E. coli nitroreductase/CB1954: in vitro studies into a potential system for feline cancer gene therapy (original) (raw)
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A mammalianized synthetic nitroreductase gene for high-level expression
BMC Cancer, 2009
The nitroreductase/5-(azaridin-1-yl)-2,4-dinitrobenzamide (NTR/CB1954) enzyme/ prodrug system is considered as a promising candidate for anti-cancer strategies by gene-directed enzyme prodrug therapy (GDEPT) and has recently entered clinical trials. It requires the genetic modification of tumor cells to express the E. coli enzyme nitroreductase that bioactivates the prodrug CB1954 to a powerful cytotoxin. This metabolite causes apoptotic cell death by DNA interstrand crosslinking. Enhancing the enzymatic NTR activity for CB1954 should improve the therapeutical potential of this enzyme-prodrug combination in cancer gene therapy.
Cancer Gene Therapy, 2000
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. Cancer Gene Therapy (2000) 7, 721-731
European Journal of Cancer, 1995
A recombinant retrovirus encoding E. coil nitroreductase (NTR) was used to infect mammalian cells. NIH3T3 cells expressing NTR were killed by the prodrug CB1954, which NTR converts to a bifunctional alkylating agent. Admixed, unmodified NIH3T3 cells could also be killed. In contrast to the Herpes simplex virus (HSV) thymidine kinase (TK)/ganciclovir(GCV) enzyme/prodrug system, NTRCB1954 cell hilling was effective in non-cycling cells. Co-operative hilling was observed when cells expressing both NTR and TK were treated with a combination of CB1954 and GCV. NTR expression in human melanoma, ovarian carcinoma or mesothelioma cells also rendered them sensitive to CB1954 killing. These data suggest that delivery of the NTR gene to human tumours, followed by treatment with CB1954, may provide a novel tumour gene therapy approach.
Evaluation of a synthetic CArG promoter for nitric oxide synthase gene therapy of cancer
Gene Therapy, 2005
Nitric oxide synthase gene therapy has been shown to be effective at inducing apoptosis in experimental tumours and sensitizing them to radiotherapy. We have also shown that expression of inducible nitric oxide synthase (iNOS) can be effectively restricted to the tumour volume by the use of the radiation inducible promoter (WAF1) to drive the transgene in clinically relevant protocols. A synthetic construct (pE9), incorporating nine radiosensitive CArG elements from the Egr1 promoter, has recently been developed for cancer gene therapy. We have now investigated basal gene expression of transgenes driven by this promoter to assess its suitablility for use in iNOS gene therapy protocols in vivo. Transfection of human microvascular endothelial cells (HMEC-1) with pE9iNOS, using a cationic lipid vector, resulted in progressively increasing (o5-fold) levels of iNOS protein expression up to 8 h after transfection. Transfection of an ex vivo rat artery preparation with pE9iNOS caused 83% inhibition of response to the vasoconstrictor phenylephrine (PE). CMVi-NOS transfection also reduced response to PE, but by only 52%. A single injection of 25 mg of pE9iNOS DNA in a lipid vector into the centre of a murine sarcoma (RIF1) induced iNOS protein expression by four-fold and increased nitrite concentration eight-fold. This caused a 7-day delay in tumour growth and was more effective than the constitutive CMVdriven construct. Our data suggest that generation of NO K , as a result of iNOS overexpression, is capable of further activating the E9 promoter, through a positive feedback loop, yielding stronger and sustained levels of NO K . This pE9iNOS combination may, therefore, be particularly useful in an anticancer gene therapy strategy as its antitumour effect in vivo was clearly superior to that of the strong constitutive promoter, CMV.
Cancer research, 2002
The efficacy of cancer gene therapy depends critically on "bystander effects" by which genetic modification of tumor cells results in killing of unmodified cells in the local microenvironment. In gene-dependent enzyme-prodrug therapy, expression of a prodrug-activating suicide gene is used to generate a cytotoxic metabolite that diffuses to nontransduced cells. The objective of this study was to develop a physiologically relevant tissue culture model for quantifying bystander effects and to validate the model using as an example the activation of dinitrobenzamide prodrugs (e.g., CB 1954) by Escherichia coli aerobic nitroreductase (NTR). Bystander effects were measured in three-dimensional multilayer cocultures of NTR+ and NTR- cells by determining clonogenic survival curves for both cell types using V79, Skov3, or WiDr as parental cell lines. Bystander killing by CB 1954 was much more efficient in multilayers than monolayers at equivalent cell:medium ratios, whereas the ch...
Cancer Research
Escherichia coli nitroreductase (NTR) activates the prodrug CB1954 to a cytotoxic derivative, allowing selective sensitization of NTR-expressing cells or tumors to the prodrug. This is one of several enzyme-prodrug combinations that are under development for cancer gene therapy, and the system has now entered clinical trials. Enhancing the catalytic efficiency of NTR for CB1954 could improve its therapeutic potential. From the crystal structure of an enzyme-ligand complex, we identified nine amino acid residues within the active site that could directly influence prodrug binding and catalysis. Mutant libraries were generated for each of these residues and clones screened for their ability to sensitize E. coli to CB1954. Amino acid substitutions at six positions conferred markedly greater sensitivity to CB1954 than did the WT enzyme; the best mutants, at residue F124, resulted in approximately 5-fold improvement. Using an adenovirus vector, we introduced the F124K NTR mutant into hum...