OBJECTIVETo investigate the effect of arsenic trioxide on multiple myeloma (MM) cell gene expression and explore the molecular mechanism of arsenic trioxide therapy for MM.

METHODSU266 cells were divided into two groups, group A as control group and group B as test group. Cells were cultured for 48 hours, and total RNA and mRNA were extracted. Suppression subtractive hybridization (SSHs) was performed to distinguish the differentially expressed genes. The products were cloned into pGEM-T Easy Vector, and transfected into the competent host JM109 to construct two subtractive libraries. Positive colonies were selected by blue-white screening, and the plasmids were extracted. Homologous comparison was conducted in GenBank.

RESULTSFive downregulated clones were isolated in the first SSH: (1) Aminopeptidase N, (2) Homosapiens tumor translationally-controlled protein 1, (3) Human ATP synthetase A chain, (4) Signal recognition particle A10, (5) Mitochondrial ATP synthetase/ATPase subunit 6. Four upregulated clones were isolated in the second SSH: (1) Calcium-binding protein A10, (2) Keratin 6A, (3) 45 kD MIP repetitive element containing splicing factor and (4) poly(A)-binding protein.

CONCLUSIONSArsenic trioxide exerts proliferation inhibition and apoptosis induction on MM cells by regulating genes expression.

Antineoplastic Agents ; pharmacology ; Arsenicals ; pharmacology ; Cell Line, Tumor ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; drug effects ; Gene Library ; Humans ; Multiple Myeloma ; genetics ; pathology ; Oxides ; pharmacology ; Plasmids ; genetics ; Transformation, Bacterial

OBJECTIVETo study the anti-myeloma activity of interleukin-2 activated bone marrow (ABM).

METHODSBone marrow mononuclear cells (BMMNC) from multiple myeloma and iron-deficiency anemia patients were cultured in the presence of rIL-2. The anti-myeloma activity of ABM against U266 cells, cells expressing surface CD45, CD38, CD138, the levels of TNF-alpha and IFN-gamma in ABM culture supernatant were measured with MTT method, flow cytometry and ELISA method respectively after bone marrow was activated with rIL-2 for 24 and 72 hours.

RESULTSThe tumor-killing activities against U266 cells of ABM were significantly increased compared with that of non-activated bone marrow (NBM) at 72 hours [(69.70 +/- 26.57)% vs (43.20 +/- 12.39)%, P < 0.05] and 24 hours [(34.25 +/- 11.93)% vs (26.53 +/- 5.48)%]. The CD45(-)CD38(+)CD138(+) cells of ABM from myeloma group at 72 hours were decreased from (8.46 +/- 3.66)% to (4.79 +/- 1.56)% (P < 0.05). TNF-alpha and IFN-gamma were detectable after cultured for 24 hours in both normal control group and myeloma group and went higher at 72 hours. The level of TNF-alpha and IFN-gamma were significantly increased in ABM compared with that in NBM (P < 0.05). Meanwhile, there was a positive relationship between the level of TNF-alpha, IFN-gamma and cytotoxicity of ABM from normal control group at 24 hours and 72 hours (P < 0.05), and was a negative relationship between TNF-alpha and IFN-gamma levels and the CD45(-)CD38(+)CD138(+) cells in myeloma group at 72 hours (P < 0.05).

CONCLUSIONNormal BMMNCs activated with rIL-2 have tumor-killing activities against U266 cells. Myeloma cells and tumor burden were decreased in myeloma bone marrow after the marrow was activated with rIL-2. Production of TNF-alpha and IFN-gamma from bone marrow cells including T cells, monocyte-macrophages and NK cells activated with rIL-2 might be involved in anti-myeloma activity of ABM.

