Cancer Res Treat.  2005 Feb;37(1):71-77.

Identification of CLP36 as a Tumor Antigen that Induces an Antibody Response in Pancreatic Cancer

• Hong SH

Abstract

PURPOSE
Pancreatic cancer has a poor prognosis, due in part to the lack of an effective approach for its early detection. The identification of tumor antigens potentially provides a means for the early diagnosis. The purpose of this study was to use a proteomic approach for the identification of proteins that commonly induce a humoral response in patients with pancreatic cancer. MATERIALS AND METHODS: Proteins from the pancreatic adenocarcinoma cell line, BxPC3, were subjected to two-dimensional polyacrylamide gel electrophoresis, followed by Western blot analysis, where individual sera were tested for autoantibodies. Sera from 36 patients with pancreatic adenocarcinoma, and 68 from control groups (14 from lung adenocarcinoma, 19 from colon adenocarcinoma and 35 from healthy subjects) were analyzed. CLP36 expression was evaluated by immunohistochemical analysis and real-time PCR. The cellular localization of CLP36 as an autoantigen was investigated by Western blot analysis. RESULTS: The autoantibody was detected against a protein, identified by mass spectrometry as CLP36, in 14 of the 36 sera (38.9%) from patients with a pancreatic adenocarcinoma, and 3 of the 68 controls (4.4%). Immunohistochemical analysis of CLP36 in a tissue array demonstrated diffuse and consistent immunoreactivity in the pancreatic adenocarcinomas. The levels of CLP36 mRNA were highest in the pancreatic cancer cell lines of the different cells analyzed. The molecular weight of the protein displayed in the membrane-rich fraction was larger than that in the cytosolic fraction, which is likely attributable to a post-translational modification. CONCLUSION: CLP36 was identified as a tumor autoantigen inducing a humoral immune response in pancreatic adenocarcinomas. More detailed studies need to be undertaken to understand whether the humoral response by CLP36 is tumor-specific.

Keyword

Pancreatic neoplasms; Autoantigens; Proteomics; Two-dimensional gel electrophoresis

MeSH Terms

Adenocarcinoma
Antibody Formation*
Antigens, Neoplasm
Autoantibodies
Autoantigens
Blotting, Western
Cell Line
Colon
Cytosol
Early Diagnosis
Electrophoresis, Gel, Two-Dimensional
Electrophoresis, Polyacrylamide Gel
Humans
Immunity, Humoral
Lung
Mass Spectrometry
Molecular Weight
Pancreatic Neoplasms*
Prognosis
Protein Processing, Post-Translational
Proteomics
Real-Time Polymerase Chain Reaction
RNA, Messenger
Antigens, Neoplasm
Autoantibodies
Autoantigens
RNA, Messenger

Figure

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  Fig. 1 Coomassie-stained image of the 2D gel from the whole cell lysate of BxPC3.

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  Fig. 2 Western blot analysis of CLP-36 from the sera of a healthy individual (A), of a pancreatic cancer patient (B) and with a polyclonal anti-CLP36 antibody (C).

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  Fig. 3 Tandem mass spectrometry identification of CLP36 after trypsin digestion. The MS/MS spectrum of CLP36 obtained after trypsin digestion is shown by analysis with ESI-Q-TOF, coupled with nanoflow capillary high-performance liquid chromatography. The precursor ion shown in the figure has an m/z of 1551.1383, and the peaks were searched against the non-redundant SwissProt protein sequence database using the ProteinLynx global server. A total of six tryptic peptides, as shown, matched the CLP36 protein.

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  Fig. 4 CLP36 mRNA levels measured by real-time PCR in pancreatic, lung, colon and ovarian cancer cell lines, expressed as the CLP36/GAPDH ratio, as described in "Materials and Methods". Each bar represents the mean (S.E. of five independent experiments.

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  Fig. 5 Immunohistochemical staining of CLP36 in a pancreatic adenocarcinoma. Samples of the pancreatic adenocarcinoma were immunostained with a rabbit polyclonal anti-CLP36 antibody.

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  Fig. 6 Western blots of cytosolic and membrane-rich fractions of the BxPC3 cell line, with (A) transferrin receptor antibody, and (B) rabbit anti-CLP36 antibody. 'Membr' means membrane-rich fraction.

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