High Expression of RhoBTB3 Predicts Favorable Chemothrapy Outcomes in non-M3 Acute Myeloid Leukemia.

Background: The expression patterns and prognostic significance of the Rho family GTPases in acute myeloid leukemia have not been systematically studied yet. Methods: In our study, we analyzed the expression patterns of 21 Rho family GTPases gene members in AML patients based on GEPIA database. 10 gene members with significant differential expression in AML tissue and healthy tissue were selected for subsequent research. Survival curve analysis in TCGA and GEO dataset preliminary showed that RhoBTB3 is related with the prognosis of non-M3 AML patients. The differential expression of RhoBTB3 on AML bone marrow and normal bone marrow was verified by RT-qPCR. We performed Kaplan-Meier survival analysis and Multivariate Cox analysis to assess the prognostic value of RhoBTB3 in non-M3 AML patients with different treatment regimens. Gene functional enrichment analysis of RhoBTB3 was performed using GO, KEGG and PPI network. Results: The AML patients from TCGA database were partitioned into 2 groups based on different treatment regimens: chemotherapy group and allo-HSCT group. In chemotherapy group, patients with higher expression level of RhoBTB3 showed relatively longer OS and EFS, multivariate Cox analysis revealed high RhoBTB3 mRNA expression as an independent favorable prognostic factor. However, in allo-HSCT group, no significant difference of OS and EFS were found between RhoBTB3 high and low subgroups. Meanwhile, allo-HSCT could circumvent the unfavorable prognosis that was associated with downregulation of RhoBTB3. Functional enrichment analysis showed the association of RhoBTB3 expression with several fundamental physiological components and pathways, including extracellular matrix components, extracellular structure organization, and cytokine-cytokine receptor interaction. Conclusions: Our study identified RhoBTB3 as a prognostic marker and may aid in the selection of the appropriate treatment options between chemotherapy and allo-HCST in non-M3 AML patients. Further researches are necessary to clarify the involvement of RhoBTB3 in the pathogenesis of AML.

SUMOylation of insulin-like growth factor 1 receptor, promotes proliferation in acute myeloid leukemia.

Current valid treatments for acute myeloid leukemia (AML) include chemotherapy and hematopoietic stem cell transplantation, which are defective and limited respectively. The insulin-like growth factor 1 receptor (IGF-1R) is up-regulated in many solid tumors; therefore, it may be a target for tumor therapy. Interestingly, IGF-1R is modified by SUMOylation, a type of reversible post-translational modification. In this study, we found that IGF-1R was increased in both cell lines and clinical samples of AML and was modified by SUMO-1. Furthermore, IGF-1, ligand of IGF-1R, induced the up-regulation of IGF-1R and increased the proliferation of leukemia cell line. After mutation of Lys(1025) and Lys(1100) in IGF-1R, the evolutionarily conserved lysine residues were identified as the SUMOylation sites of IGF-1R, because the SUMOylation of IGF-1R in these mutants was significantly inhibited. Furthermore, the cell proliferation mediated by IGF-1 was also reduced. After inhibition of UBC9, the activating enzyme of SUMOylation, co-expression of IGF-1R and SUMO-1 was down-regulated, and cell proliferation was also inhibited. However, cell apoptosis was not significantly affected. These results suggest that IGF-1R and its SUMOylation may be a new therapeutic target for strategy of AML.

Resveratrol induces human K562 cell apoptosis, erythroid differentiation, and autophagy.

Resveratrol (Res) is a naturally occurring phytoalexin with apoptotic and inducing-glob effects in leukemic cells, but the potential induction of erythroid differentiation in cells is not fully understood. Here, we investigated the effects of Res on human erythro-megakaryoblastic leukemia cell line K562. Among the treated cells, proliferation was inhibited and the occurrence of cell apoptosis and cell death were detected. Erythroid differentiation assay was explored, and we found that Res could increase the expression of glycophorin A (GPA), HBA1, HBB, and γ-globin genes and enforced the expression of GPA, CD71, and Band3 proteins. Res also induced K562 cell autophagy when the concentration of Res was increased up to 50 or 100 µM. Our findings suggested that Res possesses the potency not only inducing apoptosis but also inducing erythroid differentiation and autophagy in K562 cells. These results provide that Res may be a therapeutic candidate for chronic myelogenous leukemia treatment.

