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Proposed Mechanism of Therapeutic Selectivity for 9-ß-D-Arabinofuranosyl-2-fluoroadenine against Murine Leukemia Based upon Lower Capacities for Transport and Phosphorylation in Proliferative Intestinal Epithelium Compared to Tumor Cells¹

Memorial Sloan-Kettering Cancer Center and Sloan-Kettering Division, Graduate School of Medical Sciences, Cornell University, New York, New York 10021 [J. R. B., D. M. J., F. M. S.], and The Southern Research Institute, Birmingham, Alabama 35255 [C-H. C., R. W. B.]

Studies have examined transport and phosphorylation of 9-ß-D-arabinofuranosyl-2-fluoroadenine (F-Ara-A), a deaminase resistant adenosine analogue, as mechanisms that could mediate the observed therapeutic efficacy of this agent against murine tumor models. Earlier finds by Avramis and Plunkett (Cancer Res., 42: 2587–2591, 1982) showed markedly less accumulation in vivo of administered F-Ara-A as cytotoxic triphosphate in gastrointestinal mucosa and bone marrow compared to P388 cells. We have pursued the basis for this difference in vitro using L1210 ascites and proliferative epithelial cells (85–95% crypt cells) isolated from mouse small intestine as representative sample populations of drug-sensitive tumor and drug-limiting normal regenerative host tissue. Using a rapid sampling technique, linear initial rates of substrate uptake were established at 25°C for radiolabeled F-Ara-A and adenosine at a concentration range of 1–1000 µM. The relationship between velocity of initial transport and substrate concentration is indicative of Michaelis-Menten saturation kinetics for both substrates. Competition studies between F-Ara-A and adenosine suggest a common route of entry for both substrates in crypt epithelial cells. Results from double-reciprocal analysis of the velocity versus concentration data are consistent with a simple carrier-mediated facilitated diffusion process with K_m, V, and K_i values of 317 ± 44 (SE) µM, 49 ± 7 nmol/s/g dry weight, and 301 ± 34 µM for F-Ara-A, and 264 ± 14 µM, 44 ± 5 nmol/s/g dry weight, and 225 ± 44 µM for adenosine, respectively. The presence of a single lowaffinity carrier in the proliferative epithelial cells contrasts sharply with the high affinity (K_m, 68 ± 14 µM; V, 48 ± 4 nmol/s/g dry weight) and low-affinity (K_m, 326 ± 48 µM; V, 124 ± 44 nmol/s/g dry weight) routes of entry documented for L1210 cells. This differential in transport kinetics conveys a 7- to 8-fold greater capacity to L1210 ascites compared with crypt epithelial cells for uptake of the antitumor agent F-Ara-A. At pharmacologically achievable concentrations of F-Ara-A and in view of this differential, influx of F-Ara-A would be more rate limiting to phosphorylation of F-Ara-A in epithelial cells than in L1210 cells. Metabolism studies with L1210 ascites and proliferative intestinal epithelial cells show that intracellular phosphorylation of F-Ara-A is also elevated in L1210 cells. High-performance liquid chromatography analysis of extracts from cells incubated with 120 µM [³H]F-Ara-A showed substantial production of F-Ara-adenosine 5'-monophosphate, F-Ara-A-adenosine 5'-diphosphate, and the cytotoxic metabolite F-Ara-adenosine 5'-triphosphate in L1210 cells but little F-Ara-adenosine 5'-monophosphate, F-Ara-adenosine 5'-diphosphate, and no F-Ara-adenosine 5'-triphosphate in epithelial cells. Overall, the rate of phosphorylated product formation from [³H]F-Ara-A was 9- to 12-fold lower in proliferative intestinal epithelial cells than in L1210 cells. When resuspended in drugfree media, L1210 cells cleared [³H]F-Ara-A and dephosphorylated preformed products more rapidly than epithelial cells but still maintained higher levels of phosphorylated [³H]F-Ara-A products, particularly F-Ara-adenosine 5'-triphosphate. Analyses for nucleoside kinase activity in crude extracts prepared from each cell type documented much higher levels of F-Ara-A phosphorylation (12-fold) but lower levels of adenosine phosphorylation (7-fold) in extracts derived from L1210 cells. Our results suggest that the selective therapeutic action of F-Ara-A against L1210 and perhaps other murine tumor models can be attributed to separate transport and metabolic processes that appear more active in drugsensitive tumor than in drug-limiting host progenitor tissue.

¹ Supported in part by Grant CA 08748 from the National Cancer Institute, Grant CH-26 from the American Cancer Society, and the Elsa U. Pardee Foundation. J. R. B. was supported by training Grant CA 09207 from the National Cancer Institute. Presented in part at the 77th Annual Meeting of the American Association for Cancer Research, 1986.

² To whom requests for reprints should be addressed, at Laboratory for Molecular Therapeutics, Memorial Sloan-Kettering Cancer Center, New York, NY 10021.

Received 6/17/86. Revised 10/21/86. Accepted 10/23/86.

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