NDP kinase 7 is a conserved microtubule-binding protein preferentially expressed in ciliated cells.

Nucleoside diphosphate (NDP) kinase is an enzyme that synthesizes the nucleoside triphosphates. In mammals, nine sequences (NDK1-NDK9) have been found with domain(s) homologous to the catalytic domain of NDP kinase, and some of them have been shown to associate with sperm flagella. The present study examines the localization of NDK7, for which little information has been available. Database analysis showed that the NDK7 gene is present in organisms with cilia and flagella. Western blotting analyses of various mouse tissues consistently indicated that NDK7 is preferentially expressed in tissues with motile cilia as well as in sperm. Immunofluorescence microscopy revealed that this protein is localized along the entire length of the TritonX-100-insoluble fraction of sperm flagella, possibly in the axonemes. Unexpectedly, however, NDK7 in tracheal epithelia was found in the cell body but not in cilia. Finally, in vitro co-sedimentation assays using recombinant proteins showed that both mouse and Chlamydomonas NDK7 directly bind to microtubules.

Accumulation and effects of nickel and thallium in early-life stages of fathead minnows (Pimephales promelas).

Early-life stages of fathead minnows were exposed to environmentally relevant concentrations of aqueous and dietary nickel and thallium and metal accumulation was monitored from the embryo until the larvae reached 21 days after hatching. During and after metal exposure, 6 toxicity endpoints were measured: time to hatch, embryo survival rate, routine metabolic rate and the activity of key enzymes (lactate dehydrogenase, nucleoside diphosphate kinase (NDPK), cytochrome C oxidase (CCO)). Although both Ni and Tl bioaccumulation were significant in embryos and non-feeding larvae, water was the major source of Ni and Tl in feeding larvae. Exposure to aqueous Ni decreased time to hatch and increased aerobic and biosynthetic capacities (as indicated by a higher activity of CCO and NDPK, respectively), suggesting that aqueous Ni exposure stimulates metabolism in early-life stages of fathead minnows.

Direct binding of cytosolic NDP kinases to membrane lipids is regulated by nucleotides.

In spite of their complete lack of any structural features that characterize membrane proteins, cytosolic nucleoside-diphosphate kinases (NDPKs) have been found repeatedly to associate with membranes. In some instances the recruitment of cytosolic NDPKs to membranes was attributed to interactions with peripheral or integral membrane proteins, but in many cases the mechanism underlying the association of NDPKs with membranes remained unknown. We show here that cytosolic NDPKs bind directly to membrane lipids in a dynamic process that is controlled by its substrates, nucleoside tri- and diphosphates, and can be fully reconstituted with chemically defined, protein-free phospholipids and recombinant NDPK, or with purified NDPK. Our results uncover a novel mechanism for the reversible targeting of soluble NDPKs to membranes, where they may act as a reservoir of high energy phosphate, supporting the operation of membrane-based processes that utilize nucleotides other than ATP, such as intracellular traffic and phospholipid biosynthesis.

Measurement of human breast tumor cell-secreted shNDPK-B in a murine breast cancer model suggests its role in metastatic progression.

Human breast cancers metastasize early in tumorigenesis and distant lesions, though dormant are very likely extant at the time of diagnosis and treatment in the majority of cases. Removal of primary tumors by surgeons as an imperative of the current treatment approach, also removes inhibitory factors secreted by the primary tumor that had maintained the dormancy of the metastases. We have identified a factor secreted by human breast cancer cells that supports the formation of blood vessels and may be a principal early factor supporting the growth and development of metastases in human disease. Here we demonstrate for the first time that this factor, secreted (s) human (h) nucleoside diphosphate kinase type B (shNDPK-B), product of the nm23-h2 gene, can be detected specifically with high sensitivity (50 pg/ml; 2.5 pM) in an ELISA assay of our own design. We further demonstrate that shNDPK-B is released into the circulation in immunocompromized mice carrying the human breast carcinoma cell MDA-MB-231. These data support the hypothesis that shNDPK-B may be responsible for the early events in angiogenesis supporting both primary and metastatic tumor growth and development.

Trypanosoma cruzi: multiple nucleoside diphosphate kinase isoforms in a single cell.

