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Effects of Perfusion with Amethopterin on L1210 Leukemia Cells in Spin Filter Culture¹

Arthur D. Little, Inc., Cambridge, Massachusetts 02140 [P. S. T., P. H.], and Children's Cancer Research Foundation, Boston, Massachusetts 02115 [D. R.]

Cells of the murine leukemia L1210 have been treated with amethopterin (NSC 740) in a spin filter culture device which provides for continuous perfusion of populations of cells in suspension and removal of extracellular drug at a controlled rate. Experiments with exponential washout have involved daily doses of drug at initial concentrations of 1.0 µg/ml and a half-time of approximately 1.1 hr, providing an integrated concentration x time value of approximately 1.6 µg-hr/ml/day. Under these conditions, the viable cell population, as measured by cloning in soft agar, showed an immediate decline followed by partial recovery between drug doses. The degree of cell killing was greater after the second exposure to drug than after the first exposure. Experiments with tritiated amethopterin have shown a rapid initial uptake of drug followed by a slow decline of intracellular concentration; the ratio of intracellular to extracellular levels approaches 1000 after several hours of drug washout. Dihydrofolate reductase activity dropped sharply in the first few hours after drug addition and recovered partially in the period before the next treatment. A similar effect was seen for deoxyuridine incorporation into DNA, but thymidylate synthetase activity increased following the first drug dose. Similar effects were seen after the second dose, with the exception of thymidylate synthetase, which showed a transitory drop followed by recovery to a level higher than the initial level. Continuous perfusion with amethopterin at 0.067 µg/ml, to provide the same daily integrated concentration x time value resulted in comparable cell killing over a 2-day period, with a small population surviving for 6 days.

¹ This work was supported by Chemotherapy National Cancer Institute, NIH, Contracts PH-43-65-61 and PH-43-66-541.

² Present address: Monsanto Biodize Systems, Inc., College Point, N. Y. 11356.

³ Present address: St. Jude Children's Research Hospital, Memphis, Tenn. 38101.

Received 11/ 6/69. Accepted 2/ 5/70.