Adult ; Aged ; Bone Marrow Cells ; drug effects ; immunology ; metabolism ; Cell Line, Tumor ; Cells, Cultured ; Cytotoxicity, Immunologic ; immunology ; Enzyme-Linked Immunosorbent Assay ; Female ; Flow Cytometry ; Humans ; Interferon-gamma ; biosynthesis ; Interleukin-2 ; immunology ; pharmacology ; Male ; Middle Aged ; Multiple Myeloma ; blood ; immunology ; pathology ; Tumor Necrosis Factor-alpha ; biosynthesis

OBJECTIVETo explore the effect of 2-methoxyestradiol (2ME2) and arsenic trioxide (As2O3) on the apoptosis related gene expression in multiple myeloma cell line CZ-1.

METHODSTotal RNA was isolated from CZ-1 cells which had been treated with 2ME2 and As2O3 at an apoptosis-inducing concentration and reverse-transcribed into a cDNA probe labeled with Bio-16-dUTP, and then hybridized it with a microarray containing up to 96 key genes involved in apoptosis. The gene expression profile of the 2ME2 and As2O3 treated CZ-1 cells were analyzed with GEArray Analyzer software. The microarray results were confirmed by RT-PCR.

RESULTSAs2O3 treatment caused the alteration in the expression of 52 genes (54.2% of total genes on microarray). Among them, 42 (80.8%) were upregulated and 10 (19.2%) were downregulated. The upregulated genes were mainly involved in caspases family, P53 and ATM pathway, death effector domain family, TNF receptor family and CIDE family. 2ME2 treatment resulted in the alteration of 42 genes (43.8% of the total genes on microarray). Of them, 32 (76.2%) were downregulated and 10 (23.8%) were upregulated. The downregulated genes mainly belonged to bcl-2 family, inhibitor of apoptosis protein family (IAP), TRAF family, TNF ligand family, and CARD family.

CONCLUSION2ME2 and As2O3 induce the CZ-1 cells apoptosis by different pathways. As2O3 mainly induces upregulation of proapoptotic genes, and 2ME2 downregulation of anti-apoptotic genes expression.

Apoptosis ; drug effects ; genetics ; Arsenicals ; pharmacology ; Cell Line, Tumor ; Estradiol ; analogs & derivatives ; pharmacology ; Gene Expression Profiling ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; Multiple Myeloma ; genetics ; pathology ; Oligonucleotide Array Sequence Analysis ; Oxides ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Tubulin Modulators ; pharmacology

OBJECTIVETo investigate the differentiation induction effect of 2-methoxyestradiol (2ME2), an estrogen derivative on myeloma cell line CZ-1.

METHODSThe changes of CZ-1 cells in morphology, expression of surface CD49e and quantity of light chain secretion in the supernatant were observed when treated with 0.1 approximately 0.5 micromol/L 2ME2 for 48 h.

RESULTS2ME2 could induce differentiation of CZ-1 cells. The cells appeared decreased in size of nucleus, increased in cytoplasma, decreased in the ratio of nucleus to plasma, decreased in number or disappearance of nucleolus, and thickness and pyknosis of chromatin. The expression of CD49e was increased from (12.20 +/- 1.57)% to (24.80 +/- 1.26)% (P < 0.05). Light chain secretion in the supernatant was increased from (35.97 +/- 2.60) microg/ml to (79.67 +/- 1.88) microg/ml (P < 0.05).

CONCLUSIONLow concentrations of 2ME2 could induce differentiation of myeloma cell line CZ-1.

Cell Differentiation ; drug effects ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Estradiol ; analogs & derivatives ; pharmacology ; Flow Cytometry ; Humans ; Integrin alpha5 ; analysis ; Multiple Myeloma ; metabolism ; pathology ; Tubulin Modulators ; pharmacology

OBJECTIVETo analyse the outcome of different regimens for the treatment of patients with multiple myeloma (MM).

METHODSResponse rate, median survival time and overall survival rate of 206 MM patients treated with different protocols were retrospectively analysed.

