OBJECTIVETo study the expression of peptidyl-prolyl cis/trans isomerase (PPIase or Pin1) in malignant hematopoietic cells and its relation with cell cycle.

METHODSRealtime quantitative PCR with fluorescence probe hybridization was used to measure expression of Pin1 mRNA in malignant hematopoietic cell lines and normal mononuclear cells separated from bone marrow. HeLa cells were blocked with Thymidine and Nocodazole in different cell phases and then the expression of Pin1 mRNA and protein were detected by realtime-PCR and immunoblotting.

RESULTSThe expression of Pin1 in malignant hematopoietic cell lines was significantly higher than that in normal controls (0.339 +/-0.093 compared with 0.038 +/-0.005, P<0.01). Its expression in myeloid malignant hematopoietic cell lines was significantly higher than that in normal controls (0.388 +/-0.115 compared with 0.038 +/-0.005, P<0.01) and so was the malignant lymphocytic cell lines (0.226 +/-0.166 compared with 0.038 +/-0.005, P<0.01). The expression of Pin1 was closely correlated with cell cycle. It was the highest in G1 phase and the lowest in S phase (110.762 +/-16.737 compared with 4.080 +/-0.634, P<0.01).

CONCLUSIONPin1 is overexpressed in malignant hematopoietic cell lines and its expression is different during cell cycle that is highest in G1 phase and lowest in S phase.

Cell Cycle ; physiology ; G1 Phase ; Humans ; Leukemia, Lymphoid ; enzymology ; pathology ; Leukemia, Myeloid ; enzymology ; pathology ; Peptidylprolyl Isomerase ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics ; S Phase ; Tumor Cells, Cultured

In order to study the relationship between the expression of glutathione S-transferase (GST) in leukemic cells and the chemoresistance in patients with acute leukemia, the expressions of GST activity and GST mRNA were measured according to spectrophotometric assay based on the use of 1-choloro-2, 4-dinitro benzene and in situ hybridization. The results were studied in correlation with some clinical and pathological data. Results showed that: 1. There is no significant differences between activities of the enzyme with the different leukemia types according to the FAB classification. 2. GST activity and GST mRNA expression in the patients, both untreated and relapse, were (4.5 +/- 1.0) U, 33.3% and (7.9 +/- 15) U, 66.3% respectively. 3. In 56 patients, GST activity was 1.7 +/- 0.7, 5.9 +/- 2.0 and 9.3 +/- 1.7 U and GST mRNA expression was 13.3%, 29.7% and 76.6%, respectively, in CR, PR and NR groups. The lowest values of GST activity and GST mRNA expression were observed in those patients who achieved complete remission. The highest values of GST activity and GST mRNA expression were observed in those patients with no response to treatment. It was concluded that the expression of GST in patients with acute leukemia is closely related to the chemosensitivities clinically. Determinations of GST activity and GST mRNA are useful for predicting the chemosensitivities and the prognosis of the disease.
Adolescent ; Adult ; Aged ; Drug Resistance, Neoplasm ; Female ; Gene Expression Regulation, Enzymologic ; Gene Expression Regulation, Neoplastic ; Glutathione Transferase ; genetics ; metabolism ; Humans ; Isoenzymes ; genetics ; metabolism ; K562 Cells ; Leukemia ; drug therapy ; enzymology ; genetics ; Leukemia, Lymphoid ; drug therapy ; enzymology ; genetics ; Leukemia, Monocytic, Acute ; drug therapy ; enzymology ; genetics ; Leukemia, Myeloid, Acute ; drug therapy ; enzymology ; genetics ; Male ; Middle Aged ; Neoplasm Recurrence, Local ; enzymology ; genetics ; pathology ; RNA, Messenger ; genetics ; metabolism

To explore the change of telomerase activity in acute leukemia (AL) cells and its relationship with cell cycle, PCR-ELISA was used to detect telomerase activity of bone marrow cells from 148 AL patients, including 92 cases with acute non-lymphocytic leukemia (ANLL) and 56 cases with acute lymphocytic leukemia (ALL). Thirty-six patients without bone marrow disorders were detected as normal control. The cell cycle of 16 patients and 4 controls was detected with flow cytometry. The results showed that the positive rate of telomerase was 71.6% (106/148) in the cells from AL patients, which was higher than that in the control group 5.6% (2/36). It was 88.9% (32/36) in the relapse group and 81.3% (61/75) in the untreated group. Both rates were higher than that in the CR group (35.1%, 13/37). There was no significant difference in the ALL and ANLL groups. The cell number in various phases of cell cycle had no significant difference between telomerase positive and negative groups. It was concluded that the activation of telomerase was very common in acute leukemia cells. Telomerase positive rate was closely associated with the different stages and progress of acute leukemia, and it might be a molecular marker for increased proliferation of leukemic cells during the process of the disease. Activation of telomeras had no correlation with cell number in different phases of cell cycle, while telomerase activity is modulated by other biological factors in addition to cell cycle.
Adult ; Cell Cycle ; physiology ; Child ; Enzyme-Linked Immunosorbent Assay ; methods ; Flow Cytometry ; Humans ; Leukemia, Myeloid, Acute ; enzymology ; pathology ; physiopathology ; Neoplasm Staging ; Polymerase Chain Reaction ; methods ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; enzymology ; pathology ; physiopathology ; Telomerase ; genetics ; metabolism

