E. coli NfsA: an alternative nitroreductase for prodrug activation gene therapy in combination with CB1954.

Prodrug activation gene therapy is a developing approach to cancer treatment, whereby prodrug-activating enzymes are expressed in tumour cells. After administration of a non-toxic prodrug, its conversion to cytotoxic metabolites directly kills tumour cells expressing the activating enzyme, whereas the local spread of activated metabolites can kill nearby cells lacking the enzyme (bystander cell killing). One promising combination that has entered clinical trials uses the nitroreductase NfsB from Escherichia coli to activate the prodrug, CB1954, to a potent bifunctional alkylating agent. NfsA, the major E. coli nitroreductase, has greater activity with nitrofuran antibiotics, but it has not been compared in the past with NfsB for the activation of CB1954. We show superior in vitro kinetics of CB1954 activation by NfsA using the NADPH cofactor, and show that the expression of NfsA in bacterial or human cells results in a 3.5- to 8-fold greater sensitivity to CB1954, relative to NfsB. Although NfsB reduces either the 2-NO(2) or 4-NO(2) positions of CB1954 in an equimolar ratio, we show that NfsA preferentially reduces the 2-NO(2) group, which leads to a greater bystander effect with cells expressing NfsA than with NfsB. NfsA is also more effective than NfsB for cell sensitisation to nitrofurans and to a selection of alternative, dinitrobenzamide mustard (DNBM) prodrugs.

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

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 (IC(50)=0.35 muM) 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 gammaH2AX 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.

Building a metal-responsive promoter with synthetic regulatory elements.

A fusion gene consisting of the promoter region from the mouse metallothionein-I gene joined to the coding region of the herpes simplex virus thymidine kinase gene is efficiently regulated by zinc in a transient assay when transfected into baby hamster kidney cells. Analysis of similar plasmids in which the metallothionein-I promoter region was mutated indicated the presence of multiple metal regulatory elements (MREs) between -176 and -44 base pairs from the cap site. To further investigate the function of MREs, we inserted a synthetic DNA fragment containing the sequence of MRE-a (the element between -55 and -44 base pairs) into the nonresponsive promoter of the thymidine kinase gene in various positions and configurations. Little or no induction by zinc was observed with single insertions of the regulatory sequence, whereas many different constructions having two copies of MRE-a were inducible. The precise position of the two MREs relative to each other or to the thymidine kinase promoter elements had a relatively small effect on the efficiency of induction, but the inducibility could be further increased by the introduction of more MRE-a sequences. MRE-a can function synergistically with the thymidine kinase distal promoter elements, but in the presence of the TATA box alone it functions as a positive, zinc-dependent promoter element.

Regulation, linkage, and sequence of mouse metallothionein I and II genes.

The mouse metallothionein II (MT-II) gene is located approximately 6 kilobases upstream of the MT-I gene. A comparison of the sequences of mouse MT-I and MT-II genes (as well as those of other mammals) reveals that the coding regions are highly conserved even at "silent" positions but that the noncoding regions and introns are extremely divergent between primates and rodents. There are four blocks of conserved sequences in the promoters of mouse MT-I, mouse MT-II, and human MT-IIA genes; one includes the TATAAA sequence, and another has been implicated in regulation by heavy metals. Mouse MT-I and MT-II mRNAs are induced to approximately the same extent in vivo in response to cadmium, dexamethasone, or lipopolysaccharide. Mouse MT-I and MT-II genes are regulated by metals but not by glucocorticoids after transfection into HeLa cells.

Prostate-specific antigen promoter/enhancer driven gene therapy for prostate cancer: construction and testing of a tissue-specific adenovirus vector.

