[CANCER RESE\RCH 49, 2017-2021, April 15, 1989] A Mouse Analogue of the Human Carcinoembryonic Antigen N. Beauchrmin,2 C. Turbide, D. Afar, J. Bell, M. Raymond, C. P. Stanners, and A. Fuks McGill Cancer Ceñiré [N. B., C. T., C. P.S., A. F.¡and Department of Biochemistry [D. A., J. B., M. R., C. P. S.], McGill University, Montreal H3G l Y6, Canada ABSTRACT] Functional human Carcinoembryonic antigen (CEA)-like genes have been shown 19 be present in the mouse. Southern analyses of murine DNA using li uh human and murine CEA complementary DNA probes have revealed the presence of multiple CEA-like genes, while analyses of RNA fron1 different mouse tissues showed CEA-like transcripts in adult colon and liver. Furthermore, a CEA-like protein, immunoprecip- itable with a rabbit polyclonal serum raised against human CEA, has been detected in adult murine colon tissue. Several murine CEA comple mentary DNA clones have been isolated from a murine colon comple mentary I)N v library, and characterization of one such clone demon strates that loth the /V-terminal and the internal domains have been conserved bet vecn the two species. The existence of a murine counterpart of CEA strengthens the case for an essential function for this human tumor marker and provides an experimentally amenable system for elucidation of its biological properties. INTRODUCTION CEA3 is a large cell surface glycoprotein (M, 180,000) first described ir 1965 by Gold and Freedman as an antigen ex pressed in tumors of the human gastrointestinal tract (1) and in the fetal digestive system (2). This or related antigens have since been Ictected in a wide range of human malignancies including bieast, lung, and ovarian cancers (3, 4). Assays that measure th< serum or plasma levels of CEA have been useful in assessing, the prognosis and monitoring the progress of cancer patients (5). CEA is also a member of a large and complex gene family (6). NCA, for example, is a family member specifically 'bund in breast tumors, chronic myelogeneous leu kemia, and normal gall bladder (7, 8) as well as in normal spleen and lung (9, 10). The cDN \s corresponding to CEA and NCA mRNAs have recently been cloned, and their nucleotide sequences and pre dicted transition products have been determined (11-15). Both proteins belong to the immunoglobulin supergene family, and initial studi ;s point to an intercellular adhesion function for CEA.4 In depth investigations of the function of CEA and its role in malignancy will require animal models, preferably murine, allowing approaches such as transgenesis, controlled card no- genesis, anc //; xitu assessment of expression during develop ment. Therefore, we have undertaken a search for a mouse counterpart of CEA. Previous studies of animal models have identified putative counterparts of CEA or NCA using immune sera or spec fie monoclonal antibodies. A putative rat CEA was detected in chemically induced colonie adenocarcinomas, em bryonic tissues, and, in low concentrations, normal adult tissues Received 3/::9/88; revised 9/16/88; accepted 1/4/89. The costs of publication of this article were defrayed in part by the payment of page charge:. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by grants to C. P. S., J. B., and A. F. from the Medical Research Council of Canada and the National Cancer Institute of Canada. 2 Recipient cf a fellowship from Fonds de la Recherche en Santédu Québec. 3The abbre\ iations used are: CEA, Carcinoembryonic antigen: NCA, normal cross-reacting intigen; cDNA, complementary DNA; SSPE, 0.18 M NaCI:l mM EDTA:10 mM odium phosphate (pH 7.7); SDS, sodium dodecyl sulfate; poly A* RNA, polyader ylate-containing RNA; SSC, standard saline citrate. 