Expression Of Nucleoside Diphosphate Kinase Research Articles

Potential role of nucleoside diphosphate kinase in myricetin-induced selective apoptosis in colon cancer HCT-15 cells

The flavonoid myricetin (MYR) is derived from vegetables and fruits. It has been shown to exert anti-cancer effects in various cell lines; however, the exact mechanism underlying these effects is yet to be elucidated. In this study, we evaluated the anti-cancer effects induced by MYR treatment in colon cancer HCT-15 cells. To detect cell proliferation, we conducted MTT assay and real time-cell electronic sensing (RT-CES). We next performed comet assay and Annexin V and PI staining to detect cellular apoptotic features. After that, we conducted two-dimensional electrophoresis (2-DE) analysis to identify apoptotic proteins. The results of this analysis revealed that eight spots were differentially expressed. Among the spots, we selected nucleoside diphosphate kinase (NDPK) for further investigation, as it has been shown to regulate cancer cell apoptosis and metastasis. After that, we conducted realtime-PCR and western blot to detect the expression of NDPK mRNA and protein and wound-healing assay to detect cell migration and invasion. Finally, we performed NDPK siRNA transfection study and the results showed that NDPK knockdown inhibited apoptosis. Based on these collective results, we suggest that MYR induces apoptosis in human colon cancer HCT-15 cells selectively by increasing the expression of NDPK and other caspase-regulated apoptosis proteins.

Alteration of nuclear protein profiling for NIH‐3T3 cells exposed to H2O2

Oxidative stress is a threat to mammalian cells. To better understand the molecular response and mechanism underlying oxidative stress, we applied two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time-of-fight mass spectrometry analysis to identify differential nuclear protein profiling of mouse fibroblast NIH-3T3 cells exposed to mild-H₂O₂. Thirteen differentially expressed proteins were identified by MS and two of them were further validated by Western blot. The results revealed that exposure to mild-H₂O₂ for 12 h cause up-regulated expression of DJ-1, glutathione S-transferase P 1, DNA ligase I, dynamin 2, nucleophosmin, and down-regulated expression of nucleoside diphosphate kinase A, enolase-α, barrier-to-autointegration factor 1, metastasis associated protein 1, glycosytransferase-like domain containing protein 1, synaptonemal complex protein 1, alpha-centractin, bromodomain, and PHD finger containing 1 (BRPF1). Most of the identified proteins are supported as nuclear proteins localized by previous research. The findings may provide some clues to elucidate cell responses to H₂O₂ and the potential mechanism underlying protection against oxidative stress in fibroblast cells.

M1995 Spontaneously Hypertensive Rat Develops Pronounced Steatohepatitis Induced by Choline Deficient Diet: An Evidence for Hypertension as the Critical Enhancer for Nonalcoholic Fatty Liver Disease

