In Vitro Assay for the Evaluation of Cytotoxic Effects Provided by a Combination of Suicide and Killer Genes in a Bicistronic Vector

  • Alexey A. Komissarov
  • Sergey V. Kostrov
  • Ilya V. DemidyukEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1895)


When using bicistronic expression constructs the issue arises concerning proper evaluation of the cytotoxic efficiency of a combination of therapeutic genes. For this purpose, an approach can be applied based on the transient transfection of cultured human cells with a specifically designed set of mono- and bicistronic expression constructs and on the comparison of their cytotoxic effects. Here the application of this approach is described using an example of the evaluation of the combined cytotoxic action of bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion protein (FCU1) and hepatitis A virus 3C protease in a bicistronic plasmid construct.

Key words

Suicide gene therapy Bicistronic expression vector Transient transfection Colorimetric cell viability assay Bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion protein FCU1 Human hepatitis A virus 3C protease 



This work was supported in part by the Program of the Russian Academy of Sciences “Molecular and Cell Biology” and by the Russian Foundation for Basic Research (project no. 15-04-05169).


  1. 1.
    Misra S (2013) Human gene therapy: a brief overview of the genetic revolution. J Assoc Physicians India 61:127–133PubMedGoogle Scholar
  2. 2.
    Naldini L (2015) Gene therapy returns to centre stage. Nature 526:351–360CrossRefGoogle Scholar
  3. 3.
    Amer MH (2014) Gene therapy for cancer: present status and future perspective. Mol Cell Ther 2:27CrossRefGoogle Scholar
  4. 4.
    Duarte S, Carle G, Faneca H et al (2012) Suicide gene therapy in cancer: where do we stand now? Cancer Lett 324:160–170CrossRefGoogle Scholar
  5. 5.
    Zarogoulidis P, Darwiche K, Sakkas A et al (2013) Suicide gene therapy for cancer – current strategies. J Genet Syndr Gene Ther 4:16849PubMedPubMedCentralGoogle Scholar
  6. 6.
    Chang JW, Lee H, Kim E et al (2000) Combined antitumor effects of an adenoviral cytosine deaminase/thymidine kinase fusion gene in rat C6 glioma. Neurosurgery 47:931–938CrossRefGoogle Scholar
  7. 7.
    Qiu Y, Peng GL, Liu QC et al (2012) Selective killing of lung cancer cells using carcinoembryonic antigen promoter and double suicide genes, thymidine kinase and cytosine deaminase (pCEA-TK/CD). Cancer Lett 316:31–38CrossRefGoogle Scholar
  8. 8.
    Luo XR, Li JS, Niu Y et al (2012) Adenovirus-mediated double suicide gene selectively kills gastric cancer cells. Asian Pac J Cancer Prev 13:781–784CrossRefGoogle Scholar
  9. 9.
    Kong H, Liu C, Zhu T et al (2014) Effects of an adenoviral vector containing a suicide gene fusion on growth characteristics of breast cancer cells. Mol Med Rep 10:3227–3232CrossRefGoogle Scholar
  10. 10.
    Cao X, Ju DW, Tao Q et al (1998) Adenovirus-mediated GM-CSF gene and cytosine deaminase gene transfer followed by 5-fluorocytosine administration elicit more potent antitumor response in tumor-bearing mice. Gene Ther 5:1130–1136CrossRefGoogle Scholar
  11. 11.
    Ju DW, Wang BM, Cao X (1998) Adenovirus-mediated combined suicide gene and interleukin-2 gene therapy for the treatment of established tumor and induction of antitumor immunity. J Cancer Res Clin Oncol 124:683–689CrossRefGoogle Scholar
  12. 12.
    Ju DW, Yang Y, Tao Q et al (2000) Interleukin-18 gene transfer increases antitumor effects of suicide gene therapy through efficient induction of antitumor immunity. Gene Ther 7:1672–1679CrossRefGoogle Scholar
  13. 13.
    Nakamori M, Iwahashi M, Ueda K et al (2002) Dose of adenoviral vectors expressing interleukin-2 plays an important role in combined gene therapy with cytosine deaminase/5-fluorocytosine: preclinical consideration. Jpn J Cancer Res 93:706–715CrossRefGoogle Scholar
  14. 14.
    Sher YP, Chang CM, Juo CG et al (2013) Targeted endostatin-cytosine deaminase fusion gene therapy plus 5-fluorocytosine suppresses ovarian tumor growth. Oncogene 32:1082–1090CrossRefGoogle Scholar
  15. 15.
