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Escherichia coli nitroreductase plus CB1954 enhances the effect of radiotherapy in vitro and in vivo

Abstract

Escherichia coli nitroreductase (NTR) converts the prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) into a bifunctional alkylating agent that causes DNA crosslinks. In this study, the ability of NTR to enhance the combined effects of CB1954 and radiation has been tested in vitro and in vivo. Stably transduced ovarian cancer cells (SKOV3-NTR) that are sensitive to CB1954 (IC50=0.35 μM) demonstrated enhanced cytotoxicity when treated with CB1954 and single-fraction irradiation. The NTR–CB1954 system mediated a bystander effect in combination with radiation on transfer of conditioned medium from SKOV3-NTR, but not SKOV3, cells to SW480 target cells. The ability of CB1954 to enhance radiation-induced cytotoxicity in SKOV3-NTR (but not SKOV3) cells was also demonstrated by fluorescence-activated cell sorting (FACS) with dual staining for propidium iodide/fluorescein diacetate, 4′,6-diamidino-2-phenylindole dichloride staining of apoptotic cells and measurement of double-stranded DNA breaks by FACS and confocal microscopy for γH2AX foci. Adenoviral delivery of NTR, under constitutive cytomegalovirus or tissue-specific CTP1 promoters, increased the in vitro cytotoxicity of CB1954 plus radiation in MTT and clonogenic assays. Finally, adenoviral delivery of NTR plus CB1954 enhanced the effect of fractionated radiotherapy (12 Gy in four fractions) in SW480 xenograft tumours in nude mice.

Introduction

The results of early clinical trials of cancer gene therapy have demonstrated only modest evidence of efficacy.1 Although most of these studies have been conducted in patients with relapsed and treatment-resistant disease, the lack of a significant therapeutic breakthrough in patients with solid cancers and the inevitable occurrence of serious treatment-related toxicities have dented the credibility of cancer gene therapy.2 These findings have contributed to a shift in the perception of the potential clinical use of cancer gene therapy towards a view that, for the foreseeable future at least, it is most likely to find a role in combination with the established antitumour therapies such as surgery, radiotherapy and chemotherapy rather than as a stand-alone treatment. As a result, there is significant interest in strategies that seek to use gene transfer to enhance the effect of ionizing radiation and/or cytotoxic chemotherapy.

Radiotherapy is an extremely potent modality, offering the prospect of cure for a range of tumours including head and neck, lung, gastrointestinal and gynaecological cancers (either alone or in combination with cytotoxic chemotherapy). In addition, in patients with microscopic residual disease after surgery, adjuvant radiotherapy can secure local control and improve survival. However, radiotherapy delivery also causes normal tissue damage and this effectively imposes a ceiling on the dose of radiation that can safely be delivered to a tumour. The delivery of radiosensitizing agents has been seen as a means of circumventing this limitation, but most clinically useful agents cause significant sensitization of normal tissues to radiation.3

Gene therapy offers the prospect of selective vector-mediated generation of a radiosensitizing agent from an innocuous prodrug within tumour cells. This has the potential advantages of limiting sensitization in critical normal structures and also allowing (limited) diffusion of an activated agent to adjacent untransduced tumour cells to mediate a bystander effect.

The prodrug CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide) is a weak monofunctional alkylating agent which has activity when used alone against xenograft rat Walker 256 tumours.4 It entered limited clinical trial in 1970 but no antitumour activity was seen in humans. It was subsequently shown that CB1954 was reduced by the rat nitroreductase (NTR) enzyme DT diaphorase to form a potent bifunctional alkylating agent that crosslinked DNA and killed noncycling cells. However, the human and mouse forms of DT diaphorase are deficient in this reaction.5 The active species generated by the rat enzyme is up to 100 000 times more cytotoxic than the prodrug.6 An NTR enzyme from the bacteria Escherichia coli was found to perform the same reduction 60- to 100-fold faster than the rat DT diaphorase.7 CB1954 and its active metabolite have the potential to sensitize cells to the effects of ionizing radiation.8 This effect is mediated through the formation of intra- and interstrand DNA links that render chromosomes more susceptible to double-strand breaks (the most toxic effect of ionizing radiation).9 Adenovirus-mediated expression of the NTR enzyme under the control of tissue- or tumour-specific promoters offers a means of selectively generating a radiosensitizing agent within a tumour mass. The potential for a bystander effect associated with such virally directed enzyme prodrug therapy (VDEPT) strategies offers the prospect of also sensitizing adjacent untransduced malignant (and normal) cells to irradiation.3

In these studies, we seek to evaluate the ability of replication-defective adenovirus (E1 and E3 deleted) containing the E. coli NTR gene under the control of either a constitutively active viral cytomegalovirus or a tissue-specific (CTP1)10 promoter to enhance the cytotoxic effect of ionizing radiation against colorectal cancer cells both in vitro and in vivo.

Results

CB1954 combined with radiation increases cytotoxicity in NTR-expressing cells

The relative sensitivities of SKOV3 and SKOV3-NTR cells to treatment with CB1954 were defined using MTT assays. There was a significant sensitization of SKOV3-NTR cells to the effects of CB1954 with a greater than 200-fold reduction in the IC50 relative to wild-type SKOV3 cells (IC50=0.35 versus >100 μM, respectively; Figure 1a). By mixing the two cell lines in various ratios, it was possible to demonstrate the presence of a significant bystander component to cell killing (Figure 1b). Essentially, the IC50 values were identical for the 100:0, 90:10 and 75:25 ratios of SKOV3-NTR:SKOV3 cells, and the value for the 50:50 ratio was significantly reduced in comparison with the 0:100 mixture.