A range of luciferase reporter vectors was constructed, incorporating 5'-flanking sequences from the prostate-specific antigen (PSA), human glandular kallikrein 2 (hKLK2), and cytomegalovirus (CMV) promoters for expression control. Tissue specificity was evaluated in the PSA-positive line LNCaP and PSA-negative cells from different tissues of origin (CoLo320, DG75, EJ, A2780, and Jurkat). The minimal 628-bp PSA and hKLK2 promoters showed only low-level expression in either PSA-positive or PSA-negative cells and showed no increase with the addition of androgen. Tandem duplication of the PSA promoter slightly increased expression in PSA-positive LNCaP cells. The addition of CMV enhancer sequences upstream of a single PSA or hKLK2 promoter substantially but nonspecifically increased luciferase expression in all cell lines tested. However, placing a 1455-bp PSA enhancer sequence upstream of either the PSA or hKLK2 promoters increased expression 20-fold in the PSA-positive cell line LNCaP but not in the PSA-negative lines. Tandem duplication of the PSA enhancer increased expression to approximately 50-fold higher than either promoter alone while retaining tissue-specific control. The level of expression was reduced by the addition of a third copy of the PSA enhancer. Expression from all enhancer constructs was increased 100-fold above basal levels when induced with the androgen dihydrotestosterone, with the PSA-based constructs consistently exhibiting roughly twice the level of expression of the hKLK2-based constructs at all androgen concentrations. Adenovirus vectors were produced in which either enhanced green fluorescent protein or nitroreductase could be expressed from the optimized PSA double enhancer-promoter construct and evaluated in LNCaP cells and the bladder-derived line EJ. Control vectors with the CMV promoter gave good levels of expression in both cell lines, whereas the PSA constructs only produced detectable levels of protein in the LNCaP cells as assessed by fluorescence of enhanced green fluorescent protein or by Western blotting of nitroreductase. LNCaP but not EJ cells were selectively sensitized to the prodrug CB1954 following infection with Ad-PSA(EEP)-NR. The PSA-based nitroreductase virus produced comparable amounts of nitroreductase and sensitization to CB1954 approaching that of the CMV-driven virus. Plasmid and adenovirus constructs combining PSA enhancer and promoter sequences demonstrate selective expression of linked genes in PSA-positive cells. The expression is induced by androgen and gives therapeutically relevant levels of effector proteins.

The structure of Escherichia coli nitroreductase complexed with nicotinic acid: three crystal forms at 1.7 A, 1.8 A and 2.4 A resolution.

Escherichia coli nitroreductase is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Its ability to convert relatively non-toxic prodrugs such as CB1954 (5-[aziridin-1-yl]-2,4-dinitrobenzamide) into highly cytotoxic derivatives has led to interest in its potential for cancer gene therapy. We have determined the structure of the enzyme bound to a substrate analogue, nicotinic acid, from three crystal forms at resolutions of 1.7 A, 1.8 A and 2.4 A, representing ten non-crystallographically related monomers. The enzyme is dimeric, and has a large hydrophobic core; each half of the molecule consists of a five-stranded beta-sheet surrounded by alpha-helices. Helices F and F protrude from the core region of each monomer. There is an extensive dimer interface, and the 15 C-terminal residues extend around the opposing monomer, contributing the fifth beta-strand. The active sites lie on opposite sides of the molecule, in solvent-exposed clefts at the dimer interface. The FMN forms hydrogen bonds to one monomer and hydrophobic contacts to both; its si face is buried. The nicotinic acid stacks between the re face of the FMN and Phe124 in helix F, with only one hydrogen bond to the protein. If the nicotinamide ring of the coenzyme NAD(P)H were in the same position as that of the nicotinic acid ligand, its C4 atom would be optimally positioned for direct hydride transfer to flavin N5. Comparison of the structure with unliganded flavin reductase and NTR suggests reduced mobility of helices E and F upon ligand binding. Analysis of the structure explains the broad substrate specificity of the enzyme, and provides the basis for rational design of novel prodrugs and for site-directed mutagenesis for improved enzyme activity.

Improved titers of retroviral vectors from the human FLYRD18 packaging cell line in serum- and protein-free medium.

The influence of serum on the production of retroviral vectors by the HT1080 human fibrosarcoma-derived packaging cell line FLYRD18 was investigated. A fourfold increase in virus titer was observed under serum-free conditions, as compared with medium supplemented with 10% fetal calf serum. A similar improvement was also seen for bulk transduction efficiency. Serum had a negative and dose-dependent effect on titer without affecting cell growth, virus stability, or infectivity. In contrast to virus from NIH 3T3-derived packaging cells [Hanenberg, H., et al. (1996). Nature Med. 2, 876-882], the FLYRD18-derived virus did not adhere to fibronectin or serum proteins adsorbed at the surface of culture flasks. Electron microscopy supports the conclusion that the effect of serum is at the level of virus production by the cells. Addition of soybean trypsin inhibitor had an inhibitory effect on virus production, while pretreatment of serum with trypsin was found to enhance the retroviral titer. These results suggest that protease inhibitors present in serum may be responsible for the inhibition of virus production. The exact mechanism remains, however, to be determined. As compared with medium supplemented with 10% serum, the combination of increased virus titer and absence of exogenous protein under serum-free conditions resulted in a 300-fold increase in the virus:total protein ratio in the supernatants harvested from the FLYRD18 packaging line. This improvement enhances prospects for further concentration and purification of the virus.

