Clinical significance of nm23-H1 proteins expressed on cell surface in non-Hodgkin's lymphoma.

The nm23 gene was isolated as a metastasis suppressor gene that exhibits low expression in high-level metastatic cancer cells. Its gene is related to the prognosis of acute myelogenous leukemia (AML) and non-Hodgkin's lymphoma (NHL). In this study, we examined the expression of nm23-H1 protein on the lymphoma cell surface of NHL. In 28 of 108 cases (25.9%), we observed > or = 20% of cell surface nm23-H1 protein expression and expression was especially high in peripheral T cell lymphomas and extranodal NK/T cell lymphomas. We also observed a significant correlation between serum nm23-H1 level and cell surface nm23-H1 expression levels. In patients with high levels of cell surface nm23-H1 expression, overall and progression-free survival rates were significantly lower than those in patients with low surface nm23-H1 expression levels. When surface nm23-H1 and serum nm23-H1 were combined, patients with high levels of both exhibited a poorer prognosis than patients with a high level of one or the other. These results indicate that in addition to serum nm23-H1, cell surface nm23-H1 may be used as a prognostic factor in planning a treatment strategy. The nm23-H1 protein appears to be intimately related to biological aggressiveness of lymphoma and, therefore, might be a molecular target of NHL treatment.

Elevated plasma level of differentiation inhibitory factor nm23-H1 protein correlates with risk factors for myelodysplastic syndrome.

We measured plasma nm23-H1 level (nm23-H1), a differentiation inhibitory factor, by an enzyme-linked immunosorbent assay (ELISA) in patients with aplastic anemia (AA) and myelodysplastic syndrome (MDS). The nm23-H1 in AA was not significantly elevated when compared to normal subjects (6.66 +/- 1.20 ng/ml vs 5.13 +/- 0.81 ng/ml; P = 0.274). In contrast, MDS patients had significantly high levels of nm23-H1 compared not only to normal subjects (11.16 +/- 1.42 vs 5.13 +/- 0.81 ng/ml; P = 0.0004) but also to those of the AA group (11.16 +/- 1.42 ng/ml vs 6.66 +/- 1.20 ng/ml; P = 0.018). In the MDS group of patients, no significant difference was observed in the nm23-H1 levels between patients with refractory anemia (RA) and RA with excess blasts (RAEB)/RAEB in transformation (10.71 +/- 1.61 ng/ml vs 9.24 +/- 2.66 ng/ml; P = 0.672). Of the patients with RA, patients with low risk according to the International Prognostic Scoring System (IPSS) had significantly low levels of nm23-H1 compared to those of IPSS INT-1 level cases (6.40 +/- 1.36 ng/ml vs 13.05 +/- 2.50 ng/ml; P = 0.0028), suggesting that nm23-H1 may be useful as a prognostic marker for MDS, especially in low risk patients.

Serum nm23-H1 protein as a prognostic factor for indolent non-Hodgkin's lymphoma.

Standard chemotherapy has been ineffective for improving the poor 10-year survival rate of patients with indolent lymphoma. However, a wider choice of therapeutic modalities has become recently available, including immunotherapy with monoclonal antibodies and allogeneic peripheral blood stem cell transplantation. Accordingly, a sensitive prognostic indicator is required to identify high-risk patients and to help design new therapeutic approaches for them. We previously reported that the serum nm23-H1 protein level was an independent prognostic factor for aggressive lymphoma. The present study was performed to assess the clinical implications of this protein on indolent lymphoma and whether it can be used to classify the aggressiveness of the disease in order to assist in the individualization of therapy. A total of 130 patients with indolent lymphoma were enrolled in this multicenter study. The serum nm23-H1 protein level was significantly higher in patients with indolent lymphoma than in a normal control group. In addition, indolent lymphoma patients with higher nm23-H1 levels had worse overall and progression-free survival rate than those with lower nm23-H1 levels. Therefore, nm23-H1 in serum may be useful for identifying a distinct group of patients at high risk.

Induction of differentiation of human myeloid leukemia cells by immunosuppressant macrolides (rapamycin and FK506) and calcium/calmodulin-dependent kinase inhibitors.

