Flow cytometric studies of the nuclear matrix.
We have devised a method to measure the protein and nucleic acid content of the nuclear matrix using flow cytometry. Nuclear matrices were prepared from nuclei by DNase I digestion followed by 3 M NaCl extraction. The resulting particles were stained with fluorescein isothiocyanate (FITC) for protein and propidium iodide (PI) for double-stranded nucleic acids, and fluorescence as well as forward angle light scatter was detected. The matrices were also subjected to additional chemical or enzymatic perturbations, and changes in the above parameters were measured. Results showed that matrices from heat-shocked cells not only retained the majority of heat-induced excess nuclear protein, but also exhibited higher PI signals than controls after RNase A digestion. This observation did not hold if RNase A digestion preceded high-salt extraction, suggesting that a salt-extractable moiety had been replaced or altered by heat so that double-stranded RNA was protected from the nucleolytic attack. The residual PI fluorescence in matrices from heated cells bore a linear relationship to the increased protein content in those matrices, indicating that the excess protein sequesters matrix-associated RNA. Polyacrylamide gel electrophoresis of matrix polypeptides revealed increased amounts of many proteins as a result of heat as well as the appearance of several new proteins, one of which comigrates with the HSP72/73 heat-shock proteins. The results of these studies show that flow cytometry can be used to study the nuclear matrix and is capable of detecting changes that result from alterations in its protein composition.
Wright
W D
WD
Washington University School of Medicine, Section of Cancer Biology, St. Louis, Missouri 63108.
Higashikubo
R
R
Roti Roti
J L
JL
D002467:Cell Nucleus / Q000032:analysis*; D004273:DNA, Neoplasm / Q000032:analysis*; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D012330:RNA, Double-Stranded / Q000032:analysis*; D014407:Tumor Cells, Cultured / Q000032:analysis*
D004273:DNA, Neoplasm; D012330:RNA, Double-Stranded