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We investigated the significances of the expressions of dihydrodiol dehydrogenase (DDH) and glutathione-S-transferase (GST) in patients with esophageal squamous cell carcinoma (ESCC). By using immunohistochemistry, we measured expressions of DDH, GST, COX-2, nm23-H1, HER-2/neu and mdr-1 in 145 patients with ESCC. Expression of DDH was confirmed by immunoblotting and reverse transcription-polymerase chain reaction. Relation between DDH expression and clinicopathological parameters was analyzed by statistical analysis. Difference of survivals between different groups was compared by a log rank test. DDH overexpression was detected in 66.9% of pathological sections (97/145) and in 41.6% of metastatic lymph nodes (37/89). The nucleotide sequencing of DNA fragments from 16 tumorous specimens showed that the major isoform was DDH2 for ESCC. GST expression, however, was only detected weakly in 24 patients (16.6%). For patients with ESCC, DDH overexpression was positively correlated with smoking habit, tumor stage, number of metastatic lymph nodes, lymphovascular invasion and COX-2 expression, and inversely correlated with GST and nm23-H1 expressions, but not related to mdr-1 or HER-2/neu expressions. As compared to DDH overexpressed group, patients with low DDH expression had significantly lower incidence of tumor recurrences and better survival (p = 0.026). Using univariate analysis, prognostic factors included tumor stage, number of metastatic lymph nodes, cell differentiation, lymphovascular invasion and expressions of DDH and nm23-H1. Multivariate analysis showed significant correlation of tumor stage, number of metastatic lymph nodes and nm23-H1 expression with patient's survival. In conclusion, inverse expressions of DDH and GST may be associated with carcinogenesis and disease progression for ESCC patients, but their biological function and pathophysiological regulation in tumors require additional studies., (Copyright 2004 Wiley-Liss, Inc.)

Metastases of various malignancies have been shown to be inversely related to the abundance of nm23 protein expression. However, the downstream pathways involved in nm23-mediated suppression of metastasis have not been elucidated. In the present investigation, we used cDNA microarrays to identify novel genes and functional pathways in nm23-mediated spontaneous breast metastasis. Microarray experiments were performed in a pair of cell lines, namely, C-100 (only vector transfected; highly metastatic) and H1-177 (nm23 transfected; low metastatic), derived from human mammary carcinoma cell line MDA-MB-435. The cDNA microarray analysis using GeneSpring software revealed significant as well as consistent alterations in the expression (up- and downregulation) of 2158 genes in a total of 18889 genes between high and low metastatic cells. Some of these genes were grouped into 6 functional categories, namely, invasion and metastasis, apoptosis and senescence, signal transduction molecules and transcription factors, cell cycle and repair, adhesion, and angiogenesis to extrapolate an association between these genes and different functional pathways involved in nm23-regulated metastasis. The results suggest that nm23 gene plays a major role in metastasis and its mechanism of action of metastasis suppression may involve downregulation of genes associated with cell adhesion, motility (integrins alpha2, -8, -9, -L and -V, collagen type VIII alpha1, fibronectin 1, catenin, TGF-beta2, FGF7, MMP14 and 16, ErbB2) and possibly certain tumor/metastasis suppressors (2 members of SWI/SNF-related matrix-associated proteins 2 and 5 and PTEN)., (Copyright 2003 Wiley-Liss, Inc.)

The nm23-H1 gene is known as a potential metastasis suppressor gene in various types of carcinomas. However, the role of nm23-H1 in colorectal carcinoma still remains controversial and the cellular mechanisms by which its protein may modulate the metastatic phenotype are not yet known. We transfected nm23-H1 cDNA into the human colon cancer cell line, HT-29, to test the effects and cellular biological mechanism of nm23 protein in colon cancer. We found that nm23-H1 strongly inhibited the liver metastasis of HT-29 cells in nude mice and inhibited the epidermal growth factor (EGF)-induced cell migration in vitro. Furthermore, we clarified the regulation of the myosin light chain (MLC) phosphorylation by nm23-H1, which has been demonstrated as having potential role in cell migration., (Copyright 2003 Wiley-Liss, Inc.)

