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.
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Authors | Zhaoming Ye, Huiqin Peng, Yongming Fang, Jie Feng, Di-Sheng Yang |
Journal | Cellular & molecular biology letters
(Cell Mol Biol Lett)
Vol. 12
Issue 3
Pg. 407-21
(Sep 2007)
ISSN: 1689-1392 [Electronic] England |
PMID | 17415534
(Publication Type: Journal Article)
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Chemical References |
- BIRC5 protein, human
- Hemagglutinin Glycoproteins, Influenza Virus
- Inhibitor of Apoptosis Proteins
- RNA, Messenger
- Survivin
- bcl-2-Associated X Protein
- bcl-X Protein
- Azurin
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Topics |
- Apoptosis
- Azurin
(metabolism)
- Blotting, Western
- Cell Cycle
- Cell Line, Tumor
- DNA Fragmentation
- Eukaryotic Cells
(metabolism)
- Flow Cytometry
- Gene Expression
- Gene Expression Regulation, Neoplastic
- Hemagglutinin Glycoproteins, Influenza Virus
(metabolism)
- Humans
- Inhibitor of Apoptosis Proteins
(genetics, metabolism)
- Plasmids
(metabolism)
- RNA, Messenger
(genetics, metabolism)
- Survivin
- Transcription, Genetic
- Transfection
(methods)
- bcl-2-Associated X Protein
(genetics, metabolism)
- bcl-X Protein
(genetics, metabolism)
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