Pharmacogenomics of Gemcitabine Metabolism: Functional Analysis of Genetic Variants in Cytidine Deaminase and Deoxycytidine Kinase

Abstract

Gemcitabine (dFdC, 29, is metabolized by cytidine deaminase (CDA) and deoxycytidine kinase (DCK), but the contribution of genetic variation in these enzymes to the variability in systemic exposure and response observed in cancer patients is unclear. Wild-type enzymes and variants of CDA (Lys27Gln and Ala70Thr) and DCK (Ile24Val, Ala119Gly, and Pro122Ser) were expressed in and purified from Escherichia coli, and enzyme kinetic parameters were estimated for cytarabine (Ara-C), dFdC, and its metabolite 29,29-difluorodeoxyuridine (dFdU) as substrates. All three CDA proteins showed similar K m and V max for Ara-C and dFdC deamination, except for CDA70Thr, which had a 2.5-fold lower K m and 6-fold lower V max for Ara-C deamination. All four DCK proteins yielded comparable metabolic activity for Ara-C and dFdC monophosphorylation, except for DCK24Val, which demonstrated an approximately 2-fold increase (P < 0.05) in the intrinsic clearance of dFdC monophosphorylation due to a 40% decrease in K m (P < 0.05). DCK did not significantly contribute to dFdU monophosphorylation. In conclusion, the Lys27Gln substitution does not significantly modulate CDA activity toward dFdC, and therefore would not contribute to interindividual variability in response to gemcitabine. The higher in vitro catalytic efficiency of DCK24Val toward dFdC monophosphorylation may be relevant to dFdC clinical response. The substrate-dependent alterations in activities of CDA70Thr and DCK24Val in vitro were observed for the first time, and demonstrate that the in vivo consequences of these genetic variations should not be extrapolated from one substrate of these enzymes to another.

CDA Gene Polymorphisms and Enzyme Activity: Genotype–phenotype Relationship in an Italian–Caucasian Population

“…However, a very recent study [36], using purified recombinant proteins instead of the crude cell extracts from transiently overexpressed COS-1 cells used by Gilbert et al [16], reported no differences between the K27 and Q27 recombinant variants in kinetic parameters for ara-C and dFdC. Also, Kirch et al [37] reported a 1.3-to 3.3-fold higher in vitro deamination rate of ara-C for the recombinant K27 variant compared with Q27 variant.…”

Cytidine Deaminase as a Molecular Predictor of Gemcitabine Response in Patients with Biliary Tract Cancer

“…Three SNPs in the CDA-encoding gene, A79C (Lys27Gln), G208A (Ala70Thr), and C435T (Thr145Thr), have been correlated with differential clinical outcomes [14,15,16,17], but the data are inconclusive and conflicting. For example, recombinant enzymes harboring the Gln27 and Thr70 polymorphisms showed reduced activity, with increased K m values for gemcitabine [14,18]. However, the minor allele of this SNP was associated with higher enzymatic activity for gemcitabine in tests performed using lysates of red blood cells taken from Caucasian cancer patients [16,19].…”

PharmGKB summary

“…One in vitro study reports that the A allele is associated with decreased CDA enzymatic activity, and a second in vitro study reports that the A allele is associated with decreased CDA activity towards AraC, another deoxycytidine analog, but not towards gemcitabine [26, 36]. Although this variant has not been extensively studied the evidence of an association between it and CDA enzymatic activity is compelling.…”

“…In vivo studies show an association between decreased DCK protein levels with shorter progression free survival in patients treated with gemcitabine [14, 41, 42]. Despite some well-established correlations between DCK protein levels with resistance to gemcitabine, no SNPs in DCK have shown a consistent association with patient response, or risk of toxicity in clinical studies [21, 22, 26, 43]. The results of those studies are summarized in Table 6.…”