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Oligosaccharide moieties of cell-surface glycoconijugates are thought to be involved in recognition events associated with tumor metastasis and invasion. Using swainsonine (SW), an inhibitor of Golgi a-mannosidase II that results in the formation of hybrid-type oligosaccharides on N-linked glycoproteins, we have tested the hypothesis that specific glycan structures are required for pulmonary colonization by tumor cells. B16-F10 murine melanoma cells were treated with SW in growth medium and then injected intravenously into syngeneic C57BL/6 mice. This treatment resulted in dramatic inhibition ofcolonization, but it had no effect on B16-F1O viability or on cellular tumorigenicity after subcutaneous implantation. SW-treated radiolabeled B16-F1O cells were cleared from the lungs at a greater rate than control cells, suggesting that one effect of treatment is to alter tumor cell retention in the target organ. Our results implicate specific glycan structures in pulmonary colonization and offer a potential approach for identification of specific macromolecules involved in tumor cell-organ recognition during metastasis. MD), and passaged twice weekly by short exposure to 0.25% trypsin/0.02% EDTA (GIBCO). Only cells <60 days old were used for these studies. Cultures were routinely tested for microbial infection and verified to be free of mycoplasma.Pulmonary Colonization. Six-week-old pathogen-free C57BL/6 mice (Charles River Breeding Laboratories, Wilmington, MA) were quarantined for 1 week and used over an age range of 8-10 weeks. SW was obtained either as a gift from H. P. Broquist (Vanderbilt University, Nashville, TN) or from Boehringer Mannheim and was dissolved in water.B16-F1O cells were plated at 106 or 5 x 105 cells per 75-cm2 flask and 2 or 3 days later, respectively, were treated with SW in growth medium for 18-22 hr. The just confluent cultures were then washed with divalent cation-free Dulbecco's phosphate-buffered saline (PBS-; GIBCO), detached with 0.02% EDTA for 2 min, and resuspended gently to 2.5 x 105 cells per ml in Dulbecco's MEM containing 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (GIBCO).Prior to injection, animals were warmed for 10 min to elicit vasodilation, and 0.2-ml aliquots of each cell suspension (containing 5 X 104 cells) were then injected slowly into the lateral tail vein as described by Fidler (19 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

A microplaque reduction assay for human and mouse interferons is described, using plastic disposable multicompartmental plates with flat-bottomed wells of 6 mm in diameter. The procedure is rapid and reproducible and lends itself to a considerable degree of automation. Its potential application in other areas of virology, such as rapid screening of samples for virus content or assay of neutralizing antibodies, is also discussed.

To determine the function of the carbohydrate moiety of glycoproteins, we have used tunicamycin, an analog of N-acetylglucosamine, to inhibit the glycosylation of N-glycosidically linked glycoproteins. First, we examined the effect of this drug on the intracellular processing, export and biological activity of fibronectin-the major cell surface glycoprotein of chick embryo fibroblasts.Chick fibroblasts treated with tunicamycin produced only nonglycosylated fi-Olden et a1 differentiating muscle cells prior to fusion. The inhibition of fusion was partially prevented when tunicamycin was administered in the presence of protease inhibitors such as leupeptin and pepstatin. Both glycosylation and fusion were completely restored to normal after removal of tunicamycin from the medium. These studies provide strong support for the idea that myoblast fusion is partially mediated by surface glycoproteins with asparagine-linked oligosaccharides. However, the requirement for the carbohydrate portion of the glycoprotein appears to be indirect in that it acts to stabilize the protein moiety against proteolytic degradation.To elucidate the mechanism responsible for the enhancement of proteolysis of cell surface glycoproteins following treatment with tunicamycin, we investigated the effect of tunicamycin on the intracellular processing of proteolytic enzymes. Treatment of chick embryo fibroblasts with tunicamycin resulted in more than a 10-fold increase in the amount of protease activity released into the culture medium. The enzyme activity has been tentatively identified as cathepsin B based on substrate specificity, pH optimum and inhibition with leupeptin.These results as well as extensive work by other investigators [see references [l-111 for recent reviews] suggest that the carbohydrate moiety of surface glycoproteins is not required for their synthesis, secretion or biological function, but instead helps to protect the protein against proteolytic degradation. In contrast, in agreement with the results of Neufeld et a1 161, the carbohydrate moiety of lysosomal enzymes is required for their intracellular retention.