Modulation of c-myc oncogene expression by phorbol ester and interferon-gamma: appraisal by flow cytometry.
A flow cytometric assay was developed to examine the expression of the cellular myc oncogene in relation to cell cycle in individual cells. C-myc-oncoprotein was detected by indirect immunofluorescence using a purified sheep polyclonal antibody, anti-human-myc. Specific binding of anti-human-myc was measured by flow cytometry. C-myc oncoprotein was detected in 90% of HL-60 and 75% of Daudi cells; human hematopoietic cell lines known to express high levels of c-myc oncogene. However, c-myc protein could not be detected in the REH cell line, normal human peripheral lymphocytes or thymocytes. Nuclear DNA content was measured simultaneously using propidium iodide staining. There was an equal level of c-myc protein in G0/G1, S and G2/M phases. The extent and kinetics of c-myc oncoprotein induction have been determined following phorbol ester, 12-O tetradecanoylphorbol 13 acetate (TPA) and interferon-gamma (IFN-gamma) exposure of both HL-60 and Daudi cells. TPA produced a gradual reduction in the level of c-myc protein and arrested the cells in G0/G1 phase in HL-60 cells. However, TPA failed to reduce c-myc protein or to change cell cycle distribution in Daudi cells. Interestingly, c-myc protein levels were stimulated by exposure of both HL-60 and Daudi cells to IFN-gamma. The results indicate that flow cytometric assay of oncogene expression is feasible, fast and requires relatively few cells. It also allows for the direct correlation of modulation of oncogene expression with cell kinetics.
Mohamed
A N
AN
Department of Internal Medicine, Wayne State University School of Medicine, Detroit, Michigan.
Nakeff
A
A
Mohammad
R M
RM
KuKuruga
M
M
al-Katib
A
A
D000906:Antibodies; D002453:Cell Cycle; D002460:Cell Line; D004273:DNA, Neoplasm / Q000032:analysis; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology*; D007700:Kinetics; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000235:genetics*; D016271:Proto-Oncogene Proteins c-myc; D011519:Proto-Oncogenes / Q000187:drug effects*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000166:cytology / Q000187:drug effects / Q000201:enzymology
D000906:Antibodies; D004273:DNA, Neoplasm; D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc; D007371:Interferon-gamma; D011505:Protein-Tyrosine Kinases; D013755:Tetradecanoylphorbol Acetate