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Soluble Factor in Normal Tissues That Stimulates High-Molecular-Weight Sialoglycoprotein Production by Human Colon Carcinoma Cells1

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Soluble Factor in Normal Tissues That Stimulates High-Molecular-Weight Sialoglycoprotein Production by Human Colon Carcinoma Cells1 (CANCER RESEARCH 50, 3331-3338. June I, I990| Soluble Factor in Normal Tissues That Stimulates High-Molecular-Weight Sialoglycoprotein Production by Human Colon Carcinoma Cells1 Tatsuro Irimura,2 Andrew M. Mclsaac, Debora A. Carlson, Masato Vagita, Elizabeth A. Grimm, David G. Menter, David M. Ota, and Karen R. Cleary Departments of Tumor Biology IT. I., A. M., D. A. C., M. Y., E. A. G., D. G. M.], General Surgery /E. A. G., D. M. O.J, and Pathology [K. R. C.J, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030 ABSTRACT linked carbohydrate chains. Secreted mucins are believed to function as protective molecules and as lubricants on tissue The stimulation of high molecular »eight Sialoglycoprotein synthesis surfaces. Despite previous attempts by several laboratories to by a soluble factor derived from normal colon tissues was studied in vitro with human colon carcinoma cell lines, HT-29 P and a metastatic variant classify and characterize colorectal mucins, it remained unclear HT-29 LMM. The synthesis of all three high-molecular-weight sialo- whether a specific biological function is associated with unique glycoproteins (approximate M, 900,000, 740,000, and 450,000) by HT- carbohydrate structures in these mucins (10,13-22). Our recent 29 P cells or HT-29 LMM cells growing in vitro was enhanced by studies utilizing pathological specimens of colorectal carcinoma supplementing the culture medium with a conditioned medium of fresh suggested that at least four mucin-like glycoproteins with dif human colon organ culture. Changes were detected by polyacrylamide ferent carbohydrate chains were independently regulated and gel electrophoresis of lysates from [3H)glucosamine-labeled cells on 3% either directly or inversely correlated with the metastatic poten gels followed by fluorography, or by electrophoresis of lysates from tial of these malignant tumors (23-27).3 Thus, these changes unlabeled cells followed by incubation with '"I-labeled wheat germ were apparently associated with the progression of colon car agglutinin and autoradiography. No changes were detected in the major cinoma to the metastatic phenotype, but not simply with malig protein components or in glycoproteins at M, < 200,000 as revealed by polyacrylamide gel electrophoresis. The treated cells did not change their nant transformation. Determining a positive correlation be growth rate or morphology. The connective tissue portions of the colon tween the expression of high molecular weight sialoglycopro tissues were apparently responsible for the production of this stimulatory teins and the progression of colorectal carcinoma to the substance. The stimulatory activity was preserved at 56°C but was metastatic phenotype as we demonstrated seemed to be partic inactivated by heating at 100°C. The substance was eluted from a ularly significant because increased production of sialoglyco- Sephacryl S-200 column at a position between the elution positions of conjugates has been implicated in a variety of experimental ovalbumin and trypsinogen. The colon carcinoma cells treated with the métastasesin rodents (28-34). conditioned medium and producing increased amounts of high-molecular- Two important basic biological questions remained unan weight sialoglycoproteins were less sensitive to the cytolytic effects of recombinant interleukin 2-activated human peripheral blood lymphocytes swered after these studies. The first was what molecular and than untreated cells were. The treated colon carcinoma cells induced cellular mechanisms are involved in the increased expression of stronger platelet aggregation than their untreated counterparts did. sialoglycoproteins during the progression of colorectal carci Therefore, this substance may represent one of the normal host tissue noma to the metastatic phenotype. This question is not simple factors that can influence and modulate malignant behavior of carcinoma because the amount of sialoglycoproteins produced by colon cells growing in vivo. carcinoma cells growing in vivo appears to be influenced by the cellular origin of the carcinoma as well as by microenvironmen- tal factors (26). The second question was whether sialoglyco INTRODUCTION proteins account for the metastatic behavior of colon carcinoma Changes in the production and structures of glycoconjugates cells. In experimental animal models, various cell surface char upon malignant transformation have been described for a wide acteristics have been proposed as being potentially involved in variety of cells (1-5). In colon epithelial cells, changes in the metastatic phenotypes. For