Metabolism and RNA incorporation of cyclopentenyl cytosine in human colorectal cancer cells.
We studied the cytotoxicity and metabolism of the investigational cytidine analogue cyclopentenyl cytosine (CPE-C) in three human colorectal cancer cell lines: HCT 116, SNU-C4, and NCI-H630. CPE-C potently inhibited cell growth and decreased clonogenic capacity at concentrations achieved in murine and primate pharmacologic studies. CPE-C produced a concentration-dependent depletion of CTP, accompanied by changes in the dCTP pools. CPE-C exposure was associated with an accumulation of cells in the S phase at 48 hr. [3H]CPE-C was metabolized predominantly to the triphosphate (CPE-CTP) form. Saturation of phosphorylation to the monophosphate form occurred above 5-10 microM. Plateau CPE-CTP pools were of a magnitude similar to that of the physiologic ribonucleotide triphosphate pools. The intracellular half-life of CPE-CTP was 24 hr. After a 24-hr exposure to 0.5 microM CPE-C, CPE-CTP was detected for up to 96 hr post-drug removal, accompanied by persistent depletion of the CTP pools. Cesium sulfate density centrifugation of purified nucleic acids indicated that [3H]CPE-C incorporated into RNA, but was not detected in DNA. Agarose-gel electrophoresis of RNA from [3H]CPE-C-treated cells indicated that it localized predominantly in low molecular weight (4-8 S) RNA species. When CPE-C was administered concurrently with [3H]adenosine (Ado), the proportion of [3H]Ado migrating with low molecular weight RNA species increased. Concurrent exposure to 10 microM cytidine (Cyd), sufficient to replete CTP pools, provided essentially complete protection against lethality resulting from a 24-hr exposure to less than or equal to 0.5 microM CPE-C. While 10 microM Cyd substantially decreased CPE-CTP formation and CPE-C-RNA incorporation during the initial 3 hr of exposure compared to CPE-C alone, after 24 hr the levels were not significantly different. Cyd rescue did not affect the accumulation of [3H]CPE-C or [3H]Ado into low molecular weight RNA species after a 24-hr exposure to CPE-C. Our results indicate that depletion of CTP and dCTP pools is an important component of CPE-C cytotoxicity. While CPE-C incorporation into RNA may not be the critical cytotoxic event during a 24-hr exposure to CPE-C, it may play a role during prolonged exposure to CPE-C. CPE-C is a highly potent new agent and merits clinical evaluation in the treatment of colorectal cancer.
Yee
L K
LK
Medicine Branch, National Cancer Institute, Bethesda, MD 20892.
Allegra
C J
CJ
Trepel
J B
JB
Grem
J L
JL
D000241:Adenosine / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D015179:Colorectal Neoplasms / Q000378:metabolism; D003562:Cytidine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology; D004273:DNA, Neoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D007938:Leukemia / Q000378:metabolism; D000432:Methanol; D012334:RNA, Neoplasm / Q000378:metabolism*; D012265:Ribonucleotides / Q000378:metabolism; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000187:drug effects
D004273:DNA, Neoplasm; D012334:RNA, Neoplasm; D012265:Ribonucleotides; D003562:Cytidine; C045911:cyclopentenyl cytosine; D000241:Adenosine; D000432:Methanol