IMMUNOLOGICAL CHARACTERIZATION OF CELLS

将开发一个模型系统来研究x射线的影响。 诱发的染色体重排对肿瘤细胞克隆性的影响 具体地说，关于发展对 化疗药物通过基因扩增等方式发生突变。在.期间 癌症治疗中，耐药克隆变种的出现 肿瘤细胞过多是化疗失败的主要原因。这个 需要检验的一般假设是染色体断裂和 重排有助于诱导对化疗药物的耐药性。 为了研究这一过程，将采用新的细胞融合技术：1) 检测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 对化疗药物甲氨蝶呤(MTX)的耐药性可导致 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 重排通过更多的基因扩增而对MTX产生抗药性 比正常的CHO细胞频繁(几个数量级)。2)测试 这些差异是否是由于 易位的dhfr基因，或突变为易位的 染色体碎片或转移到其他区域。3)测试是否特定 联合治疗可以减少克隆细胞的比例。 肿瘤人群中的变异。这些程序将采用新的技术 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选择 对甲氨蝶呤的抗药性，通过杂交分析(Southern)定量， 并用正交场凝胶电泳法对其进行了表征。就地 杂交将定位引入的含有dhfr的x射线片段。 吉恩。组织培养单层和多细胞球体都将 用来监测不同治疗组合的效果 肿瘤细胞群体中克隆变异体的百分比。

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.