Figure 1
figure1

(a) SKOV3 cells stably transduced with NTR show more than 200-fold sensitivity to CB1954 prodrug when compared with parental cells. Parental SKOV3 or SKOV3-NTR cells were incubated with CB1954 at concentrations of 1 nM–500 μM and survival was assayed by MTT at 144 h. The relative IC50 values for SKOV3 and SKOV3-NTR were >100 and 0.35 μM, respectively. (b) SKOV3-NTR cells are able to mediate bystander killing of SKOV3 cells in the presence of CB1954. Mixtures of the two cell lines in ratios ranging from 100:0 to 0:100 SKOV3-NTR:SKOV3 were incubated with CB1954 at concentrations of 1 nM–100 μM and survival was measured at 144 h by MTT assay. Significant sensitization of SKOV3 cells was seen even at the 50:50 ratio at drug doses of <10 μM. (c) SKOV3-NTR cells show enhanced cytotoxicity in response to CB1954 plus single-fraction irradiation. At CB1954 concentrations of 1 and 10 nM, there was evidence of radiation dose-dependent cytotoxicity in both SKOV3-NTR and SKOV3 cells. At CB1954 concentrations of 100 nM and greater, there was increased cytotoxicity in the SKOV3-NTR cells that was both radiation- and drug-dose-dependent. Data are representative of three independent experiments. NTR, nitroreductase.

Thereafter, the effect of CB1954 on the response of SKOV3-NTR and SKOV3 cells to radiation was tested. Cells were treated with CB1954 at various concentrations for 16 h before delivery of single-fraction radiotherapy and survival was measured by MTT assay. For SKOV3-NTR cells, there was evidence of radiation dose-dependent cytotoxicity at CB1954 concentrations of <0.01 μM. Between 0.01 and 1 μM, there was evidence of increased cytotoxicity with the combination of radiation and CB1954, which was both radiation and drug dose dependent (Figure 1c). There was a horizontal shift in the curves for cells expressing NTR (as seen in Figure 1a), but this was not seen on addition of radiation. Therefore, it appears that the radiation decreases survival independently of the drug CB1954. Beyond 1 μM, SKOV3-NTR cells showed survival rates of 5%, irrespective of radiation dose. For the SKOV3 cells, there was only evidence of enhancement of radiation-induced cytotoxicity at CB1954 concentrations of 50 μM and above.

The effect of combining CB1954 and single-fraction radiation on cytotoxicity of SKOV3 and SKOV3-NTR cells was also measured by dual staining with propidium iodide (PI) and fluoroscein diacetate (FDA) for live (PI−/FDA+) and dead (PI+/FDA−) cells. For the SKOV3 cells, there was evidence of increased PI+/FDA− staining after 3 Gy of radiation but there was no enhancement of cytotoxicity in the presence of 0.01 or 0.1 μM CB1954. In contrast, in SKOV3-NTR cells, the addition of CB1954 caused an increase in the cell killing than that seen for 3 Gy alone. Therefore, for the cells irradiated in the absence of CB1954, the PI+/FDA− fraction was 22.2%, but for those irradiated with 0.01 or 0.1 μM CB1954, the values were 30.2 and 34.4%, respectively (Table 1).

Table 1 Effect of CB1954 and radiotherapy as cell death measured by PI/FDA staining

CB1954 plus radiation enhances apoptosis in NTR-expressing cells

SKOV3 or SKOV3-NTR cells were incubated overnight with CB1954 and then irradiated to a dose of 5 Gy (or mock-irradiated). Cells were stained 72 h after irradiation with 4′,6-diamidino-2-phenylindole dichloride and scored for the presence of apoptosis at confocal microscopy of four fields. Cells were scored as apoptotic if they demonstrated the typical microscopic features of DNA condensation and fragmentation. In the SKOV3 cells, there was no evidence of increased apoptosis with either CB1954 or radiation exposure (P>0.05). For the SKOV3-NTR cells, however, there was evidence of a drug-dose-dependent increase in apoptotic staining between 0.5 and 5 μM. After combining 5 Gy and CB1954, the percentage of SKOV3-NTR cells staining for apoptosis was significantly increased at CB1954 concentrations of 0.1 μM and above (P<0.05), even though radiation alone in the absence of CB1954 did not increase the level of apoptosis in these cells (Figure 2).

Figure 2
figure2

CB1954 plus radiation causes enhanced apoptosis in cells that express NTR. SKOV3 or SKOV3-NTR cells were exposed to CB1954 (0, 0.05, 0.1, 0.5, 1 and 5 μM) overnight and irradiated (0 or 5 Gy). Cells were stained 72 h after irradiation with 4′,6-diamidino-2-phenylindole dichloride and scored for the presence of apoptosis at confocal microscopy. *P<0.05; **P<0.01; ***P<0.001. Data are representative of three independent experiments. NTR, nitroreductase.

CB1954 and radiation increases γH2AX focus formation in the presence of NTR

Double-stranded DNA breaks were detected by measuring the presence of γH2AX foci in SKOV3 and SKOV3-NTR cells that were exposed to CB1954 and/or radiation. In fluorescence-activated cell sorting (FACS) analysis using primary anti-phosphohistone γH2AX antibody and secondary fluorescein isothiocyanate-conjugated donkey anti-mouse antibody, there was clear evidence of a radiation-dose response in fluorescein isothiocyanate staining after single-fraction radiation doses between 0 and 10 Gy (Figure 3a). Thereafter, it was shown that addition of CB1954 to single-fraction (3 Gy) irradiation caused an increase in γH2AX focus formation in SKOV3-NTR but not in SKOV3 cells (Figures 3b and c). This phenomenon was also assessed using confocal microscopy, which also demonstrated increased γH2AX focus formation in NTR-expressing cells exposed to CB1954 (1 μM) and radiation (1 Gy) (Figure 3d).

