Expression of the Prodrug-Activating Enzyme DT-Diaphorase Via Ad5

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

dentification of drugs that are selectively toxic to have been suggested as a means to target both cycling and Imalignant cells has been a long-term goal of chemo- quiescent tumor cells.7 For example, bioreduction of the therapy as a mode of cancer treatment.1–3 One approach is prodrug CB1954 by an E. coli nitroreductase to form an to utilize nontoxic prodrugs that are selectively activated in alkylating agent has recently given promising results in solid tumors as a consequence of microenvironmental experimental tumor models.8 differences between tumors and normal tissue (e.g., oxygen In the present study, we examined whether the widely level differences).4 Recently, gene therapy approaches have studied and clinically used drug mitomycin C (MMC) can be been have been developed in which prodrugs are used as a prodrug9 in conjunction with human DT- selectively activated following intratumor injection of viral diaphorase (DTD), which has the ability to activate MMC vectors able to express drug-activating enzymes.5,6 In in cell-free systems but whose role in cells as a key activating many cases, these ‘‘suicide genes’’ code for viral or enzyme is still controversial.10 DTD [NAD(P)H:quinone bacterial enzymes that activate novel prodrugs. Examples oxidoreductase (NQO1); Enzyme Commission no. 1.6.99.2] include the herpes simplex gene coding for a thymidine is a homodimeric, cytosolic flavoenzyme that acts on its kinase, which is 1000 times more efficient in phosphor- substrates by a concerted two-electron reduction process, ylating the antiviral nucleoside analogue ganciclovir, and avoiding an oxygen-sensitive free radical intermediate in the Escherichia coli gene coding for cytosine deaminase, producing the hydroquinone.11 The hydroquinone of MMC which converts 5-fluorocytosine to 5-fluorouracil, a undergoes chemical rearrangements, resulting in a bifunc- widely used chemotherapeutic agent clinically. Both these tional alkylating agent capable of crosslinking DNA.12 agents have been shown to exert significant bystander Elevated DTD activities have been reported in tumor relative effects,5,6 but their efficiency depends to a large extent on to normal tissue13 and in tumor cell lines.14 It has been the rate of target tumor cell growth. Prodrug systems in suggested that this difference be exploited using compounds which the active form of the drug is an alkylating agent that are DTD substrates.12 A high degree of heterogeneity in DTD enzymatic activities15 and protein levels16 has been observed both within and across normal and tumor tissue types. This is Received November 20, 2001. 17 Address correspondence and reprint requests to: Dr AM Rauth, Division due to factors such as DTD gene expression levels and of Experimental Therapeutics, Ontario Cancer Institute, 610 University a high-frequency C-to-T base change at nucleotide 609 Avenue, Toronto, Ontario M5G 2K9, Canada. of the DTD coding region. This base transition causes a E-mail: [email protected] proline-to-serine change in amino acid 187, resulting in DT-diaphorase expression via an Ad5 vector V Misra et al 210 impaired DTD catalytic activity and protein instability.15,18 Generation of replication-defective adenoviral vectors Individuals homozygous for this mutation exhibit little or no A modified Ad5 shuttle vector, pÁE1sp1 CMV, contain- DTD activity and heterozygotes have approximately half 18,19 ing a CMV promotor and a bovine growth hormone poly the DTD activity observed in wild-type cells. A signal inserted in the polylinker of pÁE1Sp1 (Micro- Homozygous mutants have a much reduced sensitivity 10,15 bix Biosystems) was used as a eukaryotic expression to MMC under aerobic exposure conditions in vitro. 20 vector. HindIII–XbaI fragments containing the human To reduce the problem of heterogeneity and to enhance DTD cDNA with a C or T nucleotide at position 609 were DTD activity in tumor cells, recombinant adenoviral 609C 609T isolated from pRc/CMV.DTD and pRc/CMV.DTD , vectors were constructed, which express DTD minigenes 21 respectively. The fragments were inserted into the cor- for both wild-type and mutant (C-to-T base change) responding restriction sites of pÁE1sp1Rc/CMV to generate DTD under the control of the cytomegalovirus (CMV) 609C 609T pÁE1sp1 .DTD and pÁE1sp1 .DTD . All plasmid promoter. These vectors were used to overexpress constructs were sequence-verified (Core Molecular Biology recombinant