柴胡皂甙d诱导K562细胞凋亡的差异基因筛选

目的 筛选柴胡皂甙d(SSd)诱导人白血病K562细胞凋亡的差异基因,为白血病的治疗奠定基础.方法 用Agilent Human 1A寡核苷酸芯片检测SSd处理前后K562细胞的基因表达谱变化,并筛选SSd诱导K562细胞凋亡的相关基因.结果 359个基因发生显著性变化,其中表达上调基因138个,表达下调基因221个.结论 SSd诱导K562细胞凋亡是一个多基因参与的过程,本文筛选出的差异基因有望成为人白血病治疗的靶基因.     

Prevention of autoimmune diabetes in NOD mice by 1,25 dihydroxyvitamin D3

Summary 1,25 dihydroxyvitamin D₃, the active form of vitamin D, has immunomodulatory properties in vitro and in vivo. We report that treatment with 1,25 dihydroxyvitamin D₃ (5 g/kg on alternate days) prevents the development of clinical diabetes in NOD mice, an animal model of human autoimmune diabetes. Diabetes incidence in female NOD mice at the age of 200 days was reduced to 8% in the 1,25 dihydroxyvitamin D treated group vs 56% in the control group (p<0.0001). In parallel, treatment with 1,25 dihydroxyvitamin D₃ resulted in a complete normalisation of the capacity to induce suppressor mechanisms in an autologous MLR, which is severely depressed in control NOD mice. The existence of such suppressor cells was confirmed in transfer experiments, whereby cotransfer of splenocytes from 1,25 dihydroxyvitamin D₃ treated NOD mice prevented diabetes transfer by splenocytes from diabetic NOD mice into irradiated, 6–8-week-old male NOD mice. Other known immune defects of the NOD mice, such as defective natural killer cell killing of YAC-1 targets and defective thymocyte activation by anti-CD3 were not corrected. The pharmacological doses of 1,25 dihydroxyvitamin D₃ were universally well tolerated as reflected by a normal weight gain of the mice. Serum calcium was increased (2.5±0.2 vs 2.2±0.2 mmol/l in the control group, P<0.005), whereas osteocalcin levels nearly doubled and bone calcium content was halved. These findings show that 1,25 dihydroxyvitamin D₃ can prevent diabetes in NOD mice, probably through the correction of their defective suppressor function.Abbreviations 1,25(OH)₂D₃ 1,25 dihydroxyvitamin D₃ - NOD non-obese-diabetic - MLR mixed lymphocyte reaction - ⁶⁰Co cobalt - ⁵¹Cr sodium chromate - SI stimulation index - NK natural killer     

Embryonic erythroid differentiation in the human leukemic cell line K562.

K562 human leukemia cells synthesize embryonic hemoglobins after culture in the presence of hemin. We have rigorously identified these hemoglobins by globin chain analysis and peptide mapping. No adult hemoglobin could be detected, and beta-globin synthesis was less than 2 ppm of total protein synthesis. Persistent embryonic globin gene expression is known to occur as a consequence of globin gene deletions. However, restriction endonuclease mapping showed that the globin gene complexes in K562 cells are indistinguishable from normal. Hemin increased the rate of embryonic globin synthesis. The pattern of hemoglobin synthesis proved to be stable when cells from different laboratories were compared. One line, however, synthesized large amounts of Hb X and very little Hb Portland in response to hemin. Hb X has been previously detected in human embryos; we show here that it has the composition epsilon 2 gamma 2 and is diagnostic of imbalanced chain synthesis or "zeta thalassemia." We have identified several agents that induce hemoglobin synthesis in K562 cells. Different inducers induced different patterns of embryonic hemoglobin synthesis but never any adult hemoglobin synthesis.     

