Effects of arsenic trioxide and ATRA on PLZF-RARalpha-positive U937 leukemic cells]

AIM: To investigate effects of arsenic trioxide (As(2)O(3)) and alltrans retinoic acid (ATRA) on PLZF-RARalpha-positive cells. METHODS: PLZF-RARalpha-positive U937 cells (U937/PLZF) were used as an in vitro model. The change of cell morphology was observed by Wright-Giemsa staining. Cell growth and proliferation were detected by methyl thiazolyl tetrazolium(MTT) assay. Cell cycle distribution and expression of cell membrane surface differentiation-related antigens (such as CD11b, CD64 and CD14) were determined by flow cytometry assay. Expression of PLZF was analyzed by immunofluorescence. Functional differentiation was reflected by nitroblue tetrazolium(NBT) reduction ability and cytochemistry staining. RESULTS: While U937/PLZF cells were incubated in tetracycline-withdrawn medium, the expression of PLZF-RARalpha; protein increased. After treated with As(2)O(3) (0.5 micromol/L) and ATRA (1 mumol/L), U937/PLZF cells presented some changes such as decreased nuclear/cytoplasm ratio, and partial disappearance of nucleoli, suggesting a certain degree of morphological differentiation. The cell growth and proliferation were inhibited in a dose- and time-dependent manner. The proportion of cells in S phage was decreased and CD11b level was increased. The expression of PLZF relocated in treated cells. However, no significant difference in NBT assay and cytochemistry staining was documented with the combination therapy. CONCLUSION: The combination of As(2)O(3) with ATRA can cause a slight tendency to morphological differentiation but is insufficient to induce functional differentiation of PLZF-RARalpha positive U937 leukemia cells.     

The death-associated protein kinase 2 is up-regulated during normal myeloid differentiation and enhances neutrophil maturation in myeloid leukemic cells

The death-associated protein kinase 2 (DAPK2) belongs to a family of Ca(2+)/calmodulin-regulated serine/threonine kinases involved in apoptosis. During investigation of candidate genes operative in granulopoiesis, we identified DAPK2 as highly expressed. Subsequent investigations demonstrated particularly high DAPK2 expression in normal granulocytes compared with monocytes/macrophages and CD34(+) progenitor cells. Moreover, significantly increased DAPK2 mRNA levels were seen when cord blood CD34(+) cells were induced to differentiate toward neutrophils in tissue culture. In addition, all-trans retinoic acid (ATRA)-induced neutrophil differentiation of two leukemic cell lines, NB4 and U937, revealed significantly higher DAPK2 mRNA expression paralleled by protein induction. In contrast, during differentiation of CD34(+) and U937 cells toward monocytes/macrophages, DAPK2 mRNA levels remained low. In primary leukemia, low expression of DAPK2 was seen in acute myeloid leukemia samples, whereas chronic myeloid leukemia samples in chronic phase showed intermediate expression levels. Lentiviral vector-mediated expression of DAPK2 in NB4 cells enhanced, whereas small interfering RNA-mediated DAPK2 knockdown reduced ATRA-induced granulocytic differentiation, as evidenced by morphology and neutrophil stage-specific maturation genes, such as CD11b, G-CSF receptor, C/EBPepsilon, and lactoferrin. In summary, our findings implicate a role for DAPK2 in granulocyte maturation.     

The Notch ligand, Delta-1, partially inhibits GM-CSF-induced differentiation and apoptosis along with reducing the cleavage of PARP in U937 cells

