Serum and tumor tissue were obtained at the time of diagnosis after obtaining the informed consent of the subjects. The experimental protocol was approved by The University of Michigan Institutional Review Board. Sera were obtained from 36 patients with a pancreatic adenocarcinoma, and from 35 healthy individuals and 33 patients with other cancers (14 with lung cancer and 19 with colon cancer) which were used as controls. The human pancreatic cancer cell line, BxPC3, was cultured in RPMI 1,640 medium supplemented with 10% fetal bovine serum, and 100 units/ml each of penicillin and streptomycin (Invitrogen).

Cultured BxPC3 pancreatic carcinoma cells were harvested in 300 µl of solubilization buffer, using a cell scraper, and stored at -80℃ until used. Proteins derived from the extracts of cultured cells were separated by 2-D PAGE, as described previously (10). Briefly, solubilized proteins were applied to isoelectric focusing gels, with focusing performed using pI 4 to 8 carrier ampholytes at 700 V for 16 h, followed by 1,000V for an additional 2 h. Following equilibration in loading buffer (125 mM Tris, pH 6.8, 10% glycerol, 2% SDS, 1% dithiothreitol and bromophenol blue), the first-dimension gel was loaded into a cassette containing the second-dimension gel. For the second-dimension separation, a gradient of 11 to 14% acrylamide (Crescent Chemical, Hauppauge, NY) was used. Proteins were transferred to a PVDF membrane (Millipore, Bedford, MA) or directly visualized by staining of the gels.

Following transfer, the membranes were incubated with blocking buffer, consisting of Tris-buffered saline, 1.8% nonfat dry milk and 0.05% Tween 20, for 2 h. The membranes were individually incubated for 1.5 h at room temperature, with serum obtained from either a patient with pancreatic cancer or a control at a 1:100 dilution. After three washes with washing buffer (Tris-buffered saline containing 0.05% Tween 20), the membranes were incubated with horseradish peroxidase-conjugated anti-human IgG antibodies (Amersham), at a dilution of 1:1,000, for 30 min at room temperature. Immunodetection was accomplished by ECL (Enhanced Chemiluminescence (Amersham)) followed by autoradiography on Hyperfilm MP (Amersham).

A rabbit anti-CLP36 polyclonal antibody was kindly provided by Dr T. Makela (Haartman Institute and Helsinki University Central Hospital, Biomedicum Helsinki, University of Helsinki, Finland). The antibody was used at a 1:2,000 dilution for Western blotting, and processed as for the incubations with patient sera, using horseradish peroxidase-conjugated anti rabbit IgG (Amersham) as secondary antibody.

For protein identification by mass spectrometry, 2-D gels were stained by a modified silver staining method, and excised proteins destained for 5 min in 15 mM potassium ferricyanide and 50 mM sodium thiosulfate, as previously described (11). Following three washes with water, the gel pieces were dehydrated in 100% acetonitrile for 5 min and then dried. Digestion was performed by the addition of 100 ng of trypsin (Promega, Madison, WI) in 200 nM ammonium bicarbonate. Following enzymatic digestion, overnight at 37℃, the peptides were extracted twice with 50 µl of 60% acetonitrile/1% trifluoroacetic acid. Following removal of the acetonitrile by centrifugation in a vacuum centrifuge, the peptides were concentrated using C18 pipette tips (Millipore, Bedford, MA) and identified by nanoflow capillary liquid chromatography coupled with electrospray quadrupole-time of flight tandem mass spectrometry (ESI Q-TOF MS/MS) in the Q-TOF micro (MicroMass, Manchester, UK). The acquired spectra were processed and searched against a non-redundant Swiss-Prot protein sequence database using the proteinLynx Global Server (www.micromass.co.uk).

Human tumor cell lines were homogenized in the presence of TRIzol reagent (Life Technologies Inc., Gaithersburg, MD) and the total cellular RNA purified according to manufacturer's procedures. RNA samples were further purified using acid phenol extraction and RNeasy spin columns (Qiagen, Valencia, CA), and the quality assessed by 1% agarose gel electrophoresis in the presence of ethidium bromide.

Five pancreatic, 4 lung, 4 colon and 2 ovarian cancer cell lines were used to compare the levels of CLP36 mRNA expression after normalizing with GAPDH mRNA. Oligonucleotide primers and TaqMan probes were designed using the Light Cycler Probe Design Software (Roche Applied Science). The forward and reverse primers for human CLP36 were 5'-AGCGTCATCCATACAAG-3' and 5'-TGGTCTAAGGGTCTGC-3', respectively, and those for GAPDH were 5'-GAAGGTGAAGGTCGGAGTC-3' and 5'-GAAGATGGTGATGGGATTTC-3', respectively. All oligonucleotide primers were purchased from Applied Biosystems.

The first-strand cDNA was synthesized using the SuperScript First-Strand Synthesis System for RT-PCR, according to the manufacturer's instructions (Invitrogen). Quantitative PCR reactions were carried out in 96-well optical reaction plates, using cDNA equivalent of 50 ng of total RNA for each sample, in a volume of 25 µl. PCR was performed on the ABI Prism 7700 Sequence Detector (Applied Biosystems). The cycling conditions were 10 min at 95℃, followed by 55 cycles at 95℃ for 30 sec, 60℃ for 45 sec and 72℃ for 45 sec.

