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Specificity of Systems Mediating Transport of Adenosine, 9-ß-D-Arabinofuranosyl-2-fluoroadenine, and Other Purine Nucleoside Analogues in L1210 Cells¹

Memorial Sloan-Kettering Cancer Center, New York, New York 10021 [F. M. S., P. L. C., D. M. D.], and Southern Research Institute, Birmingham, Alabama 35255 [J. A. M.]

In studies discriminating between transport and metabolism, we have reported results with L1210 cells which document (P. L. Chello, F. M. Sirotnak, D. M. Dorick, C-H. Yang, and J. A. Montgomery. Cancer Res., 43: 97–103, 1983) the existence of high- and low-affinity systems with a high capacity for mediated transport of adenosine. We now report on a similar kinetic duality for transport of the adenosine analogue, 9-ß-D-arabinofuranosyl-2-fluoroadenine. High-capacity systems mediating influx of this analogue in L1210 cells were delineated which exhibited high affinity [K_m = 69.4 ± 7.4 (S.E.) µM] and low affinity [K_m = 305 ± 31 (S.E.) µM] for this analogue. This analogue shares mediated entry with [³H]adenosine by high affinity [K_m = 14.9 ± 1.3 (S.E.) µM] and low-affinity [K_m = 218 ± µM] systems, and the same high values for V_max (low affinity versus high affinity, 2:1 to 3:1) are derived when either [³H]-adenosine or the analogue is used as substrate. The kinetic duality for mediated transport of either substrate was the same in L1210/ara-C cells which are not able to phosphorylate the analogue. From the results of reciprocal competition experiments between adenosine and 9-ß-D-arabinofuranosyl-2-fluoroadenine showing good agreement between values for K_m and K_i, it was concluded that mediated entry of the analogue can be entirely accounted for by these two systems which transport [³H]adenosine. During studies with a large group of related purine analogues, marked differences in specificity of each system for these analogues were shown. Purine ribosides bearing a substituent at position 6, as in the case of adenosine but not inosine, were in one case more effective and in other cases equivalent to or nearly as effective as adenosine as inhibitors of [³H]adenosine transport by either system. Halogenation at position 2 or 8 reduced 2-to 5-fold the effectiveness of adenosine as a competitive inhibitior of [³H]adenosine by the high-affinity system. Fluorination at position 2 had no effect (high-affinity system) or little effect (low-affinity system) on the action of 1-ß-D-arabinofuranosyladenine as an inhibitor of transport. 8-Bromoadenosine was 2-fold better than was adenosine as an inhibitor of [³H]adenosine transport by the low-affinity system. A variety of other structural modifications of the base and sugar of adenosine were found to reduce effectiveness as inhibitors of [³H]adenosine transport.

¹ Supported in part by Grants CA 08748, CA 24153, and CA 36218 from the National Cancer Institute, Department of Health and Human Services; Grant CH-26 From the American Cancer Society; and the Elsa U. Pardee Foundation.

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

Received 4/29/82. Accepted 10/ 5/82.

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