Determination of 6-thioguanosine diphosphate and triphosphate and nucleoside diphosphate kinase activity in erythrocytes: novel targets for thiopurine therapy?

6-Thioguanine nucleotides are the sum of 6-thioguanosine 5'-monophosphate (TGMP), -diphosphate (TGDP), and -triphosphate (TGTP) representing essential metabolites involved in drug action of thiopurines. Elevated levels of TGDP have been associated with poor response to azathioprine therapy in patients with inflammatory bowel disease. The conversion of TGDP to TGTP is supposed to be catalyzed by nucleoside diphosphate kinase (NDPK). The aim of this work was to investigate simultaneously individual 6-thioguanosine phosphate levels and NDPK activity in red blood cells (RBCs) of patients on azathioprine therapy. Ion-pair high-performance liquid chromatography methods with fluorescence and ultraviolet detection were applied to quantify individual levels of 6-thioguanosine 5'-phosphates and NDPK activity, respectively, in RBCs. Recombinantly expressed NDPK isoforms A and B were unequivocally identified to catalyze the formation of TGTP (30.6 +/- 3.88 nmol x min x mg for NDPK A versus 41.2 +/- 1.05 nmol x min x mg for NDPK B). Comprehensive analyses on the stability of TGMP, TGDP, and TGTP and the reproducibility of NDPK activity in RBCs were performed to provide a reliable sampling protocol for clinical practice. Of note, isolation of RBCs within 6 hours followed by immediate storage at -80 degrees C is crucial for prevention of degradation of 5'-phosphates. In a clinical study of 37 patients on azathioprine, TGTP was the predominant 6-thioguanosine phosphate in RBCs. In contrast, three patients showed TGTP/(TGDP + TGTP) ratios of 57.2%, 64.3%, and 66% corresponding to elevated TGDP levels. NDPK activity ranged from 4.1 to 11.3 nmol x min x mg hemoglobin. No correlation between NDPK activity and the 6-thioguanosine phosphate levels was found. The question whether interindividual variability of NDPK activity may explain differences in 6-thioguanosine 5'-phosphates levels has to be investigated in a prospective large-scale study.

Phase II study of the CPT-11, mitoxantrone and dexamethasone regimen in combination with rituximab in elderly patients with relapsed diffuse large B-cell lymphoma.

Standard treatment for elderly patients with relapsed or refractory DLBCL has not been established. CPT-11 has a broad spectrum of anticancer activities including a cytotoxic effect in a variety of malignant tumors. The results of combined treatment with CPT-11 and rituximab have not been reported. The R-CMD regimen was given to elderly patients with relapsed or refractory DLBCL. The safety and efficacy of this regimen were studied. In addition, the serum nm23-H1 level was determined to study whether or not it can serve as a prognostic factor. Thirty elderly patients with DLBCL were studied. The main non-hematological toxicities were infusion-related adverse events. Grade 3/4 hematological toxicity was seen in 19 patients. Following R-CMD treatment, the BNP and troponin T levels did not increase. The CR rate was 57%, PR rate was 17%, 2-year survival rate was 45.2%, and PFS rate was 37.2%. Patients with serum nm23-H1 levels of higher than 80 ng/mL before the treatment showed significantly poorer prognosis. The serum nm23-H1 level of the 30 subjects before the treatment was elevated at 39.4 +/- 41.3 ng/mL, but it significantly decreased only in the subset of patients who achieved CR. The R-CMD regimen was safe in elderly patients with DLBCL. No new signs of cardiotoxicity were observed with this regimen. It was also effective in patients with relapsed or refractory DLBCL who had previously used DXR.

Mass spectrometric identification of human prostate cancer-derived proteins in serum of xenograft-bearing mice.