Figure 3
figure3

CB1954 plus irradiation causes increased double-stranded DNA breaks in cells that express NTR. (a) SKOV3-NTR cells were irradiated (0, 1, 3, 5 and 10 Gy). (b) SKOV3 cells were exposed to CB1954 (0, 0.1, 0.5 and 1 μM) and irradiated (0 or 3 Gy). (c) SKOV3-NTR cells were exposed to CB1954 (0, 0.1, 0.5 and 1 μM) and irradiated (0 or 3 Gy). In each case, FACS analysis was performed with primary anti-phosphohistone γH2AX antibody and secondary FITC-conjugated donkey anti-mouse antibody. In panels b and c, for clarity of data presentation, the 0 Gy controls are not shown. In all cases, there was no evidence of positive staining for γH2AX in these 0 Gy controls. Data are representative of at least three independent experiments. (d) Confocal micrographs of SKOV3 and SKOV3-NTR cells showing increased γH2AX focus formation in NTR-expressing cells exposed to CB1954 (1 μM) and radiation (1 Gy). FACS, fluorescence-activated cell sorting; FITC, fluorescein isothiocyanate; NTR, nitroreductase.

A transferable activated metabolite of CB1954 sensitizes bystander cells to radiation

To investigate whether, or not, the NTR–CB1954 system is capable of mediating a bystander effect in combination with radiation, conditioned media from SKOV3 or SKOV3-NTR cells that had been incubated with CB1954 at concentrations of 0, 5, 12.5 and 25 μM overnight was added to SW480 cells in 96-well plates. These plates were subsequently irradiated to doses of 0, 3, 6 or 9 Gy at 16 h after addition of the conditioned media. As shown in Figure 4a, transfer of conditioned medium from SKOV3 cells did not alter the effect of radiation against SW480 cells. In contrast, the transfer of conditioned medium from SKOV3-NTR cells significantly enhanced the effect of single-fraction radiation in a drug- and radiation-dose-dependent manner (Figure 4b).

Figure 4
figure4

A transferable metabolite of CB1954 is capable of sensitizing SW480 target cells to single-fraction irradiation. Conditioned medium from (a) SKOV3 cells or (b) SKOV3-NTR cells that had been incubated overnight with CB1954 (0, 5, 12.5 and 25 μM) was added to SW480 cells that were then irradiated (0, 3, 6 or 9 Gy). Data are representative of two independent experiments. The SW480 cells irradiated in medium from SKOV3 cells demonstrated radiation dose-dependent cell kill, whereas those irradiated in medium from SKOV3-NTR cells demonstrated radiation- and drug-dose-dependent cytotoxicity. These data demonstrate that conditioned medium from cells that express NTR contains an agent that sensitizes to the effects of radiation. NTR, nitroreductase.

Adenoviral delivery of NTR plus CB1954 enhances the effect of single-fraction and fractionated radiation in vitro

Before conducting the studies of combinations of VDEPT and single-fraction irradiation, we characterized the sensitivity of HCT116 and SW480 cells to single-fraction radiation doses between 1 and 10 Gy per day and to fractionated radiation doses between 1 and 3 Gy per day for 3 days (data not shown). From these experiments, single-fraction radiation doses of 1, 2 and 3 Gy and fractionated radiation doses of 3, 4.5 and 6 Gy in three daily fractions of 1, 1.5 and 2 Gy, respectively, were selected for subsequent experiments. Thereafter, we assessed the effect of Ad.CMV-NTR at multiplicity of infection of 10 PFU (plaque-forming unit) in combination with radiation (0 or 3 Gy) in the presence of CB1954 at doses between 1 and 500 μM. In both SW480 and HCT116 cell lines there was evidence of enhanced cell kill that was both radiation- and drug-dose dependent (Figure 5a). Thereafter, we demonstrated that the addition of VDEPT to single-fraction radiation reduced the formation of viable colonies as assessed by crystal violet staining (Figure 5b). This effect was also quantitated by formal clonogenic survival assay for single-fraction radiation for both SW480 and HCT116 cell lines, (Figures 6a and b). In particular, at a prodrug dose of 10 μM, there was significant improvement in the reduction of colonies after infection with Ad.CMV-NTR and irradiation in both the HCT116 and SW480 cell lines. In the case of the Ad.CTP1-NTR virus, the effect was seen more clearly in the SW480 cell line. At the higher prodrug dose of 50 μm, the effect of the addition of VDEPT to single-fraction irradiation was less clearly seen, at least in part because of the significant effect of the prodrug/VDEPT alone.

Figure 5
figure5

Adenovirus-mediated expression of NTR increases cytotoxicity of CB1954 plus radiation. (a) HCT116 and SW480 cells were infected with AdCMV-NTR at MOI of 10 PFU. Cells were exposed to CB1954 (1, 10, 50, 100 and 500 μM) 72 h later and irradiated (0 or 3 Gy) after another 24 h. Enhanced tumour cell kill was seen in both cell lines after irradiation to 3 Gy. (b) HCT116 cells were infected with virus (MOI 0 or 50 PFU), exposed to CB1954 (0, 10 and 50 μM) 72 h later and irradiated (0, 1, 2 Gy) after another 24 h. Cell survival was measured by direct staining with 0.2% (w/v) crystal violet in a 7% (v/v) solution of ethanol/PBS. Data are representative of three independent experiments. MOI, multiplicity of infection; NTR, nitroreductase; PBS, phosphate-buffered saline.

