Apoptosis-inducing Effect of 8-Bromo-7-Methoxychrysin on K562 cells / 中国实验血液学杂志

This study was purposed to investigate the apoptosis-inducing effect of 8-bromo-7-methoxychrysin (BrMChR) on leukemia K562 cells as well as the variation of caspase-3 activity and phosphorylated Akt (p-Akt) expression of K562 cells during the process of apoptosis. MTT assay was used to determine the inhibitory effect of BrMChR on proliferation of K562 cells. Cell apoptosis was assayed by AO/EB staining under fluorescent microscope and flow cytometry with Annexin V-FITC/PI staining. The expression level of p-Akt was measured by Western blot. The results showed that BrMChR had the inhibitory effect on proliferation of K562 cells and could induce apoptosis of these cells in dose-dependent manner, and these effects were significantly stronger than ChR. After treatment of K562 cells with 3 µmol/L ChR for 12 hours, the apoptosis rate was only 3.68%, but the apoptosis rate of K562 cells treated with 3 µmol/L BrMChR was 21.8%. In the same time, the caspase-3 activity significantly increased (p < 0.05), but the expression of p-Akt was down-regulated (p < 0.01). It is concluded that BrMChR can induce apoptosis of K562 cells and with effect stronger than chR. P-Akt may participate in the apoptosis process of K562 cells induced by BrMChR.

Expression and activity of glycosylphosphatidylinositol-specific phospholipase d mRNA in bone marrow mononuclear cells isolated from patient with acute myeloid leukemia and their significance / 中国实验血液学杂志

This study was purposed to investigate the expression and significance of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) in bone marrow mononuclear cells (BMMNC) isolated from patients with acute myeloid leukemia (AML), GPI-PLD activity in BMMNC isolated from 78 patients with AML and 15 normal persons was measured by using GPI-anchored placental alkaline phosphatase (PLAP) as a substrate and Triton X-114 phase partitioning. The GPI-PLD mRNA expression was measured by semi-quantitive reverse transcription-polymerase chain reaction (RT-PCR). The results showed that the mRNA expression level and activity of GPI-PLD in BMMNC from de novo AML patients were 1.86 +/- 0.32 and 46.96 +/- 7.15% respectively; the mRNA expression level and activity of GPI-PLD in BMMNC from completely remission and refractory or relapsed patients were 1.26 +/- 0.29, 33.36 +/- 5.13%and 1.79 +/- 0.19, 44.31 +/- 7.22%, while those in BMMNC from normal controls were 1.27 +/- 0.23, 35.38 +/- 5.15% respectively. The mRNA expression level and activity of GPI-PLD in de novo and refractory or relapsed patients were obviously higher than those in normal controls with significant difference (p < 0.01), while the comparison between remitted patients and normal controls showed no statistical difference (p > 0.05). It is concluded that the expression level of GPI-PLD mRNA coincides with GPI-PLD activity. The mRNA expression and activity of GPI-PLD in de novo and refractory or relapsed patients are obviously higher than those in normal controls. It is worthy of further exploring whether GPI-PLD plays a certain role in process of leukemia pathogenesis.

The effect of ATRA-induced leukemic cell differentiation on Brd7 gene expression in leukemia cell lines / 中国实验血液学杂志

This study was purposed to investigate the relationship between brd7 gene and differentiation of leukemia cells and the role of brd7 gene in differentiation of leukemia cells. The HL-60 and K562 cell lines were induced by all-trans retinoic acid (ATRA) for 7 days, then the cell morphologic change was observed under inverted microscope with Wright-Giema staining, the expression level of CD11b was detected by flow cytometry for evaluating cell differentiation level, the expression changes of BRD7 protein before inducing differentiation and in process of cell differentiation were determined by Western blot. The results showed that ATRA could inhibit the proliferation and induce differentiation of HL-60 cells, but no differentiation in K562 cells was induced by ATRA. The level of CD11b expression in HL-60 cells was up-regulated gradually during ATRA-induced cell differentiation. The expression of BRD7 protein increased markedly along with differentiation of HL-60 cells towards granulocytes. However, BRD7 protein did not significantly alter in K562 cells in which inducing differentiation was not found. It is concluded that brd7 gene expression enhances as the HL-60 cells differentiate, underlying which the mechanism remains to clarify.

