Cell to Cell Contact Is Not Required for Bystander Cell Killing by Escherichia coli Purine Nucleoside Phosphorylase

Abstract

Expression of Escherichia coli purine nucleoside phosphorylase (PNP) activates prodrugs and kills entire populations of mammalian cells, even when as few as 1% of the cells express this gene. This phenomenon of bystander killing has been previously investigated for herpes simplex virus-thymidine kinase (HSV-TK) and has been shown to require cell to cell contact. Using silicon rings to separate E. coli PNP expressing cells from nonexpressing cells sharing the same medium, we demonstrate that bystander cell killing by E. coli PNP does not require cell-cell contact. Initially, cells expressing E. coli PNP convert the non-toxic prodrug, 6-methylpurine-2-deoxyriboside (MeP-dR) to the highly toxic membrane permeable toxin, 6-methylpurine (MeP). As the expressing cells die, E. coli PNP is released into the culture medium, retains activity, and continues precursor conversion extracellularly (as determined by reverse phase high performance liquid chromatography of both prodrug and toxin). Bystander killing can also be observed in the absence of extracellular E. coli PNP by removing the MeP-dR prior to death of the expressing cells. In this case, 100% of cultured cells die when as few as 3% of the cells of a population express E. coli PNP. Blocking nucleoside transport with nitrobenzylthioinosine reduces MeP-dR mediated cell killing but not MeP cell killing. These mechanisms differ fundamentally from those previously reported for the HSV-TK gene.

In vivo sensitization of ovarian tumors to chemotherapy by expression of E. coli purine nucleoside phosphorylase in a small fraction of cells

“…For example, it has been shown in vitro that the fraction of cells expressing E. coli PNP is a much less important predictor of tumor cell killing than the overall levels of enzymatic activity within the entire tumor cell population, since both prodrug and toxin are membrane permeant. 2,3,5 In other words, very high level PNP expression in a small number of tumor cells might liberate enough toxin to produce measurable antitumor effects.…”

“…The results in Figure 2 are attributable to sevenfold greater expression of E. coli PNP by the pXP-HIV-PNP/pSV-TAT (compared with pSV-PNP) and support previous studies demonstrating that in vitro bystander killing by E. coli PNP can be explained by the levels of enzyme expressed. 1,5 Human ovarian tumors expressing high levels of E. coli PNP were also studied after implantation into SCID mice. Mice expressing E. coli PNP in their tumors demonstrated prolonged survival and sig- nificantly less tumor tissue after treatment with MeP-dR.…”

“…coli PNP differs fundamentally from the HSVtk/ganciclovir system, since direct cell-to-cell contact or gap junctions are not required for E. coli PNP to kill cells. [1][2][3][4][5] The PNP enzyme cleaves MePdR to liberate 6-methyl purine, (MeP), a potent, membrane permeant and freely diffusible toxin. Strong bystander killing is a hallmark of this approach in human colon, melanoma, and glioma cancer cells in vitro.…”

“…Strong bystander killing is a hallmark of this approach in human colon, melanoma, and glioma cancer cells in vitro. [1][2][3][5][6][7][8][9] Furthermore, because MeP inhibits protein, RNA, and DNA synthesis, the compound kills both quiescent and proliferating tumor cells. 6 Tumor cell killing can be evoked in a specific fashion through tumor specific regulatory elements.…”

Potential therapeutic applications of recombinant, invasive E. coli

“…This compound can be metabolized into the toxin 6-methyl purine (MeP) by the product of the E. coli gene deoD: purine nucleoside phosphorylase (PNP). [20][21][22] Therefore, we hypothesized that bacterial invasion could sensitize Caco-2 cells to 6-MPDR. Caco-2 cells were invaded for 2 h with E. coli BM2710 pGB2Oinv or E. coli BM2710 pGB2Oinv-hly at an MOI of 100.…”