OBJECTIVETo investigate the mechanisms of arsenic trioxide (As(2)O(3)) induced demethylation.

METHODMethylation of p15INK4B gene in MUTZ-1 cell was detected by PCR using a methylation specific primer (MSP), the expression of P15, DNA methyltransferase (DNMT) 1, DNMT3A and DNMT3B gene by RT-PCR, the As(2)O(3) induced growth inhibition of MUTZ-1 cell by MTT method.

RESULTSP15 gene failed to express in MUTZ-1 cells after methylation. The expression was recovered after the cells exposed to As(2)O(3). As(2)O(3) could significantly down-regulate DNMT3A and DNMT3B but not DNMT1 gene on mRNA level in a dose dependent manner.

CONCLUSIONAs(2)O(3) could activate the expression of p15 gene by demethylation or/and by inhibiting DNMT3A and DNMT3B gene.

Arsenicals ; pharmacology ; Cell Line ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; Cyclin-Dependent Kinase Inhibitor p15 ; genetics ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; genetics ; DNA Methylation ; drug effects ; Gene Expression Regulation ; drug effects ; Humans ; Myelodysplastic Syndromes ; genetics ; pathology ; Oxides ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo identify the human hematopoietic stem cells from the human/goat xenogeneic model with molecular techniques.

METHODSDNA and total RNA were extracted from 11 transplanted goat peripheral blood cells. Human CD(34), GPA and SRY genes were amplified with PCR in these samples, and CD(34), GPA mRNA transcripts were detected using RT-PCR in 5 and 6 goat peripheral blood cells, respectively. Southern blot analysis was performed in 8 goat DNAs to detect the human specific alpha-satellite sequence. Meanwhile FISH was also performed to detect the human cells in goat blood with a probe of human Y chromosome.

RESULTSHuman CD(34) and GPA genes could be detected with PCR in all the 11 goats, and SRY gene did in 5 goats transplanted with hematopoietic stem cells derived from male human babies. Southern blot showed that human specific alpha-satellite sequence was present in 8 goats. By RT-PCR, human CD(34) mRNA was detected in 5 experimental goats, GPA mRNA was found in the other 6 experimental goats and FISH assay showed that some peripheral blood cells of the human/goat xenogeneic model were positive.

CONCLUSIONExistence of human cells in the recipient goats was identified by molecular detection, which was feasible for the examination of human/goat xenogeneic models.

Animals ; Antigens, CD34 ; genetics ; Blotting, Southern ; Female ; Genes, sry ; genetics ; Glycophorin ; genetics ; Goats ; Hematopoietic Stem Cell Transplantation ; methods ; Hematopoietic Stem Cells ; cytology ; metabolism ; Humans ; In Situ Hybridization, Fluorescence ; Male ; Polymerase Chain Reaction ; Transplantation Chimera ; genetics ; Transplantation, Heterologous

OBJECTIVETo prepare a monoclonal antibody against human telomeric repeat binding factor 1 (TRF1) protein and explore its biological characteristics.

METHODSBALB/c mice were immunized with GST-TRF1(33-277) fusion protein for the preparation of monoclonal antibody by hybridoma technique. The obtained antibody was used for clinical assay by Western-blot and immunohistochemical staining.

RESULTSOne strain of hybridoma was obtained. It was confirmed by Western-blot that the antibody specifically recognized the 60 kD TRF1 protein. Immunohistochemical staining of the antibody showed that TRF1 protein located in the cytoplasm of epithelial cells and bone marrow cells.

CONCLUSIONA TRF1 monoclonal antibody, with high specificity was developed. It is useful for detection of TRF1 protein in tissue specimens.

Animals ; Antibodies, Monoclonal ; immunology ; Blotting, Western ; Female ; Humans ; Hybridomas ; immunology ; Immunohistochemistry ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; immunology ; Telomeric Repeat Binding Protein 1 ; analysis ; genetics ; immunology

OBJECTIVETo study the effects of low dose sodium selenite combined with all-trans retinoic acid (ATRA) on apoptosis and differentiation of human acute promyelocytic leukemia (APL) NB4 cells.