Nucleoside diphosphate kinases (NDPKs) are multifunctional enzymes involved mainly in the conservation of nucleotides and deoxynucleotides at intracellular levels. Here we report the characterization of two NDPKs from the protozoan parasite Trypanosoma cruzi, the etiological agent of Chagas disease. TcNDPK1 and TcNDPK2 were biochemically characterized presenting different kinetic parameters and regulation mechanisms. NDPK activity was mainly detected in soluble fractions according to the digitonin extraction technique; however 20% of the activity remains insoluble at digitonin concentrations up to 5 mg ml(-1). TcNDPK1 is a short enzyme isoform, whereas TcNDPK2 is a long one containing a DM10 motif. In addition, two other putative NDPK genes (TcNPDK3 and TcNDPK4) were detected by data mining at the T. cruzi genome database. The large number and diversity of NDPK isoforms are in agreement with those previously observed for other T. cruzi phosphotransferases, such as adenylate kinases.

Proteomic analysis of Trypanosoma cruzi resistance to Benznidazole.

The first proteomic analysis of Trypanosoma cruzi resistance to Benznidazole (BZ) is presented. The differential proteome of T. cruzi with selected in vivo resistance to Benznidazole (BZR and Clone27R), its susceptible pairs (BZS and Clone9S), and a pair from a population with Benznidazole- in vitro-induced resistance (17LER) and the susceptible pair 17WTS were analyzed by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS) for protein identification. Out of 137 spots analyzed through MS, 110 were identified as 56 distinct proteins. Out of the 56 distinct proteins, 36 were present in resistant, 9 in susceptible, and 11 in both phenotypes. Among the proteins identified in resistant samples, 5 were found in Cl 27R and in BZR (calpain-like cysteine peptidase, hypothetical protein conserved 26 kDa, putative peptidase, peroxiredoxin and tyrosine amino transferase) and 4 in Cl 27R and 17LER (cyclophilin A, glutamate dehydrogenase, iron superoxide dismutase and nucleoside diphosphate kinase). As for the proteins identified in Benznidazole-susceptible samples, PGF-2a was found in BZS and 17WTS. A functional category analysis showed that the proteins involved with transcription and protein destination were overexpressed for the Benznidazole-resistant phenotype. Thus, the present study provides large-scale, protein-related information for investigation of the mechanism of T. cruzi resistance to Benznidazole.

Reduced protein expression of metastasis-related genes (nm23, KISS1, KAI1 and p53) in lymph node and liver metastases of gastric cancer.

PURPOSE: Metastasis remains an incurable common complication in patients with gastric cancer. A variety of theories have been proposed to explain the inefficiency of the metastatic process. To compare protein expression of metastasis-related genes (nm23, KISS1, KAI1 and p53) between primary tumours and metastatic tumours may be useful in illustrating these theories. METHODS: Metastasis-related tissue microarrays (including normal tissues, primary tumours, nodal metastases and liver metastases) were constructed. The protein expression of nm23, KISS1, KAI1 and p53 in lymph node and liver metastases from advanced gastric cancer specimens was mainly examined by immunohistochemical staining in relation to primary tumours. RESULTS: Immunohistochemical staining showed reduced protein expression of nm23, KISS1 and KAI1 in lymph node and liver metastases compared with primary tumours. Results for p53 were to the contrary. CONCLUSIONS: Our investigations revealed a tendency of reduced protein expression of metastasis suppressor genes nm23, KISS1 and KAI1 in gastric cancer with the progress of metastasis. This means that the progression theory is an important determinant of metastatic efficiency.

Relationship of serum levels of tumor markers with tissue expression of gene products in ovarian carcinoma.

PURPOSE: The aim of this prospective study was to determine serum levels and tissue expression of CA125, CA 15-3, p53, HER-2 and nm23 tumor markers, which are used in the detection and follow up of patients with ovarian carcinoma. PATIENTS AND METHODS: 19 patients with malignant and benign ovarian tumors were included in this study. Serum levels of CA125, CA 15-3 and p53 tumor markers were detected in preoperative and postoperative blood samples using ELISA technique. Tissue expression of p53, HER-2 and nm23 were examined using immunohistochemistry. RESULTS: All serum tumor markers were elevated in patients with ovarian carcinoma. Serum level of CA 15-3 was increased in patients with ovarian carcinoma (median 48.33 U/ml, normal range 0-36), while it was normal in patients with benign ovarian tumors (median 20.67 U/ml; p >0.05). CA125 serum values were strikingly increased in ovarian carcinoma (median 264.16 IU/ml, normal range 0-35) and benign ovarian tumors (median 119.59 IU/ml; p <0.05). Serum levels of p53 in patients with ovarian carcinoma were increased (median 0.69 U/ml, normal range 0-0.50) compared to patients with benign tumors (0.32 U/ml; p <0.05). Histological HER-2 overexpression was detected in 7 cases, including 4 with strong (score 3+ and 2+) and 3 with weak or no HER-2 expression (score 1+ and 0) in ovarian carcinoma tissue; in benign tumors HER-2 overexpression was detected in 1 case (p >0.05). Strong overexpression of p53 was detected in 3 cases with malignant and none with benign tumors (p >0.05); and strong overexpression of nm23 was detected in 5 cases with malignant and 2 with benign tumors (p >0.05). CONCLUSION: Serum levels of CA125, CA 15-3 and p53 are strikingly increased, as well as the expression of HER-2 and p53 in carcinomatous tissue. Detection and analysis of multiple tumor-specific markers in serum and tissue can give useful clinical information for the management of ovarian carcinoma and can also improve the sensitivity and specificity of these markers.