OBJECTIVE: Potent immunosuppressants, such as rapamycin, FK506, and ascomycin, are known to regulate the phosphorylation of proteins. The purpose of this study was to investigate the effects of these immunosuppressants on differentiation of several human myeloid leukemic cell lines. MATERIALS AND METHODS: Human myeloid leukemic cell lines were cultured with each immunosuppressant, and several differentiation markers were assayed. RESULTS: Rapamycin effectively induced granulocytic differentiation of human myeloid leukemic HL-60 and ML-1 cells. In addition to morphologic differentiation, it also induced nitroblue tetrazolium reduction, lysozyme activity, and expression of CD11b in HL-60 cells. The commitment to differentiation was observed after treatment with rapamycin for 1 day, indicating that the effect of rapamycin was irreversible. FK506 and ascomycin induced differentiation of HL-60 cells, but at higher concentrations than rapamycin. A calcium/calmodulin-dependent kinase (CaMK) was copurified with FKBP52 immunophilin, a binding protein of immunosuppressants. We also found that the CaMK inhibitors KN62 and KN93 induced differentiation of HL-60 cells. Rapamycin and CaMK inhibitors induced differentiation of human myeloid leukemia ML-1 and K562, but not of other cell lines such as NB4, U937, or HEL. CONCLUSION: Immunosuppressants and CaMK inhibitors induced differentiation of HL-60, ML-1, and K562 cells.

Serum nm23-H1 protein as a prognostic factor in aggressive non-Hodgkin lymphoma.

Advances in chemotherapy have led to a favorable long-term prognosis in approximately 50% of patients with aggressive non-Hodgkin lymphoma (NHL). However, the remaining patients do not enjoy such prolonged survival after standard treatment. New prognostic factors are needed to define this poor-prognosis group and to plan an appropriate treatment strategy. It has been reported that serum nm23-H1 protein may be a new prognostic factor for aggressive NHL. In the present study involving multiple institutions and a large number of patients, the level of nm23-H1 protein was compared among different types of lymphoma; it was lowest for indolent lymphoma, followed by aggressive lymphoma and then highly aggressive lymphoma. In addition, patients with aggressive NHL and higher nm23-H1 levels had worse overall and progression-free survival rates than those with lower nm23-H1 levels. The nm23-H1 level was also compared between patients with diffuse large B-cell lymphoma and patients with peripheral T-cell lymphoma. The results suggest that the level of nm23-H1 could serve as a prognostic factor in both groups. Moreover, the prognosis of lymphoma patients could be ascertained even more precisely by combining soluble interleukin-2 receptor or soluble CD44 and nm23-H1 levels. A multivariate analysis confirmed that the nm23-H1 level is an independent and important prognostic factor in aggressive NHL. Therefore, it may provide useful information for clinicians to determine the appropriate therapy for each type of lymphoma.

Induction of differentiation of human myeloid leukemia cells by novel synthetic neurotrophic pyrimidine derivatives.

OBJECTIVE: Some pyrimidine analogues have been found to induce differentiation of several human myeloid leukemia cells. Newly synthesized heterocyclic pyrimidine derivatives promote neurite outgrowth and survival in neuronal cell lines. In this study, the growth-inhibiting and differentiation-inducing effects of these pyrimidine derivatives on human myeloid leukemia cells were examined. MATERIALS AND METHODS: Several myeloid leukemia cells were cultured with novel heterocyclic pyrimidine derivatives. Cell differentiation was determined by nitroblue tetrazolium-reducing activity, morphologic changes, expression of CD11b, lysozyme activity, and hemoglobin production. RESULTS: MS-430 (2-piperidino-5,6-dihydro-7-methyl-6-oxo (7H) pyrrolo [2,3-d] pyrimidine maleate) effectively induced HL-60 cells into mature granulocytes. MS-430 activated the mitogen-activated protein kinase (MAPK) of the cells before causing granulocytic differentiation. MAPK activation was necessary for MS-430-induced differentiation, because PD98059, an inhibitor of MAPK kinase, suppressed the differentiation induced by MS-430. MS-430 also induced monocytic differentiation of THP-1, P39/Tsu, and P31/Fuj leukemia cells, but did not affect erythroid differentiation of K562 or HEL cells. CONCLUSIONS: MS-430 potently induces differentiation of some myelomonocytic leukemia cells. This novel synthesized pyrimidine compound shows promise as a therapeutic agent for treatment of leukemia and as a neurotrophic drug.

