title: A novel method to detect translation of membrane proteins following microvesicle intercellular transfer of nucleic acids.
abstract: Microvesicles (MVs) serve as vectors of nucleic-acid dissemination and are important mediators of intercellular communication. However, the functionality of packaged nucleic acids on recipient cells following transfer of MV cargo has not been clearly elucidated. This limitation is attributed to a lack of methodology available in assessing protein translation following homotypic intercellular transfer of nucleic acids. Using surface peptide shaving we have demonstrated that MVs derived from human leukaemic cells transfer functional P-glycoprotein transcripts, conferring drug-efflux capacity to recipient cells. We demonstrate expression of newly synthesized protein using Western blot. Furthermore, we show functionality of translated P-gp protein in recipient cells using Calcein-AM dye exclusion assays on flow cytometry. Newly synthesized 170 kDa P-gp was detected in recipient cells after coculture with shaven MVs and these proteins were functional, conferring drug efflux. This is the first demonstration of functionality of transferred nucleic acids between human homotypic cells as well as the translation of the cancer multidrug-resistance protein in recipient cells following intercellular transfer of its transcript. This study supports the significant role of MV's in the transfer of deleterious traits in cancer populations and describes a new paradigm in mechanisms governing the acquisition of traits in cancer cell populations.
authors.affiliation: Discipline of Pharmacy, the Graduate School of Health, University of Technology Sydney, New South Wales 2007, Australia.
authors.forename: Jamie F
authors.initials: JF
authors.lastname: Lu
authors.affiliation: Discipline of Pharmacy, the Graduate School of Health, University of Technology Sydney, New South Wales 2007, Australia.
authors.forename: Deep
authors.initials: D
authors.lastname: Pokharel
authors.affiliation: Proteomics Core Facility, University of Technology Sydney, New South Wales 2007, Australia.
authors.forename: Matthew P
authors.initials: MP
authors.lastname: Padula
authors.affiliation: Discipline of Pharmacy, the Graduate School of Health, University of Technology Sydney, New South Wales 2007, Australia mary.bebawy@uts.edu.au.
authors.forename: Mary
authors.initials: M
authors.lastname: Bebawy
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D045744:Cell Line, Tumor; D018014:Gene Transfer Techniques; D006801:Humans; D014176:Protein Biosynthesis
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1
keywords: P-glycoprotein; mRNA; microvesicles; multidrug resistance; nucleic acids