Efficient Prodrug Activator Gene Therapy by Retroviral Replicating Vectors Prolongs Survival in an Immune-Competent Intracerebral Glioma Model.

Prodrug activator gene therapy mediated by murine leukemia virus (MLV)-based retroviral replicating vectors (RRV) was previously shown to be highly effective in killing glioma cells both in culture and in vivo. To avoid receptor interference and enable dual vector co-infection with MLV-RRV, we have developed another RRV based on gibbon ape leukemia virus (GALV) that also shows robust replicative spread in a wide variety of tumor cells. We evaluated the potential of GALV-based RRV as a cancer therapeutic agent by incorporating yeast cytosine deaminase (CD) and E. coli nitroreductase (NTR) prodrug activator genes into the vector. The expression of CD and NTR genes from GALV-RRV achieved highly efficient delivery of these prodrug activator genes to RG-2 glioma cells, resulting in enhanced cytotoxicity after administering their respective prodrugs 5-fluorocytosine and CB1954 in vitro. In an immune-competent intracerebral RG-2 glioma model, GALV-mediated CD and NTR gene therapy both significantly suppressed tumor growth with CB1954 administration after a single injection of vector supernatant. However, NTR showed greater potency than CD, with control animals receiving GALV-NTR vector alone (i.e., without CB1954 prodrug) showing extensive tumor growth with a median survival time of 17.5 days, while animals receiving GALV-NTR and CB1954 showed significantly prolonged survival with a median survival time of 30 days. In conclusion, GALV-RRV enabled high-efficiency gene transfer and persistent expression of NTR, resulting in efficient cell killing, suppression of tumor growth, and prolonged survival upon CB1954 administration. This validates the use of therapeutic strategies employing this prodrug activator gene to arm GALV-RRV, and opens the door to the possibility of future combination gene therapy with CD-armed MLV-RRV, as the latter vector is currently being evaluated in clinical trials.

Overexpression of the nitroreductase NfsB in an E. coli strain as a whole-cell biocatalyst for the production of chlorinated analogues of the natural herbicide DIBOA.

Benzohydroxamic acids, such as DIBOA (2,4-dihydroxy-2 H)-1,4-benzoxazin-3(4 H)-one), are plant products that exhibit interesting herbicidal, fungicidal and bactericidal properties. A feasible alternative to their purification from natural sources is the synthesis of analogous compounds such as D-DIBOA (2-deoxy-DIBOA) and their chlorinated derivatives. Their chemical synthesis has been simplified into two steps. However, the second step is an exothermic reaction and involves hydrogen release, which makes this methodology expensive and difficult to scale up. The study reported here concerns the possibility of producing chlorobenzoxazinones by a whole-cell biocatalytic process using the ability of the engineered E. coli nfsB-/pBAD-NfsB to catalyse the synthesis of 6-Cl-D-DIBOA and 8-Cl-D-DIBOA from their respective precursors (PCs). The results show that this strain is able to grow in media that contain these compounds and to produce the target molecules with 59.3% and 46.7% biotransformation yields, respectively. Moreover, the strain is capable of processing non-purified PCs from the first chemical step to give similar yields to those obtained from the purified PCs. The kinetics of the reaction in vitro with purified recombinant NfsB nitroreductase were studied to characterise the catalysis further and evaluate the effects that several components of the non-purified PCs have on the process. The results revealed that the kinetics are that of an allosteric enzyme. The inhibitory effect of the substrate of the first step of the chemical synthesis, which is present in some non-purified PCs, was also demonstrated.

Biochemical characteristics of a nitroreductase with diverse substrate specificity from Streptomyces mirabilis DUT001.

A nitroreductase-encoded gene from an efficient nitro-reducing bacterium Streptomyces mirabilis DUT001, named snr, was cloned and heterogeneously expressed in Escherichia coli. The purified Streptomyces nitroreductase SNR was a homodimer with an apparent subunit molecular weight of 24 kDa and preferred NADH to NADPH as a cofactor. By enzyme incubation and isothermal calorimetry experiments, flavin mononucleotide (FMN) was found to be the preferred flavin cofactor; the binding process was exothermic and primarily enthalpy driven. The enzyme can reduce multiple nitro compounds and flavins, including antibacterial drug nitrofurazone, priority pollutants 2,4-dinitrotoluene and 2,4,6-trinitrotoluene, as well as key chemical intermediates 3-nitrophthalimide, 4-nitrophthalimide, and 4-nitro-1,8-naphthalic anhydride. Among the substrates tested, the highest activity of kcat(app) /Km(app) (0.234 µM-1  Sec-1 ) was observed for the reduction of FMN. Multiple sequence alignment revealed that the high FMN reduction activity of SNR may be due to the absence of a helix, constituting the entrance to the substrate pocket in other nitroreductases.

