Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Expression of the prodrug-activating enzyme DT-diaphorase via Ad5 delivery to human colon carcinoma cells in vitro

Cancer Gene Therapy volume 9, pages 209–217 (2002)Cite this article

Abstract

Intratumoral injection of recombinant adenoviral type 5 (Ad5) vectors that carry prodrug-activating enzymes like DT-diaphorase (DTD) could be used to selectively target tumor cells for chemotherapy. To demonstrate the feasibility of this approach, Ad5 vectors were constructed, which express human DTD minigenes for both wild-type and mutant (C-to-T change in nucleotide 609 in DTD cDNA) DTD under the control of the cytomegalovirus (CMV) promoter. HT29 human colon carcinoma cells express wild-type DTD, whereas BE human colon carcinoma cells express mutant DTD, have low to undetectable DTD activity, and are 4- to 6-fold more resistant to mitomycin C (MMC) than HT29 cells. A test of the ability of Ad5 to infect these cells (using a β-galactosidase CMV-driven minigene) indicated that 90–100% of BE cells were infected at a multiplicity of infection (MOI) of 100, whereas only 15–40% of HT29 cells were infected at this MOI. Infection of BE cells in vitro with recombinant Ad5 carrying a minigene for wild-type DTD at MOIs of 3–100 resulted in a progressive increase in DTD activity and a maximal 8-fold increase in sensitivity to MMC as measured by a colony-forming assay. HT29 cells were sensitized 2- to 3-fold following treatment with Ad5.DTD at an MOI of 100. These results indicate that adenovirus-mediated gene transfer and expression of wild-type DTD can sensitize resistant tumor cells to MMC and that this therapeutic strategy may exert a significant bystander effect.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6

Similar content being viewed by others

References

  1. Cox PJ, Farmer PB . Towards selectivity? Approaches to the design of new anti-tumour agents — I and II Cancer Treat Rev 1977 4: 47–63 119–134

    Article  CAS  Google Scholar 

  2. Elion GB . Selectivity — key to chemotherapy: presidential address Cancer Res 1985 45: 2943–2950

    CAS  PubMed  Google Scholar 

  3. Garrett MD, Workman P . Discovering novel chemotherapeutic drugs for the third millennium Eur J Cancer 1999 35: 2010–2030

    Article  CAS  Google Scholar 

  4. Rauth AM, Marshall RS, Kuehl BL . Cellular approaches to bioreductive drug mechanisms Cancer Metastasis Rev 1993 12: 153–164

    Article  CAS  Google Scholar 

  5. Springer CJ, Niculescu I . Prodrug-activating systems in suicide gene therapy J Clin Invest 2000 105: 1161–1167

    Article  CAS  Google Scholar 

  6. Aghi M, Hochberg F, Breakefield XO . Prodrug activation enzymes in cancer gene therapy J Gene Med 2000 30: 148–164

    Article  Google Scholar 

  7. Connors TA . The choice of prodrugs for gene-directed enzyme prodrug therapy of cancer Gene Ther 1995 2: 702–709

    CAS  PubMed  Google Scholar 

  8. Weedon SJ, Green NK, McNeish IA et al. Sensitization of human carcinoma cells to the prodrug CB1954 by adenovirus vector–mediated expression of E. coli nitroreductase Int J Cancer 2000 86: 848–854

    Article  CAS  Google Scholar 

  9. Rauth AM, Melo T, Misra V . Bioreductive therapies: an overview of drugs and their mechanisms of action Int J Radiat Oncol Biol Phys 1998 42: 755–762

    Article  CAS  Google Scholar 

  10. Rauth AM, Goldberg Z, Misra V . DT-diaphorase: possible roles in cancer chemotherapy and carcinogenesis Oncol Res 1997 9: 339–349

    CAS  PubMed  Google Scholar 

  11. Ernster L . DT-diaphorase: a historical review Chem Scr 1987 27A: 1–13

    CAS  Google Scholar 

  12. Workman P . Enzyme-directed bioreductive drug development revisited: a commentary on recent progress and future prospects with emphasis on quinone anticancer agents and quinone metabolizing enzymes, particularly DT-diaphorase Oncol Res 1994 6: 461–475

    CAS  PubMed  Google Scholar 

  13. Marin A, Lopez de Cerain A, Hamilton E et al. DT-diaphorase and cytochrome B5 reductase in human lung and breast tumours Br J Cancer 1997 76: 923–929

