We have evaluated the possible role of proteoglycans in the uptake of spermine by human lung fibroblasts. Exogenous glycosaminoglycans behaved as competitive inhibitors of spermine uptake, the most efficient being heparan sulphate (Ki=0.16+/-0.04 microM). Treatment of fibroblasts with either heparan sulphate lyase, p-nitrophenyl-O-beta-D-xylopyranoside or chlorate reduced spermine uptake considerably, whereas chondroitin sulphate lyase had a limited effect. Inhibition of polyamine biosynthesis with alpha-difluoromethylornithine resulted in an increase of cell-associated heparan sulphate proteoglycans exhibiting higher affinity for spermine. The data indicate a specific role for heparan sulphate proteoglycans in the uptake of spermine by fibroblasts. Spermine uptake by pgsD-677, a mutant Chinese hamster ovary cell defective in heparan sulphate biosynthesis, was only moderately reduced (20%) compared with wild-type cells. Treatment of mutant cells with the above-mentioned xyloside resulted in a greater reduction of endogenous proteoglycan production as well as a higher inhibition of spermine uptake than in wild-type cells. Moreover, treatment with chondroitin sulphate lyase resulted in a selective inhibition of uptake in mutant cells, indicating a role for chondroitin/dermatan sulphate proteoglycans in the uptake of spermine by these cells. Fibroblasts, made growth-dependent on exogenous spermine by alpha-difluoromethylornithine treatment, were growth-inhibited by heparan sulphate or beta-D-xyloside, which might have future therapeutical implications.

We have evaluated the possible role of proteoglycans in the uptake of spermine by human lung fibroblasts. Exogenous glycosaminoglycans behaved as competitive inhibitors of spermine uptake, the most efficient being heparan sulphate (K i l 0.16p0.04 µM). Treatment of fibroblasts with either heparan sulphate lyase, p-nitrophenyl-O-β--xylopyranoside or chlorate reduced spermine uptake considerably, whereas chondroitin sulphate lyase had a limited effect. Inhibition of polyamine biosynthesis with α-difluoromethylornithine resulted in an increase of cell-associated heparan sulphate proteoglycans exhibiting higher affinity for spermine. The data indicate a specific role for heparan sulphate proteoglycans in the uptake of spermine by fibroblasts. Spermine uptake by pgsD-677, a mutant Chinese hamster ovary cell defective in heparan sulphate biosynthesis, was only moderately reduced (20 %) compared with wild-type

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Depletion of intracellular polyamine pools inhibits cell proliferation. Polyamine pools are maintained by intracellular synthesis and by uptake from the extracellular environment. It may be expected that cationic polyamines are sequestered by the polyanionic glycosaminoglycan substituents of extracellular proteoglycans. Moreover, highly sulphated heparin-related glycans inhibit growth of human embryonic lung fibroblasts. We have therefore investigated interactions between polyamines and heparin-related glycosaminoglycans. Affinity chromatography of various polyamines on heparin-agarose indicated that spermine was the only polyamine that bound efficiently to this type of glycan. By using equilibrium dialysis we found that spermine binds to a highly sulphated heparan sulphate/heparin preparation with a dissociation constant of 3.7 x 10(-5)M. Enzymatic degradation of heparan sulphate using three different heparan sulphate/heparin lyases, separately or in combination and in the absence or presence of spermine, was used to generate spermine-binding and degradation-protected oligosaccharides. As indicated by chromatographic and electrophoretic analysis a size- and chargeheterogeneous collection was obtained. However, protected oligosaccharides derived from antiproliferative heparan sulphates were inactive. Highly sulphated, antiproliferative heparan sulphates were subfractionated on spermine-agarose yielding high-affinity material with increased antiproliferative activity. A very potent material was obtained from pig skin. Although there was generally a clear correlation between high spermine-affinity and strong growth-inhibition, no correlation with sulphate content or oligosaccharide mapping patterns could be detected. Beef lung heparan sulphate comprised naturally occurring fragments of eicosasaccharide size with substantially increased specific activity. As these fragments were longer than oligosaccharides generated by enzymatic degradation in the presence of spermine (hexa- to tetradecasaccharide), multiple spermine-binding sites in tandem may be necessary to induce antiproliferative activity.

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