Background:Mallory-Denk bodies (MDBs) are hepatocyte inclusions associated with alcoholic and nonalcoholic steatohepatitis, and some forms of drug-induced liver injury. Understanding the biological significance of MDBs and devising means to alter their formation has clinical relevance to protein aggregation diseases beyond the liver. Feeding mice 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) produces, in a strain-selective manner, inclusions resembling human MDBs. Aims: We hypothesized that proteomic comparison of livers from MDBprone C57BL/6 mice and MDB-resistant C3H mice, before and after DDC treatment, will illuminate the molecular mechanisms regulating susceptibility of C57BL/6 mice to MDB formation. Methods: 2-dimensional (2D) differential-in-gel electrophoresis and mass spectrometry of liver protein homogenates resulted in the identification of >200 charged isoforms with >2 fold differences between the two strains. The results for some of the proteins were validated in control and DDC-treated livers, and isolated hepatocytes using 1D/2D gel analysis followed by immunoblotting. Enzymatic activities were also measured. Results: Proteins involved in cellular homeostasis and survival, such as carbonic anhydrase 3 (CA3) and GAPDH are expressed at lower levels in C57BL/6 livers. Whereas DDC treatment strongly induces CA3 and GAPDH expression in isolated C3H hepatocytes, it has no effect in C57BL/ 6 hepatocytes. There is significantly higher expression of reactive oxygen species-generating cytochrome P450 and detoxifying glutathione-S-transferase enzymes, and lower expression of the antioxidant enzyme peroxiredoxin 6 in the C57BL/6 livers, indicating a state of elevated oxidative stress. Furthermore, the livers, but not other organs, of untreated and DDC-fed C3H mice have a 2-fold higher expression of nucleoside diphosphate kinase B (NDPK-B), a multifunctional protein involved in regulating intracellular energy pools and protection from oxidative stress. Additionally, isolated C3H hepatocytes have 6-fold higher NDPK expression and activity relative to C57BL/6 hepatocytes, indicating that differences in total liver NDPK levels are accounted for by differences in hepatocytes. Conclusions: Genetic predisposition to MDB formation is likely related, at least in part, to deficiencies in systems involved in regulating intracellular homeostasis, energy generation and protection from oxidative stress. NDPK may be an upstream effector functioning in a strain-dependent and tissue-selective manner to limit MDB formation. Similar molecular determinants might contribute to the genetic differences in human liver diseases that are associated with MDB formation.

M1994 Molecular Factors Involved in Determining Genetic Susceptibility to Mallory-Denk Body Formation

Background:Mallory-Denk bodies (MDBs) are hepatocyte inclusions associated with alcoholic and nonalcoholic steatohepatitis, and some forms of drug-induced liver injury. Understanding the biological significance of MDBs and devising means to alter their formation has clinical relevance to protein aggregation diseases beyond the liver. Feeding mice 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) produces, in a strain-selective manner, inclusions resembling human MDBs. Aims: We hypothesized that proteomic comparison of livers from MDBprone C57BL/6 mice and MDB-resistant C3H mice, before and after DDC treatment, will illuminate the molecular mechanisms regulating susceptibility of C57BL/6 mice to MDB formation. Methods: 2-dimensional (2D) differential-in-gel electrophoresis and mass spectrometry of liver protein homogenates resulted in the identification of >200 charged isoforms with >2 fold differences between the two strains. The results for some of the proteins were validated in control and DDC-treated livers, and isolated hepatocytes using 1D/2D gel analysis followed by immunoblotting. Enzymatic activities were also measured. Results: Proteins involved in cellular homeostasis and survival, such as carbonic anhydrase 3 (CA3) and GAPDH are expressed at lower levels in C57BL/6 livers. Whereas DDC treatment strongly induces CA3 and GAPDH expression in isolated C3H hepatocytes, it has no effect in C57BL/ 6 hepatocytes. There is significantly higher expression of reactive oxygen species-generating cytochrome P450 and detoxifying glutathione-S-transferase enzymes, and lower expression of the antioxidant enzyme peroxiredoxin 6 in the C57BL/6 livers, indicating a state of elevated oxidative stress. Furthermore, the livers, but not other organs, of untreated and DDC-fed C3H mice have a 2-fold higher expression of nucleoside diphosphate kinase B (NDPK-B), a multifunctional protein involved in regulating intracellular energy pools and protection from oxidative stress. Additionally, isolated C3H hepatocytes have 6-fold higher NDPK expression and activity relative to C57BL/6 hepatocytes, indicating that differences in total liver NDPK levels are accounted for by differences in hepatocytes. Conclusions: Genetic predisposition to MDB formation is likely related, at least in part, to deficiencies in systems involved in regulating intracellular homeostasis, energy generation and protection from oxidative stress. NDPK may be an upstream effector functioning in a strain-dependent and tissue-selective manner to limit MDB formation. Similar molecular determinants might contribute to the genetic differences in human liver diseases that are associated with MDB formation.

