A multicenter randomized open-label study of rituximab plus rhTPO vs rituximab in corticosteroid-resistant or relapsed ITP.

This study aimed to compare the efficacy and safety of rituximab (RTX) plus recombinant human thrombopoietin (rhTPO) with RTX alone in patients with immune thrombocytopenia (ITP) who had failed to respond to corticosteroids or relapsed. Recruited patients were randomized at a ratio of 2:1 into 2 groups: the combination group (RTX + rhTPO, n = 77) and the monotherapy group (RTX, n = 38). Overall response was achieved in 79.2% of patients in the combination group vs 71.1% in the monotherapy group (P = .36), and the complete response (CR) rate was 45.4% in the combination group compared with 23.7% in the monotherapy group (P = .026). The combination group had significantly shorter time to response (TTR; median and range, 7 and 4-28 days) compared with the monotherapy group (28 and 4-90 days) (P < .01). There was no difference between these 2 groups in terms of the long-term response (P = .12). Our findings demonstrated that the combination of RTX and rhTPO significantly increased the CR rate and shortened TTR compared with RTX monotherapy in the treatment of corticosteroid-resistant or relapsed ITP but failed to show a beneficial effect on the long-lasting response. This study is registered at www.clinicaltrials.gov as #NCT01525836.

[Abnormal expression of transcription factors LYL1 and LMO2 and interaction between them in myeloid leukemia].

OBJECTIVE: To explore the expression of the transcription factors LMO2 and LYL1 and the interaction between these 2 factors in myeloid leukemia cells and to analyze the significance thereof in leukemogenesis. METHODS: Samples of peripheral blood and bone marrow were collected form 51 AML patties, and 5 normal bone marrow donors to isolate mononuclear cells (MNCs) with high percentage of CD34(+) cells. Western blotting (WB) was used to detect the protein expression of LMO2 and LYL1 in the cells. Human myeloid leukemia cells of the line K562 were cultured and transfected with pcDNA3-LMO2, plasmid containing LMO2, pcDNA3-LYL1, plasmid containing LYL1, or pcDNA-GFP, blank plasmid containing green fluorescent protein. RT-PCR was used to detect the mRNA expression of LMO2 and LYL1. Co-immunoprecipitation (co-IP) and WB were used to detect the binding protein of LMO2 and LYL1. RESULTS: The MNCs of 51.1% of the patients with acute myeloblastic leukemia (AML) without remission expressed higher levels of LMO2, the MNCs of 62.2% of the AML patients expressed higher levels of LYL1, and the MNCs of 31.1% of those expressed both. The K562 cells transfected with pcDNA3-LMO2 showed higher mRNA and protein expression levels of both LMO2 and LYL1, and the K562 cells transfected with pcDNA3-LYL1 showed higher mRNA and protein expression levels of both LYL1 and LMO2 too, as indicated by RT-PCR and WB, which suggested that the expression of LMO2 and the expression of LYL1 stimulated each other in the myeloid leukemia cells. Co-IP assay detected the presence of LMO2-LYL1 complex in those cells. CONCLUSION: The abnormal expression and protein interaction of LMO2 and LYL1 may play a role in the abnormal proliferation and differentiation of myeloid hematopoietic cells.

[Epigenetic regulation of expression of retinoic acid receptor beta gene in leukemia cell].

