Nm23 Gene Family Research Articles

Expression of the nm23 homologues nm23‐H4, nm23‐H6, and nm23‐H7 in human gastric and colon cancer

Eight members of the nm23-gene family have been described. The involvement of nm23-H1 and nm23-H2 in tumour progression and metastasis, as well as in gene regulation and apoptosis, has been shown in numerous studies. Whether nm23-H4, -H6, and -H7 play a role in tumours is, however, largely unknown. This study describes data on the expression of these three nm23 homologues in human colon and gastric cancer by real-time RT-PCR and immunohistochemistry. Increased expression of these genes, most strikingly nm23-H4 and -H7, was observed in the majority of tumours analysed. No correlation with tumour stage according to the TNM classification was found. In contrast, by immunohistochemical analysis, nm23-H4 and -H6 overexpression correlated with the intestinal tumour type in gastric cancer tissues, whereas no increased immunoreactivity for the three nm23 proteins was noted in the diffuse type tumour specimens. These findings indicate that nm23-H6, and particularly nm23-H4 and -H7, may be involved in the development of colon and gastric carcinoma, the latter possibly in a type-specific manner. A contribution to tumour progression or metastasis could not, however, be proven. Elucidation of the specific mechanisms by which the nm23 homologues nm23-H4, -H6, and -H7 are involved in tumour development requires further studies.

Nm23-M2/NDP kinase B induces endogenous c- myc and nm23-M1/NDP kinase A overexpression in BAF3 cells. Both NDP kinases protect the cells from oxidative stress-induced death

The nm23 gene family encodes nucleoside diphosphate kinases (NDPKs) which supply the cell with (d)NTPs. The human NDPKB, also known as the PuF protein, binds the c- myc promoter and transactivates the c- myc protooncogene. We have now studied the effects of mouse NDPKA and NDPKB overexpression on endogenous c- myc transactivation in the mouse BAF3 and the rat PC12 cell lines. c- myc transcripts were found to be up-regulated by NDPKB only in the BAF3 line. This suggests that c- myc transcriptional control via NDPKB depends on the presence of cell-specific co-factors. Unexpectedly, NDPKB also induced NDPKA expression. This new effect was found in both cell lines, suggesting that NDPKB-dependent nm23-M1 gene transactivation requires cis and/or trans elements different from those involved in c- myc transactivation. Moreover, the BAF3 cell proliferation capacities were found to be independent of NDPKA or B cell contents. Interestingly, cell death induced by c- myc overexpression or H 2O 2 exposure was decreased in nm23-transfected compared to control BAF3 cells. These data collectively suggest that NDPKs might improve cell survival by a mechanism coupling DNA repair and transcriptional regulation of genes involved in DNA damage response.

Lbc proto-oncogene product binds to and could be negatively regulated by metastasis suppressor nm23-H2

Lbc was identified as transforming gene from human leukemic cells and encodes Rho type guanine nucleotide exchange factor with 47kDa molecular weight. We isolated overlapping cDNAs of Lbc from human lung tissue. Full-length Lbc cDNA encodes 309kDa huge protein with Ht31 PKA anchoring motif, Dof domain, C1 domain, and coiled-coil structure. In order to analyze the regulatory mechanism of its activity, we searched for binding proteins. By yeast two-hybrid screening, we identified metastasis suppressor nm23-H2 as binding protein, which interacts with amino-terminal region of Lbc containing Dof domain. nm23 gene family encodes nucleoside diphosphate kinase, however, the binding of nm23-H2 to Lbc was independent of kinase activity. nm23-H1, which binds to Rac-specific GEF Tiam1, could not bind to Lbc suggesting nm23-H2 would be specific regulator for Lbc. Expression of nm23-H2 in cells leads to decrease the amount of GTP-bound Rho and suppress stress fiber formation stimulated by expression of Lbc. Our data suggest that metastasis suppressor nm23-H2 could regulate Lbc negatively by binding to amino-terminal region of Lbc proto-oncogene product.

