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Metabolism and Actions of 2-Chloro-9-(2-deoxy-2-fluoro-ß-D-arabinofuranosyl)-adenine in Human Lymphoblastoid Cells¹

Department of Clinical Investigation, The University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030

2-Chloro-9-(2-deoxy-2-fluoro-ß-D-arabinofuranosyl)adenine (Cl-F-ara-A) is a new deoxyadenosine analogue that is resistant to phosphorolytic cleavage and deamination. Studies with a variety of cell lines demonstrated that Cl-F-ara-A is a potent cytotoxic agent; in cell-free systems, its triphosphate (Cl-F-ara-ATP) inhibited DNA polymerase and ribonucleotide reductase. To further characterize its mechanism of cytotoxicity, the present study investigated the cellular metabolism of Cl-F-ara-A and the actions of its nucleotide metabolites in human T-lymphoblast leukemia CCRF-CEM cells. The mono-, di-, and triphosphates of Cl-F-ara-A accumulated in cells, with the monophosphate as its major metabolite. After washing cells into drug-free medium, the elimination of each Cl-F-ara-A nucleotide was nonlinear with a prolonged terminal phase. Incubation of CCRF-CEM cells with Cl-F-ara-A resulted in the incorporation of Cl-F-ara-AMP into DNA; a much lesser amount was associated with RNA, suggesting that Cl-F-ara-A is a more DNA-directed compound. The site of Cl-F-ara-AMP in DNA was related to the ratio of the cellular concentrations of the analogue triphosphate and the natural substrate dATP. At low Cl-F-ara-ATP:dATP values, incorporation was mainly in phosphodiester linkages at internal sites, whereas at higher Cl-F-ara-ATP:dATP values, Cl-F-ara-AMP was principally detected at terminal sites. Clonogenicity assays showed a strong inverse correlation between cell survival and Cl-F-ara-AMP incorporation into DNA. These results suggest that the incorporation of Cl-F-ara-A monophosphate into DNA is critical for the cytotoxicity of Cl-F-ara-A.

¹ This work was supported in part by Grant DHP-1 from the American Cancer Society and Grant CA28596 from the National Cancer Institute, Department of Health and Human Services. C. X. is a Rosalie B. Hite predoctoral fellow.

² To whom requests for reprints should be addressed, at Department of Clinical Investigation, Box 71, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030.

Received 2/28/95. Accepted 5/19/95.

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