Function of OTUD7B in acute myeloid leukemia cells and its mechanism / 上海交通大学学报（医学版）

Objective·To investigate the biological effect and mechanism of OTUD7B in acute myeloid leukemia (AML) cells.Methods·The expression of OTUD7B in peripheral blood mononuclear cells and bone marrow mononuclear cells of AML patients were detected.The relationship between OTUD7B and survival of AML patients was confirmed by using TCGA database.Mouse model of M2 type AML was established,and the expression of OTUD7B in the bone marrow,spleen and liver of the mice was detected.OTUD7B was overexpressed in AML cell lines HL60 and kasumil,then the cell viability and cell cycle were measured.The AKT/mTOR pathway proteins were detected after OTUD7B overexpressed and then the cell growth inhibition was detected after overexpression ofAKT1.Results·The expression of OTUD7B was lower in primary leukemia cells from all types of AML patients and in the bone marrow,liver and spleen of M2 type AML mice,which was closely related to the survival time of AML patients.OTUD7B overexpression in HL60 and kasumil cells significantly inhibited the cell viability and decreased the percentage of S phase cells.OTUD7B significantly inhibited the phosphorylation of AKT and mTOR,and AKT1 overexpression partially reversed the inhibitory effect of OTUD7B on cell growth.Conclusion·OTUD7B expression is low in primary leukemia ceils from AML patients and in bone marrow,liver and spleen of the M2 type AML mice.The survival time of patients with low OTUD7B expression is shorter.Overexpression of OTUD7B significantly inhibited the cell viability of HL60 and kasumi 1 cells and the entry of cells into S phase.The inhibitory effect of OTUD7B overexpression on AML cells might be related to the inhibition ofAKT / mTOR signaling pathway.

Effects of Quercetin on Chronic Myeloid Leukemia Cell Line Resistant to Imatinib and Its Mechanism / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the growth inhibitory effect of quercetin on imatinib-resistant chronic myeloid leukemia cell lines and to clarify its involved mechanisms.</p><p><b>METHODS</b>The cell viability was detected by trypan blue Staining, percentage of apoptotic cells and cell cycle distribution were detected by flow cytometry, the protein expression was detected by Western blot.</p><p><b>RESULTS</b>Both inhibitory effect of proliferation and apoptosis-inducing effect were similar between the imatinib-resistant and -sensitive cell lines treated with 25 µmol/L quercetin for 24 hours and with arrest of cell cycle at G/M phase. Quercetin could not change the expression of BCR-ABL. The expression of γ-H2AX was markedly enhanced and the phosphorylation of JNK up-regulated by quercetin in both imatinib-resistant and imatinib-sensitive cell lines.</p><p><b>CONCLUSION</b>The growth of imatinib-resistant cells can be inhibited by quercetin, and the apoptosis of cells can be induced by quercetin, which may be related to cell cycle arrest in G/M. The DNA damage and up-regulation of p-JNK may be involved in these processes.</p>

Longikaurin A induces apoptosis of multiple myeloma H929 cells / 中国实验血液学杂志

The aim of this study was to investigate the biological effect of longikaurin A on multiple myeloma H929 cells. Effects of oridonin and longikaurin A on proliferation of H929 cells were evaluated by CCK-8 assay. Cell morphological features of H929 cells were examined under inverted phase contrast microscope. Apoptosis was determined by Annexin V/PI staining. Expression of caspase-3, caspase-9, and PARP were detected by Western blot. Reactive oxygen species (ROS) level was determined by DCFDA assay. The results showed that longikaurin A (IC(50) 0.85 µmol/L) was more effective than oridonin ((IC(50) 10.66 µmol/L) to inhibit the proliferation of H929 cells. Treatment with longikaurin 2 µmol/L significantly increased the percentage of Annexin V positive cells. Caspase-3 and caspase-9 were activated and PARP, one substrate of caspase-3, was cleaved into 85 kDa fragments. The ROS scavenger N-acetyl-cysteine significantly blocked apoptosis induced by longikaurin A. However, longikaurin A did not increase the ROS level in H929 cells. It is concluded that longikaurin A is more effective than oridonin to induce apoptosis in multiple myeloma H929 cells without increasing the ROS level.