METHODSApo-ptosis was detected by translocation of phosphatidylserine (PS) with a Annexin-V kit and DNA fragmentation by agarose gel electrophoresis analysis, cell differentiation was studied by flow cytometry of CD(11b) expression and NBT reduction assay.

RESULTSFive micromol/L sodium selenite or 0.1 micromol/L ATRA alone could not induce apoptosis of NB4 cells within 48 hours. However, combination of the two drugs at the same doses as above could induce significant apoptosis in 48 hours characterized by increased PS translocation and DNA ladder. Sodium selenite at concentration of 2 micromol/L was not able to induce differentiation of NB4 cells, but when combined with 0.1 micromol/L ATRA, CD(11b) expression and NBT reduction were increased as compared with that of 0.1 micromol/L ATRA alone.

CONCLUSIONLow dose sodium selenite could enhance the effects of low dose ATRA in inducing apoptosis and differentiation of NB4 cells.

Apoptosis ; drug effects ; CD11b Antigen ; analysis ; Cell Differentiation ; drug effects ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Drug Synergism ; Flow Cytometry ; Humans ; Leukemia, Promyelocytic, Acute ; metabolism ; pathology ; Sodium Selenite ; pharmacology ; Tretinoin ; pharmacology

OBJECTIVETo evaluate the feasibility and characteristics of human engraftment in HLA disparate cord blood transplantation.

METHODSTwo human HLA-haploidentical or HLA-mismatched cord blood units were transplanted into sublethally irradiated severe combined immunodeficiency (SCID) mice. The characteristics of engraftment, hematopoietic and immunological reconstitution between the two groups were compared.

RESULTSTwo mixed cord blood units can engraft in SCID mice with donor-recipient chimerism and reconstitute hematopoiesis and immunological functions. No unfavorable factors had been observed. Only one of the two cord blood units which had higher colony forming ability in vitro could engraft in most SCID mice as shown by HLA-DQB(1) gene detection. Two HLA-haploidentical cord blood units were simultaneously engrafted in 3 SCID mice.

CONCLUSIONDouble HLA-haploidentical or HLA-mismatched cord blood can engraft in SCID mice and reconstitute hematopoietic and immunological functions. HLA disparity has no significant effect on survival and engrafting rate. However, in less HLA disparity group, two cord blood units were prone to engraft simultaneously.

Animals ; Antigens, CD ; immunology ; Cord Blood Stem Cell Transplantation ; methods ; Disease Models, Animal ; Female ; Fetal Blood ; immunology ; metabolism ; Flow Cytometry ; HLA Antigens ; genetics ; immunology ; Hematopoiesis ; Humans ; Mice ; Mice, SCID ; Random Allocation ; Severe Combined Immunodeficiency ; immunology ; physiopathology ; surgery ; Survival Analysis ; Transplantation, Heterologous

OBJECTIVETo determine the ETO-interaction domain of nuclear receptor co-repressor (N-CoR) for abolishing the biological function of AML1-ETO.

METHODSTen different regions of N-CoR (N-CoRYs) were generated by means of polymerase chain reaction (PCR), and cloned into yeast expression plasmid pGADGL to construct pGADGL/N-CoRYs. The yeast two-hybrid technique and X-gal staining were used to determine the binding between the 10 different regions of N-CoR and ETO.

RESULTSIt was shown that the co-existence of 988-1,126 and 1,551-1,803 amino acid residues of N-CoRY was the ETO-interaction domains required for the binding with ETO.

CONCLUSIONTwo domains of N-CoR that interact with two zinc fingers of ETO, and keep stable binding between the two proteins were identified.

