Andreeff M, Slater D E, Bressler J, Furth M E
Blood. 1986 Mar;67(3):676-81.
Human hematopoietic malignancies provide an excellent model for the study of the activity of cellular oncogenes in a context of known defects in cell proliferation and differentiation. A flow cytometric immunofluorescence assay was developed to quantitate the expression of the cellular ras oncogenes in relation to the cell cycle in individual leukemic cells. Specific binding of a monoclonal antibody to the 21-kd protein (p21ras) encoded by the Ha-ras, Ki-ras, and N-ras genes was measured by flow cytometry and confirmed by fluorescence microscopy. P21ras was detected in 416B, a murine hematopoietic precursor cell characterized by a high level of Ki-ras expression, and in the human leukemic cell lines P-12 and KG-1. The presence of p21ras in the cell lines was also shown by immunoprecipitation. Cellular DNA content was determined simultaneously to define cell cycle phases. There was an equal distribution of p21ras in G1, S, and G2M, with considerable heterogeneity of ras gene expression in the G1 compartment. The assay allows oncogene expression to be studied in populations of intact single cells in which cell heterogeneity is maintained, requires very few cells per sample, and directly correlates oncogene expression to cell kinetic data.
人类造血系统恶性肿瘤为研究细胞癌基因在已知细胞增殖和分化缺陷背景下的活性提供了一个极佳模型。开发了一种流式细胞术免疫荧光测定法，以定量单个白血病细胞中细胞ras癌基因的表达与细胞周期的关系。通过流式细胞术测定单克隆抗体与Ha-ras、Ki-ras和N-ras基因编码的21-kd蛋白（p21ras）的特异性结合，并通过荧光显微镜进行确认。在以高水平Ki-ras表达为特征的小鼠造血前体细胞416B以及人白血病细胞系P-12和KG-1中检测到了p21ras。免疫沉淀法也证实了细胞系中存在p21ras。同时测定细胞DNA含量以确定细胞周期阶段。p21ras在G1期、S期和G2M期分布均匀，在G1期ras基因表达存在相当大的异质性。该测定法允许在维持细胞异质性的完整单细胞群体中研究癌基因表达，每个样品所需细胞极少，并将癌基因表达与细胞动力学数据直接关联起来。
