Abstract
Gemcitabine and other cytidine antimetabolites require metabolic activation by phosphorylation. Deoxycytidine kinase (DCK) and cytidine monophosphate kinase (CMPK) catalyze these reactions. We have applied a genotype-to-phenotype strategy to study DCK and CMPK pharmacogenomics. Specifically, we resequenced DCK and CMPK using 240 DNA samples, 60 each from African-American, Caucasian-American, Han Chinese-American, and Mexican-American subjects. We observed 28 DCK polymorphisms and 28 polymorphisms in CMPK, 33 of which were novel. Expression in COS-1 cells showed that variant allozyme enzyme activities ranged from 32 to 105% of the wild type (WT) for DCK and from 78 to 112% of WT for CMPK—with no significant differences in apparent Km values for either enzyme except for a DCK Val24/Ser122 double variant allozyme. Relative levels of DCK and CMPK immunoreactive protein in the COS-1 cells paralleled relative levels of enzyme activity and were significantly correlated for DCK (Rp = 0.89, P = 0.0004) but not for CMPK (Rp = 0.82, P = 0.095). The results of an analysis of DCK and CMPK structural models were compatible with the observed functional consequences of sequence alterations in variant allozymes. We also confirmed that the CMPK protein expressed in COS-1 cells and in a rabbit reticulocyte lysate was 196 rather than 228 amino acids in length. In summary, we determined common sequence variations in DCK and CMPK and systematically evaluated their functional implications. These gene sequence differences may contribute to variations in the metabolic activation of gemcitabine and other cytidine antimetabolites.
Footnotes
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This work was supported in part by National Institutes of Health Grants R01 GM28157, R01 GM35720, R01 CA132780, and U01 GM61388 (The Pharmacogenetics Research Network), by a PhRMA Foundation “Center of Excellence in Clinical Pharmacology” Award, and by a grant from the Commonwealth Foundation for Cancer Research. The gene resequencing data described in this article have been deposited in the NIH-funded database PharmGKB with submission numbers PS207098 and PS207100.
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N.A.K. and P.A. contributed equally to this work.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.108.020925.
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ABBREVIATIONS: dFdC, 2′-deoxy-2′,2′-difluorocytidine, gemcitabine; AraC, cytosine arabinoside; DCK, deoxycytidine kinase; CMPK, cytidine monophosphate kinase; dFdCDP, gemcitabine diphosphate; dFdCTP, gemcitabine triphosphate; AA, African-American; CA, Caucasian-American; HCA, Han Chinese-American; MA, Mexican-American; SNP, single nucleotide polymorphism; cSNP, coding single nucleotide polymorphism; PCR, polymerase chain reaction; 5′-FR, 5′-flanking region; MAF, minor allele frequency; ORF, open reading frame; WT, wild type; kb, kilobase(s).
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The online version of this article (available at http://dmd.aspetjournals.org) contains supplemental material. -
↵1 Current affiliation: University of Gazi, Faculty of Pharmacy, Department of Toxicology, Ankara, Turkey.
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↵2 On leave of absence from Istanbul University, Pharmacy Faculty, Department of Biochemistry, Istanbul, Turkey.
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↵3 Current affiliation: Inha University Hospital, Incheon, South Korea.
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↵4 Current affiliation: Roswell Park Cancer Institute, Buffalo, New York.
- Received February 12, 2008.
- Accepted June 11, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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