Lack of sensitivity and specificity of current tumor markers has intensified research efforts to find new biomarkers. The identification of potential tumor markers in human body fluids is hampered by large variability and complexity of both control and patient samples, laborious biochemical analyses, and the fact that the identified proteins are unlikely produced by the diseased cells but are due to secondary body defense mechanisms. In a new approach presented here, we eliminate these problems by performing proteomic analysis in a prostate cancer xenograft model in which human prostate cancer cells form a tumor in an immune-incompetent nude mouse. Using this concept, proteins present in mouse serum that can be identified as human will, by definition, originate from the human prostate cancer xenograft and might have potential diagnostic and prognostic value. Using one-dimensional gel electrophoresis, liquid chromatography, and mass spectrometry, we identified tumor-derived human nm23/nucleoside-diphosphate kinase (NME) in the serum of a nude mouse bearing the androgen-independent human prostate cancer xenograft PC339. NME is known to be involved in the metastatic potential of several tumor cells, including prostate cancer cells. Furthermore we identified six human enzymes involved in glycolysis (fructose-bisphosphate aldolase A, triose-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, alpha enolase, and lactate dehydrogenases A and B) in the serum of the tumor-bearing mice. The presence of human NME and glyceraldehyde-3-phosphate dehydrogenase in the serum of PC339-bearing mice was confirmed by Western blotting. Although the putative usefulness of these proteins in predicting prognosis of prostate cancer remains to be determined, the present data illustrate that our approach is a promising tool for the focused discovery of new prostate cancer biomarkers.

Clinical significance of serum NM23-H1 protein in neuroblastoma.

We have previously reported that NM23 genes are overexpressed in various hematological malignancies and that serum NM23-H1 protein levels are useful for predicting patient outcomes. In this study we assessed the clinical implications of serum NM23-H1 protein on neuroblastoma. We examined serum NM23-H1 protein levels in 217 patients with neuroblastoma, including 131 found by mass-screening and 86 found clinically by an enzyme-linked immunosorbent assay, and determined the association between levels of this protein, and known prognostic factors or the clinical outcome. The serum NM23-H1 protein level was higher in neuroblastoma patients than in control children (P < 0.0001). Patients with MYCN amplification had higher serum NM23-H1 levels than those with a single copy of MYCN. Overall survival was assessed in the 86 patients found clinically, and was found to be worse in patients with higher serum MN23-H1 levels (> or = 250 ng/mL) than in those with lower levels (< 250 ng/mL; P = 0.034). The higher level of NM23-H1 was correlated with a worse outcome in patients with a single MYCN copy, or in those younger than 12 months of age. Serum NM23-H1 protein levels may contribute to predictions of clinical outcome in patients with neuroblastoma.

PARK7 and nucleoside diphosphate kinase A as plasma markers for the early diagnosis of stroke.

BACKGROUND: Plasma markers for stroke could be useful in diagnosis and prognosis and in prediction of response of stroke patients to therapy. PARK7 and nucleoside diphosphate kinase A (NDKA) are increased in human postmortem cerebrospinal fluid (CSF), a model of global brain insult, suggesting that measurement in CSF and, more importantly, in plasma may be useful as a biomarker of stroke. METHODS: We used ELISA to measure PARK7 and NDKA in plasma in 3 independent European and North American retrospective studies encompassing a total of 622 stroke patients and 165 control individuals. RESULTS: Increases in both biomarkers were highly significant, with sensitivities of 54%-91% for PARK7 and 70%-90% for NDKA and specificities of 80%-97% for PARK7 and 90%-97% for NDKA. The concentrations of both biomarkers increased within 3 h of stroke onset. CONCLUSIONS: PARK7 and NDKA may be useful plasma biomarkers for the early diagnosis of stroke. In addition, this study demonstrated the utility of analysis of postmortem CSF proteins as a first step in the discovery of plasma markers of ischemic brain injury.

Medroxyprogesterone acetate elevation of Nm23-H1 metastasis suppressor expression in hormone receptor-negative breast cancer.

