhardt solution, 5.5% dextran sulfate, 0.33% dimethyl sulfoxide, 0.55% Ethoquad^® 18/25 and 44% deionized formamide) was added to each slide. After coverslipping, heating at 95°C for 5 min, and incubation overnight in a humidified chamber at 37°C, sections were rinsed for 10 min in TBST and incubated with anti-digoxygenin antibody conjugated with alkaline phosphatase (AP; Roche Diagnostics, GmbH, Penzberg, Germany) for 20 min at 37°C. The slides were then washed for 5 min and immersed in solution II (100 mmol/l Tris-HCl, pH 9.5, 100 mmol/l NaCl and 50 mmol/l MgCl₂) for 30 min followed by exposure to NBT (Nitroblue tetrazolium chloride)/BCIP (5-bromo-4-chloro-3′-indolyl phosphatase p-toluidine salt) as a chromogen. Finally, counter staining was performed using methyl green for 2 min.

As indicated in Fig. 2, the mRNA signal of SERCA3 was positively localized in the cytoplasm. One hundred cells were randomly selected and counted from 5 representative fields of each section in a blinded manner by two independent observers (W.-F.G. and H.-C.Z.). Inconsistent data were assessed by both observers until a final consensus was reached. The scoring system was the same as that for immunohistochemistry.

Serum CEA was determined using a chemiluminescence immunoassay (Diagnostic Agnostic Automation Inc.). Briefly, 50 μl of standard (0–120 ng/ml), specimens and controls was dispensed into appropriate wells. We then added 100 μl of the enzyme conjugate reagent into each well, gently mixed and incubated the plate at room temperature for 60 min. The microtiter wells were rinsed and flicked with wash buffer. After that, residual water droplets were removed by striking the well sharply onto absorbent paper. Finally, 100 μl chemiluminescence substrate solution into each well was dispensed, mixed gently and subjected to determination of absorbance.

Statistical analysis was performed using the Spearman correlation test to analyze the rank data, and the Mann-Whitney U test to differentiate the means of the different groups. Kaplan-Meier survival plots were generated, and comparisons were carried out with log-rank statistics. Cox’s proportional hazards model was employed for multivariate analysis. P<0.05 was considered to indicate a statistically significant result. SPSS 10.0 software was employed to analyze all data.

SERCA3 protein was detected in the cytoplasm of all colorectal carcinoma cell lines by western blot analysis and immunofluorescence (Fig. 3A and C). To assess SERCA3 mRNA expression, we employed RT-PCR. SERCA3 mRNA was expressed at equal levels in these carcinoma cell lines (Fig. 3B). Additionally, all of the amplicons were subjected to direct DNA sequencing and confirmed to be accurate (data not shown). Among 27 frozen samples of colorectal carcinoma, decreased SERCA3 mRNA expression was observed in 24 cases (88.9%) when compared with the mRNA expression in the adjacent mucosa by real-time PCR (Fig. 4A). SERCA3 mRNA expression was low in the carcinoma samples than that in the matched NNM (P<0.05). However, there was no difference in SERCA3 expression between carcinoma and paired NNM by western blot analysis (P>0.05; Fig. 4B).

As shown in Fig. 1, SERCA3 was expressed in the cytoplasm of the superficial mucosa, infiltrating inflammatory cells, macrophages, lymphoid follicle, adenoma, well-differentiated, moderately differentiated and poorly differentiated adenocarcinoma, signet ring cell carcinoma, metastatic carcinoma in lymph node and liver, and endothelial cells in the vein and artery. SERCA3 expression was detected in 93.3% (443/475) of the NNM, 95.5% (21/22) of adjacent adenomas, 50.8% (245/482) of primary carcinomas, 32.5% (50/154) of the metastatic carcinomas in lymph node and 29.2% (7/24) of metastatic carcinomas in the liver. When statistics was performed, we considered the 4-graded semi-quantitative scoring and employed Spearman correlation analysis. Low expression of SERCA3 was observed in the colorectal carcinoma cases when compared to that in the NNM and adenomas (P<0.05; Table I). In contrast, primary carcinoma showed higher SERCA3 expression than metastatic carcinomas in the lymph node or liver (P<0.05; Table I).

To confirm SERCA3 mRNA expression, in situ hybridization (ISH) was also employed on TMAs of colorectal carcinoma, NNM, adenoma, primary and metastatic carcinoma (Fig. 2). The positive signal of SERCA3 mRNA was detectable in the cytoplasm of colorectal epithelial cells, adenoma, carcinoma, infiltrating inflammatory cells or lymphocytes in lymphatic follicles. SERCA3 mRNA was detected in colorectal NNM (48.7%, 37/76), adenoma (57.0%, 53/93), primary carcinoma (64.0%, 71/111), metastatic carcinoma in lymph node (82.6%, 38/46) and liver (65.4%, 17/26). Statistically, its expression was lower in NNM than that in the colorectal carcinoma (P<0.05; Table II). SERCA3 mRNA was expressed at a lower level in the primary carcinoma when compared to that in the metastatic carcinoma in lymph node (P<0.05; Table II).

As shown in Table III, SERCA3 expression was negatively correlated with lymphatic invasion (P<0.05), but not with age, gender, depth of invasion, venous invasion, lymph node metastasis, distant metastasis, TNM stage or degree of differentiation (P>0.05). The serum CEA concentration was higher in the carcinoma patients with positive SERCA3 expression than that in patients without SERCA3 positivity (P<0.05; Fig. 5). Follow-up information was available for 381 colorectal carcinoma patients for a period ranging from 1.6 months to 16.8 years (median, 10.5 years). Survival curves for colorectal carcinomas were stratified according to SERCA3 expression (Fig. 6). Univariate analysis using the Kaplan-Meier method indicated that the cumulative survival rate of patients was not linked to SERCA3 expression status (P>0.05), even when the data were stratified according to the depth of invasion (data not shown). Multivariate analysis using Cox’s proportional hazard model indicated that depth of invasion and distant metastasis (P<0.05), but not age, gender, lymphatic and venous invasion, lymph node metastasis, liver metastasis, differentiation, TNM stage or SERCA3 expression (P>0.05; Table IV) are independent prognostic factors for overall survival of the colorectal carcinoma parients.