Hypoxia and its simulant CoCl(2) down-regulates Foxp3 expression independent from HIF-1alpha / 中国实验血液学杂志

This study was purposed to investigate the expression of Jurkat cell Foxp3 in hypoxia condition and the role of HIF-1alpha in this process as well as to clarify the mechanism influencing function of regulatory T cells by hypoxia. The Jurkat cells were incubated with hypoxia (1% O(>2)) and its simulant CoCl(2) for different times (0, 6, 12, 24 hours), the viability was measured by trypan blue staining, the expression of HIF-1alpha was detected by Western blot, the expression of Foxp3 was detected by real-time PCR, the expressions of HIF-1alpha and Foxp3 were assayed after HIF-1alpha in Jurkat cells was inhibited by using RNA interference technique. The results indicated that after Jurkat cells were treated with hypoxia and its simulant CoCl(2), the significant accumulation of HIF-1alpha in cells appeared, but the expression of Foxp3 was obviously down-regulated; after expression of HIF-1alpha in Jurkat cells was inhibited by siRNA interference, the CoCl(2) still could down-regulate the expression of Foxp3. It is concluded that the hypoxia and its simulant CoCl(2) can obviously down-regulate the expression of Foxp3, but this process is independent from HIF-1alpha.

Effect of TGF-beta1 on biological characteristics of umbilical cord blood hematopoietic progenitor cells during ex-vivo expansion / 中国实验血液学杂志

To investigate the effects of TGF-beta1 on biological characteristics of hematopoietic progenitor cells (HPC) in umbilical cord blood (UCB) during ex-vivo expansion and feasibility of using it for expansion of UCB HPC, different concentrations of TGF-beta1 were added in the serum-free medium containing a combination of hematopoietic growth factors for expansion of UCB CD133(+) cells and enumeration of nucleated cells (NC), progenitor colonies, immunophenotyping, cell cycle and expression of adhesion molecules of the NC were monitored at every interval. The results showed that total number and expansion of NC from all groups of TGF-beta1 were remarkably less than those in control at each interval. However the content and total numbers as well as expansion of CD34(+), CD133(+), CD34(+)CD38(-) and CD34(+)CD133(+) cells from all groups of TGF-beta1 were more than those in control at each interval during expansion; the plating efficiency and expansion of CFU-GM, CFU-mix and HPP-CFC from NC of TGF-beta1 group were more than those in control at each interval. The contents of cells in G(0)/G(1) phase of NC of TGF-beta1 group at every interval were high. Meanwhile, TGF-beta1 could elevate the expression of some adhesion molecules on NC during expansion such as CD54, CD49d and CD11a, and the contents of CD34(+) cells coexpressing these adhesion molecules in NC of TGF-beta1 group were significantly more than those in control at each interval. In conclusion appropriate dose of TGF-beta1 could accelerate expansion of CD133(+) cells, delay and decrease over-differentiation of HPC, increase the content of HPC in expanded products, upregulate the expression of adhesion molecules on expanded HPC, thus it could promote engraftment of expanded progenitor cells and advantage the ex-vivo expansion of UCB HPC.

Preliminary study on the molecular mechanism of K562 cell apoptosis induced by As2S2 / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the apoptotic inducing effect of As(2)S(2) on K562 cells.</p><p><b>METHODS</b>The apoptotic inducing effect of As(2)S(2) on K562 cells was determined by flow cytometry, DNA fragmentation analysis and morphology observation. Expression of protein was determined by Western-blot. RT-PCR was used to evaluate changes in gene expression.</p><p><b>RESULTS</b>Apoptosis of K562 cells was induced by 48 - 72 h exposure to 5 micromol/L As(2)S(2). Apoptosis was induced in (34.4 +/- 3.3)% treated cells by 72 h exposure to 3 micro mol/L As(2)S(2), in (21.8 +/- 3.6)% treated cells by 48 h exposure to 5 micromol/L As(2)S(2) and in (46.0 +/- 5.2)% treated cells by 72 h exposure to As(2)S(2) at the same concentration. With 5 micromol/L As(2)S(2), the protein level of Bcr-Abl and JAK2 decreased, while bax expression was upregulated and c-myc was downregulated both in protein and mRNA level. The activity of caspase 3 in K562 cells was increased by As(2)S(2). As(2)S(2) also induced apoptosis of fresh mononuclear cells derived from chronic myelogenous leukemia (CML) patients.</p><p><b>CONCLUSION</b>As(2)S(2) can induce apoptosis of CML cells. The decline of Bcr-Abl may play an important role. The upregulation of bax, increase of the activity of caspase 3, downregulation of c-myc and decrease of JAK2 may also be involved in the mechanism.</p>

