MECHANISM OF GENE ACTIVATION BY DNA METHYLASE INHIBITORS

DNA 甲基化酶抑制剂激活基因的机制

• 批准号: 2827904
• 负责人: EDWARD M NEWMAN
• 金额: $ 22.6万
• 依托单位: BECKMAN RESEARCH INSTITUTE/CITY OF HOPE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-22 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2827904
• 关键词: DNA damage; DNA methylation; azacitidine; deoxycytidine; enzyme activity; enzyme inhibitors; flucytosine; gene induction /repression; methyltransferase; neoplastic cell; tissue /cell culture; tumor suppressor genes

5-Aza-2'-deoxycytidine (DAC) is an investigational agent with demonstrated activity in the treatment of acute myeloid leukemia and likely therapeutic utility in other leukemias and myelodysplastic syndromes. After incorporation into DNA, DAC inhibits the enzyme DNA (cytosine-5)-methyltransferase (MT'ase). This inhibition has generally been assumed to be responsible for its activity. While this action may be necessary, our preliminary results suggest that MT'ase inhibition alone does not fully explain DAC's molecular biological effects. In neoplastic cells in culture, DAC has been shown to decrease the methylation of 5' regulatory regions and to increase the expression of tumor suppressor genes silenced by methylation. As a result, it could inhibit carcinogenesis or tumor progression. DAC has been shown to reduce polyp formation in a murine model for colonic preneoplasia when only one copy of the MT'ase gene is present. 5- Fluoro-2'-deoxycytidine (FdCyd) is another analog which, when present in DNA, binds irreversibly to MT'ase in vitro. Despite the fact that both analogs are irreversible inhibitors of the MT'ase, we have obtained preliminary evidence that FdCyd incorporated into cellular DNA does not decrease the methylation of the tumor suppressor gene p16. DAC and FdCyd do differ significantly in their chemical stability; DAC is far less stable because of its triazine ring structure. Our, Hypothesis is that, in addition in inhibiting MT'ase, unstable DAC residues in DNA contribute to the loss of methylation through DNA damage-induced excision and repair. Our Specific Aims to test this hypothesis are: (1) to compare the reactivation of p16 by DAC and FdCyd as measured by the methylation of p16 exon and the expression of p16 mRNA, (2) to compare the incorporation of DAC and FdCyd into DNA in cells, and (3) to compare the inhibition of MT'ase by DAC and FdCyd in cells as measured by the MT'ase activity in cellular extracts and the methylation of newly- incorporated deoxycytidine residues in DNA. As part of specific aim 3, we will develop a new assay for MT'ase which does not require measuring the incorporation of radioactivity from [CH3-3H]-S-adenosylmethionine into DNA. This new assay will expedite the analysis of samples in this project and other studies, including future clinical trials. If our hypothesis is correct, DNA-damaging agents combined with FdCyd should produce the same effects as DAC does through its two different actions, MT'ase inhibition and DNA damage. Increasing DNA damage with another agent might also potentiate the effects of DAC. Thus, our final specific aim in this project (4) is to determine the effects of DNA- damaging agents combined with FdCyd and DAC on the methylation and expression of specific genes. The overall goal of this proposal is to understand the actions of MT'ase inhibitors and to use this understanding to develop more effective methods of modulating DNA methylation in human diseases.

5-氮杂-2‘-脱氧胞苷(DAC)是一种具有 显示出治疗急性髓系白血病的活性和 对其他白血病和骨髓增生异常的可能治疗作用 综合症。在掺入DNA后，DAC抑制了DNA酶 (胞嘧啶-5)甲基转移酶(MT‘酶)。这种抑制通常会 被认为对其活动负责。虽然此操作可能 有必要，我们的初步结果表明，MT‘ase抑制 单靠这一点并不能完全解释DAC的分子生物学效应。在……里面 在培养的肿瘤细胞中，DAC已被证明可减少 5‘调控区的甲基化并增加其表达 肿瘤抑制基因因甲基化而沉默。因此，它可能会 抑制癌变或肿瘤进展。DAC已被证明是 减少小鼠结肠息肉形成的实验研究 只有一份MT‘ase基因存在时的创业症。5 氟-2‘-脱氧胞苷(FdCyd)是另一种类似物，当存在时 在DNA中，在体外不可逆地与MT‘酶结合。尽管事实是 这两个类似物都是MT‘酶的不可逆转的抑制剂，我们已经获得 初步证据表明，掺入细胞DNA的FdCyd不会 减少肿瘤抑制基因p16的甲基化。DAC和 FdCyd在化学稳定性方面确实有很大差异；DAC远远不同 由于其三嗪环结构，稳定性较差。我们的假设是 除了抑制MT‘酶，DNA中不稳定的DAC残基 通过DNA损伤导致甲基化缺失 切除和修复。我们检验这一假说的具体目标是：(1) 为了比较DAC和FdCyd对p16的重新激活， P16外显子甲基化与p16基因表达的比较 DAC和FdCyd在细胞DNA中的掺入；(3)比较 DAC和FdCyd对细胞MT‘酶的抑制作用 细胞抽提物中MT‘酶活性及新陈代谢产物的甲基化 在DNA中加入了脱氧胞苷残基。作为特定目标3的一部分， 我们将开发一种新的不需要测量的MT‘ase检测方法 [CH_3-~3H]-S-腺苷蛋氨酸的放射性掺入 变成DNA。这种新的化验方法将加快对这一地区样品的分析。 项目和其他研究，包括未来的临床试验。如果我们的 假设是正确的，DNA损伤剂与FdCyd联合应该 通过两个不同的动作产生与DAC相同的效果， 线粒体酶抑制和DNA损伤。与其他人一起增加DNA损伤 药剂也可能增强DAC的作用。因此，我们的决赛 这个项目(4)的具体目标是确定DNA- FdCyd和DAC联合使用对肿瘤细胞甲基化的影响 特定基因的表达。这项提案的总体目标是 了解MT‘ase抑制剂的作用并使用此 对开发更有效的DNA调控方法的理解 人类疾病中的甲基化。