Figure 6
figure6

Adenovirus-mediated NTR expression enhances the cytotoxicity of CB1954 plus single-fraction and fractionated radiation. (a) HCT116 cells treated with AdCMV-NTR or AdCTP1-NTR (MOI 0 or 10 PFU), CB1954 (0, 10 and 50 μM) and single-fraction radiation (0, 1, 2 or 3 Gy). (b) SW480 cells treated with AdCMV-NTR or AdCTP1-NTR (MOI 0 or 10 PFU), CB1954 (0, 10 and 50 μM) and single-fraction radiation (0, 1, 2 or 3 Gy). (c) SW480 cells treated with AdCMV-NTR or AdCTP1-NTR (MOI 0 or 10 PFU), CB1954 (0 or 10 μM) and fractionated radiation (0, 3, 4.5 or 6 Gy in three equal fractions). Data are representative of at least three independent experiments. MOI, multiplicity of infection; NTR, nitroreductase.

The effect of fractionated irradiation to total doses of 3, 4.5 and 6 Gy in three daily fractions of 1, 1.5 and 2 Gy, respectively, was also assessed in SW480 cells. Cells were infected with Ad.CMV-NTR or Ad.CTP1-NTR at a multiplicity of infection of 10 PFU and treated with CB1954 at either 0 or 10 μM. For both the CMV- and CTP1-promoter-driven constructs, there was evidence of enhanced activity of the combination of VDEPT and radiation (Figure 6c).

Ad.CMV-NTR- and Ad.CTP1-NTR-mediated VDEPT enhance the effect of fractionated radiation in vivo

Initial in vivo studies were conducted to characterize the effect of fractionated radiotherapy at doses of 9 Gy in three fractions over 6 days, 12 Gy in four fractions over 8 days and 15 Gy in five fractions over 10 days on the growth of SW480 tumours (data not shown). These studies confirmed the efficacy of fractionated radiotherapy in this tumour model and allowed us to select 12 Gy in four fractions as an appropriate radiation dose for subsequent analysis of VDEPT plus radiation in vivo. The effect of fractionated radiotherapy at a dose of 12 Gy in four fractions over 8 days in conjunction with the various test treatments on the growth of SW480 tumour xenografts is presented in Figures 7a and b. These data demonstrate that both Ad.CMV-NTR (P<0.007) and Ad.CTP1-NTR (P<0.01) significantly enhance the effect of CB1954 in combination with fractionated radiotherapy when compared with the corresponding virus, fractionated radiotherapy and phosphate-buffered saline (PBS). There was no evidence of exacerbation of acute normal tissue (skin, subcutaneous) reactions within the radiation fields or increased systemic toxicity as assessed by weight change (data not shown).

Figure 7
figure7

SW480 xenograft tumours were grown in nude mice. Groups of eight mice were treated with a single injection of adenoviral vector (Ad.CMV-GFP, Ad.CMV-NTR or Ad.CTP1-NTR), radiation (0 or 12 Gy in four fractions) and intraperitoneal injections of either PBS or CB1954. (a) Survival curve using time taken to reach three times the initial tumour volume (Vo) as a surrogate marker of death. (b) Relative tumour volume with time after radiotherapy. Error bars represent standard error of the mean. Radiation alone caused significant growth delay compared with unirradiated control. Enhanced tumour growth delay was seen only in animals that received an NTR-expressing adenovirus plus CB1954. NTR, nitroreductase; PBS, phosphate-buffered saline.

Discussion

A number of preclinical studies have demonstrated that VDEPT approaches can sensitize tumour cells to the effects of ionizing radiation. Such studies have used both adenoviral and herpes simplex viral vectors.11, 12, 13, 14, 15, 16, 17, 18, 19, 20 The virus-prodrug systems that have been used have focussed largely on HSVtk and ganciclovir and/or E. coli cytosine deaminase/uracil phosphoribosyltransferase and 5-fluorocytosine. These systems have shown significant advantages in terms of both in vitro and in vivo cell killing. However, in each case, the activated prodrug is specifically active in S phase of the cell cycle at which time point cells are relatively resistant to the effects of ionizing radiation. In contrast, the E. coli NTR and CB1954 system does not show S phase dependence. On these grounds, we hypothesized that this system would represent a potent radiosensitizing gene therapy approach.

Initial experiments confirmed specific cytotoxicity of CB1954 against NTR-expressing SKOV3 cells in comparison to parental non-expressing cells. Further cell-mixing experiments demonstrated a bystander effect, although it must be remembered that in vitro experiments are able to achieve levels of homogeneity of admixture of expressing and non-expressing cells that are unlikely to reflect accurately the situation in vivo. Thereafter, we examined the effect of the NTR–CB1954 system in combination with radiation in both NTR-expressing and non-expressing cells. Increased cell death was seen by both colorimetric and FACS-based analysis when CB1954 was combined with irradiation in NTR-expressing cells. This increased level of cell killing was shown to be due to an increased level of apoptosis in the NTR-expressing cells that received both CB1954 and radiation. Since radiation kills cells principally through the generation of double-stranded DNA breaks, we studied the effect of the combined treatment on the presence of phospho-γH2AX foci 2 h after irradiation. Both FACS and confocal microscopy confirmed that there were an increased number of phospho-γH2AX foci after treatment with radiation and CB1954. This effect was specific for the NTR-expressing cells and at a constant radiation dose of 3 Gy was CB1954 dose dependent. Importantly, the ability of the NTR–CB1954 system to generate a transferable metabolite that was capable of mediating a bystander radiosensitizing effect was confirmed by transferring conditioned medium from NTR-expressing or non-expressing cells onto SW480 cells and then irradiating them. Subsequent experiments were then performed to demonstrate that adenoviral delivery of NTR, under the transcriptional control of either a constitutive viral promoter or a tumour-specific promoter, plus CB1954 was able to enhance the cytotoxicity of single-fraction or fractionated radiation in colorectal cancer cells in vitro. The efficacy of this approach was also confirmed in vivo in nude mice bearing SW480 colon cancer xenografts. However, despite the increased efficacy of the triple combination of Ad-NTR, CB1954 and radiation, most of the tumours that received this treatment subsequently progressed. We do not have histological data on the intratumoural distribution of adenovirus within treated tumours, but this is certainly an area that deserves attention in future studies with both replication-defective and replication-competent adenoviral vectors.

