Cytoskeletal association of the A and B nucleoside diphosphate kinases of interphasic but not mitotic human carcinoma cell lines: Specific nuclear localization of the B subunit

The human A and B subunits of nucleoside diphosphate kinase (NDP kinase), e ncoded by the nm23-H1 and nm23-H2 genes, respectively, associate as homo- o r heterohexamers to be catalytically active for the synthesis of nucleoside triphosphates. Despite 88% identity, they appear to possess specific funct ions. The nm23-H1 gene is implicated in tumor progression and metastasis, a nd the nm23-H2 gene product is a transcription factor for c-myc. To determi ne if these distinct functions reflect different subcellular localizations, the distribution of the A and B NDP kinases was analyzed by immunocytofluo rescence microscopy in human breast cancer cell lines (MCF-7 and MDA-MB-231 ) using highly specific polyclonal and monoclonal antibodies. Interphasic c ells exhibited a granular and filamentous cytoplasmic staining, particularl y intense around nuclei, with both anti-NDP kinase A and B antibodies. The filamentous component observed with either anti-A or anti-B antibodies was altered in parallel to tubulin labeling with compounds interacting with mic rotubules, such as taxol and colchicine. Confirming published biochemical d ata, a partial colocalization with the vimentin network was observed in the MDA-231 cell line. A nuclear and nucleolar localization of NDP kinase B wa s shown by confocal microscopy which was not observed with the A enzyme. In dividing cells, NDP kinase labeling was punctiform and was not colocalized with the mitotic spindle. In conclusion, the A and B NDP kinases are simil arly distributed in cytosol, associated partly to microtubules supporting a role in nucleotide channeling. Only the B enzyme is present in nuclei in a ccord with its role as a DNA binding protein. Their altered localization in dividing cells suggests colocalization with yet unidentified structures wh ich are not intermediate filament aggregates, (C) 1999 Academic Press.