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 evaluate the effect of mycophenolate mofetile (MMF) on acute graft versus host disease (aGVHD) prophylaxis after allogeneic bone marrow transplantation (allo-BMT) in a murine model.

METHODSAcute GVHD was induced in male BALB/c mice by total-body irradiation (TBI) followed by female allogeneic (C57BL/6J) bone marrow and spleen cells transplantation. Acute GVHD was assessed both physically and histologically. Severe aGVHD was developed in the recipients and the mean survival time (MST) of untreated BM recipients was 6 days. After allo-BMT, animals were divided into 6 groups. Group 1 was given MMF (30 mg.kg(-1).d(-1)), group 2 CsA (1.5 mg.kg(-1).d(-1)) and MTX (0.4 mg.kg(-1).d(-1)), group 3 MMF (30 mg.kg(-1).d(-1)) in combination with CsA and MTX, group 4 MMF (60 mg.kg(-1).d(-1)) in combination with CsA and MTX, group 5 MMF (10 mg.kg(-1).d(-1)) in combination with CsA and MTX, and group 6 no agents for aGVHD prophylaxis. The physical signs, MST, peripheral blood counts, and aGVHD histopathologic examination were observed in all recipients.

RESULTSAnimals in control group developed typical aGVHD and 100% of mortality, with a MST of 6 days. The MSTs of group 1 approximately 5 were significantly longer than that of control, being 3.4, 8.4, 9.0, 6.1 and 8.8 days longer, respectively (P < 0.05). The MSTs of groups 2 approximately 5 were longer than that of group 1 (P < 0.05), but there was no statistical significance between groups 3 approximately 5 and 2. There was no statistical difference in peripheral blood count among groups 1 approximately 5. Histopathological examination of skin, liver, and gastrointestinal tract showed typical signs of aGVHD. Animals received immunosuppressive agents (MMF, CsA, MTX) showed the less severe signs.

CONCLUSIONSMMF markedly prolonged MST of allo-BMT recipients, delayed the onset of aGVHD signs. The prophylaxis effect of CsA + MTX with or without MMF was better than that of MMF alone, synergism between MMF of 10 or 30 mg.kg(-1).d(-1) and CsA + MTX was better than that of MMF of 60 mg.kg(-1).d(-1) and CsA + MTX.

Animals ; Bone Marrow Transplantation ; Cyclosporine ; pharmacology ; Dose-Response Relationship, Drug ; Drug Synergism ; Female ; Graft Survival ; drug effects ; Graft vs Host Disease ; pathology ; prevention & control ; Immunosuppressive Agents ; pharmacology ; Methotrexate ; pharmacology ; Mice ; Mice, Inbred C57BL ; Models, Animal ; Mycophenolic Acid ; analogs & derivatives ; pharmacology ; Time Factors ; Transplantation, Homologous

OBJECTIVETo explore the correlation between expression of surviving gene in acute leukemic cells and its clinical effects.

METHODSBy using semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) technique, surviving gene expression in 50 previously untreated acute leukemia (AL) patients was analysed. The apoptosis of primary leukemia cells cultured in vitro was assayed with terminal deoxyribonucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL).

RESULTSSurviving gene expression levels in cells of AL patients at diagnosis were significantly higher than that in normal bone marrow mononuclear cells (MNCs) (82.0% vs 33.3%, P < 0.05). The expression level was higher in ALL cells than in ANLL cells (89.5% vs 75.0%). Among 22 cases of ANLL, bone marrow remission (BMR) rate was higher in surviving gene negative expression cells from patients accepted a course of chemotherapy than in positive expression cells (83.3% vs 25.0%, P = 0.023). Among 13 ANLL patients received a course of HA regimen chemotherapy, the BMR was higher in patients surviving mRNA negative expression cells than in positive cells (100.0% vs 27.3%). Patients with surviving/beta-actin ratio>0.6 attained lower BMR.

CONCLUSIONHigher expression level of surviving mRNA in AL cells may be one of the reasons that leukemic cells are insensitive to chemotherapy.

Adolescent ; Adult ; Aged ; Antineoplastic Agents ; therapeutic use ; Chromosomal Proteins, Non-Histone ; genetics ; metabolism ; Drug Resistance, Neoplasm ; genetics ; physiology ; Female ; Gene Expression Regulation, Leukemic ; Humans ; Inhibitor of Apoptosis Proteins ; Leukemia, Myeloid, Acute ; drug therapy ; genetics ; metabolism ; Male ; Microtubule-Associated Proteins ; Middle Aged ; Neoplasm Proteins ; Precursor Cell Lymphoblastic Leukemia-Lymphoma ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; Remission Induction ; Treatment Outcome

Objective　To evaluate the efficacy and safety of mycophenolate mofetil（MMF） in combinat ion with cyclosporine A(CsA) and methotrexate(MTX) for prevention of acute graft versus host disease(GVHD) after unrelated donor allogeneic bone marrow transpla ntation (allo-BMT). Method　Twelve cases of unrelated donor allo -BMT were evaluated in a single center trial. The acute GVHD was prevented with 1 g MMF daily in addition to CsA 3?mg*kg-1*d-1 and MTX 10～15 ?mg at post BMT day1，day3，day6 and day11. Results　Acute GVHD was found in one case（Grade Ⅳ） at the seventh day and two cases （Grade Ⅱ ）at the tenth day and seventeenth day after BMT. These patients were treated wi th a combination of MMF,methyprednisolone and CsA. The common adverse hematologic events of MMF was leukopenia. Conclusion　 The preliminary study showed that MMF could be used effectively and safe ly for prevention of acute GVHD in unrelated donor allo-BMT.