example, siah lai ion of cell surface carbohydrate chains on mucin-like, very high-molecular-weight adhesion molecules may modulate the interaction of tumor glycoprotein molecules during carcinogenesis have been re cells with extracellular matrices (35). The effects of cell surface ported since the 1960s (6, 7). A variety of antigenic molecules glycosylation on invasion of embryonic muscle tissues by tumor associated with normal and malignant human colon epithelium cells have also been reported (36). Furthermore, sialoglycocon- were reported as mucin-like, high-molecular-weight glycopro jugates on tumor cell surfaces are known to interfere with the teins or mucin associated, but the epitope structures and spec cytotoxic effects of lymphocytes through nonspecific or specific ificities of the corresponding antibodies were not clear (8-12). mechanisms (37-41). In rat renal carcinoma, sialoglycoconju- Furthermore, the biochemical basis for changes in the expres gates are proposed to play an important role in tumor cell- sion of such tumor-associated antigens was not well understood. induced platelet aggregation (42). Mouse colon carcinoma cells Particularly, the effects of host microenvironments on the pro that were treated with an inhibitor of sialyltransferase showed duction of these molecules were not elucidated. Mucins are significant reduction in their ability to induce platelet aggrega major products of various epithelial cells and are defined by tion (43). their large molecular size and high content of serine/threonine- We describe a soluble factor from normal colon tissues that appears to stimulate the production of cell-associated high- Received 8/16/89; revised 2/9/90. The cosls of publication of this article «eredefrayed in part by the payment molecular-weight sialoglycoproteins by colon carcinoma cells of page charges. This article must therefore be hereby marked advertisement in in vitro. The present study is carried out with organ culture- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1This work was supported by USPHS Grant RO1-CA39319. CA45225. and conditioned media because we did not find a similar stimulatory CA50231: Texas Higher Education Program Grant 1549: and the National factor when an aqueous extract of normal colon tissues or Foundation for Cancer Research. ! To whom requests for reprints should be addressed, at Department of Tumor 3S. D. Hoff. Y. Matsushita. D. M. Ota. K. R. Cleary. T. Yamori, S. Hakomori. Biology. Box 108. The University of Texas M. D. Anderson Cancer Center, 1515 and T. Irimura. Increased expression of sialyl-dimeric Le" antigen in liver Holcombe Boulevard. Houston. TX 77030. métastasesofcolorectal carcinoma. Cancer Res., 49: 6883-6888. 1989. 3331 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1990 American Association for Cancer Research. MUCOMODULIN EFFECTS ON COLON CARCINOMA CELLS partial fractionation products of the extract were tested at by measuring absorbance at 590 nm after being dissolving in 1% SDS. various concentration. The results should eventually lead to The morphology of these cells during incubation with NCCM was answers to important questions regarding the function and examined under a phase contrast microscope. regulation of carcinoma mucins. Electrophoretic Analysis of Colon Carcinoma Cell Lysates. Cellular glycoproteins were extracted with non-ionic detergent and treated with SDS and 2-mercaptoethanol as previously described (31). Electropho MATERIALS AND METHODS retic analysis of high-molecular-weight sialoglycoproteins was per formed by using 3% polyacrylamide gels according to previously estab Preparation of Conditioned Media from Normal Colon Tissues. Hu lished methods (25, 26). Sialoglycoproteins were identified by direct man colon tissues used in this study were derived from sigmoid or left binding of I2ÕI-WGAto polyacrylamide gels. Lysates from ['Hjgluco- colon tissue resected during surgery for colon carcinoma. Normal samine-labeled and ['H]threonine-labeled cells were analyzed by fluo- mucosa at least 5 cm from the carcinoma was used. For the preparation rography with Enhance (DuPont NEN, Wilmington, DE). Lysates from of NCCM,4 the colon epithelium with attached submucosal connective ["Sejselenomethionine-labeled cells were directly autoradiographed tissues and muscularis propria was made free of fat tissues. Fragments after electrophoretic separation. of tissue (approximately 1.0 x 0.3 cm) were then rinsed five times with Quantitäten of Changes in High-Molecular-Wcight Sialoglycoprotein gentle agitation and décantationwith DPBS supplemented with 50 Production. In most of the experiments, the relative amounts of high- units/ml penicillin, 50 ng/m\ streptomycin, and 1.25 pg/m\ amphoter- molecular-weight sialoglycoprotein production were estimated by the icin B (Fungizone; Sigma, St. Louis, MO). The tissue fragments were binding of 125I-WGA(10 Mg/ml) to the
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