DTD in human colon cancer cells that are Facility, York University, Toronto, Canada). homozygous for either wild-type (HT-29, MMC-sensi- Ad5.DTD vectors were produced according to a mo- tive) or mutant DTD (BE, MMC-resistant). Assessments 22 dification of the method developed by McGrory et al. of DTD protein levels and enzymatic activities in relation 293T cells were cotransfected through the CaPO4 to MMC sensitivity, as determined by in vitro colony- 23 609C coprecipitation method using pÁE1sp1 .DTD or forming assays, demonstrated that the adenoviral vector 609T 24 pÁE1sp1 .DTD and pJM17 (Microbix Biosystems). expressing DTD/MMC combination represents a novel Individual plaques were isolated, expanded on 293T cells, and effective prodrug activation strategy for cancer 20 and recombinant adenovirus purified on CsCl gradients. treatment. Dialyzed viral stocks were stored at À708 in phosphate- buffered saline (PBS) glycerol.24 Ad5.DTD viral particle concentrations determined by Materials and methods 25 12 OD260 measurements were 5.5Â10 particles/mL for Chemicals and reagents Ad.5DTD609C and 4.4Â1012 particles/mL for Ad5DTD609T. Viral infectious titers were determined by plaque-forming All reagents used for the DTD enzymatic assay were 23 11 purchased from Sigma (St. Louis, MO). Materials used for unit (pfu) assay on 293T cells to be 1Â10 pfu/mL for both Ad5.DTD 609C and Ad5.DTD609T. Testing for wild- polymerase chain reaction, including AmpliTaq polymerase, 24 were obtained from Roche Molecular Systems (Branchburg, type Ad5 contamination using HeLa cells was negative. NJ). DTD monoclonal antibodies B771 and A180 were Polymerase chain reaction reactions on DNA isolated from supplied by Dr. David Ross (University of Colorado Health viral particles using DTD gene-specific primers or Ad5 fiber protein gene-specific primers confirmed the presence of Sciences Center, Denver, CO). Antihuman integrin V (MAB1960) was purchased from Chemicon (Temecula, both DTD (bp 261–721 of DTD cDNA) and fiber protein CA). MMC (catalog no. M4287), X-gal, potassium (bp 5861–6151 of the Ad5 genome from JM17) coding ferricyanide, and potassium ferrocyanide, used for histo- regions in the Ad5.DTD viruses, indicating successful chemical detection of -galactosidase ( -gal), were also recombination between vectors. purchased from Sigma. Infection of BE and HT29 cells with Ad5. -gal Cell culture BE and HT29 (1Â105 per well) were seeded in six-well tissue culture dishes (Nunc) in 3 mL of growth medium and 293T human embryonic kidney cells were obtained from incubated overnight in a humidified atmosphere containing Microbix Biosystems (Toronto, Ontario, Canada) for the 5% CO at 378C. Growth medium was gently aspirated from production of E1-defective recombinant adenoviral type 5 2 each well and overlaid with 0.5 mL of serum-free -MEM (Ad5). HeLa human cervical carcinoma cells, used to test containing recombinant Ad5.CMV. -gal under the control for wild-type recombinant virus, were purchased from of the CMV promoter 20 at multiplicities of infection (MOI) American Type Culture Collection (Manassas, VA). BE of 3, 10, 30, 60, or 100 pfu/cell. After 30 minutes at 378C, and HT29 human colon carcinoma cells were chosen on 3 mL of growth medium was added to each well. Histochem- the basis of previous work that showed a 6-fold ical detection of -gal–positive cells was performed difference in MMC sensitivity due to low or absent 26 15 48 hours later. Infection efficiency of Ad5. -gal was assess- DTD activation in BE cells, and that both cells can be ed by determining the mean fraction of -gal–positive cells in infected by Ad5 (unpublished data). BE and HT29 human five random 4.3 mm2 fields at 80 magnification. Positive cells were obtained from Dr. T Mulcahy (Wisconsin Â cells were scored independent of variations in color intensity. Clinical Cancer Centre, Madison, WI). All cell lines were maintained as monolayer cultures in growth medium consisting of -minimal essential medium supplemented Western blot analysis of HT29 and BE cells or BE cells with 10% fetal bovine serum (Cansera; Rexdale, Ontario, infected with Ad5.DTD Canada), growth medium, and maintained in a humidified BE or HT29 cells (50 cells/mm2) were seeded in 10-cm- 2 atmosphere containing 5% CO2 at 378C in 175 cm diameter tissue culture dishes (Nunc) containing 10 mL of polystyrene tissue culture flasks (Nunc, Life Technologies, growth medium and incubated overnight in a humidified Burlington, Ontario, Canada). atmosphere containing 5% CO2 at 378C. Cells were

