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Mechanism of Cyclophosphamide Transport by L5178Y Lymphoblasts in Vitro¹

Department of Medicine, University of Manitoba, and The Manitoba Institute of Cell Biology [G. J. G., H. B. L.] and Department of Physics, The Manitoba Cancer Treatment and Research Foundation and the Department of Medical Microbiology, University of Manitoba [D. V. C.], Winnipeg, Manitoba, R3E 0V9, Manitoba, Canada

Mechanism of transport of the alkylating agent cyclophosphamide-¹⁴C was investigated in L5178Y lymphoblasts in vitro. A time course of cyclophosphamide uptake showed a rapid, initial phase, probably due to binding of drug to the cell surface. Subsequent uptake into the cells was carrier mediated and consisted of two components. Analysis of cyclophosphamide uptake over a 40-fold range of drug concentration showed biphasic kinetics with evidence of saturation only at low drug concentrations whereas, at high drug levels, uptake occurred by a second transport system that was technically nonsaturable. After correction for binding and the interaction of two-component transport, kinetic parameters for low-dose transport consisted of a Michaelis constant K_m (mean ± S.E.) of 0.39 ± 0.03 mM and a transport capacity V_max of 0.49 ± 0.07 x 10^-17 moles/min/cell. At high-dose cyclophosphamide transport, the apparent K_m was 75 ± 29 mM, and the V_max was 49 ± 14 x 10^-17 moles/min/cell. Both low- and high-dose cyclophosphamide transport were temperature sensitive and partially dependent on sodium. In addition, low-dose transport was inhibited by oligomycin and cyanide. Other alkylating agents and several naturally occurring substrates did not inhibit cyclophosphamide transport; thus, a native substrate was not identified for the cyclophosphamide carrier, and transport was by a mechanism separate from that of other alkylating agents.

Evidence that low-dose cyclophosphamide transport was mediated by a facilitated diffusion process was that uptake obeyed saturation kinetics, was temperature and sodium dependent, was partially dependent on metabolic energy, and cell/medium concentration gradients did not exceed unity. Although high-dose drug uptake failed to show saturation kinetics, the demonstration of temperature and sodium dependence also suggested that high-dose uptake may be carrier mediated.

Cyclophosphamide uptake by chick embryo liver cells was examined also; uptake was temperature sensitive and exhibited biphasic kinetics similar to that observed in L5178Y cells, suggesting a similar mechanism of drug transport in normal liver and leukemic cells.

¹ This work was supported by a grant from the National Cancer Institute of Canada.

² Clinical Research Associate of the National Cancer Institute of Canada.

Received 6/ 3/74. Accepted 8/27/74.

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