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Understanding apoptotic signaling pathways in cytosine deaminase-uracil phosphoribosyl transferase-mediated suicide gene therapy in vitro

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Abstract

Cytosine deaminase-uracil phosphoribosyl transferase (CD-UPRT) fusion gene is known to exhibit therapeutic effect by inducing apoptosis in vitro. However, bystander effects of 5-flurocytosine (5-FC)/CD-UPRT and the molecular mechanism for apoptosis are yet to be established. In the present study, we have generated BHK21 cell line expressing both CD-UPRT and green fluorescent protein (GFP) from two separate transcripts, where GFP was used as a noninvasive probe to monitor the therapeutic effect of CD-UPRT. Enzyme activity of CD-UPRT in the stable cell line was measured by the reverse phase high-performance liquid chromatography analysis. Inhibition of cell growth and strong bystander effects of 5-FC/CD-UPRT were established, whereas characteristic surface morphology of apoptotic cell death was identified by AFM analysis. Involvement of various apoptotic signaling genes using semi-quantitative RT-PCR has been explored to substantiate the potential application of 5-FC/CD-UPRT suicide gene in therapy.

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Abbreviations

CD-UPRT:

Cytosine deaminase-uracil phosphoribosyl transferase

GFP:

Green fluorescence protein

5-FC:

5-Flurocytosine

5-FU:

5-Flurouracil

AFM:

Atomic force microscope

AO:

Acridine orange

EB:

Ethidium bromide

References

  1. Yazawa K, Fisher WE, Brunicardi FC (2002) Current progress in suicide gene therapy for cancer. World J Surg 26:783–789. doi:10.1007/s00268-002-4053-5

    Article  PubMed  Google Scholar 

  2. Denning C, Pitts JD (1997) Bystander effects of different enzyme–prodrug systems for cancer gene therapy depend on different pathways for intercellular transfer of toxic metabolites, a factor that will govern clinical choice of appropriate regimes. Hum Gene Ther 8:1825–1835. doi:10.1089/hum.1997.8.15-1825

    Article  PubMed  CAS  Google Scholar 

  3. Freeman SM, Abboud CN, Whartenby KA et al (1993) The “bystander effect”: tumor regression when a fraction of the tumor mass is genetically modified. Cancer Res 53:5274–5283

    PubMed  CAS  Google Scholar 

  4. Springer CJ, Duvaz IN (2000) Prodrug-activating systems in suicide gene therapy. J Clin Invest 105:1161–1167. doi:10.1172/JCI10001

    Article  PubMed  CAS  Google Scholar 

  5. Moolten FL (1986) Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. Cancer Res 46:5276–5281

    PubMed  CAS  Google Scholar 

  6. Goto T, Nishi T, Tamura T et al (2000) Highly efficient electro-gene therapy of solid tumor by using an expression plasmid for the herpes simplex virus thymidine kinase gene. Proc Natl Acad Sci USA 97:354–359. doi:10.1073/pnas.97.1.354

    Article  PubMed  CAS  Google Scholar 

  7. Rowley S, Lindauer M, Gebert JF et al (1996) Cytosine deaminase gene as a potential tool for the genetic therapy of colorectal cancer. J Surg Oncol 61:42–48. doi:10.1002/(SICI)1096-9098(199601)61:1<42::AID-JSO10>3.0.CO;2-Z

    Article  PubMed  CAS  Google Scholar 

  8. Trinh QT, Austin EA, Murray DM et al (1995) Enzyme/prodrug gene therapy: comparison of cytosine deaminase/5-fluorocytosine versus thymidine kinase/ganciclovir enzyme/prodrug systems in a human colorectal carcinoma cell line. Cancer Res 55:4808–4812

    PubMed  CAS  Google Scholar 

  9. Gopinath P, Ghosh SS (2007) Monitoring green fluorescent protein for functional delivery of E. coli cytosine deaminase suicide gene and the effect of curcumin in vitro. Gene Ther Mol Biol 11:219–228

    Google Scholar 

  10. Gopinath P, Ghosh SS (2008) Apoptotic induction with bifunctional E. coli cytosine deaminase-uracil phosphoribosyl transferase mediated suicide gene therapy is synergized by curcumin treatment in vitro. Mol Biotechnol 39:39–48. doi:10.1007/s12033-007-9026-3

    Article  PubMed  CAS  Google Scholar 

  11. Gopinath P, Gogoi SK, Chattopadhyay A et al (2008) Implications of silver nanoparticle induced cell apoptosis for in vitro gene therapy. Nanotechnology. doi:10.1088/0957-4484/19/7/075104

  12. Gopinath P, Ghosh SS (2008) Implication of functional activity for determining therapeutic efficacy of suicide genes in vitro. Biotechnol Lett. doi:10.1007/s10529-008-9787-1

