PLANT PHYSIOLOGY , Vol 101, Issue 2 477-483, Copyright © 1993 by American Society of Plant Biologists
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METABOLISM AND ENZYMOLOGY |
Methotrexate Resistance in Datura innoxia (Uptake and Metabolism of Methotrexate in Wild-Type and Resistant Cell Lines)
K. Wu, I. J. Atkinson, E. A. Cossins and J. King
Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 0W0 (K.W., J.K.)
A wild-type Datura innoxia cell line (Px4) was used to select
methotrexate-resistant cells through a stepwise procedure. Two
independently selected cell lines, MTX161 and MTX132, were stable and shown
to be 5 to 15 times more resistant to methotrexate than wild type. These
methotrexate-resistant cells were similar to the wild-type cells in levels
and kinetic properties of dihydrofolate reductase, the sensitivity of
dihydrofolate reductase to methotrexate, the binding of [3H]methotrexate to
soluble proteins, and the formation of methotrexate polyglutamate
derivatives. High performance liquid chromatographic analyses indicated
that methotrexate polyglutamylation is only slight and may not be
significant in the toxicity of methotrexate to Datura cells. The uptake of
methotrexate was also investigated in the wild-type and resistant cells.
The Px4 cells exhibited a linear uptake that lasted for 1 to 7 h. The
uptake was saturable, pH and energy dependent, and had a Km of 65.6 nM and
a Vmax of 12.5 nmol h-1g-1 fresh weight. Neither MTX161 nor MTX132
exhibited the sustained uptake of methotrexate shown by the Px4 cells. As a
result, there were greatly reduced concentrations of intracellular
methotrexate in resistant cells. Resistant cell lines had 2- to 3-fold
higher Km values for methotrexate uptake compared with Px4 cells. It is
proposed that these cells became resistant as a result of a stable change
in the membrane transport system for methotrexate.