Proteomics of rat prostate lobes treated with 2-N-hydroxylamino-1-methyl-6-phenylimidazo[4,5-b]pyridine, 5α-dihydrotestosterone, individually and in combination

Epidemiological and preclinical studies suggest that environmental factors, hormonal responses and lifestyle, including diet and physical inactivity, are likely contributors to the initiation and progression of prostate cancer in humans. Although the effects of the food derived carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and/or testosterone (T) in the development of prostate cancer in the rat have been reported, the extent to which such compounds impact cancer related proteins is not clear. Knowledge of cancer-related proteins impacted by PhIP and/or T is prerequisite to developing novel strategies to early-detect prostate cancer. Male F344 rats were sacrificed, the prostate tissue isolated and separated into dorsolateral, ventral, and anterior lobes. The lobes were cultured and treated with 10(-3) M NHPhIP and/or 10(-7) M DT for 24 h. NHPhIP is the genotoxic form of PhIP and DT is the more proliferative form of T. We used 2D-DIGE and LC/MS/MS technologies to study the proteome of the prostate lobes to determine if the compounds will trigger detectable changes in expression of cancer-related proteins. Analysis of the signals from 2D-DIGE revealed that about 10% of proteins were differentially expressed in the NHPhIP and/or DT treatments compared to controls. Eight candidate protein spots detected by 2D-DIGE in at least two out of three lobes showed > or =2-fold difference between treated and control samples. Five out of the eight spots contained single proteins; including, phospholipase Calpha (PLP-Calpha), Rab7, SAR1a, ribosomal protein S7 (RPS7), and nucleoside diphosphate kinase (NDPK). A survey of the literature shows that NDPK expression is altered in human cancers, including prostate cancer. Thus, we validated the altered expression of NDPK by Western blot analysis. The concordance between 2D-DIGE and Western blot analysis was 80%. The results of this study demonstrate, for the first time, that the combination of 2D-DIGE and LC/MS/MS is a powerful tool for identification of proteins in the prostate tissue that are altered by environmental carcinogens and/or hormones.

Activation of Heterotrimeric G Proteins by a High Energy Phosphate Transfer via Nucleoside Diphosphate Kinase (NDPK) B and Gβ Subunits: SPECIFIC ACTIVATION OF Gsα BY AN NDPK B·Gβγ COMPLEX IN H10 CELLS

Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to G protein activation in vitro. To study the effect of NDPK on G protein activity in living cells, the NDPK isoforms A and B were stably expressed in H10 cells, a cell line derived from neonatal rat cardiomyocytes. Overexpression of either NDPK isoform had no effect on cellular GTP and ATP levels, basal cAMP levels, basal adenylyl cyclase activity, and the expression of G(s)alpha and G(i)alpha proteins. However, co-expression of G(s)alpha led to an increase in cAMP synthesis that was largely enhanced by the expression of NDPK B, but not NDPK A, and that was confirmed by direct measurement of adenylyl cyclase activity. Cells expressing an inactive NDPK B mutant (H118N) exhibited a decreased cAMP formation in response to G(s)alpha. Co-immunoprecipitation studies demonstrated a complex formation of the NDPK with Gbetagamma dimers. The overexpression of NDPK B, but not its inactive mutant or NDPK A, increased the phosphorylation of Gbeta subunits. In summary, our data demonstrate a specific NDPK B-mediated activation of a G protein in intact cells, which is apparently caused by formation of NDPK B.Gbetagamma complexes and which appears to contribute to the receptor-independent activation of heterotrimeric G proteins.

