Development of antisense therapy for solid tumors with reciplocal translocation

1.粘液样和圆细胞脂肪肉瘤TLS-CHOP易位的遗传分析：分离出两种新型融合转录本并测序。对基因组融合点的精确分析表明，断裂点周围的序列具有一些特征。在两个病例中发现与已克隆的白血病重组热点结合蛋白Translin结合位点的序列同源性。此外，断裂点序列显示与拓扑异构酶II结合位点高度同源。2.将TLS-CHOP融合基因导入前脂肪细胞系中，将4种不同类型的TLS-CHOP融合基因编码区克隆到CMV表达载体中，转染前脂肪细胞系Swiss 3 T3 L1细胞。建立了两株表达TLS-CHOP融合基因的脂肪肉瘤细胞系。一个是KS 509，其表达一种新型的融合转录本。从亲本细胞系中也建立了SV 40转化衍生物（KS 509 SV），并将其用于评估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AON中的一种显示出约50%的抑制。另一个细胞系KS 559是从接种在SCID小鼠上的肿瘤建立的。该肿瘤的组织学结果与粘液样脂肪肉瘤相容，并且体外细胞也显示脂肪细胞形态，因此认为其是体内和体外反义治疗的合适模型。

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.