OBJECTIVETo investigate the regulation mechanism of apoptosis induced by the antisense bcl-2 treatment.

METHODSDNA content analysis and terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) were adopted to detect apoptosis. Semi-quantitative reverse transcription-PCR was performed to detect the mRNA expression of bcl-2 c-myc survivin bax s100A(2) TNFalpha TGFbeta(1) and IL-6 in the small-cell lung cancer cell line NCI-H446 treated with antisense bcl-2 oligodeoxynucliotide.

RESULTSbcl-2 AS-PS-ODN treatment could induce apoptosis, accompanied with 72.71% up-regulation of IL-6 and 65.90% down-regulation of TNFalpha, whereas little or no effect was seen on c-myc survivin bax s100A(2) and TGFbeta(1).

CONCLUSIONIL-6 and TNFalpha may be involved in the regulation of apoptosis induced by antisense bcl-2 treatment.

Apoptosis ; drug effects ; genetics ; Chemotactic Factors ; genetics ; Gene Expression Regulation, Neoplastic ; drug effects ; Humans ; In Situ Nick-End Labeling ; Inhibitor of Apoptosis Proteins ; Interleukin-6 ; genetics ; Microtubule-Associated Proteins ; genetics ; Neoplasm Proteins ; Oligonucleotides, Antisense ; pharmacology ; Proto-Oncogene Proteins ; genetics ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; Proto-Oncogene Proteins c-myc ; genetics ; RNA, Messenger ; drug effects ; genetics ; metabolism ; S100 Proteins ; genetics ; Transforming Growth Factor beta ; genetics ; Transforming Growth Factor beta1 ; Tumor Cells, Cultured ; drug effects ; Tumor Necrosis Factor-alpha ; genetics ; bcl-2-Associated X Protein

OBJECTIVETo establish a real-time quantitative RT-PCR method for the detection of expression of bcl-2 mRNA.

METHODSThe vector containing bcl-2 gene as standard template was constructed with T-A cloning technique. The fluorogenic probe (i.e.,Taq man probe) was used to establish a real-time RT-PCR. bcl-2 mRNA expression level in Burkitt's lymphoma pre-and post-treated with bcl-2 antisense phosphothioate oligodeoxynucleotides (AS-PS-ODN) was determined with real-time quantitative RT-PCR, also with semi-quantitative RT-PCR.

RESULTSThe expression of bcl-2 mRNA in Burkitt's lymphoma treated with bcl-2 AS- PS-ODN decreased significantly and no changes of bcl-2-mRNA expression in group treated with nonsense ODN were noticed. The semi-quantitative RT-PCR results also demonstrated that bcl-2 level varied as detected with real-time fluorogenic quantitative RT-PCR, but less sensitive and accurate.

CONCLUSIONDetection of bcl-2 mRNA expression with the fluorogenic real-time quantitative RT-PCR is more accurate and sensitive than semi-quantitive RT-PCR.

Electrophoresis, Agar Gel ; methods ; Gene Expression ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; RNA, Messenger ; analysis ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo study the anticancer activities of curcumin on human Burkitt's lymphoma and their molecular mechanism.

METHODSThe effect of curcumin on the growth of CA46 cells and apoptosis were studied through Trypan blue exclusion, MTT assay, cell cycle, DNA fragmentation analysis and detection of TdT-mediated dUTP nick end labeling (TUNEL). The effect of curcumin on the expression of c-myc, bcl-2, mutant-type p53 and Fas protein and mRNA was studied by flow cytometry (FCM) and reverse transcription-polymerase chain reaction (RT-PCR).

RESULTS1. Curcumin inhibited proliferation of CA46 cells in a time- and dose-dependent manner, 2. CA46 cells treated with curcumin showed G(0)/G(1) or G(2)/M phase increase and S phase decrease, 3. CA46 cells apoptosis induced by curcumin was confirmed by DNA fragmentation and TUNEL and 4. The expression of c-myc, bcl-2, mutant-type p53 protein and mRNA was decreased sharply in CA46 cells treated with curcumin, while Fas protein and mRNA was increased.

CONCLUSIONCurcumin is able to inhibit the proliferation of CA46 cells and induce the cell apoptosis by down-regulating the expression of c-myc, bcl-2, mutant-type p53 and up-regulating the expression of Fas.

