BRIEF REPORT |
Correspondence to: Kelley J. Murphy, Huntsman Cancer Inst., University of Utah, 2000 Circle of Hope, Salt Lake City, UT 84112-5550. E-mail: kelley.murphy@hci.utah.edu
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Summary |
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As techniques evolve that allow molecular characterization of disease processes such as cancer, definition of "normal" at a molecular level becomes increasingly important. Increasingly large numbers of mutations are found at the genomic level, but whether all of those mutations contribute to the malignant state of a carcinoma cell is not clear. Without knowledge of what constitutes normality on the proteomic level in an organ or cell, we cannot determine what genomic changes are physiologically important. Traditionally, colon cancer is identified and classified by histological criteria. Margins of the colon are defined as "grossly uninvolved" when the histology is indistinguishable from that of normal (free from disease) colon. By using molecular pathology techniques and working backward from colon adenocarcinoma to hypoplastic polyps to presumably normal mucosa, we defined some of those protein differences. Our results may provide a molecular basis for identifying tumor formation and progression in situ.
(J Histochem Cytochem 49:667668, 2001)
Key Words: colon cancer, molecular pathology, techniques, genomic mutations, histology
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Introduction |
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The objective of this study was to identify protein expression patterns that indicate that a cell is normal. These expression patterns were then used to differentiate "normal" colon tissue from colon cancer.
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Materials and Methods |
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Grossly uninvolved colon samples resected for reasons other than cancer, colon polyps, and matched samples of grossly uninvolved/adenocarcinoma from the same patient were screened in the pilot study (n=5 for each group of samples). Tissue samples were collected from patients who had given informed consent before surgery at the University of Utah Hospital. The tissues were embedded in paraffin by standard techniques and sections were cut at 6-µm thickness for immunohistochemistry. The human gastric mucin (HGM), mucin-1, CA242, and heat shock protein 70 (HSP70) antibodies were from Novacastra/Vector Labs (Burlingame, CA). -Catenin/plakoglobin and zonula occludins-1 (ZO-1) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). ß-Catenin antibody was from Transduction Laboratories (San Diego, CA), and the topoisomerase II
antibody was from DAKO (Carpinteria, CA). Antibodies conjugated to biotin (Jackson ImmunoResearch; West Grove, PA) were used to detect the primary antibodies, followed by use of the Vector Elite ABCHRP kit according to the manufacturer's instructions. The reactions were visualized with Vector Lab's DAB substrate kit and the sections counterstained with Gill's hematoxylin (Vector Labs) before dehydration and mounting. Relative staining intensity and distribution (pattern change) were assessed by two independent observers (KM and KN).
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Results |
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Baseline immunostaining intensity and distribution for each protein were determined by examining the grossly uninvolved (GU) samples. The proteins associated with adhesion tended to have the same relative immunostaining intensity between GU and the polyps or adenocarcinoma (CA) (Table 1), while their expression translocated from the cell membrane in the GU samples to either the cytoplasm of the cell or the nucleus in the polyp and CA samples. The mucins showed variable immunostaining patterns. None of them was highly expressed by either signal intensity or distribution in the GU samples. Muc-1 was expressed less in the polyps than in the CA, whereas HGM or CA242 proteins were expressed more in the polyps than in the CA. The HSP70 signal in GU colon epithelium was in the apical half of the crypt, whereas it was seen throughout the neoplastic epithelium. HSP70 was upregulated in the polyps and in the least dysplastic of the CAs, and was downregulated (as compared to the grossly uninvolved samples) in the most dysplastic cancers. Topoisomerase II was upregulated and generally more widespread in CA, as we expected.
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Conclusions |
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Protein expression (relative intensity and distribution) differs between grossly uninvolved colon, polyps, and adenocarcinoma. Recently, a group found approximately 11,000 genomic mutations per colon carcinoma cell (-catenin, and ZO-1, another cellcell adhesion molecule, to determine if their protein expression changes during that progression. Although their expression patterns did not change as dramatically as that of ß-catenin, other groups have found that they do play roles in pathways that may contribute to the development of the disease (
immunostaining to either metastases (
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Footnotes |
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Presented in part at the Joint Meeting of the Histochemical Society and the International Society for Analytical and Molecular Morphology, Santa Fe, NM, February 27, 2001.
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Acknowledgments |
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Supported by grants 5 P3 CA2014 and PO1 CA73992-03 from the National Institutes of Health and by the Huntsman Cancer Foundation.
Received for publication November 27, 2000; accepted February 16, 2001.
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