RESULTThe median survival time, 3- and 5-year overall survival (OS) of 200 MM patients treated with conventional therapy were 30.5 months, 32.0% and 15.8%, respectively. The total response rate and complete response (CR) rate of 195 patients treated with MP regimen and combination chemotherapy (CCT) were 45.6% and 14.9%, respectively. The response rates were higher for the patients treated with CCT than for those treated with MP (50.3% versus 30.4%, P < 0.05). The median survival time, 3- and 5- year OS in MP versus CCT group were 30.0 versus 30.5 months, 22.0% versus 35.0%, 13.2% versus 16.7%, respectively, but all of them have no statistical difference. Compared with those without IFN alpha maintenance therapy, patients received IFN alpha therapy showed a higher response rate (34.4% versus 53.6%, P < 0.05) and a longer median survival time (27 versus 52 months, P < 0.01). The total response in patients received thalidomide was 65.5%. Of the 6 patients received hematopoietic stem cell transplantation (HSCT), 5 remained alive in CR or PR with a mean survival time of (73.0 +/- 12.5) months.

CONCLUSIONSCCT yields higher response rates, but not longer survival time than MP does for the treatment of MM. The response rate as well as the overall survival rate increased when IFN alpha was used as maintenance therapy. Thalidomide can improve response rate as well. HSCT could prolong survival time in patients aged < 60 years with good status.

Adult ; Aged ; Aged, 80 and over ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Female ; Follow-Up Studies ; Hematopoietic Stem Cell Transplantation ; methods ; Humans ; Immunologic Factors ; administration & dosage ; Interferon-alpha ; administration & dosage ; Kaplan-Meier Estimate ; Male ; Middle Aged ; Multiple Myeloma ; drug therapy ; surgery ; Retrospective Studies ; Treatment Outcome

OBJECTIVETo observe the bone marrow angiogenesis in leukemia and evaluate the expression and role of endostatin (ES), vascular endothelial growth factor (VEGF) and its receptor (VEGFR) in leukemia patients.

METHODSBone marrow angiogenesis was assayed by vWF immunohistochemical method. The ES and VEGF concentrations in plasma were detected by ELISA. The expression of VEGFR in leukemia cells was determined by flow cytometry (FCM).

RESULTSThe bone marrow microvessel density (MVD) in 26 cases of acute leukemia (AL) [(20.78 +/- 7.75)/high-power field (HP)] and 5 chronic myelogenous leukemia (CML) [(28.67 +/- 7.32)/HP] at newly diagnosed stage was significantly higher than that in the control group [(9.29 +/- 3.53)/HP, P < 0.01]. The bone marrow MVD in the complete remission (CR) groups, (11.33 +/- 5.66)/HP for AL and (17.00 +/- 8.04)/HP for CML, was significantly lower than that of newly diagnosed groups (P < 0.05 and < 0.01). No significant difference was found between the remission groups and the control group (P > 0.05). The plasma ES and VEGF concentrations of newly diagnosed AL and CML groups were significantly higher than those of the control group (P < 0.05). The levels of plasma ES and VEGF in AL CR group and plasma ES in CML CR group decreased to levels comparable to normal values (P > 0.05), but the plasma VEGF in CML CR group was still higher than that in control (P < 0.01). AL bone marrow mononuclear cells had higher expression of VEGFR at various levels whereas CML and control samples had lower levels of VEGFR.

CONCLUSIONSLeukemia patients at newly diagnosed stage had remarkable angiogenesis in bone marrow and elevated plasma ES and VEGF concentrations. VEGFR was expressed at different levels in AL cells.

