title: Heparan sulfate proteoglycan-mediated polyamine uptake.
abstract: The polyamines are polycationic compounds essential for cellular proliferation and transformation. In addition to a well-defined biosynthesis pathway, polyamines are internalized into cells by as yet incompletely defined mechanisms. Numerous reports have shown that efficient polyamine uptake depends on the presence of polyanionic, cell surface-associated heparan sulfate proteoglycans (HSPGs). In this chapter, we provide protocols for studying HSPG-mediated uptake of polyamines in various cell lines, and provide instructions for the use of two different genetic models of HSPG deficiency. We describe the enzymatic reduction of cell surface HSPG through Heparinase III lyase treatment as well as the use of phage display-derived single chain variable fragment (scFv) anti-HS antibodies to block HSPGs at the cell surface. Finally, we provide a protocol for the quantitative verification of loss or reduction of cell surface HSPGs and a detailed description of polyamine uptake measurement.
authors.affiliation: Department of Clinical Sciences, Section of Oncology, Lund University and Lund University Hospital, Lund, Sweden.
authors.forename: Johanna
authors.initials: J
authors.lastname: Welch
authors.forename: Katrin
authors.initials: K
authors.lastname: Svensson
authors.forename: Paulina
authors.initials: P
authors.lastname: Kucharzewska
authors.forename: Mattias
authors.initials: M
authors.lastname: Belting
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001671:Biochemistry / Q000379:methods*; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D019812:Heparan Sulfate Proteoglycans / Q000096:biosynthesis / Q000378:metabolism*; D006801:Humans; D019426:Integrases / Q000378:metabolism; D051379:Mice; D011073:Polyamines / Q000378:metabolism*; D011133:Polysaccharide-Lyases / Q000378:metabolism; D015203:Reproducibility of Results; D013194:Staining and Labeling; D014161:Transduction, Genetic
chemical_list: D000906:Antibodies; D019812:Heparan Sulfate Proteoglycans; D011073:Polyamines; C045073:Cre recombinase; D019426:Integrases; D011133:Polysaccharide-Lyases; C021349:heparitinsulfate lyase