ABSTRACT
Cell lysates prepared from 3T3-L1 cells were analyzed by western blotting using the avidin-biotin complex system and anti-Bax antibody. The antibody interacted with bands of proteins with estimated molecular weights of 120, 74, 72, and 25 kDa. However, only the 25-kDa band was detected with the anti-Bax antibody when the direct immunoblotting method was used. Peroxidase-conjugated avidin interacted with the 120-, 74-, and 72-kDa bands. This interaction was not limited to 3T3-L1 cells, because peroxidase-avidin also interacted with these three proteins in MC3T3-E1, YROS, Saos-2, MG63, SCCKN, and SCCTF cells although the staining intensity was different in each cell type. Avidin-peroxidase also interacted with these three proteins in the mitochondria-containing fractions prepared from 3T3-L1 cells. FITC-streptavidin was also localized in mitochondria in the cultured cells. The localization of avidin/streptavidin-interacting proteins in mitochondria was confirmed by using double staining with FITC-streptavidin and Mito-tracker.
Subject(s)
Mitochondria/chemistry , Mitochondrial Proteins/analysis , Proto-Oncogene Proteins c-bcl-2 , Streptavidin , 3T3-L1 Cells/chemistry , 3T3-L1 Cells/ultrastructure , Animals , Blotting, Western , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Indicators and Reagents , Mice , Proto-Oncogene Proteins/analysis , bcl-2-Associated X ProteinABSTRACT
To investigate the behavior of nuclear proteins in apoptotic cells, we examined the changes in nucleolin and proteins of the nucleolar organizing region during apoptosis in human osteoblastic cell lines, Saos-2 and MG63. Apoptosis was induced by treatment of these cells with okadaic acid. Proteins prepared from apoptotic cells were subjected to Western blot analysis and a modified Western blot method using silver nitrate. The anti-nucleolin antibody recognized the 110-kDa band and the staining intensity of this band decreased in the proteins prepared from the okadaic acid-treated apoptotic cells. The additional band of an 80-kDa was also detected in the proteins prepared from the apoptotic cells. Two major silver nitrate-stained bands, 110-kDa and 37-kDa, were detected among the proteins obtained from control cells. Like the Western blot analysis, the intensity of the 110-kDa silver nitrate-staining band decreased; an 80-kDa band appeared and its staining intensity increased in the lysate from the okadaic acid-treated cells. The signal intensity of the 37-kDa protein did not change in the sample from the apoptotic cells. In a cell-free apoptotic system, the 80-kDa protein was also detected and the amount of the 110-kDa protein decreased in the extract of Saos-2 cell nuclei incubated with apoptotic cytosol. The change in nucleolin in Saos-2 cells induced to undergo apoptosis was examined by an immunocytochemical procedure using the anti-nucleolin antibody and Hoechst 33342. Nucleolin was visible as dots in nucleoli in the control cells; however, it was not detected in the cells undergoing apoptosis. The dual-exposure view of Hoechst 33342 and anti-nucleolin staining cells confirmed that nucleolin had disappeared from the apoptotic nuclei of Saos-2.
Subject(s)
Apoptosis/physiology , Nuclear Proteins/metabolism , Nucleolus Organizer Region/metabolism , Phosphoproteins/metabolism , RNA-Binding Proteins/metabolism , Silver Nitrate/metabolism , Blotting, Western/methods , Cell Fractionation , Cell Line , Cell Nucleolus/metabolism , Cell Nucleus/metabolism , Cell-Free System , DNA Fragmentation , Enzyme Inhibitors/pharmacology , Fluorescent Dyes/metabolism , Humans , Immunohistochemistry , Nucleolus Organizer Region/chemistry , Okadaic Acid/pharmacology , Osteoblasts/cytology , Osteoblasts/drug effects , Osteoblasts/metabolism , NucleolinABSTRACT
An unusual new sesquiterpene derivative, kuhistaferone (1), was isolated from the fruits of the Uzbekistan medicinal plant Ferula kuhistanica. The structure of 1 was established on the basis of spectroscopic evidence. Compound 1 showed moderate cytotoxicity against the human colon tumor cell line HCT116.