p53, a tumor suppressor gene involved in the G1 cell cycle checkpoint, is also the most frequently mutated gene in human cancer. In addition, p53 modifies the ability of tumor cells to metastasize. The metastasis-associated gene Nm23-H1, which encodes an 18-kDa nucleoside diphosphate kinase, was previously identified in cells with low metastatic potential. Although p53 and Nm23-H1 proteins play an important part in regulating the progression of cancer, any functional relationship between these two proteins is currently unknown. Here we report an association between p53 levels and expression of the Nm23-H1 gene. Our data indicate that wild-type (wt) p53 upregulated the expression of Nm23-H1 at protein and mRNA levels in MCF-7 and J7B cells. This capacity of wt p53 to regulate expression of Nm23-H1 was not only dependent on the endogenous but also the exogenous origin of p53, and could not be reproduced with mutant p53. Subsequently, the invasive ability of MCF-7 and J7B cells was suppressed upon induction of the Nm23-H1 protein by p53. In contrast, increased levels of p53 downregulated the expression of Nm23-H1 at the protein and mRNA levels in RKO and H1299 cells and, as a consequence, increased the invasive ability of both cell types. Thus, our results implicated the differential regulation of Nm23-H1 by p53 in different cell types as an important component in the molecular mechanisms of tumor metastasis., (Copyright 2003 Wiley-Liss, Inc.)

Prognostic parameters for axillary node-negative (ANN) breast cancer are still rare. Our aim was to establish potential markers that predict distant metastasis in ANN breast carcinoma and permit detection of the patients with high metastasis risk. A case control study was designed that comprised 64 ANN patients who developed distant metastasis during a 5-10 year follow-up period, 64 ANN patients with recurrence-free survival and 64 node-positive (ANP) comparitors. Immunohistochemistry and/or in situ hybridization were used to detect nm23, Cathepsin-D (Cath-D), Epidermal Growth Factor Receptor (EGFR) and Laminin Receptor (LR) in 192 cases. A significantly lower expression of both nm23 mRNA and protein was found in the ANN-group with poor prognosis compared with the ANN-group with good prognosis (p < 0.01). The protein levels of Cath-D, EGFR and LR were significantly higher in the ANN-group with poor prognosis and in the ANP-group compared with the ANN-group with good prognosis (p < 0.01 or p < 0.05), but no differences were found between the poor ANN-group and the ANP-group. Multiple regression analysis showed a close correlation of nm23, Cath-D and EGFR expression with occurrence of distant metastasis of ANN breast carcinoma. All markers except nm23 correlated with conventional histopathologic criteria such as tumor grade, margin and vessel invasion. The results suggest the combined detection of nm23, Cath-D and EGFR as predictive markers of distant metastasis in ANN breast cancer patients. Quantitative analysis together with clinicopathologic factors could contribute to estimate the potential risk of metastasis and select individual therapy regimen., (Copyright 2002 Wiley-Liss, Inc.)

Nm23 has been identified as a gene family encoding different isoforms of nucleoside diphosphate kinase (NDPK). This protein is a key enzyme in nucleotide metabolism and has been shown to play important roles in various cellular functions. In the present study, we have investigated the expression of three isotypes in mouse dorsal root ganglia. In situ hybridization and reverse transcriptase-polymerase chain reaction analysis demonstrated high levels of nm23-M1, -M2, and -M3 mRNA expression in peripheral nervous tissue. Moreover, in situ hybridization also displayed a specific nuclear localization for nm23-M2 mRNA. Immunohistochemistry with light and electron microscopy on isoform-specific antibodies revealed a differential subcellular distribution of NDPK isoforms. Isoform A was mainly cytosolic, showing only partial association with organelles. In contrast, isoform B was also found in the nucleus, which is in agreement with its proposed role as a transcription factor. The results also indicate a preferential association of isoform C with endoplasmic reticulum and plasma membranes in neuronal cells. Furthermore, isoform C appeared to combine with other NDPK isoforms as demonstrated by double-labeling evidence by electron microscopy and might be responsible for binding NDPK oligomers to membranes. Thus, isoform C may be considered as a protein of importance for maintaining intracellular pools of GTP in the vicinity of membranes and, hence, for transmembrane signaling. The results indicate a high expression of NDPK isoforms, not only in the central but also in the peripheral nervous system. Their different subcellular compartmentalization suggests that they have isoform-specific roles in neuronal cell physiology., (Copyright 2002 Wiley-Liss, Inc.)

