Using Bcl-xL anti-apoptotic protein for altering target cell apoptosis

Background: Altering target cell apoptosis is one of the challenging ideas of biotechnological applications. There are several applications of over expressing Bcl-xL anti-apoptotic protein from recombinant protein production to DNA vaccination strategies. The aim of the present study is to evaluate...

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Autores principales:	Iz,Sultan Gülçe, Çalimlioglu,Beste, Gürhan,Saime Ismet Deliloglu
Lenguaje:	English
Publicado:	Pontificia Universidad Católica de Valparaíso 2012
Materias:	animal cell biotechnology apoptosis Bcl-xL anti-apoptotic protein
Acceso en línea:	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582012000500002
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spelling	oai:scielo:S0717-345820120005000022012-11-29Using Bcl-xL anti-apoptotic protein for altering target cell apoptosisIz,Sultan GülçeÇalimlioglu,BesteGürhan,Saime Ismet Deliloglu animal cell biotechnology apoptosis Bcl-xL anti-apoptotic protein Background: Altering target cell apoptosis is one of the challenging ideas of biotechnological applications. There are several applications of over expressing Bcl-xL anti-apoptotic protein from recombinant protein production to DNA vaccination strategies. The aim of the present study is to evaluate the anti-apoptotic efficacy of Bcl-xL expressing dual promoter plasmid system as a candidate to be used for recombinant protein production and DNA vaccination approaches. For this purpose, Bcl-xL anti-apoptotic protein gene was inserted in a dual expressing vector system in frame with EGFP (enhanced green fluorescence protein) after IRES (internal ribosomal site). The plasmid has a multiple cloning site after CMV (cytomegalovirus promoter) left empty to be inserted a biopharmaceutical protein gene region or DNA vaccine antigens. Results: In order to determine the anti-apoptotic efficacy of Bcl-xL inserted dual expressing vector, BHK-21 cells were transfected both with this plasmid and empty vector as control. Apoptosis was stimulated by several apoptosis inducing agents and serum deprivation in the transfected cells for 48 hrs. Cells expressing Bcl-xL protein in frame with EGFP were determined by flow cytometry as an indicator of cell viability. Additionally, apoptosis were determined by intracellular cleaved Casp 3 staining in Bcl-xL expressing EGFP positive cells. The dual expression plasmid bearing Bcl-xL anti-apoptotic protein prolonged the cell survival rate and protected cells from apoptosis upon apoptosis induction by doxorubicin and camptothecin in which the anti-apoptotic efficacies are inhibited through over expressing of Bcl-xL. pIRES2EGFP/Bcl-xL transfected cell ratio was significantly higher compared to empty vector transfected cells (P < 0.001). In contrast, apoptotic cell ratio was significantly lower in pIRES2EGFP/Bcl-xL transfected cell population compared to empty vector transfected cells (P < 0.001). Conclusion: In conclusion, it was shown that in vitro transient expression of Bcl-xL efficiently inhibited apoptosis induced by serum deprivation, doxorubicin and camptothecin. Thus, the dual expression plasmid bearing Bcl-xL anti-apoptotic protein could be a good candidate for recombinant protein production and DNA vaccination applications.info:eu-repo/semantics/openAccessPontificia Universidad Católica de ValparaísoElectronic Journal of Biotechnology v.15 n.5 20122012-09-01text/htmlhttp://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582012000500002en
institution	Scielo Chile
collection	Scielo Chile
language	English
topic	animal cell biotechnology apoptosis Bcl-xL anti-apoptotic protein
spellingShingle	animal cell biotechnology apoptosis Bcl-xL anti-apoptotic protein Iz,Sultan Gülçe Çalimlioglu,Beste Gürhan,Saime Ismet Deliloglu Using Bcl-xL anti-apoptotic protein for altering target cell apoptosis
description	Background: Altering target cell apoptosis is one of the challenging ideas of biotechnological applications. There are several applications of over expressing Bcl-xL anti-apoptotic protein from recombinant protein production to DNA vaccination strategies. The aim of the present study is to evaluate the anti-apoptotic efficacy of Bcl-xL expressing dual promoter plasmid system as a candidate to be used for recombinant protein production and DNA vaccination approaches. For this purpose, Bcl-xL anti-apoptotic protein gene was inserted in a dual expressing vector system in frame with EGFP (enhanced green fluorescence protein) after IRES (internal ribosomal site). The plasmid has a multiple cloning site after CMV (cytomegalovirus promoter) left empty to be inserted a biopharmaceutical protein gene region or DNA vaccine antigens. Results: In order to determine the anti-apoptotic efficacy of Bcl-xL inserted dual expressing vector, BHK-21 cells were transfected both with this plasmid and empty vector as control. Apoptosis was stimulated by several apoptosis inducing agents and serum deprivation in the transfected cells for 48 hrs. Cells expressing Bcl-xL protein in frame with EGFP were determined by flow cytometry as an indicator of cell viability. Additionally, apoptosis were determined by intracellular cleaved Casp 3 staining in Bcl-xL expressing EGFP positive cells. The dual expression plasmid bearing Bcl-xL anti-apoptotic protein prolonged the cell survival rate and protected cells from apoptosis upon apoptosis induction by doxorubicin and camptothecin in which the anti-apoptotic efficacies are inhibited through over expressing of Bcl-xL. pIRES2EGFP/Bcl-xL transfected cell ratio was significantly higher compared to empty vector transfected cells (P < 0.001). In contrast, apoptotic cell ratio was significantly lower in pIRES2EGFP/Bcl-xL transfected cell population compared to empty vector transfected cells (P < 0.001). Conclusion: In conclusion, it was shown that in vitro transient expression of Bcl-xL efficiently inhibited apoptosis induced by serum deprivation, doxorubicin and camptothecin. Thus, the dual expression plasmid bearing Bcl-xL anti-apoptotic protein could be a good candidate for recombinant protein production and DNA vaccination applications.
author	Iz,Sultan Gülçe Çalimlioglu,Beste Gürhan,Saime Ismet Deliloglu
author_facet	Iz,Sultan Gülçe Çalimlioglu,Beste Gürhan,Saime Ismet Deliloglu
author_sort	Iz,Sultan Gülçe
title	Using Bcl-xL anti-apoptotic protein for altering target cell apoptosis
title_short	Using Bcl-xL anti-apoptotic protein for altering target cell apoptosis
title_full	Using Bcl-xL anti-apoptotic protein for altering target cell apoptosis
title_fullStr	Using Bcl-xL anti-apoptotic protein for altering target cell apoptosis
title_full_unstemmed	Using Bcl-xL anti-apoptotic protein for altering target cell apoptosis
title_sort	using bcl-xl anti-apoptotic protein for altering target cell apoptosis
publisher	Pontificia Universidad Católica de Valparaíso
publishDate	2012
url	http://www.scielo.cl/scielo.php?script=sci_arttext&pid=S0717-34582012000500002
work_keys_str_mv	AT izsultangulce usingbclxlantiapoptoticproteinforalteringtargetcellapoptosis AT calimlioglubeste usingbclxlantiapoptoticproteinforalteringtargetcellapoptosis AT gurhansaimeismetdeliloglu usingbclxlantiapoptoticproteinforalteringtargetcellapoptosis
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Using Bcl-xL anti-apoptotic protein for altering target cell apoptosis

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