Multiple Transcripts for Rat Nucleoside Diphosphate Kinase α Isoform Are Structurally Categorized into Two Groups That Exhibit Cell-specific Expression and Distinct Translation Potential

Rat nucleoside diphosphate (NDP) kinase is composed of two isoforms (alpha and beta) encoded by independent genes. The mRNAs are expressed ubiquitously; however, the level of expression is tissue-dependent and is also up- or down-regulated under certain conditions, including growth stimulation, differentiation, and tumor metastasis. To address the regulatory mechanisms of gene expression for the rat NDP kinase major isoform alpha (an nm23-H2/PuF homologue), we identified the transcription initiation sites in detail by RNase protection and 5'-rapid amplification of DNA ends and located the core promoter region by chloramphenicol acetyltransferase assay. The transcripts, initiated from an extraordinarily wide range of sites, were categorized into two groups; one transcribed from an upstream region was spliced in the untranslated region (group 1), whereas the other initiated in the downstream region was not (group 2). RNase protection demonstrated that the group 1 mRNA was the dominant form present in all tissues except heart and skeletal muscle. In situ hybridization revealed cell-specific expression of these mRNA species. Furthermore, they differed in the translational efficiency (the group 2 alpha > beta > the group 1 alpha). These findings suggest that the regulation of the NDP kinase expression at both transcriptional and posttranscriptional steps could be fundamentally governed by the selection of transcription initiation sites.

Immunohistochemical analysis of nm23-H1 in stage I non-small cell lung cancer: a useful marker in prediction of metastases

About 30% to 40% of patients with pathologic stage I non-small cell lung cancer die within 5 years after complete resection. The identification of subgroups with high risk of recurrence is important. The level of nm23-H1/nucleoside diphosphate kinase expression has been reported to correlate inversely with the metastatic potential of some tumors. We examined immunohistochemically the expression of nm23-H1 in 32 stage I non-small cell lung cancers to determine its value in predicting the outcome of the operation. Twelve (37.5%) recurrences and ten deaths occurred in this series. There were eight (25%) distant and four (12.5%) locoregional recurrences. Of the 32 pathologic stage I non-small cell lung cancers, 10 (31.3%) were positive for nm23-H1 gene product. None of the patients with a higher nm23-H1 protein level had detectable distant metastases at the end of this study (chi 2 test, p < 0.05). The median follow-up time to date is only 35 months (range, 18 to 83 months), and so far no significant difference in overall survival rate has been observed between the groups with high and low nm23-H1 protein levels. The level of nm23-H1 protein is more useful than the T status or histologic type for the prediction of distant metastases, whereby cases may be selected for postoperative adjuvant chemotherapy.

Elevated nm23 Protein Expression Is Correlated with Diminished Progression-Free Survival in Patients with Epithelial Ovarian Carcinoma

Objectives: The role of the candidate metastasis-suppressor gene nm23-H1 first characterized in breast cancer remains controversial, with both metastasis suppression and disease progression being linked to elevated nm23-H1 gene expression in different human tumor types. We sought to characterize (1) the pattern and intensity of nm23-H1/nucleoside diphosphate (NDP) kinase expression in human epithelial ovarian carcinoma (EOC) and (2) the relationship between nm23-H1/NDP kinase expression and tumor extent at diagnosis (FIGO stage) and response to treatment as defined by progression-free survival and actuarial survival. Methods: Twenty-four patients with EOC aged 61.1 ± 13.0 (mean ± SD) years were followed for 614.0 ± 289.7 days after a debulking procedure, cisplatin-based chemotherapy (19 of 24), and second-look laparotomy (9 of 24). After the primary debulking procedure, 63% of patients had no or microscopic residual disease. Overnight incubation of formalin-fixed tumor sections at 4°C with primary rabbit polyclonal IgG antibody to human nm23-H1/NDP kinase was followed by detection with standard ABC method. Nonimmune rabbit serum and normal breast tissue served as controls. Immunohistochemical staining was graded by a clinically blinded observer for intensity of staining (0, negative; 1, weak; 3, strong), pattern of staining (focal or diffuse), and histologic grade of tumor (1 through 4). Results: Of the EOCs, 54% were histologic grade 3 or 4 and 58% were FIGO stage III; 88% (21 of 24) stained positively, and 18 of 21 stained strongly and 14 of 21 stained diffusely. No correlation was found between either intensity or pattern of nm23-H1/NDP kinase immunostaining and histologic grade. No correlation was found between either staining pattern or intensity and FIGO stage. There was a trend toward decreased actuarial survival with both focal pattern (P= 0.12, log-rank test) and strong intensity (P= 0.15, log-rank test) of nm23-H1 staining. Decreased progression-free survival was likewise correlated with focal nm23-H1/NDP kinase immunostaining pattern (P= 0.02, log-rank test) and strong intensity of nm23-H1/NDP kinase staining (P= 0.08, log-rank test). Conclusions: Expression of nm23-H1/NDP kinase is strongly upregulated in most EOCs. Redundant overexpression of nm23-H1/NDP kinase may contribute to deranged cell cycle progression and EOC proliferation. Pattern and intensity of nm23-H1/NDP kinase immunostaining of EOC tissue retrieved at primary operation may identify patients at risk for tumor progression and help guide treatment strategies. These findings suggest that nm23-H1 gene expression may have distinct if not opposite biologic functions in EOC and breast carcinoma.

