RADIATION SENSITIVITY OF QUIESCENT TUMOR CELLS

该提案的总体目标是调查基本的 细胞和分子机制的辐射响应性 静止的细胞。平台期单层中存在静止细胞 培养细胞以及多细胞球体和实体肿瘤 他们被牵连到优先有能力 修复潜在的致命辐射损伤(PLD)。然而， 直到最近，这些细胞还很难识别。 和量化，以及静止时的固有辐射敏感性 细胞仍未分解，尤其是人类肿瘤细胞。 利用离心洗脱和流式细胞仪技术 我们实验室最近开发的显著丰富了 静止细胞，我们建议从平台期培养来评估 和椭球体：a)孤立静止的辐射敏感性 肿瘤细胞；b)辐射诱导的动态变化 从静止到不断扩大的招募；c) 静止期肿瘤细胞对肿瘤细胞可能的分化能力 辐射诱导的PLD的修复；以及d)特定DNA- 相关酶(包括一种新发现的酶) 修复的分子机制(S)。蜂窝网络之间的关系 静止细胞的分子修复将通过以下方法进行研究 比较DNA的修复动力学(链断裂、DNA- 蛋白质交联物、DNA-DNA交联物)和染色体损伤 以相同的放射生物学相关剂量恢复细胞。 这些研究将用碱性洗脱，DNA 解离和过早凝聚染色体技术。 使用这些化验结果将相互关联，以确定 哪些类型的损伤可以测量并与辐射相关 静止细胞的灵敏度。使用抑制剂的后续研究 的DNA复制和修复将在静止和 增殖细胞及其抑制物对细胞生长的影响 将评估辐射诱导的PLD修复的动力学。 选择抑制剂来特异性地抑制DNA聚合酶 α、β、增量I、增量II、拓扑异构酶I和II。 对静止细胞辐射机制的认识 反应性，静止状态下的招募动力学 增殖状态，以及静止细胞在细胞周期中的作用。 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.