BACKGROUND: Reestablishment of metastasis suppressor gene expression may constitute a therapeutic strategy for high-risk breast cancer patients. We previously showed that medroxyprogesterone acetate (MPA), a progestin that has been tested as treatment for advanced breast cancer, elevates expression of the Nm23-H1 metastasis suppressor gene in hormone receptor-negative metastatic human breast carcinoma cell lines in vitro via a glucocorticoid receptor-based mechanism. Here, we tested whether MPA treatment inhibits metastatic colonization of a hormone receptor-negative breast cancer cell line in vivo. METHODS: We tested the soft-agar colony-forming efficiency of untransfected MDA-MB-231T human breast carcinoma cells and MDA-MB-231T cells transfected with antisense Nm23-H1 in the presence and absence of MPA. Pharmacokinetic studies were used to establish dose and injection schedules that led to MPA serum levels in mice similar to those achievable in humans. For in vivo studies, nude mice were injected intravenously with MDA-MB-231T cells. After 4 weeks, mice were randomized to control or MPA arms. Endpoints included incidence, number, and size of gross pulmonary metastases; Nm23-H1 protein expression in gross metastases; and side effects. All statistical tests were two-sided. RESULTS: MPA reduced colony formation of MDA-MB-231T cells by 40%-50% but had no effect on colony formation of Nm23-H1 antisense transfectants. Metastases developed in 100% (95% confidence interval [CI] = 78% to 100% and 77% to 100%, respectively) of control mice injected with MDA-MB-231T cells. In two independent experiments, only 73% (95% CI = 45% to 92%) and 64% (95% CI = 35% to 87%) of mice injected with 2 mg of MPA developed metastases. Mice injected with 2 mg of MPA showed reductions in the mean numbers, per mouse, of all metastases and of large (>3 mm) metastases (P = .04 and .013, respectively). Nm23-H1 was expressed at high levels in 43% of pulmonary metastases in MPA-treated mice but only 13% of metastases in untreated mice. Mice receiving at least 1-mg doses of MPA gained more weight than control-treated mice but exhibited no bone density alterations or abnormal mammary fat pad histology. CONCLUSION: Our preclinical results show that MPA appears to elevate Nm23-H1 metastasis suppressor gene expression, thereby reducing metastatic colonization. The data suggest a new use for an old agent in a molecularly defined subset of breast cancer patients.

A monoclonal antibody driven biodiagnostic system for the quantitative screening of breast cancer.

A prospective clinical parametric study comprising women afflicted by breast cancer and otherwise healthy participants was undertaken. The mean plasmatic concentration of putative leucine amino peptidase and nucleoside diphosphate phosphotransferase enzymatic complex in breast cancer cases was significantly elevated [43.9 +/- 2.8 microg ml(-1) (n = 9)] when compared to those found in otherwise healthy women [8.07 +/- 0.14 microg ml(-1) (n = 8)]. Women without images compatible with any tumours (n = 13) had a mean concentration of 10.77 +/- 1.49 microg ml(-1). The mean value obtained in women with fibroadenomas was 10.15 +/- 0.81 microg ml(-1) (n = 6) and with cystic fibrosis mastopathy 8.75 +/- 0.28 microg ml(-1) (n = 7). The efficacy of a tandem quantitative biodiagnostic system as a parametric screening tool for the early detection of breast cancer is underlined, raising the possibility of increasing the cost effectiveness of current imaging non-parametric technologies.

Soluble purine-converting enzymes circulate in human blood and regulate extracellular ATP level via counteracting pyrophosphatase and phosphotransfer reactions.

Extracellular ATP and other purines play a crucial role in the vasculature, and their turnover is selectively governed by a network of ectoenzymes expressed both on endothelial and hematopoietic cells. By studying the whole pattern of purine metabolism in human serum, we revealed the existence of soluble enzymes capable of both inactivating and transphosphorylating circulating purines. Evidence for this was obtained by using independent assays, including chromatographic analyses with 3H-labeled and unlabeled nucleotides and adenosine, direct transfer of gamma-terminal phosphate from [gamma-32P]ATP to NDP/AMP, and bioluminescent measurement of ATP metabolism. Based on substrate-specificity and competitive studies, we identified three purine-inactivating enzymes in human serum, nucleotide pyrophosphatase (EC 3.6.1.9), 5'-nucleotidase (EC 3.1.3.5), and adenosine deaminase (EC 3.5.4.4), whereas an opposite ATP-generating pathway is represented by adenylate kinase (EC 2.7.4.3) and NDP kinase (EC 2.7.4.6). Comparative kinetic analysis revealed that the Vmax values for soluble nucleotide kinases significantly exceed those of counteracting nucleotidases, whereas the apparent Km values for serum enzymes were fairly comparable and varied within a range of 40-70 micro mol/l. Identification of soluble enzymes contributing, along with membrane-bound ectoenzymes, to the active cycling between circulating ATP and other purines provides a novel insight into the regulatory mechanisms of purine homeostasis in the blood.