Immunohistochemical localization of the NM23 protein in salivary gland neoplasms with distinct biological behavior.

The NM23 protein was shown to be associated with metastasis suppression in human malignancies with various tissue origins. However, its association with the metastatic phenotype of salivary gland neoplasms (SGN) remains unknown. To evaluate the role of NM23 in SGN, the expression patterns of NM23 in the following were compared: benign (pleomorphic adenoma) vs malignant (adenoid cystic carcinoma and mucoepidermoid carcinoma) SGN, and primary malignancies with/without evidence of metastasis vs their metastatic implants (MI). The lesions were studied immunohistochemically. NM23 protein was found in the cytoplasm of 75% of benign SGN, 73.3% of primary SGN malignancies with no evidence of metastasis, 86.6% of primary SGN malignancies with evidence of metastasis, and 60% of MI. There was no statistically significant difference in the frequency of NM23-positive cells between benign and primary malignant tumors (p = 0.79), nor between primary malignancies with/without evidence of metastasis and MI (p = 0.51). However, nuclear NM23 protein was restricted to primary SGN malignancies with evidence of metastasis and MI. The presence of nuclear NM23 protein may be a good marker for predicting the metastatic potential of SGN malignancies.

Proteomics-based strategy to identify biomarkers and pharmacological targets in leukemias with t(4;11) translocations.

Translocations and other aberrations involving the MLL (mixed lineage leukemia) gene result in aggressive forms of leukemias. Heterogeneity in partner genes, in chromosomal breakpoints, in MLL itself, and in the different partner genes results in heterogeneous fusion transcripts that can be alternatively spliced, which complicates deciphering a unifying mechanism of leukemogenesis. However, recent microarray studies completed with clinical leukemia specimens have uncovered several distinct mRNA signatures within MLL leukemia that differ from other types of leukemia. A global proteomics strategy using MV4-11 and RS4:11 cells in culture was employed to investigate possible protein signatures common to different MLL leukemias and to identify disease biomarkers and protein targets for pharmacological intervention. Initial proteomics screening experiments with two-dimensional differential in-gel electrophoresis revealed heat shock protein 90 alpha (HSP90alpha) as a potential target for pharmacological inhibition and nucleoside diphosphate kinase (nm23) as a biomarker for measuring treatment efficacy. Using a modified stable isotope labeling of amino acids in cell culture (SILAC) approach, coupled with two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS), changes in abundance for over 500 proteins were measured. In addition, decreased expression of the novel biomarker nm23 was observed during HSP90 inhibition with 17-allylamino-17-demethoxygeldanamycin (17-AAG) in the MV4-11 cell line. The present study validates the use of a global proteomics strategy to uncover novel biomarkers and pharmacological targets for leukemias with MLL translocations. Additionally, several proteins were found to be expressed in concordance with microarray studies of mRNA expression in specimens from patients showing the value in comparing mRNA transcript and proteomic profiles. This work represents one of the most comprehensive proteomics screens of MLL leukemias that have been conducted to date.

[Expression of adhesive molecules, angiogenesis markers and protein products of suppressor genes in neoplasms of oral cavity and maxillary sinus]. / Ekspresja czastek adhezyjnych, wskazników angiogenezy i produktów bialkowych genów supresorowych w nowotworach jamy ustnej i zatok szczekowych.

