Cell cycle-independent down-regulation of BCL-2 protein expression in differentiating HL-60 cells

To understand the relationship between bcl-2 protein expression and the cell cycle during the processes of differentiation, we examined bcl-2 protein levels expressed during cell cycle phases in differentiating HL-60 human leukemia cells by using a two-color flow cytometric method. In exponentially proliferating HL-60 cells bcl-2 protein was constitutively expressed throughout the cell cycle phases, but a small population of G0/G1 cells expressed decreased levels of protein as compared with other cell cycle phases. HL-60 cells can be induced to differentiate to granulocytic pathway by retinoic acid or dimethylsulfoxide, and to monocytic/macrophagic pathway by 1, 25 dihydroxyvitamin D3 or 12-O-tetradecanoylphorbol -13-acetate. During treatment with any of these inducing agents, bcl-2 protein expression was time-dependently down-regulated after 24 h. A two-color flow cytometric analysis revealed that this down-regulation occurred throughout cell cycle phases, indicating that bcl-2 protein expression was down-regulated in cell cycle-independent manner during induction of differentiation in HL-60 cells. To our knowledge, this is the first demonstration suggesting that the regulation of bcl-2 protein expression is not related to the cell cycle during induction of differentiation in HL-60 cells.     

Cooperative regulation of c-myc expression in differentiation of human promyelocytic leukemia induced by recombinant gamma-interferon and 1,25-dihydroxyvitamin D3

The biological activity of recombinant human gamma-interferon (IFN-gamma) to induce the phenotypic differentiation of human promyelocytic leukemia cell line HL-60 and to regulate c-myc expression was evaluated. Treatment with IFN-gamma increased monocyte-associated cell surface antigens detected by monocyte-specific monoclonal antibodies in a dose- and time-dependent manner. These antigenic changes were accompanied by a functional differentiation, determined by the increase of phagocytic capability and superoxide generation. IFN-gamma was also found to suppress the growth of HL-60 cells and reduce expression of a c-myc oncogene. These phenotypic and morphological changes to macrophage-like cells induced by IFN-gamma were similar to those by 1,25-dihydroxyvitamin D3, whereas the plasma membrane antigenic changes were different. Moreover, the combination of IFN-gamma and suboptimal doses of 1,25-dihydroxyvitamin D3 have synergistic effects in augmenting mature monocyte specific antigens (Mo2, 63D3, OKM1, and OKM5). In the reduction of c-myc expression by these drugs, a cooperative effect was observed with the inhibition of transferrin receptor expression and cell growth. These results indicate that human recombinant IFN-gamma induces a monocyte phenotype in the HL-60 cells via a mechanism different from the action of 1,25-dihydroxyvitamin D3.     

Levels of myc protein, as analyzed by flow cytometry, correlate with cell growth potential in malignant B-cell lymphomas

We have analyzed c-myc protein expression during the cell cycle in malignant B-cell lymphomas by dual flow cytometric detection of a fluoresceinated polyclonal anti-myc antibody and propidium iodide which binds stochiometrically to DNA. The data obtained were correlated to other parameters of cell activation such as histopathological grading, expression of the activation antigen 4F2, light scatter (proportional to cellular volume), DNA synthesis and percentage of S-phase cells. The c-myc protein level was strongly correlated to parameters of DNA synthesis/content. In addition, the oncoprotein level was largely unvarying from the late G1 phase through the rest of the cell cycle in both malignant cells and normal purified B cells stimulated to proliferate in vitro.     

曲古抑菌素A对HL-60细胞分化的影响

目的 探讨曲古抑菌素A(TSA)在体外诱导急性早幼粒白血病细胞HL-60分化的作用.方法 采用溴化四甲基偶氮唑蓝法检测TSA对HL-60细胞增殖的影响,流式细胞术检测细胞周期;通过检测细胞分化特异性抗原CD11b的表达研究细胞分化作用;逆转录-聚合酶链反应(RT-PCR)方法检测c-myc基因mRNA的表达.结果 经TSA作用后,细胞增殖受抑制,细胞周期阻滞在G0和G1期,P<0.01;TSA作用48 h后,细胞分化率达15.24%;c-myc表达随TSA作用时间延长而降低.结论 TSA对HL-60细胞具有抑制增殖和诱导分化作用.     

