Effects of transcription factor GATA-2 on transcriptive regulation of iASPP gene / 中国实验血液学杂志

iASPP can prompt the cell proliferation and inhibit the apoptosis of many cells. There are putative binding sites of transcription factor GATA-2 upstream of iASPP transcription start site. GATA-2 plays an important role in the proliferation and differentiation of hematopoietic stem cells (HSC) and progenitors. This study was aimed to explore the role of GATA-2 protein in iASPP gene transcription. Firstly, the expression of iASPP and GATA-2 protein in some leukemia cell lines was detected by Western blot. Second, The expressive vector of pCMV5-GATA2 and the luciferase reporter vectors containing possible binding sites of GATA-2 were constructed and co-transfected into HEK293 and CV-1 cells. Then the luciferase activity was assayed by luminometer. Also, ChIP assays were performed to further confirm the specific binding of GATA-2 to iASPP promoter. The results showed that GATA-2 was overexpressed in most cell lines with high level of iASPP. GATA-2 exhibited a significant effect on luciferase activity of reporter gene iASPP and in a dose-dependant manner. The relative luciferase activity was up-regulated to about two-fold of the empty vector control when the transfection dose of pCMV5-GATA2 plasmid was increased to 100 ng. While the effect was more significant in CV-1 cells and showed a 6.7-fold increase. The ChIP assay demonstrated the in vivo specific binding of GATA-2 to iASPP. The binding sites of GATA2 were located between nt -361 ∼ -334 in upstream of iASPP gene transcription start site. It is concluded that transcription factor GATA-2 can bind with the cis-regulatory region of the iASPP promoter and up-regulate iASPP expression.

Study on expression of PTEN gene and its pseudogene PTENP1 in acute leukemia and correlation between them / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the expression of PTEN (phosphatase and tension homology deletion on chromosome 10, PTEN) and its pseudogene PTENP1 in acute leukemia (AL) and correlation between them, and to explore the role of PTENP1 on the PTEN expression in AL cells.</p><p><b>METHODS</b>PTEN and PTENP1 mRNA expression were evaluated in bone marrow (BM) samples from 138 newly diagnosed AL patients and 15 healthy controls by quantitative real-time RT-PCR (qRT-PCR). pCDH1-PTENP1 3'UTR-GFP lentivirus vectors were constructed. 293T cells were transfected by calcium phosphate precipitation to produce retrovirus. HL-60 cell line was infected with the retroviral vectors expressing pCDH1-GFP and pCDH1-PTENP1 3'UTR-GFP respectively. The flow cell sorter was used to sort the HL-60 with GFP positively expressed. The mRNA expression of PTEN and PTENP1 was detected by qRT-PCR, the expression of PTEN protein by western blot, and the impact of PTENP13'UTR on the proliferation of HL-60 cells by MTT assay.</p><p><b>RESULTS</b>AML patients showed significantly lower PTEN and PTENP1 mRNA expression in BM compared to healthy controls. Correlation analysis showed that the expression of PTEN and PTENP1 mRNA were positively correlated (P < 0.05). The 108 cases of PTENP1(+) AML were classified according to the prognostic classification of 2011 NCCN Clinical Practice Guidelines in AML, there was no difference among different subgroups. HL-60 cell line was infected with the retroviral vectors expressing pCDH1-GFP (control group) and pCDH1-PTENP1 3'UTR-GFP respectively. Compared with the control group, PTENP1 mRNA level of HL-60 infected with the retroviral vectors expressing pCDH1-PTENP1 3'UTR-GFP increased significantly, and PTEN mRNA level also increased. While the PTEN protein level and the cell growth rate of the PTENP1 3'UTR group didn't change significantly.</p><p><b>CONCLUSION</b>PTEN and PTENP1 mRNA expression level of BM cells from AL patients is significantly lower. There is a positive correlation between expression of PTEN and PTENP1 mRNA. PTENP1 may regulate the expression of PTEN in mRNA level.</p>

