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Effect of Feeder Cell-released Substances on the Survival of Clonogenic 9L Cells after Treatment with Antimetabolites¹

Brain Tumor Research Center, The Department of Neurological Surgery, [M. W., D. F. D.] and Department of Radiation Oncology [D. F. D.], School of Medicine University of California, San Francisco, California 94143

Exponentially growing monolayer cultures of 9L rat brain tumor cells were treated with either 5-fluorouracil or methotrexate. The surviving fraction of cells was determined by a colony formation assay. After 5-fluorouracil treatment, 2 to 5 x 10⁵ feeder cells were required to maximize surviving fractions for each drug concentration and to generate a biphasic dose-response curve. If only 1 x 10⁴ feeder cells were used, the dose-response curve was steep. Uridine added to the dishes containing 1 x 10⁴ feeder cells restored the biphasic shape, while uridine and thymidine added to the dishes yielded the same curve obtained with 2 x 10⁵ feeder cells.

After methotrexate treatment, the surviving fraction of cells was dependent on feeder cell number when the medium in the dishes was supplemented with dialyzed fetal bovine serum, but it was not dependent on feeder cell number when nondialyzed fetal bovine serum was used. Biphasic dose-response curves were generated from methotrexate-treated cells plated in medium supplemented with either dialyzed or nondialyzed serum, but the drug was more toxic to cells plated in medium containing dialyzed serum. This additional toxicity could be reduced if either thymidine or N-5-formyltetrahydrofolate were added to the dishes and eliminated if 1 x 10⁴ feeders were added. These results suggest that any cell culture system used to evaluate antimetabolites should be optimized for possible feeder cell and serum effects.

¹ This research was supported by NIH Grant CA-13525, the Department of Neurology, Klinikum Charlottenberg, Free University of Berlin, West Germany, and a grant from the German Research Society (Deutsche Forschungsgemeinschaft), Bonn, West Germany.

² Visiting scientist at the Brain Tumor Research Center, Department of Neurological Surgery, University of California, San Francisco, Calif. 94143. Permanent address: Neuropathologisches Institut, Universitaet Düsseldorf, Moorenstr. 5, 4000 Düsseldorf 1, West Germany.

³ To whom requests for reprints should be addressed, at the Brain Tumor Research Center, Department of Neurological Surgery, 783 HSW, University of California, San Francisco, Calif. 94143.

Received 4/21/80. Accepted 6/ 5/80.

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