    Article  CAS  Google Scholar 

  14. Kepa JK, Ross D . DT-diaphorase activity in NSCLC and SCLC cell lines: a role for fos/jun regulation Br J Cancer 1999 79: 1679–1684

    Article  CAS  Google Scholar 

  15. Traver RD, Horikoshi T, Danenberg KD et al. NAD(P)H: quinone oxidoreductase gene expression in human colon carcinoma cells: characterization of a mutation which modulates DTD activity and mitomycin sensitivity Cancer Res 1992 52: 797–802

    CAS  Google Scholar 

  16. Belcourt MF, Hodnick WF, Rockwell S et al. Exploring the mechanistic aspects of mitomycin antibiotic bioactivation in Chinese hamster ovary cells overexpressing NADPH: cytochrome C (P-450) reductase and DT-diaphorase Adv Enzyme Regul 1998 38: 111–133

    Article  CAS  Google Scholar 

  17. Begleiter A, Leith MK, Curphey TJ et al. Induction of DTD in cancer chemoprevention and chemotherapy Oncol Res 1997 9: 371–382

    CAS  PubMed  Google Scholar 

  18. Siegel D, Anwar A, inski SL et al. Rapid polyubiquitination and proteasomal degradation of a mutant form of NAD(P)H: quinone oxidoreductase 1 Mol Pharmacol 2001 59: 263–268

    Article  CAS  Google Scholar 

  19. Misra V, Grondin A, Klamut HJ et al. Assessment of the relationship between genotypic status of a DT-diaphorase point mutation and enzymatic activity Br J Can 2000 83: 998–1002

    Article  CAS  Google Scholar 

  20. Li JH, LI P, Klamut H et al. Cytotoxic effects of AD5CMV–p53 expression in two human nasopharyngeal carcinoma cell lines Clin Cancer Res 1997 3: 507–514

    CAS  PubMed  Google Scholar 

  21. Misra V, Klamut H, Rauth AM . Transfection of COS-1 cells with DT-diaphorase cDNA: role of a base change at position 609 Br J Cancer 1998 77: 1236–1240

    Article  CAS  Google Scholar 

  22. McGrory WJ, Bautista DS, Graham FL . A simple technique for the rescue of early region I mutations into infectious human adenovirus type 5 Virology 1988 63: 614–617

    Article  Google Scholar 

  23. Graham FL, Van der Eb AJ . A new technique for the assay of infectivity of human adenovirus 5 DNA Virology 1973 52: 456–467

    Article  CAS  Google Scholar 

  24. Graham FL, Prevec L . Manipulation of adenovirus vectors Methods Mol Biol 1991 7: 109–128

    CAS  PubMed  Google Scholar 

  25. Sambrook J, Fritsch EF, Maniatis T . Molecular Cloning, A Laboratory Manual Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press 1989 28

    Google Scholar 

  26. Sanes JR, Rubenstein JL, Nicolas JF . Use of a recombinant retrovirus to study post-implantation cell lineage in mouse embryos EMBO J 1986 5: 3133–3142

    Article  CAS  Google Scholar 

  27. Levine AJ . Tumor suppressor genes Bioessays 1990 12: 60–66

    Article  CAS  Google Scholar 

  28. Winski SL, Hargreaves RH, Ross D . A new screening system for NAD(P)H: quinone oxidoreductase (NQO1)–directed antitumour quinones: identification of a new aziridinylbenzoquinone, RH1, as a NQO1-directed antitumor agent Clin Cancer Res 1998 4: 3083–3088

    CAS  Google Scholar 

  29. Marshall RS, Paterson MC, Rauth AM . DTD activity and mitomycin C sensitivity in non-transformed cell strains derived from members of a cancer-prone family Carcinogenesis 1991 12: 1175–1180

    Article  CAS  Google Scholar 

  30. Fitzsimmons SA, Workman P, Grever M et al. Reductase enzyme expression across the National Cancer Institute tumor cell line panel: correlation with sensitivity to mitomycin C and EO9 J Natl Cancer Inst 1996 88: 259–269

    Article  CAS  Google Scholar 

  31. Belinsky M, Jaiswal AK . NAD(P)H: quinone oxidoreductase1 (DT-diaphorase) expression in normal and tumor tissues Cancer Metastasis Rev 1993 12: 103–117

