Aryee D N, Sommergruber W, Muehlbacher K, Dockhorn-Dworniczak B, Zoubek A, Kovar H
Children's Cancer Research Institute, St. Anna Kinderspital, Vienna, Austria.
Lab Invest. 2000 Dec;80(12):1833-44. doi: 10.1038/labinvest.3780194.
Type 1 and type 2 EWS-FLI1 fusion products result from variation in breakpoint locations arising from the t(11;22)(q24;q12) recurrent chromosomal translocation in Ewing's sarcoma family tumors (EFT). Previously, studies from our institution (updated in the present communication at a median follow-up of more than 6 years) and others suggested a prognostic difference for EFT patients with localized disease depending on the type of EWS-FLI1 fusion present in the tumor. It has been suggested that the observed clinical discrepancies result from different transactivation potentials of the various EWS-FLI1 fusion proteins. In an attempt to identify genes whose expression levels are differentially modulated by structurally different EWS-FLI1 transcription factors, we have used two related PCR-based subtractive approaches, cDNA representational difference analysis (cDNA-RDA) and linker-capture subtraction (LCS) to compare transcript representations in cDNA pools of type 1 versus type 2 EFT cell lines. About 800 clones obtained by the two approaches were analyzed by dot blot hybridization to cDNA pools. Eighty-six clones showing the highest variability in signal intensities on the dot blots were further hybridized to individual EFT cell line RNAs on Northern blots, and four of them were additionally studied by real-time quantitative PCR (RTQ-PCR). Although interindividual variations in gene expression patterns in the range of one- to several-fold were observed, no correlation to specific EWS-FLI1 fusion types could be identified. Among the genes differentially expressed in individual EFT cell lines are several previously implicated in tumor growth, invasion, and metastasis. Although our data may have revealed candidate genes whose composite expression pattern may be relevant for the biology of individual EFT, they do not support a role of distinct EWS-FLI1 fusion types for EFT prognosis based on different transactivation potentials.
1型和2型EWS-FLI1融合产物源于尤因肉瘤家族肿瘤（EFT）中t(11;22)(q24;q12)复发性染色体易位导致的断点位置变异。此前，我们机构的研究（在本次通讯中更新，中位随访时间超过6年）以及其他研究表明，对于局限性疾病的EFT患者，根据肿瘤中存在的EWS-FLI1融合类型，预后存在差异。有人认为，观察到的临床差异是由各种EWS-FLI1融合蛋白不同的反式激活潜能导致的。为了鉴定其表达水平受结构不同的EWS-FLI1转录因子差异调节的基因，我们使用了两种基于PCR的相关消减方法，即cDNA代表性差异分析（cDNA-RDA）和接头捕获消减（LCS），来比较1型与2型EFT细胞系cDNA文库中的转录本表现。通过这两种方法获得的约800个克隆通过点杂交与cDNA文库进行分析。在点杂交上显示信号强度变化最大的86个克隆进一步与Northern杂交上的单个EFT细胞系RNA杂交，其中4个还通过实时定量PCR（RTQ-PCR）进行了研究。尽管观察到基因表达模式存在个体间1至几倍范围内的差异，但未发现与特定EWS-FLI1融合类型的相关性。在单个EFT细胞系中差异表达的基因中有几个先前已涉及肿瘤生长、侵袭和转移。尽管我们的数据可能揭示了其复合表达模式可能与个体EFT生物学相关的候选基因，但它们不支持基于不同反式激活潜能的不同EWS-FLI1融合类型对EFT预后的作用。
