The Bystander Effect of the Nitroreductase/CB 1954 Enzyme/Prodrug System Is Due to a Cell-Permeable Metabolite

Abstract

The bystander effect is an important part of tumor kill using gene-directed enzyme prodrug therapy (GDEPT). Recently, we have described a novel enzyme prodrug system using bacterial nitroreductase and the prodrug CB1954 (NTR/CB1954). We demonstrate here the presence of a cell-permeable cytotoxic activity in the conditioned growth medium of nitroreductase (NTR)-transduced cells treated with CB1954 and show that its appearance corresponds to the appearance of two metabolites of CB1954 previously identified (Friedlos et al., 1992). The degree of bystander effect and the degree of transferred cytotoxicity correlates with the level of NTR enzyme expression. Two other prodrugs for NTR show little bystander killing and do not produce detectable cell permeable metabolites. The elucidation of the mechanism of the bystander effect may allow the more effective use of NTR/CB1954.

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

“…4 Bystander effects have been reported for the ntr/CB1954 system in vitro 14 -16 and have been demonstrated to be mediated by a diffusible metabolite of CB1954. 20 In the present study we clearly demonstrate that antitumoral effects can be achieved in response to CB1954 administration when only a minority of tumor cells express NTR. The experiments with s.c. HepG2 tumors formed from mixtures of cells either expressing or not expressing NTR show that significant antitumoral effects and an improvement in time to compulsory sacrifice result when only 5% of the tumor cells express the enzyme.…”

“…The prodrug CB1954, which is similarly inert, 19 has been shown to mediate potent in vitro tumor cell killing of cells engineered to express E. coli NTR as well as cocultured nonexpressing tumor cells. 11,20 In this study, we investigated the in vivo efficacy of the ntr/CB1954 system using xenograft models of hepatocellular carcinoma and squamous carcinoma of the head and neck. These tumor types were selected because they will be two of the earliest clinical targets for the system, as they are amenable to targeting ntr gene delivery by direct i.t.…”

Experimental targeted radioiodide therapy following transfection of the sodium iodide symporter gene: Effect on clonogenicity in both two-and three-dimensional models

“…The choice of radionuclide will be influenced by the expected intracellular residence time of the radioactivity and the range of crossfire radiation needed to sterilize clumps of tumor cells of different sizes. Although differences in the ionic radii of the various halides appears to affect affinity for the NIS, 29 experimental evidence indicates that the alphaparticle -emitting radiohalogen 211 At, administered in the 33 and has affinity for NIS -expressing tissues. 34 Currently, we are evaluating the uptake and toxicity of Na 211 At in NIS genetransfected cells.…”

Cell surface display of a lysosomal enzyme for extracellular gene-directed enzyme prodrug therapy

“…Most GDEPT systems available at present, such as the herpes simplex virus thymidine kinase/ganciclovir system, function intracellularly and thereby limit the efficacy of the system and choice of the prodrug to membrane-permeable molecules. [4][5][6][7] In an attempt to improve this situation, prokaryotic carboxypeptidase G2 displayed on the cell surface has been developed for GDEPT. 8,9 However, the use of a non-human enzyme may provoke undesired immune responses, particularly if multiple applications are required.…”