Cancer Gene Therapy DT-diaphorase expression via an Ad5 vector V Misra et al 211 overlayed with 1 mL of serum-free -MEM containing an Statistics MOI of 10 or 100 of Ad5.DTD609C or Ad5.DTD609T for 30 One-way analysis of variance (ANOVA) was used to com- minutes. Ten milliliters of growth medium was added to each 609T pare mean DTD activities of uninfected and Ad5.DTD - dish and, after 48 hours, cells were harvested by scraping. infected HT29 cells. This procedure involves calculation of Total cell protein extracts were prepared, protein concen- (a) each activity variance from the overall mean; (b) trations determined, and Western blot and densitometry 609T variance within group (uninfected or Ad5.DTD -infected analyses carried out as described previously,19 with the cells) means; and (c) variance between group means. The exception that blots were probed for integrin expression V Student’s t test was used to compare the mean survival (using a 1:1000 dilution of antihuman integrin )asa 609C V of BE cells infected with Ad5.DTD at an MOI of 100 loading control and protease inhibitors were not used. for 48 hours and uninfected controls. ANOVA of regression was used to compare the MMC sensitivities of HT29 cells DTD assay infected with Ad5.DTD609C and uninfected controls. DTD enzyme activity was measured using the substrate, 2,6-dichlorophenolindophenol (DCPIP), as previously described.21 Results Infection of HT29 and BE cells with Ad5. -gal or Ad5.DTD DTD enzyme activity in Ad5.DTD-infected BE cells A single-base transition corresponding to nucleotide 609 of BE cells were infected at an MOI of 10 or 100 with DTD cDNA (C-to-T) has been shown to result in impaired Ad5.DTD609C and DTD enzyme activity determined 24, 48, DTD activity and decreased protein stability.18,19 HT29 cells and 72 hours postinfection. In one experiment, BE cells (homozygous for the wild-type DTD allele) displayed infected with Ad5.DTD609C at an MOI of 100 were tryp- mean±SEM (n=5) DTD activity of 1060±215 nmol/min/ sinized at the 48-hour time point, the cells were reseeded at mg protein, whereas BE cells (homozygous for the variant the 48-hour time point, and DTD activity was determined DTD allele) contained very low to undetectable DTD 48 hours later (96 hours after infection). activity (less than 20 nmol/min/mg protein). Consistent with the fact that the variant allele promotes decreased DTD Clonogenic survival of cells exposed to MMC protein stability,19 HT29 cells display Western blot reactivity BE or HT29 cells in 10-cm dishes were infected with whereas BE cells express very low to undetectable levels of Ad5.DTD609C or Ad5.DTD609T at various MOIs for 48 hours DTD protein. However, low levels of the 609T isoform can before MMC exposure. MMC stocks were prepared by be detected by increasing antibody incubation time and/or reconstitution with PBS at 1 mg/mL and stored for up to protein concentration (data not shown). 1 month at 48C in the dark. Stock concentrations were verified Preliminary studies of Ad5. -gal infection of HT29 by measuring absorbance at 363 nm ("=21,840 cm À 1 M À 1 ). and BE cells had demonstrated that BE cells were In experiments requiring MMC treatment, medium (with or transduced more efficiently than HT-29 cells (unpublished without virus) was replaced with 10 mL of growth medium data). The present work verified this observation (Fig 1). containing MMC at the desired concentrations and cells were At an MOI of 100, only 15% of HT29 cells stained incubated with drug for 1 hour in a humidified atmosphere positive for Ad5. -gal, whereas BE cells were infected at >90% efficiency. containing 5% CO2 at 378C. After MMC exposure, medium containing drug was decanted and residual drug was The levels of recombinant DTD protein expressed in removed by washing cells with 5 mL of PBS. human BE cells following Ad5 infection were assessed by Western blot analysis (Figs 2 and 3). Monolayers of BE cells Cells were harvested by trypsinization and pelleted by 609C centrifugation at 250Âg for 5 minutes at 48C. Pellets were were infected with Ad5.DTD and Western blot reactivity resuspended in 10 mL of growth medium and cell density was determined 48 hours postinfection (Fig 2A). As shown was assessed by 0.2% trypan blue (Life Technologies) in Figure 2B, densitometric analysis indicated that BE cells 4 3 infected at an MOI of 100 contained three to four times exclusion. Cell dilutions were prepared at 10 ,10, and 609C 102 cells/mL, and 1 mL of each dilution was plated in greater DTD protein levels than cells infected at an MOI 60Â15 mm dishes (Nunc) in duplicate (for drug-treated of 10 when normalized to integrin V loading controls. Similar levels of 609T isoform expression were observed cells) or in triplicate (untreated controls) and incubated as 609T before. Colonies were counted 12–14 days later after following Ad5.DTD infection of BE cells at 10 and 100 staining with 0.5% methylene blue (Sigma) dissolved in pfu/cell (Fig 3). The lower molecular weight band (0.5–1 equal parts ethanol to water. Plating efficiency was kDa smaller than wild-type DTD) observed in Figure 3A may be a result of proteolytic processing or chemical assessed by dividing the number of observed colonies by 19 the total number of cells plated. The mean±SEM (n=5) degradation of the 609T isoform and was included in the plating efficiencies of uninfected HT29 and BE control densitometric analysis. cells were 0.84±0.09 and 0.69±0.12, respectively. Fold sensitivities of BE cells infected at various MOIs of Ad5.DTD infection of human cells and enzyme activity Ad5.DTD609C to MMC were determined by comparing the To assess whether exogenous expression of the 609T DTD ratio of dose (g/mL) required to achieve 1log of cell kill isoform modulates the enzymatic activity of the wild-type relative to the initial plating efficiency to that of BE cells DTD allele, such as through a dominant negative effect,27 infected with Ad5.DTD609T. HT29 monolayer cultures were infected with Ad5.DTD609T

