REPLICATION MECHANISM OF DRUG INDUCED GENE AMPLIFICATION

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发展 以及在肿瘤细胞对各种抗癌药物产生耐药性的过程中 毒品。这项拨款申请中所描述的研究旨在 提供对药物诱导的基因扩增的各种机制的见解 在人CCRF-CEM白血病细胞中。需要检验的中心假设是 某些抗癌药物，如阿糖胞苷(Arac)或 APHIDICOIN通过诱导耐药基因扩增 异常DNA复制中间产物的形成成为早期的 扩增产物的前体。提出了几种实验方法来 评估药物诱导的DNA复制改变的重要性 CAD和DHFR基因的扩增。在具体目标1中 Arac和Aphidicolin对RNA诱导的DNA在小鼠体内积累的影响 将监测DHFR序列的下游以及累积 在c-myc复制起始处或其下游的RNA启动的DNA 吉恩。这些实验将解决这样一种假设，即基因的诱导者 扩增产物(阿司匹林、Arac)优先抑制DNA合成 位于复制源的下游，但允许源激活，这 导致异常复制分叉的产生。另一个目标是 确定扩增抑制剂氟达拉滨(FaraA)是否降低 通过抑制RNA引发的DNA形成异常复制结构 在起始处合成，从而防止激活新的复制 起源。在具体目标2中，对CAD或DHFR进行链特异性分析 在Arac或aphidiclin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 抗性细胞，起源于领先的、落后的或两者兼而有之 DNA复制叉子的链。在具体目标3中，最终目标将 以确定Arac或aphidiclin是否诱导了 复制叉与含有CAD或CAD的附体的形成有关 DHFR序列与耐药的发展。FaraA的能力 为了阻止这些耐药表型的形成，还将进行测试。

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.