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PubMedDate not supplied

Nucleoside diphosphate kinase (nm23 protein) expression in retinoblastoma.

Forty-two enucleated eyes of 42 patients with unilateral retinoblastoma were studied histologically, including histochemically examination with anti-nm23 polyclonal antibody which does not recognise cDNA but its product. Primary tumours of >15 mm diameter with less evidence of apoptosis and with the most pleomorphic and anaplastic nuclei were associated with an increased risk of distant metastasis, but rosette formation did not discriminate. A high intensity of nm23 staining also indicated a tendency to metastasize, consistent with childhood neuroblastoma but in contrast to findings in carcinoma of the breast, colon and uterine cervix.

Antigens, Neoplasm↗
PubMed2003

Plasma membrane-associated nucleoside diphosphate kinase (nm23) in the heart is regulated by beta-adrenergic signaling.

1. Receptor-independent activation of heterotrimeric G proteins by plasma membrane-associated nucleoside diphosphate kinase (NDPK) has been demonstrated in vivo, and elevated levels of NDPK were found in purified sarcolemmal membranes of patients with end-stage heart failure. 2. Among 22 consecutive patients with chronic heart failure who underwent cardiac transplantation, those treated with a beta-blocker (n=8) had a 65% lower NDPK content and activity in the cardiac sarcolemma, compared to patients with similar base line characteristics who had no beta-blocker therapy (n=14). 3. The lower NDPK was associated with a reduced NDPK-dependent, Gi-mediated inhibition of adenylyl cyclase activity, as assessed by in vitro measurement of adenylyl cyclase activity in the presence of GDP or its kinase-resistant analog guanosine 5'-O-(2-thio)diphosphate (GDPbetaS). 4. We further tested whether treatment with a beta-adrenergic agonist would induce an increase in sarcolemmal NDPK. Rats treated with isoproterenol developed myocardial hypertrophy, and NDPK in the sarcolemma rose by 60% during 14 days of treatment. The beta-blocker propranolol prevented both effects. When hypertrophy was induced with thyroid hormone, NDPK did not increase. 5. In conclusion, chronic activation of beta-adrenergic receptors increases the binding of NDPK to cardiac sarcolemma, where it may activate heterotrimeric G proteins.

Adenylyl Cyclases↗
PubMed1991

Overexpression of nucleoside diphosphate kinase (Nm23) in solid tumours.

The product of the nm23-H1 gene, reported to be related to the metastatic potential of tumour cells, was recently identified as the nucleoside diphosphate (NDP) kinase A (Gilles et al., 1991, J Biol Chem, 266, 8784-8789). An analysis of the enzyme by activity measurement and immunological techniques using polyclonal antibodies raised against the NDP kinase A purified from human erythrocytes, was performed on 39 human tissue specimens. Markedly increased activity and higher level of the protein were observed in extracts of solid tumours as compared to the corresponding normal tissues (P less than 0.01). An intense immunolabelling of tumoral cells was observed in sections of the malignant tumours and of some but not all benign neoplasia. The staining is observed in noninvasive and invasive ductal breast carcinomas with or without lymph node involvement as well as in colon and cervix carcinomas and in a case of metastatic melanoma. Therefore, NDP kinase A level is increased in neoplastic tissues but no correlation with metastatic potential could be demonstrated.

Blotting, Western↗
PubMed1998

Association of nucleoside diphosphate kinase nm23-H2 with human telomeres.

Telomeres, the ends of eukaryotic chromosomes, are essential structures formed by specific protein-DNA complexes that protect chromosomes from degradation and end-to-end fusion. TRF1, a double-stranded telomeric TTAGGG-repeat binding protein, is associated with mammalian telomeres and controls telomere length by inhibiting the action of telomerase. We identified human nucleoside diphosphate kinase nm23-H2 as a human TRF1-interacting protein by yeast two-hybrid screening. In vitro-binding assays using different recombinant nucleoside diphosphate kinases showed that TRF1 predominantly binds the nm23-H2 isoform rather than nm23-H1. Electrophoretic mobility shift analysis revealed that the recombinant nm23-H2 protein can bind the single-stranded telomeric TTAGGG-repeat while it cannot bind the double-stranded telomeric repeat. The synthetic 20 base oligoribonucleotide, which consists of the template sequence CUAACCCUAAC and the adjacent region of the RNA component of human telomerase, was also found to form the complex with the recombinant nm23-H2 protein. Furthermore, the affinity of telomerase for its substrate was increased in vitro by presence of the plentiful nm23-H2 protein. These findings indicate a close relationship between nm23-H2 and human telomeres and suggest a new biological role for nucleoside diphosphate kinase.