Nitroreductase gene-directed enzyme prodrug therapy: insights and advances toward clinical utility.

This review examines the vast catalytic and therapeutic potential offered by type I (i.e. oxygen-insensitive) nitroreductase enzymes in partnership with nitroaromatic prodrugs, with particular focus on gene-directed enzyme prodrug therapy (GDEPT; a form of cancer gene therapy). Important first indications of this potential were demonstrated over 20 years ago, for the enzyme-prodrug pairing of Escherichia coli NfsB and CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. However, it has become apparent that both the enzyme and the prodrug in this prototypical pairing have limitations that have impeded their clinical progression. Recently, substantial advances have been made in the biodiscovery and engineering of superior nitroreductase variants, in particular development of elegant high-throughput screening capabilities to enable optimization of desirable activities via directed evolution. These advances in enzymology have been paralleled by advances in medicinal chemistry, leading to the development of second- and third-generation nitroaromatic prodrugs that offer substantial advantages over CB1954 for nitroreductase GDEPT, including greater dose-potency and enhanced ability of the activated metabolite(s) to exhibit a local bystander effect. In addition to forging substantial progress towards future clinical trials, this research is supporting other fields, most notably the development and improvement of targeted cellular ablation capabilities in small animal models, such as zebrafish, to enable cell-specific physiology or regeneration studies.

Hepatocyte-specific ablation in zebrafish to study biliary-driven liver regeneration.

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

Overexpression of reactive cysteine-containing 2-nitrobenzoate nitroreductase (NbaA) and its mutants alters the sensitivity of Escherichia coli to reactive oxygen species by reprogramming a regulatory network of disulfide-bonded proteins.

The effects of redox-sensitive proteins on Escherichia coli were investigated by overexpressing Pseudomonas 2-nitrobenzoate nitroreductase (NbaA) and its mutants. Overexpression of wild-type and mutant NbaA proteins significantly altered the sensitivity of E. coli to antibiotics and reactive oxygen species regardless of the enzyme activity for reduction of 2-nitrobenzoic acid. The overexpressed proteins rendered cells 100-10000-fold more sensitive to superoxide anion (O2(•-))-generating paraquat and 10-100-fold more resistant to H2O2. A significant increase in intracellular levels of O2(•-), but not H2O2, was observed during expression of wild-type and truncated (Δ65-74, Δ193-216, and Δ65-74Δ193-216) NbaA. From two-dimensional nonreducing/reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometry analyses, 29 abundant proteins in the cytoplasm were identified to form interchain disulfide bonds, when cells were exposed to polymyxin B. Of them, down-regulation and modifications of SodB, KatE, and KatG were strongly associated with elevated cellular O2(•-) levels. Western blotting showed up-regulation of cell death signal sensor, CpxA, and down-regulation of cytoplasmic superoxide dismutase, SodB, with ∼2-fold up-regulation of heterodimeric integration host factor, Ihf. Activity gel assays revealed significant reduction of glyceraldehyde-3-phosphate dehydrogenase with constant levels of 6-phosphogluconate dehydrogenase. These changes would support a high level of NADPH to reduce H2O2-induced disulfide bonds by forced expression of thioredoxin A via thioredoxin reductase. Thus, overexpression of wild-type and truncated NbaA partially compensates for the lack of KatE and KatG to degrade H2O2, thereby enhancing disulfide bond formation in the cytoplasm, and modifies a regulatory network of disulfide-bonded proteins to increase intracellular O2(•-) levels.

Nitroreductase-mediated cell ablation in transgenic zebrafish embryos.

Prodrug dependent cell ablation is a method that allows inducible and spatially restricted cell destruction. We describe transgenic methods to express the Escherichia coli nfsB in a tissue restricted manner in the zebrafish. This bacterial gene encodes a nitroreductase (NTR) enzyme that can render prodrugs such as metronidazole (Met) cytotoxic. Using the expression of NTR fused to a fluorescent protein, one can simultaneously make cells susceptible to prodrug treatment and visualize cell ablation as it occurs.

Genotoxic activation of the environmental pollutant 3,6-dinitrobenzo[e]pyrene in Salmonella typhimurium umu strains expressing human cytochrome P450 and N-acetyltransferase.