Background and Objectives: Tumor suppressor gene MTS1/p16 (cyclin-dependent kinase-4 inhibitor) and a putative tumor metastasis suppressor gene nm23 (nucleoside diphosphate A kinase) have been identified in a variety of human tumors but have not been well studied in mesenchymal neoplasms., Methods: Expression of nm23 and MTS1 mRNA was determined by quantitative analysis from paraffin-embedded tumor tissue. The series comprised 31 patients with localized primary synovial sarcoma of soft tissues who were followed for a median of 83 months., Results: Neither MTS1 nor nm23 expression levels correlated with the patient's age or sex, tumor type, depth, size, mitotic rate, or extent of tumor necrosis. In addition, there was no correlation between MTS1 and nm23 levels. Patients' survival was not related to sex, age, tumor type, location, mitotic rate, or MTS1 mRNA level. The only factors that correlated with poor survival in multivariate analysis were the presence of extensive tumor necrosis (> 15%) and higher levels of nm23 mRNA., Conclusions: Our results suggest that increased expression level of nm23 mRNA may be implicated in the mechanism of tumor progression and is associated with poor survival in patients with synovial sarcoma.

Background and Objectives: A number of evidence indicate that downregulation of the nm23-H1 gene may be relevant to metastatic progression of many kinds of human cancer. However, its role in colorectal cancers remains controversial. To address the issue, this study was performed to investigate the clinical relevance of nm23-H1 in patients with colorectal cancers., Methods: Immunohistochemical expression of nm23-H1 protein product (NM23-H1) was studied in a total of 146 colorectal cancer patients and compared for its prognostic value at a mean follow-up of 54 months., Results: There was no apparent correlation between NM23-H1 expression and clinicopathological indicators, including Dukes category, lymphatic metastasis, distant metastasis, histological grading, and tumor location (P < 0.1, respectively). In addition, determination of NM23-H1 expression status did not provide independent prognostic information compared with conventional pathological staging., Conclusions: The results indicate that nm23-H1 gene does not play an important part in the progression of colorectal carcinogenesis.

The effects of transfection of the metastasis suppressor gene nm23-H1 and cell-cycle related tumor-suppressor gene p16 on the activity of N-acetylglucosaminyltransferase V (GnT-V) and their relations to cancer metastatic potential were investigated. After transfection of nm23-H1 into 7721 human hepatocarcinoma cells and A549 human lung cancer cells, the activities of GnT-V were decreased by 28%-42% in the cells. In contrast, when p16 was transfected into these two cell lines, the decrease of GnT-V activity was only observed in A549 cells. This was probably to be due to the obvious expression of p16 gene in parental 7721 cells and the deletion of p16 in A549 cells. The decrease of GnT-V mRNA was only observed in nm23-H1-transfected cells, but not in p16-transfected A549 cells, suggesting that these two genes regulated GnT-V via different mechanisms. Horseradish peroxidase (HRP)-lectin staining showed that the 7721 cells transfected with nm23-H1 or the A549 cells transfected with p16 displayed a decreased intensity with HRP-leucoagglutinating phytohemagglutinin and increased intensity with HRP-concanavalin A, indicating the decline of beta1,6 N-acetylglucosamine branching structure on the asparagine-linked glycans of cell-surface and intracellular glycoproteins. The nm23-H1 transfected 7721 cells also displayed some changes in metastasis-related phenotypes, including the increase in cell adhesion to fibronectin (Fn), the decline in cell adhesion to laminin (Ln), and the decreased cell migration and invasion through matrigel. Transfection of antisense GnT-V cDNA into 7721 cells resulted in a decrease of GnT-V activity, an increase of cell adhesion to Fn or Ln, and a decrease in cell migration and invasion through matrigel. These phenotypes bore similarity to those of the 7721 cells transfected with nm23-H1. Our findings indicate that the down-regulation of GnT-V by nm23-H1 contributes to the alterations in metastasis-related phenotypes, and is an important molecular mechanism of metastasis suppression mediated by nm23-H1., (Copyright 2000 Wiley-Liss, Inc.)

Background and Objectives: Anatomic extent is not the sole axis of classification of tumors and of tumor patients relevant to treatment planning and estimation of prognosis. This results in the need to demonstrate an improvement in prognostic assessment and choice of therapy achieved by consideration of factors other than TNM. nm23 protein does prevent tumor from metastasizing and may also play a role in the control of growth and development. The purpose of this study was to elucidate the clinical significance of nm23 expression in human anal canal carcinoma and to evaluate its influence on the outcome of patients after surgery or radiochemotherapy., Methods: Twenty-two patients affected by anal canal carcinoma were evaluated. Each section was incubated with monoclonal antibody nm23 NDPK-A. Immunostaining was considered positive when at least 10% of the tumor cells were immunostained., Results: nm23 immunoreactivity was detected in 6/22 (27.3%) tumors. No significant association was found between nm23 expression and prognosis., Conclusions: The mechanisms causing enhanced nm23-H1 expression in anal canal carcinoma are unknown. Although the level and expression were not correlated with prognosis, activation of nm23-H1 gene might be a prerequisite for oncogenesis in this type of tumor, while an alternate possibility is the modification of cellular characteristics in relation to proliferation and/or differentiation as a consequence of oncogenesis.

