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: 6124688
• 负责人: EDWARD M NEWMAN
• 金额: $ 22.3万
• 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/6124688
• 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）是一种研究药物， 在治疗急性髓性白血病中表现出活性， 在其他白血病和骨髓增生异常中可能的治疗效用 综合征 DAC掺入DNA后，可抑制DNA酶 （胞嘧啶-5）-甲基转移酶（MT'ase）。这种抑制通常 他们被认为是对他们的活动负责。 虽然这一行动可能 我们的初步结果表明，MT'酶抑制 单靠这一点并不能完全解释DAC的分子生物学效应。 在 在培养的肿瘤细胞中，DAC已被证明可以减少 5'调控区的甲基化，并增加 肿瘤抑制基因沉默甲基化。 因此，它可以 抑制癌发生或肿瘤进展。 DAC已被证明 减少结肠癌小鼠模型中息肉的形成 肿瘤前病变，仅存在一个MT'ase基因拷贝。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'ase结合。 尽管事实上 这两种类似物都是MT'酶的不可逆抑制剂，我们获得了 初步证据表明，FdCyd掺入细胞DNA并不 降低抑癌基因p16的甲基化。 DAC和 FdCyd在化学稳定性方面确实存在显著差异; DAC远 由于其三嗪环结构而不太稳定。 我们的…假设是 除了抑制MT'酶，DNA中不稳定的DAC残基 通过DNA损伤诱导的甲基化丧失 切除修复 我们检验这一假设的具体目标是：（1） 比较DAC和FdCyd对p16的再激活，如通过 p16基因外显子甲基化和p16基因mRNA表达水平的比较 DAC和FdCyd掺入细胞DNA中，以及（3）比较 DAC和FdCyd对细胞中MT'ase的抑制，如通过 细胞提取物中的MT'酶活性和新的- 在DNA中掺入脱氧胞苷。作为具体目标3的一部分， 我们将开发一种新的MT'ase检测方法， [CH 3 - 3 H]-S-腺苷甲硫氨酸放射性掺入 转化成DNA 这种新的分析方法将加快这一领域的样本分析。 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组合应 通过两种不同的作用产生与DAC相同的效果， MT酶抑制和DNA损伤。 增加DNA损伤 药物也可能增强DAC的作用。 因此，我们的最终 该项目的具体目标（4）是确定DNA的影响- 与FdCyd和DAC组合的损伤剂对甲基化的影响， 特定基因的表达。 本提案的总体目标是 了解MT'ase抑制剂的作用，并使用这种 了解如何开发更有效的调节DNA的方法 甲基化与人类疾病