Antileukemic efficacy of 2-deoxycoformycin in monocytic leukemia cells.

2'-Deoxycoformycin (dCF) as a single agent has been reported to be less effective against myeloid than against lymphoid malignancies in clinical trials. However, previous studies have shown that in the presence of 2'-deoxyadenosine (dAd), human monocytoid leukemia cell lines are much more sensitive to dCF with regard to the inhibition of cell proliferation. Thus, dCF might be useful for treating monocytoid leukemia with the aid of dAd analogs. The antiproliferative effects of dCF in combination with dAd or its derivatives were examined on normal and malignant blood and bone marrow cells. In the presence of 10 micromol/L dAd, the concentration of dCF required to inhibit the viability of primary monocytoid leukemia cells was much lower than that required to inhibit normal or non-monocytoid leukemic cells. Among the dAd analogs, 9-beta-D-arabinofuranosyladenine (AraA) was also effective in combination with dCF. Athymic nude mice were inoculated with human monocytoid leukemia U937 cells and treated with dCF or a dAd analog or both. Although dCF alone slightly but significantly prolonged the survival of mice inoculated with U937 cells, combined treatment with dCF and AraA markedly prolonged their survival. These data suggest that the combination of dCF and AraA may be useful for the clinical treatment of acute monocytic leukemia. (Blood. 2000;96:1512-1516)

Plasma levels of the differentiation inhibitory factor nm23-H1 protein and their clinical implications in acute myelogenous leukemia.

A previous study reported that a nondifferentiating myeloid leukemia cell line produced differentiation-inhibiting factors. One of the factors was purified as a homologue of the nm23 genes. The nm23 genes were overexpressed in acute myelogenous leukemia (AML) cells, and a higher level of nm23 gene expression was correlated with a poor prognosis in AML. The present study determined the plasma levels of nm23-H1 protein by enzyme-linked immunosorbent assay and assessed the association between this level and the clinical outcome in 102 patients with AML. The plasma concentration of nm23-H1 was higher in patients with AML than in normal controls (P =.0001). Plasma nm23-H1 levels were correlated with the product of the intracellular nm23 messenger RNA (mRNA) level and the white blood cell count, but not with the mRNA level alone. Therefore, nm23-H1 plasma levels probably depend on the total mass of leukemic cells overexpressing the nm23-H1 gene. Overall survival was lower in patients with higher plasma nm23-H1 levels than in those with lower levels. Multivariate analysis using the Cox proportional hazard model showed that elevated plasma nm23-H1 levels significantly contributed to the prognosis of AML patients. Furthermore, the plasma nm23-H1 levels were investigated in 70 patients with other hematologic neoplasms, including 6 with acute lymphoblastic leukemia, 13 with chronic myelogenous leukemia, and 12 with myelodysplastic syndrome. Plasma nm23-H1 levels were significantly higher in all of these hematologic neoplasms than in normal controls. Increased plasma levels of nm23-H1 may have prognostic value in these hematologic malignancies as well as in AML.

Anticancer derivative of butyric acid (Pivalyloxymethyl butyrate) specifically potentiates the cytotoxicity of doxorubicin and daunorubicin through the suppression of microsomal glycosidic activity.

Pivalyloxymethyl butyrate (AN9) is an anticancer derivative of butyric acid. In this study, doxorubicin (DXR) and AN9 synergistically inhibited the growth of lymphoma and lung carcinoma cells, whereas there was no synergy between AN9 and antimetabolites. AN9 did not affect the intracellular uptake of DXR. Among anthracyclines and their derivatives, the synergistic effect was prominent in compounds with a daunosamine moiety, suggesting that AN9 may affect the catabolism of these compounds. The degradation of DXR in the extract from AN9-treated cells was much less than that in extract from untreated cells. AN9 did not directly inhibit the enzyme activity but rather suppressed expression of the enzyme. With respect to the expression of drug resistance-related genes, there was no significant difference between untreated and AN9-treated cells. However, AN9 significantly down-regulated the levels NADPH-cytochrome P450 reductase and DT-diaphorase mRNA in the presence of DXR but not the level of xanthine oxidase mRNA. The enhancement of the sensitivity to anthracyclines was closely associated with the suppression of the mRNA expression.

