Gemcitabine pharmacogenomics: deoxycytidine kinase and cytidylate kinase gene resequencing and functional genomics.
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 K(m) 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 (R(p) = 0.89, P = 0.0004) but not for CMPK (R(p) = 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.
Department of Molecular Pharmacology and Experimental Therapeutics, Mayo Clinic, Rochester, Minnesota 55905, USA.
Neslihan Aygun
NA
Kocabas
Pinar
P
Aksoy
Linda L
LL
Pelleymounter
Irene
I
Moon
Jeong-Seon
JS
Ryu
Judith A
JA
Gilbert
Oreste Ezequel
OE
Salavaggione
Bruce W
BW
Eckloff
Eric D
ED
Wieben
Vivien
V
Yee
Richard M
RM
Weinshilboum
Matthew M
MM
Ames
D000970:Antineoplastic Agents / Q000494:pharmacology*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D003842:Deoxycytidine Kinase / Q000737:chemistry / Q000235:genetics*; D006239:Haplotypes; D006801:Humans; D007700:Kinetics; D015810:Linkage Disequilibrium; D008958:Models, Molecular; D009703:Nucleoside-Phosphate Kinase / Q000737:chemistry / Q000235:genetics*; D010597:Pharmacogenetics; D000093542:Gemcitabine
D000970:Antineoplastic Agents; D003841:Deoxycytidine; D003842:Deoxycytidine Kinase; C019979:cytidylate kinase; D009703:Nucleoside-Phosphate Kinase; D000093542:Gemcitabine