ヒト骨芽細胞のオカダ酸誘導アポトーシスと核内蛋白の変化

概要

論文の詳細を見る
Alterations of nucleolar proteins during apoptosis are not well understood. We examined the AgNORs in apoptotic cells using double staining with Hoechst 33342 and silver nitrate. Apoptosis was induced by treatment of the human osteosarcoma cell line, Saos-2 cells and MG63 cells with okadaic acid. AgNORs, visible as dots in the nucleus of the control cells, disappeared from the apoptotic nuclei in both cell lines. Proteins prepared form Saos-2 cells treated with 20 nM okadaic acid for various time periods were prepared and subjected to SDS-PAGE followed by transferring to transfer membranes and stained with silver nitrate. Two major bands, 110 kDa and 37 kDa, were detected in the proteins obtained from the control cells. The 110 kDa protein level was decreased in the apoptotic cells. However, an additional band, 80 kDa, appeared and the level of the 80 kDa protein increased in the proteins prepared from okadaic acid-induced apoptotic cells. The reaction intensity of the 37 kDa protein did not change in the apoptotic cells. Cellular fractionation of Saos-2 cells was done in the control and apoptotic cells. Two major bands, 110 kDa and 37 kDa, were detected in the nuclear fraction prepared from the control cells. The 80 kDa protein was detected and the 110 kDa protein level was decreased in the nuclear fraction from the apoptotic cells. In the cell-free apoptotic system, the 80 kDa protein also was detected while the 110 kDa protein was decreased in the proteins extracted from the nuclei of Saos-2 cells, which underwent apoptosis in vitro. The changes of these AgNOR proteins in vitro were identical in the nuclei prepared from MG63 cells. We deduced that the 110 kDa and 37 kDa proteins are nucleolin and nucleophosmin, respectively, from their AgNORs staining features including their molecular weights. The 80 kDa protein may be the cleavage product of the 110 kDa protein. Our results indicate that the nucleolar AgNOR proteins are associated with the induction of DNA fragmentation or the final active phase of apoptosis. In the present study, We also examined the cytolocalization of AgNORs, nucleolin, and nucleophosmin and the expression of these proteins in Saos-2 cells by immunofluorescent technique, Western blot analysis and RT-PCR method. Staining pattern of nucleolin and nucleophosmin in Saos-2 cells and MG63 cells is similar to that of the AgNORs. Nucleolin and nucleophosmin were specifically stained as dots in the nucleus in both cell lines. The dual fluorescence image revealed that nucleolin and nucleophosmin represent the same localization in nucleolus. In the apoptotic Saos-2 cells nucleolin and nucleophosmin redistributed to the whole nucleus. The levels of nucleolin decreased in the apoptotic cells whereas that of nucleophosmin did not change. The levels of mRNAs of nucleolin and nucleophosmin did not change in the apoptotic cells. These findings suggest that degradation of nucleolin is regulated at the post-translational level.
九州歯科学会の論文
2002-02-25

ヒト骨芽細胞のオカダ酸誘導アポトーシスと核内蛋白の変化

スポンサーリンク

概要

著者

関連論文

スポンサーリンク