Prognostic implications of the differentiation inhibitory factor nm23-H1 protein in the plasma of aggressive non-Hodgkin's lymphoma.

The outcome of patients with non-Hodgkin's lymphoma has been improved by current approaches to treatment. Nevertheless, many patients either do not have a complete remission or ultimately relapse. To identify such patients, it is important to be able to predict the outcome. We previously found that the differentiation inhibitory factor/nm23 was correlated with the prognosis of acute myeloid leukemia. To examine the prognostic effect of nm23 on non-Hodgkin's lymphoma, we established an enzyme-linked immunosorbent assay procedure to determine nm23-H1 protein levels in plasma and assessed the association of this protein level with the response to chemotherapy, overall survival, and progression-free survival in patients with aggressive non-Hodgkin's lymphoma. The plasma concentration of nm23-H1 was significantly higher in patients with malignant lymphoma than in normal controls, especially in aggressive non-Hodgkin's lymphoma. The complete remission rate in patients with higher nm23-H1 levels was significantly worse than that in patients with lower nm23-H1 levels. Overall survival and progression-free survival were also lower in patients with higher nm23-H1 levels than in those with lower levels. The 3-year survival rates in patients with low and high nm23-H1 levels were 79.5% and 6. 7% (P =.0001). A multivariate analysis of prognostic factors showed that the plasma nm23-H1 level was independently associated with the survival and progression-free survival. An elevated plasma nm23-H1 concentration predicts a poor outcome of advanced non-Hodgkin's lymphoma. Therefore, nm23-H1 in plasma may be useful for identifying a distinct group of patients at very high risk.

Role of CD14 expression in the differentiation-apoptosis switch in human monocytic leukemia cells treated with 1alpha,25-dihydroxyvitamin D3 or dexamethasone in the presence of transforming growth factor beta1.

Transforming growth factor beta (TGF-beta) enhanced the growth-inhibitory activities of dexamethasone (Dex) and 1alpha,25-dihydroxyvitamin D3 (VD3) on human monocytoid leukemia U937 cells. TGF-beta and VD3 synergistically increased the expression of differentiation-associated markers such as the CD11b and CD14 antigens, whereas TGF-beta and Dex did not. On the other hand, TGF-beta and Dex synergistically increased the number of Apo2.7-positive cells, which represents the early stage of apoptosis, whereas TGF-beta and VD3 did not, suggesting that TGF-beta enhanced apoptosis with Dex and enhanced monocytic differentiation with VD3. In the presence of TGF-beta, the retinoblastoma susceptibility gene product, pRb, was synergistically dephosphorylated by Dex as well as VD3. TGF similarly enhanced the expression of the p21Waf1 gene in U937 cells treated with Dex and VD3. TGF-beta dose-dependently increased the expression of Bcl-2 and Bad and decreased the expression of Bcl-X(L) in U937 cells. Dex enhanced the down-regulation of Bcl-X(L) expression in TGF-beta-treated cells, whereas VD3 blocked this down-regulation of Bcl-X(L). However, the down-regulation of Bcl-X(L) by treatment with the antisense oligomer did not affect the apoptosis or differentiation of U937 cells. The apoptosis of CD14-positive cells was suppressed in the VD3 plus TGF-beta-treated cultures. These results suggest that the expression of CD14 is involved in the survival of differentiated cells.

Identity of human normal counterpart (MmTRA1b) of mouse leukemogenesis-associated gene (MmTRA1a) product as plasma membrane phospholipid scramblase and chromosome mapping of the human MmTRA1b/phospholipid scramblase gene.

