Nm23/nucleoside Diphosphate Kinase Research Articles

Mechanisms of non-metastatic 2 (NME2)-mediated control of metastasis across tumor types

Non-metastatic 23 [NM23/nucleoside diphosphate kinases (NDPK)] genes are the first discovered metastasis suppressor genes. More than two decades of research has demonstrated their roles in a variety of biological processes with NME1 and NME2 being most studied in the context of metastasis suppression. Although NME1 and NME2 share >85% homology at amino acid level, they show redundant as well as unique molecular functions. Phenotypic analyses of knockout (KO) mice for NM23 members (NDPK-A, B) and compound KO (A as well as B) showed requirement of both proteins in hematopoiesis suggesting shared functions in development disease. Several reviews have discussed NME1, however the role of NME2 appears to be relatively less understood in the context of metastasis suppression. Here, we focus on NME2 and by meta-analysis of gene expression from multiple tumor types, and survey of in vivo and vitro studies, suggest the possibility that NME2 may be one of the key factors in metastasis. This along with the relevance of normal physiological functions of NME2 in the context of metastasis is discussed. We further examined the genetic and epigenetic features of NME2 and NME1 gene promoters and found aspects of transcription control that could be unique to NME2/NME1. Findings on signaling pathways and small molecules which regulate the expression of NME2 that could be therapeutically important are also discussed.

The application of proteomic technology in differentiation and early diagnosis of myelocytic leukemia

Objective To figure out the differentially expressed proteins using proteome technology in leukemia cells induced into different lineages and investigate the application value in early screening of leukemia.Methods With induction of ATRA and NSC67657, the differentiation models was constructed using HL60 cells which has the potentiality to be induced into different lineages by different inducers.Then the differentially expressed proteins in the process of differentiation was separated using two-dimensional electrophoresis and identified using MALDI-TOF MS.The expression of 3 proteins FE1A1, TLE1, NME3 were chosen to be verified in myeloid samples of 5 leukemia patients and 1 normal volunteer using RT-PCR and WB.Results WB showed that NME3 was differentially expressed after both granulocytic and monocytic differentiation( Normal A value = 0.227, NSC67657 A value= 0.079, ATRA A value = 0.064, P ＜ 0.01 ).However, only in 4 of 5 tested patients' myeloid samples, the NME3 protein expression were differentially expressed compared to the normal myeloid sample( Normal A value = 0.082,2 acute leumia transferred from chronic granulocytic leumia A value = 0.274,0.269, acute monocytic leukemia A value = 0.297, one patient with chronic granulocytic leukemia A value = 0.258.There was significant difference between normal and leukemia group, P ＜0.05 ).A value was 0.121 for another patents with chronic granulocytic leukemia The NME3 protein expression was not differentially expressed compared to the normal myeloid sample,P ＞0.05.Conclusions It is still a long way to go for proteome technology from basic research to clinical application.However, the identification of NME3 protein related to differentiation in leukemia patients' myeloid samples had set the foundation for the early diagnosis of leukemia using proteome technology. Key words: Leukemia, promyelocytic, acute; Proteomics; NM23 nucleoside diphosphate kinases; Electrophoresis,gel,two-dimensional; Cell differentiation

The Upregulation of Expressed Proteins in HepG2 Cells Transfected by the Recombinant Plasmid‐containing HBx Gene

It is known that the hepatitis B virus X protein (HBx) plays a crucial role in the pathogenesis of HCC, but the exact functions and molecular mechanisms of HBx in HCC are not well understood. In the present study, HepG2 cell lines were cultured and transfected with pEGFP-N1 and pEGFP-N1-X. Twenty-four hours after transfection, cells were harvested and total RNA was extracted using TRIzol reagent. The expression of HBx in HepG2 cell line was assayed by real-time polymerase chain reaction and was detected by Western blotting. Moreover, proteomic analysis was performed for the HepG2-pEGFP-X cells and HepG2-pEGFP control cells. The combination of 2DE and MALDI-TOF-MS/MS revealed that SEC13L1 (SEC13-like 1 isoform b), PA28 alpha (proteasome activator REG alpha), serine-threonine kinase receptor-associated protein (STRAP) and nm23/nucleoside diphosphate kinase (NME) were upregulated in HepG2-pEGFP-X cells. STRAP is known to be a WD40 domain-containing protein, which interacts with TbetaR-I and TbetaR-II and negatively regulates TGF-beta signalling, was also found increased in human cancers. NME is known to be involved in the regulation of cancer cell progression and metastasis. These results would help the understanding of how HBx maintains tumorigenicity and progression of HCC.