Cancer Gene Therapy DT-diaphorase expression via an Ad5 vector V Misra et al 212 which is similar to that observed in uninfected HT29 cells. These results demonstrate that infection of BE cells with Ad5.DTD609C at an MOI of 100 results in a linear increase in DTD activity over time and within the range of DTD activity in HT29 cells.

Clonogenic survival of Ad5.DTD-infected cells exposed to MMC HT29 and BE cells have been previously used to demonstrate the association of the variant DTD allele with MMC resistance.15,28 In accordance with previous acute 1-hour MMC exposure studies comparing drug sensitivity of human cells containing wild-type and variant DTD alleles,29 BE cells displayed a 4- to 6-fold resistance to MMC compared to HT29 cells at the 10% survival level when assessed for colony-forming ability 12–14 days after exposure (Fig 5A). To examine whether exogenous expression of wild-type DTD could sensitize resistant cells Figure 1 Infection efficiency of HT29 and BE cells by Ad5 -gal as a 609C function of MOI. The infection efficiency of Ad5 -gal was assessed to MMC, BE cells were infected with Ad5.DTD at an by determining the fraction of -gal–positive cells. HT29 cell results are open bars and BE cell results are gray bars. Each bar represents the mean±SD of the fraction of -gal–positive cells in five randomly chosen 4.3 mm2 fields. Results are from one repre- sentative experiment.

