AbstractSwainsonine is an indolizidine alkaloid that inhibits glycoprotein processing by inhibiting mannosidase II. Thus, cells grown in the presence of this alkaloid exhibit a decreased amount of complex types of oligosaccharides at their cell surface, and instead have hybrid types of structures. Since this compound could be useful for studying functional roles of glycoproteins, it was important to determine whether it affected the growth of mammalian cells in culture, and whether it was cytotoxic to these cells. At levels of up to 1 μg/ml, swainsonine did not affect the growth rate of Madin‐Darby canine kidney (MDCK) cells, Chinese hamster ovary (CHO), simian virus‐181 (SV‐101), B‐16 melanoma, or intestine 407 cells, as measured by the increase in cell numbers over a 5‐day period. There was also no apparent change in cell size or cell shape in cells grown in the presence of this inhibitor. Swainsonine also did not appear to be cytotoxic, nor to cause alterations in cell morphology, as evidenced by comparison of thin sections of normal and swainsonine‐grown cells in the electron microscope. Since alterations in the oligosaccharide chains of cell surface glycoproteins could greatly affect cell surface properties, we examined the binding of various lectins and bacteria to cells grown in swainsonine as a measure of changes in their cell surface carbohydrates. Thus, when MDCK cells, CHO cells, or B‐16 melanoma cells were grown for several days in the presence of swainsonine (100–500 ng/ml), these cells showed a 50–100% increase in their ability to bind [3H]concanavalin A, and a substantial decrease in the binding of [3H]wheat germ agglutinin. These alterations suggested an increase in high‐mannose (or hybrid) types of receptors and a decrease in the complex types. The adhesion of E. coli B‐886, a bacterium that binds to high‐mannose glycoproteins, was also increased 1.5‐to twofold, in cells grown in swainsonine. However, the binding of E. coli SS‐142, another bacterial strain that does not bind to high‐mannose receptors, was not altered by growth in swainsonine. In addition to the decrease in wheat germ agglutinin binding, another indication of a decrease in complex chains was the finding that CHO cells grown in swainsonine were more resistant to the toxic effects of the lectin, ricin. This increased resistance could be measured microscopically by the decrease in the number of cells remaining attached to the plates, or by the inhibition of amino acid incorporation, at various ricin concentrations. The effect of swainsonine on the incorporation of amino acids and sugars into protein was also examined. When MDCK cells were grown overnight in swainsonine (1 μg/ml), or were incubated in the alkaloid for several hours before the start of the experiment, there was no alteration in the incorporation of [3H]leucine or [3H]proline into protein. There was, however, a significant inhibition in the incorporation of [3H]fucose, [3H]glucosamine, and [3H]galactose caused by this alkaloid. Fucose incorporation was decreased by about 40%, glucosamine by about 40 or 50%, and galactose by about 50%. In many cases (but not all), the incorporation of mannose was enhanced about 20–30% in cells grown in swainsonine.
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Jun 1, 1984
S.P.R Rose + 1
Open Access