INTRODUCTION: Adhesion molecules, angiogenesis markers and protein products of suppressor genes are potential prognostic indicators in different type of tumours. The purpose of this study was to analyze relations between expression of these markers and clinical as well as histological features of oral cavity and maxillary sinus tumours. MATERIAL AND METHODS: Measurements of expression of CD44, TP53, Nm23 oncoproteins and angiogenesis markers were performed. Forty-three patients treated in years 1985-2000 in the Department of Maxillofacial Surgery of the University Hospital in Wroclaw entered our study. Thirty-two of them were treated for oral cavity cancers. Remaining eleven patients were treated for maxillary sinus cancers. RESULTS: We have shown, there was a positive correlation between the number of the vessels and presence of the nodal metastases; between histological grading and VCAM expression; between CD44 expression and total surface area of the blood vessels. There was also correlation between total surface area of the blood vessels and patients' survival time.

Molecular and functional interactions between Escherichia coli nucleoside-diphosphate kinase and the uracil-DNA glycosylase Ung.

Escherichia coli nucleoside-diphosphate kinase (Ndk) catalyzes nucleoside triphosphate synthesis and maintains intracellular triphosphate pools. Mutants of E. coli lacking Ndk exhibit normal growth rates but show a mutator phenotype that cannot be entirely attributed to the absence of Ndk catalytic activity or to an imbalance in cellular triphosphates. It has been suggested previously that Ndk, similar to its human counterparts, possesses nuclease and DNA repair activities, including the excision of uracil from DNA, an activity normally associated with the Ung and Mug uracil-DNA glycosylases (UDGs) in E. coli. Here we have demonstrated that recombinant Ndk purified from wild-type E. coli contains significant UDG activity that is not intrinsic, but rather, is a consequence of a direct physical and functional interaction between Ung and Ndk, although a residual amount of intrinsic UDG activity exists as well. Co-purification of Ung and Ndk through multicolumn low pressure and nickel-nitrilotriacetic acid affinity chromatography suggests that the interaction occurs in a cellular context, as was also suggested by co-immunoprecipitation of endogenous Ung and Ndk from cellular extracts. Glutathione S-transferase pulldown and far Western analyses demonstrate that the interaction also occurs at the level of purified protein, suggesting that it is specific and direct. Moreover, significant augmentation of Ung catalytic activity by Ndk was observed, suggesting that the interaction between the two enzymes is functionally relevant. These findings represent the first example of Ung interacting with another E. coli protein and also lend support to the recently discovered role of nucleoside-diphosphate kinases as regulatory components of multiprotein complexes.

Light-dependent subcellular localization of nucleoside diphosphate kinase-1 in Neurospora crassa.

Nucleoside diphosphate kinase (NDK) is a housekeeping enzyme localized in cellular organelles and distributed in various organs in prokaryotes and eukaryotes. In Neurospora crassa, NDK-1 is suggested to control catalases in response to heat, oxidative stress and light. In this study, we identified the presence of NDK-1 during most developmental stages in submerged mycelia, aerial hyphae, asexual conidia and perithecia, and the localization of it in soluble, mitochondrial, nuclear and membrane fractions in the mycelial cell. A light-dependent localization of NDK-1 was shown by Western blotting and immunohistochemical analysis using anti-NDK-1 antibody. In the mycelia, NDK-1 was compartmentalized on the plasma membrane in darkness, while it was relocated in the cytoplasm under light. These results suggest that NDK-1 protein was translocated from the plasma membrane to cytoplasm in response to light, and may interact with catalase.

Influence of the c-erb B-2, nm23, bcl-2 and p53 protein markers on colorectal cancer.

BACKGROUND/AIMS: Under stringent intra-laboratory conditions we evaluated the relationship between the expression of four protein markers and clinicopathologic characteristics of colorectal tumors. METHODS: 124 patients with colorectal cancer from 1999 to 2002 were assessed. RESULTS: The expression of cerb B-2, nm23 and p53 was mostly determined in tumors located in the rectum. However, about 20% of the rectal lesions had bcl-2 expression. p53 and c-erb B-2 expression was significantly demonstrated in the lesions with vascular and lymph node involvement. However, the difference between the markers and staging was not statistically significant (p=0.388, p=0.301). Cerb B-2 and p53 were more frequently expressed in the patients with large tumors (more than 5 cm) with moderate and poor differentiation grade. About half of the tumors expressing c- erb B- 2 and p53 had vascular invasion and more than 70% had N1 and N2 lymphatic invasion as well. In the patients with tumors expressing c-erb B-2 and p53, recurrences often occurred and both disease-free survival (DFS) and overall survival (OS) in the first two years after surgery were shorter than of the patients with tumors expressing nm23 and bcl-2. CONCLUSION: In this study, c-erb B-2 and p53 were frequently expressed in the Astler- Coller stage C large tumors located in the rectum and a high degree of vascular and lymphatic invasion was observed. In the patients with tumors expressing c-erb B-2 and p53, recurrences were determined more frequently and DFS and OS were shorter than in patients with tumors expressing nm23 and bcl-2. Thus, two different protein markers should be taken into consideration when evaluating the clinical outcome of patients with colorectal cancer.