Decreased expression of nucleoside diphosphate kinase α isoform, annm23-H2 gene homolog, is associated with metastatic potential of rat mammary-adenocarcinoma cells

The nm23 gene [encoding nucleoside diphosphate kinase (NDPK)] may act as a metastasis suppressor in certain tumor cells. We investigated the role of NDPK isoforms (alpha and beta) in the metastatic processes, using rat mammary-adenocarcinoma cell lines of poor (MTC) and high (MTLn3) spontaneous metastatic potential respectively. In these cell lines, as in most rat tissues, the alpha isoform (nm23-H2 homolog) was more highly expressed than the beta isoform (nm23-H1 homolog) at the mRNA and protein levels. When examined by Northern- and Western-blot analyses, expression of the 2 isoforms was reduced in highly metastatic MTLn3 cells compared with poorly metastatic MTC cells. The reduced expression was also associated with diminished NDPK-enzyme activity in the cell extracts. Southern-blot and RT-PCR-SSCP analyses suggested that the 2 genes were not grossly altered or mutated in their translation regions. MTLn3 cell clones transfected with NDPKalpha or NDPKbeta cDNA were all tumorigenic when implanted into the mammary fat pad of syngeneic rats. Among those, only clones transfected with the NDPKalpha gene exhibited reduced lung metastasis in a spontaneous metastasis assay.

Nm23 gene expression and its correlation with lymph node metastasis in human lung cancer

Using labelled streptavidin-biotin (LSAB) method, we examined the expression of nucleoside diphosphate kinase(NDPK), the product of metastasis suppressor gene nm23, in human lung cancer. Of 88 patients tested, 48 (54.5%) showed positive staining. The positive staining rate was higher in adenocarcinoma (28/42, 66.7%) than in squamous cell carcinoma (20/46, 43.5%; P < 0.05). Higher incidence of positive staining was also found in squamous cell carcinoma without hilar or mediastinal lymph node metastasis (16/27, 59.3%) than in that with hilar or mediastinal lymph node involvement (4/19, 21.1%; P < 0.05). NDPK/nm23 was equally expressed in adenocarcinoma irrespective of lymph node status. In both cell types of carcinoma, expression of NDPK/nm23 was not correlated with tumor cell differentiation, nor was it correlated with the P-TNM staging. Our results suggest that NDPK/nm23 may play different roles in the pathogenesis and metastasis of human pulmonary squamous cell carcinoma and adenocarcinoma. Its expression levels are inversely correlated with lymph node metastasis in squamous cell carcinoma.

Loss of Heterozygosity and Decreased Expression of [formula omitted] Genes Correlate with Teratomatous Differentiation in Human Male Germ Cell Tumors

Embryonal carcinoma in the human male is a pluripotential germ cell tumor (GCT), which is suggested to further differentiate to teratoma which displays somatic differentiation representing all three germinal layers. In a panel of 37 GCTs we determined frequency of loss of heterozygosity (LOH) and the level of expression of nucleoside diphosphate kinase (NDPK) genes NME 1 and NME 2. The frequency of LOH in teratomas (86%) was found to be highly significant (P<0.01) compared to embryonal carcinomas (17%). We also found that the NME encoded proteins are expressed at a 4-5 fold lower level in teratomas compared to embryonal carcinomas. These findungs lead us to hypothesize that a critical level of NDPK may be necessary for suppression of aberrant somatic differentiation.