This study was aimed to investigate the mechanism of indoleamine 2, 3-dioxygenase (IDO) activity in acute myeloid leukemia cells contributing to tumor immune escape. Myeloid leukemia cells were isolated from bone marrow of 23 patients with acute myeloid leukemia (AML) and IDO expression was detected by immunochemistry and RT-PCR methods. Then mixed lymphocyte reaction (MLR) of one way was carried out, leukemia cells were used as stimulating cells and T-lymphocytes were used as reactive cells in culture with or without 1-MT. T-lymphocyte proliferation rate was determined by MTT assay and IDO activity in supernatant of MLR was detected by high-performance liquid chromatography (HPLC). The results showed that IDO expression was found in 17 out of 23 cases of acute myeloid leukemia cells; IDO enzyme activity in leukemia cells inhibited T-lymphocyte proliferation in MLR cultures. It is concluded that IDO activity expressing in leukemia cells can suppress T-lymphocyte proliferation responses, which may be contributing to tumor immune escape.
Cell Proliferation ; Humans ; Immune Tolerance ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; metabolism ; Leukemia, Myeloid, Acute ; enzymology ; immunology ; pathology ; T-Lymphocytes ; cytology ; immunology ; Tumor Cells, Cultured ; Tumor Escape ; immunology

Leukemia seems to depend on a small population of "leukemia stem cells (LSCs)" for its growth and metastasis. However, the precise surviving mechanisms of LSCs remain obscure. Cellular senescence is an important obstacle for production and surviving of tumor cells. In this study we investigated the activated state of a pathway, in which reactive oxygen species (ROS) induces cellular senescence through DNA damage and phophorylation of p38 MAPK (p38), in myeloid leukemic CD34(+)CD38(-) cells. Bone marrow samples were obtained from patients with acute myeloid leukemia (AML, n=11) and chronic myeloid leukemia (CML, n=9). CD34(+)CD38(-) cells were isolated from mononuclear cells from these bone marrow samples, and K562 and KG1a cells (two kinds of myeloid leukemia cell lines) by mini-magnetic activated cell sorting. Hematopoietic stem cells (HSCs) from human cord blood served as controls. Intracellular ROS level was detected by flow cytometry. DNA damage defined as the γH2AX level was measured by immunofluorescence staining. Real-time RT-PCR was used to detect the expression of p21, a senescence-associated gene. Western blotting and immunofluorescence staining were employed to determine the p38 expression and activation. The proliferation and apoptosis of CD34(+)CD38(-) cells were detected by MTT assay and flow cytometry. Our results showed that ROS and DNA damage were substantially accumulated and p38 was less phosphorated in myeloid leukemic CD34(+)CD38(-) cells as compared with HSCs and H(2)O(2)-induced senescent HSCs. Furthermore, over-phosphorylation of p38 by anisomycin, a selective activator of p38, induced both the senescence-like growth arrest and apoptosis of CD34(+)CD38(-) cells from K562 and KG1a cell lines. These findings suggested that, although excessive accumulation of oxidative DNA damage was present in LSCs, the relatively decreased phosphorylation of p38 might help leukemic cells escape senescence and apoptosis.
ADP-ribosyl Cyclase 1 ; metabolism ; Antigens, CD34 ; metabolism ; Cellular Senescence ; Female ; Humans ; Leukemia, Myeloid, Acute ; enzymology ; pathology ; Male ; Neoplastic Stem Cells ; metabolism ; pathology ; Oxidative Stress ; Phosphorylation ; Reactive Oxygen Species ; metabolism ; Tumor Cells, Cultured ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo study the effect of transforming growth factor-β activated kinase-1 (TAK1) gene silencing on the proliferation and apoptosis of Kasumi-1 cells induced by arsenic trioxide (As₂O₃).

METHODSAcute myeloid leukemia with t(8;21) cell line Kasumi-1 cells were treated with As₂O₃ or in combination with TAK1 siRNA interference technology. The experiment was divided into four groups: Kasumi-1 cells without any treatment, TAK1 specific siRNA transfection alone, Kasumi-1 cells treated with different concentration of As₂O₃, TAK1siRNA transfection combined with As₂O₃. CCK-8 was used to detect the cell viability. The expression of phosphorylated c-Jun N-terminal kinase (P-JNK) was determined by Western Blot. Cell apoptosis and growth were examined by morphological and colony formation assay.

