Cellular ras oncogene expression and cell cycle measured by flow cytometry in hematopoietic cell lines

Abstract

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.