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Deoxynojirimycin (DNJ) analogues are inhibitors of ceramide glucosyltransferase (CGT), which catalyses the first step in the glucosphingolipid (GSL) biosynthetic pathway. We have synthesized a series of DNJ analogues to study the contribution of N-alk(en)yl side chains (C4, C9 or C18) to the behaviour of these analogues in cultured HL60 cells. When cells were treated for 16 h at non-cytotoxic concentrations of inhibitor, a 40-50% decrease in GSL levels was measured by HPLC analysis of GSL-derived oligosaccharides following ceramide glycanase digestion of GSL and 2-aminobenzamide labelling of the released oligosaccharides. Using a novel technique for short-term [14C]galactose labelling of cellular GSL, we used compound inhibition of GSL biosynthesis as a marker for compound uptake into cells. Surprisingly, the uptake of all three of the DNJ analogues was extremely rapid and was not dependent upon the length of the N-alk(en)yl moiety. Compound uptake occurred in less than 1 min, as shown by the complete inhibition of GSL labelling in cells treated with all the DNJ analogues. Greatly increased cellular retention of N-cis-13-octadecenyl-DNJ was observed relative to the shorter-chain compounds, N-butyl-DNJ and N-nonyl-DNJ, as indicated by complete inhibition of CGT 24 h after removal of inhibitor from the culture medium. The present study further characterizes the properties of N-alk(en)ylated DNJs, and demonstrates that increasing the length of the side chain is a simple way of improving imino sugar retention and therefore inhibitory efficacy for CGT in cultured cells.

Original publication

DOI

10.1042/BJ20031822

Type

Journal article

Journal

Biochem J

Publication Date

01/08/2004

Volume

381

Pages

861 - 866

Keywords

1-Deoxynojirimycin, Carbon Radioisotopes, Cell Line, Tumor, Chromatography, High Pressure Liquid, Enzyme Inhibitors, Glycosphingolipids, HL-60 Cells, Humans, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Staining and Labeling