RESULTSAfter Kasumi-1 cells were treated with As₂O₃, the rate of cell inhibition was concentration-dependent, and the 50% inhibitory concentration was 3.5 μmol/L. The highest expression level of P-JNK appeared in 30 minutes after cells were treated with As₂O₃. The apoptosis rates of Kasumi-1 cells without any treatment, TAK1 siRNA interference alone group, As₂O₃ alone group and the combined group were (5.02 ± 1.13)%, (6.18 ± 0.28)%, (48.33 ± 2.70)% and (86.07 ± 2.21)%; colony formation rates were (73.83 ± 2.78)%, (76.03 ± 1.46)%, (55.07 ± 1.50)% and (22.20 ± 1.15)%; apoptosis rate of TAK1 siRNA group and the untreated group has no significant difference (P = 0.052); colony formation rate between TAk1 siRNA group and the untreated group has no significant difference (P = 0.179), but the difference in other groups was significant (P = 0.000).

CONCLUSIONSilencing the expression of TAK1 can enhance the anti-proliferative and pro-apoptotic effect of As₂O₃ on Kasumi-1 cells, and its mechanism may be through the TAK1 downstream JNK signal pathway.

Apoptosis ; drug effects ; Arsenicals ; pharmacology ; Cell Line, Tumor ; Humans ; JNK Mitogen-Activated Protein Kinases ; metabolism ; Leukemia, Myeloid, Acute ; enzymology ; pathology ; MAP Kinase Kinase Kinases ; genetics ; metabolism ; Oxides ; pharmacology ; RNA Interference ; RNA, Small Interfering ; genetics ; Signal Transduction

Ornithine decarboxylase (ODC) is the rate-limiting enzyme in polyamine biosynthesis and a target for chemoprevention. Hydroxydibenzoylmethane (HDB), a derivative of dibenzoylmethane of licorice, is a promising chemopreventive agent. In this paper, we investigated whether HDB would inhibit the ODC pathway to enhance apoptosis in human promyelocytic leukemia HL-60 cells. We found ODC enzyme activity was reduced during HDB treatment. Overexpression of ODC in HL-60 parental cells could reduce HDB-induced apoptosis, which leads to loss of mitochondrial membrane potential (Deltapsim), through lessening intracellular ROS. Furthermore, ODC overexpression protected cytochrome c release and the activation of caspase-3 following HDB treatment. The results demonstrated HDB-induced apoptosis was through a mechanism of down-regulation of ODC and occurred along a ROS-dependent mitochondria-mediated pathway.
Apoptosis/*drug effects ; Caspase 3/metabolism ; Chalcones/metabolism/*pharmacology ; Chemoprevention ; Cytochromes c/biosynthesis/secretion ; Down-Regulation ; Gene Expression ; HL-60 Cells ; Humans ; Immunoblotting ; Leukemia, Myeloid/*enzymology/pathology ; Membrane Potential, Mitochondrial/drug effects ; Mitochondria/enzymology ; Ornithine Decarboxylase/antagonists & inhibitors/genetics/*metabolism ; Reactive Oxygen Species/analysis/metabolism ; Reverse Transcriptase Polymerase Chain Reaction

Senescence is an important obstacle to cancer development. Engaging a senescent response may be an effective way to cure acute myeloid leukemia (AML). The aim of this study was to examine the effect of resveratrol-downregulated phosphorylated liver kinase B1 (pLKB1) on the senescence of acute myeloid leukemia (AML) stem cells. The protein expressions of pLKB1 and Sirtuin 1 (SIRT1), a regulator of pLKB1, were measured in CD34(+)CD38(-) KG1a cells treated with resveratrol (40 μmol/L) or not by Western blotting. Senescence-related factors were examined, including p21 mRNA tested by real-time PCR, cell morphology by senescence-associated β-galactosidase (SA-β-gal) staining, cell proliferation by MTT assay and cell cycle by flow cytometry. Besides, apoptosis was flow cytometrically determined. The results showed that pLKB1 was highly expressed in CD34(+)CD38(-) KG1a cells, and resveratrol, which could downregulate pLKB1 through activation of SIRT1, induced senescence and apoptosis of CD34(+)CD38(-) KG1a cells. It was concluded that resveratrol-downregulated pLKB1 is involved in the senescence of AML stem cells.
Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Cellular Senescence ; Cyclin-Dependent Kinase Inhibitor p21 ; genetics ; Down-Regulation ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Leukemia, Myeloid, Acute ; enzymology ; genetics ; pathology ; Neoplastic Stem Cells ; drug effects ; Phosphorylation ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Sirtuin 1 ; genetics ; metabolism ; Stilbenes ; pharmacology