title: The construction of the eukaryotic expression plasmid pcDNA3.1/azurin and the increased apoptosis of U2OS cells transfected with it.
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.
authors.affiliation: Department of Orthopedics, Second Affiliated Hospital, Medical College, Zhejiang University, 88 Jie Fang Road, Hangzhou, 310009, Zhejiang, P.R. China.
authors.forename: Zhaoming
authors.initials: Z
authors.lastname: Ye
authors.forename: Huiqin
authors.initials: H
authors.lastname: Peng
authors.forename: Yongming
authors.initials: Y
authors.lastname: Fang
authors.forename: Jie
authors.initials: J
authors.lastname: Feng
authors.forename: Di-Sheng
authors.initials: DS
authors.lastname: Yang
mesh_terms: D017209:Apoptosis; D001400:Azurin / Q000378:metabolism*; D015153:Blotting, Western; D002453:Cell Cycle; D045744:Cell Line, Tumor; D053938:DNA Fragmentation; D005057:Eukaryotic Cells / Q000378:metabolism*; D005434:Flow Cytometry; D015870:Gene Expression; D015972:Gene Expression Regulation, Neoplastic; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000378:metabolism; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000077022:Survivin; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods*; D051028:bcl-2-Associated X Protein / Q000235:genetics / Q000378:metabolism; D051020:bcl-X Protein / Q000235:genetics / Q000378:metabolism
chemical_list: C107489:BIRC5 protein, human; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D051034:Inhibitor of Apoptosis Proteins; D012333:RNA, Messenger; D000077022:Survivin; D051028:bcl-2-Associated X Protein; D051020:bcl-X Protein; D001400:Azurin