title: Endocytosis and intracellular localisation of type 1 ribosome-inactivating protein saporin-s6.
abstract: Saporin-S6 is a single-chain ribosome-inactivating protein (RIP) that has low toxicity in cells and animals. When the protein is bound to a carrier that facilitates cellular uptake, the protein becomes highly and selectively toxic to the cellular target of the carrier. Thus, saporin-S6 is one of the most widely used RIPs in the preparation of immunoconjugates for anti-cancer therapy. The endocytosis of saporin-S6 by the neoplastic HeLa cells and the subsequent intracellular trafficking were investigated by confocal microscopy that utilises indirect immunofluorescence analysis and transmission electron microscopy that utilises a direct assay with gold-conjugated saporin-S6 and an indirect immunoelectron microscopy assay. Our results indicate that saporin-S6 was taken up by cells mainly through receptor-independent endocytosis. Confocal microscopy analysis showed around 30% co-localisation of saporin-S6 with the endosomal compartment and less than 10% co-localisation with the Golgi apparatus. The pathway identified by the immunofluorescence assay and transmission electron microscopy displayed a progressive accumulation of saporin-S6 in perinuclear vesicular structures. The main findings of this work are the following: i) the nuclear localisation of saporin-S6 and ii) the presence of DNA gaps resulting from abasic sites in HeLa nuclei after intoxication with saporin-S6.
authors.affiliation: Department of Experimental Pathology, Alma Mater Studiorum, University of Bologna, Italy.
authors.forename: A
authors.initials: A
authors.lastname: Bolognesi
authors.forename: L
authors.initials: L
authors.lastname: Polito
authors.forename: V
authors.initials: V
authors.lastname: Scicchitano
authors.forename: C
authors.initials: C
authors.lastname: Orrico
authors.forename: G
authors.initials: G
authors.lastname: Pasquinelli
authors.forename: S
authors.initials: S
authors.lastname: Musiani
authors.forename: S
authors.initials: S
authors.lastname: Santi
authors.forename: M
authors.initials: M
authors.lastname: Riccio
authors.forename: M
authors.initials: M
authors.lastname: Bortolotti
authors.forename: M G
authors.initials: MG
authors.lastname: Battelli
mesh_terms: D004249:DNA Damage; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D011500:Protein Synthesis Inhibitors / Q000493:pharmacokinetics; D054789:Ribosome Inactivating Proteins, Type 1 / Q000378:metabolism* / Q000493:pharmacokinetics; D000076985:Saporins
chemical_list: D011500:Protein Synthesis Inhibitors; D054789:Ribosome Inactivating Proteins, Type 1; D000076985:Saporins