OBJECTIVETo study the effect of matrix metalloproteinase 2 (MMP-2), membrane type 1 MMP (MT1-MMP) and tissue inhibitor of metalloproteinase 2 (TIMP-2) expressions on the in vitro invasive capacity of acute monocytic leukemia SHI-1 cells.

METHODSSHI-1, NB4, K562, M937 and THP-1 human leukemia cell lines were cultured in vitro. The mRNA and protein expressions of TIMP-2, MMP-2 and MT1-MMP in different cells were detected by quantitative RT-PCR and western blot. A retroviral vector carrying human TIMP-2 cDNA was constructed and transfected into SHI-1 cells. Three subclone cells (S1, S2 and S3) were screened by G418 and selected by limiting dilution. RNA interference (RNAi) was used to knock down the expression of MMP-2, MT1-MMP and TIMP-2. Cell invasion capacity was performed through a reconstituted human basement membrane assays. Zymography was used to analyze the expression of MMP-2 in the supernatant of co-culture.

RESULTSThe expressions of MMP-2, MT1-MMP and TIMP-2 in SHI-1 cells were higher than that in other leukemic cells at both mRNA and protein levels (P < 0.05). The amount of proMMP-2 and activated MMP-2 in the conditioned media from SHI-1 cells co-cultured with bone marrow stromal cells (BMSCs) was more than that from other cells (P < 0.05). The in vitro invasive capacity of SHI-1 cells were higher than that of other cells (P < 0.05). The mRNA levels of TIMP-2 were increased by about 3 fold, 2 fold and 1.5 fold in S1, S2 and S3 cells, respectively (P < 0.05), while the protein levels were by about 2.6 fold, 1.5 fold and 1.3 fold than that of SHI-1 cells, respectively (P < 0.01). The invasion rates of subclone cells demonstrated a 1.5 - 2.5 fold' elevation (P < 0.05) and activated MMP-2 from their supernatants increased by 1.5 - 2.0 fold (P < 0.01). The knock-down efficiency of siRNA was 85% to 98%. The down-regulation of TIMP-2, MMP-2 and MT1-MMP decreased the invasion rates of SHI-1 cells by 60% - 70%, 50% - 60% and 40% - 50%, respectively (P < 0.05). No activated MMP-2 in the supernatants from any knock-down cells could be found.

CONCLUSIONSSHI-1 cells constitutively overexpress MMP-2, MT1-MMP and TIMP-2 at both mRNA and protein levels. After co-cultured with BMSCs the SHI-1 cells increased MMP-2 activation and cell invasion. An increase of TIMP-2 expression in SHI-1 cells reflects an activating effect on cells invasion and MMP-2 activation.

Human monocyte leukemia cell line THP-1 was stimulated with lipopolysaccharide (LPS) to simulate the sepsis model and the expression of human glucocorticoid receptor-alpha (GR-alpha) mRNA in montocytes with endotoxin tolerance was investigated. THP-1 cells were cultured in serum-free medium, randomly divided into groups A, B, C, D and E, and stimulated with 0, 10, 10, 100, 0 ng/mL LPS for 24 h followed with 100, 100, 10, 100, 0 ng/mL LPS for another 24 h respectively. The expression of GR-alpha mRNA was detected by semi-quantitative reverse transcriptional polymerase chain reaction. Tumor necrosis factor-alpha (TNF-alpha) was determined by enzyme linked immunosorbent assay (ELISA). The results showed that the A values of GR-alpha/beta-actin in groups A, B, C, D and E was 0.607 +/- 0.006, 0.368 +/- 0.005, 0.484 +/- 0.008, 0.509 +/- 0.004 and 0.564 +/- 0.014 respectively with the difference being significant among the groups (P < 0.05). The GR-alpha mRNA expression was negatively correlated with the TNF-alpha expression (P < 0.01). It was concluded that the down-regulation of the expression of GR-alpha mRNA in endotoxin tolerance THP-1 cells might play an important role in the development of endotoxin tolerance in THP-1 cells.
Drug Tolerance ; Endotoxins/*pharmacology ; Leukemia, Monocytic, Acute/*metabolism ; Leukemia, Monocytic, Acute/pathology ; Monocytes/*metabolism ; RNA, Messenger/biosynthesis ; RNA, Messenger/genetics ; Receptors, Glucocorticoid/*biosynthesis ; Receptors, Glucocorticoid/genetics ; Tumor Cells, Cultured