Tumor suppressor genes have a pivotal role in normal cells regulating cell cycle processes negatively. Furthermore, the inhibition of cell proliferation is a crucial step in the achievement of cell differentiation. Increasing evidence suggests that the nm23 genes, initially documented as suppressors of the invasive phenotype in some cancer types, are involved in the control of normal development and differentiation. In this review, we summarize some data concerning the involvement of the nm23 genes in development and differentiation, attempting to delineate an overall view of many facets of their biological role., (Copyright 2000 Wiley-Liss, Inc.)

Genomic alterations at the long arm of chromosome 17, and in particular at the nm23 locus, are still controversial in colorectal cancer (CRC). Our aim was to investigate the possible relationship of loss of heterozygosity (LOH) and microsatellite instability (MI), at 4 microsatellite loci spanning the 17q21-23 region, to the risk of liver metastasis and nm23 protein expression. Genomic DNA extracted from 58 primary and 54 liver secondary formalin-fixed and paraffin-embedded CRCs was obtained from 82 patients. A fluorescent PCR coupled with an automated DNA sequencer was applied. Increasing fraction of loci showing LOH was positively associated with risk of liver metastases (logrank test for trend, p = 0.005); this remained independent after adjusting to T-stage (Cox regression, p = 0.022), N-stage (p = 0.007), or Dukes' stage (p = 0.012). Conversely, increasing frequency of MI was associated with a reduced risk of liver metastases in Dukes' B tumours (logrank test for trend, p = 0.032). When comparing 30 primary and matched liver secondary lesions, we found concordant genomic alteration in 72% (NME1) to 43% (D17S579). Finally, we observed a trend in association between the proportion of loci with LOH and nm23 positivity (chi2 test for trend, p = 0.024). Our findings suggest that genomic alterations in the 17q21-23 region may affect prognosis of CRC as well as regulation of the nm23 protein expression via an unknown underlying mechanism.

Background and Objectives: Predicting the prognosis in gastric carcinoma patients with intermediate stages is difficult. We investigated the prognostic impacts of CD44 and nm23 expression in a homogeneous group of patients with stage II and IIIA gastric carcinoma who had undergone curative resections., Methods: A total of 261 paraffin-embedded gastric carcinomas were stained with the monoclonal antibodies CD44 and nm23 using the labeled streptovidin biotin method., Results: The expression of CD44 and nm23 was detected, respectively, in 31.0% (81/261) and 70.1% (183/261) of all tumors. There was no correlation between CD44 expression and clinicopathological variables. However, nm23 was more frequently expressed in older patients with differentiated adenocarcinoma. A significant difference in 5-year survival rates was found between patients with CD44-positive (43.2%) and CD44-negative tumors (63.4%), (P = 0.0018). However, there was no significant difference in 5-year survival rates between patients with nm23-positive (54.7%) and nm23-negative tumors (62.7%) (P = 0.2734)., Conclusions: CD44 expression was a significant adverse prognostic factor in gastric carcinoma and may be a predictor of metastatic potential of the primary tumor. By contrast, immunohistochemical detection of nm23 expression was not a predictor of outcome of patients with gastric carcinoma.

The expression levels of nm23-H1 mRNA and its protein in human nasopharyngeal carcinoma (NPC) were detected to clarify the relationship between nm23-H1 and metastasis and prognosis of patients with NPC. nm23-H1 mRNA expression in fresh tissues from 78 patients with NPC was investigated by in situ hybridization and RT-PCR. Routine labeling streptavidin-biotin immuno-histochemistry with the nm23-H1 murine monoclonal antibody was employed to study the expression of nm23-H1 protein in paraffin-embedded specimens from 231 patients with NPC treated in our hospital. The clinical pathologic data and results of follow-up were collected. Comparisons between expression of nm23-H1 protein or mRNA and clinical outcome were performed using the chi2 test. Multivariate prognostic analyses were performed by the Cox regression model. We found that nm23-H1-negative tumors were associated with a higher incidence of lymph-node metastasis (84.2%) than nm23-H1-positive ones (32.8%, p < 0.01). The distant metastasis and loco-regional recurrence rates in the nm23-H1-negative group were 55.8% and 31.68%, respectively but only 17.2% and 11.5%, respectively, in the nm23-H1-positive group (p < 0.01). A significant association was found between expression of nm23-H1 protein and prognosis (p < 0.01). Expression of nm23-H1 protein indicated favorable prognosis, suggesting that the absence of nm23-H1 protein expression was significantly associated with lymph-node metastasis, recurrence and distant metastasis in NPC. Expression of the nm23-H1 gene may be valuable for assessing the prognosis of NPC.