NM-23 gene loss of heterozygosity and protein expression in high-stage laryngeal squamous cell carcinomas.

Tumor suppressor genes that reduce metastatic potential have been described in a variety of different tumor types. One of the main tumor metastasis suppressor genes is nm-23, which is a nucleoside diphosphate kinase. Two isotypes, nm-23H1 and nm-23H2, have been cloned and map to chromosome 17q21.3. In a variety of tumors, including colon cancer and breast cancer, loss of expression of nm-23 is associated with lymph node metastasis. In other organ systems, however, this relationship is not seen. In head and neck squamous cell carcinomas (HNSCC), there have been conflicting results regarding the association between nm-23 protein expression and metastatic potential. To further explore the tumor metastasis suppressor function of nm-23 in HNSCC, we studied high-stage laryngeal carcinomas, tumors with and without cervical lymph node metastasis for nm-23 protein expression and loss of heterozygosity of the gene locus. Twenty-five cases were included (11 cases with and 14 cases without metastasis). Loss of heterozygosity for the nm-23 gene locus was seen in 7 of 22 (32%) informative tumors. Using immunohistochemistry, most tumors expressed nm-23, though decreased expression was seen in 10 of 25 (40%) cases. Only 2 tumors showed negative expression. We did not find a correlation between either protein expression or loss of heterozygosity with metastatic disease or any other adverse prognostic factors in this group of high-stage laryngeal squamous cell carcinomas. These data imply that nm-23 may be tumor suppressor gene involved in HNSCC but that it may not function as a tumor metastasis suppressor in high-stage laryngeal carcinoma.

nm23 is expressed in reactive mesothelium and is not useful for detection of malignant cells in serous effusions.

The cytological dilemma of distinguishing malignant cells from proliferating mesothelium has generated volumes of research but no clear consensus regarding an optimal staining panel. nm23 has been frequently described in malignant cells as a metastasis suppressor, but its mechanism of action and the relationship between nm23 expression, metastases, and prognosis is controversial. This is the first study to apply nm23 immunostaining in the setting of effusion cytology. One hundred samples of effusions (56 malignant and 44 benign) were immunostained with nm23 using the biotin-avidin technique and diaminobenzidine as a chromogen. Additionally, a mucicarmine stain was performed on most samples, all of which were evaluated for nm23 expression and mucin in a blinded fashion. After all the samples were reviewed, the diagnoses were disclosed and staining patterns evaluated. From the malignant cases, 51 of 56 cases were positive for nm23 in at least 5% of malignant cells, while 27 of 44 reactive mesothelium cases were also positive in at least 5% of cells. Of the malignant cases, all non-adenocarcinomas expressed nm23. We conclude that nm23 is a highly sensitive marker for detecting malignant cells. Its relatively high rate of expression in reactive mesothelium supports previous studies that suggest nm23 is expressed in proliferating cells.

Analysis of gene and protein expression in healthy and carious tooth pulp with cDNA microarray and two-dimensional gel electrophoresis.

Complementary DNA (cDNA) microarray and two-dimensional (2-D) gel electrophoresis, combined with mass spectrometry, enable simultaneous analysis of expression patterns of thousands of genes, but their use in pulp biology has been limited. Here we compared gene and protein expression of pulp tissues from sound and carious human teeth using cDNA microarray and 2-D gel electrophoresis to evaluate their usefulness in pulp biology research and to identify the genes with changes in carious teeth. The cDNA microarray revealed several differentially expressed genes and genes with a high expression in both tissues. These genes have various functions, e.g. effects on vascular and nerve structures, inflammation, and cell differentiation. Variability between cDNA hybridizations indicates that the overall gene expression pattern may vary significantly between individual teeth. The 2-D gel electrophoresis revealed no change between healthy and diseased tissue. The identification of 96 proteins in the pulp tissue revealed none of the gene products with corresponding high/different mRNA expression in cDNA microarray. Interestingly, we detected also a hypothetical protein (putative nucleoside diphosphate kinase), and present therefore the first evidence for the existence of this protein. Even though the methods reveal potentially important gene expression, they may currently have only limited value in in vivo pulp biology research.