Expression of Escherichia coli B nitroreductase in established human tumor xenografts in mice results in potent antitumoral and bystander effects upon systemic administration of the prodrug CB1954.

Expression of the Escherichia coli enzyme nitroreductase (NTR) in mammalian cells enables them to activate the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954), leading to interstrand DNA cross-linking and apoptosis in both proliferating and quiescent cells. In the work reported here, we used human hepatocellular carcinoma and squamous carcinoma cell lines constitutively expressing NTR to demonstrate that the ntr/CB1954 system results in potent, long-lasting antitumoral effects in mice. We also demonstrate that this enzyme/prodrug combination results in antitumoral effects in vivo when only a minority of tumor cells express the enzyme, using either cells constitutively expressing NTR or ntr gene delivery in situ.

Sensitization of colorectal and pancreatic cancer cell lines to the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) by retroviral transduction and expression of the E. coli nitroreductase gene.

Expression of genes encoding prodrug-activating enzymes can increase the susceptibility of tumor cells to prodrugs, and may ultimately achieve a better therapeutic index than conventional chemotherapy. CB1954 is a weak, monofunctional alkylating agent which can be activated by Escherichia coli nitroreductase to a potent dysfunctional alkylating agent which crosslinks DNA. We have inserted the nitroreductase gene into an LNCX-based retroviral vector, to allow efficient gene transfer and expression in colorectal (LS174T) and pancreatic (SUIT2, BxPC3, and AsPC1) cancer cell lines. A clone of LS174T cells expressing nitroreductase showed > 50-fold increased sensitivity to CB1954, and nitroreductase-expressing clones of pancreatic tumor lines were up to approximately 500-fold (SUIT2) more sensitive than parental cells. Concentrations of CB1954 minimally toxic to nontransduced cells achieved 100% cell death in a 50:50 mix of parental cells with SUIT2 cells expressing nitroreductase; and marked "bystander" cell killing was seen with just 10% of cells expressing nitroreductase. Significant bystander cell killing was dependent on a high cell density. In conjunction with regional delivery of vectors and tumor selectivity of cell entry and/or gene expression, nitroreductase and CB1954 may be an attractive combination for prodrug-activating enzyme gene therapy of colorectal and pancreatic cancer.

Rapid estrogen metabolism and vitellogenin gene expression in Xenopus hepatocyte cultures.

Male hepatocytes metabolized estradiol-17 beta, 17 alpha-ethinylestradiol and mestranol extremely rapidly (t 1/2 = 40, 60 and 300 min, respectively), whereas these were more stable in cultures of female hepatocytes (t 1/2 = 120, 150 and 640 min, respectively). Vitellogenin mRNA accumulated for only 12 h after a single addition of 10(-6) M estradiol to male hepatocyte cultures; mestranol, but not 17 alpha-ethinylestradiol or diethylstilbestrol, was more potent than the natural hormone. The level and rate of accumulation of vitellogenin mRNA were 5-15 times higher in female than in male hepatocytes, mestranol and estradiol being more potent than 17 alpha-ethinylestradiol and diethylstilbestrol. Ovariectomy, 60 days prior to cell culture, did not alter the metabolism of estradiol or the vitellogenic response of female hepatocytes. On the other hand, a single administration of estradiol in vivo to male Xenopus caused a long-lasting shift (at least 16 weeks) to the female pattern of its metabolism, although the enhanced inducibility of vitellogenin genes was partially reversed between 4 and 16 weeks after hormonal treatment. The addition of fresh estradiol every 4 h to male hepatocyte cultures to compensate for its rapid metabolism resulted in a continuous and sustained accumulation of vitellogenin mRNA at rates comparable to those attained in vivo. Our findings explain the requirement for high levels of estrogen to activate vitellogenin genes and establish Xenopus hepatocyte cultures as a reproducible system for analysing the expression of this multigene family.