MicroRNA-20b and ERK1/2 pathway independently regulate the expression of tissue factor in hematopoietic and trophoblastic differentiation of human embryonic stem cells.

INTRODUCTION: Tissue factor (TF) is expressed in various types of cells. TF expression is essential for many biological processes, such as blood coagulation and embryonic development, while its high expression in stem cells often leads to failure of transplantation. In this study, we used the human embryonic stem cell (hESC) culture system to understand the molecular mechanisms by which TF expression is regulated in hESC-derived hematopoietic and trophoblastic cells. METHODS: hESCs were induced in vitro to differentiate into hematopoietic and trophoblastic cells. TF expression in various types of cells during these differentiation processes was examined by quantitative real-time polymerase chain reaction analysis and western blot analysis. The regulatory mechanisms of TF expression were investigated by miRNA expression analysis, luciferase report assay, TF mRNA and protein analysis, and pathway phosphorylation analysis. RESULTS: We first found that TF was expressed only in trophoblasts and granulocyte-monocyte (G-M) cells differentiated from hESCs; and then demonstrated that miR-20b downregulated and Erk1/2 signaling pathway upregulated the TF expression in trophoblasts and G-M cells. Finally, we found that miR-20b downregulated the TF expression independently of the Erk1/2 signaling pathway. CONCLUSIONS: The miR-20b and Erk1/2 pathway independently regulate expression of TF in trophoblasts and G-M cells differentiated from hESCs. These findings will open an avenue to further illustrate the functions of TF in various biological processes.

[Screening and expression of CD34(+) cell-specific microRNA in acute myelogenous leukemia].

OBJECTIVE: To screen and analyze CD34(+) cell specific microRNAs (miRNAs) from the patients with acute myelogenous leukemia (AML) and their expression. METHODS: CD34(+) cells were sorted from AML patients or the mobilized peripheral blood of the donors of hematopoietic stem cell transplantation (normal control subjects) and followed by the extraction of the cell total RNAs. The differentially expressed microRNAs (miRNAs, miR) were selected after hybridizing with miRNA microarray, real time polymerase chain reaction (real-time PCR) was subsequently applied to confirm the expression of the selected miRs, and PCR products were further cloned and sequenced to check their specificity. RESULTS: Of the differentially expressed miRNAs, 191 were found to be at least one-fold change in the CD34(+) cells between the AML patients and the normal control subjects. Of the 191 miRNAs, the expression difference of 94 was significant (P < 0.05). Among these 94 miRNAs, the expression of 44 miRNAs was increased and the other 50 miRNAs was decreased in the CD34(+) cells from the bone marrow of AML patients compared with the CD34(+) cells from the mobilized peripheral blood of the normal control subjects. Real time PCR verified that the expression level of miR-10a and miR-220c in the CD34(+) cells from the bone marrow of AML patients was 19.6% and 19.0% of that of CD34(+) cells from mobilized peripheral blood of the normal control subjects. DNA sequencing and BLAST DNA database searching results indicated that the PCR products were really miR-10a and miR-220c. CONCLUSION: A variety of differentially expressed-miRNAs are existed between AML and normal control subjects CD34(+) cells, the expression of miR-10a and miR-220c was significantly down-regulated in the CD34(+) cells from the bone marrow of AML patients.

[Expression of DNA-dependent protein kinase catalytic subunit in adult acute leukemia and its significance].

