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The Relationship among Transport, Intracellular Binding, and Inhibition of RNA Synthesis by Actinomycin D in Ehrlich Ascites Tumor Cells in Vitro¹

Departments of Pharmacology and Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27514 [D. B., I. D. G.], and Department of Medicine, Medical College of Virginia, Richmond, Virginia 23298 [I. D. G.]

³ Supported by Research Career Development Award CA-50281 from the National Cancer Institute. To whom requests for reprints should be addressed, at Department of Medicine, Medical College of Virginia, 1200 East Broad Street, Richmond, Va. 23298.

Uptake of actinomycin D (AD) was characterized in Ehrlich ascites tumor cells in vitro. The time course of uptake consists of an initial component that represents a unidirectional flux of AD into the cells, following which the net uptake rate falls to achieve a velocity that is constant over a 45-min interval of observation. The velocity of influx exceeds that of the late uptake phase by a factor of 2. Influx is 1st order over an AD concentration range of 0.02 to 14 µM. Influx is highly temperature sensitive (Q₁₀ = 4.5); this is related only in part to the high temperature dependence (Q₁₀ = 2) of the oil-aqueous partition coefficient for this agent. Within 4 min after exposure of cells to AD, a component of intracellular AD appears which exceeds the concentration of extracellular AD and rapidly leaves the cells (t 3 min) when the cells are resuspended into an AD-free medium. The level of this component of intracellular drug is unaffected by metabolic poisons, and the apparent chemical gradient is attributed to loose binding. While transport of AD does not appear to be energy-dependent, the data do not clarify whether translocation across the cell membrane is diffusional or carrier mediated. In addition to a loosely bound intracellular component, analysis of the unidirectional net efflux of AD indicates at least 3 other exit components; one with a t of 35 min, a very tightly bound component, and another representing drug that is free within the intracellular water. The bulk of AD taken up is tightly bound to intracellular constituents, and the rate of accumulation of this cellular component accounts almost entirely for the net rate of uptake of AD during the late uptake phase.

Because influx exceeds the rate of tight binding within the cell by a factor of only 2, it is unlikely that osmotically active AD achieves thermodynamic equilibrium across the cell membrane over the interval of these experiments. Rather, prior to saturation of binding sites within the cell, the level of free AD achieved is probably below the extracellular concentration. Since the rate of binding should be influenced by the level of free intracellular AD, factors that increase influx and bring the intracellular AD level closer to equilibrium should also increase the rate of intracellular binding. Tween 80 augments both influx and AD binding within the cell, an effect that may be related to augmented transport alone, but an additional stimulatory effect on the binding process is also possible.

Association of AD with only a small number of the very-high-affinity intracellular binding sites, in the absence of free or loosely bound AD, results in 35% inhibition of [2-¹⁴C]uridine incorporation into RNA within 50 sec after exposure of cells to 1 µM AD. Within 10 min, 75% inhibition is achieved indicating that association of AD with only a small fraction of the total high-affinity sites is sufficient to suppress RNA synthesis.

¹ This investigation was supported by USPHS Grants CA-11725 and CA-16906 from the National Cancer Institute.

² Recipient of USPHS Pre-doctoral Fellowship ES-00129.

Received 2/28/75. Accepted 7/30/75.