at an MOI of 100. DTD activity was determined 48 hours later and compared to uninfected controls. No significant differences were observed in DTD enzymatic activities in Ad.DTD609T-infected HT29 cells and uninfected controls (P>0.1, one-way ANOVA, data not shown). The ability of Ad5.DTD609C to express functional exogenous DTD was tested by measuring DTD activities in BE cells at different times postinfection. In particular, this kinetic study was used to assess whether sufficient exogenous DTD activity can be achieved in human cells for in vitro MMC sensitization. 106 BE cells were infected with Ad5.DTD609C at an MOI of 10 or 100 and DTD activities were assessed at various time points postinfection. As shown in Figure 4, BE cells infected at 100 MOI with Ad5.DTD609C displayed a linear increase in DTD enzyme activity up to 72 hours postinfection. Activity peaked at around 2400 nmol of DCPIP reduced/min/mg protein. However, beyond 72 hours, a significant proportion of cells began to lift (monolayers reached confluency at 48 hours postinfection) and DTD activity declined. In one experiment, BE cells infected with Ad5.DTD609C at an MOI of 100 were trypsinized and reseeded at a 1:2 dilution and activity was determined 48 hours later (96 hours postinfection). Enzyme activity at this point remained high (around 2000 nmol of DCPIP reduced/min/mg protein), indicating that Figure 2 Expression of wild-type DTD in BE cells 48 hours after exogenous DTD continued to be expressed after trypsiniza- 609C tion and plating (data not shown). infection with Ad5.DTD . A: Western blot analysis of cell However, a slower rate of increase in DTD activity was extracts probed with DTD monoclonal antibodies and v integrin as a protein loading control. Lane 1: uninfected cells. Lanes 2 and observed in cells infected at an MOI of 10 and activity 3: Ad5DTD609C (wild type)-infected cells at an MOI of 10 and 100, plateaued after 24 hours at around 250 nmol of DCPIP respectively. B: Densitometry analysis of DTD band intensities

reduced/min/mg protein. At 48 hours postinfection, cells normalized for integrin v levels. Values represent mean±SD of infected with an MOI of 100 displayed DTD activities of five densitometric readings for each lane standardized to uninfected around 1320±70 nmol of DCPIP reduced/min/mg protein, cells.

Cancer Gene Therapy DT-diaphorase expression via an Ad5 vector V Misra et al 213 indicated that HT29 cells infected with Ad5.DTD609C at an MOI of 100 were significantly more sensitive to MMC than uninfected controls (P<.01). These results show that Ad5.DTD609C can be used to sensitize BE cells to MMC. At an MOI of 100, DTD activities are similar to HT29 cells after 48 hours. Ad5.DTD609C infection also sensitizes HT29 cells to MMC despite the low infection efficiency compared to BE cells. To assess the effect that different levels of exogenous wild-type DTD expression would have on MMC sensitivity, BE cells were infected with Ad5.DTD609C at MOIs ranging from 3 to 100 (Fig 6A). DTD activities 48 hours postinfection increased linearly with MOI (Fig 6B). As before, BE cells infected with Ad5.DTD609C at an MOI of 100 displayed reduced plating efficiency (mean PE±SEM was 0.42±0.06) compared to uninfected controls (mean plating efficiency 0.8±0.1). Also, as shown in Figure 6A, drug sensitivity was dependent on MOI. Similar acute MMC toxicities were observed between BE cells infected with Ad5.DTD609C at an MOI of 60 and uninfected HT29 cells. However, BE cells displayed less than 50% of the DTD activity (mean±SEM was 495±13 nmol of DCPIP Figure 3 Expression of mutant DTD in BE cells 48 hours after reduced/min/mg protein; Fig 6B) of HT29 cells infection with Ad5.DTD609T. A: Western blot analysis of cell extracts (1060±215 nmol of DCPIP reduced/min/mg protein) probed with DTD monoclonal antibodies and integrin as a protein measured previously. v 609C loading control. Lanes 1 and 2: Ad5.DTD609T -infected cells at an In these experiments, BE cells infected with Ad5.DTD