  13. Ye H, Gan L, Yang X et al (2006) Membrane-associated cytotoxicity induced by realgar in promyelocytic leukemia HL-60 cells. J Ethnopharmacol 103:366–371. doi:10.1016/j.jep.2005.08.014

    Article  PubMed  CAS  Google Scholar 

  14. Ribble D, Goldstein NB, Norris DA et al (2005) A simple technique for quantifying apoptosis in 96-well plates. BMC Biotechnol 5:12. doi:10.1186/1472-6750-5-12

    Article  PubMed  CAS  Google Scholar 

  15. Khatri A, Zhang B, Doherty E et al (2006) Combination of cytosine deaminase with uracil phosphoribosyl transferase leads to local and distant bystander effects against RM1 prostate cancer in mice. J Gene Med 8:1086–1096. doi:10.1002/jgm.944

    Article  PubMed  CAS  Google Scholar 

  16. Wang J, Lu X, Chen D et al (2004) Herpes simplex virus thymidine kinase and ganciclovir suicide gene therapy for human pancreatic cancer. World J Gastroenterol 10:400–403

    PubMed  CAS  Google Scholar 

  17. Le Grimellec C, Lesniewska E, Cachia C et al (1994) Imaging of the membrane surface of MDCK cells by atomic force microscopy. Biophys J 67:36–41

    Article  PubMed  Google Scholar 

  18. Parpura V, Fernandez JM (1996) Atomic force microscopy study of the secretory granule lumen. Biophys J 71:2356–2366

    Article  PubMed  CAS  Google Scholar 

  19. Hoganson DK, Batra RK, Olsen JC et al (1996) Comparison of the effects of three different toxin genes and their levels of expression on cell growth and bystander effect in lung adenocarcinoma. Cancer Res 56:1315–1323

    PubMed  CAS  Google Scholar 

  20. Lee KC, Hamstra DA, Bullarayasamudram S et al (2006) Fusion of the HSV-1 tegument protein vp22 to cytosine deaminase confers enhanced bystander effect and increased therapeutic benefit. Gene Ther 13:127–137. doi:10.1038/sj.gt.3302631

    Article  PubMed  CAS  Google Scholar 

  21. Pandey S, Smith B, Walker PR et al (2000) Caspase dependent and independent cell death in rat hepatoma 5123tc cells. Apoptosis 5:265–275. doi:10.1023/A:1009608630145

    Article  PubMed  CAS  Google Scholar 

  22. Cohen GM (1997) Caspases: the executioners of apoptosis. Biochem J 326:1–16

    PubMed  CAS  Google Scholar 

  23. Duyao MP, Kessler DJ, Spicer DB et al (1990) Binding of NF-kB-like factors to regulatory sequences of the c-myc gene. Curr Top Microbiol Immunol 166:211–220

    PubMed  CAS  Google Scholar 

  24. Merino R, Grillot DA, Simonian PL et al (1995) Modulation of anti-IgM-induced B cell apoptosis by Bcl-xL and CD40 in WEHI-231 cells. Dissociation from cell cycle arrest and dependence on the avidity of the antibody-IgM receptor interaction. J Immunol 155:3830–3838

    PubMed  CAS  Google Scholar 

  25. Sun XM, MacFarlane M, Zhuang J et al (1999) Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. J Biol Chem 274:5053–5060. doi:10.1074/jbc.274.8.5053

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This research work was supported by the Department of Biotechnology (No. BT/PR9988/NNT/28/76/2007), Council of Scientific and Industrial Research [No. 37 (1248)/06/EMR-II] Government of India. Assistance from Central instruments Facility (CIF) IIT Guwahati, for confocal analysis is gratefully acknowledged. We are also thankful to Mr. Vijay Kumar Ravi (Department of Biotechnology, IIT Guwahati) for AFM imaging. Our sincere thanks to www.imagemet.com for allowing us to use the software SPIP 4.6.3.

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  1. Department of Biotechnology, Centre for Nanotechnology, Indian Institute of Technology Guwahati, Guwahati, 781039, India

    P. Gopinath & Siddhartha Sankar Ghosh

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  1. P. Gopinath
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  2. Siddhartha Sankar Ghosh
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Correspondence to Siddhartha Sankar Ghosh.

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Gopinath, P., Ghosh, S.S. Understanding apoptotic signaling pathways in cytosine deaminase-uracil phosphoribosyl transferase-mediated suicide gene therapy in vitro. Mol Cell Biochem 324, 21–29 (2009). https://doi.org/10.1007/s11010-008-9980-5

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  • DOI: https://doi.org/10.1007/s11010-008-9980-5

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