Hemin-induced erythroid differentiation changes the sensitivity of K562 cells to tumor necrosis factor-alpha

Tumor necrosis factor-alpha (TNF) can inhibit the growth of erythroid progenitors (erythroid colony-forming units [CFU-E] and erythroid burst-forming units [BFU-E]) at picomolar concentrations, but only if added within the first 48 h of culture. These data suggested that cells undergoing erythroid differentiation become resistant to TNF. To test this hypothesis, K562 cells were treated with hemin to induce erythroid differentiation and then tested for their sensitivity to TNF in terms of growth and TNF receptor expression. TNF inhibited the growth of untreated K562 cells, but not hemin-treated K562 cells. Untreated K562 cells expressed TNF receptors, whereas few hemin-treated K562 cells expressed TNF receptors within 24 h of exposure to hemin. These data show that K562 cells induced to differentiate along the erythroid pathway are resistant to TNF because they lack TNF receptors and suggest that the resistance of erythropoietin-treated human bone marrow cells to TNF added after 48 h of culture may also reflect loss of TNF receptors associated with erythroid differentiation.     

1,25 dihydroxyvitamin D3 limits monocyte maturation in lupus sera

In this study we examined whether treatment with 1,25-dihydroxyvitaminD(3) (1,25(OH)(2)D(3)) affects human APC maturation in vitro under multiple cytokine milieus. Human monocytes were elutriated from whole blood, incubated with human sera in the presence or absence of 1,25(OH)(2)D(3), and stimulated with IFNα, GM-CSF/IL-4, or were cultured without stimulatory cytokines. Incubation of control monocytes with 1,25(OH)(2)D(3) limited expression of cell surface makers of maturation whether monocytes were stimulated in IFNα, GM-CSF/IL-4, or in serum alone. The ability of 1,25(OH)(2)D(3) to affect human monocyte phenotype was assessed by incubating cells with sera from 15 patients with SLE and from 5 healthy volunteers. Addition of 1,25(OH)(2)D(3) resulted in significant reductions in the expression of MHC Class II, CD40, and CD86 and increases in expression of CD14 in both types of sera. Overall, 1,25(OH)(2)D(3) limited human APC activation via IFNα-induced and independent mechanisms. 1,25(OH)(2)D(3) inhibited APC activation by systemic lupus erythematosus (SLE) sera, suggesting that it may be possible for 1,25(OH)(2)D(3) to reduce the immunostimulatory effects of the SLE milieu by interfering with the soluble cytokine mediators in the sera of SLE patients.     

Butyrate-induced erythroid differentiation of human K562 leukemia cells involves inhibition of ERK and activation of p38 MAP kinase pathways

Butyrate induces cytodifferentiation in many tumor cells of different origin, suggesting that an as yet unidentified common mechanism inherent to malignant cells is the target of butyrate action. This study determined the role of different mitogen-activated protein (MAP) kinase signal transduction pathways in butyrate-induced erythroid differentiation of K562 human leukemia cells. Using a panel of anti-ERK, JNK, and p38 phosphospecific antibodies, the study showed that phosphorylation of ERK and JNK is decreased following treatment of cells with butyrate, whereas phosphorylation of p38 is increased. In contrast, a K562 subline defective in butyrate-mediated induction of erythroid differentiation did not reveal these changes in phosphorylation patterns. Inhibition of ERK activity by UO126 induces erythroid differentiation and acts synergistically with butyrate on hemoglobin synthesis and inhibition of cell proliferation, whereas inhibition of p38 activity by SB203580 completely abolished induction of hemoglobin expression by butyrate. Taken together, our data suggest a model in which butyrate induces erythroid differentiation of K562 cells by inhibition of ERK and activation of p38 signal transduction pathways.     

Basic fibroblast growth factor antagonizes transforming growth factor beta-mediated erythroid differentiation in K562 cells

Basic fibroblast growth factor (bFGF) and transforming growth factor- beta 1 (TGF-beta) have both been shown to act on hematopoietic progenitor cells. bFGF is a hematopoietic cytokine that acts on progenitor cells in concert with other cytokines to promote their proliferation. TGF-beta induces erythroid differentiation in K562 cells. To determine whether bFGF might act on progenitor cells by antagonizing the effects of cytokines that induce differentiation, we determined the effects of bFGF on the TGF-beta-mediated induction of hemoglobin synthesis in K562 cells. bFGF antagonized the TGF-beta- mediated induction of hemoglobin in a dose-dependent manner, with 0.1 ng/mL bFGF inhibiting hemoglobin induction by 40% and 10 ng/mL bFGF completely abrogating hemoglobin production. bFGF was most effective at antagonizing the TGF-beta-mediated induction of hemoglobin if it and TGF-beta were added simultaneously to K562 cells, but delayed addition of bFGF to TGF-beta-treated cultures still resulted in significant inhibition of hemoglobin synthesis. The inhibitory effects of bFGF on hemoglobin production were fully reversible, showing that bFGF did not permanently alter the phenotype of K562 cells. The hemin-mediated induction of hemoglobin synthesis in K562 cells was only partially negated by bFGF. bFGF also diminished the expression of glycophorin A on the surface of K562 cells. These results indicate that bFGF might increase progenitor/stem cell numbers by antagonizing the effects of cytokines that induce differentiation, thereby increasing the pool of proliferating progenitor/stem cells.     

