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.
BACKGROUND
Recombinant 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.


RESULTS
We 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.


CONCLUSIONS
Widely-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.
Andersson
Monica
M
Department of Biology, R&D, Biovitrum AB, SE-11276 Stockholm, Sweden. monica.andersson@biovitrum.com <monica.andersson@biovitrum.com>
Warolén
Malin
M
Nilsson
Joakim
J
Selander
Martin
M
Sterky
Catharina
C
Bergdahl
Katrin
K
Sörving
Christina
C
James
Stephen R
SR
Doverskog
Magnus
M
D000818:Animals; D016367:Baculoviridae; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D008099:Liver / Q000166:cytology; D009447:Neuroblastoma / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D010770:Phosphotransferases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*
D007328:Insulin; D011994:Recombinant Proteins; D010770:Phosphotransferases; D051057:Proto-Oncogene Proteins c-akt