The issue of potential exacerbation of normal tissue toxicity by the triple therapy (adenovirus, prodrug and radiation) has not been modelled well in the current studies. The in vivo data demonstrated no increase in normal tissue toxicity in the radiation fields with the addition of virotherapy and CB1954, but it must be remembered that murine cells are poorly infected by adenovirus and so the observed toleration of the treatment may be an underrepresentation of what might occur in a clinical study. However, it is also worth considering that activated CB1954 diffusing locally from infected human tumour cells would have been able to interact with the dose of radiation that was delivered. Clearly, before clinical studies can be undertaken with this approach, it will be necessary to conduct detailed analyses of the effect of VDEPT plus radiation on normal tissue toxicity.

CB1954 has been shown to be safe and tolerable in a phase I dose-escalation and pharmacokinetic trial in which the drug was administered to 30 patients at doses between 3 and 37.5 mg m−2 every 3 weeks. The dose-limiting toxicities were diarrhoea and hepatotoxicity.21 That study has been followed by a phase I trial of a replication-defective adenovirus encoding NTR (CTL102) in patients with resectable liver tumours in which high levels of NTR expression were documented after viral doses of 1–5 × 1011 virus particles.22 A subsequent combination study of CTL102 in combination with CB1954 is in progress. The data in this report suggest that the NTR–CB1954 represents an extremely promising system for enhancing the effects of therapeutic irradiation in the clinic. Suitable targets would include prostate, lung, head and neck, and cervix cancers and other tumour types in which radiation can play a curative role. A number of similar VDEPT systems have also been developed, including the carboxypeptidase G2 (CPG2)/ZD2767P combination.23 ZD2767P has been used in clinical trials of antibody-directed enzyme prodrug therapy,24 and a future trial of a replication-competent adenoviral vector expressing CPG2 is in the planning stages. Interestingly, ZD2767P is metabolized by CPG2 into a potent alkylating agent that is highly likely to enhance the effects of radiation. Therefore, this combination would also represent a potential means of developing strategies that aim to use VDEPT as a means of enhancing radiotherapy in the clinic.

Materials and methods

Cell lines

A range of tumour cell lines were grown in the appropriate medium containing penicillin 100 U ml−1 and streptomycin 100 μg ml−1, supplemented with 10% fetal calf serum (FCS) (Gibco, Paisley, UK) at 37 °C in a humidified atmosphere of 5% CO2 in air. SKOV3-NTR cells were maintained under selection in Dulbecco's modified Eagle's medium supplemented with 10% FCS, antibiotics as above and G418 400 μg ml−1.

Adenoviral vectors

Ad.CMV-NTR and Ad.CTP1-NTR were provided by ML Laboratories (Keele, UK). The CTP1 promoter is a synthetic promoter that is preferentially active in tumour cells with dysregulated β-catenin expression.10 Briefly, it consists of multiple consensus TCF-4-binding sites upstream of the minimal SV40 large T-antigen promoter. It has previously been shown to direct E. coli β-galactosidase expression from an adenovirus in infected colorectal cancer cells.

MTT assay

SKOV3 and SKOV3-NTR cells grown to 80% confluence in 175 cm2 flasks (Falcon, Franklin Lakes, NJ, USA) were harvested, counted and plated in 96-well plates at a density of 1 × 104 cells per well. After 24 h, CB1954 was added at various concentrations from 1 nM to 500 μM. Control wells received an equivalent volume of unmodified medium. After 144 h incubation, 3-(4,5-dimethylthiazol-2-yl)-2,-5-diphenyl tetrazolium bromide (MTT) reagent (20 μl of a solution of 5 mg ml−1) was added for 5 h and the assay was developed by solubilizing in 100 μl dimethyl sulphoxide and reading at 550 nm on a SPECTRAmax 384 plate reader (Molecular Devices, Sunnyvale, CA, USA).

Radiation dosimetry

All in vitro and in vivo irradiations were delivered using a Pantak HF 320 kV X-ray machine with samples placed 27 cm beneath the X-ray source. The dose rate was determined using a Farmer Sub-Standard X-ray dosimeter Mk.2/S3 according to the manufacturer's instructions. Typically, the dose rate for irradiations was between 6.6 and 6.8 Gy min−1 at 240 kVp and 10 mA. As an additional quality assurance check, the dosimetry of the system was measured by thermoluminescent dosimetry (TLD) as follows. TLDs were irradiated to doses of 1, 2, 3, 5 and 10 Gy on a 6 MV linear accelerator (Varian, Crawley, UK) in the Radiotherapy Department, Royal Marsden Hospital NHS Trust. The absorbed radiation dose was determined by reading light output in a Toledo 654 TLD reader (DA Pitman, Weybridge, UK) to yield a standard curve (data not shown). For dosimetry of the in vitro studies, 25 cm2 tissue culture flasks and 24-well plates containing TLDs from the same batch were irradiated to radiation doses up to 10 Gy. For dosimetry of the in vivo studies, phantom mice (25 g) were constructed from tissue equivalent material and placed in a specially constructed irradiation jig that ensured that, during in vivo irradiation, the tumour on the animal's flank received a homogeneous radiation dose, but the rest of the animal's body received less than 10% of the radiation dose. TLDs were attached to the surface of a ‘tumour’ made of tissue equivalent material on the right flank of the phantom. Other TLDs were placed on the undersurface of the ‘tumour’, the contralateral flank and in a cavity within the body of the phantom. Using this setup, the ‘tumour’ was irradiated to doses up to 10 Gy. In each case, the absorbed radiation dose was determined by comparison of the measured light output against the standard curve as described above.