To control the variation in the amount of starting RNA in the samples, amplification of the GAPDH mRNA was performed, as an internal reference, against which the other RNA values were normalized. The real-time PCR products were purified using a QIAQuick Gel Extraction Kit (Qiagen) and subjected to DNA sequencing to verify the identity of the real-time PCR products.

A tissue array containing triplicates of 4 normal pancreas, 12 non-pancreas normal tissues, 78 pancreatic and ampullary adenocarcinomas and 2 large anaplastic carcinomas was constructed, as previously described (12). Immunohistochemistry for CLP36 was performed using the same rabbit polyclonal antibody (30-minute incubation at RT), at a dilution of 1:100 using Tris buffer (pH 9.0), with microwave antigen retrieval (15 minutes). The primary antibody was detected using the Dako Envision kit.

Both the cytosolic and membrane-rich fractions were analyzed to determine the subcellular location of the CLP36 as an autoantigen. To obtain the cytosolic and membrane-rich fractions of BxPC3 whole cell lysates, the cells were lysed in a buffer containing 50 mM Tris-HCl (pH 7.4), 0.32 M sucrose, 1mM EDTA, protease inhibitors and 10 mM iodoacetamide. The cells were passed 15 times through a 25-gauge needle and centrifuged at 20,000×g for 2 min at 4℃, after which the supernatant was collected. The supernatant was again centrifuged at 100,000×g for 20 min at 4℃. The resulting supernatant (cytosolic fraction) and pellet (membrane-fraction) were collected. The membrane fraction was solubilized in homogenization buffer containing 1% Triton-X 100. The fractions were resolved by 4~15% SDS-PAGE and transferred onto a nitrocellulose membrane. A transferrin receptor (CD 71) antibody (Santa Cruz Biotechnology) was used as a positive control for the membrane-rich fraction.

The sera obtained from the 36 newly diagnosed patients with pancreatic adenocarcinoma, 33 patients with other types of cancers and the 35 healthy donors were screened individually for the presence of antibodies to BxPC3 proteins (Fig. 1). α-Actin, a well recognizable landmark in 2-D patterns, exhibited frequent reactivity with the sera of cancer patients relative to those of the controls, but exhibited no specificity for pancreatic cancer (Table 1). Another polypeptide, with an estimated molecular mass of 36 kDa and pI of 7.5, showed reactivity with sera from 14/36 (38.9%) patients with pancreatic adenocarcinomas and 3/33 (9.1%) of the other cancer patients, but no reactivity with the sera from the 35 healthy donors (Table 1). No further protein spots on the 2-D gels showed specific reactivity against the sera from the cancer patients compared with those from the healthy subjects, so these two spots were evaluated.

Both the presumed α-actin and second reactive spots were excised from the silver-stained 2-D PAGE gels. The proteins were digested with trypsin, and the resulting peptides analyzed by ESI Q-TOF Tandem MS spectrometry. The acquired spectra were processed and searched against a non-redundant Swiss-Prot protein sequence database using the proteinLynx Global Server (www.micromass.co.uk). The identity of α-actin was confirmed (data not shown), and the protein in the second reactive spot identified as CLP36 (Fig. 2). The identity of this protein was further confirmed by 2-D Western blotting using BxPC3 whole-cell extracts and the anti-CLP36 rabbit polyclonal antibody (Fig. 3).

To try and determine the RNA level for the CLP36 gene, real-time PCR was performed. For normalization with the expression of the endogenous gene, the CLP36/GAPDH ratio was calculated from different cancer cell lines (Fig. 4). The ratio of CLP36/GAPDH measured in the BxPC3 was 1.2×10^-4, the highest expression level among the different cell lines tested. Other pancreatic cancer cell lines also exhibited a relatively high ratio. Three of the 4 colon cancer cell lines showed high CLP36 mRNA levels. The CLP36 mRNA levels in the four lung and two ovarian cancer cell lines tested were relatively reduced in comparison to the other cell lines.

The CLP36 protein expression was investigated using immunohistochemistry and a pancreas/duodenal adenocarcinoma tissue array. Diffuse and consistent cytoplasmic immunoreactivities were observed in the large majority of pancreatic and ampullary adenocarcinomas (Fig. 5). Immunoreactivities were also observed in normal endothelial cells and various other normal tissues (not shown).

The cytosolic and membrane-rich fractions were analyzed to determine the distribution of CLP36 showing immunoreactivity with the sera from pancreatic cancer patients. The transferrin receptor antibody, which was utilized as a positive control for plasma membrane protein, reacted strongly with the membrane-rich fraction. The rabbit anti-CLP36 antibody showed reactivity with both the cytosolic and membrane-rich fractions, but the molecular weight of the protein displayed in the membrane-rich fraction was higher than that in the cytosolic fraction (Fig. 6), which is likely attributable to a post-translational modification. To determine whether CLP36 was post-translationally modified by glycosylation, a putative cause of autoantigenicity, the whole cell lysate of BxPC3 was simultaneously treated with N-glycosidase F, Endo-α-N-acetylgalactosaminidase and α-2-3,6,8,9-Neuraminidase. BxPC3 whole cell lysates were separated by 1-D SDS PAGE and transferred to PVDF membrane for Western blotting with anti-CLP36 antibody. As the deglycosylating enzyme treatment resulted in no mobility shifts of the CLP36 in the SDS-polyacrylamide gels (data not shown), it was concluded that the CLP36 present in the membrane fraction was not glycosylated.