Prevention of methotrexate cytotoxicity by asparaginase inhibition of methotrexate polyglutamate formation.

Escherichia coli asparaginase (Asnase) pretreatment of Asnase-sensitive L5178Y cells in vitro is thought to antagonize methotrexate (MTX) cytotoxicity through nonspecific inhibition of protein synthesis and MTX uptake. We have reexamined the mechanism of this interaction in view of recent data demonstrating the importance of MTX metabolism to polyglutamate derivatives (MTXPGs) in the cytotoxic effects of the antifolate. After a 3-hr exposure to 0.5 microM MTX, 67% of intracellular drug was in the form of MTXPGs containing a total of 2 to 5 glutamyl residues (MTX-Glu2-5), and cloning efficiency in drug-free medium was only 7% of untreated control. After a 3-hr pretreatment with E. coli Asnase (0.1 unit/ml), [3H]thymidine incorporation dropped by 29%, MTXPG formation during subsequent MTX exposure decreased by more than one-half (MTX-Glu2 unchanged; MTX-Glu3 and 4 decreased to 51.7 and 18.5% of levels achieved in cells not pretreated with Asnase; no MTX-Glu5 formed), and cloning efficiency increased to 71% of untreated control. This effect was not due to decreased MTX uptake into L5178Y cells or to decreased intracellular free L-glutamate or L-glutamine levels. A 3-hr exposure of L5178Y cells to media lacking L-isoleucine, an essential amino acid for cell growth, prior to MTX exposure inhibited [3H]thymidine incorporation by 37%, decreased subsequent MTXPG formation by 62%, and increased subsequent cloning in drug-free medium to control levels. Decreased MTXPG formation was responsible for the prevention of MTX cytotoxicity seen after both pretreatments. Unmetabolized MTX rapidly left L5178Y cells after removal of extracellular MTX. Consequently, lower levels of unbound intracellular drug, a prerequisite of drug activity, were maintained in pretreated than in control cells after passage in drug-free medium. Asnase pretreatment protects L5178Y cells from the cytotoxic effects of MTX, possibly through inhibition of cell growth which nonspecifically decreases MTXPG formation.