RNA干扰对白血病细胞系HL-60增生的影响

 目的 研究针对c-myc基因的小分子干扰RNA片断（small interfering RNA, siRNA）对白血病细胞系HL-60细胞增生的影响,探讨应用siRNA进行白血病基因治疗的可能性。方法 制备特异性对应c-myc基因的siRNA转染HL-60细胞,倒置显微镜观察细胞形态学改变,MTT法检测细胞增生状态,流式细胞仪进行细胞周期分析。结果 siRNA转染HL-60细胞后,细胞生长受抑,增生能力减弱,其增生抑制作用于转染后48 h、72 h最明显,增生抑制率分别为53.2 %和51.5 %;S期细胞比值由（56.1±4.7）%降至（17.8±3.2）%,细胞阻滞在G0／G1期。结论 针对c-myc基因的siRNA对HL-60细胞增生有明显抑制作用,有望为白血病提供新的治疗途径。     

Cell Cycle-Independent Down-Regulation of BCL-2 Protein Expression in Differentiating HL-60 Cells

To understand the relationship between bcl-2 protein expression and the cell cycle during the processes of differentiation, we examined bcl-2 protein levels expressed during cell cycle phases in differentiating HL-60 human leukemia cells by using a two-color flow cytometric method. In exponentially proliferating HL-60 cells bcl-2 protein was constitutively expressed throughout the cell cycle phases, but a small population of G0/G1 cells expressed decreased levels of protein as compared with other cell cycle phases. HL-60 cells can be induced to differentiate to granulocytic pathway by retinoic acid or dimethylsulfoxide, and to monocytic/macrophagic pathway by 1, 25 dihydroxyvitamin D3 or 12-O-tetradecanoylphorbol-13-acetate. During treatment with any of these inducing agents, bcl-2 protein expression was time-dependently down-regulated after 24h. A two-color flow cytometric analysis revealed that this down-regulation occurred throughout cell cycle phases, indicating that bcl-2 protein expression was down-regulated in cell cycle-independent manner during induction of differentiation in HL-60 cells. To our knowledge, this is the first demonstration suggesting that the regulation of bcl-2 protein expression is not related to the cell cycle during induction of differentiation in HL-60 cells.     

羧甲基茯苓多糖对IL-2促诱生效应的实验与临床研究

采用0.5％～1.0％低血清培养法较好地模拟了小剂量rhTNF-α(50U/ml)对人早幼粒白血病细胞株HL-60的增殖抑制效应,但未发现促分化作用,对细胞株c-myc基因的表达亦无明显影响;而TNF(50μ/ml)具有诱导HL-60细胞分化和抑制细胞增殖的作用,且显著抑制其c-myc癌基因的表达水平。结果表明c-myc基因表达减低主要与TNF诱导HL-60白血病细胞分化相关,并非细胞增殖受抑的结果,对癌基因表达水平的调控可能是小剂量TNF发挥分化诱导作用的重要机制。     

环氧合酶-2抑制剂尼美舒利对白血病HL-60细胞增殖的抑制作用

 目的　探讨选择性环氧合酶-2（COX-2）抑制剂尼美舒利对人类急性髓系白血病HL-60细胞的增殖抑制作用。方法　以不同浓度的尼美舒利体外处理HL-60细胞,采用CCK-8、流式细胞术、Western blotting、ELISA等方法,检测尼美舒利对HL-60细胞增殖的作用及对细胞凋亡、细胞周期、COX-2、前列腺素E2（PGE2）、Bax、bcl-2、c-myc的影响。结果　尼美舒利对HL-60细胞增殖的抑制呈剂量、时间依赖性,可诱导细胞凋亡,将细胞周期阻滞于G0～G1期。尼美舒利作用HL-60细胞48 h后,细胞COX-2表达下调,100、200、400 μmol／L尼美舒利处理组与对照组HL-60细胞总凋亡率分别为（24.97±6.36）%、（34.22±5.76）%、（44.59±6.69）%及（4.11±1.26）%,差异有统计学意义（P＜0.05）。HL-60细胞合成PGE2减少,同时bcl-2、c-myc蛋白表达明显减少,Bax蛋白表达明显上调。结论　尼美舒利可抑制HL-60细胞增殖,诱导细胞凋亡,其作用与抑制COX-2表达,减少PGE2合成,阻滞细胞周期和调节凋亡相关蛋白bcl-2、c-myc、Bax的表达等有关。     