3,6-Dinitrobenzo[e]pyrene (DNBeP) is a potent mutagen identified in surface soil in two metropolitan areas of Japan. We investigated whether DNBeP can cause genotoxicity through any metabolic activation pathway in bacteria using the parental strain Salmonella enterica serovar Typhimurium (S. typhimurium) TA1535/pSK1002, nitroreductase (NR)-deficient strain NM1000, the O-acetyltransferase (O-AT)-deficient strain NM2000, bacterial O-AT-overexpressing strain NM2009, and bacterial NR- and O-AT-overexpressing strain NM3009 established in our laboratory. To further clarify the role of human cytochrome P450 (P450 or CYP) and N-acetyltransferase (NAT) enzymes in the bioactivation of DNBeP to genotoxic metabolites, we determined the genotoxicity of DNBeP using a variety of umu tester strains expressing human P450 and NAT enzymes. The dose-dependent induction of umuC by DNBeP was observed at concentrations between 0.01 and 1nM in the O-AT-expression strain, but not in the O-AT-deficient strain. In the CYP3A4-, CYP1A2-, CYP1A1-, and CYP1B1-expressing strains, DNBeP was found to be activated to reactive metabolites that cause the induction of umuC gene expression compared with the parent strain. The induction of DNBeP in the NAT2-expressing strain had a 10-fold lower concentration than that in the NAT1-expressing strain. Collectively, these results suggest that nitroreduction by human CYP1A2, CYP3A4, and CYP1A1 and O-acetylation by human NAT2 contributed to the genotoxic activation of DNBeP to its metabolites.

Characterization of the CopR regulon of Lactococcus lactis IL1403.

To identify components of the copper homeostatic mechanism of Lactococcus lactis, we employed two-dimensional gel electrophoresis to detect changes in the proteome in response to copper. Three proteins upregulated by copper were identified: glyoxylase I (YaiA), a nitroreductase (YtjD), and lactate oxidase (LctO). The promoter regions of these genes feature cop boxes of consensus TACAnnTGTA, which are the binding site of CopY-type copper-responsive repressors. A genome-wide search for cop boxes revealed 28 such sequence motifs. They were tested by electrophoretic mobility shift assays for the interaction with purified CopR, the CopY-type repressor of L. lactis. Seven of the cop boxes interacted with CopR in a copper-sensitive manner. They were present in the promoter region of five genes, lctO, ytjD, copB, ydiD, and yahC; and two polycistronic operons, yahCD-yaiAB and copRZA. Induction of these genes by copper was confirmed by real-time quantitative PCR. The copRZA operon encodes the CopR repressor of the regulon; a copper chaperone, CopZ; and a putative copper ATPase, CopA. When expressed in Escherichia coli, the copRZA operon conferred copper resistance, suggesting that it functions in copper export from the cytoplasm. Other member genes of the CopR regulon may similarly be involved in copper metabolism.

The nitroreductase prodrug SN 28343 enhances the potency of systemically administered armed oncolytic adenovirus ONYX-411(NTR).

Conditionally replicating adenoviruses (CRAd) 'armed' with prodrug-activating genes have the potential to augment the efficacy of virotherapy. An Escherichia coli nitroreductase (NTR) gene (nfsB) was introduced into the E3B region of the systemically active CRAd ONYX-411, to produce ONYX-411(NTR), which had single agent oncolytic activity equivalent to unarmed virus in vitro and in vivo. A fluorogenic probe (SN 29884) developed to monitor NTR expression revealed robust, durable NTR expression in ONYX-411(NTR) infected neoplastic but not primary human cell lines. NTR expression occurred >24 h post-infection in parallel with fiber and was sensitive to ara-C indicating transcriptional linkage to viral replication. A novel NTR prodrug, the 3,5-dinitrobenzamide-2-bromomustard SN 27686, was shown to be more dose potent and selective than CB 1954 and provided a superior bystander effect in 3D multicellular layer cultures. Its water-soluble phosphate ester SN 28343 was substantially more active than CB 1954 against xenografts containing a minority of stable NTR-expressing cells. A single intravenous dose of ONYX-411(NTR) (10(8) PFU) to nude mice bearing large H1299 xenografts (>350 mm(3)) resulted in tumor-specific NTR expression which increased over time. Despite extensive viral spread by day 14, this conservative virus dose and schedule was unable to control such well-established tumors. However, subsequent administration of SN 28343 resulted in the majority of mice (62.5%) being tumor-free on day 120.