MOI of 10 and 100 (respectively normalized for) integrin v levels. at an MOI of 100 exhibited four to five times higher Values represent mean±SD of five densitometric reading for each sensitivity to MMC relative to cells treated at an MOI of lane standardized to cells infected at an MOI of 10. 60 in terms of the ratio of doses corresponding to 1log cell kill from initial PE. Because DTD activity determinations in HT29 cells can vary by as much as 2-fold from one MOI of 100 and MMC exposure was carried out 48 hours experiment to another, it is possible that MOIs greater than postinfection. As shown in Figure 5B, BE cells infected with Ad5.DTD609C at an MOI of 100 were 8-fold more sensitive to MMC compared to cells infected with Ad5.DTD609T,as determined by the ratio of MMC doses required to achieve 1log cell kill from the initial PE. However, untreated BE cells infected with Ad5.DTD609C had a reduced plating efficiency compared to uninfected controls (mean PE±SEM, n=3, 0.45±0.11 vs 0.69±0.12, respectively, P<.05, Student’s t test) indicating toxicity due to expression of exogenous wild-type DTD. This toxicity is not due to the vector itself because untreated BE cells infected with Ad5.DTD609T at an MOI of 100 had a plating efficiency of 0.81±0.14 (Fig 5B), which is in the range of uninfected control cells. BE cells infected with Ad5.DTD609T exhibited no major increases in MMC sensitivity, consistent with previous observations of impaired catalytic activity and protein instability of the DTD 609T isoform.18,19 As shown in Figure 5C, exogenous expression of DTD was confirmed in cells infected with Ad5.DTD609C by assessing DTD activities in cells that were infected in parallel with the MMC sensitivity studies described above. HT29 cells infected with Ad5.DTD609C were 2-fold more sensitive to MMC than uninfected controls (Fig 5B), although no significant increase in DTD activity was seen Figure 4 DTD enzyme activity in BE cells infected with Ad5.DTD609C compared to uninfected cells (Fig 5C). There was no effect as a function of time after infection. Cells were mock-infected of the virus on the plating efficiency of control cells, which (triangles) or infected at MOIs of 10 (circles) or 100 (squares). was in the range of uninfected cells. ANOVA of regression Values represent the mean±SEM of three independent experiments.

Cancer Gene Therapy DT-diaphorase expression via an Ad5 vector V Misra et al 214

Figure 5 Clonogenic survival of cells exposed to increasing concentrations of MMC and DTD activity 48 hours after viral infection. A: Plating efficiency of uninfected HT29 (squares) and BE (circles) cells exposed to MMC. B: Plating efficiency of HT29 cells infected with Ad5.DTD609C (circles) and BE cells infected with Ad5.DTD609C (squares) or Ad5.DTD609T (triangles) versus MMC concentration. All viral infections were at 100 MOI. Arrow on points indicates a less-than-this-value plating efficiency. The dashed curves are taken from panel A and represent the uninfected survival response of HT29 and BE cells to MMC. C: DTD activities of cells in panel B at the time of MMC exposure. The activity of untransfected HT29 cells was 1060±215 nmol DCPIP reduced/min/mg protein. Each value in panels A, B, and C represents the mean±SEM of three to five independent experiments.

Cancer Gene Therapy DT-diaphorase expression via an Ad5 vector V Misra et al 215 Ad5.DTD609C at MOIs of 10–100 pfu/cell provides for a continual increase in the sensitization to MMC.

