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  • Open Access

The construction of the eukaryotic expression plasmid pcDNA3.1/azurin and the increased apoptosis of U2OS cells transfected with it

  • 1,
  • 2,
  • 3,
  • 1 and
  • 1Email author
Cellular & Molecular Biology LettersAn International Journal200712:12

https://doi.org/10.2478/s11658-007-0012-3

  • Received: 10 August 2006
  • Accepted: 11 January 2007
  • Published:

Abstract

In our previous study, we demonstrated that azurin could selectively trigger apoptosis in human osteosarcoma cell line U2OS cells. However, the rate of apoptosis (35.8 ± 3.2%) is not very high, and azurin is too expensive to obtain readily. To solve these problems, we constructed a eukaryotic expression plasmid containing the azurin gene with an influenza virus haemagglutinin 9 peptide HA epitope tag, and transfected the recombinant plasmid pcDNA3.1(+)/azurin into U2OS cells. RT-PCR and Western blot analysis validated the successful transfection and the expression of the azurin-HA protein. Conspicuous apoptosis of the transfected cells was detected by flow cytometry (FCM) and the DNA ladder test. The apoptosis rate reached 64.3 ± 13.1%. The transcriptional levels of the Bax and p53 genes increased significantly in U2OS cells transfected with pcDNA3.1(+)/azurin, but the Bcl-2 mRNA level decreased. There was no difference in the levels of Bcl-xl mRNA and Survivin mRNA. We propose that the transfection of the recombinant plasmid pcDNA3.1(+)/azurin can significantly induce apoptosis in U2OS cells. This is closely associated with the up-regulation of the transcriptional level of the Bax and p53 genes, and the down-regulation of that of the Bcl-2 gene.

Keywords

  • Azurin
  • Transfection
  • Osteosarcoma
  • Apoptosis

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