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State of nucleolar proteins B23/nucleophosmin and UBF in HeLa cells during apoptosis induced by tumor necrosis factor

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Abstract

The structural state of two major nucleolar proteins, UBF and B23/nucleophosmin (both monomeric and oligomeric forms), was for the first time established in HeLa cells treated with apoptosis inducers: tumor necrosis factor (TNF-α), emetine, and their combination. The treatment of the cells with either TNF-α or emetine did not induce apoptosis and affect the state of UBF and nucleophosmin (both monomers and oligomers). Apoptosis was rather pronounced only if HeLa cells were treated with a mixture of TNF-α and emetine. States of the UBF and B23 proteins were analyzed in samples containing 25, 45, and 100% of cells with apoptotic nuclei. It was shown by immunoblotting that TNF-α-induced apoptosis of HeLa cells was associated with proteolysis of UBF and production of a 76-kD fragment, the content of which increased in correlation with the fraction of apoptotically changed cells. The N-and C-terminal amino acid sequences of UBF and its 76-kD fragment were characterized, and the site of the apoptosis-induced specific proteolysis was identified. As differentiated from UBF, protein B23 did not undergo proteolytic degradation during the TNF-α-induced apoptosis of HeLa cells and its content was unchanged even in the cell fraction with fragmentation of virtually all nuclei. However, the ratio between the monomeric and oligomeric states of B23 protein was changed in apoptotic cells, and apoptosis-specific forms of nucleophosmin were detected.

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Abbreviations

DAB:

3,3′-diaminobenzidine

DAPI:

dihydrochloride 4′,6-diamidino-2-phenylindole dihydrochloride

PBS:

phosphate-buffered saline

TNF-α:

tumor necrosis factor α

Versene solution:

PBS containing 0.02% EDTA

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Authors and Affiliations

  1. Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997, Moscow, Russia

    E. N. Sautkina, N. A. Potapenko & N. M. Vladimirova

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  1. E. N. Sautkina
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  2. N. A. Potapenko
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  3. N. M. Vladimirova
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Correspondence to N. M. Vladimirova.

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Original Russian Text © E. N. Sautkina, N. A. Potapenko, N. M. Vladimirova, 2006, published in Biokhimiya, 2006, Vol. 71, No. 6, pp. 786–797. Originally published in Biochemistry (Moscow) On-Line Papers in Press, as Manuscript BM05-170, April 2, 2006.

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Sautkina, E.N., Potapenko, N.A. & Vladimirova, N.M. State of nucleolar proteins B23/nucleophosmin and UBF in HeLa cells during apoptosis induced by tumor necrosis factor. Biochemistry (Moscow) 71, 634–643 (2006). https://doi.org/10.1134/S0006297906060071

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  • DOI: https://doi.org/10.1134/S0006297906060071

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