To verify the differential expression of non-metastasis cell 3 (NME3) protein in HL-60 cells when they were induced to differentiate into monocyte and granulocyte like cells, and study its value in diagnosis of acute myeloid leukemia, all-trans retinoic acid (ATRA) and a new steroidal drug NSC67657 were employed to induce acute myeloid leukemia HL-60 cells into monocyte and granulocyte like cells. Then the cell differentiating direction was observed by chemical staining, the degree of differentiation was determined by surface antigen CD11b/CD14 detection, and the apoptosis was excluded by phosphatidylserine valgus analysis, by which cellular differentiating model was constructed. Furthermore, RT-PCR and Western blot were employed to verify the differentially expression of NME3 before and after differentiation of HL-60 cells. At last, samples from bone marrow nucleated cells of 26 patients with myeloid leukemia, which were diagnosed definitely by clinical doctors, and 5 normal people were chosen. Then the expressing trend of NME3 protein in these testing groups was analyzed by means of comparison. The results showed that ATRA (2 µmol/L for 5 d) and NSC67657 (10 µmol/L for 5 d) could induce HL-60 cells to differentiate into monocyte and granulocyte like cells above 90% without cell apoptosis. The expression of NME3 gene and protein were down-regulated by the inducers, which was accorded with the screening results that was got using proteomics technology in the former research. The expression of NME3 protein in bone marrow from acute myeloid leukemia patients was elevated significantly as compared to normal persons. It is concluded that the expression level of NME3 protein is down-regulated after cellular differentiation, according with the changing trend in leukemia patients, which imply that NME3 protein may be a potential biomarker for diagnosis of acute myeloid leukemia.
Adolescent ; Adult ; Aged ; Bone Marrow ; metabolism ; Case-Control Studies ; Child ; Child, Preschool ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; genetics ; metabolism ; Mesylates ; pharmacology ; Middle Aged ; NM23 Nucleoside Diphosphate Kinases ; metabolism ; Steroids ; pharmacology ; Tretinoin ; pharmacology ; Young Adult

NM23 is a family of structurally and functionally conserved proteins known as nucleoside diphosphate kinases (NDPK). There is abundant mRNA expression of NM23-H1, NM23-H2, or a read through transcript (NM23-LV) in the primary sites of hepatocellular carcinoma (HCC). Although the NM23-H1 protein is implicated as a metastasis suppressor, the role of NM23-H2 appears to be less understood. Thus, the aim of this study was to examine whether NM23-H2 is associated with hepatocarcinogenesis. The level of NM23-H2 expression in tumor tissues and the surrounding matrix appeared to be independent of etiology and tumor differentiation. Its subcellular localization was confined to mainly the cytoplasm and to a lesser extent in the nucleus. Ectopic expression of NM23-H2 in NIH3T3 fibroblasts and HLK3 hepatocytes showed a transformed morphology, enhanced focus formation, and allowed anchorage-independent growth. Finally, NIH3T3 fibroblasts and HLK3 hepatocytes stably expressing NM23-H2 produced tumors in athymic mice and showed c-Myc over-expression. In addition, NF-kappaB and cyclin D1 expression were also increased by NM23-H2. Lentiviral delivery of NM23-H2 shRNA inhibited tumor growth of xenotransplanted tumors produced from HLK3 cells stably expressing NM23-H2. Collectively, these results indicate that NM23-H2 may be pro-oncogenic in hepatocarcinogenesis.
Animals ; Carcinoma, Hepatocellular/*enzymology/genetics/pathology ; Cell Line ; Cell Line, Tumor ; *Gene Expression Regulation, Neoplastic ; Humans ; Liver/*enzymology/metabolism/pathology ; Liver Neoplasms/*enzymology/genetics/pathology ; Mice ; Mice, Nude ; NIH 3T3 Cells ; NM23 Nucleoside Diphosphate Kinases/*genetics/metabolism

OBJECTIVETo study the chemosensitivity of lung adenocarcinoma cell line A549 cells to liposome-encapsulated paclitaxel after treatment by nm23-H1-small interference RNA (nm23-H1-siRNA) in vitro.

