The expression of bacterial nitroreductase in transgenic mice results in specific cell killing by the prodrug CB1954

Abstract

The enzyme nitroreductase, isolated from Escherichia coli a much higher concentration (500 M). We subsequently B, converts CB1954 ((5-aziridin-1-yl)-2,4-dinitro-benzgenerated transgenic mice to test the prodrug system in amide) into a cytotoxic DNA interstrand cross-linking agent.vivo. Nitroreductase was expressed specifically in T cells The E. coli B gene (nfnB, NTR) encoding nitroreductase driven by the control elements of the human CD2 locus. (NTR) was cloned into eukaryotic expression vectors.Upon CB1954 treatment, transgenic mice show extensive When driven by a CMV promoter, 5-10% of the stably cell depletion in thymus and spleen (14-16% of normal cell transfected mouse fibroblasts expressed the NTR enzyme.numbers), whereas all other tissues are unaffected by proThese cells were killed at a concentration of 20 M CB1954 drug administration. These results raise the possibility of in comparison to nonexpressing cells which were killed at using the NTR gene in anticancer therapy.

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

“…The results of histological examination during tumor regression were consistent with previous reports 12,13 that the ntr/CB1954 system kills cells through induction of apoptosis. Our results also showed, however, that apoptotic cell death was followed by necrosis at later timepoints, suggesting that the macrophage infiltration Figure 9.…”

“…After confirmatory DNA sequencing, the gene was recloned as a NotI-HindIII fragment into pJG1. 13 The resultant plasmid, pTX0147, expresses NTR from the human cytomegalovirus (CMV) early promoter/enhancer and also carries the ␤-globin second intron and poly(A) sequences and a G418 selectable marker. The plasmid pTX0340 was derived from pTX0147 by subcloning the NTR expression cassette into pBluescript (Stratagene, Amsterdam, The Netherlands) as a BglII-XbaI fragment and recircularization of a 4-kb BspHI fragment from the resultant plasmid, pTX0160.…”

“…11,12 At the molecular level, this cell killing involves induction of interstrand DNA cross-links and apoptosis. 10,12,13 Tumor cell lines expressing NTR in vitro after stable transfection with the ntr gene are usually at least a hundred times more sensitive to CB1954 than untransfected cells. 14,15 Furthermore, bystander cell killing has been clearly demonstrated for tumor cells in vitro.…”

“…14 -16 Expression of NTR restricted either to the thymus and spleen (using the CD2 locus control region) or to the mammary gland (using the ␤-lactoglobulin promoter) in transgenic mice has been used to investigate cell killing in vivo with the ntr/CB1954 system. 12,13 Both studies demonstrated selective cell killing after CB1954 administration but gave little information regarding bystander killing effects in vivo.…”

Testing double mutants of the enzyme nitroreductase for enhanced cell sensitisation to prodrugs: Effects of combining beneficial single mutations

“…The ability of activated CB1954 to kill both dividing and non-dividing cells may be an advantage when the target cells are not proliferating rapidly [7,14,17,18]. Besides cancer gene therapy, NTR and CB1954 are also being used increasingly for other applications requiring conditional cell killing, including killing of cells responsible for loosening of orthopaedic implants in patients [19], and targeted, controllable tissue ablation in transgenic animals [20][21][22][23][24].…”