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State of oncomarker protein B23/nucleophosmin in HeLa cells

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Abstract

Western blot after SDS-PAGE for protein separation showed two immunoreactive bands corresponding to monomers (38–40 kDa) and oligomers (210–230 kDa) of nucleophosmin in HeLa cell lysates. Decreasing the buffer ionic strength during the incubation of cells and nuclei destabilized these oligomers. We also showed the existence of two B23/nucleophosmin pools in nuclei of HeLa cells with different sensitivity to hypotonic buffer treatment: one extractable from the nucleus and the other non-extractable and tightly bound to the nucleus. A detailed structural analysis of the extractable B23 pool was carried out: two closely related nucleophosmin isoforms (B23.1 and B23.2) were identified as a result of analysis of C-terminal amino acid sequences using carboxypeptidase hydrolysis; the N-termini of both isoforms are blocked by an acetyl group. As a result of sequencing of the deacetylated proteins, it has been established that the N-terminal amino acid sequence of nucleophosmin in these preparations is truncated by nine amino acid residues and the acetylated residue is Ser. The truncated monomer of nucleophosmin (represented only by the extractable part of the protein) on addition of magnesium ions to low ionic strength buffer or increase in buffer ionic strength was shown to form oligomers with molecular weights (210–230 kDa) similar to those revealed in the total cell lysate. It should be noted that the set of oligomers in this case differs from the one in total cell lysate. Our strategy of characterization of B23 forms for HeLa cells can be applied for other tumor cells.

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Abbreviations

cp A:

carboxypeptidase A

DAPI:

4′,6-diamidino-2-phenylindole

DMEM:

Dulbecco’s modified Eagle’s medium

DTT:

dithiothreitol

FITC:

fluorescein isothiocyanate

MA:

monoclonal antibodies

MALDI-MS:

matrix assisted laser desorption ionization mass spectrometry

2-ME:

2-mercaptoethanol

PBS:

phosphate buffered saline

PFA:

paraformaldehyde

PMSF:

phenylmethylsulfonyl fluoride

PVP-40:

polyvinyl pyrolidone-40

RSB:

reticulocyte standard buffer

References

  1. Mattern, K. A., Humbel, B. M., Muijsers, A. O., de Jong, L., and van Driel, R. (1996) J. Cell Biochem., 62, 275–289.

    Article  CAS  PubMed  Google Scholar 

  2. Borer, R. A., Lehner, C. F., Eppenberger, H. M., and Nigg, E. A. (1989) Cell, 56, 379–390.

    Article  CAS  PubMed  Google Scholar 

  3. Yun, J. P., Chew, E. C., Liew, C. T., Chan, J. Y., Jin, M. L., Ding, M. X., Fai, Y. H., Li, H. K., Liang, X. M., and Wu, Q. L (2003) J. Cell Biochem., 90, 1140–1148.

    Article  CAS  PubMed  Google Scholar 

  4. Zatsepina, O. V., Todorov, I. T., Philipova, R. N., Krachmarov, C. P., Trendelenburg, M. F., and Jordan, E. G. (1997) Eur. J. Cell. Biol., 73, 58–70.

    CAS  PubMed  Google Scholar 

  5. Lim, M. J., and Wang, X. W. (2006) Cancer Detect. Prev., 30, 481–490.

    Article  CAS  PubMed  Google Scholar 

  6. Grisendi, S., Mecucci, C., Falini, B., and Pandolfi, P. P. (2006) Nature Rev. Cancer, 6, 493–505.

    Article  CAS  Google Scholar 

  7. Brady, S. N., Yu, Y., Maggi, L. B., Jr., and Weber, J. D. (2004) Mol. Cell. Biol., 24, 9327–9338.

    Article  CAS  PubMed  Google Scholar 

  8. Bertwistle, D., Sugimoto, M., and Sherr, C. J. (2004) Mol. Cell. Biol., 24, 985–996.

    Article  CAS  PubMed  Google Scholar 

  9. Herrera, J. E., Correia, J. J., Jones, A. E., and Olson, M. O. (1996) Biochemistry, 35, 2668–2673.

    Article  CAS  PubMed  Google Scholar 

  10. Namboodiri, V. M., Akey, I. V., Schmidt-Zachmann, M. S., Head, J. F., and Akey, C. W. (2004) Structure, 12, 2149–2160.

