Translocations induced by abortive apoptosis

流产细胞凋亡诱导的易位

• 批准号: 6672060
• 负责人: ANDREW T. VAUGHAN
• 金额: $ 21.09万
• 依托单位: LOYOLA UNIVERSITY CHICAGO
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-01 至 2004-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6672060
• 关键词: DNA repair; apoptosis; cancer risk; carcinogenesis; chemical cleavage; chromosome translocation; clinical research; cysteine endopeptidases; enzyme activity; fusion gene; human subject; leukemia; neoplasm /cancer genetics; nonHodgkin's lymphoma; oncogenes; patient oriented research; polymerase chain reaction; tissue /cell culture; transfection

DESCRIPTION (provided by applicant): The following hypothesis is to be tested. "Transient activation of apoptotic nucleases initiate leukemogenic translocations of MLL through DNA cleavage and error prone repair". It is proposed that apoptotic execution may be arrested, after targeted DNA damage has occurred, in cells that survive. Preliminary data shows that apoptotic triggers initiate cleavage of MLL, which is subsequently translocated to AF9, creating the leukemogenic MLL-AF9 fusion gene that is transcribed in cells capable of division. The relevance to health care is that this process would represent a novel pathway for the generation of fusion genes implicated in leukemogenesis that would be open to therapeutic intervention. Three Aims are proposed that sequentially address the mechanism controlling cleavage within MLL, the growth of cells that survive and validation of these in-vitro data with a clinical model of early stage leukemogenesis. Aim 1 Mechanism of site-specific cleavage in MLL. It is proposed that apoptotic cleavage of MLL is regulated by adjacent sequence motifs, including ATC tracts or topoisomerase II binding sites. These possibilities will be tested by modifying each motif-using site directed mutagenesis within an MLL containing episome (pREP4MLL) that reproduces genomic MLL cleavage in ceils undergoing apoptosis. Aim 2. Selection of cells that survive apoptotic activation. Cell lines will be selected using the pREP4 episome containing the minimal MLL apoptotic cleavage motif intemal to one of two corrupt antibiotic resistance genes. Apoptotic cleavage within pREP4MLL min will be used to stimulate selection of surviving cells containing a stable copy ofpREP4MLL min by gene conversion repair of the resistance gene. Aim 3. Detection ofMLL translocations in patients at risk of therapy related leukemia. Blood taken from patients after treatment for non-Hodgkins lymphoma will be analyzed for the presence of MLL-AF9 message by RT-PCR and MLL rearrangements at the apoptotic cleavage site using inverse PCR. In each case the ability of cells containing such aberrations to divide will be determined by the presence of duplicated genomic breakpoint junctions within each patients sample.

描述（由申请人提供）：以下假设将被检验。 “凋亡核酸酶的瞬时激活通过 DNA 切割和易错修复启动 MLL 的白血病易位”。有人提出，在存活的细胞中发生靶向 DNA 损伤后，细胞凋亡的执行可能会被阻止。初步数据显示，细胞凋亡触发因素会启动 MLL 的裂解，随后 MLL 易位至 AF9，产生在能够分裂的细胞中转录的致白血病 MLL-AF9 融合基因。与医疗保健的相关性在于，这一过程将代表一种与白血病发生有关的融合基因生成的新途径，并且可以接受治疗干预。提出了三个目标，依次解决 MLL 内的裂解控制机制、存活细胞的生长以及通过早期白血病发生的临床模型验证这些体外数据。目标 1 MLL 中位点特异性切割的机制。有人提出，MLL 的凋亡裂解受到相邻序列基序的调节，包括 ATC 片段或拓扑异构酶 II 结合位点。这些可能性将通过修改包含MLL的附加体（pREP4MLL）中的每个基序使用定点诱变来测试，该附加体在经历凋亡的细胞中再现基因组MLL切割。目标 2. 选择在凋亡激活后存活的细胞。将使用 pREP4 附加体来选择细胞系，该附加体包含两个腐败抗生素抗性基因之一内部的最小 MLL 凋亡裂解基序。 pREP4MLL min 内的凋亡裂解将用于刺激通过抗性基因的基因转换修复来选择含有稳定拷贝的pREP4MLL min 的存活细胞。目标 3. 检测有治疗相关白血病风险的患者中的 MLL 易位。将通过 RT-PCR 分析从非霍奇金淋巴瘤治疗后患者采集的血液中是否存在 MLL-AF9 信息，并使用反向 PCR 在凋亡裂解位点进行 MLL 重排。在每种情况下，含有此类畸变的细胞分裂的能力将取决于每个患者样本中是否存在重复的基因组断点连接。