Background: The estramustine binding protein (EMBP) specifically binds to estramustine and was first discovered in the rat ventral prostate. However, the physiological property of EMBP in the human prostate still remains to be elucidated. To elucidate whether EMBP is interrelated with cellular proliferation in human prostatic carcinoma (PC), the change in EMBP immunostaining during luteinizing hormone-releasing hormone (LH-RH) analog administration or during Cis-platinum-based chemotherapy, and the difference in EMBP immunostaining between hormone refractory (hr-PC) and untreated PC were analyzed., Methods: Forty-six patients with histologically proven untreated PCs (34 were treated with LH-RH analog and 12 were treated with chemotherapy as an initial therapy) and 14 with hr-PC were used in this study. PC tissues were obtained before and 3 months after the initial therapy. The changes in immunostainings for EMBP, proliferating cell nuclear antigen (PCNA), and nm23 protein were compared with the change in serum prostate-specific antigen (PSA) level and the histological response during the treatment., Results: The increased EMBP expression was observed in tumors with high histological grade and high clinical stage as well as in hr-PC. In untreated PC, EMBP expression weakly correlated with PCNA or nm23 protein immunoreactivity. In PC receiving LH-RH analog, EMBP expression was significantly reduced after treatment, however, no significant changes were observed in PCNA or nm23 protein immunoreactivity. In addition, EMBP expression before the treatment significantly correlated with the serum PSA change, while PCNA expression and nm23 protein immunoreactivity did not. On the other hand, no significant relationship was observed between histological changes induced by the LH-RH analog and immunostainings for EMBP, PCNA, and nm23 protein before treatment. In PC patients receiving chemotherapy, immunostainings for EMBP, PCNA, and nm23 protein were not significantly changed during the treatment. EMBP immunoreactivity was significantly higher in hr-PC than in untreated PC with paralleled change of PCNA expression and nm23 protein immunoreactivity., Conclusions: These observations indicate that EMBP is androgen regulated in some PCs. However, EMBP expression is demonstrated even in hr-PC and is interrelated with cellular proliferation especially in hr-PC.

Background and Objective: The purpose of this retrospective study was to test whether the expression of p53, nm23, and bcl-2 in T3-4N1M0 oesophageal carcinoma is associated with patient survival., Methods: Immunohistochemical localisation of p53, nm23, and bcl-2 was performed on formalin-fixed, paraffin-embedded tissue sections (N = 46). The observed range of follow-up period was 0.2-24.0 months with a median of 11.0 months. A total of 85% (39/46) of the patients died within 24.0 months, which could be due to advanced disease at presentation. The immunohistochemical signal was expressed as the proportion of positive cells. The immunostaining for p53 was nuclear, whereas that for nm23 and bcl-2 was cytoplasmic in the neoplastic cells., Results: p53 was expressed in 70% (32/46) of cases; nm23 in 29% (13/45), and bcl-2 in 67% (29/43) of tumours. The univariate analysis showed that the expression of two markers, i.e., expression of p53 and absence of nm23 were independently associated with unfavourable overall survival time. Despite a small number of patients treated with adjuvant therapy, we observed that tumours positive for p53 had an unfavourable prognosis when compared with tumours negative for p53., Conclusions: Our preliminary findings suggest: expression of p53 and nm23 negativity may be related to an unfavourable prognosis in patients with advanced oesophageal carcinoma.

Background: In view of the cumulative results to date, p53, nm23, erbB-2, and cathepsin D are the most promising investigational prognostic factors in breast cancer., Objectives: The clinical utility of these molecular markers to predict recurrence was evaluated., Methods: Archival pathology tissues of 100 breast cancer patients were analyzed by immunohistochemical assay. Molecular biologic data were merged with clinicopathologic variables., Results: Thirty-two patients (32%) had recurrence of disease at a median follow-up of 48 months (range 26-72 months). Investigational factor expression had statistical correlation for recurrence with increasing coexpression: one variable 20.6%, two variables 34.2%, three variables 47.1%, four variables 80.0% (P = 0.003). In univariate analysis, lymph node metastasis, tumor size, erbB-2 protein overexpression, and loss of nm23 protein expression were significant variables to determine recurrence; in multivariate analysis, node status and tumor size emerged as the most significant variables for recurrence., Conclusions: Coexpression of the studied investigational variables functioned as significant prognostic correlates for recurrence. These findings suggest that the studied investigational prognostic factors possess the ability to discriminate a highly aggressive phenotype in breast cancer.