    Article  CAS  PubMed  Google Scholar 

  11. Chan, P. K., and Chan, F. Y. (1995) Biochim. Biophys. Acta, 1262, 37–42.

    PubMed  Google Scholar 

  12. Enomoto, T., Lindstrom, M. S., Jin, A., Ke, H., and Zhang, Y. (2006) J. Biol. Chem., 281, 18463–18472.

    Article  CAS  PubMed  Google Scholar 

  13. Kerr, L. E., Birse-Archbold, J. L., Short, D. M., McGregor, A. L., Heron, I., MacDonald, D. C., Thompson, J., Carlson, G. J., Kelly, J. S., McCulloch, J., and Sharkey, J. (2006) Oncogene, 26, 2554–2562.

    Article  PubMed  Google Scholar 

  14. Wang, W., Budhu, A., Forgues, M., and Wang, X. W. (2005) Nat. Cell Biol., 7, 823–830.

    Article  CAS  PubMed  Google Scholar 

  15. Yu, Y., Maggi, L. B., Brady, S. N., Apicelli, A. J., Dai, M. S., Lu, H., and Weber, J. D. (2006) Mol. Cell Biol., 26, 3798–3809.

    Article  CAS  PubMed  Google Scholar 

  16. Negi, S. S., and Olson, M. O. (2006) J. Cell Sci., 119, 3676–3685.

    Article  CAS  PubMed  Google Scholar 

  17. Okuwaki, M., Tsujimoto, M., and Nagata, K. (2002) Mol. Biol. Cell, 13, 2016–2030.

    Article  CAS  PubMed  Google Scholar 

  18. Okuwaki, M. (2008) J. Biochem., 143, 441–448.

    Article  CAS  PubMed  Google Scholar 

  19. Hingorani, K., Szebeni, A., and Olson, M. O. (2000) J. Biol. Chem., 275, 24451–24457.

    Article  CAS  PubMed  Google Scholar 

  20. Umekawa, H., Chang, J. H., Correia, J. J., Wang, D., Wingfield, P. T., and Olson, M. O. (1993) Cell. Mol. Biol. Res., 39, 635–645.

    CAS  PubMed  Google Scholar 

  21. Herrera, J. E., Savkur, R., and Olson, M. O. (1995) Nucleic Acids Res., 23, 3974–3979.

    Article  CAS  PubMed  Google Scholar 

  22. Naoe, T., Suzuki, T., Kiyoi, H., and Urano, T. (2006) Cancer Sci., 97, 963–969.

    Article  CAS  PubMed  Google Scholar 

  23. Falini, B., Mecucci, C., Saglio, G., Coco, F., Diverio, D., Brown, P., Pane, F., Mancini, M., Martelli, M. P., Pileri, S., Haferlach, T., Haferlach, C., and Schnittger, S. (2008) Haemotologica, 93, 439–442.

    Article  CAS  Google Scholar 

  24. Grummit, C. G., Townsley, F. M., Johnson, C. M., Warren, A. J., and Bycroft, M. (2008) J. Biol. Chem., 283, 23326–23332.

    Article  Google Scholar 

  25. Colombo, E., Martinelli, P., Zamponi, R., Shing, D. C., Bonetti, P., Luzi, L., Volorio, S., Bernard, L., Pruneri, G., Alcalay, M., and Pelicci, P. G. (2006) Cancer Res., 66, 3044–3050.

    Article  CAS  PubMed  Google Scholar 

  26. Castro, I. P., Carcer, G., and Malumbres, M. (2007) Carcinogenesis, 28, 899–912.

    Article  Google Scholar 

  27. Ulanet, D. B., Torbenson, M., Dang, C. V., Carciola-Rosen, C., and Rosen, A. (2003) Proc. Natl. Acad. Sci. USA, 100, 12361–12366.

    Article  CAS  PubMed  Google Scholar 

  28. Vladimirova, N. M., Sautkina, E. N., Tabdanov, E. D., Bulycheva, T. I., Ovchinnikova, T. V., Zatsepina, O. V., and Potapenko, N. A. (2004) Biol. Membr. (Moscow), 21, 94–101.