Regulatory roles for nm23/nucleoside diphosphate kinase-like enzymes in insulin secretion from the pancreatic islet beta cell

Recent studies from multiple laboratories, including our own, provided fresh insights into the contributory roles for GTP-binding proteins (G-proteins) in glucose-stimulated insulin secretion (GSIS) from the islet beta cell. However, the precise mechanisms underlying the activation of this class of signaling proteins by insulin secretagogues remain only partially understood. We recently proposed that nm23/nucleoside diphosphate kinase (NDPK) catalyzes an alternate, non-receptor-dependent activation of islet endogenous G-proteins. In further support of this proposal, we report, herein, that overexpression of wild type (WT) nm23-H1 mutant in INS cells markedly potentiated GSIS. However, an inactive mutant of nm23-H1(H118F), which is deficient in histidine kinase and NDPK activities, was considerably less effective in potentiating GSIS from these cells, suggesting that both of these activities may be relevant for the potentiating effects of nm23-H1. Potential significance of these findings in relation to contributory roles for nm23/NDPK-like enzymes in the stimulus-secretion coupling of GSIS is discussed.

In vivo cross-linking of nm23/nucleoside diphosphate kinase to the PDGF-A gene promoter.

Human isoforms A and B of nm23/nucleoside diphosphate (NDP) kinase, functionally important in development and cancer, have been reported to bind to DNA, and in particular isoform A to the PDGF-A promoter and isoform B to the c-myc promoter and to telomeric repeats. However, no direct proof of the binding in vivo has yet been obtained. To demonstrate this interaction, human erythroleukemic K562 cells were incubated with two different cross-linking reagents, formaldehyde or cis-diammine dichloro platinum H. The DNA-protein covalent complexes were isolated and analyzed by Western blotting. The positive immunochemical staining showed that in both conditions NDP kinase isoforms A and B were efficiently cross-linked to DNA in vivo. NDP kinase-linked DNA fragments obtained by immunoprecipitation, subjected to hybridization with different probes, showed a definite enrichment in the nuclease-hypersensitive silencer element of the PDGF-A promoter. No conclusive evidence was found by this technique of preferential hybridization with a nuclease-hypersensitive element of the c-myc promoter and with the telomeric TTAGGG repeats. The immunoprecipitated NDP kinase-DNA complexes are a promising material for the detection of other specific DNA sequences interacting with NDP kinase.

The Human nm23-H4 Gene Product Is a Mitochondrial Nucleoside Diphosphate Kinase

We demonstrate here the catalytic activity and subcellular localization of the Nm23-H4 protein, product of nm23-H4, a new member of the human nm23/nucleoside diphosphate (NDP) kinase gene family (Milon, L., Rousseau-Merck, M., Munier, A., Erent, M., Lascu, I., Capeau, J., and Lacombe, M. L. (1997) Hum. Genet. 99, 550-557). Nm3-H4 was synthesized in escherichia coli as the full-length protein and as a truncated form missing the N-terminal extension characteristic of mitochondrial targeting. The truncated form possesses NDP kinase activity, whereas the full-length protein is inactive, suggesting that the extension prevents enzyme folding and/or activity. X-ray crystallographic analysis was performed on active truncated Nm23-H4. Like other eukaryotic NDP kinases, it is a hexamer. Nm23-H4 naturally possesses a serine residue at position 129, equivalent to the K-pn mutation of the Drosophila NDP kinase. The x-ray structure shows that the presence of Ser(129) has local structural effects that weaken subunit interactions. Site-directed mutagenesis shows that the serine is responsible for the lability of Nm23-H4 to heat and urea treatment, because the S129P mutant is greatly stabilized. Examination of human embryonic kidney 293 cells transfected with green fluorescent protein fusions by confocal microscopy shows a specific mitochondrial localization of Nm23-H4 that was also demonstrated by Western blot analysis of subcellular fractions of these cells. Import into mitochondria is accompanied by cleavage of the N-terminal extension that results in NDP kinase activity. Submitochondrial fractionation indicates that Nm23-H4 is associated with mitochondrial membranes, possibly to the contact sites between the outer and inner membranes.

