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SUBCELLULAR LOCALIZATION OF NM23/NDPK SUBUNITS IN HEp-2 CELLS

Introduction: Since the day of its discovery Nm23/NDPK has been assigned a number of different functions in the cell apart from its well-known ability to catalyze the conversion of (d)NDPs to (d)NTPs through a high-energy His118 intermediate. Eukaryotic NDP kinases have been reported to be catalytically active only as homo- or heterohexamers. In humans the hexamers consist of two highly homologous subunits Nm23-H1/NDPK A and Nm23-H2/NDPK B, having an 88% amino acid sequence identity. On the basis of its reduced expression in melanoma cell lines with low versus high metastatic potential nm23-H1 was proposed to constitute a potential metastatic suppressor gene. Apart from being involved in metastasis formation, Nm23 proteins have been linked to several biological processes such as proliferation, differentiation, and development. Although forming a functional enzyme together, Nm23-H1 and Nm23-H2 have also been assigned separate cellular functions. The aim of this work was to determine whether the distinct functions of the two subunits, Nm23-H1/NDPK A and Nm23-H2/NDPK B, reflect their subcellular localization. Methods: The issue was addressed using GFP fusion proteins and immunofluorescent techniques. nm23-H1, nm23-H2, and nm23-H1H118N (carrying a point mutation in the kinase domain) full-length cDNA fragments were subcloned from pcDNA3nm23 constructs into pEGFPC1 (Clontech) and pyRed generating in frame fusion proteins. Head and neck tumor cell line HEp-2 was transfected with pEGFPC1-nm23-H1, pEGFPC1-nm23-H2 and pEGFPC1-nm23-H1H118N, as well as with the pEGFPC1 control vector and analyzed using fluorescent microscopy to determine the subcellular localization of fusion proteins. Fluorescent immunocytochemical methods were used to assess the possible colocalization of GFP-Nm23 proteins with microtubules, vimentin intermediate filaments, and the centrosome as well as with the endoplasmic reticulum and lysosomes. Cell-cycle dependent distribution of Nm23 proteins was revealed by treating transfected cells with dihydroxyurea and nocodazole and by staining them with BrdU. Results and conclusions: HEp-2 cells transfected with pEGFPC1-nm23-H1, – H2, and pEGFPC1-nm23-H1H118N exhibited green fluorescent staining in both, the cytoplasm and the nucleus. The distribution of fluorescent staining could be seen throughout the cytosol with its intensity decreasing from the perinuclear region towards the periphery of the cell. Approximately 15-20% of the nuclei were also stained. In some cells granum-like structures were observed: mostly one or two such structures per cell, intensely fluorescent in most cases and positioned adjacent to the nucleus. Interestingly, cells transfected with the mutant variant of nm23-H1 never displayed any granum-like structures. Cotransfection experiments with a combination of a “ green” and a “ red” fluorescent protein confirmed our previous observation that GFP-Nm23-H1, GFP-Nm23-H2 and the Nm23-H1H118N adopt similar subcellular distributions. In combination with Nm23-H1 or Nm23-H2, the mutated form of the Nm23-H1 protein also formed granum-like structures. These structures were analyzed by applying laser scanning confocal microscopy. A cell-wide distribution of single, separate particles having various sizes and/or a conglomeration of more uniformly sized granules attached to each other and typically positioned adjacent to the nucleus were observed. Optical sectioning of several individual particles revealed that they typically have a spherical shape and are filled with the fluorescent material. The colocalization signal (yellow staining) was observed in a very small portion of the cells expressing either of the fusion proteins with vimentin or tubulin, and it was always adjacent to the nuclear wall. The granum-like structures in the perinuclear region did not colocalize with the centrosome. GFP-Nm23 proteins, however, colocalized with the endoplasmic reticulum (Figure 1A). S-phase HEp-2 cells (stained with BrdU) were only stained in the cytosol. The cells arrested late in the G1 phase exhibited fluorescent staining in the cytosol and in up to 80% of the nuclei transfected with either of the pEGFPC1-nm23 constructs (Figure 1B). In conclusion it seams that even if Nm23-H1 and Nm23-H2 proteins have some distinct functions in the cell they perform them in the same or similar locations. The functional significance of the ER and cell cycle-dependent nuclear localization for Nm23 remains to be elucidated. Out present work is concentrated in revealing the possible changes in Nm23 localization in cells undergoing stress and apopotosis.

nm23; GFP; oral squamous cell carcinoma

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