This study was aimed to explore the expression of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in adult acute leukemia and its correlation with clinical characteristics, karyotype and prognosis. Indirect immunofluorescent cytometry was used to detect the expression of DNA-PKcs in bone marrow mononuclear cells of 105 patients with acute leukemia before chemotherapy and 41 of them after 2 cycles of chemotherapy. Cytogenetic data were obtained from 26 of them by R band karyotypic analysis. The results showed that the expression of DNA-PKcs was correlated with higher WBC count level in peripheral blood (p < 0.05), but was not obviously associated with median age, gender, percentage of bone marrow blasts, clinical classification, median hemoglobin level and median platelet count (p > 0.05). The middle and strong positive expression of DNA-Pkcs in non-remission group was significantly higher than that in remission group (p < 0.05). The positive rate of DNA-PKcs in abnormal chromosome group was significantly higher than that in chromosome normal group (p < 0.05). It is concluded that the DNA-PKcs expression level is closely related with the increased WBC count, and the expression of DNA-PKcs is correlated also with karyotype and clinical prognosis in adult acute leukemia.

[Screening and structure analysis of nucleic acid aptamers binding to surface of CD33(+)/CD34(+) cells from patients with acute myeloid leukemia subtype M2].

A little is known about the specific marker on the surface of acute leukemia cells, leading to the lack of the specific diagnosis method for acute leukemia. Therefore, in this study, cell-systematic evolution of ligands by exponential enrichment (cSELEX) was performed to screen the aptamers binding to CD33(+)/CD34(+) cells from the patients with acute myeloblastic leukemia (AML) of M(2) subtype (AML-M2) so as to provide the basis for finding the specific marker on the surface of AML-M(2) CD33(+)/CD34(+) cells. Firstly, AML-M2 CD33(+)/CD34(+) cells were sorted and used as targeted cells, and normal CD33(+)/CD34(+)cells were used as counter-targeted cells; the aptamers binding to CD33(+)/CD34(+) cells from patients with AML-M2 were screened from the single strand deoxyribonucleic acid (ssDNA) library by cSELEX. Subsequently, each aptamer structure was analyzed after cloning and sequencing. The results indicated that after 13 round of screenings, the enrichment of aptamers in the ssDNA library was ranged from 0.7% to 52.9%, and reached steady state at 13th round screening. Sequence analysis for 30 aptamers showed that most of the aptamers born one of the three conserved sequences of CCCCT, CTCTC, and CTCAC. Secondary structure analysis indicated that three different secondary structures existed in these aptamers. It is concluded that the aptamers binding to the AML-M(2) CD33(+)/CD34(+) cells are successfully screened, which lay the basis for further looking for the specific marker on the surface of AML-M2 CD33(+)/CD34(+) cells, and the molecular diagnosis of the AML-M2 leukemia.

[Expression of angiotensin-II type 1 receptor at1 mRNA in myeloid leukemia].

This study was aimed to explore the expression level of angiotensin-II type 1 receptor (AT1) mRNA in bone marrow of myeloid leukemic patients, and its correlation with the proportion of leukemia cells in samples and Hb, WBC, Plt counting in peripheral blood. 51 samples, including 36 AML, 7 CML, and 8 samples of non-malignant hematological diseases as control group were collected. The expression of at1 mRNA was detected by real time-PCR; the expression levels of at1 gene in AML and CML groups were relatively quantitatively analyzed by using 2(-ΔΔCT) and were compared with control group. The results showed that the expression levels of at1 mRNA in AML, CML and control groups were 0.038 ± 0.076, 0.033 ± 0.039, 0.281 ± 0.366, respectively. at1 gene expression in the myeloid leukemic group was significantly lower than that in the control group. The expression level of at1 mRNA in AML was negatively correlated with the proportion of leukemia cells and positively with Hb level in peripheral blood. It is concluded that at1 gene may play a minor role in leukaemogenesis, however, may promote erythropoiesis.

[Effect of acetyl-11-keto-ß-boswellic acid on proliferation, apoptosis and cell cycle of human acute myeloid leukemia cell line HL-60].

