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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.

Andersson M, Warolén M, Nilsson J, Selander M, Sterky C, Bergdahl K, Sörving C, James SR, Doverskog M - BMC Cell Biol. (2007)

Bottom Line: Therefore, we investigated the effect of adenovirus infection on insulin unresponsiveness by means of Akt activation in cultured cells.Our data further indicate that the use of recombinant baculovirus does not increase the phosphorylation of Akt in SHSY-5Y and C3A cells.In addition, our data may contribute to an understanding of the molecular mechanisms underlying baculovirus infection of human cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, R&D, Biovitrum AB, SE-11276 Stockholm, Sweden. monica.andersson@biovitrum.com <monica.andersson@biovitrum.com>

ABSTRACT

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.

Conclusion: 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.

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Transduction of SHSY-5Y and C3A cells 48 hours post infection with recombinant adenovirus expressing green fluorescent protein. Flow cytometry scatter plots of SHSY-5Y and C3A cells infected at MOI 10, 20, and 50; cells corresponding to the upper areas of the respective scatterplot (upper panels) were analysed for transduction efficiency by GFP expression (lower panels).
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Figure 1: Transduction of SHSY-5Y and C3A cells 48 hours post infection with recombinant adenovirus expressing green fluorescent protein. Flow cytometry scatter plots of SHSY-5Y and C3A cells infected at MOI 10, 20, and 50; cells corresponding to the upper areas of the respective scatterplot (upper panels) were analysed for transduction efficiency by GFP expression (lower panels).

Mentions: Adenoviral gene delivery is commonly utilized for functional over-expression and characterization of a gene-of-interest in mammalian cells. However, it is widely recognized that viral infection of human cell lines with homologous adenovirus vectors induces the activation of phosphatidylinositol 3-kinase (PI3-kinase), subsequently leading to the phosphorylation of protein kinase B/Akt on Serine 473 [12,11,14]. Akt is central for cellular responses mediated by insulin (reviewed in [43]) and activation of Akt is hence used as a common marker in insulin signalling. Therefore, while studying the impact of over-expression of various protein kinases on insulin signalling in experimental human cell lines, we investigated the influence of recombinant adenovirus on Akt phosphorylation in SHSY-5Y and C3A cells. Cultured cells were first infected with recombinant adenovirus expressing green fluorescent protein (AdGFP) as a model protein, and phosphorylation of Akt was studied with specific Ser473-phosho-Akt antibodies (ELISA and western blot). Viral titration was performed at multiplicity of infection (MOI) between 10 and 50 and GFP expression was determined 48 hours post infection (p.i.) by flow cytometry gated for intact adenovirus infected cells. Figure 1 displays side scatter against forward scatter for SHSY-5Y and C3A cells infected with AdGFP at MOI 10, 20, and 50, respectively. The resulting transduction efficiencies were determined from expression histograms of the populations of intact cells (illustrated as the upper cell population in respective scatter plot) which are shown in the lower panels of Figure 1 for each respective cell line, and was found to be 26.5, 58.6, and 86.9 % for SHSY-5Y cells, and 26.2, 47.8, and 67.3 % for C3A cells infected at MOI 10, 20, and 50, respectively.


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.

Andersson M, Warolén M, Nilsson J, Selander M, Sterky C, Bergdahl K, Sörving C, James SR, Doverskog M - BMC Cell Biol. (2007)

Transduction of SHSY-5Y and C3A cells 48 hours post infection with recombinant adenovirus expressing green fluorescent protein. Flow cytometry scatter plots of SHSY-5Y and C3A cells infected at MOI 10, 20, and 50; cells corresponding to the upper areas of the respective scatterplot (upper panels) were analysed for transduction efficiency by GFP expression (lower panels).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1808450&req=5

Figure 1: Transduction of SHSY-5Y and C3A cells 48 hours post infection with recombinant adenovirus expressing green fluorescent protein. Flow cytometry scatter plots of SHSY-5Y and C3A cells infected at MOI 10, 20, and 50; cells corresponding to the upper areas of the respective scatterplot (upper panels) were analysed for transduction efficiency by GFP expression (lower panels).
Mentions: Adenoviral gene delivery is commonly utilized for functional over-expression and characterization of a gene-of-interest in mammalian cells. However, it is widely recognized that viral infection of human cell lines with homologous adenovirus vectors induces the activation of phosphatidylinositol 3-kinase (PI3-kinase), subsequently leading to the phosphorylation of protein kinase B/Akt on Serine 473 [12,11,14]. Akt is central for cellular responses mediated by insulin (reviewed in [43]) and activation of Akt is hence used as a common marker in insulin signalling. Therefore, while studying the impact of over-expression of various protein kinases on insulin signalling in experimental human cell lines, we investigated the influence of recombinant adenovirus on Akt phosphorylation in SHSY-5Y and C3A cells. Cultured cells were first infected with recombinant adenovirus expressing green fluorescent protein (AdGFP) as a model protein, and phosphorylation of Akt was studied with specific Ser473-phosho-Akt antibodies (ELISA and western blot). Viral titration was performed at multiplicity of infection (MOI) between 10 and 50 and GFP expression was determined 48 hours post infection (p.i.) by flow cytometry gated for intact adenovirus infected cells. Figure 1 displays side scatter against forward scatter for SHSY-5Y and C3A cells infected with AdGFP at MOI 10, 20, and 50, respectively. The resulting transduction efficiencies were determined from expression histograms of the populations of intact cells (illustrated as the upper cell population in respective scatter plot) which are shown in the lower panels of Figure 1 for each respective cell line, and was found to be 26.5, 58.6, and 86.9 % for SHSY-5Y cells, and 26.2, 47.8, and 67.3 % for C3A cells infected at MOI 10, 20, and 50, respectively.

Bottom Line: Therefore, we investigated the effect of adenovirus infection on insulin unresponsiveness by means of Akt activation in cultured cells.Our data further indicate that the use of recombinant baculovirus does not increase the phosphorylation of Akt in SHSY-5Y and C3A cells.In addition, our data may contribute to an understanding of the molecular mechanisms underlying baculovirus infection of human cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biology, R&D, Biovitrum AB, SE-11276 Stockholm, Sweden. monica.andersson@biovitrum.com <monica.andersson@biovitrum.com>

ABSTRACT

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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus