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Transport and Metabolism of Methotrexate in Normal and Resistant Cultured Rat Hepatoma Cells¹

Division of Laboratories and Research, New York State Department of Health, Albany, New York 12201

The transport and metabolism of methotrexate (MTX) have been examined in monolayer cultures of a cell line derived from the Reuber H35 hepatoma. The H35 cells have a doubling time of approximately 19 hr and cannot grow when MTX is present at 3 x 10^–8 M. The cells exhibit linear uptake of MTX which lasts for 4 to 6 hr and reaches a steady state after approximately 16 hr. The uptake was saturable with a maximal rate of 1.1 nmol/min/g cell protein at an MTX concentration of 18 µM or higher. 5-Methyltetrahydrofolate markedly inhibited uptake at equimolar concentration but folic acid exerted no effect at 5-fold excess. Once inside the cell, MTX was rapidly converted to its polyglutamate derivatives with a conversion in excess of 90% when the intracellular concentration was greater than 2 µM. Resistant sublines were developed by culturing H35 cells in increasing concentrations of MTX. These cells are similar to the parent cells in levels of dihydrofolate reductase, thymidylate synthetase, and the extent that MTX can be converted to its polyglutamate derivatives. They appear to differ from normal cells in not having the capacity for the extended linear uptake that is observed with normal cells. As a result, there is a greatly reduced concentration of MTX and its polyglutamates at steady state. These results suggest that the cells become resistant as a result of a stable change in the transport system for MTX, but the mechanism of this process is not yet understood.

¹ Supported in part by Grant NIA AG00207 from the National Institute on Aging.

Received 8/11/78. Accepted 11/14/78.

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