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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.

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

  1. 1.

    Sinha, G. Bacterial battalions join war against cancer. Nat. Med. 9 (2003) 1229.

    PubMed  Article  CAS  Google Scholar 

  2. 2.

    Chakrabarty, A.M. Microorganisms and cancer: quest for a therapy. J. Bacteriol. 185 (2003) 2683–2686.

    PubMed  Article  CAS  Google Scholar 

  3. 3.

    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.

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    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.

    PubMed  Article  CAS  Google Scholar 

  5. 5.

    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.

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    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.

    PubMed  Article  CAS  Google Scholar 

  7. 7.

    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.

    PubMed  CAS  Google Scholar 

  8. 8.

    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.

    PubMed  Article  CAS  Google Scholar 

  9. 9.

    Feng, J., Zhao, L. and Yu, Q. Receptor-mediated stimulatory effect of oligochitosan in macrophages. Biochem. Biophys. Res. Commun. 317 (2004) 414–420.

    PubMed  Article  CAS  Google Scholar 

  10. 10.

    Reed, J.C. Apoptosis-regulating proteins as targets for drug discovery. Trends. Mol. Med. 7 (2001) 314–319.

    PubMed  Article  CAS  Google Scholar 

  11. 11.

    Wyllie, A.H. Apoptosis (the 1992 Frank Rose Memorial Lecture). Br. J. Cancer. 67 (1993) 205–208.

    PubMed  CAS  Google Scholar 

  12. 12.

    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.

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Zhang, H., Heim, J. and Meyhack, B. Novel BNIP1 variants and their interaction with BCL2 family members. FEBS Lett. 448 (1999) 23–27.

    PubMed  Article  CAS  Google Scholar 

  14. 14.

    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.

    PubMed  Article  CAS  Google Scholar 

  15. 15.

    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.

    PubMed  CAS  Google Scholar 

  16. 16.

    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.

    PubMed  Article  CAS  Google Scholar 

  17. 17.

    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.

    PubMed  Article  CAS  Google Scholar 

  18. 18.

    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.

    PubMed  Article  CAS  Google Scholar 

  19. 19.

    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.

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    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.

    PubMed  Article  CAS  Google Scholar 

  21. 21.

    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.

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    Vogelstein, B., Lane, D. and Levine, A.J. Surfing the p53 network. Nature 408 (2000) 307–310.

    PubMed  Article  CAS  Google Scholar 

  23. 23.

    Haupt, Y., Maya, R., Kazaz, A. and Oren, M. Mdm2 promotes the rapid degradation of p53. Nature 387 (1997) 296–299.

    PubMed  Article  CAS  Google Scholar 

  24. 24.

    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.

    PubMed  Article  CAS  Google Scholar 

  25. 25.

    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.

    PubMed  Article  CAS  Google Scholar 

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Correspondence to Di-Sheng Yang.

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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

Keywords

  • Azurin
  • Transfection
  • Osteosarcoma
  • Apoptosis