OBJECTIVE: To study the reactivation of retinoic acid receptor beta (RARbeta) expression in myeloid leukemia cells by a combination of all-trans retinoic acid (ATRA) with a DNA demethylating agent, decitabine (DAC), and valproic acid (VPA, a histone deacetylase inhibitor),and their effects on cell differentiation and proliferation. METHODS: Human myeloid leukemia cells of the line U937 were cultured and treated with all-trans retinoic acid (ATRA), DAC, and VPA. 72 h later cell differentiation test, cloning formation test, and chromatin immunoprecipitation test were performed. Bone marrow specimens were collected from 56 patients with acute myeloblastic leukemia (AML) were cultured and treated with and 10 bone marrow specimens were used as controls. Methylation of RARbeta promoter was detected with methylation specific polymerase chain reaction (MSP) after bisulfite treatment. Relative levels of RARbeta mRNA were assessed with real time quantitative PCR assay. Flow cytometry assay was used to detect the myeloid differentiation marker CD11b in U937 cells. Chromatin immuno-precipitation assay (ChIP) was used to analyze the acetylated histone 3 bound to the retinoic acid response element (RARE) at the promoter region of RARbeta. RESULTS: Methylation of RARbeta was positive in 36 of the 56 (64.3%) AML patients and the U937 myeloid leukemia cells, however, was negative in the marrow mononuclear cells from the 10 healthy donors. The expression of RARbeta in U937 cells was up-regulated after treatment with ATRA (1 micromol/L) plus DAC (1 micromol/L) or VPA (0.5 mmol/L) for 72 hours, especially when the three drugs were used together. ChIP assay showed that the acetylated histone 3 bound to the RARE promoter region was increased after the cells were exposed to ATRA plus DAC/VPA. The data indicated that the reactivated expression of RARbeta might be secondary to the drug-induced histone acetylation as well as DNA demethylation. Treatment of U937 cells with ATRA and DAC/VPA also resulted in increased myeloid differentiation (CD11b expression) and decreased plating efficiency. CONCLUSION: The repressed expression of RARbeta in myeloid leukemia cells is closely related to both DNA hypermethylation and histone deacetylation, and a combination of ATRA with epigenetic modulators can be beneficial in the treatment of myeloid malignancies.

[Reactivated Expression of MicroRNA-124 in Patients with Myelodysplastic Syndromes after Demethylating Therapy].

OBJECTIVE: To explore the role of microRNA-124(miR-124) in the pathogenesis of myelodysplastic syndromes(MDS) through detecting the expression level of miR-124 in bone marrow mononuclear cells(MNC) of MDS patients before and after demethylating therapy with decitabine. METHODS: The expression levels of miR-124 in the MNC of 35 MDS patients and 10 healthy donors were detected with stem-loop quantitative real time polymerase chain reaction assay. RESULTS: The expression level of miR-124 was lower in MDS patients than that in healthy donors. The difference was not statistically significant between patients with low-risk MDS subtypes (RA and RCMD) and control, but statistically significant between patients with high-risk MDS subtypes (RAEB1, RAEB2 and CMML) and control. This study also proved that expression of miR-124 was reactivated in 7 out of 18 MDS patients after treatment with low dose decitabine. CONCLUSION: The hypermethylation and silencing of miR-124 may be an important factor in the clonal transformation of MDS cells.

Downregulation of alpha-fetoprotein siRNA inhibits proliferation of SMMC-7721 cells.

AIM: To study the function of alpha-fetoprotein (AFP) in SMMC-7721 hepatoma cells. METHODS: A hairpin siRNA expressing plasmid pSilencer3.0-H1-afp was constructed and transfected into SMMC-7721 cells with Lipofectamine 2000. The expression of AFP was monitored by real-time RT-PCR and immunoassays, its effect on SMMC-7721 cell proliferation and cell death was detected by MTT and fluorescence activated cell sorter (FACS). RESULTS: The AFP-siRNA expressing plasmid downregulated the expression of AFP obviously (about 34%), and inhibited SMMC-7721 cell proliferation, but did not induce apoptosis. CONCLUSION: Downregulation of AFP siRNA inhibits proliferation of SMMC-7721 cells, but cannot cause apoptosis.

Proteomic analysis on multi-drug resistant cells HL-60/DOX of acute myeloblastic leukemia.

