Modulation by budesonide of a CpG endonuclease in mouse lung tumors.
Modulation by budesonide of a CpG endonuclease in mouse lung tumors.
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DOI：
10.1093/carcin/bgm056
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发表时间：
2007-07
期刊：
影响因子：
4.7
通讯作者：
Long Li;L. Tao;R. Lubet;V. Steele;Michael A. Pereira
中科院分区：
文献类型：
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作者：
Long Li;L. Tao;R. Lubet;V. Steele;Michael A. Pereira
在从小鼠肺肿瘤获得的核提取物中鉴定了CpG内切核酸酶活性。酶活性测定使用333 bp的聚合酶链反应产物的雌激素受体-α基因，含有放射性标记的胞嘧啶或氚标记的甲基在CpG位点。活性测量为从底物释放的放射性。当底物中的CpG位点被甲基化时，通过高压液相色谱（HPLC）将核酸酶活性的产物鉴定为5-甲基-2 '-脱氧胞苷，或者当CpG位点未被甲基化时，鉴定为2'-脱氧胞苷。CpG内切酶的活性依赖于核蛋白和温度，有倾向于双链的单链DNA和抑制乙二胺四乙酸或2-巯基乙醇。乙烯基氨基甲酸酯诱导的A/J系小鼠肺肿瘤具有比未受累肺组织更高水平的CpG内切酶活性。布地奈德是一种有效的小鼠肺化学预防剂，不仅可以防止肺肿瘤中CpG核酸内切酶活性的增加，而且当给予已建立肿瘤的小鼠时，还可以降低肿瘤中核酸内切酶活性的水平。布地奈德对肺肿瘤中CpG核酸内切酶活性的影响与其对小鼠肺肿瘤中DNA甲基化的公开影响呈负相关，即药物降低CpG核酸内切酶活性并增加DNA甲基化。小鼠肺肿瘤中增加的CpG内切酶活性和布地奈德对其的抑制表明这种内切酶是化学预防的潜在分子靶点。
CpG endonuclease activity was identified in nuclear extracts obtained from mouse lung tumors. Enzyme activity was determined using a 333 bp polymerase chain reaction product of the estrogen receptor-alpha gene that contained either radiolabeled cytosine or tritium-labeled methyl groups at CpG sites. Activity was measured as the release of radioactivity from the substrate. The product of the nuclease activity was identified by high pressure liquid chromatography (HPLC) as either 5-methyl-2'-deoxycytidine when the CpG sites in the substrate were methylated or 2'-deoxycytidine when the CpG sites were not methylated. The CpG endonuclease activity was dependent on nuclear protein and temperature, had a proclivity for double-stranded over single-stranded DNA and was inhibited by ethylenediaminetetraacetic acid or 2-mercaptoethanol. Strain A/J mouse lung tumors induced by vinyl carbamate had a greater level of CpG endonuclease activity than non-involved lung tissue. Budesonide, a potent chemopreventive agent in mouse lung, not only prevented an increase in CpG endonuclease activity in lung tumors but, when administered to mice with established tumors, also decreased the level of endonuclease activity in the tumors. The effect of budesonide on CpG endonuclease activity in lung tumors was inversely related to its published effect on DNA methylation in mouse lung tumors, i.e. the drug decreased CpG endonuclease activity and increased the methylation of DNA. The increased CpG endonuclease activity in mouse lung tumors and its inhibition by budesonide would suggest this endonuclease as a potential molecular target for chemoprevention.