Molecular cloning, sequence determination and heterologous expression of nucleoside diphosphate kinase from Pisum sativum

Protein sequence data derived from the N-terminal region of a 17 kDa polypeptide associated with the microsomal membrane fraction from Pisum sativum was used to design degenerate oligonucleotides which were used to amplify P. sativum cDNA via the polymerase chain reaction (PCR). Amplified cDNA was used as a probe to screen a P. sativum cDNA library and a cDNA clone, NDK-P1 was isolated and sequenced. The protein encoded by NDK-P1 had a calculated molecular mass of 16,485 Da and possessed substantial homology with nucleoside diphosphate kinases (NDKs) isolated and cloned from other sources. High levels of expression of NDK-P1 protein were achieved in Escherichia coli using a T7-driven expression system. Recombinant NDK-P1 protein was shown to possess NDK activity and had similar biochemical characteristics to NDKs isolated from other sources. The Michaelis constants for a variety of nucleoside diphosphate (NDP) substrates were found to be broadly similar to those reported for other NDKs, with thymidine nucleotides being the substrates of greatest affinity.

Clinicopathological features and prognostic significance of nucleoside diphosphate kinase/nm23 gene product in human pancreatic exocrine neoplasms.

Expression levels of nucleoside diphosphate (NDP) kinase/nm23 gene product in the surgically resected 59 human pancreatic exocrine neoplasms were examined immunohistochemically using antiNDP kinase antibody. Immunoreactivity for NDP kinase varied from one tumor to the other. Out of the 47 invasive pancreatic duct cell carcinomas examined, 31 (66%) tumors showed strong immunoreactivity for NDP kinase, whereas ten (83%) of 12 benign or less invasive tumors (in situ adenocarcinoma and mucin-producing tumor) showed negative or weak immunoreactivity (p < 0.01; Chi-square test). Overall survival of invasive pancreatic duct cell carcinomas with strong immunoreactivity for NDP kinase was poorer than those with negative or weak immunoreactivity (p < 0.03; generalized Wilcoxon test). Strong immunoreactivity for NDP kinase was associated with the type of histological differentiation, the presence of lymph node metastases (p < 0.05, respectively; Chi-square test), and the number of argyrophilic nucleolar organizer regions (p < 0.01; Student's t-test). These results suggest that NDP kinase/nm23 gene product expression was positively associated with tumor aggressiveness and poor survival of patients in human pancreatic exocrine neoplasms. They also suggest that NDP kinase expression is related to cell proliferation activity represented by the number of argyrophilic nucleolar organizer regions. Therefore, examining the level of NDP kinase/nm23 gene product could serve as a marker for malignant potentiality of pancreatic exocrine neoplasms.

Nucleoside diphosphate kinase expression in human skin

Nucleoside diphosphate kinase expression in human skin

Immunohistochemical analysis of expression of nm23-H1/nucleoside diphosphate kinase in human thyroid carcinomas: lack of correlation between its expression and lymph node metastasis.

