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.

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.

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.

Expression and SNP analysis of BRD7 gene in acute leukemia cells / 中南大学学报(医学版)

OBJECTIVE@#To evaluate the expression level of BRD7 gene in bone marrow mononuclear cells (BMNCs) in patients with acute leukemia (AL) and to analyze BRD7 single nucleotide polymorphism(SNP).@*METHODS@#RT-PCR was used to detect BRD7 expression in patients with AL and normal bone marrow subjects. Single-strand conformation polymorphism and DNA sequence analysis were also used to identify BRD7 mutation or SNP to investigate the relation between BRD7 and AL.@*RESULTS@#BRD7 mRNA in BMNCs from 52 patients with AL and 30 control subjects was expressed. The mRNA relative expression of BRD7 in patients with AL was higher than that of the control group (P=0.001). Three SNPs (C657A,C495T and A737G) in BRD7 gene coding region (447 approximately 844 bp) were found, and A737G was coupled with C495T . The allele frequencies of SNP C657A were not significantly different between AL and the control group. The genotype and the allele frequencies of the 2 coupled SNPs were significantly different (P0.05).@*CONCLUSION@#Expression of BRD7 gene is up-regulated in AL cells. The 2 coupled SNPs (C495T and A737G ) in BRD7 gene coding region (447 approximately 844 bp) are correlated with AL, indicating that SNPs may be one of the genetic susceptibility factors of AL.

In vitro differentiation into megakaryocytes and generation of platelets from CD34+ cells of umbilical cord blood / 中南大学学报(医学版)

OBJECTIVE@#To induce hematopoietic progenitor/stem cells of umbilical cord blood to differentiate into mature megakaryocytes and platelets in vitro and to investigate the mechanism of production of platelets.@*METHODS@#The CD34+ cells were sorted from umbilical cord blood by magnetic activated cell sorting (MACS) and then cultured in vitro with optimized medium to be differentiated into mature megakaryocytes and platelets. The cultured cells and the platelet-like particles were isolated from the culture and were checked by the fluorescence-activated cell sorter (FACS), immunohistochemistry assays, light microscope,electron microscope and platelet aggregation tests.@*RESULTS@#The cultured megakaryocytes were detected with proplatelets and both the cultured cells and the platelet-sized particles were found to have the same structure with the normal megakaryocytes and platelets by light and electron microscope. The immunohistochemistry assays revealed the cultured cells expressed GP II b III a with a positivity of 95% which was a special antigen for platelets and megakaryocytes. Culture-derived platelet-sized particles aggregated in response to thrombin as the plasma derived-platelets did. The cultured platelets had the same positivity of CD41 as the platelets from platelet rich plasma.@*CONCLUSION@#The hematopoietic progenitor/stem cells can be induced to differentiate into purified and mature megakaryocytes and platelets. It provides a practical way to study the mechanism of platelets production.

Effects of integrin alpha IIb(R995A) mutation on receptor affinity and pp125 (FAK) phosphorylation / 中国医学科学杂志(英文版)

<p><b>OBJECTIVE</b>To investigate the role of cytoplasmic domain of integrin alpha IIb in platelet signal transduction.</p><p><b>METHODS</b>Binding capacity of integrin alpha IIb(R995A) to antibody platelet activation complex-1 (PAC-1) and pp125 focal adhesion kinase (FAK) phosphorylation of cells were detected by flow cytometry, immune precipitation, and Western blotting.</p><p><b>RESULTS</b>Without activation, wild-type alpha IIb beta3 Chinese hamster ovary (CHO) cells failed to bind to PAC-1, but mutant chimera alpha IIb(R995A)beta3 CHO cells were able to bind with PAC-1. Furthermore, phosphorylation of pp125 (FAK) in wild-type alpha IIb beta3 CHO cells occured only when cells were adhered to fibrinogen, but could not be detected in bovine serum albumin suspension. However in the mutant chimera group, it could be detected in both conditions.</p><p><b>CONCLUSION</b>The mutation in integrin alpha IIb(R995A) alters its affinity state as a receptor, thus also mediating cytoplasmic signal transduction leading to the phosphorylation of pp125 (FAK) without ligand binding.</p>