Notch signaling plays an important role in the regulation of self-renewal and differentiation of hematopoietic cells. Human monoblastic U937 cells undergo differentiation into macrophage-like cells, growth suppression, and apoptosis following stimulation with GM-CSF. We examined the effects of Notch activation induced by Notch ligands on GM-CSF-induced differentiation and apoptosis in U937 cells. Furthermore, the molecular mechanism of the effects was investigated. A recombinant Notch ligand, Delta-1 protein did not affect the growth of U937 cells by itself. GM-CSF-induced growth suppression and apoptosis of U937 cells were partially rescued by incubation with Delta-1. Delta-1 also reduced the GM-CSF-induced differentiation. Incubation with Delta-1 did not affect the expression of GM-CSF receptor. GM-CSF stimulation induced the phosphorylation of ERK1/2 and STAT5 and the cleavage of caspase-8, which were not affected by Delta-1 incubation, either. GM-CSF stimulation induced the cleavage of PARP, which is the key molecule for differentiation and apoptosis. We found that incubation with Delta-1 significantly suppressed the GM-CSF-induced cleavage of PARP. Taken together, we found that Notch activation induced by Delta-1 partially inhibited GM-CSF-induced differentiation, growth suppression, and apoptosis, along with reducing the GM-CSF-induced cleavage of PARP. These findings suggest one of the mechanisms by which Notch activation inhibits differentiation and apoptosis.     

PKC-ζ参与调节基质细胞来源的因子-1诱导的人U937细胞VEGF mRNA表达

目的：探讨用短发夹RNA表达质粒抑制人U937细胞的蛋白激酶C-ζ（PKC-ζ）表达后，对基质细胞来源的因子-1刺激的U937细胞VEGFmRNA表达的影响。方法: 设计合成一对针对人PKC-ζ的mRNA的寡核苷酸序列，退火后将其连入载体。对重组质粒pSIRENp进行酶切鉴定。用脂质体介导的方法分别将质粒pSIRENp和对照质粒转染U937细胞，用RT-PCR检测PKC-ζ和U937细胞VEGFmRNA。结果: 酶切证实目的寡核苷酸片段已经被克隆到pSIRENp，转染后几乎完全抑制U937细胞的PKC-ζ表达；VEGF mRNA表达在SDF-1作用前后均明显低于对照。结论: shRNA方法可成功抑制U937细胞的PKC-ζ表达。PKC-ζ可能参与调节U937细胞VEGF的表达。     

Glycans and proteoglycans are involved in the interactions of human immunodeficiency virus type 1 envelope glycoprotein and of SDF-1alpha with membrane ligands of CD4(+) CXCR4(+) cells

We demonstrate that human immunodeficiency virus HIV-1(LAI) envelope glycoprotein 120 (gp120(LAi)) specifically interacts with several membrane ligands on lymphoid CEM or monocytic U937 cells in addition to its previously identified receptor, CD4, and CXCR4, its coreceptor. In its native state, gp120(LAI) is able to elicit specific multimolecular complexes with these membrane ligands at the surface of the cells; most of the interactions are abolished by mannan or heparin but not by dextran. Similarly, stromal cell-derived factor (SDF)-1alpha interacts not only with CXCR4 expressed by CXCR4(+) CD4(+) U937, CEM, and HOS-CD4(+) CXCR4(+) cells but also with CD4 expressed by intact U937, CEM, and HOS-CD4(+) CXCR4(+/-) cells or electroblotted onto Immobilon. SDF-1alpha binding to CD4(+) CXCR4(+/-) cells, or soluble CD4 electroblotted onto Immobilon, is significantly inhibited by sCD4, whereas truncated sCD4 lacking D3 and D4 domains had no significant effect, which indicates that SDF-1 binds to CD4 but at regions different from the HIV-gp120-binding site. Heparin and mannan also inhibit SDF-1alpha binding to intact CD4(+) CXCR4(+/-) cells, and electroblotted soluble CD4. Heparitinase treatment of such cells reduced SDF-1alpha binding. These data demonstrate that glycans and glycosaminoglycans are directly or indirectly involved in the interactions of HIV-1 gp120(LAI) and of SDF-1alpha with membrane ligands of CD4(+) CXCR4(+) cells and thus could play a role both in HIV-1 infection and in the physiology of SDF-1alpha.     

