engleski

Nucleophosmin (NPM) and telomerase immunopositivity in CLL lymphoid cells

Background: Nucleophosmin (NPM) is phosphoprotein that is ubiquitously expressed in tissues, resides in nucleolus and shuttles continuously between nucleus and cytoplasm. Genetic locus of human NPM1 is 5q35.1. The wild-type protein is mainly located in the nucleolus and on the nuclear membrane and in cytoplasm of mitotic cells. Major role of NPM is to mediate nuclear export of ribosome components to the cytoplasm and to control centrosome duplication and it also interacts with oncosupressors p53 and alternate–reading-frame protein (ARF) thus controlling cell proliferation and apoptosis. NPM promotes cell growth by enhancing ribosome biogenesis and transport to the cytoplasm, as its expression increase in response to mitogenic stimuli and above normal amounts are detected in highly proliferating and malignant cell. Telomerase (TE) is an RNA-dependent DNA polymerase that catalyzes the addition of telomeric repeat sequences to chromosome ends, and its genetic locus is 5p15.33. In most human somatic cells, TE activity is undetectable, and telomeres shorten with successive cell divisions. However, telomerase activity is detectable in immortal cells and in many human tumors. It was found that TE is sequestrated within the nucleolus during most of the cell cycle of normal cells and released into the nucleus only just before cell division. TE is not sequestrated in tumor cells and continually interacts with the chromosomes. Aims: The aim of the study was to analyze NPM and TE immunocytochemical positivity in lymphoid cells and lymphocytes of patients with chronic lymphoid leukemia (CLL). Methods: Immunocytochemical procedure was performed on cytological bone marrow (BM) specimens of five CLL patients and on effusion sediments in patient with benign reactive effusion lymphocytosis. Cytological specimens collected for immunostaining were obtained in patients at diagnosis, only as a part of standard diagnostic procedure and patients signed informed consent. Cytological specimens were stained with anti-NPM mouse monoclonal antibody (clone FC82291) raised against full length NPM, useful for identification of both wild-type and mutated NPM, and with anti-human goat polyclonal antibody (clone C-20) to TERT (telomerase reverse transcriptase). Immunostaining was further done with LSAB immuno peroxidase technique. NPM and TE nucleus immunopositivity was analyzed by microscope, determined were percentages of immunopositive cells, as well as score values of immunoreaction. Score values of NPM and TE immunopositivity were obtined by rating intensity of 100 cell immunopositivity using a scale of 0 to 4+ and then the number of cells counted in each cell rating is multiplied by the cell rating value and these values were summarized. Results: Nuclei of almoust all CLL lymphoid cells were strongly NPM immunopostive (percentages median 100%, range 92-100% ; score median 257, range 222-375). Telomerase was weakly immunopositive in most of nuclei of CLL lymphoid cells (percentages median 74%, range 54-93% ; score median 110 ; range 58-150). Opposite, in most of nuclei of reactive effusion lymphoid cells NPM was weakly positive (61%, score 91), and TE was completely negative. NPM and TE immunopostivity pattern in CLL lymphoid cells was different: TE was positive in form of fine small dots, and NPM was mostly positive in form of numerous coarse granules, coarse network or as diffuse coarse immunoreaction. Opposite, in most of benign reactive effusion lymphocytes NPM was weakly immunopositive in form of one fine dot. Summary/Conclusion: In our five CLL patients most lymphoid cells were NPM and TE immunopositive, but in most of CLL lymphoid cells intensity of TE immunopositivity was weak in comparison to strong NPM immunopositivity. Moreover, benign reactive effusion lymphocytes were negative for telomerase and NPM was weakly immunopostive mostly in form of one fine dot. These results point that telomerase activity is very low and undetectable in benign lymphocytes and that NPM immunopositivity pattern is different in CLL lymphiod cells in comparison to benign reactive effusion lymphocytes. However, the number of analyzed patients is small and further analysis in larger groups of patients with CLL and patients with benign reactive effusion lymphocytes is needed to clearly determine NPM and TE cell immunopositivity differences between CLL lymphoid cells and benign lymphocytes.

Gene expression ; Immunohistochemistry ; Telomerase

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