Antineoplastic Agents ; pharmacology ; Apoptosis ; Burkitt Lymphoma ; Cell Cycle ; drug effects ; Cell Division ; drug effects ; Curcumin ; pharmacology ; Gene Expression Regulation ; drug effects ; Humans ; Proto-Oncogene Proteins c-bcl-2 ; biosynthesis ; genetics ; Proto-Oncogene Proteins c-myc ; biosynthesis ; genetics ; Tumor Cells, Cultured ; Tumor Suppressor Protein p53 ; biosynthesis ; genetics ; fas Receptor ; biosynthesis ; genetics

Objective: To study the effect of combining bcr-abl Aspo and c-myb Aspo on K562 cells. Methods: Cellswere exposed to oligomers. Cell inhibitory rate was determined by typan blue dye exclusion. CFU-K562 cells were culturedin 0.8% methyleellulose. P210 was measured by flow cytometry. Cellular bcr-abl mRNA was detected by RT-PCR semiquantitative analysis. Cell apoptosis was measured by flow cytometry and observed by electron microscope. Results: When the concentration of both bcr-abl Aspo and c-myb Aspo was 5 μmol/L, K562 cells were still growth in clone state. The growth inhibitory rate was 61.7% at 120 h. P210 was depressed at 24 h and went up to 25.7% at 120 h. The apoptosis rate was 22.5%. While K562 cells were dealt with 10 μmol/L bcr-abl Aspo and 10 μmol/L c-myb Aspo, the cells were growth in dispersal. The cell growth inhibitory rate reached to 92.2% and 64.3% of K562 cells were induced to apoptosisat 120 h. P210 was complelely depressed untill 120 h. The decrease of bcr-abl mRNA was from 69.2% to 85.3% after incubation 48 h with 5 μmol/L Aspo and 10 mol/L. Conclusion: It emerges coordination to combine bcr-abl Aspo and c-myb Aspo on K562 cells, and enhances the anti-leukemia effect.

AIM: To study the expression of cytokines and their receptors in leukemia cell lines and normal blood cells. METHODS: RT-PCR was used to detect expression of mRNA for cytokines in leukemia cell lines(HL-60,U937,K562,HEL,DAMI,MEG-01,HUT78 and CA) and normal blood cells, including CD34 + cells, megakaryocytes,platelets, peripheral mononucleates cells and granulocytes. RESULTS: ①CD34 + cells simultaneously expressed mRNA for IL-1(?,?),IL-3, IL-6 , G-CSF, GM-CSF and their receptors and SCFR,MPL as well. The granulocytes only expressed IL-6,IL-6R,G-CSFR,GM-CSF. Megakaryocytes and platelets only expressed IL-3R,IL-6,IL-6R,MPL.Interestingly, TGF? 1 ,TNF? and their receptors sustained to express in normal cells.②Most leukemia cell lines were found to simultaneously express at least two or more stimulating cytokines and receptors ,while TGF? 1 , TNF? and their receptors were expressed in all the leukemia cell lines we observed. CONCLUSIONS: ①Multi-autocrine loops exist in leukemia cells;②Imbalance of autocrine loops of positive and negative cytokines may be related to leukemia.

To accelerate the identification of HLA-DPB1 matched marrow donors from unrelated population, a very simple HLA-DPB1 genotyping method called PCR-fingerprinting (PCRF) was developed according to the theory about homoduplex and heteroduplex formation from different PCR coding strands and non-coding ones. Unlike PCR-SSCP, strict laboratory condition is not needed in the PCRF. After denaturing at 94℃ for 2 min and cooling at 37℃ for 8 min, the PCR product was separated by 8% PAGE for 5 h and polymorphism band patterns would appear when the gel staining was completed with either EB or silver staining procedure. To confirm its reliability, 21 individuals from 9 family whose DPB1 genotypes assigned by PCR-RFLP were verified. It was found that there were 8 PCRF patterns corresponding to the 9 HLA-DPB1 genotypes from the 21 cases and the same DPB1 genotypes produced identical PCRF pattern except one pair.The factors on efficient separation of heteroduplexes and homoduplexes were also discussed.