METHODSThe A549 cells were divided into two groups: non-transfected group and nm23-H1-siRNA-transfected group. Western blot analysis was used to detect the expression of nm23-H1. MTT and flow cytometry were used to determine the cell mortality rate, apoptosis rate and cell cycle after liposome-encapsulated paclitaxel treatment in both groups.

RESULTSThe expression of nm23-H1 in A549 cells was significantly decreased after transfection with nm23-H1-siRNA. After treatment for 48 hours with liposome-encapsulated paclitaxel, the cell mortality rate was increased with the increasing concentration of liposome-encapsulated paclitaxel in both groups, but increased higher in the nm23-H1-siRNA-transfected group. When the concentration of liposome-encapsulated paclitaxel was above 5 µg/ml, the cell mortality rate was significantly higher than that in the non-transfected group (P < 0.05). The proportion of apoptotic cells also increased in the nm23-H1-siRNA-transfected group, compared with that of the non-transfected group (t = 3.812, P < 0.05), while the proportion of cells at S and G(2)/M phase decreased after transfection with nm23-H1-siRNA (S phase:t = 8.356, P < 0.05; G(2)/M phase:t = 7.256, P < 0.05).

CONCLUSIONSNm23-H1 is related with the chemoresistance to liposome-encapsulated paclitaxel in lung adenocarcinoma cell line A549 cells. Inhibition of the expression of nm23-H1 by nm23-H1-siRNA can improve the in vitro chemosensitivity of A549 cells to liposome-encapsulated paclitaxel.

Adenocarcinoma ; metabolism ; pathology ; Antineoplastic Agents, Phytogenic ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Cell Cycle ; drug effects ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Humans ; Lung Neoplasms ; metabolism ; pathology ; NM23 Nucleoside Diphosphate Kinases ; genetics ; metabolism ; Paclitaxel ; administration & dosage ; pharmacology ; RNA, Small Interfering ; genetics ; Transfection

This experimental study sought to find out the inhibitory effects of Ad-GFP-nm23-H1 on proliferation and metastasis of human colorectal carcinoma cell line Lovo, and, further, to gain an insight into some theoretical and methodical basis for instituting nm23-H1 gene therapy of cancers. MTT assay and Transwell chamber were used to detect the rates of proliferation and invasion as well as the adhesion of Lovo cells in vitro. The results demonstrated that the proliferation inhibition rates of Lovo cells treated with Ad-GFP-nm23-H1 of 10(10) PFU/ml, 10(9) PFU/ml and 10(8) PFU/ml were 84.9% +/- 1.51%, 48.5% +/- 7.23% and 22.5% +/- 5.47%, that the adherence inhibition rates of Lovo cells treated with Ad-GFP-nm23-H1 of 10(10) PFU/ml, 10(9) PFU/ml and 10(8) PFU/ml were 70.3% +/- 2.40%, 60.1% +/- 5.68% and 18.5% +/- 3.61%, and that the invasiveness inhibition rates of Lovo cells treated with Ad-GFP-nm23-H1 of 10(10) PFU/ml, 10(9) PFU/ml and 10(8) PFU/ml were 83.2% +/- 5.71%, 52.2% +/- 6.94% and 28.1% +/- 8.21%. These data suggested that Ad-GFP-nm23-H1 exerted significant inhibitory effects on the proliferation and metastasis of human colorectal carcinoma cell line Lovo in a dose-dependent way.
Adenoviridae ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; genetics ; pathology ; Gene Expression Regulation, Neoplastic ; Genetic Therapy ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; NM23 Nucleoside Diphosphate Kinases ; genetics ; metabolism