Acute Disease ; Adolescent ; Adult ; Aged ; Aged, 80 and over ; Bone Marrow ; blood supply ; metabolism ; Endostatins ; blood ; Enzyme-Linked Immunosorbent Assay ; Female ; Flow Cytometry ; Humans ; Immunohistochemistry ; Leukemia ; blood ; pathology ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; blood ; pathology ; Male ; Microvessels ; metabolism ; pathology ; Middle Aged ; Neovascularization, Pathologic ; blood ; pathology ; Receptors, Vascular Endothelial Growth Factor ; metabolism ; Vascular Endothelial Growth Factor A ; blood ; Young Adult ; von Willebrand Factor ; metabolism

OBJECTIVETo explore the effects of chloride channels on the regulation of platelet cytoplasmic free calcium concentration ([Ca2+]i) and platelet aggregation (PAG).

METHODSFreshly separated platelets were activated by thrombin. Chloride channel blockers DIDS or NFA and calcium channel blockers SK&F96365 or nifedipine were added to study the effects on platelet [Ca2+]i and PAG by a single reagent or the combination of reagents and find out the interactions among DIDS, NFA, SK&F96365 and nifedipine.

RESULTSBoth DIDS and NFA could inhibit the thrombin (1 U/ml) induced PAG in a dose-dependent manner, whereas had little effect on resting [Ca2+]i. As compared with the control group, DIDS, SK&F96365 and Nifedipine could significantly reduce the PAG, Ca2+ release and Ca2+ influx in thrombin activated platelet (P < 0.05). The combination of DIDS and SK&F96365 had greater effects in reducing the PAG, Ca2+ release and Ca2+ influx than either reagent alone (P < 0.05). The combination of DIDS and nifedipine also had greater effect than each alone in reducing Ca2+ release (P < 0.05). The combination of NFA and SK&F96365 weakened each other's effect on Ca2+ release (P < 0.05), while NFA and nifedipine weakened each other's effects on PAG, Ca2+ release and Ca2+ influx in thrombin activated platelet (P < 0.05).

CONCLUSIONDIDS and NFA have no effect on the resting [Ca2+]i and the leak calcium influx of platelet. DIDS can inhibit the Ca2+ release, Ca2+ influx and PAG of platelet induced by thrombin, while NFA can only inhibit the Ca2+ release. The chloride channel and calcium channel blockers have interactions in affecting resting [Ca2+]i and PAG of platelet. The opening of chloride channel can influence the cellular calcium movement of platelet.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid ; pharmacology ; Adult ; Blood Platelets ; cytology ; drug effects ; metabolism ; Calcium ; metabolism ; Calcium Channel Blockers ; pharmacology ; Cells, Cultured ; Chloride Channels ; antagonists & inhibitors ; physiology ; Cytoplasm ; drug effects ; metabolism ; Drug Interactions ; Humans ; Imidazoles ; pharmacology ; Nifedipine ; pharmacology ; Niflumic Acid ; pharmacology ; Platelet Aggregation ; drug effects ; Thrombin ; pharmacology

OBJECTIVETo evaluate the clinical usefulness of direct monoclonal antibody immobilization of platelet antigen (MAIPA) technique in the differential diagnosis of immune and non-immune thrombocytopenia.

METHODSPlatelet-bound autoantibodies in thrombocytopenic patients (immune and non-immune) were measured by direct MAIPA. Monoclonal antibodies against GP II b/III a, GPIb and GP I a/II a were used.

RESULTSThe positive rates of platelet-bound GP-specific autoantibodies between immune (76.4%) and non-immune thrombocytopenia (3.6%) were significantly different (P < 0.05). The direct MAIPA had a sensitivity of 76.4%, a specificity of 96.4%, and a positive predictive value of 97.1% for the diagnosis of immune thrombocytopenia. There was a significant inverse correlation between platelet-bound GP II b/III a specific autoantibody levels and platelet counts (r = -0.338, P < 0.05).

CONCLUSIONThe direct MAIPA technique can be used to differentiate immune from non-immune thrombocytopenias.

