Effects of 5-fluorouracil on dihydrofolate reductase and dihydrofolate reductase mRNA from methotrexate-resistant KB cells

• Published in: The Journal of biological chemistry Vol. 260; no. 5; pp. 3006 - 3014
• Main Authors: Dolnick, B J, Pink, J J
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 10.03.1985; American Society for Biochemistry and Molecular Biology
• Subjects: Animals; Biological and medical sciences; Cell physiology; Drug Resistance; Fluorometry; Fluorouracil - pharmacology; Fundamental and applied biological sciences. Psychology; Gene Amplification; KB Cells - enzymology; Methotrexate - pharmacology; Miscellaneous; Molecular and cellular biology; RNA, Messenger - metabolism; Tetrahydrofolate Dehydrogenase - genetics; Tetrahydrofolate Dehydrogenase - metabolism; Cell culture; Translation; Enzyme; Oxidoreductase; Cell line KB; Animal origin; Tetrahydrofolate dehydrogenase; Cytotoxicity; Error; Antifolate; Resistance; Antagonism; Messenger RNA; Mechanism of action
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)89465-0

Growth of methotrexate-resistant dihydrofolate reductase gene-amplified KB cells in the presence of 5-fluorouracil results in an increase in dihydrofolate reductase mRNA. This increase can be solely attributed to a species of RNA of approximately 3.5 kilobase pairs in size. Although dihydrofolate reductase enzyme activity increases per cell with increasing 5-fluorouracil, there is a decrease of enzyme activity per mg of protein (Dolnick, B. J., and Pink, J. J. (1983) J. Biol. Chem. 258, 13299-13306). The rate of in vivo enzyme synthesis, as assayed by immunoprecipitation and supported by gel electrophoresis, does not decrease and may in fact increase with increasing 5-fluorouracil. Translation of purified dihydrofolate reductase mRNA in vitro shows that the rate of translation is unaffected by 5-fluorouracil incorporation into mRNA. The inhibition of dihydrofolate reductase by a monospecific polyclonal antiserum is reduced with extracts from 5-fluorouracil-treated cells. Inhibition of dihydrofolate reductase by methotrexate is significantly reduced in extracts from 5-fluorouracil-treated cells compared to control extracts. Tight binding of [3H]methotrexate is also different in extracts from 5-fluorouracil-treated cells. This data supports the hypothesis of translational miscoding during protein synthesis as a major mechanism of 5-fluorouracil-mediated cytotoxicity and suggests a new mechanism of 5-fluorouracil-methotrexate antagonism.