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On the Poor Correlation between the Inhibition by Methotrexate of Dihydrofolate Reductase and of Deoxynucleoside Incorporation into DNA¹

Laboratories of Pharmacology, Children's Cancer Research Foundation, and Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115 and Arthur D. Little, Inc., Cambridge, Massachusetts

Inhibition by Methotrexate of dihydrofolate reductase and of the incorporation of deoxynucleosides into DNA has been examined in three mouse ascites cell leukemias which had differences in uptake and sensitivity. Dihydrofolate reductase activity in all cell lines was completely inhibited in less than five minutes after the optimal therapeutic dose of Methotrexate. The rate of recovery of enzyme activity was not related to the effectiveness of the drug in prolonging the life of the host. Methotrexate blocked the in vivo incorporation of deoxyuridine into DNA in the sensitive line and increased the ultilization of thymidine without provoking a statistically significant change in the drug-induced resistant or the innately insensitive lines. Although dihydrofolate reductase activity was severely inhibited in cells from Methotrexate-treated animals, the in vitro incorporation of deoxyuridine into DNA was less effectively inhibited than in vivo. The addition of Methotrexate in vitro to cells from Methotrexate-treated animals increased the inhibition of deoxyuridine incorporation in the sensitive and innately insensitive cell lines. Although dihydrofolate reductase had been inhibited in vivo by the drug, Methotrexate concentrations in excess of 3 x 10^-5 M were required in vitro to inhibit deoxyuridine incorporation by the resistant cells. Presumably, Methotrexate not bound to the enzyme was washed from the cells during isolation. To achieve in vitro an inhibition of deoxyuridine similar to that observed in vivo after treatment with Methotrexate required an excess of drug beyond that bound to the enzyme.

¹ Supported by contracts PH-43-66-541 and PH-43-65-61 from the Cancer Chemotherapy National Service Center, National Cancer Institute, NIH.

² Reprint requests should be addressed to this author in care of St. Jude Children's Research Hospital, 332 N. Lauderdale, P. O. Box 318, Memphis, Tennessee 38101.

Received 1/ 4/68. Accepted 5/21/68.