Effect of metformin on acute promyelocytic leukemia cell line NB4 and its mechanism / 中国实验血液学杂志

The purpose of this study was to investigate the effect and molecular mechanism of metformin (Met) on biological characteristics of acute promyelocytic leukemia (APL) cell line NB4. NB4 cells were treated with various concentrations of Met for different time, MTT method was used to detect cell proliferation, the alteration of cell apoptosis was analyzed by flow cytometry, and the change of cell adhesion ability was examined by cell adhesion assay. NB4 cells were pretreated with U0126, a specific inhibitor for extracellular signal-regulated kinase (ERK) phosphorylation, ERK phosphorylation was assessed by Western blot analysis, apoptosis and cell adhesion ability were evaluated by flow cytometry and cell adhesion test respectively. The results showed that Met could inhibit the cell proliferation, induce the cell apoptosis and increase the ability of cell adhesion. The pretreatment of NB4 cells with 5 µmol/L U0126 could effectively inhibit the phosphorylation of ERK, and reduce cell apoptosis and adhesion induced by 5 mmol/L Met. It is concluded that Met can inhibit the proliferation and promote the apoptosis and adhesion of NB4 cells. MEK/ERK signaling pathway may be one of the molecular mechanisms of metformin on NB4 cells.

Proportion of CD34(+)CD38(-) cell population in bone marrow of patients with de novo AML as prognostic factor of complete remission at first course of induction chemotherapy / 中国实验血液学杂志

This study was to investigate the relationship between the CD34(+)CD38(-) cell population and its proportion in G(0) phase of de novo AML non-M(3) at diagnosis and the clinical and experimental characteristics. The flow cytometry was used to detect the expression of the cell surface antigen CD34 and CD38 in the bone marrow mononuclear cells (MNC) of the AML non-M(3) at diagnosis and investigate the cell cycle of the subpopulations, and then the relationships between the proportion of CD34(+)CD38(-)cell population and its G(0) state and the complete remission (CR) rate after the first induction chemotherapy was analyzed. The results showed that the proportion of the CD34(+)CD38(-) cell population and its G(0) phase had no relationship with the karyotypes and WBC count at new diagnosis and the Flt3/ITD status, but correlate with the blasts in the bone marrow after the first course induction chemotherapy. The proportion of the CD34(+)CD38(-) cells in patients who have visible blasts in the bone marrow at day 7 after completion of the first course induction chemotherapy was (12.47 ± 26.26)%, but the counterparts was (2.62 ± 7.20)% in the group of patients whose bone marrow had no visible blasts (p = 0.031). The proportion of the CD34(+) cell population in patients who had visible blasts in the bone marrow at day 1 after completion of the first course induction chemotherapy was (17.40 ± 21.20)%, yet the proportion of the CD34(+) cell populations was (5.64 ± 6.96)% in the patients who had no visible blasts in the bone marrow (p = 0.001). The proportion of the CD34(+)CD38(-) cell populations in the patients who achieved CR after the first course induction chemotherapy was (2.51 ± 9.72)%, which was lower than the proportion (24.92 ± 27.04%) of the non-CR patients (p = 0.001). Furthermore, the proportion (1.60 ± 4.82%) of the CD34(+)CD38(-) cell population in the AML non-M(2b) CR patients was more obviously lower than that in the non-CR patients (p < 0.001). In univariate analysis, whether or not achieved CR after the first course induction chemotherapy correlated with age (p = 0.022), the proportion of the CD34(+)CD38(-) cell population (p = 0.008) and the proportion of the visible blasts in the bone marrow at day 7 after induction therapy (p = 0.011). Multivariate analysis showed that only the proportion of the CD34(+)CD38(-) cells had correlation tendency with CR rate. It is concluded that the proportion of the CD34(+)CD38(-) cells in bone marrow of de novo AML non-M(3) is a prognostic factor to anticipate the CR rate of the first course for induction therapy.