Inhibitory effects of prostaglandin A2 on c-myc expression and cell cycle progression in human leukemia cell line HL-60

The effect of prostaglandin A2 (PGA2) on c-myc expression was investigated in a human promyelocytic leukemia cell line, HL-60, which responded to PGA2 with a dose-dependent growth inhibition. Northern blot analysis indicated that treatment with PGA2 at 0.5 to 5.0 micrograms/ml remarkably reduced the steady state level of c-myc mRNA within 3 h, and then it gradually recovered according to the order of concentration of the drug. In contrast to c-myc, the level of class I HLA mRNA, as an internal control, was not diminished by PGA2 treatment. Further, this reduction of c-myc was not disturbed by cycloheximide, suggesting that this PGA2 action on c-myc expression is independent of de novo protein synthesis. Cytofluorometric analysis revealed that the exposure of HL-60 cells to PGA2 at 0.5 or 5.0 micrograms/ml arrested the cells in the G0-G1 phase of the cell cycle. This accumulation of the cells in G0-G1 phase continues until 24 or 36 h at 0.5 or 5.0 micrograms/ml, respectively. The G0-G1 arrest of the cell cycle was also recovered as the inhibition of c-myc was released. This recovery may be due to the loss of activity of PGA2 in culture medium. This study clearly showed that PGA2 treatment arrested HL-60 cells in the G0-G1 phase of the cell cycle and was associated with the reduction of c-myc mRNA.     

F1t3 receptor expression on the surface of malignant hematopoietic cells and responses to F1t3 ligand stimulation

Hematopoiesis is a complex process in which cell growth and differentiation are controlled by a number of hematopoietic growth factors or cytokines, acting as either positive or negative regulators. Disruption of balanced hematopoiesis may lead to the uncontrolled clonal expansion of a cell lineage, causing the leukemic transformation. Among them, class III receptor-type tyrosine kinases (RTKs) and their ligands seem to play an important role in hematopoiesis, especially in the early stages …     

全反式维甲酸对HL-60细胞VEGF表达与分泌影响的分子机制研究

目的 探讨人髓系白血病细胞株HL-60在诱导分化过程中血管内皮生长因子(VEGF)表达及分泌水平变化的分子机制.方法 四甲基偶氮唑蓝(MTT)法检测HL-60细胞被全反式维甲酸(ATRA)诱导分化后的增殖水平变化,流式细胞术测定HL-60细胞CD11b表达和细胞周期的改变,半定量逆转录聚合酶链式反应(RT-PCR)检测HL-60细胞VEGF、c-myc、缺氧诱导因子(HIF)-1α、基质金属蛋白酶(MMP)-9和MMP-2 mRNA表达水平,应用酶联免疫吸附试验(ELISA)法检测药物作用前后HL-60细胞培养上清液中VEGF蛋白的含量.结果 经ATRA诱导分化后,HL-60细胞增殖水平明显降低,CD11b表达水平明显升高,出现粒系定向分化趋势,且分化程度明显升高(P<0.05),c-myc与VEGFmRNA表达水平均明显下调(P<0.05),HIF-1α mRNA表达水平则明显升高(P<0.05).用MMP-2及MMP-9抑制因子Ⅰ阻断HL-60细胞MMP-9和MMP-2表达后,细胞培养上清中VEGF蛋白分泌量明显降低(P<0.05).结论 HL-60细胞被诱导分化过程中VEGF的表达与c-myc表达正相关,与HIF-1α表达负相关.MMP-9、MMP-2可能是调控HL-60细胞分泌VEGF的主要原因之一.     