Cyclosporin A induces erythroid differentiation of K562 cells through p38 MAPK and ERK pathways

We studied the effects of cyclosporin A (CsA) on the erythroid differentiation of human erythroid leukemia cell line K562. After K562 was treated with CsA for 4 days, the percentage of hemoglobinized cells was increased by 3.3 times. Because it was reported p38 MAPK (p38) and ERK are involved in erythropoietin-induced erythroid differentiation, we studied their roles using specific inhibitors. p38 inhibitor (SB203580) prevented CsA-induced hemoglobin synthesis in K562 cells, although MEK/ERK inhibitor (U0126) enhanced it by 3.3 times in K562 cells. These results indicate activation of p38 and inactivation of ERK are involved in CsA-induced erythroid differentiation of K562 cells.     

Human leukemic K562 cells treated with cytosine arabinoside: enhancement of erythroid differentiation by retinoic acid and retinol

Abstract: Human leukemia K562 cells can be induced to erythroid differentiation when treated with a variety of compounds, including hemin, cytosine arabinoside and 5-azacytidine. Following erythroid induction, K562 cells express at high level γ-globin and accumulate both Hb Portland and Hb Gower 1. In this paper we determined whether a combination treatment of K562 cells with suboptimal concentrations of cytosine arabinoside and retinoids lead to full expression of differentiated functions. Cell growth kinetics studies, intracellular detection of hemoglobin by benzidine staining and hemoglobin analysis by cellulose acetate were performed. The results obtained show that (a) retinoic acid and retinol are not able to induce differentiaton of K562 cells and (b) cytosine arabinoside induces differentiation only when used at 100–300 nmol/l concentrations. In addition, our data demonstrate that erythroid differentiation of K562 occurs when 40 μmol/l of retinoic acid or retinol are added together with 75 nmol/l cytosine arabinoside.     

Contribution of GPR30 for 1,25 dihydroxyvitamin D3 protection in EAE

Previous studies have demonstrated that vitamin D3-mediated protection in EAE occurs only in females and is dependent on the presence of diestrus levels of 17β-estradiol (E2). To evaluate the role of estrogen receptors in vitamin D3 treatment of EAE, we compared disease severity, CNS histopathology and immunological responses in vehicle and calcitrol (1,25 dihydroxyvitamin D₃) treated WT C57BL/6 mice vs. GPR30 membrane estrogen receptor (MER) knockout mice with MOG-35-55 peptide-induced EAE. Our results demonstrated that vitamin D₃-mediated prevention of clinical signs, CNS cellular lesions and demyelination observed in WT mice was abrogated in GPR30-KO mice with EAE. Regulatory effects of vitamin D₃ treatment that were MER dependent included increased levels of IL-10 and IL-6 secreted by MOG peptide-reactive splenocytes and increased expression of CCL5, CCR1 & CCR3 in spleen tissue. These results demonstrate for the first time that the MER is a key contributor to the E2-dependent effects of vitamin D₃-mediated protection in EAE.     

盐酸肾上腺素对白血病细胞株K562增殖及分化的影响

目的探讨盐酸肾上腺素在白血病发生、发展中的作用。方法采用MTT法集落培养实验观察盐酸肾上腺素对白血病细胞株K562细胞增殖的影响;采用Gimesa染色观察细胞形态学变化,采用流式细胞术检测CD71分化抗原表达。结果盐酸肾上腺素可明显降低K562细胞增殖、集落生长,并呈浓度依赖性;在形态上可诱导细胞趋向红系分化,同时上调K562细胞膜CD71分化抗原的表达。结论盐酸肾上腺素在白血病发生、发展中起重要作用,其机制为抑制K562细胞增殖和诱导其分化。     

