Simultaneous detection of cellular ras p21 oncogene product and DNA content by two-parameter flow cytometry.
The rapid identification of the expression of oncogene products in specific cell types could be useful to investigate normal and malignant cell proliferation. We have developed a sensitive fixation - permeabilization technique (with 70% ethanol and 0.01% Triton x 100) for the detection of p21 ras oncoprotein and DNA content. Cell suspensions with negligible cell clumping, bright specific immunofluorescent staining were obtained with this method. Bivariate flow cytometry was then used to quantitate simultaneously the distribution of anti p21 ras oncoprotein (with a specific FITC-labeled antibody) and of total DNA (with propidium iodide). The study was carried out in human leukemic cell lines HL-60 and K562, human breast carcinoma cell line MCF-7 and fresh neoplastic cells from human acute leukemia. The p21 ras oncoprotein was found in all phases of cell cycle. The degree of its expression, however, varied widely in diploid (C0/G1) cells from different samples, which could be related to differences in the relative proportion of G0 and G1 cells. Compared to the conventional gel electrophoretic technique, the use of bivariate FCM is feasible, fast, requires fewer cells per sample (2 x 10(6] and allows both the ras oncogene expression in intact cell populations as well as its relationship with cell cycle phases to be studied.
Giordano
M
M
Dipartimento di Medicina Interna e Terapia Medica, Universitá and I.R.C.C.S., S. Matteo, Pavia, Italy.
Danova
M
M
Riccardi
A
A
Mazzini
G
G
D002453:Cell Cycle; D004273:DNA, Neoplasm / Q000032:analysis*; D005434:Flow Cytometry; D006801:Humans; D011518:Proto-Oncogene Proteins / Q000032:analysis*; D016283:Proto-Oncogene Proteins p21(ras); D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D011518:Proto-Oncogene Proteins; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)