Simultaneous detection of cellular ras p21 oncogene product and DNA content by two-parameter flow cytometry

The rapid identification of the expression of oncogene products in specific cell types could be useful to investigate normal and malignant cell proliferation. We have developed a sensitive fixation - permeabilization technique (with 70% ethanol and 0.01% Triton x 100) for the detection of p21 ras oncoprotein and DNA content. Cell suspensions with negligible cell clumping, bright specific immunofluorescent staining were obtained with this method. Bivariate flow cytometry was then used to quantitate simultaneously the distribution of anti p21 ras oncoprotein (with a specific FITC-labeled antibody) and of total DNA (with propidium iodide). The study was carried out in human leukemic cell lines HL-60 and K562, human breast carcinoma cell line MCF-7 and fresh neoplastic cells from human acute leukemia. The p21 ras oncoprotein was found in all phases of cell cycle. The degree of its expression, however, varied widely in diploid (C0/G1) cells from different samples, which could be related to differences in the relative proportion of G0 and G1 cells. Compared to the conventional gel electrophoretic technique, the use of bivariate FCM is feasible, fast, requires fewer cells per sample (2 x 10(6] and allows both the ras oncogene expression in intact cell populations as well as its relationship with cell cycle phases to be studied.     

Relationship between G-CSF and hyperleukocytosis in patients with APL after treatment with all-trans retinoic acid

Thepatientswithacutepromyelocyticleukemia(APL)oftendiedfromearlyinfectionorcoagulationdisorders.lllCompleteremission(CR)ratesof50%-60%duringthechemotherapywerereportedinrecentyears.Studiesinvitrohaveshownthatall-transretinoicacid(ATRA)wascapableoflettingAPLcellstodifferentiateatsomelowconcentrations.ThispropertyhasbeenappliedinvivoinnewlydiagnosedandinrelapsingAPL.Otherwise,withlesscoagulationofdisordersandbonemarrowaplasia.DevelopmentofATRAtreatmenthasresultedinincreasingCRratestoabo…     

c-myc oncogene product expression and prognosis in operable breast cancer

The 62 kDa protein product of the c-myc oncogene (p62 c-myc) is thought to be involved in the control of normal cellular proliferation and differentiation. We have measured oncoprotein levels using a flow cytometric assay in 141 operable breast cancers and have correlated levels with prognostic variables, patient survival and disease free intervals. High levels of p62 c-myc were associated with well differentiated tumours. There was no correlation with tumour DNA index, lymph node or oestrogen receptor status. C-myc oncoprotein levels were not predictive of patient survival or disease free interval. This relationship of oncoprotein levels with tumour histological grade is in keeping with the suggestion that the c-myc oncogene is important in the control of cellular differentiation. The other findings imply that measurement of c-myc oncoprotein levels does not yield useful prognostic information.     

The c-myc oncogene is regulated independently of differentiation in myeloid cell lines

Human myeloid leukaemia (U-937 and HL-60) cells when incubated at low cell densities with human recombinant gamma-interferon underwent functional maturation without any loss of proliferative potential relative to uninduced cells. In addition, the proportion of cells in S,G2/M and levels of c-myc oncogene (mRNA and protein) were maintained at the same level as those of untreated control cells. However, cells grown under similar conditions but with retinoic acid matured to the same extent but became growth inhibited with concomitant reductions in the proportion of cells in S,G2/M and levels of c-myc mRNA and protein. These studies indicate firstly that c-myc levels are regulated independently from differentiation in myeloid (non lymphoid) cells, secondly that gamma-interferon can induce differentiation without growth arrest under conditions of low cell density and thirdly emphasise the close association of c-myc expression with proliferative capacity.     