Cloning, Molecular↗
PubMed1993

Human c-myc transcription factor PuF identified as nm23-H2 nucleoside diphosphate kinase, a candidate suppressor of tumor metastasis.

A human gene encoding the c-myc purine-binding transcription factor PuF was identified by screening of a cervical carcinoma cell complementary DNA library with a DNA fragment containing PuF binding sites. The 17-kilodalton bacterially produced PuF was shown to have biological activity and properties similar to that of human PuF. DNA sequence analysis of recombinant PuF revealed perfect identity with the human nm23-H2 nucleoside diphosphate kinase gene, a potential negative regulator of cancer metastasis. These results provide a link between nm23 and the c-myc oncogene and suggest that the nm23 protein can function in vitro in the transcriptional regulation of c-myc expression.

DNA-Binding Proteins↗
PubMed1995

The crystal structure of a human nucleoside diphosphate kinase, NM23-H2.

The 2.8 A resolution X-ray structure of NM23-H2 has been determined by molecular replacement using the structure of Myxococcus xanthus nucleoside diphosphate (NDP) kinase. NM23-H2 is a human NDP kinase. The enzyme catalyses phosphoryl transfer, binds DNA, and can activate the transcription of the c-myc oncogene in vitro. NM23 has also been reported to be a suppressor of metastasis in some types of tumours. Whereas the M. xanthus NDP kinase is a tetramer, NM23-H2 is a hexamer. The fold of NM23-H2 is identical to the fold of other NDP kinases. Two antiparallel helices joined by a turn form one edge of the nucleotide binding cleft. This region moves in a hinge-like fashion in response to substrate binding and crystal packing forces. Additional differences in conformation among the NDP kinases are principally in regions involved in protein-protein contacts within the oligomers. The only protein-protein interaction conserved among all NDP kinases is a dimeric interaction. Several mutations of NM23-H2 have been detected in tumour tissues. These mutations do not involve residues interacting with the substrates, and probably destabilise the enzyme without directly affecting the catalytic activity. Low level phosphorylation of serines has been reported for NM23 both in vitro and in vivo. The structure of the hexamer indicates that two serine residues that have been reported as being phosphorylated, Ser44 and Ser122, are on the surface of the hexamer, and are likely to be phosphorylated by exogenous kinases. In contrast, Ser120 is buried, and is most likely phosphorylated by a direct transfer from the phosphohistidine intermediate of the reaction mechanism.

Amino Acid Sequence↗
PubMed1999

A novel human nucleoside diphosphate (NDP) kinase, Nm23-H6, localizes in mitochondria and affects cytokinesis.

Nucleoside diphosphate kinases (NDP kinases) are enzymes known to be conserved throughout evolution and have been shown to be involved in various biological events, in addition to the "housekeeping" phosphotransferase activity. We present the molecular cloning of a novel human NDP kinase gene, termed Nm23-H6. Nm23-H6 gene has been mapped at chromosome 3p21.3 and is highly expressed in heart, placenta, skeletal muscle, and some of the cancer cell lines. Recombinant Nm23-H6 protein has been identified to exhibit functional NDP kinase activity. Immunolocalization studies showed that both endogenous and inducibly expressed Nm23-H6 proteins were present as short, filament-like, perinuclear radical arrays and that they colocalized with mitochondria. Cell fractionation study also demonstrated the presence of Nm23-H6 protein in a mitochondria-rich fraction. Moreover, induction of overexpression of Nm23-H6 in SAOS2 cells, using the Cre-loxP gene activation system, resulted in growth suppression and generation of multinucleated cells. Flow cytometric analysis also demonstrated that the proportion of cells with more than 4N DNA content increased to 28.1% after induction of Nm23-H6, coinciding with the appearance of multinucleated cells. These observations suggest that Nm23-H6, a new member of the NDP kinase family, resides in mitochondria and plays a role in regulation of cell growth and cell cycle progression.

Amino Acid Sequence↗
PubMed1994

Measurement of nucleoside diphosphate kinase-Nm23 activity by anion-exchange high-performance liquid chromatography.