The aim of this study was to investigate the effect of 3-O-acetyl-11-keto-ß-boswellic acid (AKBA) on the proliferation, apoptosis and cell cycle of human acute myeloid leukemia (AML) cell line HL-60. HL-60 cells were treated by AKBA at various concentrations. The inhibitory effects of AKBA on the proliferation of HL-60 were analyzed by MTT assay. Morphologic changes of HL-60 cells were observed by fluorescence microscopy with Hochest33342 staining. Cell apoptosis rate was determined by flow cytometry with Annexin-V-FITC/PI double staining. The cell cycle was measured by flow cytometry with PI staining. The results showed that AKBA inhibited the proliferation of HL-60 and the apoptosis rate of HL-60 cells was gradually enhanced when AKBA dose increased. AKBA changed the cell cycle of HL-60, resulting in cell increase at G(1) phase and decrease at S phase. It is concluded that the AKBA has anti-proliferation and apoptosis-inducing effects on HL-60 cells, that seems a promising therapeutical approach for AML.

[Apoptosis of Burkitt's lymphoma Raji cell line induced by bortezomib].

The aim of this study was to clarify whether bortezomib might induce apoptosis in Burkitt's lymphoma Raji cell line and its mechanism. Different concentrations of bortezomib were used to treat Raji cells and its effects of time and dose were observed. Cell morphology was observed under light microscope; flow cytometry was used to analyze cell apoptosis; RT-PCR was used to detect the expressions of NF-kappaB and p53 gene mRNAs. The results showed that the bortezomib could inhibit Raji cell growth within a certain range of treating time and dose. Apoptosis were induced in relation to time and dose. The expression of NF-kappaB mRNA and p53 mRNA decreased after treatment with bortezomib. It is concluded that the bortezomib can induce Raji cell apoptosis, which provides a theoretical basis for clinical treatment. NF-kappaB and p53 gene are supposed to participate in the bortezomib induced apoptosis of Raji cells.

[Renin angiotensin system in bone marrow of patients with aplastic anemia].

Renin-angiotensin system (RAS) has been shown to be involved in the growth, production, proliferation and differentiation of the bone marrow (BM) hematopoietic cells, while aplastic anemia (AA) is a disease in which proliferation ability of the BM hematopoietic cells is damaged with defective hematopoietic microenvironment. To investigated the pathogenesis of AA, the rennin activity, angiotensin I (Ang I) and angiotensin II (Ang II) concentration in peripheral blood and BM of 22 AA patients were detected by radioimmunoassay, 16 nonhematological disease patients with normal blood counts and BM picture were used as control, and the difference between two groups was compared. The results showed that BM Ang II concentration in the AA patients was significantly lower than that in the control (P < 0.01). In nonhematological disease patients, Ang II concentration in BM was significantly higher than that in peripheral blood, the renin activities and Ang I concentrations were not significantly different in the two groups (P > 0.05). In conclusion, the decreased BM Ang II concentration in AA patients may be involved to the pathogenesis of AA.

[Study on platelet-associated tissue factor and its significance].

OBJECTIVE: To explore whether normal platelet contains tissue factor (TF), and the significance of platelet-associated TF (PATF). METHODS: Platelets were isolated by Sepharose 2B gel column. ELISA was used to detect the TF content in the lysates of washed platelets. Procoagulant activity of PATF was measured by one stage clotting time assay. The mRNA of TF was detected by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: A certain amount of TF antigen (16.37 +/- 6.39) ng/L was detected in the washed-platelet lysates. Upon activation by collagen, platelets released TF and caused a marked increase in TF level in plasma (P <0.05). Resting platelets had no TF procoagulant activity, while procoagulant activity of platelets activated by collagen increased significantly, which could be blocked by TF McAb and poor VII plasma. TF mRNA could not be detected in washed platelets. TF content in platelets from patients with coronary heart disease was significantly higher than that from normal controls (P < 0.05). Resting platelets from the patients showed a higher procoagulant activity, which could be inhibited by TF McAb. CONCLUSION: Platelets contain TF and the latter released by activated platelet was functionally active. Platelet itself might not synthesize TF. Protein content and procoagulant activity of PATF in patients with coronary heart disease were higher than that in controls. All these indicate that platelet may be involved in coagulation and thrombosis by releasing TF.