Enhanced transformation of tnt by tobacco plants expressing a bacterial nitroreductase.

The manufacture, disposal, and detonation of explosives have resulted in the pollution of large tracts of land and groundwater. Historically, 2,4,6-trinitrotoluene (TNT) is the most widely used military explosive and is toxic to biological systems and recalcitrant to degradation. To examine the feasibility of enhancing the ability of plants to detoxify the explosive TNT, we created transgenic tobacco (Nicotiana tabacum) constitutively expressing the nsfI nitroreductase gene from Enterobacter cloacae. The product of TNT reduction by the nitroreductase was found to be 4-hydroxylamino-2,6-dinitrotoluene (4-HADNT). Characterization of the transgenic lines in sterile, aqueous conditions amended with TNT demonstrated that these plants were able to remove all of the TNT from the medium at an initial concentration of 0.5 mM (113 mg L(-1)) TNT. In contrast, growth was suppressed in wild-type plants at 0.1 mM (23 mg L(-1)). Following uptake, transgenic seedlings transformed TNT predominantly to 4-HADNT and its high levels appeared to correlate with enhanced tolerance and transformation of TNT. Transformation products of TNT were subsequently conjugated to plant macromolecules to a greater degree in transgenic tobacco, indicating enhanced detoxification compared to the wild type.

Structural basis of 5-nitroimidazole antibiotic resistance: the crystal structure of NimA from Deinococcus radiodurans.

5-Nitroimidazole-based antibiotics are compounds extensively used for treating infections in humans and animals caused by several important pathogens. They are administered as prodrugs, and their activation depends upon an anaerobic 1-electron reduction of the nitro group by a reduction pathway in the cells. Bacterial resistance toward these drugs is thought to be caused by decreased drug uptake and/or an altered reduction efficiency. One class of resistant strains, identified in Bacteroides, has been shown to carry Nim genes (NimA, -B, -C, -D, and -E), which encode for reductases that convert the nitro group on the antibiotic into a non-bactericidal amine. In this paper, we have described the crystal structure of NimA from Deinococcus radiodurans (drNimA) at 1.6 A resolution. We have shown that drNimA is a homodimer in which each monomer adopts a beta-barrel fold. We have identified the catalytically important His-71 along with the cofactor pyruvate and antibiotic binding sites, all of which are found at the monomer-monomer interface. We have reported three additional crystal structures of drNimA, one in which the antibiotic metronidazole is bound to the protein, one with pyruvate covalently bound to His-71, and one with lactate covalently bound to His-71. Based on these structures, a reaction mechanism has been proposed in which the 2-electron reduction of the antibiotic prevents accumulation of the toxic nitro radical. This mechanism suggests that Nim proteins form a new class of reductases, conferring resistance against 5-nitroimidazole-based antibiotics.

Mechanism of metronidazole-resistance by isolates of nitroreductase-producing Enterococcus gallinarum and Enterococcus casseliflavus from the human intestinal tract.

Enterococcus casseliflavus and Enterococcus gallinarum strains resistant to metronidazole, nitrofurantoin and nitrofurazone were isolated from fecal samples of a patient with recurrent ulcerative colitis treated with metronidazole. Unlike other metronidazole-resistant bacteria, these strains produced nitroreductase but metabolized metronidazole to compounds that could not be detected by liquid chromatography with UV or mass spectral analysis. Metronidazole-susceptible Clostridium perfringens grew equally well in spent cultures of Enterococcus spp. incubated with or without metronidazole. These data indicate that the nitroreductases produced by these Enterococcus strains did not activate metronidazole to bactericidal metabolites and these bacteria may reduce the effectiveness of metronidazole. We have indirect evidence for an alternative pathway that results in metronidazole resistance. These strains of enterococcus had nitroreductase so resistance should not have occurred.

The fdxA ferredoxin gene can down-regulate frxA nitroreductase gene expression and is essential in many strains of Helicobacter pylori.

