REPLICATION MECHANISM OF DRUG INDUCED GENE AMPLIFICATION

药物诱导基因扩增的复制机制

• 批准号: 2011979
• 负责人: DANIEL James FERNANDES
• 金额: $ 17.8万
• 依托单位: MEDICAL UNIVERSITY OF SOUTH CAROLINA
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-04-15 至 2001-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2011979
• 关键词: DNA replication; aphidicolin; chemical carcinogenesis; circular DNA; cytosine arabinoside; dihydrofolate reductase; fludarabine; human genetic material tag; natural gene amplification; neoplasm /cancer genetics; nucleic acid sequence; pharmacogenetics; plasmids; tissue /cell culture

DESCRIPTION: Gene amplification is of major relevance to the cancer problem, since it is directly involved in tumor initiation and progression as well as in the development of tumor cell resistance to various anticancer drugs. The studies described in this grant application are designed to provide insights into various mechanisms of drug-induced gene amplification in human CCRF-CEM leukemia cells. The central hypothesis to be tested is that certain anticancer agents, such as cytosine arabinoside (araC) or aphidicolin, promote amplification of drug resistance genes by inducing the formation of aberrant DNA replication intermediates that become the early precursors of amplicons. Several experimental approaches are proposed to evaluate the importance of drug-induced alterations in the DNA replication fork to the amplifications of CAD and DHFR genes. In Specific Aim 1 the effects of araC and aphidicolin on accumulation of RNA-primed DNA at or downstream of DHFR sequences will be monitored as well as the accumulation of RNA-primed DNA at or downstream of the replication origin of the c-myc gene. These experiments will address the hypothesis that inducers of gene amplification (aphidicolin, araC) preferentially inhibit DNA synthesis downstream of the replication origin but allow origin activation, which leads to generation of abnormal replication forks. Another goal is to determine if the amplification inhibitor fluodarabine (FaraA) decreases formation of abnormal replication structures by inhibiting RNA-primed DNA synthesis at the origin and thereby prevents activation of new replication origins. In Specific Aim 2, strand-specific analysis of the CAD or DHFR sequences synthesized during araC or aphidicolin treatment will determine whether these sequences are preferentially recovered in PALA or MTX resistant cells and originate from either the leading, lagging, or both strands of the DNA replication fork. In Specific Aim 3 the final goal will be to determine whether the araC or aphidicolin induced alterations in the replication fork are related to the formation of episomes containing CAD or DHFR sequences and the development of drug resistance. The ability of FaraA to block formation of these drug resistance episomes will also be tested.

描述：基因扩增与癌症密切相关 问题，因为它直接参与肿瘤的发生和进展 以及肿瘤细胞对各种抗癌药物产生耐药性的过程 药物。 本拨款申请中描述的研究旨在 提供对药物诱导基因扩增的各种机制的见解 在人类 CCRF-CEM 白血病细胞中。 要检验的中心假设是 某些抗癌药物，例如阿糖胞苷 (araC) 或 aphidicolin 通过诱导耐药基因的扩增来促进耐药基因的扩增 形成异常的DNA复制中间体，成为早期的DNA复制中间体 扩增子的前体。 提出了几种实验方法 评估药物诱导的 DNA 复制改变的重要性 分叉至 CAD 和 DHFR 基因的扩增。 在具体目标 1 中 araC 和 aphidicolin 对 RNA 引发的 DNA 积累的影响 DHFR 序列的下游以及累积情况将受到监测 c-myc 复制起点或其下游的 RNA 引发的 DNA 基因。 这些实验将解决基因诱导剂的假设 扩增（aphidicolin、araC）优先抑制 DNA 合成 复制起点的下游，但允许起点激活，这 导致异常复制叉的产生。 另一个目标是 确定扩增抑制剂氟达拉滨 (FaraA) 是否减少 通过抑制 RNA 引发的 DNA 形成异常复制结构 在起点合成，从而防止新复制的激活 起源。 在具体目标 2 中，CAD 或 DHFR 的链特异性分析 araC 或 aphidicolin 处理过程中合成的序列将决定 这些序列是否优先在 PALA 或 MTX 中恢复 耐药细胞源自领先细胞、滞后细胞或两者 DNA复制叉的链。 在具体目标 3 中，最终目标将 确定 araC 或 aphidicolin 是否诱导了 复制叉与包含 CAD 或的附加体的形成有关 DHFR 序列和耐药性的发展。 法拉A的能力 还将测试阻止这些耐药附加体形成的方法。