Vaughn J P, Iglehart J D, Demirdji S, Davis P, Babiss L E, Caruthers M H, Marks J R
Department of Surgery, Duke University Medical Center, Durham, NC 27710, USA.
Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8338-42. doi: 10.1073/pnas.92.18.8338.
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 G1 phase of the cell cycle.
已推断出ERBB2过表达在乳腺癌及其他上皮性恶性肿瘤的病因中起因果作用。开发抑制这种酪氨酸激酶细胞表面受体的治疗方法仍然是当务之急。本报告描述了通过使用硫代磷酸反义DNA对乳腺癌细胞系SK-BR-3中ERBB2蛋白和mRNA进行特异性下调。开发了一种方法来检测反义效应，该方法允许在单个细胞基础上同时测量反义剂量和基因特异性调控。将异硫氰酸荧光素末端标记的示踪寡核苷酸与反义DNA共同递送，随后对ERBB2蛋白表达进行免疫荧光染色。双色流式细胞术测量细胞内寡核苷酸和ERBB2蛋白的量。此外，对接受不同剂量核酸的细胞群体进行物理分离并研究。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%，但仅在摄取范围相对较窄的细胞中。稳态ERBB2 mRNA水平选择性降低，表明存在特异性反义效应。对接受最佳反义剂量的细胞进行分选并分析细胞周期变化。在ERBB2抑制2天后，乳腺癌细胞在细胞周期的G1期出现积累。