    CAS  Google Scholar 

  29. Chou, Y. H., and Yung, B. Y. M. (1995) Biochem. Biophys. Res. Commun., 217, 313–325.

    Article  CAS  PubMed  Google Scholar 

  30. Bulycheva, T. I., Dergunova, N. N., Artemenko, E. G., Dudnik, O. A., Shpakova, A. P., Malashenko, O. S., and Zatsepina, O. V. (2000) Tsitologiya, 42, 944–954.

    CAS  Google Scholar 

  31. Sautkina, E. N., Potapenko, N. A., Bulycheva, T. I., and Vladimirova, N. M. (2008) Prikl. Biokhim. Mikrobiol., 44, 287–295.

    CAS  PubMed  Google Scholar 

  32. Lowry, O. H., Rothenbrough, N. Y., Farr, A. H., and Randall, R. G. (1951) J. Biol. Chem., 193, 265–275.

    CAS  PubMed  Google Scholar 

  33. Laemmli, U. K. (1970) Nature, 227, 680–685.

    Article  CAS  PubMed  Google Scholar 

  34. Vladimirova, N. M., Potapenko, N. A., Sachs, G., and Modyanov, N. N. (1995) Biochim. Biophys. Acta, 1233, 175–184.

    Article  PubMed  Google Scholar 

  35. Sautkina, E. N., Potapenko, N. A., and Vladimirova, N. M. (2006) Biokhimiya, 71, 786–797.

    Google Scholar 

  36. Bergman, T., Gheorghe, M. T., Hjelmqvist, L., and Jornvall, H. (1996) FEBS Lett., 390, 199–202.

    Article  CAS  PubMed  Google Scholar 

  37. Sautkina, E. N., Vladimirova, N. M., Tabdanov, E. D., and Potapenko, N. A. (2005) Biol. Membr. (Moscow), 22, 373–382.

    Article  Google Scholar 

  38. Zatsepina, O. V., Dudnic, O. A., Todorov, I. T., Thiry, M., Spring, H., and Trendelenburg, M. F. (1997) Chromosoma, 105, 418–430.

    Article  CAS  PubMed  Google Scholar 

  39. Andersen, J. S., Lyon, C. E., Fox, A. H., Leung, A. K., Lam, Y. W., Steen, H., Mann, M., and Lamond, A. I. (2002) Curr. Biol., 12, 1–11.

    Article  PubMed  Google Scholar 

  40. Sherl, A., Coute, Y., Deon, C., Calle, A., Kindbeiter, K., Sanchez, J., Greco, A., Hochstrasser, D., and Diaz, J. (2002) Mol. Biol. Cell, 13, 4100–4109.

    Article  Google Scholar 

  41. Coute, Y., Burgess, J. A., Diaz, J. J., Chichester, C., Lisacek, F., Greco, A., and Sanchez, J. C. (2006) Mass Spectrom. Rev., 25, 215–234.

    Article  CAS  PubMed  Google Scholar 

  42. Kellermayer, M., Olson, M. O., Smetana, K., Daskal, I., and Busch, H. (1974) Exp. Cell Res., 85, 191–204.

    Article  CAS  PubMed  Google Scholar 

  43. Peters, K. E., and Commings, D. E. (1980) J. Cell Biol., 86, 135–155.

    Article  CAS  PubMed  Google Scholar 

  44. Li, X. Z., McNeilage, L. J., and Whittingham, S. (1989) Biochem. Biophys. Res. Commun., 163, 72–78.

    Article  CAS  PubMed  Google Scholar 

  45. Okuwaki, M., Iwamatsu, A., Tsujimoto, M., and Nagata, K. (2001) J. Mol. Biol., 311, 41–55.

    Article  CAS  PubMed  Google Scholar 

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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

    N. M. Vladimirova, N. V. Lobanova & N. A. Potapenko

Authors
  1. N. M. Vladimirova
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  2. N. V. Lobanova
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  3. N. A. Potapenko
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Correspondence to N. M. Vladimirova.

Additional information

Original Russian Text © N. M. Vladimirova, N. V. Lobanova, N. A. Potapenko, 2010, published in Biokhimiya, 2010, Vol. 75, No. 7, pp. 966–977.

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Vladimirova, N.M., Lobanova, N.V. & Potapenko, N.A. State of oncomarker protein B23/nucleophosmin in HeLa cells. Biochemistry Moscow 75, 851–860 (2010). https://doi.org/10.1134/S0006297910070060

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  • DOI: https://doi.org/10.1134/S0006297910070060

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