6-methylpurine-2--deoxyriboside and 6-methylpurine

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 non-expressing 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.

Topics: Affinity Labels; Animals; Cell Communication; Cell Death; Cell Division; Culture Media; Escherichia coli; Humans; Mice; Prodrugs; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Purines; Thioinosine; Tumor Cells, Cultured

6MPDR causes the death of cells lightly infected with mycoplasmas. This is brought about by the action of the mycoplasmal enzyme adenosine phosphorylase which converts 6MPDR to highly toxic metabolites. If the medium conditions are adjusted to favour the growth of mycoplasmas by the addition of pig serum to the medium, infections as low as 1 mycoplasma per 200,000 cells can be detected within 7 days. This finding enables mycoplasma tests to be carried out rapidly and reliably by untrained personnel.

Topics: Cell Survival; Cells, Cultured; Culture Media; Deoxyribonucleosides; Humans; Mycoplasma; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Purines

Topics: Animals; Bacillus subtilis; Cells, Cultured; Fibroblasts; Mice; Mice, Inbred Strains; Mycoplasma; Pentosyltransferases; Purine Nucleosides; Purine-Nucleoside Phosphorylase; Purines

Topics: Animals; Cell Line; Leishmania; Macrophages; Mice; Purine Nucleosides; Purines; Spectrophotometry, Ultraviolet