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Development of antisense therapy for solid tumors with reciplocal translocation

相互易位实体瘤反义疗法的发展

基本信息

批准号：
08557085
负责人：
TOGUCHIDA Junya
金额：
$ 11.26万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1997
项目状态：
已结题

项目摘要

1.Genetic analysis of TLS-CHOP translocations in myxoid and round-cell liposarcomas.Two novel types of fusion transcripts were isolated and sequenced. Precise analysis of genomic fusion points revealed that there are several characteristics on sequences around the breakpoints. Sequence homology with Translin binding site, which has been cloned as a recombination hot spot binding protein in leukemias, was found in two cases. In addition, the breakpoint sequence showed high homology with topoisomerase II binding sites. These results suggested the importance of two proteins in the process of translocation.2.Introduction of TLS-CHOP fusion genes into preadipocytic cell line.Coding region of four different types of TLS-CHOP fusion genes were cloned into CMV expression vector and transfected to preadipocytic cell line, Swiss 3T3 L1 cells. Transformed cell lines were established and characterized.3.Establishment of liposarcoma cell lines expressing TLS-CHOP fusion genes.Two liposarcoma cell lines were estabilshed. One is KS509, which expressed a novel type of fusion transcript. SV40 transformed derivative (KS509SV) was also established from the parental cell line, and it was used to evaluate the growth inhibitory effects of antisense oligonucleotides (AONs), which were designed to target the fusion point sequences. One of three AONs showed approximately 50% inhibition. The other cell line, KS559, was established from tumors inoculated on SCID mouse. Histological findings of this tumor were compatible with myxoid liposarcomas, and in vitro cells also showed adipocytic morphology, and therefore it was considered to be a suitable model for in vivo and in vitro antisense therapy.

1.黏液样和圆细胞脂肪肉瘤中TLS-CHOP易位的遗传分析。分离并测序了两种新型的融合转录本。基因组融合点的精确分析表明，在断点周围的序列上有几个特征。在2例白血病中发现与已克隆为重组热点结合蛋白的Translin结合位点序列同源。此外，断点序列与拓扑异构酶II结合位点具有较高的同源性。这些结果提示了两种蛋白在易位过程中的重要性。将TLS-CHOP融合基因导入前脂肪细胞。将4种不同类型TLS-CHOP融合基因的编码区克隆到CMV表达载体中，转染到前脂肪细胞Swiss 3T3 L1细胞。建立了转化细胞系并对其进行了鉴定。表达TLS-CHOP融合基因的脂肪肉瘤细胞系的建立。建立了2个脂肪肉瘤细胞系。一个是KS509，它表达了一种新型的融合转录物。从亲本细胞系中建立了SV40转化衍生物（KS509SV），并用于评价针对融合点序列设计的反义寡核苷酸（AONs）的生长抑制作用。三种AONs中的一种显示出约50%的抑制作用。另一个细胞系KS559是由接种在SCID小鼠上的肿瘤建立的。该肿瘤的组织学表现与黏液样脂肪肉瘤一致，体外细胞也表现为脂肪细胞形态，因此被认为是体内和体外反义治疗的合适模型。