Regulation of phosphorylation of deoxycytidine and 2',2'-difluorodeoxycytidine (gemcitabine); effects of cytidine 5'-triphosphate and uridine 5'-triphosphate in relation to chemosensitivity for 2',2'-difluorodeoxycytidine.
Deoxycytidine kinase(dCK) and deoxycytidine deaminase (dCDA) are two key enzymes in the activation and inactivation, respectively, of deoxycytidine and its antiviral and anticancer analogues. One purpose of this study was to determine whether or not the deoxycytidine-converting activity of both enzymes would correlate with growth inhibition by 2',2'-difluorodeoxycytidine (dFdC), a deoxycytidine analogue with established antitumour activity in solid tumours. Another aim of this work was to determine the effects of normal nucleotides on dCK. dCK and dCDA activities were measured with both deoxycytidine and dFdC as substrates in 5 solid tumour cell lines, but no correlation with cellular sensitivity to dFdC was found with either substrate. The normal dCK activities with deoxycytidine as substrate varied between 0.8 and 13 nmol/hr/10(6) cells. The activities determined with dFdC as substrate were remarkably similar in all 5 cell lines (1.1-1.6 nmol/hr/10(6) cells). dCDA activities varied considerably with both substrates (20-30 fold). Because dFdC markedly affected intracellular concentrations of cytidine 5'-triphosphate (CTP) and uridine 5'-triphosphate (UTP), we studied their effect on deoxycytidine-and dFdC-phosphorylating activities in 3 cell lines (i.e., A2780, WiDr and C26-10) with similar dCK activity but major differences in dFdC sensitivity, 1 mM CTP inhibited deoxycytidine phosphorylation (at 230 muM) by 20-30% in A2780 and C26-10 cells, but increased that of WiDr cells by approximately 70%. CTP did not++ affect dFdC phosphorylation (at 230 muM) in A2780 cells, but did increase it by 40% in WiDr cells. At 1 and 10 muM of deoxycytidine the effects of CTP on dCK activity in A2780, C26-10 and WiDr cells were less pronounced. 1mM UTP enhanced deoxycytidine phosphorylation at 230 muM in WiDr cells by approximately 40%, whereas dFdC phosphorylation was increased 40% by UTP in C26-10 cells but decreased by 70-80% in WiDr cells. UTP caused a more pronounced increase in dCK activity at 1 and 10 muM deoxycytidine in C26-10 cells, but provoked a higher inhibition in A2780 and WiDr Cells at 10 muM. Because of these complex results, dCK kinetics were studied in greater detail. Biphasic kinetics for deoxycytidine were observed in all 3 cell lines, with Km values of 23.2 and 0.4 muM for A2780 cells, 15.9 and 1.5 muM for C26-10 cells, and 27.2 and 0.9 muM for WiDr cells. In all 3 cell lines, adenosine 5'-triphosphate (ATP) was the optimal phosphate donor, as compared to CTP and UTP. In conclusion, the efficiency of dCK (Vmax/Km ratio) seems to correlate with accumulation of dFdCTP, the active metabolite of dFdC, and with cellular sensitivity. UTP and CTP, which are seriously affected in cells exposed to dFdC, display varying effects in these solid tumour cell lines. Both activation and inhibition have been observed; the physiologically low CTP pools and the relatively minor effect on dCK in A2780 cells seem to favour dFdC phosphorylation in these cells, which are the most sensitive.
van Haperen
V W
VW
Department of Medical Oncology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands.
Veerman
G
G
Vermorken
J B
JB
Pinedo
H M
HM
Peters
G
G
D003564:Cytidine Deaminase; D003570:Cytidine Triphosphate / Q000494:pharmacology*; D003841:Deoxycytidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D003842:Deoxycytidine Kinase / Q000187:drug effects*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D006801:Humans; D007700:Kinetics; D009700:Nucleoside Deaminases / Q000187:drug effects*; D010051:Ovarian Neoplasms / Q000188:drug therapy; D010766:Phosphorylation; D014407:Tumor Cells, Cultured; D014544:Uridine Triphosphate / Q000494:pharmacology*; D000093542:Gemcitabine
D004791:Enzyme Inhibitors; D003841:Deoxycytidine; D003570:Cytidine Triphosphate; D003842:Deoxycytidine Kinase; D009700:Nucleoside Deaminases; D003564:Cytidine Deaminase; C011530:deoxycytidine deaminase; D014544:Uridine Triphosphate; D000093542:Gemcitabine