Reduced metastasis-suppressor gene mRNA-expression in breast cancer brain metastases.

PURPOSE: Brain metastases are an increasingly common complication in breast cancer patients. The Metastasis Suppressor Genes (MSG) Nm23, KISS1, KAI1, BRMS1, and Mkk4 have been associated with the metastatic potential of breast cancer in vitro and in vivo. METHODS: The mRNA expression of Nm23, KISS1, KAI1, BRMS1, and Mkk4 in fresh frozen tissue samples of brain metastases from ductal invasive breast cancer specimens was examined in relation to primary tumors. In a first step, mRNA expression screening was carried out using a semi-quantitative RT-PCR approach, in a second step quantitative real-time RT-PCR was performed on selected specimens. By immunohistochemical staining, gene products were visualized on the protein level. RESULTS: Semi-quantitative RT-PCR revealed reduced mRNA expression of Nm23, KISS1, KAI1, BRMS, and Mkk4 in brain metastases. Results for KISS1, KAI1, BRMS, and Mkk4 were confirmed by real-time RT-PCR. In detail, mRNA expression reduction in breast cancer brain metastases was tenfold. Expression of MSG could be confirmed by immunohistochemical staining on protein level. CONCLUSIONS: Our investigations revealed significantly reduced mRNA expression of metastases suppressor genes KISS1, KAI1, BRMS1, and Mkk4 in breast cancer brain metastasis. Particularly, in the case of KISS1 and Mkk4, an important role for future treatment of patients with breast cancer brain metastatic lesions can be assumed.

Oral alveolar soft-part sarcoma: review of literature and case report with immunohistochemistry study for prognostic markers.

OBJECTIVE: To describe a case of alveolar soft-part sarcoma (ASPS) affecting the tongue of a child and to study prognostic imunohistochemical markers for the disease. STUDY DESIGN: Tissue sections were incubated with primary antibodies reactive to neuron-specific enolase (NSE), vimentin, desmin, S-100 protein, cytokeratins AE1-AE3, EMA, neurofilament, synaptophysin, and muscle-specific actin (MSA), and for prognostic markers, including Ki-67, p53, bcl-2, bax, and nm23. RESULTS: Histologically, the tumor showed a proliferation of large polygonal cells with PAS-positive diastase-resistant intracytoplasmatic material, arranged in an alveolar growth pattern. Diffuse positive reaction for neuron specific enolase (NSE), focal reactivity for desmin and S-100 protein, strong positivity for nm23 and bax, but weak reaction for p53 and Ki-67 were found. No bcl-2-positive cells were noted. CONCLUSION: These immunohistochemical findings may reflect the less aggressive behavior of ASPS in oral tissues.

nm23-H1 expression and loss of heterozygosity in colon adenocarcinoma.

BACKGROUND: The discovery that genetic alterations in oncogenes and tumour suppressor genes accompany tumour formation in many human tumours has encouraged the search for genes that promote or suppress tumour spread and metastasis; nm23 is a promising candidate for a metastasis suppressing gene. AIMS: To evaluate whether expression of nm23-H1 protein or loss of heterozygosity (LOH) of the nm23-H1 gene is associated with colon cancer progression. MATERIALS/METHODS: Paraffin wax embedded tissue sections were analysed immunohistochemically. DNA isolated from normal and tumour tissue was used for LOH analysis using a variable nucleotide tandem repeat (VNTR) marker located in the untranslated 5' region of the nm23-H1 gene. RNA isolated from tumour and normal tissue was used for "real time" RT-PCR. RESULTS: Of 102 adenocarcinomas examined, 58.8% stained weakly for nm23-H1 protein. There was a negative correlation between nm23-H1 positivity and tumour histological grade. In VNTR analysis, 70.2% of patients were informative and 27.4% of tumours had nm23-H1 LOH. There was a positive correlation between nm23-H1 LOH and both tumour histological grade and Dukes's stage. Expression of nm23-H1 mRNA was increased in 22 of 30 colon tumours compared with normal tissue. No significant correlation was found between nm23-H1 mRNA expression and histological grade or Dukes's stage of tumours. CONCLUSIONS: These findings suggest that nm23-H1 protein expression in early stages may have a role in suppressing metastasis in sporadic colon cancer, whereas at a later stage both reduced nm23-H1 protein expression and LOH of the nm23-H1 gene may play role in colon cancer progression and metastasis.