NME6: a new member of the nm23/nucleoside diphosphate kinase gene family located on human chromosome 3p21.3.

The NME (nm23/nucleoside diphosphate kinase) gene family in human is involved in the phosphorylation of nucleoside diphosphates and a variety of regulatory phenomena associated with development, oncogenic transformation, and metastasis. Here we report the cDNA sequence for a sixth member of this family, NME6. The cDNA sequence predicts a 186-residue protein that includes the characteristic active site motif of a nucleoside diphosphate (NDP) kinase, as well as the other residues previously identified as crucial for nucleotide binding and catalysis. The NME6 protein sequence is only 34-41% identical to the five previously reported human NME proteins, and is similarly related to prokaryotic and primitive eukaryotic NDP kinases. Compared to typical proteins of this family such as NME1 and NME2, NME6 has three additional residues located in the Kpn loop, and a 22-residue extension at the COOH-terminal. Using radiation hybrid mapping, the NME6 gene was localized to chromosome 3p21.3. The 1.3-kb transcript of NME6 is expressed at a moderately low level in many human tissues, and is most abundant in kidney, prostate, ovary, intestine, and spleen. Homologous cDNAs were also cloned and sequenced for rat and mouse. The sequence of the first 171 residues of the mouse homologue (Nm23-M6) is 94% identical to the deduced human NME6 protein.

A new human nm23 homologue ( nm23-H5) specifically expressed in testis germinal cells

We have identified a cDNA encoding a 212 amino acid protein (Nm23-H5) with 27–31% identity to the human members of the nm23/nucleoside diphosphate (NDP) kinase gene family. The nm23-H5 gene is located on chromosome 5q23–31 and is transcribed as one main transcript of 1.1 kb which is highly and specifically expressed in testis, in the spermatogonia and early spermatocytes. Nm23-H5 possesses most of the residues conserved among NDP kinases plus an additional COOH-terminus end of 55 amino acids unique to this protein. However, under usual assay conditions, Nm23-H5 expressed in Escherichia coli did not exhibit NDP kinase activity. These results suggest that Nm23-H5 is specifically involved in early stages of spermatogenesis.

Nm23 -H4, a new member of the family of human nm23 /nucleoside diphosphate kinase genes localised on chromosome 16p13

A novel human nm23/nucleoside diphosphate (NDP) kinase gene, called nm23-H4, was identified by screening a human stomach cDNA library with a probe generated by amplification by reverse transcription-polymerase chain reaction. The primers were designed from publicly available database cDNA sequences selected according to their homology to the human nn23-H1 putative metastasis suppressor gene. The full-length cDNA sequence predicts a 187 amino acid protein possessing the region homologous to NDP kinases with all residues crucial for nucleotide binding and catalysis, strongly suggesting that Nm23-H4 possesses NDP kinase activity. It shares 56, 55 and 60% identity with Nm23-H1, Nm23-H2 and DR-Nm23, respectively, the other human Nm23 proteins isolated so far. Compared with these proteins, Nm23-H4 contains an additional NH2-terminal region that is rich in positively charged residues and could indicate routing to mitochondria. The nm23-H4 gene has been localised to human chromosomal band 16p13.3. The corresponding 1.2 kb mRNA is widely distributed and expressed in a tissue-dependent manner, being found at very high levels in prostate, heart, liver, small intestine and skeletal muscle tissues and in low amounts in the brain and in blood leucocytes. Nm23-H4 naturally possesses the Pro-Ser substitution equivalent to the K-pn mutation (P97S) of Drosophila.

A 16-kDa Protein Functions as a New Regulatory Protein for Hsc70 Molecular Chaperone and Is Identified as a Member of the Nm23/Nucleoside Diphosphate Kinase Family

