Is uracil misincorporation into DNA of mammalian cells a consequence of methotrexate treatment?
We have carried out an exhaustive analysis to determine if uracil is incorporated into DNA of different mammalian cells exposed to methotrexate. Both HeLa and human lymphoblastoid cells (CCRF-HSB2) were incubated in medium containing [5-3H] deoxyuridine and 10 microM or 100 microM methotrexate. In some experiments non-radioactive 10mM uracil was present to inhibit uracil-DNA-glycosylase and thus facilitate the subsequent detection of uracil in the DNA. This was extracted and freed of RNA, ribonucleotides and protein with the use of phenol, RNAase, pronase, ethanol precipitation and Sephadex chromatography. DNA was enzymically degraded to nucleosides which were analysed directly by HPLC. We did not detect uracil in the DNA in over 12 different experiments under various conditions and times of drug-treatment. In view of this we caution against ready acceptance of the notion that uracil is incorporated to any significant extent, or indeed at all, in all types of cells exposed to methotrexate.
D002460:Cell Line; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000096:biosynthesis*; D006801:Humans; D008727:Methotrexate / Q000494:pharmacology*; D014498:Uracil / Q000378:metabolism*
D014498:Uracil; D004247:DNA; D008727:Methotrexate