Discussion

Bioreductive enzyme–prodrug therapy involves the use of compounds that undergo metabolic reduction to cytotoxic metabolites.4 One barrier to this approach is insufficient levels of activating enzymes in tumor tissue.16 DTD is a bioreductive enzyme that can activate a variety of prodrugs, including quinone-based drugs, to DNA alkylating agents. DTD activity has been reported to be elevated in a range of tumor types30 such as in human non–small cell lung cancer,13,14 breast, liver,31 and colon tumors relative to surrounding normal tissue. It has been proposed that various prodrugs that are substrates for DTD could be useful for the treatment of tumors with high DTD activity.32 However, not all tumors express high levels of DTD. Heterogeneity in DTD expression has been reported in the National Cancer Institute (USA) human tumor cell line panel, which includes cell lines derived from leukemic, lung, colon, and breast tumors.30 This conclusion was based on both DTD mRNA expression levels and Western blot analysis. Heterogeneity was observed both within and across tumor cell types, but a correlation was observed between DTD expression and MMC sensitivity in this panel. DTD enzymatic activity has also been shown to be comparable in breast and lung tumor samples relative to corresponding normal tissue.31,32 Further complicating the heterogeneous nature of DTD expression across tissue types is the role of a single-base change, a C-to-T transition in nucleotide 609 of DTD cDNA, conferring a predicted proline-to-serine change in amino acid 187. This base change is frequent in the population33 and has been associated with reduced catalytic activity, protein instability, and MMC resistance.15,18,21 One way to address this problem is to introduce a bioreductive enzyme like DTD into tumor cells using gene transfer techniques.34 Gene therapy strategies involving the delivery of activating enzymes to sensitize human tumors to prodrugs have been explored using nonmammalian enzymes, such as herpes simplex virus thymidine kinase35 and E. coli cytosine deaminase36 in combination with the nucleoside analogues ganciclovir and 5-fluorocytosine, Figure 6 Clonogenic survival of BE cells after infection with Ad5.DTD609C (wild-type DTD) followed by exposure to MMC and respectively. In the current study, human DTD coding cellular DTD activity. A: Cells were infected at MOIs of 3 sequences were delivered using a first generation replica- (diamonds), 10 (inverted triangles), 30 (triangles), 60 (circles), tion-defective adenoviral vector to increase DTD activity or 100 (squares). Forty eight hours later, infected cells were and sensitize tumor cells to MMC. exposed for 1 hour to different concentrations of MMC and their BE cells express low to undetectable levels of DTD plating efficiency determined. The dashed curves are from Figure activity and protein15,21 and are good hosts for Ad5 5, panel A showing the sensitivity of uninfected HT29 and BE infection (Fig 1). Infection of BE cells with Ad5.DTD609C cells. Arrow on point indicates a less-than-this-value plating (wt DTD) or Ad5.DTD609T (mt DTD) produced DTD efficiency. B: DTD activity of cell cultures 48 hours after infection protein levels that increased with MOI (Figs 2 and 3) and versus their MOI. All values are the mean ±SEM of three DTD activities consistent with these protein levels (and the independent experiments. known activities) of wild-type and mutant DTD (Fig 4). HT29 cells infected with wild-type DTD did not show a significant increase in DTD activity, probably due to the 60 are required to achieve equivalent MMC toxicity in BE lower infection efficiency of these cells by Ad5 (Fig 1). cells. These results show that transduction of BE cells with Conversely, they did not show any loss of activity when