Binding Sites ; genetics ; Humans ; Nuclear Proteins ; chemistry ; genetics ; metabolism ; Nuclear Receptor Co-Repressor 1 ; Plasmids ; genetics ; Protein Binding ; Proto-Oncogene Proteins ; genetics ; metabolism ; RUNX1 Translocation Partner 1 Protein ; Recombinant Fusion Proteins ; genetics ; metabolism ; Repressor Proteins ; chemistry ; genetics ; metabolism ; Transcription Factors ; genetics ; metabolism ; Transfection ; Two-Hybrid System Techniques

OBJECTIVETo prepare a single chain antibody (ScFv) of mAb SZ-21 against platelet GPIIIa for its future clinical application.

METHODSThe expression vector pET20b-SZ-21ScFv was constructed and the fusion protein was expressed in E. coli BL21 (DE3) PlysS. The activated fusion protein was obtained after a series of purification steps, including cell breakage, inclusion body solubilization, His-bind resin affinity chromatography and protein refolding.

RESULTSThe fusion protein yields were up to 21% of the total amount of bacteria protein. The ScFv fragment could inhibit ADP-induced platelets aggregation in a dose-dependent manner in vitro and the maximal inhibition rate was obtained at a concentration of 20 micro g/ml. It also reacted with endothelial cells as detected by flow cytometry. Moreover, the ScFv fragment was able to inhibit the binding of fibrinogen to platelet.

CONCLUSIONThe SZ-21ScFv fragment had the activity to inhibit platelets aggregation and the binding of fibrinogen to platelet, being potentially useful for the treatment of thrombotic diseases.

Antibodies, Monoclonal ; pharmacology ; Blood Platelets ; metabolism ; Endothelium, Vascular ; cytology ; Fibrinogen ; metabolism ; Humans ; Immunoglobulin Fragments ; pharmacology ; Platelet Aggregation ; Platelet Glycoprotein GPIIb-IIIa Complex ; immunology ; Recombinant Fusion Proteins ; isolation & purification ; pharmacology

OBJECTIVESequences analysis of Ig variable regions from the peripheral blood mononuclear cells (PBMC) of patients with chronic B lymphocytic leukemia.

METHODSTotal RNA was isolated from PBMC of patients with chronic B lymphocytic leukemia, oligo-dT-primed cDNA was synthesized from RNA. The cDNA was amplified by Taq DNA polymerase with a set of specific 5' primers corresponding to Ig FR1 and 3' primers corresponding to CH1 (C micro /C) or CL (Ckappa/Clambda), the PCR products of variable regions of Ig heavy (IgH) and light (IgL) chains were sequenced by ABI PRISM Dye terminator cycle sequencing ready reaction kit and ABI PRISM 310 Genetic Analyzer. The gene homology of variable regions of IgH and IgL chains was compared by using DNA tools 5.1 system and "the international immunogenetics database".

RESULTSFour light chains and 3 heavy chains were amplified from 4 and 3 patients respectively. Homology analysis of the sequences of 4 light chains and 3 heavy chains were performed by DNA tools system. The sequences of light chains are high homologous. And the sequences of heavy chains are quite different. The homologous analysis of the sequences of variable region by using "the international immunogenetics database" showed that the sequences were higher homologous to idiotype gene of some B lymphocytic leukemia. Four VL genes belong to human Ig Vkappa subgroup I, 2 of 3 VH genes belong to VH3 family and 1 belongs to VH5 family.

CONCLUSIONIg genes have idiotype and same disease may have same idiotype.

Base Sequence ; Cloning, Molecular ; DNA, Complementary ; chemistry ; Gene Rearrangement ; Genes, Immunoglobulin ; Humans ; Immunoglobulin Fab Fragments ; genetics ; Immunoglobulin Heavy Chains ; genetics ; Immunoglobulin Light Chains ; genetics ; Immunoglobulin Variable Region ; genetics ; Leukemia, Lymphocytic, Chronic, B-Cell ; genetics ; immunology ; Molecular Sequence Data ; Polymerase Chain Reaction

OBJECTIVETo study the effect of DNR on HL-60 cells apoptosis in vitro and the related mechanism.

