Abstract
Cytosine deaminase (EC 3.5.4.1), a non-mammalian enzyme, catalyzes the deamination of cytosine and 5-fluorocytosine to form uracil and 5-fluorouracil, respectively. Eukaryotic cells have been genetically modified with a bacterial cytosine deaminase gene to express a functional enzyme. When the genetically modified cells are combined with 5-fluorocytosine, it creates a potent negative selection system, which may have important applications in cancer gene therapy. In this paper, we introduce a novel positive selection method based upon the expression of the cytosine deaminase gene. This method utilizes inhibitors in the pyrimidine de novo synthesis pathway to create a condition in which cells are dependent on the conversion of pyrimidine supplements to uracil by cytosine deaminase. Thus, only cells expressing the cytosine deaminase gene can be rescued in a positive selection medium.
Highlights
The current challenge for the successful clinical exploitation of this approach is to routinely achieve cytosine deaminase (CDase) gene transfer at the required specific activity in a solid tumor mass
We have attempted to make mammalian cells depend upon CDase activity by blocking de novo pyrimidine synthesis
We have explored the combination of PALA and CDase as a positive selection method for cells expressing CDase
Summary
Cytosine deaminase (EC 3.5.4.1), a non-mammalian enzyme, catalyzes the deamination of cytosine and 5-fluorocytosine to form uracil and 5-fluorouracil, respectively. We introduce a novel positive selection method based upon the expression of the cytosine deaminase gene. This method utilizes inhibitors in the pyrimidine de novo synthesis pathway to create a condition in which cells are dependent on the conversion of pyrimidine supplements to uracil by cytosine deaminase. We have attempted to make mammalian cells depend upon CDase activity by blocking de novo pyrimidine synthesis Once blocked, these cells will depend on the activity of CDase to convert extracellular cytosine into uracil for growth. Such a system makes it possible to use the CDase gene as a positive selection marker gene
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