Bystander assay

SKOV3 and SKOV3-NTR cells were harvested as above, counted and mixed in different ratios from 100:0 to 0:100 at a total cell concentration of 5 × 104 cells ml−1. The resulting cell mixtures were plated in 96-well plates and treated with CB1954 at various concentrations between 1 nM and 500 μM. Subsequently, cell survival was measured by means of MTT assay as detailed above.

DAPI staining for apoptosis

SKOV3 or SKOV3-NTR cells were plated in 6-well plates at a density of 1 × 105 cells per well, and after 24 h they were exposed to CB1954 at concentrations between 0 and 5 μM. Plates were irradiated to a dose of 5 Gy (or mock irradiated) 16 h later. At 72 h post-irradiation, cells were fixed in 3.8% formaldehyde (BDH Laboratory Supplies, Poole, UK) in PBS and stored at 4 °C. At the time of assay, cells were washed twice in PBS and stained with 2 μl of a 1 mg ml−1 solution of 4′,6-diamidino-2-phenylindole dichloride (Molecular Probes, Eugene, OR, USA) in 1 ml of PBS. After 20 min, a further 1 ml of PBS was added and cells were viewed under a Nikon Eclipse E500 confocal microscope at × 10 magnification. Cells were scored as apoptotic if they demonstrated the typical microscopic features of DNA condensation and fragmentation. The proportion of apoptotic cells in four microscopic fields was counted.

FACS analysis of cell survival

SKOV3 or SKOV3-NTR cells were treated with CB1954 (0, 0.01 and 0.1 mM) and irradiated (0 or 3 Gy), washed thrice with PBS and resuspended at 1 × 106 cells in 500 μl PBS. Cells were stained with 5 μl of PI at 1 mg ml−1 and 50 μl of FDA (Molecular Probes) at 100 ng ml−1 and incubated for 10 min. Twenty thousand events were collected and analysed on a FACScalibur flow cytometer (Becton Dickinson, BD Europe (UK), Oxford, UK) using CellQuest Pro version 5.2 (BD Biosciences, BD Europe (UK), Oxford, UK).

Measurement of γH2AX focus formation

SKOV3 or SKOV3-NTR cells at a density of 5 × 106 cells per ml in a 175 cm2 flask were exposed to 0.1–1 μM. CB1954 (or PBS control) and irradiated 16 h later to doses between 0 and 10 Gy. After irradiation, cells were incubated at 37 °C for 2 h and then fixed in 4 ml of 70% ice-cold ethanol. Cells were subsequently stained with 200 μl mouse anti-phosphohistone γH2AX antibody (Upstate Biotechnology, Millipore, UK, Watford, UK), which was diluted 1:500 in TST (4% FCS+0.1% Triton X-100 in PBS) for 90 min at room temperature with agitation. After washing twice with PBS, cells were treated with 200 μl of fluorescein isothiocyanate-conjugated donkey anti-mouse secondary antibody diluted 1:200 in TST and incubated for 1 h at room temperature. Thereafter, cells were washed, counted and resuspended at 1 × 106 cells in 500 μl PBS. Twenty thousand events were collected and analysed on a FACScalibur flow cytometer.

Confocal microscopy

SKOV3 or SKOV3-NTR cells were grown on coverslips in 6-well plates. They were subsequently irradiated and returned to the incubator at 37 °C for 2 h before being fixed in 4% paraformaldehyde in PBS for 1 h at room temperature. Following fixation, cells were kept in IFF (1% bovine serum albumin and 2% FCS in PBS) at 4 °C until stained. At the time of staining, cells at room temperature were permeabilized with a covering volume of 0.2% (v/v) Triton × 100 in PBS for 10 min, washed and incubated with IFF in PBS for 10 min. Coverslips were removed and inverted onto 50 μl of mouse anti-phosphohistone γH2AX antibody diluted 1:2000 in IFF for 1 h at room temperature. Coverslips were washed three times and then inverted onto 50 μl of fluorescein isothiocyanate-conjugated donkey anti-mouse secondary antibody diluted 1:250 in IFF for 40 min. Cells were washed thrice with TO-PRO-3 iodide (Molecular Probes) diluted 1/10 000 in PBS in a 6-well plate on a rocking platform. Coverslips were inverted onto a drop of DakoCytomation Fluorescent mounting medium on glass slides and viewed with a Nikon Eclipse E500 confocal microscope.

In vitro clonogenic assays

In vitro clonogenic assays were performed according to the following general schema. Briefly, 2.5 × 105 colorectal cancer cells (SW480 or HCT116) were grown in 25 cm2 flasks (Falcon) for 24 h before infection. At that time point the test adenoviruses (Ad.CMV-lacZ, Ad.CMV-NTR and Ad.CTP1-NTR) were added at a multiplicity of infection of 50 PFU in 3 ml of Dulbecco's Modified Eagle's Medium containing 1% FCS for 90 min. Thereafter, the medium was removed, the flask was washed with Dulbecco's PBS and 5 ml of fresh medium containing 10% FCS was added. After 48 h, the cells were harvested, counted and plated in 24-well plates at cell densities between 5 × 101 and 5 × 103 cells per well. The following day, CB1954 was added to the wells at a concentration of 10 and 50 μM (for the single-fraction irradiations) and 10 and 25 μM (for the fractionated irradiations). The irradiation schedule commenced 24 h after addition of CB1954 to the medium. Colonies were stained 14 days later by addition of 100 μl of a 0.2% solution of crystal violet in 2% ethanol and incubation at 37 °C for 30 min. Colonies that were visible to the naked eye were counted.

