On the binding of growth-promoting polyamines to proteoglycans: Implications for growth-regulation and polycation-mediated gene transfer

Sammanfattning: Initial investigations were directed at studying the interaction between polyamines and various glycosaminoglycans (GAGs). The polyamine spermine displayed binding to dermatan sulphate (DS) and heparan sulphate (HS) with similar (Kd, 3.9 x 10-4 M) and higher (Kd, 0.37 x 10-6 M) affinity, respectively, than to DNA. Antiproliferative spermine-binding DS fragments (tetra- to decasaccharides), and affinity-subfractionated HS chains were obtained by enzyme protection and affinity chromatography experiments, respectively. A clear correlation between high spermine-affinity and strong growth-inhibition was observed. Subsequent studies addressed the possible functional roles of the interaction. Pre-treatment of cells with GAG lyases, chlorate, or xylosides, all of which reduce the amount of cell-associated proteoglycans (PGs), resulted in diminished polyamine uptake. Mutant cells, deficient in PG, exhibited i) reduced polyamine uptake, ii) increased sensitivity to inhibition of polyamine biosynthesis, and iii) decreased growth-restoration by extracellular polyamines, as compared with wild-type cells. Moreover, one of the mutants exhibited the wild-type phenotype upon ectopic expression of the defective gene. The fact that several, widely used non-viral gene delivery vehicles, i.e. cationic lipids (CLs), make use of the physiological interaction between DNA and the polyamines, prompted us to investigate the possible role for PGs in CL-mediated gene transfer. Secreted PGs were shown to compete with DNA plasmid for binding to CL, leading to exchange of DNA for PG, intracellular accumulation of CL-PG complexes, nuclear deposition of GAG, and, consequently, a 100- 1000-fold decrease in reporter gene expression. In contrast, cell-associated PGs were shown to serve a protective role against CL cytotoxicity, thus allowing optimal transfection efficiency. In conclusion, the results suggest a role for PGs in 1) polyamine uptake, 2) polyamine-dependent cell growth, and 3) non-viral gene transfer.