Cancer Gene Therapy DT-diaphorase expression via an Ad5 vector V Misra et al 216 infected with mutant DTD. The possibility that the mutant possibility of a MMC bystander effect is under study allele might have a dominant negative effect27 on wild-type currently. DTD activity has been eliminated by recent work showing DTD activity increased linearly in BE cells as a func- that a 5-fold excess of the mutant isoform over the wild- tion of Ad5.DTD609C dose up to an MOI of 100 pfu/cell. type form has no effect on DTD activity.37 The linear (Fig 6B). Also, sensitization of BE cells was shown to increase in DTD activity with time following infection at an be dose-dependent because a series of Ad5.DTD609C MOI of 100 (Fig 4) is consistent with previous work titrations (Fig 6A) at MOIs of 3, 10, 30, 60, or 100 demonstrating that wild-type DTD protein has a half-life resulted in 0.7-, 1.0-, 1.1-, 3-, or 8-fold increases in that is greater than 24 hours.18 The leveling-off of DTD MMC sensitivity. It is important to point out that enzyme activities after 24 hours in BE cells infected with increased cell killing with increasing MOI may be a Ad5.DTD wild type at an MOI of 10 may reflect a balance function of both increased DTD expression per cell as between the rates of recombinant DTD production and well as an increasing fraction of cells transfected (Fig 1). degradation. In BE cells infected at an MOI of 100, the Some caution must also be exercised in comparing HT29 degradation pathway may be overwhelmed by the produc- and BE cell responses to MMC. Though differences in tion pathway, resulting in a continuous increase in DTD. the MMC sensitivity appear to be mediated to a large Expression of exogenous wild-type DTD in BE cells likely degree by DTD levels, these cells are not isogenic and extends beyond 72 hours postinfection. Trypsinization of may express different levels of other activating or repair cells 48 hours postinfection at an MOI of 100, dilution, enzymes.40 and replating, to avoid cell confluence and lifting beyond The present results support further studies in an in vivo 72 hours (data not shown), demonstrated high DTD activity model. However, the results of a recent independent study 96 hours postinfection (2000 nmol/min/mg protein). using a BE cell stably transfected with human DTD were In MMC sensitization studies, BE cells were treated with disappointing because wild-type DTD expressing BE cells MMC 48 hours postinfection with wild-type DTD at an growing as a solid tumor in vivo were not sensitized to MMC MOI of 100. Of the two MOIs and various times relative to control BE cells.40 However, the levels of DTD postinfection of Ad5.DTD609C tested, an MOI of 100 for activity were so low in these tumors that the interpretation of 48 hours was chosen because it results in maximal DTD the data was difficult. This reduction in DTD activity may activity and optimal cell viability (Fig 4). Untreated BE cells reflect extinction or clonal variations in DTD expression were 4- to 6-fold more resistant to MMC than HT29 cells with tumor growth. Clearly, the ability to express high levels (Fig 5A). However, treatment with Ad5.DTD609C at an MOI of DTD in vivo will be crucial to the success of this of 100 resulted in an 8-fold increase in MMC sensitivity therapeutic strategy. The use of DTD isoforms that are more relative to BE cells infected with Ad5.DTD609T at an MOI of catalytically efficient than wild-type human DTD,41,42 and 100 when assessed by clonogenic survival 12–14 days the use of drugs that exhibit a substantial bystander effect43 subsequent to a 1-hour exposure to MMC (Fig 5B). could also significantly improve the therapeutic potential of Increased sensitivity to MMC can be attributed to expression this strategy. of exogenous wild-type DTD, and not to the Ad5 vector In summary, first generation recombinant Ad5 vectors itself because transduction of BE cells with Ad5.DTD609C have been constructed, which express both the wild-type resulted in only a 33% reduction in plating efficiency and and mutant isoforms of DTD. Treatment of MMC-resistant treatment with Ad5.DTD609T had no effect on BE cell BE cells with the wild-type isoform increased MMC survival. No evidence for resistant BE cells was seen sensitivity 8-fold, raising the possibility that gene therapy down to 4–5logs of cell kill, suggesting that all cells were strategies, which sensitize tumors to drugs bioreductively infected. activated by DTD, can be developed. Interestingly, HT29 cells infected with Ad5.DTD609C at 100 MOI were 2-fold more sensitive to MMC compared to uninfected controls (Fig 5B) despite exhibiting similar DTD References activities (Fig 5C). Recent work, using cell lines expressing different levels of DTD, has shown that bioreductive 1. Cox PJ, Farmer PB. Towards selectivity? Approaches to the activation of the quinone-containing compound RH1 has design of new anti-tumour agents — I and II. Cancer Treat both a threshold and an apparent saturation level as a func- Rev. 1977;4:47–63, 119–134. tion of enzyme activity.38 In addition, it has been shown that 2. Elion GB. 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