Expression of the prodrug-activating enzyme DT-diaphorase via Ad5 delivery to human colon carcinoma cells in vitro Veet Misra, Henry J Klamut, and AM Rauth Division of Experimental Therapeutics, Ontario Cancer Institute, Toronto, Ontario, Canada; and Department of Medical Biophysics, University of Toronto, Toronto, Ontario, Canada. 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  - 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. Cancer Gene Therapy (2002) 9, 209 – 217 DOI: 10.1038 / sj / cgt / 7700430 Keywords: adenovirus ( Ad5 ); DT - diaphorase ( DTD ); mitomycin C ( MMC ); HT - 29; BE; clonal assay I dentification of drugs that are selectively toxic to malignant cells has been a long-term goal of chemo- therapy as a mode of cancer treatment. 1–3 One approach is to utilize nontoxic prodrugs that are selectively activated in solid tumors as a consequence of microenvironmental differences between tumors and normal tissue ( e.g., oxygen level differences ). 4 Recently, gene therapy approaches have been have been developed in which prodrugs are selectively activated following intratumor injection of viral vectors able to express drug - activating enzymes. 5,6 In many cases, these ‘‘suicide genes’’ code for viral or bacterial enzymes that activate novel prodrugs. Examples include the herpes simplex gene coding for a thymidine kinase, which is 1000 times more efficient in phosphor- ylating the antiviral nucleoside analogue ganciclovir, and the Escherichia coli gene coding for cytosine deaminase, which converts 5 - fluorocytosine to 5 - fluorouracil, a widely used chemotherapeutic agent clinically. Both these agents have been shown to exert significant bystander effects, 5,6 but their efficiency depends to a large extent on the rate of target tumor cell growth. Prodrug systems in which the active form of the drug is an alkylating agent have been suggested as a means to target both cycling and quiescent tumor cells. 7 For example, bioreduction of the prodrug CB1954 by an E. coli nitroreductase to form an alkylating agent has recently given promising results in experimental tumor models. 8 In the present study, we examined whether the widely studied and clinically used drug mitomycin C ( MMC ) can be used as a prodrug 9 in conjunction with human DT- diaphorase (DTD), which has the ability to activate MMC in cell - free systems but whose role in cells as a key activating enzyme is still controversial. 10 DTD [NAD(P)H:quinone oxidoreductase ( NQO1 ); Enzyme Commission no. 1.6.99.2 ] is a homodimeric, cytosolic flavoenzyme that acts on its substrates by a concerted two-electron reduction process, avoiding an oxygen - sensitive free radical intermediate in producing the hydroquinone. 11 The hydroquinone of MMC undergoes chemical rearrangements, resulting in a bifunc- tional alkylating agent capable of crosslinking DNA. 12 Elevated DTD activities have been reported in tumor relative to normal tissue 13 and in tumor cell lines. 14 It has been suggested that this difference be exploited using compounds that are DTD substrates. 12 A high degree of heterogeneity in DTD enzymatic activities 15 and protein levels 16 has been observed both within and across normal and tumor tissue types. This is due to factors such as DTD gene expression levels 17 and a high-frequency C-to-T base change at nucleotide 609 of the DTD coding region. This base transition causes a proline-to-serine change in amino acid 187, resulting in Received November 20, 2001. Address correspondence and reprint requests to: Dr AM Rauth, Division of Experimental Therapeutics, Ontario Cancer Institute, 610 University Avenue, Toronto, Ontario M5G 2K9, Canada. E- mail: rauth@uhnres.utronto.ca Cancer Gene Therapy (2002) 9, 209–217 D 2002 Nature Publishing Group All rights reserved 0929-1903 / 02 $25.00 www.nature.com/cgt