Establishment of xenograft tumours

All husbandry and investigational procedures were conducted in accordance with UK Home Office regulations. Solid subcutaneous tumours were established in the right flanks of athymic nude mice (MF1 nu/nu, Charles Rivers plc, UK) by injecting a single-cell suspension of 1 × 106 SW480 cells in 100 μl PBS. Tumour growth was observed until the mean diameter was approximately 6–8 mm. At this time, animals were stratified by tumour size and allocated randomly in to treatment groups such that the mean diameters in the various groups were equivalent.

Starting 10 days after tumour inoculation, the tumours were measured on at least two occasions before the start of the treatment. Tumour volume was estimated as described previously.25 Briefly, three orthogonal diameters corresponding to the length, breadth and height (d1, d2 and d3) were measured using Vernier calipers and the tumour volume was calculated from the formula: V=π/6 × d1 × d2 × d3. The tumour volume on the day of injection of the therapeutic agent was designated as the initial volume or Vo. Tumour volume was assessed two to three times per week. The absolute and relative (as compared to Vo) tumour volumes were calculated and mice were killed when the tumour had increased to more than three times its original volume (3Vo). The time taken to reach 3Vo was recorded and used as a surrogate measure of animal survival, on the assumption that those tumours that had tripled their original volume were destined to increase in size inexorably. Use of this measure was designed to spare the animals from the physical distress of unnecessarily large tumour burdens and to comply with the Medical Research Council guidelines (Responsibility in the Use of Animals for Medical Research (1993)).

Intratumoural adenoviral delivery

Mice received a single injection of the various adenoviral vectors (Ad.CMV-GFP, Ad.CMV-NTR and Ad.CTP1-NTR) at a dose of 1 × 109 PFU per tumour diluted in 100 μl of sterile PBS. A single passage was made through the tumour, but attempts were made to ensure wide distribution of the injectate throughout the tumour.

In vivo irradiation

Before therapeutic radiation, the animals were anaesthetized with an intraperitoneal injection of 100 μl of a 1:1:4 mixture of Hypnorm (fentanyl citrate 0.315 mg ml−1, fluanisone 10 mg ml−1) (Janssen-Cilag Ltd, High Wycombe, UK), Hypnovel (midazolam 5 mg ml−1) (Roche Products Ltd, Welwyn Garden City, UK) and water for injection BP (Fresenius Health Care Group, Basingstoke, UK). Anaesthetized animals were positioned in the irradiation jig with the subcutaneous xenograft tumours under the radiation aperture in the 3 mm lead sheet that shielded the rest of the animal's body.

Statistical analysis

For analysis of apoptotic cell death, comparisons between groups were performed using the t-test. The Kaplan–Meier method was used to plot survival curves and the log-rank test was used to analyse differences between groups. Statistical analysis was performed using the Statistical Program for Social Sciences (SPSS 2006 SPSS Inc., Chicago, IL, USA) v 14.0.

References

  1. 1

    Harrington KJ, Melcher AA, Bateman AR, Ahmed A, Vile RG . Cancer gene therapy: part 2. Candidate transgenes and their clinical development. Clin Oncol (R Coll Radiol) 2002; 14: 148–169.

    Article  Google Scholar 

  2. 2

    Relph K, Harrington K, Pandha H . Recent developments and current status of gene therapy using viral vectors in the United Kingdom. BMJ 2004; 329: 839–842.

    Article  Google Scholar 

  3. 3

    Harrington KJ, Nutting CM . Interactions between ionising radiation and drugs in head and neck cancer—how can we maximize the therapeutic index? Curr Opin Investig Drugs 2002; 3: 807–811.

    CAS  PubMed  Google Scholar 

  4. 4

    Cobb LM, Connors TA, Elson LA, Khan AH, Mitchley BC, Ross WC et al. 2,4-dinitro-5-ethyleneiminobenzamide (CB1954): a potent and selective inhibitor of growth of the Walker carcinoma 256. Biochem Pharmacol 1969; 18: 1519–1527.

    CAS  Article  Google Scholar 

  5. 5

    Boland MP, Knox RJ, Roberts JJ . The differences in kinetics of rat and human DT diaphorase result in a differential sensitivity of derived cell lines to CB1954 (5-(aziridin-1-yl)-2,4-dinitrobenzamide). Biochem Pharmacol 1991; 41: 867–875.

    CAS  Article  Google Scholar 

  6. 6

    Roberts JJ, Friedlos F, Knox RJ . CB 1954 (2,4-dinitro-5-aziridinyl benzamide) becomes a DNA interstrand crosslinking agent in Walker tumour cells. Biochem Biophys Res Commun 1986; 140: 1073–1078.

    CAS  Article  Google Scholar 

  7. 7

    Anlezark GM, Melton RG, Sherwood RF, Coles B, Friedlos F, Knox RJ . The bioactivation of 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954)—I. Purification and properties of a nitroreductase enzyme from Escherichia coli—a potential for antibody-directed enzyme prodrug therapy (ADEPT). Biochem Pharmacol 1992; 44: 2289–2295.

    CAS  Article  Google Scholar 

  8. 8

    Walling JM, Stratford IJ, Adams GE . Radiosensitization by the 2,4-dinitro-5-aziridinyl benzamide CB 1954: a structure/activity study. Int J Radiat Biol Relat Stud Phys Chem Med 1987; 52: 31–41.

    CAS  Article  Google Scholar 

  9. 9

    Hennequin C, Favaudon V . Biological basis for chemo–radiotherapy interactions. Eur J Cancer 2002; 38: 223–230.

    CAS  Article  Google Scholar 

  10. 10

    Lipinski KS, Djeha AH, Ismail T, Mountain A, Young LS, Wrighton CJ . High-level, beta-catenin/TCF-dependent transgene expression in secondary colorectal cancer tissue. Mol Ther 2001; 4: 365–371.

    CAS  Article  Google Scholar 

  11. 11

    Valerie K, Brust D, Farnsworth J, Amir C, Taher MM, Hershey C et al. Improved radiosensitization of rat glioma cells with adenovirus-expressed mutant herpes simplex virus-thymidine kinase in combination with acyclovir. Cancer Gene Ther 2000; 7: 879–884.