The purpose of this study was to investigate a prognostic indicator that can differentiate node negative breast cancer patients (N = 39, T2N0M0) with high risk and low risk for the development of recurrence or metastases. Preoperative plasma prolactin (PRL) was estimated by radioimmunoassay. The expression of PRL, p53, nm23, and c-erbB2 was investigated by immunohistochemical (IHC) localization; cathepsin D (CD, Enzyme Linked Sorbant Assay) and estrogen- and progesterone-receptors (ER and PR, Dextran coated charcoal method) were estimated in the tumor cytosols. The follow-up period was 2-6 years. Statistical comparisons were made between each marker for relapse-free survival (RFS) and overall survival (OS). Of the 39 patients, 18 had hyperprolactinemia (PRL > 20.0 ng/ml plasma), whereas overexpression of p53 was observed in 55% (17/31) tumors. These were independently and in combination associated with a reduced RFS and OS. The rest of the investigated markers did not show promising results. Hyperprolactinemia and/or overexpression of p53 were associated with aggressiveness of the tumor, early disease relapse or metastases, and poor OS in patients with node negative breast cancer. These two markers may enhance our ability to identify node negative breast cancer patients with aggressive tumors, for whom the use of adjuvant chemo and/or endocrine therapy is unequivocally justified.

The nm23 gene has been described as a potential metastasis suppressor gene in certain rodent and human tumors. We previously demonstrated that tyrosine and phenylalanine restriction suppresses metastatic heterogeneity of B16-BL6 murine melanoma and selects for tumor variants with decreased metastatic potential. In this study, we investigated nm23 expression in the highly metastatic B16-BL6 (ND) melanoma, its nutritionally derived poorly metastatic (LT) variant, and the syngeneic non-tumorigenic Mel-ab melanocytes. No differences in nm23 expression were observed between ND and LT cells, and nm23 expression varied between different isolates. Previously, we showed that metastatic potential of 1-ND cells decreases and is not altered in 1-LT cells after prolonged in vitro cell passage; however, nm23 expression is equivalently increased by 2-fold. In 2-ND and 2-LT cells, expression of nm23 is not different at higher in vitro cell passage. Expression of nm23 decreased about 2-fold when phorbol 12-myristate 13-acetate (PMA) was removed from Mel-ab cells, which induces these cells to become quiescent. Although membrane-associated protein kinase C (PKC) activity decreased after prolonged PMA treatment in all cells, neither nm23 expression nor proliferation of ND and LT cells was affected by PMA. These data indicate that nm23 expression is related to proliferative activity rather than to the suppression of metastatic potential.

The genetic events involved in the development of metastases of epithelial ovarian cancer are largely unknown. One gene postulated to play a role in tumour metastasis suppression is NME1 (nm23-H1), and an inverse relationship between NME1 expression and metastatic potential has been observed for some solid tumours. In this study we have investigated the levels of mRNA expression of the 2 isoforms of the NME gene, NME1 and NME2. A maximum of 45 tumours samples from 33 patients were available for Northern blot analysis. We observed variable levels expression of NME1 and NME2 mRNA. The average level of NME1, but not NME2, mRNA expression was statistically higher in metastatic biopsies when compared with primary tumour biopsies. To examine the possible tumour suppressor gene role of NME1 in ovarian tumours, 76 patients were investigated by Southern blot analysis to determine the rate of allelic deletion. Allele loss at 5 other chromosome 17 loci (D17S5, TP53, NF1, D17S74, D17S4) was also evaluated for many of these 76 patients. Allele loss was observed in 22/30 (73%) informative patients at the NME1 locus. We also observed high rates of allele loss at the other loci evaluated. No correlations with clinical stage, histological subtype or patient survival were observed in either mRNA or DNA analyses. We have established that tumour progression in ovarian cancer is accompanied by over-expression of the NME1 gene; however, despite high rates of allele loss at the NME1 locus, the concept that NME1 may be a candidate tumour suppressor gene in ovarian cancer cannot be confirmed by this study.

