Gemcitabine pharmacogenomics: cytidine deaminase and deoxycytidylate deaminase gene resequencing and functional genomics.

ABSTRACT

PURPOSE: Gemcitabine is a nucleoside analogue with activity against solid tumors. Gemcitabine metabolic inactivation is catalyzed by cytidine deaminase (CDA) or, after phosphorylation, by deoxycytidylate deaminase (DCTD). We set out to study the pharmacogenomics of CDA and DCTD. EXPERIMENTAL DESIGN: The genes encoding CDA and DCTD were resequenced using DNA from 60 African American and 60 Caucasian American subjects. Expression constructs were created for nonsynonymous coding single nucleotide polymorphisms (cSNP) and reporter gene constructs were created for 5'-flanking region polymorphisms. Functional genomic studies were then conducted after the transfection of mammalian cells. RESULTS: CDA resequencing revealed 17 polymorphisms, including one common nonsynonymous cSNP, 79 A>C (Lys27Gln). Recombinant Gln27 CDA had 66 +/- 5.1% (mean +/- SE) of the wild-type (WT) activity for gemcitabine but without a significant decrease in level of immunoreactive protein. The apparent Km (397 +/- 40 micromol/L) for the Gln27 allozyme was significantly higher than that for the WT (289 +/- 20 micromol/L; P < 0.025). CDA 5'-flanking region reporter gene studies showed significant differences among 5'-flanking region haplotypes in their ability to drive transcription. There were 29 SNPs in DCTD, including one nonsynonymous cSNP, 172 A>G (Asn58Asp), in Caucasian American DNA. Recombinant Asp58 DCTD had 11 +/- 1.4% of WT activity for gemcitabine monophosphate with a significantly elevated level of immunoreactive protein. No DCTD polymorphisms were observed in the initial 500 bp of the 5'-flanking region. CONCLUSIONS: These results suggest that pharmacogenomic variation in the deamination of gemcitabine and its monophosphate might contribute to variation in therapeutic response to this antineoplastic agent.