Effect of anti-S phase agents on human chromosomes

说明(申请人提供)：阿霉素、吉西他滨、阿糖胞苷和羟基脲等抗S相药广泛用于癌症和血液病的治疗，如原发性血小板减少症、真性红细胞增多症和镰状细胞性贫血。所有这些药物都被认为会导致复制分叉停滞，导致抑制细胞增殖的检查点通路的激活。然而，关于这些抑制剂如何抑制复制的起源，负责检查点激活的部位/DNA损伤，以及这些损伤对染色体结构或基因表达的影响，人们还知之甚少。此外，已知其中许多药物通过未知的机制影响基因表达。例如，羟基尿素已被用于治疗镰状细胞性贫血，因为它增加了胎儿血红蛋白的表达，降低了细胞表面黏附分子的表达。我们最近发表了研究复制时间、复制起源和抗S相剂对人类细胞复制起始点影响的基因组方法。我们发现，用于治疗的两种抗S相剂导致聚集的新起始点的激发，导致在基因组中指定位置的高密度停滞的复制叉子。 在这项建议中，我们计划确定这是否是其他抗S期药物的一般特征，这些聚集的新起点在不同细胞系中出现的位置是否相同，或者当细胞暴露于这些药物时，S期所有部分的细胞是否发生改变。我们将调查在聚集的新起始点的高密度停滞的复制叉子是否使基因组的这些位置容易受到双链断裂(DSB)和染色体重排的影响。我们将检查由于聚集的新起源的高密度而引起的染色质的紊乱状态是否导致基因表达的抑制。最后，我们将研究哪些检查点激活物和dna修复蛋白被招募到由抗S相剂产生的停滞的复制叉中，在哪里，以什么顺序被招募。

DESCRIPTION (provided by applicant): Anti-S phase agents like doxorubicin, gemcitabine, cytarabine and hydroxyurea are in extensive use for the treatment of cancers and of hematological diseases such as essential thrombocytopenia, polycythemia vera and sickle cell anemia. All these agents are believed to cause replication fork stalling leading to activation of checkpoint pathways that inhibit cell proliferation. Yet, not much is known about how these inhibitors inhibit the origins of replication, of the sites/DNA lesions responsible for checkpoint activation and of the effect of these lesions on chromosome structure or gene expression. In addition, many of these agents are known to affect gene expression by unknown mechanisms. For example, hydroxyurea has been used in sickle cell anemia because it increases the expression of fetal hemoglobin and decreases the expression of cell-surface adhesion molecules. We have recently published genomic approaches to study time of replication, origins of replication and effects of anti-S phase agents on the replication origins in human cells We have discovered that two anti-S phase agents used for therapy lead to the firing of clustered neo-origins, leading to a high density of stalled replication forks at defined sites in the genome. In this proposal we plan to determine whether this is a general feature of other anti-S phase agents, whether the sites where these clustered neo-origins occur are the same in different cell lines or whether the sites change when cells are exposed to these agents in all parts of the S phase. We will investigate whether the high density of stalled replication forks at clustered neo-origins make these sites of the genome susceptible to double-strand breaks (DSB) and chromosomal rearrangements. We will examine whether the disturbed state of the chromatin due to the high density of clustered neo-origins leads to suppression of gene expression. Finally, we will examine which, where and in what order checkpoint activators and DNA repair proteins are recruited to the stalled replication forks created by anti-S phase agents.