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The construction of the eukaryotic expression plasmid pcDNA3.1/azurin and the increased apoptosis of U2OS cells transfected with it
Cellular & Molecular Biology Letters volume 12, pages 407–421 (2007)
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.
Abbreviations
- FCM:
-
flow cytometry
- GFP:
-
green fluorescent protein
- HA:
-
haemagglutinin 9 peptide HA epitope
- hCMV:
-
human cytomegalovirus
- IAP:
-
inhibitor apoptosis protein
- PCR:
-
polymerase chain reaction
- RT:
-
reverse transcription
- UV:
-
ultraviolet
References
Sinha, G. Bacterial battalions join war against cancer. Nat. Med. 9 (2003) 1229.
Chakrabarty, A.M. Microorganisms and cancer: quest for a therapy. J. Bacteriol. 185 (2003) 2683–2686.
Kukimoto, M., Nishiyama, M., Tanokura, M., Murphy, M.E., Adman, E.T. and Horinouchi, S. Site-directed mutagenesis of azurin from Pseudomonas aeruginosa enhances the formation of an electron-transfer complex with a copper-containing nitrite reductase from Alcaligenes faecalis S-6. FEBS Lett. 394 (1996) 87–90.
Yamada, T., Fialho, A.M., Punj, V., Bratescu, L., Gupta, T.K. and Chakrabarty, A.M. Internalization of bacterial redox protein azurin in mammalian cells: entry domain and specificity. Cell. Microbiol. 7 (2005) 1418–1431.
Yamada, T., Hiraoka, Y., Ikehata, M., Kimbara, K., Avner, B.S., Das Gupta, T.K. and Chakrabarty, A.M. Apoptosis or growth arrest: Modulation of tumor suppressor p53’s specificity by bacterial redox protein azurin. Proc. Natl. Acad. Sci. 101 (2004) 4770–4775.
Yang, D.S., Miao, X.D., Ye, Z.M., Feng, J., Xu, R.Z., Huang, X. and Ge, F.F. Bacterial redox protein azurin induce apoptosis in human osteosarcoma U2OS cells. Pharmacol. Res. 52 (2005) 413–421.
Miao, X.D., Ye, Z.M., Yang, D.S., Xu, R.Z., Li, W.X. and Tao, H.M. Cytotoxicity and apoptosis of human osteosarcoma U2OS cells induced by recombinant soluble AZURIN. Zhejiang Da Xue Xue Bao Yi Xue Ban. 34 (2005) 384–389.
Wilson, I.A., Niman, H.L., Houghten, R.A., Cherenson, A.R., Connolly, M.L. and Lerner, R.A. The structure of an antigenic determinant in a protein. Cell 37 (1984) 767–778.
Feng, J., Zhao, L. and Yu, Q. Receptor-mediated stimulatory effect of oligochitosan in macrophages. Biochem. Biophys. Res. Commun. 317 (2004) 414–420.
Reed, J.C. Apoptosis-regulating proteins as targets for drug discovery. Trends. Mol. Med. 7 (2001) 314–319.
Wyllie, A.H. Apoptosis (the 1992 Frank Rose Memorial Lecture). Br. J. Cancer. 67 (1993) 205–208.
Zhou, M., Gu, L., Yeager, A.M. and Findley, H.W. Sensitivity to Fas-mediated apoptosis in pediatric acute lymphoblastic leukemia is associated with a mutant p53 phenotype and absence of Bcl-2 expression. Leukemia 12 (1998) 1756–1763.
Zhang, H., Heim, J. and Meyhack, B. Novel BNIP1 variants and their interaction with BCL2 family members. FEBS Lett. 448 (1999) 23–27.
Schlesinger, P.H., Gross, A., Yin, X.M., Yamamoto, K., Saito, M., Waksman, G. and Korsmeyer, S.J. Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2. Proc. Natl. Acad. Sci. 94 (1997) 11357–11362.
Tu, Y., Xu, F.H., Liu, J., Vescio, R., Berenson, J., Fady, C. and Lichtenstein, A. Upregulated expression of BCL-2 in multiple myeloma cells induced by exposure to doxorubicin, etoposide, and hydrogen peroxide. Blood 88 (1996) 1805–1812.
Salomons, G.S., Brady, H.J., Verwijs-Janssen, M., Van Den Berg, J.D., Hart, A.A., Van Den Berg, H., Behrendt, H., Hahlen, K. and Sme, L.A. The Bax alpha:Bcl-2 ratio modulates the response to dexamethasone in leukaemic cells and is highly variable in childhood acute leukaemia. Int. J. Cancer. 71 (1997) 959–965.
Li, F., Ambrosini, G., Chu, E.Y., Plescia, J., Tognin, S., Marchisio, P.C. and Altieri, D.C. Control of apoptosis and mitotic spindle checkpoint by Survivin. Nature 396 (1998) 580–584.
Yamada, T., Goto, M., Punj, V., Zaborina, O., Chen, M.L., Kimbara, K., Majumdar, D., Cunningham, E., Das Gupta, T.K. and Chakrabarty, A.M. Bacterial redox protein azurin, tumor suppressor protein p53, and regression of cancer. Proc. Natl. Acad. Sci. 99 (2002) 14098–14103.
Punj, V., Bhattacharyya, S., Saint-Dic, D., Vasu, C., Cunningham, E.A., Graves, J., Yamada, T., Constantinou, A.I., Christov, K., White, B., Li, G., Majumdar, D., Chakrabarty, A.M. and Das Gupta, TK. Bacterial cupredoxin azurin as an inducer of apoptosis and regression in human breast cancer. Oncogene 23 (2004) 2367–2378.
Yamada, T., Goto, M., Punj, V., Zaborina, O., Kimbara, K., Das Gupta, T.K. and Chakrabarty, A.M. The bacterial redox protein azurin induces apoptosis in J774 macrophages through complex formation and stabilization of the tumor suppressor protein p53. Infect. Immun. 70 (2002) 7054–7062.
Punj, V., Das Gupta, T.K. and Chakrabarty, A.M. Bacterial cupredoxin azurin and its interactions with the tumor suppressor protein p53. Biochem. Biophys. Res. Commun. 312 (2003) 109–114.
Vogelstein, B., Lane, D. and Levine, A.J. Surfing the p53 network. Nature 408 (2000) 307–310.
Haupt, Y., Maya, R., Kazaz, A. and Oren, M. Mdm2 promotes the rapid degradation of p53. Nature 387 (1997) 296–299.
Gross, A., Jockel, J., Wei, M.C. and Korsmeyer, S.J. Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J. 17 (1998) 3878–3885.
Budhram-Mahadeo, V., Morris, P.J., Smith, M.D., Midgley, C.A., Boxer, L.M. and Latchman, D.S. p53 suppresses the activation of the Bcl-2 promoter by the Brn-3a POU family transcription factor. J. Biol. Chem. 274 (1999) 15237–15244.
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Ye, Z., Peng, H., Fang, Y. et al. The construction of the eukaryotic expression plasmid pcDNA3.1/azurin and the increased apoptosis of U2OS cells transfected with it. Cell Mol Biol Lett 12, 407–421 (2007). https://doi.org/10.2478/s11658-007-0012-3
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DOI: https://doi.org/10.2478/s11658-007-0012-3