Grünwald S, Driever P H, Hoelzer D, Drahovsky D
Zentrum der Biologischen Chemie, Universität Frankfurt, F.R.G.
Biochim Biophys Acta. 1988 Sep 7;950(3):366-73. doi: 10.1016/0167-4781(88)90133-9.
Previous studies have shown that 1-beta-D-arabinofuranosylcytosine (ara-C) can induce differentiation of various malignant cells and that DNA methylation patterns become altered under ara-C treatment of those cells. The aim of this study was to investigate whether this influence on DNA methylation is caused by a direct effect of DNA-incorporated ara-C molecules on nuclear DNA methylase. For this reason, we constructed various ara-C-substituted DNA polymers and used them as substrates for highly purified eukaryotic DNA methylase isolated from murine P815 mastocytoma cells. The ara-C incorporation into DNA polymers was measured by either an ara-C-specific radioimmunoassay or by use of radioactive-labelled ara-C during the synthesis of those polymers. We found an inverse correlation between the level of ara-C substitution of the DNA polymers and their methyl group acceptance. Kinetic experiments performed with ara-C-modified DNA polymers pointed out that the mode of action of DNA methylase remains unaltered. DNA methylase is neither detached nor fixed at an ara-C site, but is somehow hindered in its enzymatic activity, probably by slowing down the walking mechanism. Hence, the previously observed hypermethylation of DNA of some eukaryotic cells, propagated in the presence of ara-C, is apparently not due to a direct effect of DNA-incorporated ara-C molecules on endogenous DNA methylase.
先前的研究表明，1-β-D-阿拉伯呋喃糖基胞嘧啶（阿糖胞苷）可诱导多种恶性细胞分化，并且在这些细胞接受阿糖胞苷治疗时，DNA甲基化模式会发生改变。本研究的目的是调查这种对DNA甲基化的影响是否由掺入DNA的阿糖胞苷分子对核DNA甲基化酶的直接作用引起。因此，我们构建了各种阿糖胞苷取代的DNA聚合物，并将它们用作从鼠P815肥大细胞瘤细胞中分离出的高度纯化的真核DNA甲基化酶的底物。通过阿糖胞苷特异性放射免疫测定法或在这些聚合物合成过程中使用放射性标记的阿糖胞苷来测量阿糖胞苷掺入DNA聚合物的情况。我们发现DNA聚合物的阿糖胞苷取代水平与其甲基接受能力之间呈负相关。用阿糖胞苷修饰的DNA聚合物进行的动力学实验指出，DNA甲基化酶的作用方式保持不变。DNA甲基化酶既不会从阿糖胞苷位点脱离也不会固定在该位点，但其酶活性受到某种程度的阻碍，可能是通过减缓移动机制。因此，先前观察到的在阿糖胞苷存在下某些真核细胞DNA的高甲基化显然不是由于掺入DNA的阿糖胞苷分子对内源性DNA甲基化酶的直接作用。