Gene therapy for colorectal cancer: therapeutic potential.

Colorectal cancer is a leading cause of cancer mortality in Western countries. Gene therapy has been proposed as a potential novel treatment modality for colorectal cancer, but it is still in an early stage of development. The preclinical data have been promising and numerous clinical trials are underway. This brief review aims to summarise the current status of clinical trials of different gene therapy strategies, including immune stimulation, mutant gene correction, prodrug activation and oncolytic virus therapy, for patients with colorectal cancer. Data from phase I trials have proven the safety of the reagents but have not yet demonstrated significant therapeutic benefit. In order to achieve this and extend the scope of the treatment, continuing efforts should be made to improve the antitumour potency, efficiency of gene delivery and accuracy of gene targeting.

Production of retroviral vectors for gene therapy with the human packaging cell line FLYRD18.

The development of gene therapy is hampered by the difficulty of producing large stocks of retroviral vectors at high titer. This study aimed to improve culture conditions and to intensify the production of retroviruses by FLYRD18, a packaging cell line derived from the HT1080 human fibrosarcoma line. Batch virus production proved to be feasible in unsupplemented basal medium and provided significantly higher titers and productivities than medium supplemented with 10% serum. For longer-term production, however, AIM-V complete serum-free medium and basal medium supplemented with 2% serum gave superior results. Serum supplementation should nevertheless be optimized to take into account the presence of inhibitors of viral production. In monolayer cultures with 0.2 mL/cm(2), the cell concentration was increased up to 2 x 10(6) cells/mL without loss of cell productivity. A semicontinuous production process, which enables the collection of larger amounts of viruses from the same culture, has also been successfully used. Suspension culture processes were prevented by the anchorage dependency of the FLYRD18 cell line. Microcarrier cultures were able to produce viruses but will require further investigation and optimization for their performance to become competitive with monolayer cultures. In the course of this study, more than a 10-fold increase of titer has been achieved.

Combining gene and immunotherapy for prostate cancer.

The nitroreductase (NR)/CB1954 enzyme prodrug system has given promising results in pre-clinical studies and is currently being assessed in phase I and II clinical trials in prostate cancer. Enhanced cell killing by apparent immune-mediated mechanisms has been shown in pancreatic and colorectal cancer models, by co-expressing murine granulocyte macrophage colony-stimulating factor (GM-CSF) with NR in a single replication deficient adenoviral vector. This consists of the CMV immediate early promotor driving expression of NR, with an internal ribosome entry site (IRES) and the gene for murine GM-CSF (mGM-CSF). To examine if similar enhancement of tumour cell killing could be produced in prostate cancer, the TRAMP model was chosen. Results illustrate that the combination of suicide gene therapy using NR and CB1954, with cytokine stimulation with mGM-CSF gives an improved response compared with either modality alone. The mechanism of this improved response is however likely to be non-immune based as it lacks a memory effect.

Direct positive selection for improved nitroreductase variants using SOS triggering of bacteriophage lambda lytic cycle.

Expression of prodrug-activating enzymes that convert non-toxic substrates to cytotoxic derivatives is a promising strategy for cancer gene therapy. However, their catalytic activity with unnatural, prodrug substrates is often suboptimal. Efforts to improve these enzymes have been limited by the inability to select directly for increased prodrug activation. We have focussed on developing variants of Escherichia coli (E. coli) nitroreductase (NTR) with improved ability to activate the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954), and describe here a novel, direct, positive selection for improved enzymes that exploits the alternative life cycles of bacteriophage lambda. In lambda lysogens of E. coli, the activation of the prodrug CB1954 by NTR triggers the SOS response to DNA damage, switching integrated lambda prophages into lytic cycle. This provides a direct, positive selection for phages encoding improved NTR variants, as, upon limiting exposure of lysogenized E. coli to CB1954, only those encoding the most active enzyme variants are triggered into lytic cycle, allowing their selective recovery. We exemplify the selection by isolating highly improved 'turbo-NTR' variants from a library of 6.8 x 10(5) clones, conferring up to 50-fold greater sensitivity to CB1954 than the wild type. Carcinoma cells infected with adenovirus expressing T41Q/N71S/F124T-NTR were sensitized to CB1954 concentrations 40- to 80-fold lower than required with WT-NTR.