The nm23 gene that encodes nucleoside diphosphate (NDP) kinase has been proposed as a candidate tumor metastasis suppressor in rodent experimental carcinoma models and several types of human carcinomas. The present studies were designed to investigate, by immunohistochemical analysis, whether thyroid tissues express the nm23-H1/NDP kinase and, if so, whether the nm23-H1/NDP kinase expression is correlated to tumor metastasis suppressor potential in thyroid tumors. We found that normal thyroid epithelial cells were stained weakly but homogeneously by mouse monoclonal anti-nm23-H1/NDP kinase antibody. The staining intensity of the nm23-H1/NDP kinase in benign thyroid tumors tended to be weaker than that in normal thyroid tissues. In contrast, a series of malignant thyroid tumors expressed differently the nm23-H1/NDP kinase: the intensity of the nm23-H1/NDP kinase staining was stronger in 22 of 49 malignant tumors (45%), similar in 24 (49%), and weaker in 3 (6%) compared to that in normal tissues. The comparison of the staining intensity of the nm23-H1/NDP kinase in primary lesions of tumors with lymph node metastases and those without metastases revealed no statistically significant difference. In addition, there was also no significant difference in the nm23-H1/NDP kinase staining intensity of primary and metastatic lymph node lesions of tumors with lymph node metastases. These data indicate that the nm23-H1/NDP kinase may not be a good predictive marker for tumor regional metastatic potential in malignant thyroid tumors. We conclude that in thyroid tumors the nm23-H1/NDP kinase expression may be dissociated from metastasis suppressor activity.

Expression in human hepatocellular carcinoma of nucleoside diphosphate kinase, a homologue of the nm23 gene product.

Expression of nucleoside diphosphate (NDP) kinase, which is highly homologous to the nm23 gene product in a variety of species, has been found to be inversely associated with metastatic potential in human breast cancer. The present study was conducted to clarify the association of NDP kinase expression with metastatic potential in human hepatocellular carcinoma. The immunohistochemical expression of NDP kinase was analyzed in 30 patients with histopathologically proven hepatocellular carcinoma. These patients included nine with distant metastases and 21 without distant metastases. Tissue specimens were reacted with rabbit anti-rat NDP kinase antibody and stained by the biotin-streptavidin complex method. The relative staining intensities were evaluated by comparing primary tumor sites with adjacent nontumorous liver tissue or with metastatic sites, The expression of NDP kinase in primary sites in patients with distant metastases was significantly less intense than that in patients without distant metastases (P = .018). NDP kinase was expressed significantly less intensely in metastatic sites than in primary sites (P = .005). The intensity of NDP kinase expression did not statistically correlate with tumor size or number of lesions in the liver, histopathological classification of tumor, associated liver diseases, hepatitis virus markers, or tumor markers. These results suggest that the reduced expression of NDP kinase is closely associated with distant metastatic potential in hepatocellular carcinoma. It is possible that both NDP kinase and the nm23 gene product may be active in the progression and differentiation of tumor cells and that their reduced expression induces a high metastatic potential in tumor cells. Studies using Northern blotting or in situ hybridization should be planned to confirm our findings.

Regulatory GTP-binding proteins (ADP-ribosylation factor, Gt, and RAS) are not activated directly by nucleoside diphosphate kinase.

The expression of nucleoside diphosphate kinase (NDK) genes has been implicated as a negative regulator of murine and human tumor metastases and is critical to proper development in Drosophila melanogaster. Molecular mechanisms for the role(s) of NDK in these complex processes have not yet been elucidated, but several reports have suggested that these and many other signal transduction pathways may be activated by NDK acting directly on a regulatory GTP-binding protein(s). To test this hypothesis, we examined the ability of NDK to catalyze the phosphorylation of the GDP bound to the following three members of the superfamily of regulatory GTP-binding proteins: Gt, Ha-ras p21, and ARF. We have found no evidence to support the hypothesis that NDK can directly activate any GTP-binding protein. Rather, evidence is presented which clearly shows that all of the GTP formed upon incubation of GTP-binding proteins with NDK is the result of NDK utilizing free GDP as substrate. The GDP bound to the regulatory proteins is not a substrate for NDK under conditions in which free nucleotides are rapidly and efficiently phosphorylated. The importance of appropriate controls for dissociation of GDP from the regulatory proteins both during the NDK reaction and during the analysis of product is demonstrated. We believe there is currently no experimental evidence to support the hypothesis that NDK can directly activate a regulatory GTP-binding protein.