Effect of GPI-PLD on adhesion function of bone marrow mononuclear cell from patients with myeloid leukemia and its mechanism / 中国实验血液学杂志

To explore the effect of glycosyl-phosphatidyl inositol-specific phospholipase D (GPI-PLD) on the adhesion function of bone marrow mononuclear cell from patients with myeloid leukemia and analyze its mechanism, the activity of GPI-PLD in bone marrow mononuclear cell from the patients were measured by using GPI-anchored placental alkaline phosphatase (PLAP) as substrate and Triton-X114 partitioning; the adhesion rate and CD24 expression of these cells were measured by MTT and immunohistochemical method respectively, when these cells were or were not treated by 1 mmol/L 1,10-phenanthroline for 5 hours. The results showed that the GPI-PLD activity of bone marrow mononuclear cells from the patients was significantly inhibited after being treated by 1 mmol/L 1, 10-phenanthroline for 5 hours [(42.08 +/- 7.21)% vs (5.4 +/- 2.96)%], while the adhesion rate and the expression of CD24 of these cells were increased [(49.78 +/- 26.73)% vs (61.19 +/- 29.14)%, (16.02 +/- 9.68)% vs (18.5 +/- 11.14)%, respectively)]. It is concluded that depression of GPI-PLD activity can increase the adhesion rate of bone marrow mononuclear cells from the patients while the CD24 expression is enhanced.

GFP fused to the cytoplasmic tail of integrin alphaIIb allows the normal expression of alphaIIb beta3 compound in CHO cells / 中国实验血液学杂志

To investigate the effect of GFP fused to C terminal of integrin alpha(IIb) on the biosynthesis and expression of alpha(IIb) beta(3) compound, the alpha(IIb) GFP expression plamid, named palpha(IIb) GFP, the cDNA of alpha(IIb) was constructed from p3.1-2b and fused to pEGFP-N1 in frame. When the sequence of palpha(IIb) GFP was confirmed by sequencing it was transferred to Chinese Hamster Ovary (CHO) cells with or without p3.1-3a expressing integrin beta(3). Then the expression of alpha(IIb) GFP fusion protein was confirmed by Western blot and then its subcellular localization was determined with laser confocal scanning microscopy. The results showed that the target gene was cloned into recombinant vector by restriction analysis and sequencing. Overexpression of the fusion protein in the transfected CHO cells was identified with Western blot. Subcellular localization analysis confirmed that alpha(IIb) GFP was expressed in CHO cells and could be transferred from endoplasmic reticulum to Golgi apparatus. It is concluded that the eukaryotic expression plasmid containing alpha(IIb) GFP fusion gene is successfully constructed. GFP fused to the cytoplasmic tail of integrin alpha(IIb) allows the normal expression of alpha(IIb) beta(3) in CHO cells.

Effect of glycoprotein alpha II bA2334C mutation on the biosynthesis and transportation of alpha II bbeta3 complex / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the effect of glycoprotein (GP) alpha II bA2334C mutation on the biosynthesis and expression of alpha II bbeta3 complex.</p><p><b>METHODS</b>The GP alpha II bA2334C eukaryotic expression plasmid pc3.1-2334M2b was constructed. Chinese hamster ovary (CHO) cells were transfected with the plasmid with or without integrin beta3 expression plasmid pc3.1-3a. The whole expression of alpha II bA2334C was confirmed by Western blot and the membrane expression was analyzed by flow cytometry. A newly constructed alpha II bA2334C GFP fusion protein expressing plasmid was used to determine its subcellular localization by laser confocal scanning microscopy.</p><p><b>RESULTS</b>Expression of the mutant protein, alpha II bA2334C, in the transfected CHO cells was confirmed by Western blot with a lower rate of the mature type than the wild type control. The expression on membrane was only 25% of the normal. Subcellular localization analysis showed that alpha II bA2334C GFP was able to be expressed in CHO cells and could be transported from endoplasmic reticulum to Golgi apparatus.</p><p><b>CONCLUSIONS</b>The mutant alpha II bA2334C can be synthesized in CHO cells and form alpha II bbeta3 complex. However, only a small fraction of the premature alpha II bA2334C can be transported to Golgi apparatus and transformed to mature alpha II b. The possible pathogenesis of this type II thrombasthenia may be that the misfolded alpha II bA2334C is partially degraded in the endoplasmic reticulum causing lower expression of alpha II bbeta3 complex on the membrane and resulting in impared function of platelets than normal alpha II b.</p>

p73 gene expression in apoptotic process of acute myeloid leukemia cell line U937 induced by methotrexate / 中国实验血液学杂志

The purpose of this investigation was to study the variation of p73 gene expression in the apoptotic process of acute myeloid leukemia (AML) cell line U937 induced by methotrexate (MTX). Morphological changes of apoptotic cells were observed with microscopy and Wright's + Giemsa staining. DNA ladder and cell cycle were examined by agarose gel electrophoresis and flow cytometry respectively. Using semi-quantitive reverse transcription-polymerase chain reaction (RT-PCR), the expression of p73 mRNA was examined. Results showed that MTX could induce U937 cell apoptosis effectively. Condensed nuclei, fragmentation of chromosome and DNA ladder were seen after 6 hour following treatment of MTX 5 micro mol/L. Sub-G(1) peak and S + G(2)/M arrest were also determined by FCM, but the quantity of p73 expression was generally constant. In conclusion, U937 cell apoptosis induced by MTX did not change p73 mRNA level.