INFIDELITY OF CYTOSINE METHYLATION AND HUMAN CANCER

新出现的数据有力地表明，DNA胞嘧啶甲基转移酶的不忠可能导致胞嘧啶甲基化模式的扰动，并有助于人类癌症的发展。胞嘧啶甲基转移酶活性的改变可能导致癌基因的激活，肿瘤抑制基因的沉默，或者通过诱导胞嘧啶脱氨而发生突变。已经开发了几种方法，可以确定来自细胞系和肿瘤标本的DNA中特定胞嘧啶残基的甲基化状态。然而，对甲基化模式可能被改变或干扰的机制的关注要少得多。我们开发了一种新的体外试验，它允许我们定量地确定DNA胞嘧啶甲基转移酶的DNA底物偏好。这种方法，我们称之为质量标记，是基于用稳定的同位素标记合成的寡核苷酸中的目标胞嘧啶残基。用气相色谱/质谱仪(UC/MS)检测甲基酶介导的碱基修饰。用这种方法，我们可以检查DNA结构扰动，如错配和碱基损坏，对甲基酶速率和碱基选择性的影响。我们还可以检查带有基于机制的抑制剂的寡核苷酸的效果，如5-氟胞嘧啶和5-氮胞嘧啶，以及维持甲基酶对半甲基化位点附近的非甲基化位点甲基化的倾向。我们建议研究纯化的哺乳动物维持甲基酶，一类新发现的“从头”甲基酶，以及从甲基化模式中表现出扰动的细胞的提取物。这些拟议研究的完成将大大增加我们对胞嘧啶甲基化保真度降低的机制的了解，并为测试试剂提供工具，这些试剂可能会提高体内甲基化的保真度或作为选择性甲基酶抑制剂。

Emerging data strongly suggests that the infidelity of DNA cytosine methyltransferase may result in the perturbation of cytosine methylation patterns and contribute to the development of cancer in man. Altered cytosine methytransferase activity may result in activation of oncogenes, silencing of tumor suppressor genes, or mutation through induced cytosine deamination. Several methods have been developed which allow determination of the methylation status of specific cytosine residues in DNA derived from cell lines and tumor specimens. Substantially less attention, however, has been focused on the mechanisms by which methylation patterns may become altered or perturbed. We have developed a novel in vitro assay, which allows us to determine quantitatively the DNA substrate preferences for DNA cytosine methyltransferases. This method, which we call mass tagging, is based upon labelling target cytosine residues in synthetic oligonucleotides with stable isotopes. Methylase-mediated base modification is examined by gas chromatography/mass spectrometry (UC/MS). With this method, we can examine the effects of DNA structural perturbations, such as mispairs and damaged bases, on methylase rate and base selectivity. We can also examine the effects of oligonucleotides bearing mechanism-based inhibitors such as 5-fluorocytosine and 5-azacytosine, and the tendency of the maintenance methylase to methylate unmethylated sites adjacent to hemimethylated sites. We propose to study purified mammalian maintenance methylases, a newly identified class of "de novo" methylases, as well as extracts from cells with demonstrated perturbations in methylation patterns. Completion of the proposed studies will substantially increase our understanding of the mechanisms by which the fidelity of cytosine methylation may be reduced and provide tools for testing agents which may increase the fidelity of methylation in vivo or act as selective methylase inhibitors.