Objective: To study the effect of combining bcr abl Aspo and c myb Aspo on K562 cells. Methods: Cells were exposed to oligomers. Cell inhibitory rate was determined by typan blue dye exclusion. CFU K562 cells were cultured in 0.8% methylcellulose. P210 was measured by flow cytometry. Cellular bcr abl mRNA was detected by RT PCR semiquantitative analysis. Cell apoptosis was measured by flow cytometry and observed by electron microscope. Results: When the concentration of both bcr abl Aspo and c myb Aspo was 5 ?mol/L, K562 cells were still growth in clone state. The growth inhibitory rate was 61 7% at 120 h. P210 was depressed at 24 h and went up to 25.7% at 120 h. The apoptosis rate was 22.5%. While K562 cells were dealt with 10 ?mol/L bcr abl Aspo and 10 ?mol/L c myb Aspo, the cells were growth in dispersal. The cell growth inhibitory rate reached to 92.2% and 64.3% of K562 cells were induced to apoptosis at 120 h. P210 was complelely depressed untill 120 h. The decrease of bcr abl mRNA was from 69.2% to 85.3% after incubation 48 h with 5 ?mol/L Aspo and 10 ?mol/L. Conclusion: It emerges coordination to combine bcr abl Aspo and c myb Aspo on K562 cells, and enhances the anti leukemia effect.

Aim To study the inhibitory effects of F951,a novel bcl-2 antisense oligodeoxynucleotide,on expression of bcl-2,growth of tumor and survival time of nude mice transplanted subcutaneously with acute myeloid leukemia.Methods HL-60 cells with high expression of bcl-2 were proliferated in vitro.The models of the nude mice with HL-60 cells were established by subcutaneous transplantation with drugs directly injected.The effects of F951 and F951 with low dose Ara-c on growth of tumor and survival time of mice with tumor were observed.The expressions of bcl-2 mRNA in the tumors implanted were detected by fluorescent quantitation RT-PCR.The morphologic structure of tumor tissues was assayed by light microscope.Results After each group mice with tumors were treated for 14 days,the volume,the weight of tumor and the bcl-2 mRNA expression of tumor tissue were shown respectively as follows: NS control group(15.17?3.40)cm3、(12.69?0.92)g、9.79?104 Copies??g-1;FNS group(15.91?3.77)cm3,(12.38?1.21)g;8.31?104 Copies??g-1;Ara-C group(1.24?0.55)cm3,(2.32?0.49)g,2.59?104 Copies??g-1;F951 group(2.6?1.55)cm3,(3.53?0.67)g;1.01?103 Copies??g-1;F951+Ara-C group(0.62?0.48)cm3,(1.05?0.63)g,9.5?102 Copies??g-1.The data above showed that F951 could downregulate the expression of bcl-2 in nude mice with HL-60 cells xenograft and inhibit growth of tumor.The growth of tumor of F951 group was reduced,and the inhibitory rate was 72.18%,there was significant difference comparing control groups with NS and FNS(P

AIM and METHOD: The relationship between the evolution of the reticulin fibres(RF) or the collagen fibres(CF) and the growing of hematopoietic cells in long-term bone marrow culture (LTBMC) from 15 paticnts with acute myeloid leukemia (AML) and form 6 normal subjects was observed by inverted microscope, Gomoris staining and Massons staining were used. RESULTS: (1)The amount of RF contents of 8 AMLs,with self- maintained(AMLsm) in the 1～8 weeks-old-culture was significant less than that of normal control and 7 AMLs,without self-maintained(AMLnsm) ( P

AIM:To study the effect of bcr - abl gene antisense phosphorothioate oligonucleotides(Aspo) on K562 cell line and explore its significance in chronic myelogencous leukemia (CML) gene therapy. METHODS: Cells were exposed to oligomers, observed by inverted microscope. Cells inhibitory rate were determined by 0.4% trypan blue exclusion. CFU - K562 were cultured in 0.8% methylcellulose. P210 was measured by flow cytometry. RESULTS:K562 cells were treated with Aspo, they still grew in clone state and show antisense sequence specific and dose dependent. When the concentration of Aspo was more than 5?mol/L, the growth of cells was inhibited and P210 was down regulated or completely suppressed, and the greatest growth inhibition was at 120h. There was significant inhibition of cell proliferation in a rang of cells number from 1 ? 10-4/mL to 5 ? 10-4/mL after treatment with 10?mol/L Aspo. b2a2 Aspo was also effect on K562 cells which expressing b3a2 mRNA. CONCLUSION: bcr - abl Aspo has a specific growth inhibition effect on K562 cells, and worths further study in CML gene therapy.