OBJECTIVE: To study the proteins related to paclitaxel-resistant of ovarian cancer cell line. METHODS: The total proteins of paclitaxel-resistant and paclitaxel-sensitive human ovarian cancer cell lines were separated by 2-dimensional gel electrophoresis (2-DE). The differentially expressed proteins were analyzed using image analysis software. The differential proteins were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Western blot was used to determine the differential expression levels of the 2 proteins. RESULTS: Forty differentially expressed proteins were found by image analysis software, and 24 differential proteins were identified by mass spectrometry. These proteins included proliferation cell nuclear antigen (PCNA), nm23, prohibitin (PHB), annexin, alpha-enolase, heat shock protein (HSP), and so on. CONCLUSION Twenty-four proteins in human ovarian cancer cell lines of paclitaxel-resistant and paclitaxel-sensitive are found by proteomic techniques, which may be involved in the paclitaxel-resistance of human ovarian cancer cells.
Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Line, Tumor ; Drug Resistance, Neoplasm ; genetics ; Electrophoresis, Gel, Two-Dimensional ; Female ; Humans ; NM23 Nucleoside Diphosphate Kinases ; analysis ; Ovarian Neoplasms ; genetics ; metabolism ; pathology ; Paclitaxel ; pharmacology ; Proliferating Cell Nuclear Antigen ; analysis ; Proteome ; analysis ; Proteomics ; methods ; Repressor Proteins ; analysis

A single-stranded oligonucleotides containing a 6 histidine sequence, a hydroxylamine cleavage site, a thrombin cleavage site, and stop codon TAA were inserted into the polylinker's downstream of prokaryotic expression vector pBV220 between BamHI and PstI. The resultant vector is named pBV223. Proteins expressed in this vector will have a 6 histidine tail as affinity handy fused to their C terminus and can be quickly purified by one step immobilized metal affinity chromatography (IMAC). This plasmid is verified by restriction map and DNA sequencing. Subsequently, the metastasis suppressor gene nm23-H1 cDNA (without the stop codon) was cloned into vector PBV223 in frame with the 6-histidine sequence, hydroxylamine and thrombin cleavage sites. The soluble nm23-H1 fusion protein was successfully induced in the bacterial DH5a and easily purified with Ni chromatograph. These results indicated that the strategy to clone the single-stranded oligonucleotides directly into the restriction sties between BamH I and Pst I in the pBV220 vector is the simplest and cost-effective method.
Base Sequence ; Chromatography, Affinity ; methods ; Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Molecular Sequence Data ; Mutation ; NM23 Nucleoside Diphosphate Kinases ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

Carcinoma ; metabolism ; Cell Differentiation ; genetics ; Gallbladder Neoplasms ; genetics ; metabolism ; Gene Expression Regulation, Neoplastic ; Humans ; Kangai-1 Protein ; genetics ; metabolism ; NM23 Nucleoside Diphosphate Kinases ; genetics ; metabolism

To purify recombinant human nucleoside diphosphate kinase A (rhNDPK-A) efficiently in pilot scale, cells of rhNDPK-A producing E. coli were homogenized by high pressure under 4 degrees C, 950 Pa. The insoluble debris was removed by microfiltration and the soluble portion was concentrated by ultrafiltration. The resulted crude sample was loaded on DEAE-sepharose Fast Flow. The target fraction was collected and then load on Cibacron Blue 3GA Sepharose CL-4B. Eluted with buffer containing ATP from the AC column, rhNDPK-A was polished with ultrafiltration. The results showed that after homogenized 2 rounds, 1500g cells of E. coli brought crude sample containing 47.6g NDPK-A. Treated with microfiltration and ultrafiltration, 27.3g of NDPK-A were recovered from this bacteria homogenate. After 2-step purification with column chromatography and then polished with ultrafiltration, 17.2 g rhNDPK-A were collected with purity of 96.3%. The recovery of the whole purification process was 36.2%, and the productivity of rhNDPK-A was 1.15 g per 100 g wet cells. Comparing the recovery of each purification step, it was found that the recovery of polish is higher than that of affinity chromatography, which is higher than that of ion exchange chromatography. The limit step was the process of sample pretreatment among the 4 purification steps. Combine with the fermentation results reported before, it was deduced that the productivity of rhNDPK-A was 510 mg/L. In conclusion, an easily controlled purification condition with high yield provides material for the translation researches of NDPK; In addition, it was suggested the crucial step determine the recovery of non-secretive recombinant proteins might be the process of sample pretreatment, not be the process of column chromatography.
Chromatography, Affinity ; Chromatography, Ion Exchange ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Humans ; NM23 Nucleoside Diphosphate Kinases ; genetics ; metabolism ; Pilot Projects ; Recombinant Proteins ; isolation & purification ; metabolism ; Ultrafiltration