Very few examples of metabolic regulation are known in the gastric pathogen Helicobacter pylori. An unanticipated case was suggested, however, upon finding two types of metronidazole (Mtz)-susceptible strains: type I, in which frxA (which encodes a nitroreductase that contributes to Mtz susceptibility) is quiescent, and type II, in which frxA is well expressed. Here we report that inactivation of the fdxA ferredoxin gene (hp277) in type I strains resulted in high-level frxA expression (in effect, making them type II). However, fdxA null derivatives were obtained from only 6 of 32 type I strains tested that were readily transformed with an frxA::aphA marker. This suggested that fdxA is often essential. This essentiality was overcome in 4 of 20 strains by inactivating frxA, which suggested both that frxA overexpression is potentially deleterious and also that fdxA has additional, often vital roles. With type II strains, in contrast, fdxA null derivatives were obtained in 20 of 23 cases tested. Thus, fdxA is dispensable in most strains that normally exhibit (and tolerate) strong frxA expression. We propose that restraint of frxA expression helps maintain balanced metabolic networks in most type I strains, that other homeostatic mechanisms predominate in type II strains, and that these complex results constitute a phenotypic manifestation of H. pylori's great genetic diversity.

[Expression of a new cloned nitroreductase gene NOR1 and purification of expressed product].

BACKGROUND & OBJECTIVE: NOR(1)is a good candidate of tumor suppressor/susceptibility gene associated with nasopharyngeal carcinoma. This study was designed to construct the prokaryotic expression vector and to investigate the expression of nitroreductase gene NOR(1)in Escherichia coli and to purify expressed product. METHODS: Total RNA was subtracted from normal nasopharyngeal carcinoma tissue. The full length of NOR(1)gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and digested with BamHIand XhoI restriction endonucleases. The plasmid pGEX-4T-2 was also digested with BamHI and XhoI,then the NOR(1)gene was inserted into vector pGEX-4T-2. The recombinant expression vector pGEX-4T-2/NOR(1)was identified by sequencing and digested with restriction enzymes. E.coli Jm105 transformed with the recombinant plasmid was induced by IPTG to express GST fusion protein. The result was confirmed by Western blot analysis and the purified targeted protein was obtained by affinity chromatography. RESULTS: The 1.25kb NOR(1)gene was successfully isolated. After induction, a new anticipated protein of 74 kDa appeared on sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE). It existed not only in supernatant but also in precipitation of broken bacteria. The result was confirmed by Western blot analysis,and the purified targeted protein was obtained by affinity chromatography. CONCLUSION: The successes in construction of expression vector of NOR(1), expression and purification of GST/NOR(1)fusion protein make it possible to prepare for the polyantibodies for NOR(1).

Salmonella typhimurium mutagenicity tester strains that overexpress oxygen-insensitive nitroreductases nfsA and nfsB.

We have designed and constructed a series of plasmids that contain the major and/or minor Escherichia coli nitroreductase genes, nfsA and nfsB, in different combinations with R plasmid mucA/B genes and the Salmonella typhimurium OAT gene. The plasmid encoded gene products are necessary for both the metabolic activation of a range of structurally diverse nitrosubstituted compounds, and for mutagenic translation bypass. Introduction of these plasmids into S. typhimurium TA1538 and TA1535 has created several new tester strains which exhibit an extremely high mutagenic sensitivity and a broad substrate specificity towards a battery of nitrosubstituted test compounds that included 4-nitroquinoline-1-oxide (4-NQO), nitrofurazone (NF), 1-nitropyrene (1-NP), 2-nitronaphthalene (2-NN), 2-nitrofluorene (2-NF), and 1,6-dinitropyrene (1,6-DNP). Our studies show that the nfsA gene encodes a product that is extremely effective in the metabolic activation of a range of structurally diverse nitrosubstituted compounds. Several of the new tester strains are more than two orders of magnitude more sensitive to nitrosubstituted compounds than the Ames tester strains TA100 or TA98. In addition to enhancing mutagenic sensitivity, plasmids encoding both metabolic and mutagenesis functions on a single plasmid provide considerable flexibility for future mechanistic studies or tester strain development, in which it may be necessary to introduce additional plasmids containing different antibiotic resistance markers.

Production of the RdxA protein in metronidazole-susceptible and -resistant isolates of Helicobacter pylori cultured from treated mice.

The objective of this study was to use immunoblotting with RdxA antisera to examine the production of the RdxA protein in mouse-derived metronidazole-susceptible and -resistant isolates of Helicobacter pylori. A 24 kDa immunoreactive band corresponding to RdxA was observed in all 15 metronidazole-susceptible and five of 50 metronidazole-resistant isolates. The rdxA gene of these five isolates contained missense mutations and transformation experiments confirmed that these mutations were associated with inactivation of the rdxA gene. No RdxA protein was produced in the other 45 metronidazole-resistant strains, including one in which the nucleotide sequence of the rdxA gene was unchanged. These results demonstrate a high correlation between production of the RdxA protein and susceptibility of H. pylori to metronidazole. Testing for the absence of the RdxA protein identifies the majority of strains that will respond poorly to metronidazole-containing eradication regimens.