Inhibition of NF-kappaB enhances the cytotoxicity of virus-directed enzyme prodrug therapy and oncolytic adenovirus cancer gene therapy.

Virus-directed enzyme prodrug therapy utilizing the bacterial enzyme nitroreductase delivered by a replication-defective adenovirus vector to activate the prodrug CB1954 is a promising strategy currently undergoing clinical trials in patients with a range of cancers. Similarly, selectively replicating oncolytic adenoviruses are entering clinical trials. An understanding of interactions between vector and target cell are critical to the development of these strategies. We demonstrate that adenovirus vectors activate cellular pathways that promote cell survival in an NF-kappaB-dependent manner, and consequently have a negative effect on the efficacy of cell killing induced by cancer gene therapy strategies. This provides a potential therapeutic target to enhance the cytotoxicity of these approaches.

Zinc dependent binding of a liver nuclear factor to metal response element MRE-a of the mouse metallothionein-I gene and variant sequences.

Metallothionein gene transcription is inducible by zinc and other heavy metals, and several metal response elements (MREs) have been mapped within about 200 bp upstream of the site of transcription initiation in several metallothionein genes. Comparison of a number of MREs defined a 15 bp consensus sequence containing a more highly conserved MRE core sequence TGCRCNCG. I have used the proximal MRE of the mouse metallothionein-I gene (MRE-a) in DNA fragment mobility shift assays to detect a protein in rat liver nuclear extracts which binds specifically to the MRE in a zinc-regulated manner. Use of a comprehensive series of variant MRE sequences established that the binding was strongly dependent on the MRE core sequence, whereas changes at the less highly conserved positions had minor effects on binding. This provides strong evidence that the protein detected is responsible for the zinc-responsiveness of the MT genes in liver, and provides a more detailed picture of the regulatory protein:MRE interaction than was previously available.

Vitellogenin gene expression in male Xenopus hepatocytes during primary and secondary stimulation with estrogen in cell cultures.

Primary cultures of male Xenopus liver parenchymal cells that retained their competence to respond to estrogen were used to study the hormone-induced activation of the vitellogenin gene in vitro. The accumulation of vitellogenin mRNA in these cells was monitored by a quantitative diazotized paper disc hybridization procedure with a sensitivity of at least 6 pg of sequences complementary to the probe in total RNA samples of 10 micrograms. A short-term time-course analysis showed that vitellogenin mRNA was detectable within 3 hr of exposure to estrogen during primary stimulation, and that the maximum rate of accumulation was reached at 5--6 hr. A long-term time-course analysis of the accumulation of vitellogenin mRNA showed that it is possible to obtain a primary response, a hormone withdrawal effect and an enhanced secondary response in the same batch of cells in a manner analogous to that observed in vivo. Measurement of hormone concentration dependence showed a response at 10(-9) M estradiol, which continued to increase up to at least 10(-6) M estradiol. This requirement for large doses of estradiol for maximal response can be explained by the rapid metabolism of estradiol by the cultured cells.

Immunotherapy II: Antigens, receptors and costimulation.

To generate a cytotoxic T-lymphocyte (CTL) response to cancer cells requires tumour-specific antigens appropriately processed and displayed by the MHC proteins; T-lymphocytes with receptors of appropriate specificity to recognise these; and initial antigen presentation to the immune system in an immunogenic context. In vitro, autologous tumour-specific CTL have been raised against a number of tumours, thus at least some patients have a suitable combination of antigen and receptor. Vaccination with antigen, or with DNA or viral vectors encoding the antigen, leading to the presentation of identified antigens in an immunogenic context, can activate T-cells which provide protection from tumour in animal models. An alternative approach uses gene transfer to T-cells, causing them to express novel receptors which direct their cytotoxic activity towards the tumour. Non-specific immune adjuvants, and expression of novel antigens on tumour cells, are briefly discussed. Recent advances in understanding the requirements for T-cell activation suggest that failure to efficiently present antigen in an immunogenic context may explain the apparent lack of tumour-specific CTL activation in vivo. In mice, expression of the costimulatory molecule B7-1 on tumour cells, following gene transfer, allows the modified tumour cells to act as antigen-presenting cells, inducing protective and therapeutic CTL responses in some cases. Clinical trials of some approaches have commenced, with some encouraging results which provide a basis for further development of immunological gene therapy.