Expression of the T24ras oncogene induces malignancy (tumor growth, invasion and metastasis) in cloned rat embryo fibroblasts (CREF T24). In CREF T24, the rate of phosphorylation of eukaryotic translation initiation factor 4E (eIF-4E) is increased, resulting in increased protein synthesis rates. We have recently shown that reducing the protein levels of eIF-4E in CREF T24 (AS4E line) markedly decreases soft-agar colonization, increases tumor latency periods and increases tumor doubling times without significantly altering monolayer growth. In this study, cells with reduced eIF-4E had delayed and reduced invasiveness and decreased experimental metastasis. Furthermore, reduced eIF-4E levels correlated with decreased expression of the metastasis-associated 92-kDa collagenase type-IV and exon-6 variants of the CD44 adhesion molecule [CD44(6v)]. Reduced eIF-4E levels correlated inversely with increased levels of the putative metastasis-suppressor protein nm23. Cell lines established from AS4E tumors and lung metastases exhibited increased levels of eIF-4E protein and protein synthesis rates compared to the AS4E line. Tumor-derived AS4E had the shortened tumor latency periods of CREF T24 but displayed the slow tumor-growth rates of AS4E. Tumor-derived AS4E exhibited the metastatic capacity of CREF T24 controls. Furthermore, tumor- and lung-nodule-derived AS4E expressed levels of CD44 (6v) and the 92-kDa collagenase type IV comparable to CREF T24 and displayed reduced levels of nm23 relative to AS4E. These results demonstrate that eIF-4E is an important effector molecule involved in oncogenic p21ras-induced malignant transformation.

The molecular mechanisms of tumor invasion and metastasis are yet to be fully elucidated. A potential tumor-metastasis-suppressor gene nm23 has been described in certain rodent and human tumors. In the present study, we examined the potential anti-invasive and anti-metastatic effect of nm23 gene in B16F10 cells, a malignant murine melanoma cell line. Transfection of nm23 gene into B16F10 melanoma cells resulted in significant suppression of the invasiveness and metastatic ability of melanoma cells and significantly enhanced the survival of tumor-bearing mice. B16F10 melanoma cells transfected with nm23 produced significantly less soluble ICAM-I and were more susceptible to LAK-cell-mediated cytotoxicity. Co-culture of B16F10 melanoma cells with IL-2 had no effect on nm23 expression, whereas treatment with PGE2, TNF-alpha and IFN-gamma resulted in down-regulation of nm23 expression. Concomitantly, in vivo treatment with TNF-alpha or IFN-gamma in experimental mice increased pulmonary metastases and lowered the overall survival period, as compared with IL-2 treatment alone. These results provide evidence that nm23, in addition to its anti-metastatic function, could also be involved in modulating tumor-target-structure expression, in down-regulating invasive potential and in production of soluble intracellular adhesion molecules. The down-regulation of nm23 by TNF-alpha, IFN-gamma and particularly by PGE2 warrants re-examination of current immunotherapeutic protocols and of the role played by PGE2 in tumor progression.

Thrombospondin (TSP) is a member of a family of extracellular matrix glycoproteins that may participate in multiple aspects of the metastatic cascade. We report an inverse correlation of steady-state Thbs-1 mRNA and protein expression with malignant progression among murine melanoma and human lung and breast carcinoma cell lines. Murine K-1735 melanoma cell lines of low metastatic potential, including K-1735 lines transfected with the murine nm23-1 cDNA, expressed higher TSP levels than related highly metastatic lines. In a model system of lung carcinoma malignant progression, immortalized human bronchial epithelial cells expressed higher TSP levels than v-Ki-ras, v-Ha-ras or n-ras transfectants, which in turn expressed higher TSP levels than tumor-derived, more aggressive variants. Among 3 unrelated breast carcinoma cell lines, Thbs-1 steady-state mRNA levels were greater in the 2 non-metastatic lines than the metastatic line. Our data show that malignant progression in some cell lines is associated with reduced TSP expression. The suppressive effects of nm23-1 transfection on metastatic potential are also associated with increased TSP expression; ras transfection, which results in increased tumorigenesis, is associated with decreased TSP expression.

Biological functions proposed for the ST3 and nm23 genes in tumour development and progression seem to be directly opposed. Stromelysin-3 (ST3) is a putative member of the matrix metalloproteinase family. ST3 has been implicated in the progression of epithelial malignancies, specifically with regard to an invasive (and therefore potentially metastasizing) phenotype. The nm23 gene, on the other hand, encodes a nucleoside diphosphate kinase which allegedly has a metastasis-suppressor-type function. It was therefore of interest to compare the expression of ST3 and nm23 in various surgically excised normal and neoplastic breast tissues. RNA was isolated from over 200 surgical specimens and studied by Northern blots. Normal breast tissues did not express ST3, and ST3 expression was detected in only 1 of 20 normal axillary lymph nodes. None of 7 fibroadenomas expressed ST3. In contrast, 60% of primary and metastatic breast carcinomas contained ST3-mRNA. The expression of ST3 was mainly confined to invasive carcinomas and was observed less frequently in pure ductal carcinoma in situ (DCIS) lesions. Our results support the suggestion that ST3 expression is related to the malignant process in breast cancer. The role of nm23 is far less clear-cut.

