CANCER RESEARCH55, 5628-5631. December I. 19951 ABSTRACT A gene called deleted in colon cancer (DCC) has been identified on a region of chromosome 18, which is deleted in 70% of colorectal cancers. The DCC gene encodes a protein belonging to the immunoglobulin super family with similarity to the N-CAM transmembrane proteins and is a putative tumor-suppressor gene. Alternative splicing of transcripts of transmembrane proteins, including N-CAM, is known to occur, resulting in different isoforms of the protein. Using five antibodies against the DCC gene product (three monoclonal antibodies raised in our laboratory, one commercially available antibody, and a rabbit polyclonal antibody), we have demonstrated by immunostaining a DCC protein isoform in reticu loendothelial cells in human thymus, tonsil, and lymph node. This can be distinguished from another isoform described in normal colonic epithe hum, because this latter is not demonstrable with the antibodies we have used. It could not be detected in normal colonic epithelium, polyps or colorectal carcinomas. This restrictive distribution suggests that not all DCC gene products are important in colonic cancer. INTRODUCTION Tumors originate from the accumulation of genetic defects causing either activation of genes, promoting growth or survival, or inhibition of genes, repressing growth or inducing cell death. These latter genes are called tumor suppressors, and the proteins they encode are either inactivated or not expressed as a consequence of genetic damage in a variety of tumors (1, 2). One putative suppressor gene, DCC,4 has been identified and cloned (3) from a region of the long arm of chromosome 18 that is deleted in 70% of colorectal tumors. This gene encodes for a mem brane-bound protein with immunoglobulin and fibronectin domains and a unique cytoplasmic domain. The first two domains are charac teristic of an adhesion molecule belonging to the immunoglobulin superfamily, the members of which are expressed mainly on cells of the nervous and immune systems (4). As has been shown for other members of this family of genes, alternative splicing can occur, resulting in the expression of related but structurally different proteins (5, 6). These isoforms can have different biological characteristics, cellular localization, and tissue distributions. Sometimes such differ ences can be caused by minimal variations in the sequence leading to major changes in glycosylation, which will affect the reactivity of antibodies. Investigation of proteins coded by genes such as DCC, therefore, can be complex. The DCC gene seems to be no exception. Using one panel of antisera in a Western blot analysis, two forms with Mr of approxi mately 170,000 and 180,000 have been demonstrated recently (7, 8). Using immunohistochemistry, these antisera stained goblet cells in Received 1/3 1/95; accepted 10/3/95. 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 18U.S.C.Section1734solelyto indicatethisfact. I This work was supported by the Imperial Cancer Research Fund. 2 Present address: Department of Pathology, European Institute of Oncology, Via Ripamonti, Milan, 20141 Italy. 3 To whom requests for reprints should be addressed, at University Department of Cellular Science, Level 4, Academic Block, John Radcliffe Hospital, Headinglon, Oxford OX3 9DU, United Kingdom. 4 The abbreviation used is: DCC, deleted in colon cancer. colon and neurons (7, 8). Reale et a!. (9), with a different panel of antisera (including the antibody 723 used in our study), have demon strated, in human and rodent cells, proteins ranging in molecular weight from Mr 175,0002l0,000. A commercial monoclonal anti body from Oncogene Science has been shown to recognize a Mr 180,000 protein. Preliminary immunocytochemical studies using rabbit polyclonal antibodies to recombinant DCC protein and parallel in situ hybridiza tion suggested that both the DCC protein and mRNA were present on colonic goblet cells (8). However, no further information was given on the tissue distribution of DCC, nor was its pattern of loss in colonic carcinoma studied in detail. We, therefore, considered that it would be important to extend these early studies by raising and characterizing monoclonal antibodies against the protein(s) coded for by the DCC gene. Our aim was to confirm these previous, more limited studies and to identify whether other normal cells in addition to colon cells express the DCC protein and to provide supportive evidence for a tumor suppressor function by demonstrating protein loss in colonic and possibly other carcinomas. The antibodies raised reacted strongly with COS cells transfected with full-length DCC cDNA but did not give the expected expression on other tissues tested. We therefore tried to obtain other DCC antibodies to compare with these results. We were able to look at two other antibodies from two different sources and found that they gave similar results to that of our mouse antibodies. MATERIALS AND METHODS Fresh frozen tissue was obtained via the routine diagnostic histopathology laboratories at the John Radcliffe Hospital. These included lymph node, tonsil, thymus, spleen, liver, kidney, pancreas, brain, testis, ovary, lung, and colon. Twenty-seven colorectal carcinomas were also selected from the tumor tissue bank. Frozen samples were stored at —70°C until use. Cryostat sections were cut at a thickness of 8 p@m, and the slides were dried overnight at room temperature and were fixed in acetone for 10 mm at room temperature prior to staining or storage at —20°C. Cytospin preparations of the transfected COS cells were made and treated in the same way as the sections. Other fixatives, including 3.7% formalin in PBS and 50:50 acetone methanol, were investi gated on colon, thymus, and the transfectant cytospins. Immunostaining was performed using the DAKO Strept avidin-biotin corn plex horseradish peroxidase kit (code K377; Dakopatts A/S, Copenhagen, Denmark) or Strept II avidin-biotin complex AP (Dakopatts K39l). The peroxidase reaction was developed using dimethylaminoazobenzene substrate tablets (Sigma Chemical Co.), and the alkaline phosphatase reaction was developed using Napthol-AS-MX-phosphate and fast red (TR-Salt; Sigma) as substrate. The slides were then counterstained with hematoxylin, washed, and mounted in aqueous mountant (Aquarnount; BDH, Inc.). Monoclonal antibodies were raised as previously described (10) against recombinant DCC protein produced by transfecting COS cells with a pIG-l vector containing a part of the DCC gene region coding for the three imrnu noglobulin domains fused to the Fc domain of human immunoglobulin; the protein being harvested from the supernatant and affinity purified. The fusion was screened on COS cells transfected with a full-length DCC cDNA (courtesy of Dr. B. Vogelstein). Three antibodies (designated DC3/l30, DC3/159, and DC3/l68) were produced and used in this study alongside the commercially available anti-DCC antibody Ab-l (clone AF5; Oncogene Science, New York, NY) and the rabbit polyclonal antisera 723 (a gift of Dr. M. A. Reale). 5628 The Distribution of the Deleted in Colon Cancer (DCC) Protein in Human Tissues1 Helen Turley, Francesco Pezzella,2 Sylvia Kocialkowski, Margaret Comley, Loukas Kakiamanis, Jon Fawcett, David Simmons, Adrian L. Harris, and Kevin C. Gatt& University Department of Cellular Science (H. T., F. P., S. K., M. C., K C. G.J, Nuffield Department of Pathology fL K.J, and Imperial Cancer Research Fund Molecular Oncology Laboratory. Institute of Molecular Medicine Ii. F., D. S., A. L Hi, John Radcliffe Hospital, Oxford 0X3 9DU. United Kingdom Research. on December 14, 2021. © 1995 American Association for Cancer cancerres.aacrjournals.org Downloaded from