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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >ANTISENSE DNA DOWNREGULATION OF THE ERBB2 ONCOGENE MEASURED BY A FLOW CYTOMETRIC ASSAY
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ANTISENSE DNA DOWNREGULATION OF THE ERBB2 ONCOGENE MEASURED BY A FLOW CYTOMETRIC ASSAY

机译：流式细胞术检测ERBB2癌基因的反义DNA下调。

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A causal role has been inferred for ERBB2 overexpression in the etiology of breast cancer and other epithelial malignancies, The development of therapeutics that inhibit this tyrosine kinase cell surface receptor remains a high priority, This report describes the specific downregulation of ERBB2 protein and mRNA in the breast cancer cell line SK-BR-3 by using antisense DNA phosphorothioates. An approach was developed to examine antisense effects which allows simultaneous measurements of antisense dose and gene specific regulation on a per cell basis. A fluorescein isothiocyanate end-labeled tracer oligonucleotide was codelivered with antisense DNA followed by immunofluorescent staining for ERBB2 protein expression. Two-color flow cytometry measured the amount of both intracellular oligonucleotide and ERBB2 protein. In addition, populations of cells that received various doses of nucleic acids were physically separated and studied. In any given transfection, a 100-fold variation in oligonucleotide dosage was found. ERBB2 protein expression was reduced greater than 50%, but only in cells within a relatively narrow uptake range, Steady-state ERBB2 mRNA levels were selectively diminished, indicating a specific antisense effect. Cells receiving the optimal antisense dose were sorted and analyzed for cell cycle changes. After 2 days of ERBB2 suppression, breast cancer cells showed an accumulation in the G(1) phase of the cell cycle. [References: 35]
机译：已经推断出ERBB2过表达在乳腺癌和其他上皮恶性肿瘤的病因中起着因果作用，抑制该酪氨酸激酶细胞表面受体的治疗剂的开发仍然是高度优先的，该报告描述了ERBB2蛋白和mRNA在乳腺癌中的特异性下调。乳腺癌细胞系SK-BR-3通过使用反义DNA硫代磷酸酯。已经开发出一种方法来检查反义作用，该方法允许在每个细胞的基础上同时测量反义剂量和基因特异性调控。荧光素异硫氰酸酯末端标记的示踪寡核苷酸与反义DNA共转运，然后进行免疫荧光染色以鉴定ERBB2蛋白的表达。双色流式细胞仪测量了细胞内寡核苷酸和ERBB2蛋白的量。另外，物理分离并研究了接受各种剂量的核酸的细胞群。在任何给定的转染中，发现寡核苷酸剂量有100倍的变化。 ERBB2蛋白表达降低超过50％，但仅在相对较窄吸收范围内的细胞中，稳态ERBB2 mRNA水平选择性降低，表明有特定的反义作用。对接受最佳反义剂量的细胞进行分类并分析细胞周期变化。 ERBB2抑制2天后，乳腺癌细胞在细胞周期的G（1）相中显示出积累。 [参考：35]

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