METHYLATION IN AML

产品说明：申请人已经优化了用于检测人类癌症中的遗传和表观遗传改变的限制性标志基因组扫描（RLGS）方法。RLGS是一种二维凝胶电泳技术，其中在单个凝胶中显示多达2，000个末端标记的NotI限制性位点。RLGS斑点的快速克隆是在阵列NotI-EcoRV文库的帮助下完成的。甲基化敏感性限制酶（RLGS-M）的使用允许扫描改变的DNA甲基化模式。肿瘤抑制基因的失活可以导致肿瘤转化，这是公认的。失活的机制还不完全清楚，但包括基因启动子序列的DNA甲基化。这具有重要的治疗意义，因为某些成功用于治疗癌症的药物会导致DNA的去甲基化。在此，申请人和他的同事提出利用RLGS-M的独特能力来扫描急性髓性白血病（AML）中基因组的启动子甲基化。他们假设DNA甲基化促进抵抗强化治疗的AML细胞的存活。在初步研究中，他们使用RLGS-M来鉴定WIT 1，这是一种在复发性AML中甲基化但在诊断时或正常组织中不甲基化的基因。申请人和他的同事提出了四个具体目标：I）筛选具有正常核型的AML的额外病例以获得DNA甲基化的证据; II）克隆以高频率甲基化的那些基因座; III）通过用甲基化敏感性PCR测试更大组的AML来将发现与临床结果相关联;以及IV）克隆与甲基化位点相关联的新基因。申请人希望这项工作将成为治疗试验的基础，以使这些基因去甲基化，以期通过强化治疗提高治愈率。

DESCRIPTION: (adapted from the applicant's abstract) The applicant has optimized the Restriction Landmark Genomic Scanning (RLGS) method for the detection of genetic and epigenetic alterations in human cancers. RLGS is a two-dimensional gel electrophoresis technique, in which up to 2,000 end-labeled NotI restriction sites are displayed in a single gel. Rapid cloning of RLGS spots is accomplished with the help of an arrayed NotI-EcoRV library. The use of methylation sensitive restriction enzymes (RLGS-M) allows scanning for altered DNA methylation pattern. It is well established that inactivation of tumor suppressor genes can lead to neoplastic transformation. The mechanisms of inactivation are incompletely understood, but include DNA methylation of the gene's promoter sequence. This has important therapeutic implications, because certain drugs used successfully to treat cancer result in demethylation of DNA. Here, the applicant and his colleagues propose to utilize the unique ability of RLGS-M to scan the genomes for promoter methylation in acute myeloid leukemias (AML). They hypothesize that DNA methylation promotes the survival of AML cells that resist intensive treatment. In preliminary studies, they have used RLGS-M to identify WIT1, a gene methylated in relapsed AML but not methylated at diagnosis or in normal tissue. The applicant and his colleagues propose four specific aims: I) to screen additional cases of AML with normal karyotype for evidence of DNA methylation; II) to clone those loci which are methylated at high frequencies; III) to correlate the findings with clinical outcome by testing a larger set of AML with methylation-sensitive-PCR; and IV) to clone novel genes linked to the methylated sites. The applicant hopes that the work will form the basis for therapeutic trials to demethylate such genes in the hope of improving the rate of cure with intensive treatment.