Baculovirus-mediated gene transfer and recombinant protein expression do not interfere with insulin dependent phosphorylation of PKB-Akt in human SHSY-5Y and C3A cellsReportar como inadecuado




Baculovirus-mediated gene transfer and recombinant protein expression do not interfere with insulin dependent phosphorylation of PKB-Akt in human SHSY-5Y and C3A cells - Descarga este documento en PDF. Documentación en PDF para descargar gratis. Disponible también para leer online.

BMC Cell Biology

, 8:6

First Online: 19 February 2007Received: 23 October 2006Accepted: 19 February 2007

Abstract

BackgroundRecombinant adenovirus vectors and transfection agents comprising cationic lipids are widely used as gene delivery vehicles for functional expression in cultured cells. Consequently, these tools are utilized to investigate the effects of functional over-expression of proteins on insulin mediated events. However, we have previously reported that cationic lipid reagents cause a state of insulin unresponsiveness in cell cultures. In addition, we have found that cultured cells often do not respond to insulin stimulation following adenovirus treatment. Infection with adenovirus compromises vital functions of the host cell leading to the activation of protein kinases central to insulin signalling, such as protein kinase B-Akt. Therefore, we investigated the effect of adenovirus infection on insulin unresponsiveness by means of Akt activation in cultured cells. Moreover, we investigated the use of baculovirus as a heterologous viral gene delivery vehicle to circumvent these phenomena. Since the finding that baculovirus can efficiently transduce mammalian cells, the applications of this viral system in gene delivery has greatly expanded and one advantage is the virtual absence of cytotoxicity in mammalian cells.

ResultsWe show that infection of human neuroblastoma SHSY-5Y and liver C3A cells with recombinant adenovirus results in the activation of Akt in a dose dependent manner. In addition, this activation makes treated cells unresponsive to insulin stimulation as determined by an apparent lack of differential phosphorylation of Akt on serine-473. Our data further indicate that the use of recombinant baculovirus does not increase the phosphorylation of Akt in SHSY-5Y and C3A cells. Moreover, following infection with baculovirus, SHSY-5Y and C3A cells respond to insulin by means of phosphorylation of Akt on serine-473 in the same manner as uninfected cells.

ConclusionWidely-used adenovirus vectors for gene delivery cause a state of insulin unresponsiveness in human SHSY-5Y and C3A cells in culture due to the activation of central protein kinases of the insulin signalling pathway. This phenomenon can be avoided when studying insulin signalling by using recombinant baculovirus as a heterologous viral expression system. In addition, our data may contribute to an understanding of the molecular mechanisms underlying baculovirus infection of human cells.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2121-8-6 contains supplementary material, which is available to authorized users.

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Autor: Monica Andersson - Malin Warolén - Joakim Nilsson - Martin Selander - Catharina Sterky - Katrin Bergdahl - Christina Sörv

Fuente: https://link.springer.com/article/10.1186/1471-2121-8-6



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