Characterization of the nm23-M2, nm23-M3 and nm23-M4 mouse genes: comparison with their human orthologs

The nm23 gene family is thought to be involved in physiopathological processes such as growth, differentiation and cancer promotion, progression or metastasis. We report here the mouse nm23-M3 and nm23-M4 complementary DNA sequences and the genomic cloning, characterization and tissue expression pattern of the nm23-M2, nm23-M3 and nm23-M4 genes, in comparison with their human and rat orthologs and with the human nm23-H1 and mouse nm23-M1 genes. The organization and structure of the members of this gene family are remarkably similar in human and rodents. Accordingly, the striking similarities between the human and mouse nm23 genes enable the use of mouse transgenic and knock-out models for studying the role of nucleoside diphosphate kinase isoforms in human physiopathology.

DR-nm23 expression affects neuroblastoma cell differentiation, integrin expression, and adhesion characteristics.

Nm23 gene family has been associated with metastasis suppression and differentiation. We studied DR-nm23 during neuroblastoma cells differentiation. DR-nm23 expression increased after retinoic acid induction of differentiation in human cell lines SK-N-SH and LAN-5. In several cell lines, overexpression of DR-nm23 was associated with more differentiated phenotypes. SK-N-SH cells increased vimentin expression, increased deposition of collagen type IV, modulated integrin expression, and underwent growth arrest; the murine neuroblastoma cell line N1E-115 showed neurite outgrowth and a striking enhancement of beta1 integrin expression. Up-regulation of beta1 integrin was specifically responsible for the increase in the adhesion to collagen type I-coated plates. Finally, cells overexpressing DR-nm23 were unable to growth in soft agar. In conclusion, DR-nm23 expression is directly involved in differentiation of neuroblastoma cells, and its ability to affects the adhesion to extracellular substrates and to inhibit growth in soft agar suggests an involvement in the metastatic potential of neuroblastoma.

Molecular cloning of an Atlantic salmon nucleoside diphosphate kinase cDNA and its pattern of expression during embryogenesis

To gain insight into the process of development in Atlantic salmon ( Salmo salar), we sought to identify genes that were differentially expressed at gastrulation. A polymerase chain reaction-based differential screening strategy allowed for the isolation of an Atlantic salmon nucleoside diphosphate kinase cDNA ( nm23). Structural characterisation showed a high degree of homology with a large number of previously isolated nucleoside diphosphate kinases (NM23s), both prokaryote and eukaryote, though it represents the first teleost nucleoside diphosphate kinase identified. Highest similarities were found with the type 1 and type 2 NM23 isoforms of mammals. Phylogenetic analysis indicates that the duplication event that gave rise to these isoforms occurred after the splitting of tetrapods and fish, suggesting that the salmon NM23 represents a more ancestral isoform. The position of the salmon sequence on the phylogenetic tree indicates that the salmon genome is expected to have at least three copies of genes from the nm23 gene family. Northern blot analysis showed a single transcript of approximately 0.7 kb in both embryonic and adult tissues. Examination of the temporal pattern of expression of salmon nucleoside diphosphate kinase during embryonic development revealed that this gene is first expressed at the time of gastrulation. Nucleoside diphosphate kinases are thought to have a vital role in regulatory processes such as signal transduction, proliferation and differentiation. Taken together, these results suggest that nucleoside diphosphate kinases have an important role to play in early embryogenic development in vertebrates.

Cleavage of DNA by Human NM23-H2/Nucleoside Diphosphate Kinase Involves Formation of a Covalent Protein-DNA Complex