We recently cloned a new leukemogenesis-associated gene MmTRA1a (Mm-1 cell derived transplantability-associated gene 1a, former name "TRA1") from a mouse leukemogenic and monocytic Mm-P cell cDNA library and also cloned its normal counterpart MmTRA1b (former name "NOR1") from a normal mouse kidney cDNA library. The mouse MmTRA1a is a truncated form of mouse MmTRA1b. Here we report the cloning of a cDNA (human MmTRA1b) homologous to the mouse MmTRA1b from a human monocytic U937 cell cDNA library. The human MmTRA1b cDNA predicts a peptide containing 318 amino acids with a calculated molecular weight of 35,047 Da. The predicted human MmTRA1b protein sequence shared 78% amino acid identity with the mouse counterpart (328 amino acids). Both the human homologue and mouse MmTRA1b protein but not MmTRA1a protein possess a proline-rich domain at the N-terminal end. The human MmTRA1b gene was mapped to chromosome 3q23. Expression of the human homologue was increased during differentiation of U937 cells induced by most typical differentiation inducers. Moreover, predicted amino acid sequence analysis of human MmTRA1b cDNA revealed perfect identity with the human plasma membrane phospholipid scramblase that is required for transbilayer movement of membrane phospholipids. These results provide new information on the possible roles of MmTRA1b/phospholipid scramblase and the truncated MmTRA1a in the leukemogenesis and differentiation of monocytic leukemia cells.

Induction of differentiation of myelogenous leukemia cells by humulone, a bitter in the hop.

The active form of vitamin D, 1alpha,25-dihydroxyvitamin D3 (VD3), inhibits proliferation and induces differentiation of myelomonocytic leukemia cells, but its clinical use is limited by the adverse effect of hypercalcemia. VD3 mobilizes calcium stores from bone by inducing the dissolution of bone mineral and matrix. We have recently found that humulone, a bitter in the hop extract for beer brewing, effectively inhibits bone resorption. In this study we examined the effect of humulone on the differentiation of human myelogenous leukemia cells. Humulone alone inhibited the growth of monoblastic leukemia U937 cells while only slightly increasing differentiation markers such as nitroblue tetrazolium (NBT)-reducing and lysozyme activities. Humulone effectively enhanced the differentiation-inducing action of VD3. Other myelomonocytic leukemia cells were induced to differentiate by VD3 and this was also enhanced by humulone. Since humulone is a less-toxic inhibitor of bone resorption, the combination of humulone and VD3 may be useful in differentiation therapy of myelomonocytic leukemia.

Elevated expression of differentiation inhibitory factor nm23 mRNA in monoblastic crisis of a patient with chronic myelogenous leukemia.

Differentiation inhibitory factor nm23 gene has been found to be expressed in high quantities in acute myelogenous leukemia (AML), especially in acute monocytic leukemia (AML-M5) and is suggested as a new prognostic factor in AML-M5. We report an example of elevated expression of nm23 mRNA in a patient with chronic myelogenous leukemia (CML) who developed monoblastic crisis. Relative levels of nm23-H1 and -H2 mRNA extracted from the patient's peripheral blood mononuclear cells and bone marrow mononuclear cells were measured by quantitative reverse transcriptase polymerase chain reaction. The level of nm23-H1 mRNA in CML cells at the chronic phase was as high as that in bone marrow cells from healthy volunteers. The mRNA level of nm23-H2 was slightly below the normal level. At blastic crisis, however, expression of both nm23-H1 and -H2 mRNA was elevated to about three to nine times of that at the chronic phase. Proliferated blastic cells were positive for non-specific esterase, and the serum lysozyme level was elevated and diagnosed as monoblastic crisis. The patient received combined chemotherapy but response was partial. These findings are compatible with our previous report that nm23 gene is overexpressed in monocytic leukemia.

Combined analysis of differentiation inhibitory factor nm23-H1 and nm23-H2 as prognostic factors in acute myeloid leukaemia.

