Cytosine deaminase as an enzyme for monitoring suicide gene therapy

Introduction
Besides the herpes simplex virus type-1 thymidine kinase (HSV-1 TK), cytosine deaminase (CD) presents another system for gene therapy. CD catalyses the hydrolytic deamination of cytosine to form uracil. Its lack in mammalian cells and the ability to convert 5-fluorocytosine to the cell-toxic 5-fluorouracil makes it suitable for use in several gene therapy protocols [1,2]. An appropriate radiolabelled cytosine derivative would make it possible to monitor gene expression with positron emission tomography (PET), providing a sufficient amount of the tracer is trapped in the tumor cells. For this purpose, several cytosine derivatives were tested.

Results
CD was obtained by over-expression and following purification from transformed E. coli bacteria in good yields. The specific activity and K_M were determined using cytosine and 5-fluorocytosine. Some compounds (listed in table 1) were incubated in tris-buffer solution containing the isolated enzyme. Samples were taken and the rate of metabolites formation determined by HPLC.

For cytosine, the specific activity of 38 U/mg enzyme (one unit is the amount of enzyme catalysing the deamination of 1 µmol substrate per minute at 25°C) and a K_M of 0.63 mM falls in the range of further data published [3,4]. For 5-fluorocytosine, the decrease in specific activity (2.1 U/mg protein) and the increase of K_M (5.2 mM) of one dimension is also in good agreement with published data [1]. Additionally, it was shown that the CD tolerates some structural variations of its possible substrates. Beside substances which are varied at the 5-position (5-fluorocytosine, 5-methylcytosine), N-substituted derivatives like N-methylcytosine and N-hydroxycytosine are also accepted. This results offer a number of fluorine-18 labelled compounds as candidates for monitoring gene therapy.

Table 1
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Turnover to the corresponding product in % after
Compound............10 min.....30 min.....60 min
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Cytosine...............88.84......100..........100
5-Fluorocytosine......9.39.......22.42........28.61
5-Methylcytosine.....1.07.........5.94........10.25
N-Methylcytosine....1.47..........5.22.........8.53
N-Hydroxycytosine.20.11.......31.23........42.88

References
[1] Rowley, S. et al. J. Surgical Oncology 1996, 61, 42-48.
[2] Haberkorn, U. et. al. J. Nucl. Med. 1996, 37, 87-94.
[3] Ipata, P. L. Methods Enzymol. 1978, 51, 394-401.
[4] Balestreri, E. et al. Biochim Biophys Acta 1973, 315, 443-448.