METHODSThe apoptosis of HL-60 was observed by microscope, flow cytometry (FCM) and DNA electrophoresis and various apoptosis-associated proteins expression by immunocytochemistry (IC) and FCM assays; the changes of apoptosis in HL-60 cells treated with DNR or suppressors PDTC or FB1 were also observed.

RESULTSWhen treated with 0.2 approximately 2.0 micro mol/L DNR, the percentage of apoptotic HL-60 cells increased with the dose increasing and the time extending, and the typical apoptotic cells and the appearance of apoptotic DNA ladder were observed. It was shown that after treatment with 1 micro mol/L DNR, the fluorescence intensity index (FI) of both bcl-2 and c-myc in HL-60 cells decreased, the FI of Bax, caspase-3 increased at 2 h, but decreased at 5 h, the FI of NF-kappaB increased. After adding PDTC, the apoptosis percentage of HL-60 cells decreased, but FB1 didn't present these effect.

CONCLUSIONIt suggested from the results that at certain concentration, DNR can induce the apoptosis of HL-60 cells in vitro. The mechanism was supposed by suppressing the expression of bcl-2 and c-myc and activating the expression of Bax and caspase-3, NF-kappaB and ROS had the marked correlation with the apoptosis process, but the ceramide synthase wasn't associated with it.

Apoptosis ; drug effects ; Caspase 3 ; Caspases ; analysis ; Ceramides ; biosynthesis ; Daunorubicin ; pharmacology ; Dose-Response Relationship, Drug ; HL-60 Cells ; Humans ; Immunohistochemistry ; NF-kappa B ; analysis ; Proline ; analogs & derivatives ; pharmacology ; Proto-Oncogene Proteins c-myc ; analysis ; Reactive Oxygen Species ; Thiocarbamates ; pharmacology

OBJECTIVETo study the relationship between HLA-DRB1 alleles and idiopathic thrombocytopenic purpura (ITP) in children.

METHODSPCR-SSO was used to identify DRB1 alleles of 42 children with ITP. Among them, anti-GPIIb/IIIa and anti-GPIb/IX autoantibody were detected in 36 cases by modified monoclonal antibody specific immobilization of platelet antigens (MAIPA).

RESULTS(1) Compared with healthy controls, HLA-DRB1 * 17 was significantly increased (relative risk = 2.76, P < 0.05, etiologic factor = 0.106 4) and HLA-DRB1 * 1202 decreased (relative risk = 0.20, P < 0.025, prophylactic factor = 0.761 6) in children with ITP. (2) In comparison with patients with good response to steroids and IgG therapy, HLA-DRB1 * 11 was significantly increased (P < 0.025) in patients with a poor response, furthermore, most (5/6) of HLA-DRB1 * 11-positive patients were female teen-ager. (3) Twenty-seven patients (75%) had anti-GPIIb/IIIa and seventeen (47.22%) had anti-GPIb/IX autoantibodies, the positivity rates of both anti-GPIIb/IIIa (P = 0.02) and anti-GPIb/IX (P = 0.01) were associated with HLA-DRB1 * 02. However, the pos./itivity rates of autoantibodies between refractory and non-refractory patients showed no significant difference.

CONCLUSION(1) The DRB1 * 17 seems to predict susceptibility to ITP in children, while DRB1 * 1202 appears to be protective to against ITP. (2) The DRB1 * 11 plays an important role in resistance to steroid and IgG therapy in children with ITP. (3) It seems that the response to the antigenic epitope of GPIIb/IIIa and GPIb/IX is restricted by DRB1 * 02, while the presence of the autoantibodies couldn't predict prognosis. Our preliminary findings indicate that genetic factors influence the clinical course of ITP, but its exact mechanism needs to be further investigated.

Alleles ; Child ; Female ; Gene Frequency ; HLA-DR Antigens ; genetics ; HLA-DRB1 Chains ; Humans ; Male ; Purpura, Thrombocytopenic, Idiopathic ; genetics ; immunology ; therapy