Differentiation inhibitory factor (nm23 protein) inhibited the induction of the differentiation of various leukaemic cell lines. We previously reported that nm23 genes (H1 and H2) were overexpressed in acute myelogenous leukaemia (AML) and nm23-H1 expression predicted the prognosis of AML, especially AML-M5. To clarify the correlation between French-American-British (FAB) classification and nm23 expression level and to clarify the involvement of nm23-H2 and nm23-H1 in patient survival, we investigated the relative levels of nm23-H1 and -H2 mRNA in 76 AML samples using the reverse transcriptase-polymerase chain reaction. We confirmed that the expression of both nm23-H1 and -H2 genes in AML samples from three different hospitals was significantly higher than that in normal blood cells (P < 0.0005). Overexpression of nm23-H1 was observed in each FAB AML-M1, -M2, -M3, -M4 or -M5 subtype, and the predictive effect of nm23-H1 expression on AML prognosis was shown in FAB AML-M2 and -M5 cases. Although overexpression of nm23-H2 was also found in each FAB subtype, the expression of nm23-H2 in AML-M1 and -M3 cells was not significantly higher than that in normal cells. Among AML subtypes, AML-M3 showed the lowest expression levels of both nm23 genes. To understand the relationship between nm23-H1 and -H2 expression levels, nm23 expression levels for all the AML cases were plotted and divided into four groups (group A, nm23-H1 and -H2 both high; B, both low; C, only nm23-H1 high; D, only nm23-H2 high). A statistically significant correlation between the levels of expression of nm23-H1 and -H2 was observed (r= 0.726). Most AML-M3 cases belonged to group B, but not other types of AML. Analysis of survival probability between the groups showed that group B survived for significantly longer compared with group A. Furthermore, AML-M3 cases survived for significantly longer compared with non-M3 cases in the same group B. These data suggest that low expression levels of both nm23-H1 and -H2 are associated with good prognosis in AML patients.

Evaluation by multivariate analysis of the differentiation inhibitory factor nm23 as a prognostic factor in acute myelogenous leukemia and application to other hematologic malignancies.

The differentiation inhibitory factor nm23 can inhibit the differentiation of murine and human myeloid leukemia cells. We recently reported that nm23 genes were overexpressed in acute myelogenous leukemia (AML), and a higher level of nm23-H1 expression was correlated with a poor prognosis in AML, especially in AML-M5 (acute monocytic leukemia). To evaluate the importance of nm23 expression as a prognostic factor in AML, we compared it with other putative prognostic factors in AML. An analysis of the correlation between nm23 expression and the clinical parameters of 110 patients with AML demonstrated that increased nm23-H1 mRNA levels were associated with resistance to initial chemotherapy and with reduced overall survival. Multivariate analysis using Cox's proportional hazard model also showed that elevated nm23-H1 mRNA levels significantly contributed to the prognosis of patients with AML. Especially in AML-M5, nm23-H1 status was the most important prognostic factor. Furthermore, to determine whether we can apply the results observed in AML to other hematologic malignancies, we investigated the relative levels of nm23-H1 and nm23-H2 transcripts in 149 patients with hematologic neoplasms, including 110 with de novo AML, 9 with de novo acute lymphoblastic leukemia, 14 with myelodysplastic syndrome, 16 with chronic myelogenous leukemia (CML), and 5 normal subjects by the reverse transcriptase-polymerase chain reaction. Expression of nm23-H1 was significantly higher in all the hematologic neoplasms, except CML in chronic phase, than in normal blood cells. nm23 may have a prognostic effect in these hematologic malignancies as well as in AML.

Growth inhibition and differentiation induction in human monoblastic leukaemia cells by 1alpha-hydroxyvitamin D derivatives and their enhancement by combination with hydroxyurea.

The active form of vitamin D, 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3), is a potent inducer of differentiation in myeloid leukaemia cells, but its clinical use is limited because of its hypercalcaemic activity. We examined the ability of 1,25(OH)2D3 in combination with several anti-cancer drugs to inhibit the proliferation of, and induce differentiation in, human monoblastic leukaemia U937 cells. Hydroxyurea (HU), cytarabine and camptothecin showed effective synergism with 1,25(OH)2D3 with regard to growth inhibition, while daunorubicin and etoposide had only modest synergistic effects. HU and cytarabine effectively enhanced nitroblue tetrazolium-reducing activity induced by 1,25(OH)2D3. HU also enhanced the morphological maturation and expression of CD11b and CD14 in cells treated with 1,25(OH)2D3. Among the anti-cancer drugs examined, HU had the greatest synergistic effects with 1,25(OH)2D3 with regard to growth inhibition and differentiation induction in U937 cells. HU also enhanced the differentiation of other myeloid leukaemia HL-60, ML-1, THP-1, P39/TSU, P31/FUJ and NB4 cells induced by 1,25(OH)2D3 and that of U937 cells induced by 24-epi-1,25(OH)2D2 and 1,25(OH)2D7. Interestingly, 1alpha(OH)D derivatives (1alpha-hydroxyvitamin D3, D2, D4 and D7) effectively induced the differentiation of monoblastic leukaemia U937, P39/TSU and P31/FUJ cells. HU also enhanced the growth inhibition and differentiation of U937 cells induced by 1alpha(OH)D derivatives. As 1alpha(OH)D derivatives preferentially act on monocytic cells, they may be useful in the treatment of acute monocytic leukaemia, both alone and in combination with HU.

