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Resistance to Cyclopentenylcytosine in Murine Leukemia L1210 Cells

Laboratory of Medicinal Chemistry, Developmental Therapeutics Program, Division of Cancer Treatment, National Cancer Institute, NIH, Bethesda, Maryland 20892

¹ To whom requests for reprints should be addressed, at Bldg. 37, Rm. 5B22, National Cancer Institute, NIH, Bethesda, MD 20892.

Cyclopentenyl cytosine (CPEC) exhibits oncological activity in murine and human tumor cells and has now entered Phase I clinical trials. Its mode of action as an antitumor agent appears to be inhibition by its triphosphate (CPEC-TP) of CTP synthase, the enzyme which converts UTP to CTP. In an attempt to elucidate the mechanism of resistance to CPEC, a murine leukemia cell line resistant to CPEC (L1210/CPEC) was developed by N-methyl-N-nitro-N-nitrosoguanidine-induced mutagenesis and subsequent selection by cultivation of the L1210 cells in the presence of 2 µM CPEC. Resistant clones were maintained in CPEC-free medium for 6 generations before biochemical studies were performed. The resistant clone selected for further studies was 13,000-fold less sensitive to growth inhibition by CPEC than the parental cells, and the concentration of CPEC required to deplete CTP in the resistant cells was 50-fold higher than in the sensitive cells.

A comparison of the kinetic properties of CTP synthase from sensitive and resistant cells indicated alteration in the properties of the enzyme from the latter; the median inhibitory concentration for CPEC-TP increased from 2 to 14 µM, K_m for UTP decreased from 126 to 50 µM, and V_max increased 12-fold from 0.2 to 2.3 nmol/mg/min. Northern blot analyses of polyadenylated RNA from the resistant and sensitive cells indicated a 3-fold increase in transcripts of the CTP synthase gene in the resistant line. Consistent with these alterations in the properties of the enzyme, the resistant cells exhibited significantly expanded CTP and dCTP pools (4–5-fold) when compared with the sensitive cells. No change was observed, however, in the properties of uridine-cytidine kinase, the enzyme responsible for the initial phosphorylation of CPEC; despite this, however, cellular uptake of CPEC was greatly decreased, and phosphorylation of CPEC and its incorporation into RNA were 10-fold less than in the parental cells. These latter observations are most readily explained by feedback inhibition by the increased CTP levels of the resistant cells of uridinecytidine kinase and/or of the membrane transport process used for initial entry of CPEC.

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Received 9/ 2/92. Accepted 9/30/93.

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