Transfer of the bacterial gene for cytosine deaminase to mammalian cells confers lethal sensitivity to 5-fluorocytosine: a negative selection system.

Abstract

Expression of the bacterial gene for cytosine deaminase (CD; EC 3.5.4.1) in mammalian cells was evaluated as a negative selection system or suicide vector for potential use in gene transfer studies and therapies. Mammalian cells, unlike certain bacteria and fungi, do not contain the enzyme CD and do not ordinarily metabolize cytosine to uracil. Nor do they metabolize the innocuous compound 5-fluorocytosine to the highly toxic compound 5-fluorouracil. The Escherichia coli CD gene underwent PCR oligonucleotide-directed mutagenesis to enhance its expression in a eukaryotic system and it was then cloned into an expression vector, pLXSN, that also contains a neomycin-resistance gene. Murine fibroblast lines were transfected with the plasmid and subjected to brief selection in the neomycin analogue G418. Lysates from these cell populations exhibited significant CD activity detected by conversion of radiolabeled cytosine to uracil. In clonogenic assays transfected cells expressing CD were selectively killed by incubation in 5-fluorocytosine, whereas control cell lines were not. Doseresponse studies evaluating [3Hlthymidine incorporation or cloning efficiency demonstrated profound inhibition at and above 65

Gene therapy in the CNS

“…205 This prodrug activation mode continues to be a mainstay of brain tumor therapy, but is limited by the antigenic nature of the gene product, which can lead to nonspecific or premature death of cells expressing it, and by the non-diffusibility of the nucleotide analogue, such that it can only be transferred between cells by gap junctions. In the intervening years the number of prodrug activation/drug enhancement strategies has expanded rapidly to include, for example: bacterially derived cytosine deaminase activation of 5-fluorocytosine to 5-fluorouracil, the latter being a commonly used chemotherapeutic agent, 206 which acts synergistically with ganciclovir; 207 mammalian cytochrome P450 2B1 activation of cyclophosphamide, a chemotherapeutic agent normally activated in the liver, but with poor transfer of the active metabolites across the bloodtumor barrier; 208 and the mammalian deoxycytidine kinase gene that activates cytosine arabinoside. 209 Antiangiogenesis inhibitors can be used to block the growth of tumors, with examples including an antagonist for Tie2 210 and a dominant-negative acting VEGF receptor.…”

Development and application of adenoviral vectors for gene therapy of cancer

“…The principle of this approach has been shown in several systems by transferring or transducing the gene for a relevant prodrug enzyme to tumor cells in vitro and then demonstrating the suppression of growth of the genetically modified cells in vitro and in vivo. [111][112][113][114][115][116][117][118] Herpes simplex virus-thymidine kinase (HSV-tk) is the most representative prodrug enzyme studied thus far; HSV-tk converts the nontoxic nucleotide analog ganciclovir (GCV) into a phosphorylated compound that acts as a chain terminator in DNA synthesis, selectively killing dividing cells. The therapeutic efficacy of Ad-mediated HSV-tk gene after in vivo transduction of rat C 6 glioma cells followed by GCV administration was studied in tumors generated in the brains of nude mice.…”

“…The cancer cells engineered with the cytosine deaminase gene have been shown to have increased sensitivity to treatment with 5-FC either in vitro or in vivo. 115,116,122 To transduce the cytosine deaminase gene effectively to the tumor, an Ad vector was chosen for construction of AdCMV.CD. 123 The AdCMV.CD vector suppressed HT29 cell growth in vitro in the presence of 5-FC in a dose-dependent manner.…”

Therapeutic effect of suicide gene-transferred mesenchymal stem cells in a rat model of glioma

“…12,13 Expression of the CD gene within the target cell produces an enzyme that converts the prodrug, 5-FC, to the toxic metabolite, 5-fluorouracil (5-FU). The use of 5-FC may be particularly suitable for brain tumors, because it can readily cross the blood--brain barrier.…”