METHOTREXATE-INDUCED MISINCORPORATION OF URACIL INTO DNA
METHOTREXATE-INDUCED MISINCORPORATION OF URACIL INTO DNA
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DOI：
10.1073/pnas.77.4.1956
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发表时间：
1980-01-01
期刊：
影响因子：
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通讯作者：
TSENG, BY
中科院分区：
文献类型：
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作者：
GOULIAN, M;BLEILE, B;TSENG, BY
在使用或未使用二氢叶酸还原酶抑制剂甲氨蝶呤处理的情况下，对人类淋巴样细胞系的 DNA 中 dUMP 的存在进行了测试。用甲氨蝶呤处理并用[3H]dUrd[脱氧尿苷]标记的细胞在DNA中含有容易检测到的量的dUMP（每μmol总DNA核苷酸约0.8pmol dUMP），并且该值增加了约10μmol。如果同时用 Ura [尿嘧啶]处理细胞，则为 3 倍。无论 Ura 是否存在，通过这些方法在未用甲氨蝶呤处理的细胞的 DNA 中均未检测到 dUMP（< 1 fmol/.mu.mol DNA）。用甲氨蝶呤处理的细胞 DNA 中存在 dUMP，是由于药物抑制胸苷酸合成酶（5,10-亚甲基四氢叶酸：dUMP C-甲基转移酶；EC 2.1.1.45）导致细胞内 dUTP 浓度大幅增加和 dTTP 下降的结果。这些变化显然足以克服将 dUMP 从 DNA 中排除的正常机制，而 Ura 的增强反映了对其中一种机制的抑制，即通过 Ura-DNA 糖基化酶从 DNA 中去除 Ura。提示细胞中存在 DNA 活跃损伤，其中胸苷酸合成酶受到抑制。在这些条件下，似乎存在 dUMP 的循环掺入和去除，这是由于在 Ura 去除位点的间隙修复过程中重新插入 dUMP 造成的。当细胞内 dUTP 水平接近 dTTP 时，正常切除修复过程的结果可能与胸苷酸合成酶药物抑制剂的细胞毒性、叶酸和维生素 B-12 的临床缺乏以及无胸腺嘧啶死亡有关。
A line of human lymphoid cells was tested for the presence of dUMP in DNA with or without treatment with the dihydrofolate reductase inhibitor, methotrexate. Cells treated with methotrexate and labeled with [3H]dUrd [deoxyuridine] contained dUMP in DNA in readily detectable amounts (.apprx. 0.8 pmol of dUMP per .mu.mol of total DNA nucleotide), and this was increased .apprx. 3-fold if the cells were also treated with Ura [uracil] at the same time. No dUMP (< 1 fmol/.mu.mol of DNA) was detected by these methods in DNA from cells not treated with methotrexate, regardless of whether Ura was present or absent. The presence of dUMP in DNA from cells treated with methotrexate is a result of the great increase in intracellular concentration of dUTP and the fall in dTTP that accompnay inhibition of thymidylate synthetase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase; EC 2.1.1.45) by the drug. These changes are apparently sufficient to overcome the normal mechanisms that exclude dUMP from DNA, and the enhancement by Ura reflects suppression of one of the mechanisms, Ura removal from DNA by the enzyme Ura-DNA glycosylase. An active lesion of DNA in cells in which thymidylate synthetase in inhibited is suggested. Under these conditions there appears to be a cyclic incorporation and removal of dUMP resulting from reinsertion of dUMP during gap repair at sites of Ura removal. This consequence of the normal excision-repair process, which occurs when intracellular levels of dUTP approach those of dTTP, may have effects related to the cytotoxicity of drug inhibitors of thymidylate synthetase, clinical deficiencies of folate and vitamin B-12, and thymineless death, in general.