This study was aimed to investigate the clinical features of CD56(+) patients with acute monocytic leukemia (AML-M5) and their prognostic significance. The data of 76 newly-diagnosed patients from our hospital were analyzed retrospectively. Patients were divided into two groups: CD56(+) group (21 patients) and CD56(-) group (55 patients). The clinical features, CR rate, relapse rate, the duration of CR, and survival time of patients between the two groups were compared. The results indicated that the CD56(+) antigen was observed in 21 patients (27.6%), their median age was 51.5 years and with a range 16 - 70 years. Of the 21 CD56(+) patients, the high WBC count was found in 57.1% CD56(+) patients (12/21), but it only in 15% CD56(-) patients (P < 0.05). The extramedullary infiltration was seen in 13 CD56(+) patients, and accounted for 62% (13/21), meanwhile this infiltration was found in 18 CD56(-) patients (18/55) and accounted for 33% (P < 0.05). All cases immunophenotypically highly expressed CD13, CD33, CD64, CD11b, cMPO, CD38, in which only the expression frequency of CD11b was positively related with CD56 (r = 0.59, P < 0.05). The CR rate in CD56(+) group accounted for 60.0%, and had no significant difference in comparison with that in CD56(-) group. In CD56(+) group the relapse rate was 75% (P = 0.042), the mean duration of CR was 5.5 months (95%CI, 3.1 - 8.6, P = 0.002), the median overall survival time was 10.1 months (95%CI, 2.3 - 16.3, P = 0.001). and all these had statistical significance as compared with that in CD56(-) group. It is concluded that CD56(+) AML-M5 patients always complicate with high WBC count and extramedullary infiltration, their CR rate and duration of CR are lower and shorter respectively, their relapse rate and prognosis are high and poor respectively.
Adolescent ; Adult ; Aged ; CD56 Antigen ; metabolism ; Female ; Humans ; Immunophenotyping ; Leukemia, Monocytic, Acute ; diagnosis ; immunology ; Male ; Middle Aged ; Prognosis ; Young Adult

To explore CD34(+) antigen expression in new diagnosed acute myeloid leukemia (AML) and analyze the prognosis for CD34(+) AML patients, the expression of antigen CD34 in 238 AML patients was detected by indirect immunofluorescence assay. The results showed that CD34 in 92 out of the 238 patients (38.7%) were positive, there was relationship between the CD34(+) expression and FAB subtypes (M(0), M(1)), and no CD34(+) expression was observed in M(3) subtypes. The complete remission rate of CD34(+) AML patients was 32%, which was lower than that of CD34(-) AML (61%). The lymphoid-associated antigen (CD7) was significantly increased in CD34(+) AML patients, compared with CD34(-) patients (P < 0.05). It is concluded that CD34(+) AML patients show poor prognosis and lower CR rate. The detection of CD34 expression is of some value in predicting prognosis in AML.
Adolescent ; Adult ; Aged ; Antigens, CD34 ; biosynthesis ; Antigens, CD7 ; biosynthesis ; Female ; Fluorescent Antibody Technique, Indirect ; Humans ; Leukemia, Monocytic, Acute ; metabolism ; pathology ; Leukemia, Myeloid, Acute ; metabolism ; pathology ; Leukemia, Myelomonocytic, Acute ; metabolism ; pathology ; Male ; Middle Aged ; Prognosis