Adolescent ; Adult ; Aged ; Antibodies, Monoclonal ; immunology ; Autoantibodies ; blood ; Diagnosis, Differential ; Female ; Humans ; Male ; Middle Aged ; Platelet Membrane Glycoproteins ; immunology ; Purpura, Thrombocytopenic ; diagnosis ; immunology ; Young Adult

OBJECTIVETo monitor the changes of hemolysis parameters and endothelial cell markers in thrombotic thrombocytopenic purpura (TTP) and reveal the clinical significance of these changes.

METHODSvWF-cleaving protease (vWF-CP) activity in 3 cases of TTP was detected by Western blot. The percentages of fragmented red cells (FRC) were counted throughout the entire clinical course. Levels of plasma thrombomodulin were detected by Western blot combined with density screening in TTP and healthy individuals (n = 3). Concentration of plasma VEGF was measured by enzyme-linked immunosorbent assay in TTP and healthy individuals (n = 9). Fundus fluorescein angiography was performed to search the evidence of microvascular thrombosis in one TTP patient with impaired visual acuity.

RESULTSThe lower vWF-CP activity was observed in TTP patients; the percentages of FRC in 3 cases of TTP were 1.65%, 2.50%, 3.32% respectively with an average of 2.49% at the onset of and decreased with the improvement of the disease. The levels of plasma TM and VEGF were significantly elevated in TTP than those in healthy individuals, and related to the severity of TTP. Fundus photography in one TTP patient with impaired visual acuity revealed vascular occlusion in fundus arteriole and venulae.

CONCLUSIONSA decreased vWF-CP activity is in favour of TTP diagnosis. Dynamic monitoring of plasma TM and VEGF as well as percentages of FRC are useful indexes for reflecting the severity and evaluating therapeutic response of TTP. Selective fundus fluorescein angiography is useful for the judgement of microvascular thrombosis in TTP.

ADAM Proteins ; blood ; ADAMTS13 Protein ; Biomarkers ; blood ; Blotting, Western ; Endothelial Cells ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Hemolysis ; Humans ; Male ; Middle Aged ; Purpura, Thrombotic Thrombocytopenic ; blood ; pathology ; Thrombomodulin ; blood ; Time Factors ; Vascular Endothelial Growth Factor A ; blood ; Young Adult

OBJECTIVETo study the effect of glycoprotein (GP) alpha II bA2334C mutation on the biosynthesis and expression of alpha II bbeta3 complex.

METHODSThe GP alpha II bA2334C eukaryotic expression plasmid pc3.1-2334M2b was constructed. Chinese hamster ovary (CHO) cells were transfected with the plasmid with or without integrin beta3 expression plasmid pc3.1-3a. The whole expression of alpha II bA2334C was confirmed by Western blot and the membrane expression was analyzed by flow cytometry. A newly constructed alpha II bA2334C GFP fusion protein expressing plasmid was used to determine its subcellular localization by laser confocal scanning microscopy.

RESULTSExpression of the mutant protein, alpha II bA2334C, in the transfected CHO cells was confirmed by Western blot with a lower rate of the mature type than the wild type control. The expression on membrane was only 25% of the normal. Subcellular localization analysis showed that alpha II bA2334C GFP was able to be expressed in CHO cells and could be transported from endoplasmic reticulum to Golgi apparatus.

CONCLUSIONSThe mutant alpha II bA2334C can be synthesized in CHO cells and form alpha II bbeta3 complex. However, only a small fraction of the premature alpha II bA2334C can be transported to Golgi apparatus and transformed to mature alpha II b. The possible pathogenesis of this type II thrombasthenia may be that the misfolded alpha II bA2334C is partially degraded in the endoplasmic reticulum causing lower expression of alpha II bbeta3 complex on the membrane and resulting in impared function of platelets than normal alpha II b.

Animals ; Biological Transport ; Blotting, Western ; CHO Cells ; Cricetinae ; Cricetulus ; Female ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Liposomes ; Microscopy, Confocal ; Middle Aged ; Mutation ; Plasmids ; genetics ; Platelet Glycoprotein GPIIb-IIIa Complex ; genetics ; metabolism ; Transfection