Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter to the Editor
  • Published:

The concurrent use of N- and C-terminal antibodies anti-nucleophosmin 1 in immunofluorescence experiments allows for precise assessment of its subcellular localisation in acute myeloid leukaemia patients

Leukemia volume 26, pages 159–162 (2012)Cite this article

The delocalisation of the nucleophosmin 1 (NPM1) nucleolar protein to the cytoplasm, denoted as NPMc+, arises as a consequence of genetic mutations that modify the protein's C-terminus introducing a nuclear export signal and abolishing a nucleolar localisation signal.1 The mutations of NPM1 are found in approximately 30% of cases of acute myeloid leukaemia (AML),2 however, despite their clinical relevance, there are only a few reagents (Table 1) suitable for diagnostic applications and research.3 The NA24, 322 and 376(refs 2, 4) monoclonal antibodies recognise the N-terminus of both wild-type (wt) NPM1 and NPMc+ and have been used extensively in immunohistochemistry and immunofluorescence to identify the cytoplasmic displacement of the NPM mutant,2, 5, 6 whereas the SilA and SilC rabbit polyclonal antibodies that specifically recognise the mutated C-terminal region have been used in western blotting and immunoprecipitation.5, 7, 8 Successful flow cytometric detection of NPM1 mutants has been obtained using the 5E3 monoclonal antibody.3 Recently, T26, a monoclonal antibody specifically directed against the de novo sequence present in most of the NPMc+ mutants has been generated and shown as being potentially efficient in different immunotechniques used for the diagnostic work up of the disease.9

Table 1 Summary of the anti-NPM1 antibodies working in different immunotechniques and reported in the literature
Full size table

The mutations in the NPM1 locus encountered in AML are always heterozygous, and thus in the NPMc+AML blasts, wt NPM and NPMc+ co-exist. However, the two proteins show a different subcellular localisation, with the wt protein present predominantly in the nucleoli and the mutant dispersed in the cytoplasm. The situation is complicated by the fact that NPM1 protein forms oligomers, and given that the domains of oligomerisation are retained in the various mutants, hetero-oligomers consisting of wt NPM1 and NPMc+ occur as well. In this way, the wt protein is taken out of nucleolus by the mutant that exerts a dominant-negative effect over the wt protein as observed by Colombo et al.10 Similarly, Bolli et al.11 observed that in transfected cells, excess doses of NPMc+ relocate completely wt NPM1 from the nucleoli to the cytoplasm, a distribution pattern seen also in a proportion of NPMc+AML patients. The excess of NPM1wt in transfected cells, on the other hand, relocated NPMc+ from the cytoplasm to the nucleoli. Notably, this distribution pattern is extremely rare in patients' samples, although it has been described in small percent of cells in some of the cases tested with the use of the T26 antibody by immunofluorescence.9

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1

References

  1. Falini B, Nicoletti I, Martelli MF, Mecucci C . Acute myeloid leukemia carrying cytoplasmic/mutated nucleophosmin (NPMc+ AML): biological and clinical features. Blood 2007; 109: 874–885.

    Article  CAS  Google Scholar 

  2. Falini B, Mecucci C, Tiacci E, Alcalay M, Rosati R, Pasqualucci L et al. Cytoplasmic nucleophosmin in acute myelogenous leukemia with a normal karyotype. N Engl J Med 2005; 352: 254–266.

    Article  CAS  Google Scholar 

  3. Oelschlaegel U, Koch S, Mohr B, Schaich M, Falini B, Ehninger G et al. Rapid flow cytometric detection of aberrant cytoplasmic localization of nucleophosmin (NPMc) indicating mutant NPM1 gene in acute myeloid leukemia. Leukemia 2010; 24: 1813–1816.

    Article  CAS  Google Scholar 

  4. Cordell JL, Pulford KAF, Bigerna B, Roncador G, Banham A, Colombo E et al. Detection of normal and chimeric nucleophosmin in human cells. Blood 1999; 93: 632–642.

    CAS  Google Scholar 

  5. Falini B, Martelli MP, Bolli N, Bonasso R, Ghia E, Pallotta MT et al. Immunohistochemistry predicts nucleophosmin (NPM) mutations in acute myeloid leukemia. Blood 2006; 108: 1999–2005.