OBJECTIVETo study the metastasis feature of the primary and metastatic lymph node lesions in supraglottic or hypopharyngeal cancer.

METHODSThe expression of CD44 and nm23-H1 in specimens from the primary and metastatic lymph node lesions of the 41 cases with supraglottic or hypopharyngeal cancer were studied with immunohistochemistry method and flow cytometry.

RESULTSNo correlation was found between the expression of CD44, nm23-H1 and the tumor differentiation of the supraglottic or hypopharyngeal cancer, but their expression related with the clinical staging. The CD44 and nm23-H1 positive expression rates in the primary and metastatic lymph node lesions were 75.6% (31/41), 85.4% (35/41) and 34.1% (14/41), 26.8% (11/41) respectively (P >0.05). The average fluorescence index of CD44 and nm23-H1 in the primary and metastatic lymph node lesions were 1.27 +/- 0.18, 1.33 +/- 0.16 and 1.11 +/- 0.19, 1.08 +/- 0.15 (x +/- s) respectively (P >0.05).

CONCLUSIONSThe expressions of CD44 and nm23-H1 in the metastatic lymph node tumor had no difference compared with that in primary tumor of the supraglottic or hypopharyngeal cancer. The difference of metastasis potentials between the primary and metastatic lymph node lesions in the same patient was not proved in this study and should be further investigated from multiple oncogens markers.

Adult ; Aged ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Female ; Humans ; Hyaluronan Receptors ; genetics ; Hypopharyngeal Neoplasms ; metabolism ; pathology ; Laryngeal Neoplasms ; metabolism ; pathology ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; NM23 Nucleoside Diphosphate Kinases ; genetics ; Neoplasm Staging

OBJECTIVETo study the prognostic predictor for nasopharyngeal carcinoma (NPC).

METHODSThe expressions of nm23-H1 and vessel endothelium growth factor (VEGF) protein were examined by immunohistochemistry S-P staining in 108 NPC tissues, the expression of nm23-H1 and VEGF protein in NPC tissues with clinical stage of NPC, radiosensitivity of tumor, survival rate of patients, relapse and metastasis of carcinoma were studied.

RESULTSThe positive rate of nm23-H1 and VEGF was 48.1% and 59.3% respectively. The clinical staging, metastatic potential of lymph nodes were correlated with low-level expression of nm23-H1 protein. The patients with negative nm23-H1 expression had worse prognosis than those with positive nm23-H1 expression. The clinical staging, metastatic potential and poor sensitivity of radiotherapy were correlated with high level expression of VEGF protein. The patients with positive VEGF expression had worse prognosis than those with negative VEGF expression. The expression of nm23-H1 protein was negatively correlated with the expression of VEGF protein (r = -0.577, P < 0.05).

CONCLUSIONSThe low level expression of nm23-H1 protein and the high level expression of VEGF protein may be associated with the development and poor prognosis of NPC.

Adolescent ; Adult ; Aged ; Carcinoma, Squamous Cell ; metabolism ; pathology ; Female ; Humans ; Lymphatic Metastasis ; Middle Aged ; NM23 Nucleoside Diphosphate Kinases ; genetics ; metabolism ; Nasopharyngeal Neoplasms ; metabolism ; pathology ; Neoplasm Staging ; Prognosis ; Survival Rate ; Vascular Endothelial Growth Factor A ; genetics ; metabolism ; Young Adult