4 S. Benchin ol et al., manuscript in preparation. (16). An antigen which is immunologically closely related to human NCA and which cross-reacts with CEA has also been identified in lung and spleen tissues of Macaca monkeys (17), and blood samples from other primates including gorilla, or angutang, black ape, mangabey ape, baboon, and chimpanzee exhibited CEA-related substances (18). We report here the identification and initial characterization of a mouse counter part of CEA present in normal adult colon and in liver tissues. MATERIALS AND METHODS Cell Culture. Cells of the human colonie adenocarcinoma line (LS- 180) (19) and a line of L-strain mouse fibroblasts (LTA) were grown at 37°C in monolayer in n minimal essential medium (20) supplemented with 10% fetal bovine serum and asparagine •¿ H2O at 50 Mg/m'- 32P-labeled Probes. Hybridization probes for Northern and Southern analyses consisted of either an 800-base pair Nco\-Bam\\\ restriction fragment comprising the .V terminus and most of the first internal repeating domain of the human CEA cDNA ( 11), a 1.43-kilobase EcoRl restriction fragment specific for the 3'-untranslated region of the human NCA cDNA (7), or a 1.45-kilobase EcoRl restriction fragment of a murine CEA cDNA clone. These fragments were extracted from 0.8% low-melting agarose gels and 32P-labeled using the random primer technique of Feinberg et al. (21). DNA Preparation and Southern Analyses. Genomic DNA was pre pared (22) from two mouse leukemic lines L1210SS and L1210DN (23), a mouse fibroblast line LTA 42.2 (24), and normal human liver tissues. DNA was digested with EcoRl restriction endonuclease, and the digestion products were electrophoresed on a 1% agarose gel. The gels were transferred to a hybridization membrane (Zeta-probe, Bio- Rad; or Hybond-N, Amersham). Identical mouse Southern filters were hybridized with the 32P-labeled CEA probe in 5x SSPE, 1x Denhardt's solution (22), 100 ¿ig/mlof heat-denatured salmon testis DNA, and either 30%, 40%, or 50% formamide at 40°Cfor 48 h. The human genomic filters were hybridized at 42°Cfor 18 h under the same conditions but with 0.5% SDS, 10% dextran sulfate, and 30% form- amide. The murine fibroblast genomic DNA was hybridized using the above conditions with 50% formamide at 42°C.Filters were exposed to Kodak XRP X-ray films for 18 to 36 h after washing. RNA Preparation and Northern Analyses. Fresh tissues were collected from either pregnant or nonpregnant female CD-I mice and immedi ately frozen in liquid nitrogen. Approximately 2 g of tissue were powdered using a mortar and pestle kept at —¿40°C, and the fine powder was vortexed into a solution of 4.0 M guanidium isothiocyanate con taining 25 mM sodium citrate (pH 7.0), 0.5% Sarkosyl, and 0.1 M ß- mercaptoethanol (25). Poly A+ RNA was purified on oligo dT columns (26). Total or poly A* RNA was electrophoresed on 1.5% agarose gels containing 1.1 M formaldehyde, 10 mM sodium phosphate buffer (pH 7.4), and 1 mM EDTA; stained with acridine orange; and transferred to either nitrocellulose or Hybond-N (Amersham Corp.) membranes. Hybridization was done for 18 h at 37°Cor 42°Cin 5x SSPE, Ix Denhardt's solution, 30% or 50% formamide, 150 ^g/ml of heat- denatured salmon testis DNA, 2 mM sodium pyrophosphate, 0.1% SDS, 10% dextran sulfate for the nitrocellulose membranes, or 5% dextran sulfate for the Hybond-N membranes using 2.5 x IO6to 4.0 x IO6cpm/ml of the 32P-labeled probes. The filters were washed up to a final stringency of 0.1x SSC and 0.1% SDS at 50°C.18S rRNA (2.0 kilobases) and 28S rRNA (4.5 kilobases) were used as markers. cDNA Library and Isolation of Murine CEA cDNA Clones. cDNA was synthesized, as described previously (10), from 5 ^g of poly A+ RNA isolated from murine adult colon, ligated to dephosphorylated £coRI-digested XgtlO DNA, and packaged with Gigapack Plus extracts (Stratagene) to yield infectious virus. XgtlO recombinant bacteriophages 2017 Research. on February 25, 2016. © 1989 American Association for Cancer cancerres.aacrjournals.org Downloaded from