Mechanism of cyclophosphamide transport by L5178Y lymphoblasts in vitro.

Mechanism of transport of the alkylating agent cyclophos phamide-' 4C was investigated in L5 178Y 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 cyclophos phamide 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 Km (mean ±S.E.) of 0.39 ±0.03 mM and a transport capacity Vmax of 0.49 ± 0.07 X l0' @ moles/mm/cell. At high-dose cyclophosphamide transport, the apparent K@ was 75 ±29 mM , and the Vmax was 49 ±14 X l0' @ moles/mm/cell. Both lowand 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 L5 178Y cells, suggesting a similar mechanism of drug transport in normal liver and leukemic cells.