IMMUNOLOGICAL CHARACTERIZATION OF CELLS

将开发一个模型系统来研究x射线照射的影响， 诱导染色体重排对肿瘤细胞克隆变异性的影响 特别是关于耐药性的发展 通过基因扩增和其他突变的化疗剂。 期间 癌症治疗中出现耐药克隆变异体， 肿瘤细胞群是化疗失败的主要原因。 的 有待检验的一般假设是染色体断裂和 重排有助于诱导对化疗剂的抗性。 为了研究这一过程，将采用新的细胞融合技术：1） 测试电离辐射是否诱发染色体易位 包含二氢叶酸还原酶（DHFR）基因的区域（与 对化疗剂甲氨蝶呤（MTX）的抗性可导致 DHFR基因扩增频率的改变。 初步 有证据表明，携带这些染色体的CHO细胞系 通过基因扩增重排变得对MTX耐药， 比正常CHO细胞更频繁地（几个数量级）。 2)测试 这些差异是否是由于新的染色体位置的 DHFR基因的突变，或者是易位的DHFR基因的突变， 染色体片段或其他区域。 3)测试是否特定 可以采用处理组合来降低克隆的分数， 肿瘤群体中的变异。 手术将采用新技术 基于细胞融合，允许产生无限数量的CHO， 仅在CHO染色体的染色体位置上不同的细胞株 携带DHFR基因的片段。 基因扩增将通过以下方式进行选择： 甲氨蝶呤抗性，通过杂交分析（Southern）定量， 并通过正交场凝胶电泳进行表征。 原位 杂交将定位引入的含有DHFR的X射线片段 基因 组织培养单层和多细胞球体都将被 用于监测不同治疗组合对 肿瘤细胞群中克隆变体的百分比。

A model system will be developed to study the effects of x-irradiation- induced chromosomal rearrangements on tumor cell clonal variability specifically with respect to the development of resistance to chemotherapeutic agents by gene amplification, and other mutations. During cancer treatment, the emergence of drug resistant clonal variants in the population of tumor cells is a major cause of failure of chemotherapy. The general hypothesis to be tested is that chromosome breakage and rearrangement facilitate induction of resistance to chemotherapeutic agents. To study this process, novel cell fusion techniques will be employed to: 1) Test whether ionizing radiation-induced translocations of the chromosomal region encompassing the dihydrofolate reductase (DHFR) gene (associated with resistance to the chemotherapeutic agent methotrexate (MTX) can lead to alterations in the frequency of DHFR gene amplification. Preliminary evidence indicates that CHO cell lines carrying these chromosomal rearrangements become resistant to MTX by gene amplification much more frequently (several orders of magnitude) than do normal CHO cells. 2) Test whether these differences are due to the new chromosomal location of the translocated DHFR gene, or to mutations either to the translocated chromosomal fragment or to other regions. 3) Test whether specific treatment combinations can be employed to decrease the fraction of clonal variants in tumor populations. The procedures will employ novel techniques based on cell fusion that allow the generation of unlimited numbers of CHO cell strains differing only in the chromosomal location of a CHO chromosome fragment carrying the DHFR gene. Gene amplification will be selected by resistance to methotrexate, quantified by hybridization analysis (Southern), and characterized by orthogonal field gel electrophoresis. In-situ hybridization will locate the introduced x-ray fragment containing the DHFR gene. Both tissue culture monolayer and multicellular spheroids will be used to monitor the effects of different treatment combinations on percentage of clonal variants in tumor cell populations.