Identification of a Mycobacterium tuberculosis putative classical nitroreductase gene whose expression is coregulated with that of the acr aene within macrophages, in standing versus shaking cultures, and under low oxygen conditions.

Tuberculosis remains a leading killer worldwide, and new approaches for its treatment and prevention are urgently needed. This effort will benefit greatly from a better understanding of gene regulation in Mycobacterium tuberculosis, particularly with respect to this pathogen's response to its host environment. We examined the behavior of two promoters from the divergently transcribed M. tuberculosis genes acr/hspX/Rv2031c (alpha-crystallin homolog) and Rv2032/acg (acr-coregulated gene) by using a promoter-GFP fusion assay in Mycobacterium bovis BCG. We found that Rv2032 is a novel macrophage-induced gene whose expression is coregulated with that of acr. Relative levels of intracellular induction for both promoters were significantly affected by shallow standing versus shaking bacterial culture conditions prior to macrophage infection, and both promoters were strongly induced under low oxygen conditions. Deletion analyses showed that DNA sequences within a 43-bp region were required for expression of these promoters under all conditions. Multiple sequence alignment and database searches performed with PROBE indicated that Rv2032 is one of eight M. tuberculosis genes of previously unknown function that belong to an unusual superfamily of classical nitroreductases, which may have a role for bacteria within the host environment. These findings show that mycobacterial culture conditions can greatly influence the results and interpretation of subsequent gene regulation experiments. We propose that these differences might be exploited for dissection of the regulatory factors that affect mycobacterial gene expression within the host.

Phytodetoxification of TNT by transgenic plants expressing a bacterial nitroreductase.

There is major international concern over the wide-scale contamination of soil and associated ground water by persistent explosives residues. 2,4,6-Trinitrotoluene (TNT) is one of the most recalcitrant and toxic of all the military explosives. The lack of affordable and effective cleanup technologies for explosives contamination requires the development of better processes. Significant effort has recently been directed toward the use of plants to extract and detoxify TNT. To explore the possibility of overcoming the high phytotoxic effects of TNT, we expressed bacterial nitroreductase in tobacco plants. Nitroreductase catalyzes the reduction of TNT to hydroxyaminodinitrotoluene (HADNT), which is subsequently reduced to aminodinitrotoluene derivatives (ADNTs). Transgenic plants expressing nitroreductase show a striking increase in ability to tolerate, take up, and detoxify TNT. Our work suggests that expression of nitroreductase (NR) in plants suitable for phytoremediation could facilitate the effective cleanup of sites contaminated with high levels of explosives.

Sensitisation of human carcinoma cells to the prodrug CB1954 by adenovirus vector-mediated expression of E. coli nitroreductase.

The enzyme nitroreductase from E. coli can reduce the weak, monofunctional alkylating agent 5-(aziridin-1-yl)-2, 4-dinitrobenzamide (CB1954) to a potent cytotoxic species that generates interstrand crosslinks in DNA. Nitroreductase therefore has potential as a "suicide enzyme" for cancer gene therapy, as cells that express nitroreductase become selectively sensitive to the prodrug CB1954. We have incorporated a nitroreductase expression cassette into a replication-defective adenovirus vector (Ad-CMV-ntr), which allowed efficient gene transfer to SK-OV-3 or IGROV-1 ovarian carcinoma cells. Nitroreductase levels increased in line with multiplicity of infection, and this was reflected in increasing sensitisation of the cells to CB1954, reaching an optimum (approx. 2, 000-fold sensitisation) with 25-50 p.f.u. per cell. Similar Ad-CMV-ntr-dependent sensitisation to CB1954 was seen in 3 of 6 low-passage primary ovarian tumour lines. Cells grown at low-serum concentration to inhibit proliferation remained equally susceptible to the Ad-CMV-ntr-dependent cytotoxicity of CB1954, indicating a distinct advantage over retroviral gene delivery and other popular enzyme-prodrug systems for human tumours with a low rate of cell proliferation. Additionally, cisplatin-resistant cells were sensitised towards CB1954 by Ad-CMV-ntr as efficiently as the parental cells, indicating that the system could be effective in patients with cisplatin-resistant tumours. In a murine xenograft model for disseminated peritoneal carcinomatosis with ascites, treatment of nude mice bearing intraperitoneal SUIT2 tumours with Ad-CMV-ntr and CB1954 almost doubled the median survival from 14 to 26 days (p < 0.0001).