Se-methylselenocysteine enhances PMA-mediated CD11c expression via phospholipase D1 activation in U937 cells

CD11c/CD18 is expressed primarily on myeloid cells, where its expression is regulated both during differentiation and during monocyte maturation into tissue macrophages, and is also a receptor for fibrinogen and lipopolysaccharide (LPS). We focused on the molecular mechanisms leading to the activation of CD11c expression in differentiating U937 cells. During phorbol myristate acetate (PMA)-induced differentiation of U937 cells, we found that the mRNA expression of CD11c was increased. Se-methylselenocysteine (Se-MSC) potentiated up-regulation of CD11c expression and its promoter activity and increased PLD1 activity without affecting the level of PLD1 protein in PMA-treated cells. To examine the regulation mechanism of PMA and Se-MSC on CD11c gene expression through the activation of PLD1, we analyzed changes in the CD11c mRNA level and the promoter activity following treatment of a selective PLD inhibitor n-butanol. The combinatory effect of PMA and Se-MSC on CD11c gene expression was abolished by n-butanol in a dose-dependent manner. Further, introduction of PLD1 gene into U937 cells increased CD11c mRNA expression and activated CD11c promoter activity in a dose-dependent manner. These results showed that Se-MSC increased PMA-induced CD11c expression through the activation of PLD1 signaling pathway. To our knowledge, this is the first report that expression of the CD11c gene is regulated by PLD1 and is enhanced by Se-MSC during PMA-induced U937 differentiation.     

Intermediate- and high-affinity interleukin-2 receptors expressed in an IL-4-dependent T-cell line induce different signals.

We have previously described a synergism between the two physiological hormones, retinoic acid (RA) and 1 alpha,25-dihydroxyvitamin D3 (VD) in the induction of U937 cell differentiation towards a more mature state. Herein, we investigated the regulation of cytokine production during RA and/or VD treatment of U937 cells. Cell differentiation was followed by measurement of their capacity to give oxidative responses, and interleukin-1 beta (IL-1 beta), tumour necrosis factor-alpha (TNF-alpha) and IL-6 gene and protein expression were determined in RA/VD-treated cells, activated or not with lipopolysaccharide (LPS). The undifferentiated and RA-treated U937 cells were unable to produce monokines even when they were stimulated by LPS. VD induced the monokine mRNA expression in U937 cells but failed to induce protein release. However, unlike RA, it primed the cells to secrete monokines upon endotoxin stimulation. A large enhancement of the production of the monokines both at mRNA and protein levels was observed in the U937 cells exposed to the combination of RA + VD. Nevertheless, protein release required a further step of activation of the RA + VD-primed cells. The co-inducer effect of RA and VD was not observed in HL-60 or THP-1 cells and seems to be restricted to U937 cells. These results on cytokine expression support our previous finding that a combination of RA and VD brings the U937 cells to a high stage of myeloid differentiation with major characteristics of monocytes/macrophages.     

Studies on CD11a and CD18 molecules with two new monoclonal antibodies: differential myelomonocytic antigen expression of PMA treated HL60 and U937 cell lines

Biochemical and functional aspects as well as features of cellular distribution of the differentiation groups CD11a and CD18 were studied in the course of a detailed characterization of two new monoclonal antibodies which recognize the alpha (GRS3) and beta (GRF1) chains of the LFA-1 antigen. Both MAbs inhibited homotypic aggregation of an EBV cell line. In contrast, only GRF1 (anti-beta chain) was able to inhibit granulocyte aggregation as well. Different myeloid-monocyte antigen modulation was noted in PMA induced macrophage differentiation of the U937 and HL60 cell lines. PMA treated HL60 cells showed increased expression of alpha M (CD11b) and alpha X (CD11c) antigens but no change in HLA-DR or CD14 antigen expression. No variation in the expression of LFA complex antigen (CD11a, b and c, or CD18) was observed on U937 cells, which on the other hand presented de novo expression of HLA-DR and CD14 antigens.