The DNA-binding drugs mithramycin and chromomycin are powerful inducers of erythroid differentiation of human K562 cells

The human leukaemic K562 cell line can be induced in vitro to undergo erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine and cytosine arabinoside. Differentiation of K562 cells is associated with an increased expression of embryo-fetal globin genes, such as the zeta, epsilon and gamma globin genes. Therefore the K562 cell line has been proposed as a useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation which stimulate gamma-globin synthesis could be considered for possible use in the experimental therapy of those haematological diseases associated with a failure in the expression of adult beta-globin genes. In this paper we demonstrated that the G + C selective DNA-binding drugs chromomycin and mithramycin were powerful inducers of erythroid differentiation of K562 cells. Erythroid differentiation was associated with an increase in the accumulation of (a) Hb Gower 1 and Hb Portland and (b) gamma-globin mRNA.     

1,25-二羟基维生素D3和维生素K2联合诱导HL-6O细胞分化及凋亡的研究

目的研究1,25-二羟基维生素D3[1,25(OH)2D3,简称D3]与维生素K2(VK2)联合应用对HL-60细胞分化及凋亡的影响.方法通过四唑氮蓝(MTT)比色,细胞形态,流式细胞仪(FCM)测定细胞周期、凋亡率及CD14的表达,观察D3、VK2对HL-60细胞的影响.结果D3与VK2都能抑制HL-60细胞增殖,并且联合使用抑制作用显著.10-8mol/L D3与10 μmol/L VK2联合处理HL-60细胞72 h后CD14的表达率为63.15%,且G0/G1期细胞显著增多,与单独一种比较差异有统计学意义(P＜0.05).10 μmol/L、20 μmol/L VK2作用于HL-60细胞72 h凋亡率分别为11.31%、20.36%,与对照组比较差异有统计学意义(P＜0.01);而10 μmol/LVK2与10-8 mol/L D3联用72 h细胞的凋亡率为5.41%,与对照组比较差异无统计学意义(P＞0.05).结论D3与VK2联合使用可以使D3诱导分化作用加强,而VK2诱导凋亡作用受到抑制.     

Inhibition of interleukin-1 production by 1,25-dihydroxyvitamin D3

The hormonal form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], inhibits the proliferation of T lymphocytes and production of growth-promoting factors (including interleukin-2) (IL2) in CTLL2 murine cells. In this study, we investigated the role of monocytes in this hormone-mediated inhibitory effect, by testing the effects of 1,25-(OH)2D3 on the ability of the mitogenic lectin phytohemagglutinin (PHA) to induce T cell activation in either a monocyte-dependent or phorbol myristate acetate (PMA)-driven (monocyte-independent) system. The results indicate that proliferation of T cells and production of growth-promoting factors are inhibited by 1,25-(OH)2D3 only in the monocyte-dependent system. Preincubation of monocytes with 1,25-(OH)2D3 for various periods of time and subsequent removal of the hormone resulted in inhibition of the PHA-driven proliferation of T cells. Preincubation for 2 h resulted in 20% inhibition, while preincubation for 36 h reduced proliferation to 50% of the control value [no 1,25-(OH)2D3 exposure]. These data suggested that monocytes are important participants in 1,25-(OH)2D3-mediated events. Therefore, we tested the effects of the hormone on the production of IL1, a monocyte-derived product thought to be involved in the induction of IL2 release and the subsequent development of the T cell proliferative response. 1,25-(OH)2D3 inhibited the production of both extracellular and cell-associated immunoreactive IL1 alpha and IL1 beta. Indomethacin, a prostaglandin synthetase inhibitor, did not alter the inhibitory properties of 1,25-(OH)2D3, suggesting that prostaglandins are not responsible for the inhibitory phenomenon. We conclude that part of the ability of 1,25-(OH)2D3 to inhibit T cell proliferation may be due to direct effects on monocytes by down-regulating IL-1 production. However, it is unlikely that the immunoregulatory properties of 1,25-(OH)2D3 on T cells are mediated solely through monocytes, and it is possible that the hormone also exerts its influence directly on T cells.     