    Article  CAS  Google Scholar 

  32. Beall HD, Murphy AM, Siegel D et al. Nicotinamide adenine dinucleotide (phosphate): quinone oxidoreductase (DT-diaphorase) as a target for bioreductive antitumor quinones: quinone cytotoxicity and selectivity in human lung and breast cancer cell lines Mol Pharmacol 1995 48: 499–504

    CAS  PubMed  Google Scholar 

  33. Gaedigk A, Tyndale RF, Jurima-Romet M et al. NAD(P)H: quinoneoxidoreductase: polymorphisms and allele frequencies in Caucasian, Chinese and Canadian Native Indian and Inuit populations Pharmacogenetics 1998 8: 305–313

    Article  CAS  Google Scholar 

  34. Searle PF, Weedon SJ, McNeish IA et al. Sensitisation of human ovarian cancer cells to killing by the prodrug CB1954 following retroviral or adenoviral transfer of the E. coli nitroreductase gene Adv Exp Med Biol 1998 451: 107–113

    Article  CAS  Google Scholar 

  35. Esandi MC, van Someren GD, Vincent AJ et al. Gene therapy of experimental malignant mesothelioma using adenovirus vectors encoding the HSVtk gene Gene Ther 1997 4: 280–287

    Article  CAS  Google Scholar 

  36. Block A, Freund CT, Chen SH et al. Gene therapy of metastatic colon carcinoma: regression of multiple hepatic metastases by adenoviral expression of bacterial cytosine deaminase Cancer Gene Ther 2000 7: 438–445

    Article  CAS  Google Scholar 

  37. Begleiter A, Leith MK, Doherty GP et al. Factors influencing the induction of DT-diaphorase activity by 1,2-dithiole-3-thione in human tumor cell lines Biochem Pharmacol 2001 61: 955–964

    Article  CAS  Google Scholar 

  38. Winski SL, Swann E, Hargreaves RHJ et al. Relationship between NAD(P)H: quinone oxidoreductase 1 (NQO1) levels in a series of stably transfected cell lines and susceptibility to antitumor quinones Biochem Pharmacol 2001 61: 1509–1516

    Article  CAS  Google Scholar 

  39. Spooner RA, Martin J, Friedlos F et al. In suicide gene therapy, the site of subcellular localization of the activating enzyme is more important than the rate at which it activates prodrug Cancer Gene Ther 2000 7: 1348–1356

    Article  CAS  Google Scholar 

  40. Sharp SY, Kelland LR, Valenti MR et al. Establishment of an isogenic human colon tumor model for NQO1 gene expression: application to investigate the role of DT-diaphorase in bioreductive drug activation in vitro and in vivo Mol Pharmacol 2000 58: 1146–1155

    Article  CAS  Google Scholar 

  41. Vile RG, Russell SJ, Lemoine NR . Cancer gene therapy: hard lessons and new courses Gene Ther 2000 7: 2–8

    Article  CAS  Google Scholar 

  42. Chen S, Knox R, Wu K et al. Molecular basis of the catalytic differences among DT-diaphorase of human, rat, and mouse J Biol Chem 1997 272: 1437–1439

    Article  CAS  Google Scholar 

  43. Wu K, Eng E, Knox R et al. Demonstration of the activation of prodrug CB 1954 using human DT-diaphorase mutant Q104Y-transfected MDA-MB-231 cells and mouse xenograft model Arch Biochem Biophys 2001 385: 203–208

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Division of Experimental Therapeutics, Ontario Cancer Institute, Toronto, Ontario, Canada

    Veet Misra, Henry J Klamut & AM Rauth

  2. Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada

    Veet Misra, Henry J Klamut & AM Rauth

Authors
  1. Veet Misra
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Henry J Klamut
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. AM Rauth
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to AM Rauth.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Misra, V., Klamut, H. & Rauth, A. Expression of the prodrug-activating enzyme DT-diaphorase via Ad5 delivery to human colon carcinoma cells in vitro. Cancer Gene Ther 9, 209–217 (2002). https://doi.org/10.1038/sj.cgt.7700430

Download citation

  • Received:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/sj.cgt.7700430

Keywords

This article is cited by

Search

Advanced search

Quick links