Decrease in nucleoside diphosphate kinase (NDPK/nm23) expression during hematopoietic maturation.

The nucleoside diphosphate kinase (NDPK/nm23) isoforms H1 and H2 were localized in hematopoietic tissues. Flow cytometric analysis and enzymatic assays were used to quantify the intracellular and extracellular concentrations of NDPK. Bone marrow CD34(+) progenitors contained the highest intracellular levels of both nm23-H1 and nm23-H2. Lower levels were measured in more mature bone marrow cells, whereas peripheral blood leukocytes had the lowest expression of nm23. These data suggest a function of NDPK in early hematopoiesis and a down-regulation of NDPK upon differentiation. In addition, an up-regulation of nm23 expression was observed in lymphocytes after induction of proliferation with phytohemagglutinin. Multiparameter flow cytometry demonstrated that this up-regulation occurred during the G0/G1-transition. Flow cytometric analysis also revealed a weak surface expression of nm23 on a number of hematopoietic cell lines, which was not detected on normal hematopoietic cells. Our data also demonstrated the presence of NDPK in human plasma, probably due to a limited in vivo lysis of red blood cells.

Rapid purification and characterization of nucleoside diphosphate kinase isoforms using ATP-sepharose affinity column chromatography.

Nucleoside diphosphate kinases (NDP kinases), products of the nm23 gene, catalyze the transfer of the terminal phosphate group of the nucleoside triphosphate to the corresponding diphosphate and may be involved in tumor metastasis suppression, development, and signal transduction. NDP kinase from various sources including human erythrocytes, rat brain tissue and E. coli strain BL21 transformed with pET3C expression plasmids containing nm23-H1 or nm23-H2, were purified in one step to homogeneity using ATP-sepharose affinity column chromatography. This method was applicable for the purification of various NDP kinases which show the same enzymatic activity and immunodetection, but have various molecular weight and quaternary structures.

The nucleoside diphosphate kinase of human neutrophils.

Nucleoside diphosphate kinase (NDP kinase) catalyses the phosphate transfer between nucleoside triphosphates and nucleoside diphosphates. As formation of guanosine triphosphate could be dependent on ATP in neutrophils, the presence of NDP kinase was tested in these phagocytic cells. Both membrane and cytosolic fractions of human neutrophils were found to contain NDP kinase activity. The specific activity measured in the cytosol appeared 10-fold higher than in the membrane and was not modified when the cells were activated with phorbol 12-myristate 13-acetate. Interestingly, stimulation with N-formylmethionyl leucylphenylalanine in the presence of cytochalasin B showed an increase in membrane NDP kinase activity together with the translocation of the enzyme from the cytosol to the membrane, suggesting a possible role of NDP kinase in regulating G-proteins as previously reported. In addition, activation with opsonized zymosan induced an increase in cytosolic activity, suggesting different regulation depending on the signal transduction pathway. The neutrophil enzyme consisted of two subunits of 21 kDa (NDPKA) and 18 kDa (NDPKB) again essentially present in the cytosol of the cell. Separation of proteins by two-dimensional PAGE demonstrated that each subunit consisted of at least four isoforms, indicating post translational modifications. A characteristic of this family of enzymes is the stability of the phosphorylated intermediate. In neutrophils, only one acidic isoform of each NDPKA and NDPKB was labelled in the presence of EDTA. In addition, non-denatured complexes were apparent between 91 and 130 kDa, suggesting a hexameric structure as was also proposed for NDP kinases from other eukaryotic cells. These complexes were found to differ in their isoelectric points, indicating the existence of various isoenzymes probably resulting from combination between several isoforms of each subunit.

Expression of the nucleoside diphosphate kinase in human skin cancers: an immunohistochemical study.