In order to investigate the role of tryptase in angiogenesis of acute leukemia (AL), the expressions of tryptase and vascular endothelial growth factor (VEGF) in leukemic cells from 61 patients with AL were examined by using immunocytochemical method, and the correlation between tryptase and VEGF was analyzed. The results showed that tryptase positive expression was found in 15 out of 51 patients with acute myeloid leukemia (AML) (M(1) 1/3, M(2) 7/15, M(3) 5/20, M(5) 2/8). Tryptase positive expression was 29.4% in AML. However, none of 10 patients with acute lymphocytic leukemia (ALL) showed tryptase expression. There were no correlations between the amounts of cells with tryptase expression and patient age, WBC count, numbers of blood or marrow myeloblasts and neutrophil POX. VEGF expression was revealed in 41 patients with AML (80.4%) and only 3 with ALL (30%). Significant correlation has been found between the expression of tryptase and that of VEGF in AML-M(2) (r = 0.65, P < 0.05). It is concluded that tryptase appears to be a myeloid-specific marker in AML and may be involved in the angiogenesis of AML-M(2).
Biomarkers, Tumor ; biosynthesis ; Humans ; Immunohistochemistry ; Leukemia, Monocytic, Acute ; metabolism ; pathology ; Leukemia, Myeloid, Acute ; metabolism ; pathology ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; Neovascularization, Pathologic ; Tryptases ; biosynthesis ; Vascular Endothelial Growth Factor A ; biosynthesis

The aim of this study was to investigate the expression levels of genes psma6 and slc25a4 in bone marrow of patients with acute monocytic leukemia and their correlation with clinical features and prognosis. The expression levels of genes psma6 and slc25a4 in AML-M5 leukemia cells, normal blood cells and non-leukemia cells were detected by real-time quantitative RT-PCR and compared each other. The expression levels of psma6-encoding protein P27K was assayed by using immunohistochemistry method. The results showed that the expression levels of psma6 mRNA in AML-M5 leukemia cells was lower than that in non AML-M5 leukemia cells, non-leukemia cells and normal blood cells. The results obtained by immunohistochemistry assay were consistent with above-mentioned results. The expression level of psma6 in AML-M5 patients with complete remission was higher than that in AML-M5 patients without remission. The expression level of P27K protein in AML-M5 and AL correlated to leukocyte count in peripheral blood and LDH content. The overexpression of slc25a4 mRNA was found in AML-M5, but there was no significant difference in slc25a4 mRNA expression between the patients with complete remission and those without remission. It is concluded that the expression level of psma6 is probably a new prognostic indicator of acute monocytic leukemia, slc25a4 may be a novel gene of antigen associated with acute monocytic leukemia.
Adenine Nucleotide Translocator 1 ; genetics ; metabolism ; Adult ; Bone Marrow ; metabolism ; Female ; Humans ; Leukemia, Monocytic, Acute ; genetics ; metabolism ; Male ; Proteasome Endopeptidase Complex ; genetics ; metabolism

The purpose of this study was to construct a lentiviral vector carrying IK6 gene and to observe the expression of IK6 as well as related biologic feature in THP1 cells, so as to provide an effective method to further investigate the role of this gene in leukemia. The IK6 gene was obtained by using reverse transcription polymerase chain reaction (RT-PCR). Then IK6 was recombined with the pGC-FU vector to construct a recombinant lentiviral vector named pGC-FU-IK6 gene-GFP,which was confirmed by PCR and sequencing. The 293T cells were transfected with pGC-FU- IK6-GFP by using Lipofectamine 2000. After examining the titer of the virus, pGC-FU- IK6-GFP was used to transfect THP1 cells. The transfection efficiency was detected by flow cytometry, and the expression level of mRNA and IK6-GFP fusion protein were confirmed by RT-PCR and Western blot respectively. Then the impact of IK6 on apoptosis and cell cycle was analyzed. The results showed that the IK6 gene was obtained by RT-PCR and connected into the linearized lentiviral vector to successfully constructed target plasmid named pGC-FU-IK6-GFP with Amp resistant. The target plasmid was transfected into 293T cells and the virus titer was 2.0×10(9)TU/ml. Next, THP1 cells were transfected with pGC-FU-IK6-GFP and the efficiency was up to 90%. The detection of the IK6 mRNA and IK6-GFP fusion protein in target cells showed that IK6 could promote target cell clone formation and inhibit apoptosis, but had no significant effect on the cell cycle. It is concluded that virus vector carrying IK6 gene had been successfully constructed and expressed in THP1 stably. Biology studies of target THP1 cell shows that the IK6 is likely to interfere with the function of normal Ikaros protein as tumor suppressor, and it exerts a potential anti-apoptotic effect. Thus, IK6 can promote leukemia cell growth. However, there is no significant effect on the cell cycle. It provides an effective method for exploring the function of IK6 in acute myeloid leukemia.
Apoptosis ; Cell Cycle ; Cell Line, Tumor ; Gene Expression ; Genetic Vectors ; Humans ; Ikaros Transcription Factor ; genetics ; metabolism ; Lentivirus ; genetics ; Leukemia, Monocytic, Acute ; metabolism ; Plasmids ; Transfection