Differentiation inhibitory factor Nm23 as a prognostic factor for acute myeloid leukemia.

The differentiation inhibitory factor nm23 inhibits the differentiation of murine and human myeloid leukemia cells. The inhibition of differentiation may be associated with the aggressive behavior of leukemia. To clarify the role of nm23 in human myeloid leukemia, we investigated the relative levels of nm23-H1, nm23-H2 and c-myc transcripts in bone marrow and blood samples from 110 patients with acute myeloid leukemia (AML) using the reverse transcriptase polymerase chain reaction. The expression levels of nm23-H1 and nm23-H2 in these AML samples were significantly higher than in normal blood cells, and a higher level of nm23-H1 expression was correlated with poor prognosis for AML patients. Analysis of the correlation between nm23 expression and clinical parameters demonstrated that increased nm23-H1 mRNA levels were associated with resistance to initial chemotherapy and reduced overall survival. Multivariate analysis of putative prognostic factors revealed that elevated nm23-H1 mRNA levels significantly influenced the prognosis of patients with AML, particularly in AML-M5.

TRA1, a novel mRNA highly expressed in leukemogenic mouse monocytic sublines but not in nonleukemogenic sublines.

Mouse monocytic Mm-A, Mm-P, Mm-S1, and Mm-S2 cells are sublines of mouse monocytic and immortalized Mm-1 cells derived from spontaneously differentiated, mouse myeloblastic M1 cells. Although these subline cells retain their monocytic characteristics in vitro, Mm-A and Mm-P cells are highly leukemogenic to syngeneic SL mice and athymic nude mice, whereas Mm-S1 and Mm-S2 cells are not or are only slightly leukemogenic. To better understand the molecular mechanisms of these levels of leukemogenicity, we investigated putative leukemogenesis-associated genes or oncogenes involved in the maintenance of growth, especially in vivo, by means of differential mRNA display. We isolated a fragment clone (15T01) from Mm-P cells. The mRNA probed with 15T01 was expressed at high levels in leukemogenic Mm-P and Mm-A cells but not in nonleukemogenic Mm-S1 and Mm-S2 cells. The gene corresponding to 15T01, named TRA1, was isolated from an Mm-P cDNA library. The longest open reading frame of the TRA1 clone predicts a peptide containing 204 amino acids with a calculated molecular weight of 23,049 D. The predicted TRA1 protein is cysteine-rich and contains multiple cysteine doublets. A putative normal counterpart gene, named NOR1, was also isolated from a normal mouse kidney cDNA library and sequenced. NOR1 cDNA predicts a peptide containing 234 amino acids. The sequence of 201 amino acids from the C-terminal NOR1 was completely identical to that of TRA1, whereas the remaining N-terminal amino acids (33 amino acids) were longer than that (3 amino acids) of TRA1 and the N-terminus of NOR1 protein contained proline-rich sequence. A similarity search against current nucleotide and protein sequence databases indicated that the NOR1/TRA1 gene(s) is conserved in a wide range of eukaryotes, because apparently homologous genes were identified in Caenorhabditis elegans and Saccharomyces cerevisiae genomes. Northern blotting using TRA1-specific and NOR1-specific probes indicated that TRA1 mRNA is exclusively expressed in leukemogenic but not in nonleukemogenic Mm sublines and normal tissues and also indicated that NOR1 mRNA is expressed in normal tissues, especially in kidney, lung, liver, and bone marrow cells but not in any Mm sublines. After leukemogenic Mm-P cells were induced to differentiate into normal macrophages by sodium butyrate, the normal counterpart, NOR1, was expressed, whereas the TRA1 level decreased. Furthermore, transfection of TRA1 converted nonleukemogenic Mm-S1 cells into leukemogenic cells. These results indicate that the TRA1 gene is associated at least in part with the leukemogenesis of monocytic Mm sublines.