    Article  CAS  Google Scholar 

  6. Luo J, Qi C, Xu W, Kamel-Reid S, Brandwein J, Chang H . Cytoplasmic expression of nucleophosmin accurately predicts mutation in the nucleophosmin gene in patients with acute myeloid leukemia and normal karyotype. Am J Clin Pathol 2010; 133: 34–40.

    Article  CAS  Google Scholar 

  7. Quentmeier H, Martelli MP, Dirks WG, Bolli N, Liso A, MacLeod RAF et al. Cell line OCI/AML3 bears exon-12 gene mutation-A and cytoplasmic expression of nucleophosmin. Leukemia 2005; 19: 1760–1767.

    Article  CAS  Google Scholar 

  8. Martelli MP, Manes N, Liso A, Pettirossi V, Verducci Galletti B, Bigerna B et al. A western blot assay for detecting mutant nucleophosmin (NPM1) proteins in acute myeloid leukaemia. Leukemia 2008; 22: 2285–2288.

    Article  CAS  Google Scholar 

  9. Gruszka AM, Lavorgna S, Consalvo MI, Ottone T, Martinelli C, Cinquanta M et al. A monoclonal antibody against mutated nucleophosmin1 for the molecular diagnosis of acute myeloid leukemias. Blood 2010; 116: 2096–2102.

    Article  CAS  Google Scholar 

  10. Colombo E, Martinelli P, Zamponi R, Shing DC, Bonetti P, Luzi L et al. Delocalization and destabilization of the Arf tumor suppressor by the leukemia-associated NPM mutant. Cancer Res 2006; 66: 3044–3050.

    Article  CAS  Google Scholar 

  11. Bolli N, De Marco MF, Martelli MP, Bigerna B, Pucciarini A, Rossi R et al. A dose-dependent tug of war involving the NPM1 leukaemic mutant, nucleophosmin, and ARF. Leukemia 2009; 23: 501–509.

    Article  CAS  Google Scholar 

  12. Konoplev S, Huang X, Drabkin HA, Koeppen H, Jones D, Kantarjian HM et al. Cytoplasmic localization of nucleophosmin in bone marrow blasts of acute myeloid leukemia patients is not completely concordant with NPM1 mutation and is not predictive of prognosis. Cancer 2009; 115: 4737–4744.

    Article  CAS  Google Scholar 

  13. Mattsson G, Turner SH, Cordell J, Ferguson DJP, Schuh A, Grimwade LF et al. Can cytoplasmic nucleophosmin be detected by immunocytochemical staining of cell smears in acute myeloid leukemia? Haematologica 2010; 95: 670–673.

    Article  Google Scholar 

  14. Monegal A, Ami D, Martinelli C, Huang H, Aliprandi M, Capasso P et al. Immunological applications of single domain llama recombinant antibodies isolated from a naïve library. Prot Engineer Des Sel 2009; 22: 273–280.

    Article  CAS  Google Scholar 

  15. Aliprandi M, Sparacio E, Pivetta F, Ossolengo G, Maestro R, de Marco A . The availability of a recombinant anti-SNAP antibody in VHH format amplifies the application flexibility of SNAP-tagged proteins. J Biomed Biotechnol 2010; 2010: 658954.

    Article  Google Scholar 

Download references

Acknowledgements

We thank Tor Vergata University Hospital of Rome for patients' samples and AIRC for financial support.

Author information

Authors and Affiliations

  1. Department of Experimental Oncology, European Institute of Oncology, Milan, Italy

    A M Gruszka, C Martinelli, E Sparacio, P G Pelicci & A de Marco

  2. Dipartimento di Medicina, Chirurgia e Odontoiatria, University of Milano, Milan, Italy

    P G Pelicci

  3. Department of Environmental Sciences, University of Nova Gorica (UNG), Rožna Dolina, Nova Gorica, Slovenia

    A de Marco

Authors
  1. A M Gruszka
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. C Martinelli
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. E Sparacio
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. P G Pelicci
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. A de Marco
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to A de Marco.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Additional information

Supplementary Information accompanies the paper on the Leukemia website

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gruszka, A., Martinelli, C., Sparacio, E. et al. The concurrent use of N- and C-terminal antibodies anti-nucleophosmin 1 in immunofluorescence experiments allows for precise assessment of its subcellular localisation in acute myeloid leukaemia patients. Leukemia 26, 159–162 (2012). https://doi.org/10.1038/leu.2011.177

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/leu.2011.177

This article is cited by

Search

Advanced search

Quick links