Nuclear localization of nucleoside diphosphate kinase type B (nm23-H2) in cultured cells.
Nucleoside diphosphate (NDP) kinases are metabolic enzymes found ubiquitously in cells. Recently, two known human isoforms of NDP kinase (A and B), identical to the protein products of the genes nm23-H1 and nm23-H2, respectively, have been implicated in cancer metastasis and transcriptional regulation. To date, NDP kinase has been studied extensively in tissue sections and its cellular localization was described as being cytoplasmic. However, the recently discovered role of the nm23-H2 gene product in transcriptional activation of the c-myc proto-oncogene also suggests a nuclear localization of the protein. In this study, we used isoform-specific antibodies against NDPK-B to examine the subcellular localization of the nm23-H2 gene product. The cytoplasmic fluorescence is intense and homogeneous with pronounced labeling in the centromere region. The distribution of NDPK-B in interphase nuclei exhibits a pattern of numerous uniformly dispersed fine dots with reduced staining of the nucleoli. To further characterize the nuclear localization of NDPK-B, in situ sequential extraction of nuclear components was performed. Brief exposure to Triton X-100 and subsequent treatment with RNase A do not change the nuclear staining pattern of NDPK-B. In contrast, treatment of Triton X-100-permeabilized nuclei with DNase I results in a significant loss of fluorescence. In mitotic prophase cells, the protein segregates from forming chromosomes and reappears in newly formed daughter nuclei after cell division. Taken together, the results indicate an association of NDPK-B with chromatin in interphase nuclei, supporting its proposed role in transcription.
Kraeft
S K
SK
Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
Traincart
F
F
Mesnildrey
S
S
Bourdais
J
J
Véron
M
M
Chen
L B
LB
D000818:Animals; D000911:Antibodies, Monoclonal; D002455:Cell Division; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology*; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000201:enzymology; D003850:Deoxyribonuclease I; D003902:Detergents; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology; D005347:Fibroblasts / Q000201:enzymology; D019084:Fluorescent Antibody Technique, Indirect / Q000379:methods; D006801:Humans; D008938:Mitosis; D020559:Monomeric GTP-Binding Proteins; D054778:NM23 Nucleoside Diphosphate Kinases; D009701:Nucleoside-Diphosphate Kinase / Q000032:analysis*; D017830:Octoxynol; D000090063:Proto-Oncogene Mas; D016707:Tissue Fixation / Q000379:methods; D014157:Transcription Factors / Q000032:analysis*
D000911:Antibodies, Monoclonal; D003902:Detergents; C000715470:MAS1 protein, human; D054778:NM23 Nucleoside Diphosphate Kinases; D000090063:Proto-Oncogene Mas; D014157:Transcription Factors; D017830:Octoxynol; C517938:NME1 protein, human; D009701:Nucleoside-Diphosphate Kinase; D003850:Deoxyribonuclease I; D020559:Monomeric GTP-Binding Proteins