Expression of nucleoside diphosphate(NDP) kinase, which is homologous to the nm23 gene product in a variety of species, has been found to be inversely associated with metastatic potential. However, the relationship remains controversial according to the tumor cell types and experimental system, with conflicting results from different research groups. In order to determine whether NDP kinase expression serves as a marker for metastatic potential in human skin cancer, we assessed the levels of NDP kinase expression in 9 keratoacanthomas (KAs), 26 squamous cell carcinomas (SCCs), and 25 basal cell carcinomas (BCCs) using immunohistochemistry. The expression of NDP kinase was intense in KA and SCC compared with BCC. However, the difference of NDP kinase expression between KA and SCC was not statistically significant. And there was no statistically significant difference in NDP kinase expression between SCC with metastasis and SCC without metastasis. Our results contradict the hypothesis concerning the possible role of nm23 gene as a metastatic suppressor gene in human skin cancer. The mechanism of overexpression in various tumor cell types and its biological significance in cutaneous carcinogenesis remain to be determined.

Nucleoside diphosphate kinase from human erythrocytes. Structural characterization of the two polypeptide chains responsible for heterogeneity of the hexameric enzyme.

Human erythrocyte nucleoside-diphosphate kinase (NDP kinase) is a hexameric enzyme consisting of two kinds of polypeptide chains, A and B. By random association (A6, A5B...AB5, B6) these polypeptides form isoenzymes differing in their isoelectric point. Chains A and B of NDP kinase were purified by ion-exchange chromatography under denaturing conditions. Upon mixing and renaturation, the isozymic pattern of NDP kinase obtained by conventional methods was restored. Antibodies raised against purified chains showed significant cross-reactivity, both in immunoblot experiments and activity inhibition studies. Sequence determination showed that both chains consisted of 152 amino acid residues corresponding to Mr or 17,143 (chain A) and 17,294 (chain B), respectively. There was high homology between the two sequences (88% identity). The phosphorylation site on the enzyme is located at His-118. Chain A was identical with human Nm23 protein, which has been reported as a potential suppressor protein in tumor metastasis and chain B was identical with Nm23-H2 protein.

Permeabilization of platelets: an investigation of biochemical, ultrastructural and functional aspects.

The biochemical, ultrastructural and functional aspects of digitonin-permeabilized platelets were investigated. Human platelets were permeabilized by exposure to the steroid glycoside digitonin. A 60 microM concentration of this permeabilizer produced a very substantial release of cytosolic enzymes from the platelets. Release from subcellular granules was relatively low and did not inhibit the response of platelets to a series of agonists. Although digitonin-permeabilized platelets required higher threshold concentrations of the usual stimulants, both primary and secondary aggregation as well as the release of nucleotides and enzymes from their respective granules remained intact. Transmission electron micrographs revealed discontinuities in the plasma membrane of digitonin-treated platelets, but scanning electron microscopy showed no difference between control and permeabilized platelets. No substantial loss of structural or membrane proteins could be detected by one- and two-dimensional gel electrophoresis. The pore size produced by digitonin treatment was sufficient to allow entry of 125I-labeled IgG into the platelet cytosolic space.

Isolation and kinetic studies of nucleoside diphosphokinase from human platelets and effects of cAMP phosphodiesterase inhibitors.

Nucleoside diphosphokinase (NDK) of human platelets has been purified by chromatography on Blue Sepharose CL-6B gel (purification factor of 950) and shown to be free of adenylate kinase, ATPase and adenylate cyclase. The molecular weight was 70,000 with subunits of 17,000. The pH optimum was 8.0 Km values for ATP and dTDP were determined in two ways using the pyruvate kinase-lactate dehydrogenase coupled enzyme assay. Values of 0.38 and 0.20 mM were obtained for ATP and 0.29 and 0.21 mM for dTDP. Km values for ADP (0.024 mM) and GTP (0.12 mM) were determined with the hexokinase-glucose-6-phosphate dehydrogenase coupled enzyme assay. These values are in agreement with those reported for NDK from other sources. Theophylline, which inhibits the NDK activity of intact platelets and platelet membrane preparations and inhibits the ADP-induced shape change of platelets, was shown to be a competitive inhibitor of both the free and phosphorylated forms of NDK with competitive inhibition constants (Kic) of 9.3 and 9.6 mM respectively. Papaverine, another cAMP phosphodiesterase inhibitor, which also inhibits the ADP-induced shape change of platelets, had no inhibitory effect on platelet NDK. It was concluded that the inhibitory effect of theophylline on the activity of the purified enzyme was due to the structural similarity between the methylxanthine and the adenine moiety of ADP.