Expression of the candidate metastasis-suppressor gene nm23-H1 has been shown to correlate inversely with metastatic potential in some human tumors, but not in all. Until now, few studies have been carried out on the activity of the homologous nm23-H2 gene in human cancer. No nm23 transcription studies exist for human lung cancer so far. To determine whether the nm23 genes could have a metastasis-suppressor function in non-small-cell lung carcinoma (NSCLC), pulmonary sarcoma and carcinoids, we analysed both nm23-HI and nm23-H2 mRNA levels in 37 tumor samples obtained from patients who underwent potentially curative resection between 1986 and 1990, and in 4 metastatic tumors obtained from autopsy. As compared to corresponding healthy lung parenchyma, both nm23-HI and nm23-H2 transcript levels were elevated in 37 of 41 tumors. The increases in nm23 mRNA expression were stronger in advanced stages of squamous-cell carcinoma, large-cell carcinoma, sarcoma and carcinoids than in early stages of the respective tumor types. Within stages I and II of squamous-cell carcinoma, significantly higher nm23 mRNA levels were found in poorly differentiated tumors than in moderately differentiated ones. Moreover, an inverse correlation between nm23 expression and disease-free survival of the patients was observed. In conclusion, our results indicate that the increased nm23 expression in the analysed tumors is not consistent with the proposed metastasis-suppressor function, but the 2 nm23 genes nevertheless may be implicated in the mechanism of tumor progression.

The nm23 gene is a potential metastasis-suppressor gene originally identified in a murine melanoma line. Several investigators have reported the probable inverse association of nm23 expression with disease prognosis and/or metastasis. Since there are now 2 known isotypes of human nm23, namely nm23-HI and -H2, we immunohistochemically examined expression of these isotypes in human breast-cancer tissues using monoclonal antibodies (MAbs) specific for each isotype protein. We also analyzed expression of c-erbB-2 in the same collection of cancer tissues, in order to examine the significance of nm23 expression in comparison with c-erbB-2 expression. Of 130 tumors from breast-cancer patients, 73 (56%) and 69 (53%) positively expressed nm23-HI and -H2 respectively. Expression of c-erbB-2 was positive in 36 (28%). Expression of nm23-HI, but not nm23-H2, was inversely associated with lymph-node metastasis (p < 0.01). Expression of c-erbB-2 was associated with Tnm stage, tumor size and lymph-node metastasis (p < 0.01, p < 0.05 and p < 0.05 respectively). Overall survival was better (p = 0.014) in patients in whom expression of nm23-HI was positive than in those in whom it was negative. In multivariate analyses using a Cox's proportional-hazards regression model with 9 variables, nm23-HI showed the fourth greatest contribution to patient survival following lymph-node metastasis, Tnm stage and menopausal status. No significant contribution was shown for c-erbB-2 expression. nm23-HI, but not nm23-H2, may perform a role in disease prognosis in addition to its participation in cancer metastasis. It may have value for predicting long-term survival of human breast-cancer patients.

The product of the nm23-H1 gene, reported to be a metastatic suppressor gene, was recently identified as the nucleoside diphosphate (NDP) kinase A, and was found to be overexpressed in several types of malignant tumors as compared with normal tissues. In order to determine whether NDP-kinase expression serves as a marker for metastatic potential and whether hyperproliferation of neoplastic cells would correlate with expression, we analyzed NDP-kinase levels and activity by immunohistochemical staining and by an enzymatic assay in 13 benign and 98 malignant breast-tissue specimens. Our results confirm that NDP-kinase expression increases in malignant cells of breast carcinomas, but cannot be considered as a biological marker of metastatic dissemination. No correlation was found between NDP-kinase activity and S phase, taken as an index of cell proliferation. Moreover, no correlation was observed between NDP-kinase activity and tumor size, histoprognostic index, estrogen receptors or progesterone receptors. The mechanism of over-expression of NDP in malignant cells and its role in tumor progression remain to be determined.

The NM23-Hl gene is a putative tumor suppressor gene that may be important in the metastasic process. Recent genetic and immunological data indicate that the NM23-Hl gene encodes a protein with nucleoside diphosphate (NDP) kinase activity. The mapping of NM23-Hl by panels of rodent-human somatic cell hybrids and in situ hybridization showed that the gene is located in human chromosome band 17q22. A two-allele polymorphism with BglII was demonstrated.