Objective　Investigation of the role of mitochondrial membrane potential (MMP) in the homoharringtonine (HHT)-induced apoptosis. Methods　Annexin V staining, flow cytometry and confocal laser scan microscopy were used to observe the relationship between Bax, cytochrome C and MMP in the HHT-induced apoptosis of leukemic T lymphocytic line Molt-3. Results　The induction of apoptosis by HHT resulted in the translocation of Bax from cytosol to mitochondrial membrane and the decrease of cellular MMP, followed by the release of cytochrome C from mitochondria to cytosol. Conclusions　Changes of mitochondrial membrane potential might play a critical role in the HHT-induced apoptosis of leukemic T-cells.

Objective　To explore the antitumor activity of human telomerase RNA antisense oligodeoxynucleotide (As-ODN) to leukemic cells and its mechanisms. Methods　As-ODN was transfected into HL-60 cells by liposomal transfection. Telomerase activity of HL-60 cells was examined by telomeric repeat amplification protocol (TRAP)-enzyme-linked immunosorbent assay(ELISA). Apoptosis was analyzed by morphology, DNA agarose gel electrophoresis and flow cytometry. Results　The telomerase activity in HL-60 cells, being transfected with 0.1～2.0 μmol/L of As-ODN for 1～6 days, varied from 1.043±0.045 to 0.063±0.011 in a dose- and time-dependent manner. The proliferation of As-ODN-transfected HL-60 cells was lower than that of control, and the cells underwent apoptosis. The Ms-ODN(missense ODN)-transfected HL-60 cells didn′t show these changes. Conclusion　As-ODN can inhibit the telomerase activity of HL-60 cells specifically and induce apoptosis.

AIM: To explore the effect of berbamine(BER) on apoptosis in K562 cells and its possible molecular mechanisms. METHODS: The apoptosis rate was measured by flow cytometry while electron microscopy and DNA electrophoresis were used to evaluate the characteristic changes of apoptosis, RT-PCR and Western blot were used to examine the expression levels of apoptosis related gene bcr/abl and BCR/ABL protein. RESULTS: By FCM, the apoptosis rate of K562 cells treated with 8.0 mg/L BER for 24 h and 72 h increased from (29.20?3.82)% to (61.77?4.35)% (P

AIM: To explore the relationship between hypermethylation of p15INK4B gene and the pathogenesis of hematopoietic malignances. METHODS: The expression and methylation of p15INK4B gene and the expression of DNA methyltransferase genes (DNMTs) in bone marrow cells from 54 cases with hematopoietic malignances were detected by RT-PCR, Western blot, and methylation-specific PCR. RESULTS: The p15INK4B gene was methylated more often in high-risk myelodysplastic syndrome (MDS) patients, patients at blast phase of chronic myeloid leukemia (CML-BP) and acute leukemia patients than that in low-risk MDS patients (P

AIM: To explore the influence of CD47 molecules on the maturation and function of cultured dendritic cells (DCs). METHODS: Monocyte cell-derived DCs were propagated in granulocyte-macrophage colony-stimulating factor (GM-CSF) and lipopolysaccharide (LPS) plus interleukin (IL)-4, in the presence or absence of anti CD47 monoclonal antibodies (anti-CD47 mAbs). Flow cytometry was used to detect the cell surface phenotype. The concentration of IL-12P70 in supernatant was measured by ELISA technique. The antigen-presenting functions of DCs were determined in one-way mixed leukocyte reaction by Brdu-ELISA. Electrophoretic mobility shift assays (EMSA) was used to examine NF-?B activity. RESULTS: The anti-CD47 mAbs markedly suppressed the expression of CD80,CD86,CD83,CD1a,HLA-DR on the surface of DCs (P

AIM: To investigate the effects of transforming growth factor ?1 (TGF-?1) on murine-derived dendritic cells (DC). METHODS: Murine bone marrow cells were cultured with GM-CSF and TGF-?1 to develop TGF ?-DC. Then they were stimulated by lipopolysaccharide (LPS). Their phenotypes were assessed by flow cytometry (FCM). The allogeneic stimulating capacity of DC was measured by mixed lymphocyte reaction (MLR) using BrdU ELISA method. IL-12 p70 protein was detected by ELISA and the expressions of Toll like receptor 4 (TLR4) on DCs were measured by semi-quantitative RT-PCR and FCM. RESULTS: Compared to immature DC (imDC) cultured with GM-CSF alone, the expressions of CD_80, CD_86, I-A~b and CD_40 in TGF ?-DC were lower. The TGF ?-DC was resistant to maturation by LPS. Maturation resistance was evident from a failure to up-regulate CMs, to stimulate larger T cell proliferation and to increase secretion of IL-12 p70. Down-regulation of TLR4 expression on TGF ?-DC was also found. CONCLUSION: TGF-?1 inhibits the expression of co-stimulatory molecules on DC. It is resistant to maturation stimulus (LPS) and might be linked with TLR4 down-regulation.