    Yang XP, Liu L, Wang P et al (2015) Human sulfatase-1 improves the effectiveness of cytosine deaminase suicide gene therapy with 5-fluorocytosine treatment on hepatocellular carcinoma cell line HepG2 in vitro and in vivo. Chin Med J 128:1384–1390CrossRefGoogle Scholar
  16. 16.
    Erbs P, Regulier E, Kintz J et al (2000) In vivo cancer gene therapy by adenovirus-mediated transfer of a bifunctional yeast cytosine deaminase/uracil phosphoribosyltransferase fusion gene. Cancer Res 60:3813–3822PubMedGoogle Scholar
  17. 17.
    Chung-Faye GA, Chen MJ, Green NK et al (2001) In vivo gene therapy for colon cancer using adenovirus-mediated, transfer of the fusion gene cytosine deaminase and uracil phosphoribosyltransferase. Gene Ther 8:1547–1554CrossRefGoogle Scholar
  18. 18.
    Richard C, Duivenvoorden W, Bourbeau D et al (2007) Sensitivity of 5-fluorouracil-resistant cancer cells to adenovirus suicide gene therapy. Cancer Gene Ther 14:57–65CrossRefGoogle Scholar
  19. 19.
    Kreiss P, Cameron B, Rangara R et al (1999) Plasmid DNA size does not affect the physicochemical properties of lipoplexes but modulates gene transfer efficiency. Nucleic Acids Res 27:3792–3798CrossRefGoogle Scholar
  20. 20.
    Ribeiro S, Mairhofer J, Madeira C et al (2012) Plasmid DNA size does affect nonviral gene delivery efficiency in stem cells. Cell Reprogram 14:130–137CrossRefGoogle Scholar
  21. 21.
    Akita H, Kurihara D, Schmeer M et al (2015) Effect of the compaction and the size of DNA on the nuclear transfer efficiency after microinjection in synchronized cells. Pharmaceutics 7:64–73CrossRefGoogle Scholar
  22. 22.
    Thomas CE, Ehrhardt A, Kay MA (2003) Progress and problems with the use of viral vectors for gene therapy. Nat Rev Genet 4:346–358CrossRefGoogle Scholar
  23. 23.
    Glover DJ, Lipps HJ, Jans DA (2005) Towards safe, non-viral therapeutic gene expression in humans. Nat Rev Genet 6:299–310CrossRefGoogle Scholar
  24. 24.
    Yin H, Kanasty RL, Eltoukhy AA et al (2014) Non-viral vectors for gene-based therapy. Nat Rev Genet 15:541–555CrossRefGoogle Scholar
  25. 25.
    Komissarov A, Demidyuk I, Safina D et al (2017) Cytotoxic effect of co-expression of human hepatitis A virus 3C protease and bifunctional suicide protein FCU1 genes in a bicistronic vector. Mol Biol Rep 44(4):323–332. CrossRefPubMedGoogle Scholar
  26. 26.
    Shubin AV, Demidyuk IV, Lunina NA et al (2015) Protease 3C of hepatitis A virus induces vacuolization of lysosomal/endosomal organelles and caspase-independent cell death. BMC Cell Biol 16:4CrossRefGoogle Scholar
  27. 27.
    Szymczak-Workman AL, Vignali KM, Vignali DA (2012) Design and construction of 2A peptide-linked multicistronic vectors. Cold Spring Harb Protoc 2012:199–204PubMedGoogle Scholar
  28. 28.
    Hsu CY, Uludag H (2008) Effects of size and topology of DNA molecules on intracellular delivery with non-viral gene carriers. BMC Biotechnol 8:23CrossRefGoogle Scholar
  29. 29.
    Rose JK (2003) Optimization of transfection. In: Curr Protoc Cell Biol, chapter 20. John Wiley & Sons, New York, unit 20.7. CrossRefGoogle Scholar
  30. 30.
    Komissarov AA, Karaseva MA, Safina DR et al (2016) Comparative evaluation of the transgene expression efficiency provided by the model genetic constructs of different structure. Mol Gen Mikrobiol Virusol 31:156–162Google Scholar
  31. 31.
    Galluzzi L, Vitale I, Abrams JM et al (2012) Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ 19:107–120CrossRefGoogle Scholar
  32. 32.
    Galluzzi L, Bravo-San Pedro JM, Vitale I et al (2015) Essential versus accessory aspects of cell death: recommendations of the NCCD 2015. Cell Death Differ 22:58–73CrossRefGoogle Scholar
  33. 33.
    Berridge MV, Herst PM, Tan AS (2005) Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol Annu Rev 11:127–152CrossRefGoogle Scholar
  34. 34.
    Sebaugh JL (2011) Guidelines for accurate EC50/IC50 estimation. Pharm Stat 10:128–134CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Alexey A. Komissarov
    • 1
  • Sergey V. Kostrov
    • 1
  • Ilya V. Demidyuk
    • 1
    Email author
  1. 1.Institute of Molecular Genetics, Russian Academy of SciencesMoscowRussia

Personalised recommendations