This study was aimed to investigate the influence of PPARalpha agonist on the expression of TF (tissue factor) in THP-1 cells. THP-1 cells were pretreated with different concentrations of PPARalpha agonist (fenofibrate) for definite time. Lipopolysaccharide (LPS)-induced TF mRNA and protein levels were detected by RT-PCR and Western blot respectively. The results showed that fenofibrate decreased tissue factor protein and mRNA expression in supernatants of LPS-stimulated human monocytes in a concentration-dependent manner (P < 0.05 - 0.01, n = 5). It is concluded that fenofibrate inhibit TF expression induced by LPS in THP-1 cells, which may be involved in the anti-atherosclerotic effects of PPARalpha agonist.
Depression, Chemical ; Fenofibrate ; pharmacology ; Humans ; Leukemia, Monocytic, Acute ; metabolism ; pathology ; Lipopolysaccharides ; antagonists & inhibitors ; pharmacology ; PPAR alpha ; agonists ; RNA, Messenger ; biosynthesis ; genetics ; Thromboplastin ; biosynthesis ; genetics ; Tumor Cells, Cultured

This study was aimed to investigate the effect of small interfering RNA (siRNA) on the expression of mll-af9 oncogene and the proliferation of human acute monocytic leukemia cell line THP-1. One group of siRNA was designed targeting mll-af9 mRNA and finally obtained by chemosynthesis. Then the obtained siRNA was transfected into cultured human acute monocytic leukemia cell line THP-1 by lipofectamine. Flow cytometry was used to detect siRNA transfection efficiency. The level of mll-af9 mRNA expression was analyzed by reverse transcription polymerase chain reaction (RT-PCR). The cell proliferation rate was assayed by MTT. The change of cell cycles and apoptosis rate was detected by flow cytometry. The results showed that the siRNA transfection efficiency was 69.1%+/-1.8%. The level of mll-af9 mRNA expression was significantly inhibited in siRNA-transfected cells as compared with the controls. mll-af9-targeted siRNA inhibited the proliferation of THP-1 cells and induced cell apoptosis effectively after transfection. The percentage of G0/G1 phase cells significantly increased in siRNA-transfected cells in comparion with the control cells, but the percentage of S phase cells significantly decreased. It is concluded that the mll-af9-targeted siRNA can effectively inhibit the proliferation of human acute monocytic leukemia cell line THP-1.
Cell Line, Tumor ; Cell Proliferation ; Down-Regulation ; Humans ; Leukemia, Monocytic, Acute ; genetics ; pathology ; Myeloid-Lymphoid Leukemia Protein ; genetics ; metabolism ; Oncogene Proteins, Fusion ; genetics ; metabolism ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transfection

The DNA segment of the human acyl coenzyme A: cholesterol acyltransferasel (ACAT1) gene P7 promoter was amplified by PCR from human monocytic leukemia cell line (THP-1) and cloned to TA vector, then the positive clone was confirmed by restriction enzymes and sequencing. The targeted segment was subcloned to Firefly luciferase report vector pGL3-Enhancer. The recombinant plasmid pGL3E-P7 was transfected transiently into THP-1, then the expression of luciferase could be detected in THP-1 by pGL3E-P7 transfection. We successfully constructed luciferase reporter vector containing P7 promoter of the human ACAT1 gene, and established a new means to study the transcriptional regulation mechanisms of ACAT1 during atherosclerosis.
Cell Line, Tumor ; Chromosomes, Human, Pair 7 ; genetics ; Gene Expression Regulation ; Genes, Reporter ; genetics ; Genetic Vectors ; genetics ; Humans ; Leukemia, Monocytic, Acute ; pathology ; Luciferases, Firefly ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Sterol O-Acyltransferase ; genetics ; metabolism ; Transfection