PCC + CHROMOSOME PAINTING IN RADIATION AND DRUG STUDIES

这个项目的总体目标是使用荧光技术 原位杂交(FISH，或“染色体涂抹”)结合了 染色体过早凝聚(PCC)或中期扩散 作为预测实体瘤对辐射反应的一种手段。不是 预测人体个体辐射敏感性的方法 肿瘤目前是可用的，最近公布的数据表明 其他因素，除了对辐射的内在敏感性外， 肿瘤细胞在人类肿瘤的体内反应中起着重要作用。 由于这些因素可能涉及肿瘤环境(例如，细胞-细胞 接触和肿瘤缺氧)，放射敏感性的原位检测 必填项。这应该是在服用了几剂中的第一剂之后 放射治疗方案。尽管基于染色体的分析 损害是唯一合适的损害是不切实际的。通过允许 间期细胞中染色体的可视化--PCC技术 克服了体外培养肿瘤细胞的需要，但 技术问题仍然是计算少量过剩的中断数 前处理的染色体数目往往很大。然而， FISH和PCC的结合极大地简化了问题 将分析集中在单个染色体上。它还允许交换 像差很容易得分。这个项目的目的是 将PCC+FISH技术发展到可以使用的程度 临床上可作为一种预测性分析。最初，最简单的系统将是 使用G1二倍体人AG1522成纤维细胞，其存活和 PCC诱发率和辐射后的修复是已知的。下一首, 非整倍体人类肿瘤细胞(鳞癌和黑色素瘤)，覆盖 将使用广泛的辐射敏感度，最后是 这些肿瘤株的体内反应将被确定。在所有情况下， 这一假设将得到检验，即修复后的染色体损伤 (休息和交流)PCC+FISH评分接近预测 克隆形成法测定辐射敏感性。 FISH技术还将用于评估辐射敏感性 从同一细胞中期的稳定相互易位 与上述PCC+FISH一起使用的线条。将整体结合起来的能力 用二次彩色杂交技术绘制1-4条染色体 对着丝粒共有的重复序列允许明确的 辨别对称性和非对称性易位。 因为稳定易位的频率应该与 最初的染色体损伤，因为这些易位不是 从受照射的肿瘤细胞群中逐个细胞优先丢失 1-5剂量组分对肿瘤细胞杀伤率的估计 应该是可能的。 这两种方法都有各自的优点，仔细研究 两个人应该确定哪个更适合日常使用来确定 肿瘤的原位放射敏感性。

The overall goal of this project is to use the technique of fluorescence in situ hybridization (FISH, or "chromosome painting") combined either with premature chromosome condensation (PCC) or with metaphase spreads as a means of predicting the response of solid tumors to radiation. No method of predicting the radiation sensitivity of individual human tumors is presently available, and recently published data show that other factors, in addition to the intrinsic radiosensitivity of the tumor cells, play a major role in the in vivo response of human tumors. Since these factors likely involve the tumor milieu (e.g., cell-cell contact and tumor hypoxia), an in situ assay of radiosensitivity is required. This should be made following the first one of few doses in the radiotherapy regimen. Although an analysis based on chromosome damage is the only suitable damage is impractical. By allowing the visualization of chromosomes in interphase cells, the PCC technique overcomes the need to culture the tumor cells in vitro, but the technical problem remains of counting a small excess number of breaks over the often large pretreatment chromosome number. However, the combination of FISH with PCC enormously simplifies the problem by focusing the analysis on a single chromosome. It also allows exchange aberrations to be scored easily. The purpose of this project is to develop the PCC + FISH technique to the point at which it could be used clinically as a predictive assay. Initially, the simplest system will be used, that of G1 diploid human AG1522 fibroblasts, whose survival and PCC induction rate and repair following irradiation are known. Next, aneuploid human tumor cells (squamous carcinoma and melanoma), covering a wide range of radiation sensitivities, will be used, and finally the in vivo response of these tumor lines will be determined. In all cases, the hypothesis will be tested that chromosome damage after repair (breaks and exchanges) scored by PCC + FISH closely predict the radiation sensitivity as determined by clonogenic assay. The FISH technology will also be used to estimate radiation sensitivity from stable reciprocal translocations in metaphase in the same cell lines as used with PCC + FISH above. The ability to combine whole chromosome painting of 1-4 chromosomes with a second color hybridization to the repeat sequences common to the centromeres allows the unambiguous discrimination between symmetrical and asymmetrical translocations. Since the frequency of stable translocations should correlate with initial chromosome damage and since these translocations are not preferentially lost from the irradiated tumor cell population by cell death an estimate of tumor cell killing following 1-5 dose fractions should be possible. Each of these two methods has its advantages, and a careful study of the two should establish which is superior for routine use to determine tumor radiosensitivity in situ.