Loss of Rna and Protein, And Changes in Dna During A 30-Hour Cold Perchloric Acid Extraction of Cultured Cells

Abstract

KB cells derived from human carcinoma were fixed in acetic-alcohol (1:3) and extracted with 10% perchloric acid (PCA) at 4 C for 1, 3, 6, 9, 12, 24 and 30 hr. Cells were then washed in water and stained for nucleic acids, proteins, polysaccharides, and lipids. Control cells were kept in water for 30 hr prior to staining. Acridine orange (AO) fluorochroming revealed color changes in residual cytoplasmic and nucleolar RNA as well as DNA during extraction–-interpreted as indicative of molecular alterations. All nucleic acid stains (AO, gallocyanin chromalum, and azure B bromide) demonstrated a differential extraction of RNA, with cytoplasmic RNA being removed in about 6 hr and nucleolar RNA requiring 6 more hours for complete extraction. Large granules appeared early in nuclei. These were positive for DNA by azure B, gallocyanin chromalum, Feulgen, and fluorescent-Feulgen. These same granules stained for protein by mercuric bromphenol blue and alkaline Biebrich scarlet. At 24 hr, there was visual and Feulgen-cytophotometric evidence for a slight loss of DNA, which may amount to 10–20%. There was a progressive loss of cytoplasmic and nuclear but not nucleolar protein during PCA treatment. Concurrently, large protein-positive granules appeared in the cytoplasm. Apparently, PCA treatment in combination with an aqueous wash was responsible for some protein loss. Glycogen was gradually lost (fluorescent PAS) and redistributed in cells. Lipids were unaffected (Sudan black B).