Normal serum concentrations of 1,25 dihydroxyvitamin D in osteoporosis

Summary With the aim of evaluating the role of 1,25 dihydroxyvitamin D in the pathogenesis of osteoporosis, this hormone was studied in 90 subjects. They were divided into three groups: the first group consisted of 30 normal female subjects (aged 30 to 45); the second group comprised 30 elderly female subjects (aged 67 to 90) the third group consisted of female patients (aged 65 to 87) with clinical and radiological evidence of osteoporosis. Variations of serum 1,25 dihydroxyvitamin D, after stimulation with oral P04 (1.000 mg/day for 10 days) and stimulation with oral Ca (1.000 mg/day for 10 days), were studied in 16 osteoporotic females (10 of them received P04) and six Ca (Controls). This second study was performed to evaluate the renal 1-alpha-hydroxylation. No significant variation between serum 1,25 dihydroxyvitamin D of elderly and osteoporotic females was observed when compared with normal young healthy subjects. Moreover, a significant increase in serum 1,25(OH)2D after oral P04 was found in accordance with a significant increase in serum parathormone. These data demonstrate that changes in serum 1,25(OH)2D levels did not seem to play a significant part in the pathogenesis of osteoporosis in our population neither could a deficit of renal 1-alpha-hydroxylation be demonstrated.     

Apoptosis induced by erythroid differentiation of human leukemia cell lines is inhibited by Bcl-XL

The induction of tumor cell differentiation represents an attractive strategy for the treatment of a wide range of malignancies. Differentiation of HL-60 promyelocytic leukemia cells towards neutrophils or monocytes has been shown to induce apoptotic cell death, which is inhibited by bcl-2 over-expression. However, the role of the bcl-2 gene family during erythroid differentiation of human leukemia cells remains unknown. We found that human erythroleukemia (HEL) and K562, two leukemia cell lines that undergo erythroid differentiation do not express Bcl-2, but express Bcl-XL, a related protein that functions as an inhibitor of apoptosis. Differentiation of HEL or K562 cells with inducers of erythroid differentiation (hemin, retinoic acid, or transforming growth factor-beta) was accompanied by progressive cell death and degradation of genomic DNA into oligonucleosomal fragments. The loss of cellular viability was associated with downregulation of bcl-xL mRNA and protein. In contrast, the levels of Bax, another Bcl-2 family member implicated in apoptosis remained unaltered. Constitutive expression of Bcl-XL by gene transfer inhibited apoptosis triggered by erythroid differentiation of HEL K562 cells. Yet, Bcl-XL did not alter the expression of epsilon-globin, which is induced during erythoid differentiation of HEL and K562 cells, arguing that apoptosis and differentiation can be uncoupled by Bcl-XL. These results indicate that Bcl-XL acts as an antiapoptosis protein in leukemia cells that undergo erythroid differentiation and that downregulation of bcl-x is a component of the apoptotic response that is coupled to differentiation in human leukemia cells.     

Enhanced expression of transforming growth factor beta during megakaryoblastic differentiation of K562 leukemia cells

Platelet alpha granules contain several growth factors such as the transforming growth factor beta (TGF-beta) that are released during blood clotting and are thought to participate in the repair of tissue injury; however, the site of synthesis of platelet TGF-beta has not been demonstrated. We studied TGF-beta expression during megakaryoblastic differentiation of the chronic myeloid leukemia cell line K562 in vitro. These cells have mainly erythroid characteristics but acquire several megakaryoblastic properties when treated with the phorbol diester 12-0-tetradecanoyl-13-phorbolacetate (TPA). During four subsequent days of megakaryoblastic differentiation the amount of the 2.5-kilobase (kb) TGF-beta mRNA increased about eightfold, and a novel 2.3-kb mRNA species was induced in the K562 cells. This occurred concomitantly with distinct induction patterns of platelet-derived growth factor A (PDGF-A) and c-sis (PDGF-B chain) RNAs and several platelet antigens. The expression of erythroid markers such as glycophorin A decreased. Culture media of TPA-differentiated K562 cells also contained TGF-beta polypeptides as shown by a sensitive radioreceptor assay and by immunoprecipitation after metabolic labeling of the cells. These polypeptides were not seen in culture media from dimethyl sulfoxide- or sodium butyrate-treated cells. Unlike in several other cells, exogenously added TGF-beta 1 or 2 affected neither TGF- beta nor PDGF RNA expression in K562 cells.