Effect of prostaglandin E2 on gamma-interferon and 1,25(OH)2D3 vitamin D3-induced c-myc reduction during HL-60 cell differentiation

The effect of prostaglandin E2 (PGE2) on the reduction of c-myc expression during the differentiation of the human leukemic cell line, HL-60, was examined. PGE2, a potent inducer of intracellular cyclic AMP (cAMP) in HL-60 cells, augmented monocyte-associated cell surface antigens induced by human gamma-interferon (IFN-gamma) or 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) in these cells. The elevation of intracellular cAMP was induced dose-dependently by PGE2, but not by IFN-gamma or 1,25(OH)2D3. Changes were also seen in functional differentiation, such as, the increase of phagocytic capability and superoxide generation. PGE2 also enhanced the reduction of c-myc expression and the down-regulation of transferrin receptor by IFN-gamma or 1,25(OH)2D3, whereas PGE2 alone did not induce these phenotypic changes. These data suggest that IFN-gamma and 1,25(OH)2D3 reduce c-myc expression of HL-60 cells by a mechanism other than the augmentation of intracellular cAMP.     

膜结合型前列腺素E2合酶 1抑制剂MK886对白血病HL-60／A细胞周期的影响

 【摘要】　目的　探讨膜结合型前列腺素E2合酶 1（mPGES-1）抑制剂MK886对人类急性髓系白血病耐药细胞HL-60／A细胞周期的作用。方法　采用流式细胞术、Western blot、酶联免疫吸附（ELISA）等方法,检测HL-60／A及健康人单个核细胞（MNC）、敏感细胞株HL-60的细胞周期、 mPGES-1蛋白、细胞周期素D1（cyclin D1）的差异,检测不同浓度的MK886对HL-60／A细胞周期的作用及对cyclin D1、mPGES-1、PGE2、P-Akt、c-myc蛋白的影响。结果　与健康人MNC及敏感细胞株HL-60比较,HL-60／A细胞cyclin D1、mPGES-1表达最高,G0～G1期细胞比例为（30.53±2.15）％,较正常MNC[（62.63±6.58）%]及HL-60细胞[（38.86±2.25）%]减少（均P＜0.05）;S期比例为（57.56±1.54）％,较正常MNC[（12.18±4.43）%]及HL-60细胞[（47.70±1.88）%]明显增多（均P＜0.05）。MK886作用后,被阻滞于G0～G1期的细胞增多,同时mPGES-1表达下调,合成PGE2减少,cyclin D1、P-Akt、c-myc蛋白表达下降。结论　MK886对白血病HL-60／A细胞有细胞周期阻滞作用,其机制与抑制mPGES-1表达,减少PGE2的合成,抑制cyclin D1、P-Akt、c-myc表达有关。     

Decreased expression of type II protein kinase C in HL-60 variant cells resistant to induction of cell differentiation by phorbol diester

To evaluate the molecular basis for susceptibility of the cell differentiation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), we examined biochemical activities and expression of subspecies of protein kinase C from HL-60 cells that are susceptible to differentiation induced by TPA and HL-60R cells, HL-60 variant cells that are resistant to such induction. Analysis of the subcellular distribution of protein kinase C revealed that the activity of this kinase in the cytosol from HL-60R cells was 30% of that from parental HL-60 cells but that the enzyme activities in the membrane showed similar values in these cells. Treatment of HL-60 cells with 100 nM TPA for 30 min resulted in a 75% decrease in protein kinase C activity in the cytosol and a 300% increase in this activity in the membrane. A minor subcellular redistribution of the enzyme activity was found in HL-60R cells after TPA treatment. Based on analysis of protein kinase C isozymes by hydroxyapatite column chromatography, the relative activities of types I, II, and III in the cytosol of HL-60 cells were 11, 80, and 9%, whereas those in HL-60R cells were 27, 36, and 37%, respectively. Type II isozyme was a major protein kinase C in the cytosol of HL-60 cells, but type II was less in the HL-60R cells. Among the three isozymes, type II enzyme was most sensitive to TPA with histone H1 as the substrate, although all three isozymes were activated Ca2+-dependently in the presence of phosphatidylserine. We suggest that the acquired resistance of HL-60R cells toward induction of cell differentiation by TPA may be associated with a decrease in the expression of the type II isozyme of protein kinase C.