A first-order assay to detect the activity of nucleoside diphosphate kinase (NDP-kinase; EC 2.7.4.6) was developed. In this assay, the activity of NDP-kinase is measured using various deoxy- and ribonucleotide triphosphates as phosphate donors and dADP as phosphate acceptor. The enzyme activity is determined by quantifying, after anion-exchange HPLC, the amount of newly synthesized dATP. Contrary to the most common coupled enzymic assays or isotopic assays the use of different donor-acceptor pairs is not restricted. The resolution of the procedure described is limited only by the chromatographic separation of substrate and product pairs participating in the reaction.

Animals↗
PubMed1996

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.

Animals↗
PubMed2006

Down-regulated nucleoside diphosphate kinase nm23-H1 expression is unrelated to high-risk human papillomavirus but associated with progression of cervical intraepithelial neoplasia and unfavourable prognosis in cervical cancer.

OBJECTIVE: One of the factors leading to an invasive phenotype is the nm23 family of metastases-associated genes. Of the six known members, nm23-H1 is the most frequently studied potential anti-metastatic gene in cervical cancer. However, the possible molecular links to oncogenic human papillomavirus (HPV) are completely unexplored as yet. MATERIALS AND METHODS: As a part of the HPV-Pathogen Istituto Superiore di Sanità study, a series of 150 squamous cell carcinomas (SCCs) and 152 cervical intraepithelial neoplasia (CIN) lesions were examined by immunohistochemical staining for nm23-H1, and tested for HPV by polymerase chain reaction (PCR) with three sets of primers (MY09/11, GP5(+)/GP6(+) and short PCR fragment). Follow-up data were available on all patients with SCC, and 67 CIN lesions were monitored by serial PCR for clearance or persistence of HPV after cone treatment. RESULTS: A linear decrease (p = 0.001) was observed in nm23-H1 expression, starting from CIN1 (85% with normal expression), with the most dramatic down regulation on transition from CIN2 (70% normal) to CIN3 (39%) and further to SCC (25%). Reduced expression was associated with CIN3 or cancer at an odds ratio 8.72 (95% confidence interval 4.13 to 18.41). Nm23-H1 was of no use as a marker of the high-risk human papillomavirus (HR-HPV) type, and it did not predict clearance or persistence of HR-HPV after treatment of CIN. Importantly, nm23-H1 expression was a significant prognostic factor in cervical cancer, reduced expression being associated with lower survival (p = 0.022) in univariate analysis. In the multivariate (Cox) regression model, however, only the International Federation of Gynecology and Obstetrics stage (p = 0.001) and age (p = 0.011) remained independent prognostic predictors. CONCLUSIONS: Down-regulated nm23-H1 expression is markedly associated with progression from CIN2 to CIN3, and predicts poor prognosis in cervical cancer. Nm23-H1 down regulation is probably orchestrated by mechanisms independent of HR-HPV oncoproteins and is possibly related to the emergence of a proteolytic phenotype.

Adolescent↗
PubMed1994

A plant nucleoside diphosphate kinase homologous to the human Nm23 gene product: purification and characterization.

Nucleoside diphosphate kinases (NDPKs) catalyze the transfer of high-energy phosphates from nucleoside triphosphates to nucleoside diphosphates and may be involved in the regulation of growth, development, and signal transduction processes. We report here the purification and characterization of NDPK from detergent-solubilized extracts of dark-grown oat (Avena) tissue. The purification was achieved primarily through adsorption to GTP-agarose, followed by elution with ATP. SDS-polyacrylamide gel electrophoresis and gel filtration chromatography indicated that the purified protein is composed of six 18 kDa subunits. Substrate specificity experiments indicated that the purified kinase is capable of using all tested nucleosides as substrates. N-terminal sequencing of the Avena protein revealed that 87% of the 23 amino acids sequenced were identical to the human Nm23 protein, a nucleoside diphosphate kinase identified as a possible tumor metastasis suppressor and transcriptional activator of the myc oncogene.

Amino Acid Sequence↗
PubMed2000

Nucleoside diphosphate kinase beta (Nm23-R1/NDPKbeta) is associated with intermediate filaments and becomes upregulated upon cAMP-induced differentiation of rat C6 glioma.

