MECHANISM OF RESISTANCE TO CYCLOPENTENYL CYTOSINE (CPE-C) IN MOLT-4 LYMPHOBLASTS

Cyclopentenyl cytosine (CPE-C), a carbocyclic analogue of cytidine. ha s preclinical anti-neoplastic activity against ara-C resistant murine leukemias and a broad spectrum of human tumor xenografts. CPE-C is a p rodrug and requires intracellular phosphorylation to cyclopentenyl cyt osine triphosphate (CPE-CTP) which depletes endogenous CTP pools. The initial step in this activation process is catalyzed by uridine/cytidi ne kinase. We studied the mechanism of resistance to CPE-C in a Molt-4 T-cell leukemia line made resistant to CPE-C (Molt-4R) by culturing i t in the continuous presence of increasing concentrations of CPE-C. Us ing a tetrazolium based colorimetric assay to assess cytotoxicity, the IC90 for the parent Molt-4 cells (Molt-4WT) was 0.5 muM after a 24 hr drug exposure. In contrast, cytotoxicity was not observed at concentr ations as high as 1 mM in the Molt-4R cells. Following a brief exposur e to 1 muM CPE-C, parent drug could be detected intracellularly in the resistant and sensitive cell lines. However, CPE-CTP formation was re duced markedly in the resistant cell line. Measurement of the activity of anabolic and catabolic enzymes in the Molt-4WT and Molt-4R cells r evealed equivalent activities of alkaline and acid phosphatases as wel l as cytidine and dCMP deaminase but there was a significant reduction in uridine/cytidine kinase activity in Molt-4R cells. Endogenous ribo nucleotide pools and CPE-CTP pools were measured in the absence and pr esence of CPE-C. CTP pools were reduced markedly in Molt-4WT cells fol lowing exposure to CPE-C. However, CTP pools in Molt-4R cells exposed to 100 muM CPE-C were two times greater than in the untreated Molt-4WT cells. At high concentrations of CPE-C (10 and 100 muM), Molt-4R cell s were able to generate amounts of CPE-CTP equivalent to that seen in Molt-4WT cells exposed to 1 muM CPE-C (a cytotoxic concentration of dr ug in Molt-4WT cells), but no cytotoxic effect was seen in Molt-4R cel ls. Therefore, in addition to decreased uridine/cytidine kinase activi ty, a second mechanism of resistance that is the result of alterations in CTP synthetase activity also appears to be operative. Elucidation of the mechanism of resistance in vitro may provide insight into the m echanism of action of the drug and potential mechanisms of resistance in vivo.