Cellular Responses to Cancer Drug-Induced Genomic Breaks

描述（由申请方提供）：电离辐射（IR）诱导的DNA双链断裂（DSB）的细胞反应涉及通过磷脂酰肌醇3-激酶样激酶（PIKK）ATM激活细胞周期检查点蛋白，延迟细胞周期进程以允许DNA修复并保持染色体完整性。对拟放射性烯二炔C-1027诱导的DNA双链断裂的反应偏离该模型。例如，IR诱导ATM依赖性和C-1027（ATM依赖性和非依赖性）反应。此外，用等细胞毒性水平的IR或C-1027处理分别导致极低（2.9%）或极高（92%）百分比的有丝分裂细胞显示异常染色体重组。FISH分析显示，C-1027诱导的染色体畸变通常与端粒的破坏有关。 对抗肿瘤药物C-1027的这些独特反应背后的机制应该有助于扩展我们对DSB的DNA损伤反应的理解。首先，将确定参与对C-1027应答的关键检查点途径蛋白，重点是ATM依赖性和独立性应答。接下来，将鉴定C-1027诱导广泛染色体融合的条件，其特征在于异常末端连接和片段化。NHEJ DNA结合蛋白Ku不仅参与修复反应，而且在调节染色体融合中起作用，还将检查其在C-1027诱导的异常末端连接中的作用。最后，我们将测试是否C-1027，诱导DSB优先内的GTTA基序，靶向端粒串联重复序列（GGGTTA），并有助于异常末端连接诱导端粒功能障碍。将选择性地测试具有不同于C-1027的切割特征的其他烯二炔，以提供关于C-1027诱导的损伤的性质与DNA损伤反应相关的额外的机理见解。具体目标将检验以下假设： 1. C-1027与其他烯二炔和IR相比是独特的，因为它诱导ATM依赖性和独立的DNA损伤反应。 2. C-1027与其他烯二炔和IR相比是独特的，因为它会导致非常高水平的重排染色体。 3. C-1027与其他烯二炔和IR相比是独特的，因为它靶向端粒。

DESCRIPTION (provided by applicant): Cellular responses to ionizing radiation (IR)-induced DNA double strand breaks (DSBs) involve activation, via the phosphatidylinositol 3-kinase-like kinase (PIKK) ATM, of cell cycle checkpoint proteins that delay cell cycle progression to allow DNA repair and to preserve chromosomal integrity. Responses to DNA DSBs induced by the radiomimetic enediyne C-1027 deviate from this model. For example, IR induces ATM-dependent, and C-1027, both ATM- dependent and independent, responses. Furthermore, treatment with equi-cytotoxic levels of IR or C-1027 results in either a very low (2.9%), or an extraordinarily high (92%), percentage, respectively of mitotic cells showing aberrant chromosomal recombination. FISH analysis revealed that C-1027-induced chromosomal aberrations are frequently associated with disruption of telomeres. The mechanism(s) behind these unique responses to the antitumor drug, C-1027 should help extend our understanding of DNA damage responses to DSBs. First, pivotal checkpoint pathway proteins involved in the response to C-1027 will be identified, with emphasis on both ATM-dependent and independent responses. Next the conditions under which C-1027 induces extensive chromosomal fusions, characterized by aberrant end-joining and fragmentation, will be identified. The NHEJ DNA binding protein Ku, which not only is involved in repair responses but also plays a role in regulating chromosomal fusions, will also be examined for its role in C-1027 induced aberrant end-joining. Finally, we will test whether C-1027, which induces DSBs preferentially within a GTTA motif, targets the telomere tandem repeat sequence (GGGTTA) and contributes to aberrant end-joining by inducing telomere dysfunction. Other enediynes with cleavage characteristics different from C-1027, will be tested selectively to provide additional mechanistic insights into which properties of C-1027-induced damage are associated with the DNA damage responses. The specific aims will test the following hypotheses: 1. C-1027 is unique compared to other enediynes and to IR in that it induces both ATM-dependent and independent DNA damage responses. 2. C-1027 is unique compared to other enediynes and to IR in that it causes extraordinarily high levels of rearranged chromosomes. 3. C-1027 is unique compared to other enediynes and IR in that it targets telomeres.