Cellular pharmacology of cyclopentenyl cytosine in Molt-4 lymphoblasts.
The toxicity, uptake, and metabolism of the oncolytic nucleoside cyclopentenyl cytosine (CPEC) have been examined in the Molt-4 line of human lymphoblasts. This compound is known to be converted to its 5'-triphosphate, which inhibits CTP synthetase and depletes the pools of cytidine nucleotides. In the Molt-4 system, the concentration of drug reducing proliferation by 50% in a 24-h incubation was between 50 and 100 nM. Cytidine, uridine, and nitrobenzylthioinosine almost fully prevented the cytotoxicity of CPEC when introduced shortly before or together with the drug, but only cytidine was effective as an antidote when added 12 h after 200 nM CPEC. Studies of the cellular entry of CPEC revealed that nitrobenzylthioinosine fully blocked this process over a 60-s interval and for as long as 2 h, suggesting that the initial interiorization was mediated by facilitated diffusion. In Molt-4 cells incubated with tritiated CPEC, 9 metabolites could be distinguished: prominent among these was cyclopentenyl uridine (CPEU), the deamination product of CPEC; other major metabolites included the 5'-mono-, di-, and triphosphates of CPEC, and of CPEU, along with two phosphodiesters provisionally identified as CPEC-diphosphate choline and CPEC-diphosphate ethanolamine. When the accumulation of CPEC-5'-triphosphate was measured as a function of concentration of the drug in the medium, the process was found not to be saturable by levels of CPEC up to 1000 nM. In cells incubated with 200 nM drug, CPEC-5'-triphosphate accumulated rapidly and linearly for approximately 4 h, the time for doubling of the concentration being 2 h. After a 16-h incubation with 100 nM CPEC, the concentration of CPEC-5'-triphosphate was 50-fold that of the parent drug in the medium and could be readily monitored spectrophotometrically in high-pressure liquid chromatography effluents without recourse to radiolabeled nucleoside. In 2-h incubations, the concentration of free CPEC required to reduce CTP by 50% was 150 nM; this corresponded to a CPEC-5'-triphosphate level of 750 nM. After washout of extracellular CPEC, CPEC-5'-triphosphate decayed with a half-life that ranged from 9 to 14 h. Twenty-four h after washout of 200 nM CPEC (the concentration of drug reducing proliferation by 80%), cells had not resumed proliferation, and CTP pools were still depressed by 90%. Cytidine, uridine, and nitrobenzylthioinosine all strongly repressed the anabolic phosphorylation of CPEC when added to Molt-4 cells along with the drug.(ABSTRACT TRUNCATED AT 400 WORDS)
Ford
H
H
Laboratory of Medicinal Chemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Cooney
D A
DA
Ahluwalia
G S
GS
Hao
Z
Z
Rommel
M E
ME
Hicks
L
L
Dobyns
K A
KA
Tomaszewski
J E
JE
Johns
D G
DG
D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology*; D002460:Cell Line; D003562:Cytidine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D003570:Cytidine Triphosphate / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D006801:Humans; D008214:Lymphocytes / Q000378:metabolism*; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism
D000970:Antineoplastic Agents; D003562:Cytidine; D012313:RNA; D003570:Cytidine Triphosphate; C045911:cyclopentenyl cytosine; D004247:DNA