A novel method to detect translation of membrane proteins following microvesicle intercellular transfer of nucleic acids.

Publisher:
Oxford Acadmic
Publication Type:
Journal Article
Citation:
Journal of biochemistry, 2016, 160 (5), pp. 281 - 289
Issue Date:
2016-05-06
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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 co-culture 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.
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