    CAS  Article  Google Scholar 

  12. 12

    Anello R, Cohen S, Atkinson G, Hall SJ . Adenovirus-mediated cytosine deaminase gene transduction and 5-fluorocytosine therapy sensitises mouse prostate cancer cells to irradiation. J Urol 2000; 164: 2173–2177.

    CAS  Article  Google Scholar 

  13. 13

    Kievit E, Nyati MK, Ng E, Stegman LD, Parsels J, Ross BD et al. Yeast cytosine deaminase improves radiosensitization and bystander effect by 5-fluorocytosine of human colorectal cancer xenografts. Cancer Res 2000; 60: 6649–6655.

    CAS  PubMed  Google Scholar 

  14. 14

    Stackhouse MA, Pederson LC, Grizzle WE, Curiel DT, Gebert J, Haack K et al. Fractionated radiation therapy in combination with adenoviral delivery of the cytosine deaminase gene and 5-fluorocytosine enhances cytotoxic and antitumor effects in human colorectal and cholangiocarcinoma models. Gene Ther 2000; 7: 1019–1026.

    CAS  Article  Google Scholar 

  15. 15

    Vlachaki MT, Chhikara M, Aguilar L, Zhu X, Chiu KJ, Woo S et al. Enhanced therapeutic effect of multiple injections of HSV-TK+GCV gene therapy in combination with ionizing radiation in a mouse mammary tumor model. Int J Radiat Oncol Biol Phys 2001; 51: 1008–1017.

    CAS  Article  Google Scholar 

  16. 16

    Rosenberg E, Hawkins W, Holmes M, Amir C, Schmidt-Ullrich RK, Lin PS et al. Radiosensitization of human glioma cells in vitro and in vivo with acyclovir and mutant HSV-TK75 expressed from adenovirus. Int J Radiat Oncol Biol Phys 2002; 52: 831–836.

    CAS  Article  Google Scholar 

  17. 17

    Freytag SO, Paielli D, Wing M, Rogulski K, Brown S, Kolozsvary A et al. Efficacy and toxicity of replication-competent adenovirus-mediated double suicide gene therapy in combination with radiation therapy in an orthotopic mouse prostate cancer model. Int J Radiat Oncol Biol Phys 2002; 54: 873–885.

    CAS  Article  Google Scholar 

  18. 18

    Freytag SO, Khil M, Stricker H, Peabody J, Menon M, DePeralta-Venturina M et al. Phase I study of replication-competent adenovirus-mediated double suicide gene therapy for the treatment of locally recurrent prostate cancer. Cancer Res 2002; 62: 4968–4976.

    CAS  PubMed  Google Scholar 

  19. 19

    Kambara H, Tamiya T, Ono Y, Ohtsuka S, Terada K, Adachi Y et al. Combined radiation and gene therapy for brain tumors with adenovirus-mediated transfer of cytosine deaminase and uracil phosphoribosyltransferase genes. Cancer Gene Ther 2002; 9: 840–845.

    CAS  Article  Google Scholar 

  20. 20

    Chung SM, Advani SJ, Bradley JD, Kataoka Y, Vashistha K, Yan SY et al. The use of a genetically engineered herpes simplex virus (R7020) with ionizing radiation for experimental hepatoma. Gene Ther 2002; 9: 75–80.

    CAS  Article  Google Scholar 

  21. 21

    Chung-Faye G, Palmer D, Anderson D, Clark J, Downes M, Baddeley J et al. Virus-directed, enzyme prodrug therapy with nitroimidazole reductase: a phase I and pharmacokinetic study of its prodrug, CB1954. Clin Cancer Res 2001; 7: 2662–2668.

    CAS  PubMed  Google Scholar 

  22. 22

    Palmer DH, Mautner V, Mirza D, Oliff S, Gerritsen W, van der Sijp JR et al. Virus-directed enzyme prodrug therapy: intratumoral administration of a replication-deficient adenovirus encoding nitroreductase to patients with resectable liver cancer. J Clin Oncol 2004; 22: 1546–1552.

    CAS  Article  Google Scholar 

  23. 23

    Schepelmann S, Hallenbeck P, Ogilvie LM, Hedley D, Friedlos F, Martin J et al. Systemic gene-directed enzyme prodrug therapy of hepatocellular carcinoma using a targeted adenovirus armed with carboxypeptidase G2. Cancer Res 2005; 65: 5003–5008.

    CAS  Article  Google Scholar 

  24. 24

    Mayer A, Francis RJ, Sharma SK, Tolner B, Springer CJ, Martin J et al. A phase I study of single administration of antibody-directed enzyme prodrug therapy with the recombinant anti-carcinoembryonic antigen antibody fusion protein MFECP1 and a bis-iodo phenol mustard prodrug. Clin Cancer Res 2006; 12: 6509–6516.

    CAS  Article  Google Scholar 

  25. 25

    Harrington KJ, Rowlinson-Busza G, Syrigos KN, Peters AM, Uster PS, Vile RG et al. Pegylated liposome encapsulated doxorubicin and cisplatin enhance the effect of radiotherapy in a tumor xenograft model. Clin Cancer Res 2000; 6: 4939–4949.

    CAS  PubMed  Google Scholar 

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Correspondence to K J Harrington.

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White, C., Menghistu, T., Twigger, K. et al. Escherichia coli nitroreductase plus CB1954 enhances the effect of radiotherapy in vitro and in vivo. Gene Ther 15, 424–433 (2008). https://doi.org/10.1038/sj.gt.3303081

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Keywords

  • adenovirus
  • CB1954
  • nitroreductase
  • radiotherapy
  • VDEPT

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