Cytoplasmic Hsc70 is a multifunctional molecular chaperone. It is hypothesized that accessory proteins are used to specify the diverse chaperone activities of Hsc70. A 16-kDa cytosolic protein (p16) co-purified with Hsc70 obtained from a fish hepatocyte cell line, PLHC-1. Hsc70 also co-immunoprecipitated with p16 from PLHC-1 cells and fish liver. p16 was identified as a member of the Nm23/nucleoside diphosphate (NDP) kinase family based on its amino acid sequence similarity, NDP kinase activity, and recognition by anti-human NDP kinase-A antibody. This antibody also co-immunoprecipitated Hsc70 and NDP kinase from human HepG2 cells. p16 monomerized Hsc70 and released Hsc70 from pigeon cytochrome c peptide (Pc) but not from FYQLALT, a peptide specifically designed for high affinity binding. Therefore, p16 may modulate Hsc70 function by maintaining Hsc70 in a monomeric state and by dissociating unfolded proteins from Hsc70 either through protein-protein interactions or by supplying ATP indirectly through phosphate transfer. p16 did not affect basal or unfolded protein-stimulated ATPase activity of bovine brain Hsc70 using in vitro assays. Interestingly, bovine liver NDP kinase did not dissociate the Hsc70.Pc complex. In addition, two nonconservative amino acid subsitutions were found near the amino terminus of p16. Therefore, p16 may be a unique Nm23/NDP kinase that functions as an accessory protein for cytosolic Hsc70 in eukaryotes.

Site-directed Mutagenesis of nm23-H1: MUTATION OF PROLINE 96 OR SERINE 120 ABROGATES ITS MOTILITY INHIBITORY ACTIVITY UPON TRANSFECTION INTO HUMAN BREAST CARCINOMA CELLS

We report the first correlation of Nm23 sequence and its tumor metastasis-suppressive capacity using site-directed mutagenesis and an in vitro tumor cell motility assay. MDA-MB-435 human breast carcinoma cells were transfected with a control expression vector (pCMVBamneo), the vector containing the wild type nm23-H1, or the nm23-H1 vector encoding mutations at the following amino acids: serine 44, a phosphorylation site; proline 96, the k-pn mutation in the Drosophila nm23 homolog that causes developmental defects; histidine 118, involved in Nm23's nucleoside diphosphate kinase activity; and serine 120, a site of mutation in human neuroblastomas and phosphorylation. The wild type nm23-H1 transfectants were 44-98% less motile to serum and 86-99% less motile to autotaxin than control vector transfectants. The proline 96 k-pn, serine 120 to glycine, and to a lesser extent serine 120 to alanine mutant nm23-H1-transfected cell lines exhibited motility levels at or above the control transfectants, indicating that these mutations can abrogate the motility-suppressive phenotype of nm23-H1. No effect was observed on cellular proliferation, nor were the serine 44 to alanine nm23-H1 mutant transfectants motile, demonstrating the specificity of the data. The data identify the first structural motifs of nm23-H1 that influence its metastasis suppressive effect and suggest complex biochemical associations or activities in the Nm23 suppressive pathway.

Evaluation of the Prognostic Significance of nm23/NDP Kinase Protein Expression in Cervical Carcinoma: An Immunohistochemical Study

The objective of this study was to evaluate the prognostic significance of immunohistochemical staining for nm23/nucleoside diphosphate (NDP) kinase in cervical carcinoma. A retrospective analysis of 176 patients with cervical carcinoma FIGO stage IB treated with radical hysterectomy and pelvic lymphadenectomy from 1987 to 1990 was conducted. Immunohistochemical staining using the polyclonal nm23-H1/NDP kinase A antibody was correlated to various histopathological and morphological characteristics (tumor size, histologic type, grade of differentiation, vessel invasion, invasion into parametria, and lymph node metastasis) and relapse-free survival. For controls, sections were obtained from 10 hysterectomy specimens with normal cervical epithelium. Staining for nm23/NDP kinase was observed in 90% of control cases and in 70.5% of cases of cervical carcinoma, more frequent in squamous and adenosquamous cell carcinoma than in adenocarcinoma and more frequent in poorly differentiated than in more highly differentiated tumors. There were no differences related to size of tumor or invasion into vessels or parametria or occurrence of lymph node metastasis. The relapse-free survival was lower for patients with squamous cell and adenosquamous tumors with positive immunostaining for nm23/NDP kinase than for those with negative tumors when evaluated in univariate analysis. In multivariate analysis with tumor size, vessel invasion, invasion into parametria, grade of differentiation, and lymph node metastasis included, this difference was no longer significant. In patients with adenocarcinoma no difference was found. In conclusion, we did not find immunostaining for nm23/NDP kinase to be a useful indicator for prognosis in cancer of the uterine cervix.