RADIATION SENSITIVITY OF QUIESCENT TUMOR CELLS

本提案的总体目标是调查基本的 辐射反应的细胞和分子机制 静止细胞 静止期细胞以平台期单层存在 培养物以及多细胞球状体和实体瘤， 他们被认为是优先能够 修复潜在致命辐射损伤（PLD）。 然而，在这方面， 直到最近，这些细胞还很难识别， 和量化，和固有的辐射敏感性的静态 尤其是对于人肿瘤细胞仍然没有解决。 采用离心淘洗和流式细胞术技术 最近在我们的实验室开发，以显着丰富 静止细胞，我们建议从平台期培养物中评估 和球体：a）孤立的静态辐射敏感性 B）辐射诱导的肿瘤细胞动力学的改变， 从静止区室到增殖区室的募集; c） 静止期肿瘤细胞对肿瘤细胞的 辐射诱导的PLD的修复;和d）特异性DNA的作用， 相关酶（包括一种新发现的酶）， 修复的分子机制。 细胞之间的关系 和分子修复在静止细胞将研究， 比较DNA的修复动力学（链断裂，DNA- 蛋白质交联、DNA-DNA交联）和染色体损伤 在相同的放射生物学相关剂量下细胞恢复。 这些研究将使用碱性洗脱、DNA 解旋和过早染色体凝聚技术。 使用这些测定的结果将相互关联，以确定 哪些类型的损害可以测量并与辐射有关 静止细胞的敏感性。 使用抑制剂的后续研究 的DNA复制和修复将进行静止和 增殖细胞和这些抑制剂对细胞增殖的影响。 将评价辐射诱导的PLD的修复动力学。 选择抑制剂以特异性抑制DNA聚合酶 α、β、δ I、δ II、拓扑异构酶I和II。 更大 了解静止细胞辐射的机制 反应性，静止细胞募集到 增殖状态，以及静止细胞在 PLD的恢复，可以提供与两者相关的基本信息， 放射治疗和化疗。

The overall objective of this proposal is to investigate the basic cellular and molecular mechanisms of radiation responsiveness of quiescent cells. Quiescent cells exist in plateau phase monolayer cultures as well as multicellular spheroids and solid tumors and they have been implicated to be preferentially capable of repairing potentially lethal radiation damage (PLD). However, these cells have until recently been extremely difficult to identify and quantify, and the inherent radiation sensitivity of quiescent cells still remains unresolved especially for human tumor cells. Using the centrifugal elutriation and flow cytometry techniques recently developed in our laboratory to significantly enrich the quiescent cells, we propose to assess from plateau phase cultures and spheroids for: a) the radiation sensitivity of isolated quiescent tumor cells; b) radiation-induced alterations in the dynamics of recruitment from quiescent to proliferating compartments; c) the possible differential capacity of quiescent tumor cells for the repair of radiation-induced PLD; and d) the role of specific DNA- related ezymes (including one newly discovered enzyme) in the molecular mechanism(s) of repair. Relationships between cellular and molecular repair in quiescent cells will be studied by comparing the repair kinetics of DNA (strand breaks, DNA- protein crosslinks, DNA-DNA crosslinks) and chromosome damage to cell recovery at the same radiobiologically relevant doses. These studies will be performed with alkaline elution, DNA unwinding and premature chromosome condensation techniques. The results using these assays will be interrelated to determine which types of damage can be measured and related to radiation sensitivity of quiescent cells. Subsequent studies using inhibitors of DNA replication and repair will be performed on quiescent and proliferating cells and the effect of these inhibitors on the kinetics of the repair of radiation-induced PLD will be evaluated. Inhibitors are chosen to specifically inhibit DNA polymerases alpha, beta, delta I, delta II, topoisomerase I and II. A greater understanding of the mechanisms of quiescent cell radiation responsiveness, the kinetics of recruitment of quiescent to proliferating status, and the role of quiescent cells in the recovery of PLD, may provide basic information relevant to both radiation therapy and chemotherapy.