Nucleoside diphosphate kinases (Nm23/NDPK) are enzymes functional in cell proliferation, differentiation, development, tumor progression, and metastasis. Nevertheless, no consensus exists about the molecular mechanism by which Nm23/NDPK isoforms exert their role in these processes. We investigated the expression of the rat Nm23-R1/NDPKbeta and Nm23-R2/NDPKalpha isoforms, homologues of the human Nm23-H1/NDPK A and Nm23-H2/NDPK B proteins, respectively, upon cAMP-induced differentiation of rat C6 glioma cells and demonstrated a differential interaction with intermediate filaments. Semiquantitative RT-PCR, immunoblotting, and flow cytometry showed a constitutive expression of both Nm23 isoforms. After induction of differentiation in C6 cells with cAMP analogs or isoproterenol, a dose-dependent 2- and 2.5-fold upregulation of the Nm23-R1 mRNA and protein, respectively, was observed. In contrast, the expression of Nm23-R2 remained unchanged. Localization of both isoforms with confocal laser scanning microscopy demonstrated a punctate reticular staining pattern for both Nm23 isoforms in the cytosol and processes of the cells which was particularly intense in the perinuclear region. In addition, while Nm23-R2 was colocalized and coimmunoprecipitated with vimentin in nondifferentiated cells, both isoforms were associated with GFAP in differentiated cells. The significance of these findings in relation to a possible function of Nm23 isoforms in cell proliferation, differentiation, and tumor-associated mechanisms is discussed.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

Human nucleoside diphosphate kinase B (Nm23-H2) from melanoma cells shows altered phosphoryl transfer activity due to the S122P mutation.

The Ser122 --> Pro mutation in human nucleoside diphosphate kinase (NDK)-B/Nm23-H2 was recently found in melanoma cells. In comparison to the wild-type enzyme, steady state activity of NDKS122P with ATP and TDP as substrates was slowed down 5-fold. We have utilized transient kinetic techniques to analyze phosphoryl transfer between the mutant enzyme and various pairs of nucleoside triphosphates and nucleoside diphosphates. The two half-reactions of phosphorylation and dephosphorylation of the active site histidine residue (His118) were studied separately by making use of the intrinsic fluorescence changes which occur during these reactions. All apparent second order rate constants are drastically reduced, falling 5-fold for phosphorylation and 40-200-fold for dephosphorylation. Also, the reactivity of the mutant with pyrimidine nucleotides and deoxy nucleotides is more than 100-fold reduced compared with the wild-type. Thus, the rate-limiting step of the NDK-BS122P-catalyzed reaction is phosphoryl transfer from the phospho-enzyme intermediate to the nucleoside diphosphate and not phosphoryl transfer from the nucleoside triphosphate to the enzyme as was found for the wild-type protein. This results in a pronounced shift of the equilibrium between unphosphorylated and phosphorylated enzyme. Moreover, like the Killer-of-prune mutation in Drosophila NDK and the neuroblastoma Ser120 --> Gly mutation in human NDK-A/Nm23-H1, the Ser122 --> Pro substitution in NDK-B affects the stability of the protein toward heat and urea. These significantly altered properties may be relevant to the role of the mutant enzyme in various intracellular processes.

Adenosine Triphosphate↗
PubMed1996

A nucleoside diphosphate kinase A (nm23-H1) serine 120-->glycine substitution in advanced stage neuroblastoma affects enzyme stability and alters protein-protein interaction.

A high level of nucleoside diphosphate kinase A (NDPK A/nm23-H1) in neuroblastoma is associated with advanced stage disease. We have also found a serine 120-->glycine substitution in NDPK A and/or amplification of the nm23-H1 gene in advanced stage neuroblastomas. Serine 120, a highly conserved residue, is located in proximity to histidine 118 which forms a phosphorylated intermediate essential for NDPK activity. The effect of Ser120-->Gly substitution on the biochemical properties of NDPK A was investigated. Phosphate-transferase activity was lower in the recombinant mutant NDPK A and in the immunoprecipitated complex consisting of NDPK A and NDPK B prepared from a neuroblastoma tumor containing the mutation, relative to the wild-type. There was a significant decrease in the enzyme stability toward urea- or temperature-induced denaturation for the recombinant mutant NDPK A and in an immunoprecipitate from a tumor containing the mutation. Recombinant NDPK A containing the Ser120-->Gly mutation exhibited reduced hexameric and increased dimeric oligomerization relative to the wild-type. Moreover a 28 kDa cellular protein was detected, that co-precipitated with the mutant but not wild-type NDPK A. The altered properties of the mutant protein may have relevance to a role for NDPK A in neuroblastoma progression.

Base Sequence↗