REPLICATION MECHANISM OF DRUG INDUCED GENE AMPLIFICATION

描述：基因扩增与癌症有很大关系。 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的抗性的发展中， 毒品 本资助申请中描述的研究旨在 深入了解药物诱导的基因扩增的各种机制 在人类CCRF-CEM白血病细胞中。 待检验的中心假设是 某些抗癌剂，如阿糖胞苷（araC）或 aphidicolin，通过诱导 形成异常的DNA复制中间体， 扩增子的前体。 提出了几种实验方法， 评价药物诱导的DNA复制改变的重要性 与CAD和DHFR基因扩增有关。 具体目标1 阿糖胞苷和阿非迪霉素对RNA引发的DNA在细胞内积累的影响 将监测DHFR序列的下游以及 在c-myc复制起点处或下游的RNA引发的DNA 基因 这些实验将解决基因诱导物 扩增（aphidicolin，araC）优先抑制DNA合成 下游的复制起点，但允许起点激活， 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 在起点合成，从而防止激活新的复制 起源. 在特定目标2中，CAD或DHFR的链特异性分析 在araC或阿非迪霉素处理期间合成的序列将决定 这些序列是否优先在PALA或MTX中恢复 抵抗细胞和起源于领先，落后，或两者兼而有之 DNA复制叉的链。 在《目标3》中， 是为了确定阿糖胞苷或阿非迪霉素是否诱导了 复制叉与含有CAD的附加体的形成有关， DHFR序列和耐药性的发展。 FaraA的能力 以阻断这些药物抗性附加体的形成。

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.