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The use of antisense oligonucleotides in evaluating survivin as a therapeutic target for radiation sensitization in lung cancer

Abstract

Elucidating the mechanism of over and under expression of proteins is critical in developing a better understanding of cancer. Multiple techniques are used to examine differential expression of proteins in cells and assess changes in protein expression in response to therapies such as radiation. Reduced expression can be caused by protein inactivation, mRNA instability, or reduced transcription. The following protocol was used to determine the mechanism for the reduced expression of an antiapoptotic factor, survivin, in normal tissues in response to radiation and the defect in cancer cells that prevents this reduction. We also examined ways to overcome survivin over expression in cancer cells in order to sensitize them to radiation. We will focus on the use of antisense oligonucleotides, cell cycle analysis, and luciferase reporter genes.

Abbreviations

HUVEC:

human umbilical vein endothelial cells

ASO:

antisense oligonucleotides

MDM2:

Murine Double Minute 2

PBS:

Phosphate Buffer Saline

IAP:

inhibitors of apoptosis family

7-AAD:

7-Aminoactinomycin D

DN:

Dominant Negative

MTT:

3-(4,5-methylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide

EBM-2:

endothelial basal medium-2

EGM-2:

endothelial growth media

References

  1. Lu B, Mu Y, Cao C, Zeng F, Schneider S, Tan J, Price J, Chen J, Freeman M, Hallahan DE. Survivin as a therapeutic target for radiation sensitization in lung cancer. Cancer Res 2004; 64(8):2840–2845.

    Article  PubMed  CAS  Google Scholar 

  2. Bao R, Connolly DC, Murphy M, Green J, Weinstein JK, Pisarcik DA, Hamilton TC. Activation of cancer-specific gene expression by the survivin promoter. J Natl Cancer Inst 2002; 94(7):522–528.

    PubMed  CAS  Google Scholar 

  3. Altieri DC. Validating survivin as a cancer therapeutic target. Nat Rev Cancer 2003; 3(1):46–54.

    Article  PubMed  CAS  Google Scholar 

  4. Altieri DC, Marchisio PC, Marchisio C. Survivin apoptosis: an interloper between cell death and cell proliferation in cancer. Lab Invest 1999; 79(11):1327–1333.

    PubMed  CAS  Google Scholar 

  5. Shih AH, Dai C, Hu X, Rosenblum MK, Koutcher JA, Holland EC. Dose-dependent effects of platelet-derived growth factor-B on glial tumorigenesis. Cancer Res 2004; 64(14):4783–4789.

    Article  PubMed  CAS  Google Scholar 

  6. Zhang Z, Li M, Wang H, Agrawal S, Zhang R. Antisense therapy targeting MDM2 oncogene in prostate cancer: Effects on proliferation, apoptosis, multiple gene expression, and chemotherapy. Proc Natl Acad Sci USA 2003; 100(20):11636–11641.

    Article  PubMed  CAS  Google Scholar 

  7. Weiss RH, Randour CJ. Attenuation of matrix protein secretion by antisense oligodeoxynucleotides to the cyclin kinase inhibitor p21(Waf1/Cip1). Atherosclerosis 2002; 161(1):105–112.

    Article  PubMed  CAS  Google Scholar 

  8. Hasholt L, Abell K, Norremolle A, Nellemann C, Fenger K, Sorensen SA. Antisense downregulation of mutant huntingtin in a cell model. J Gene Med 2003; 5(6):528–538.

    Article  PubMed  CAS  Google Scholar 

  9. Kamiyama M, Ichikawa Y, Ishikawa T, Chishima T, Hasegawa S, Hamaguchi Y, Nagashima Y, Miyagi Y, Mitsuhashi M, Hyndman D, Hoffman RM, Ohki S, Shimada H. VEGF receptor antisense therapy inhibits angiogenesis and peritoneal dissemination of human gastric cancer in nude mice. Cancer Gene Ther 2002; 9(2):197–201.

    Article  PubMed  CAS  Google Scholar 

  10. Lord RV, Salonga D, Danenberg KD, Peters JH, DeMeester TR, Park JM, Johansson J, Skinner KA, Chandrasoma P, DeMeester SR, Bremner CG, Tsai PI, Danenberg PV. Telomerase reverse transcriptase expression is increased early in the Barrett’s metaplasia, dysplasia, adenocarcinoma sequence. J Gastrointest Surg 2000; 4(2):135–142.

    Article  PubMed  CAS  Google Scholar 

  11. Gibson UE, Heid CA, Williams PM. A novel method for real time quantitative RT-PCR. Genome Res 1996; 6(10):995–1001.

    Article  PubMed  CAS  Google Scholar 

  12. Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996;6(10):986–994.

    Article  PubMed  CAS  Google Scholar 

  13. Ormerod MG. Investigating the relationship between the cell cycle and apoptosis using flow cytometry. J Immunol Methods 2002; 265(1–2):73–80.

    Article  PubMed  CAS  Google Scholar 

  14. Otaki M, Hatano M, Kobayashi K, Ogasawara T, Kuriyama T, Tokuhisa T. Cell cycle-dependent regulation of TIAP/msurvivin expression. Biochim Biophys Acta 2000; 1493(1– 2):188–194.

    PubMed  CAS  Google Scholar 

  15. Zhou M, Gu L, Li F, Zhu Y, Woods WG, Findley HW. DNA damage induces a novel p53-survivin signaling pathway regulating cell cycle and apoptosis in acute lymphoblastic leukemia cells. J Pharmacol Exp Ther 2002; 303(1):124–131.

    Article  PubMed  CAS  Google Scholar 

  16. Gewirtz AM, Stein CA, Glazer PM. Facilitating oligonucleotide delivery: helping antisense deliver on its promise. Proc Natl Acad Sci USA 1996; 93(8):3161–3163.

    Article  PubMed  CAS  Google Scholar 

  17. Yuen AR, Halsey J, Fisher GA, Holmlund JT, Geary RS, Kwoh TJ, Dorr A, Sikic BI. Phase I study of an antisense oligonucleotide to protein kinase C-alpha (ISIS 3521/CGP 64128A) in patients with cancer. Clin Cancer Res 1999; 5(11):3357–3363.

    PubMed  CAS  Google Scholar 

  18. Orr RM. Technology evaluation: fomivirsen, Isis Pharmaceuticals Inc/CIBA vision. Curr Opin Mol Ther 2001; 3(3):288–294.

    PubMed  CAS  Google Scholar 

  19. Pisarev V, Yu B, Salup R, Sherman S, Altieri DC, Gabrilovich DI. Full-length dominant-negative survivin for cancer immunotherapy. Clin Cancer Res 2003; 9(17):6523–6533.

    PubMed  CAS  Google Scholar 

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Correspondence to Bo Lu.

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Published: October 25, 2004.

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Shinohara, E.T., Hallahan, D.E. & Lu, B. The use of antisense oligonucleotides in evaluating survivin as a therapeutic target for radiation sensitization in lung cancer. Biol. Proced. Online 6, 250–256 (2004). https://doi.org/10.1251/bpo95

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  • DOI: https://doi.org/10.1251/bpo95

Indexing terms

  • Survivin
  • Antisense oligonucleotides
  • Luciferase