The NM23 gene family in humans is implicated in differentiation and cancer, but the biochemical mechanisms are unknown. Most NM23 proteins have phosphotransferase (nucleoside diphosphate kinase) activity, and the second human isoform, NM23-H2, also binds to a nuclease-hypersensitive c-MYC promoter element through which it activates c-MYC transcription. It is shown here that this DNA binding can result in double-stranded breaks. The DNA breaks occur within repeated sequence elements in the linear nuclease-hypersensitive duplex and leave staggered ends with 5-nucleotide-long 3'-extensions. The enzyme also cleaves supercoiled plasmid DNA to yield nicked circular and unit length linear products. The cleavage reaction requires only NM23-H2, DNA, Mg(2+), and buffer, occurs in the absence of denaturing conditions, and can be reversed by EDTA. The cleaved DNA strands have free 3'-OH groups, and protein is attached to the 5'-phosphoryl ends. Transfer of (32)P radioactivity from DNA to NM23-H2 has been observed, and a covalent polypeptide-DNA complex has been isolated and identified by Western blotting as NM23-H2. Since covalent protein-DNA complexes are known to serve the role of breaking and rejoining DNA strands, the present findings suggest that NM23-H2 is involved in DNA structural transactions necessary for the activity of the c-MYC promoter.

DR-nm23 gene expression in neuroblastoma cells: relationship to integrin expression, adhesion characteristics, and differentiation.

Neuroblastoma, a childhood tumor originating from cells of the embryonic neural crest, retains the ability to differentiate, yielding cells with epithelial-Schwann-like, neuronal, or melanocytic characteristics. Since nm23 gene family members have been proposed to play a role in cellular differentiation, as well as in metastasis suppression, we investigated whether and how DR-nm23, a recently identified third member of the human nm23 gene family, might be involved in neuroblastoma differentiation. Three neuroblastoma cell lines (human LAN-5, human SK-N-SH, and murine N1E-115) were used in these experiments; cells from two of the lines (SK-N-SH and N1E-115) were also studied after being stably transfected with a plasmid containing a full-length DR-nm23 complementary DNA. Cellular expression of specific messenger RNAs and proteins was assessed by use of standard techniques. Cellular adhesion to a variety of protein substrates was also evaluated. DR-nm23 messenger RNA levels in nontransfected LAN-5 and SK-N-SH cells generally increased with time after exposure to differentiation-inducing conditions; levels of the other two human nm23 messenger RNAs (nm23-H1 and nm23-H2) remained essentially constant. Transfected SK-N-SH cells overexpressing DR-nm23 exhibited some characteristics of differentiated cells (increased vimentin and collagen type IV expression) even in the absence of differentiation-inducing conditions. Compared with control cells, DR-nm23-transfected cells exposed to differentiation-inducing conditions showed a greater degree of growth arrest (SK-N-SH cells) and greater increases in integrin protein expression, especially of integrin beta1 (N1E-115 cells). DR-nm23-transfected N1E-115 cells also showed a marked increase in adhesion to collagen type I-coated tissue culture plates that was inhibited by preincubation with an anti-integrin beta1 antibody. DR-nm23 gene expression appears to be associated with differentiation in neuroblastoma cells and may affect cellular adhesion through regulation of integrin protein expression.

Quantitation of NM23 Expression in Human Prostate Tissues

The NM23 gene family (nm23-Hl and nm23-H2) has been reported as a measure of metastatic potential. The goal of this study was to discriminate nm23-Hl and nm23-H2 gene expression in benign and malignant human prostate tissue and to determine the relationship of their expression to tumor stages. Specimens included 5 benign prostatic hyperplasias (BPH), 11 primary prostate adenocarcinomas (CaP) (5 stage B, 5 stage C and 1 stage Dl), 2 pelvic lymph nodes with metastases and 3 prostate cancer cell lines derived from metastatic lesions. Polymerase chain reaction analysis of mRNA (RNA/PCR) was used to amplify transcripts of both NM23 genes and a normalizing gene (c-N-ras) to determine the relative levels of expression. A significant difference was shown between the BPH specimens and the cell lines from metastatic prostate cancer for nm23-H2 expression (p = 0.037) and the nm23-Hl/nm23-H2 gene expression ratio (p = 0.037). The nm23-Hl/nm23-H2 ratio increased significantly (p = 0.026, tau-b = 0.377) from BPH, through the CaP stages, to the cell lines. The expression of nm23-H2 decreased significantly (p = 0.002, tau-b = —0.517) from BPH, through the CaP stages, to the cell lines. Thus, while nm23-H2 appears to be significant for characterizing stages of CaP, an understanding of the metastatic phenotype will require further analysis of both NM23 genes.