Effect of cAMP phosphodiesterase inhibitors on ADP-induced shape change, cAMP and nucleoside diphosphokinase activity of rabbit platelets.

The effects of cAMP phosphodiesterase inhibitors on ADP-induced shape change and cAMP concentrations have been studied. Caffeine (10 mM), theophylline (8 mM), dipyridamole (0.2 mM), or papaverine (0,05 mM) prevented the shape change of washed rabbit platelets induced by 0.4 microM ADP. At these concentrations, none of these cAMP phosphodiesterase inhibitors increased 14C-cAMP in platelets in which the cytoplasmic adenine nucleotides had been labelled with 14C-adenine. By a protein binding assay, only papaverine by itself increased platelet cAMP above its basal level. These results indicate that two pools of cAMP may exist in platelets. Both methods showed that stimulation of platelet adenylate cyclase with PGE1 (1 microM) resulted in an increase in platelet cAMP and all these cAMP phosphodiesterase inhibitors potentiated this increase caused by PGE1. By themselves, some of these compounds may act through mechanisms that do not involve platelet cAMP. The effects of these cAMP phosphodiesterase inhibitors on platelet nucleoside diphosphokinase (NDK) activity were also investigated. At concentrations that prevented ADP-induced shape change, papaverine and dipyridamole had no effect on the formation of 14C-ATP from 14C-ADP by washed rabbit platelets. The methylxanthines partially inhibited NDK activity of washed rabbit platelets and of isolated platelet membranes, probably due to the structural similarity between the adenine ring of ADP and these substances. However, adenine (8 mM) inhibited ADP-induced shape change and platelet NDK activity but was a less effective inhibitor of ADP-induced platelet aggregation. Thus it seems unlikely that interference with platelet NDK or the ADP receptor is the major mechanism by which the methylxanthines inhibit platelet functions.

Binding of adenosine diphosphate to human blood platelets and to isolated blood platelet membranes.

The equilibrium binding of 14C-labeled ADP to intact washed human blood platelets and to platelet membranes was investigated. With both intact platelets and platelet membranes a similar concentration dependence curve was found. It consisted of a curvilinear part below 20 microM and a rectilinear part above this concentration. At high ADP concentrations, the rectilinear part appeared to be saturable. Because of this, two classes of saturable ADP binding sites were proposed. ADP was partly converted to ATP and AMP with intact platelets while this conversion was virtually absent in isolated platelet membranes. ADP was bound to platelet membranes with the same type of curves found for intact platelets. The ADP binding to the high affinity system, which was stimulated by calcium ions, was nearly independent of temperature and had a pH optimum at 7.8. A number of agents were investigated for inhibiting properties. Of the sulfhydryl reagents only p-chloromercuribenzene sulfonate inhibited both high and low affinity binding systems while iodoacetamide and N-ethylmaleimide were without effect. Compounds acting via cyclic AMP on platelet aggregation, such as adenosine and cyclic AMP itself, had no influence on binding. Some nucleosidediphosphates and nucleotide analogs at a concentration of 100 microM had no, or only a slight, effect on high affinity ADP binding. For some other nucleotides inhibitor constants were determined for both platelet ADP aggregation and ADP binding. The inhibitor constants of ATP, adenyl-5'-yl-(beta,gamma-methylene)diphosphate, IDP, adenosine-5'(2-O-thio)diphosphate, for aggregation and high affinity binding were in good correlation with each other. Exceptions formed fluorosulfonylbenzoyl adenosine and AMP. The ATP formation found with intact platelets could be attributed to a nucleosidediphosphate kinase. It was investigated in some detail. The enzyme was magnesium dependent, had a Q10 value of 1.41, a pH optimum at 8.0, was competitively inhibited by AMP and reacted via a ping pong mechanism. All findings described in this paper indicate that platelets as well as platelet membranes bind ADP with the same characteristics and they suggest that the high affinity binding of ADP is involved in platelet aggregation induced by ADP. The results on nucleosidediphosphate kinase did not permit a firm conclusion about the role of the enzyme in induction of platelet aggregation by ADP.