Synergism of As2S2 and STI 571 in inducing apoptosis of K562 cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To investigate the synergistic effect of As(2)S(2) and STI 571 on K562 cells and its mechanism.</p><p><b>METHODS</b>The inhibitive effect of As(2)S(2) on the proliferation of K562 cells was determined by cell number count. Cell apoptosis was assessed by flow cytometry, DNA fragmentation and morphology. Protein expression was determined by Western-blot and gene expression by RT-PCR.</p><p><b>RESULTS</b>As(2)S(2) could significantly inhibit the proliferation and induce apoptosis of K562 cells in a dose and time-dependent manner at concentrations from 1 micromol/L to 5 micromol/L for 24 approximately 72 h. 34.4%, 21.8% and 46.0% of the treated-cells displayed apoptosis at 3 micro mol/L for 72 h, 5 micromol/L for 48 h and 5 micromol/L for 72 h, respectively. Compared to treatment with STI571 (0.25 approximately 1.00 micromol/L) or As(2)S(2) (1 approximately 5 micromol/L) alone, treatment of K562 cells with As(2)S(2) and STI571 combination induced more cell apoptosis. (18.4 +/- 1.4)% and (15.8 +/- 1.2)% cells underwent apoptosis at 1 micromol/L STI571 for 48 h and 5 micromol/L As(2)S(2) for 48 h, respectively, and (40.6 +/- 2.0)% cells did in combination treatment (P < 0.05). For U937 cells, the percentages of apoptotic cells were (6.0 +/- 1.1)% at 1 micromol/L STI571 for 48 h, (4.5 +/- 1.2)% at 5 micromol/L As(2)S(2) for 48 h, and (7.3 +/- 1.0)% in combination treatment. As(2)S(2) decreased the bcr-abl fusion protein expression and PTK activity of c-abl and bcr-abl, but not for bcr-abl expression.</p><p><b>CONCLUSION</b>Combination treatment with As(2)S(2) and STI 571 induced more apoptosis of K562 cells. The reduction of PTK activity may be involved in the mechanisms.</p>

Studies on the dynamics of biological characteristics of CD133+ cells from human umbilical cord blood during short-term culture / 中国实验血液学杂志

This study was to investigate dynamics of biological properties of CD133(+) cells from human umbilical cord blood (UCB) during short-term culture containing the combination of hematopoietic growth factors and the feasibility of in vitro expansion of CD133(+) cells. The biology activities including analysis of cell cycle, immunophenotype, telomerase activity, expression of adhesion molecules and expansion potential of CD133(+) cells were monitored during ex-vivo expansion, and compared with those of CD34(+) cells. The results showed that the contents of CD133(+) and CD34(+) cells in fresh UCB were (1.05 +/- 0.73)% and (1.40 +/- 0.56)% respectively. About 79.62% of CD34(+) cells expressed CD133, and more than 97% of CD133(+) cells were CD133(+)CD34(+), markedly higher than that in CD34(+) fraction (P < 0.01). No significant differences were observed in content of cells expressing CD38, CD13, CD14, CD61 and glycophorin-A between the two fractions. Expansion of CD133(+), CD133(+)CD34(+) and CD34(+)CD38(-) cells at 10 days and those of CFU-mix, HPP-CFC and CD34(+)CD38(-) cells at 6 days from CD133(+) cells group were significantly higher than those from the CD34(+) cell group (P < 0.05). Analysis of immunophenotype showed that CD133(+)CD34(+) cells declined gradually while CD133(-)CD34(+) and CD133(-)CD34(-) cells increased during ex-vivo expansion; basal telomerase activities of fresh UCB CD133(+) and CD34(+) cells were low but significantly exceeded that of CD34(-) fraction (P < 0.05). At first week of expansion, telomerase activity was significantly upregulated, after two weeks, telomerase activity remarkably declined, and decreased to baseline or below the limits of detection in day 20. More than 90% of CD133(+) cells expressed CD49d and CD11a, and, more than 85% of the cells expressed CD54, about 50% of cells expressed CD62L. At the early stage of expansion, expression of CD49d was upregulated, expression of CD11a remaining no change, while as expression of CD54 and CD62L was downregulated. Expression of all adhesion molecules was decreased gradually with extend of culture. But expression of these adhesion molecules on CD34(+) subsets were not affected significantly during expansion. It is concluded that CD133(+) population may be a more primitive hematopoietic stem/progenitor cells (HSPC) than CD34(+) cells, CD133(+) cells have great expansion potential for ex-vivo expansion and is a suitable target cell for ex-vivo expansion of HSPC. Downregulation of adhesion molecules and telomerase activity may be one of the reasons for delayed engraftment of expanded products.

