PCC + CHROMOSOME PAINTING IN RADIATION AND DRUG STUDIES

本项目的总体目标是利用荧光技术 原位杂交（FISH，或“染色体彩绘”）结合了以下两种方法： 伴有过早染色体凝聚（PCC）或中期扩散 作为预测实体肿瘤对放射的反应的一种手段。没有 预测个体辐射敏感性的方法 肿瘤是目前可用的，最近公布的数据显示， 其他因素，除了固有的放射敏感性的 肿瘤细胞在人肿瘤的体内反应中起主要作用。 由于这些因素可能涉及肿瘤环境（例如，细胞间 接触和肿瘤缺氧），放射敏感性的原位测定被 必需的.这应该在第一次几剂之后进行， 放疗方案。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 间期细胞染色体的可视化，PCC技术 克服了体外培养肿瘤细胞的需要， 技术问题仍然是计算少量过量的断裂数 在通常较大的预处理染色体数目上。但 FISH与PCC的结合极大地简化了问题， 集中分析单个染色体。它还允许交换 畸变易于评分。该项目的目的是 将PCC + FISH技术发展到可以使用的程度 临床上作为预测性分析。最初，最简单的系统将是 使用G1二倍体人AG 1522成纤维细胞，其存活和 辐照后PCC诱导率和修复是已知的。下一个是 非整倍体人肿瘤细胞（鳞状细胞癌和黑色素瘤），包括 广泛的辐射敏感性，将被使用，最后， 将测定这些肿瘤系的体内反应。在所有情况下， 将检验染色体损伤修复后 PCC + FISH评分（断裂和交换）密切预测 通过克隆形成试验测定的辐射敏感性。 FISH技术还将用于估计辐射敏感性 来自同一细胞中期稳定的相互易位 与上述PCC + FISH一起使用的细胞系。将整个联合收割机 1-4条染色体的染色体涂染与第二次彩色杂交 与着丝粒共有的重复序列相结合， 区分对称易位和不对称易位。 由于稳定易位的频率应该与 最初的染色体损伤，由于这些易位不是 受照射的肿瘤细胞群体中， 死亡1-5个剂量分次后肿瘤细胞杀伤的估计值 应该是可能的。 这两种方法各有其优点，仔细研究 两个应该建立哪一个是上级的日常使用，以确定 原位肿瘤放射敏感性。

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.