Effects of MIP-1β and TGF-β antibody on the expansion of CD34+ cells from cord blood in stroma-contact culture system / 第三军医大学学报

Objective　To study the effects of MIP-1β and TGF-β antibody on CD34+ cells from cord blood in stroma-contact culture system. Methods　Immunomagnetic selected CD34+ cells were inoculated onto the pre-established irradiated human stroma layer. MIP-1β, TGF-β antibody (20 μg/ml) and MIP-1β+anti-TGF-β (5 μg/ml) were added on day 0 and day 5. On day 5 and day 10, cell counting, hematopoietic progenitor cells count were made by semi-solid culture and CD34+ cells were assayed by FACS. Results　On day 5, no significant difference of total cell number was observed as compared with the control group (P>0.05), but the number of CD34+cells, CFC, and HPP-CFC in TGF-β antibody and MIP-1β+ TGF-β antibody groups was significantly higher than that in the control (P<0.05). On day 10, the number of total cell, CD34+ cells, CFC, and HPP-CFC in TGF-β antibody and MIP-1β+ TGF-β antibody groups was significantly higher than that in the control (P<0.05). No significant difference was observed between groups MIP-1β and control either on day 5 or day 10 (P>0.05). Conclusion　MIP-1β has no significant effect on the CD34+ cells as compared with the control while the CD34+ cells can be expanded 1-3 folds with TGF-β antibody (20 μg/ml) or MIP-1β+ TGF-β antibody (5 μg/ml) in 10 days. There are synergic interactions between MIP-1β and TGF-β antibody.

Effects of MIP-1β and TGF-β antibody on the expansion of CD34+ cells from cord blood in stroma-contact culture system / 第三军医大学学报

Objective　To study the effects of MIP-1β and TGF-β antibody on CD34+ cells from cord blood in stroma-contact culture system. Methods　Immunomagnetic selected CD34+ cells were inoculated onto the pre-established irradiated human stroma layer. MIP-1β, TGF-β antibody (20 μg/ml) and MIP-1β+anti-TGF-β (5 μg/ml) were added on day 0 and day 5. On day 5 and day 10, cell counting, hematopoietic progenitor cells count were made by semi-solid culture and CD34+ cells were assayed by FACS. Results　On day 5, no significant difference of total cell number was observed as compared with the control group (P>0.05), but the number of CD34+cells, CFC, and HPP-CFC in TGF-β antibody and MIP-1β+ TGF-β antibody groups was significantly higher than that in the control (P<0.05). On day 10, the number of total cell, CD34+ cells, CFC, and HPP-CFC in TGF-β antibody and MIP-1β+ TGF-β antibody groups was significantly higher than that in the control (P<0.05). No significant difference was observed between groups MIP-1β and control either on day 5 or day 10 (P>0.05). Conclusion　MIP-1β has no significant effect on the CD34+ cells as compared with the control while the CD34+ cells can be expanded 1-3 folds with TGF-β antibody (20 μg/ml) or MIP-1β+ TGF-β antibody (5 μg/ml) in 10 days. There are synergic interactions between MIP-1β and TGF-β antibody.