Multi-drug resistance (MDR) is an important factor that causes treatment failure in acute leukemia. However, the full development mechanisms of MDR still await [corrected] investigation. The purpose of this study is to investigate differentially expressed proteins in the multi-drug resistant acute myeloblastic leukemia (AML) cell line HL-60/DOX and the drug sensitive cell line HL-60, and to identify new potential multi-drug resistant related molecules with the proteomic approach. Two-dimensional gel electrophoresis (2-DE) maps of the proteins, extracted from two AML cell lines, HL-60/DOX and HL-60, were established respectively. The extracted proteins were digested by enzymes and identified with the matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The data of the peptide mass fingerprinting (PMF) was matched with databases of proteomics available on the Internet. Results showed that 16 proteins were identified to be differentially expressed between HL-60/DOX and HL-60 cells. They involved the protein disulfide isomerase precursor (PDI), the proteasomes alpha1 and other proteins which are related to drug resistance or cell metabolism, but their functional significances are required further investigation. Nevertheless, it is clear that this proteomic approach for studing the biology and development of MDR is a prerequisite in leukemia.

[Abnormal expression of microRNA-124 in patients with leukemia or myelodysplastic syndrome and its significance].

This study was aimed to investigate the abnormal expression of microRNA-124 (miR-124) in bone marrow cells of patients with leukemia or myelodysplastic syndrome (MDS) and its significance. The relative expression levels of miR-124 in bone marrow mononuclear cells from 33 patients with newly diagnosed leukemia or MDS, and 10 normal donors (as controls) were detected by stem-loop fluorescence real-time quantitative RT-PCR. The methylation levels of miR-124 promoter were detected by quantitative methylation specific PCR in partial MDS samples. The results indicated that as compared with normal control, lower levels of miR-124 (≤ 1/3) were found in 2/18 of leukemia patients and in 5/15 of MDS patients (among them ≤ 1/4 in 3/15 MDS patients). No statistically significance difference was observed between leukemia patients and normal controls (P = 0.725). However the difference was statistically significant between MDS group and control group (P = 0.031). Furthermore, an elevated methylation level of miR-124 promoter region in some of MDS patients (7/11) was detected by using quantitative methylation-specific PCR. The expression level of miR-124 was related with methylation level of promoter region (R(2) = 0.339, P = 0.018). It is concluded that the expression of miR-124 in partial MDS patients is inhibited, which may be associated with the abnormal methylation of its promoter.

[Expression of miR-21 in multiple myeloma and its clinical significance].

This study was aimed to explore the expression of microRNA-21 (miR-21) in multiple myeloma (MM), and its correlation with plasma ß2-microglobulin (ß2-MG) and staging of MM by Durie-Salmon (D-S) classification. The expression level of miR-21 in bone marrow mono-nuclear cells (BMMNC) of 43 patients with MM and 20 healthy individuals was examined by real-time polymerase chain reaction (real-time PCR), and the correlations among the expression level of miR-21 and related clinical pathologic features, plasma ß2-MG and staging of MM by D-S classification were analyzed. The results showed that the expression of miR-21 in BMMNC of MM patients was obviously higher than that in normal controls (P < 0.05). The expression of miR-21 in BMMNC of relapsed/refractory MM patients was obviously higher than that in newly diagnosed MM patients. The expression of miR-21 in MM patients decreased after chemical therapy, especially in effective group (P < 0.05), there was no significant change of miR-21 expression level in ineffective/progressive group before and after chemical therapy (P > 0.05). The expression of miR-21 was related with staging of MM and plasma ß2-MG level. It is concluded that the expression levels of miR-21 in BMMNC of MM patients are significantly higher than in normal bone marrow, these data indicated that miR-21 may play an important role in the development of MM. Super expression of miR-21 is positively correlated with level of plasma ß2-MG and staging of MM by D-S classification.

[Expression of transcription factor LYL1 in leukemia and its possible role in leukemogenesis].

OBJECTIVE: To study the expression of transcription factor LYL1 in leukemia and its possible role in leukemogenesis. METHODS: Fluorescence real time quantitative polymerase chain reaction was used to detect the expression levels of LYL1 in leukemias. Specific siRNA was used to silence the expression of LYL1 in K562 cells. RESULTS: Compared to CD34 positive cells from normal bone marrow, the expression of LYL1 was significantly elevated in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). LYL1 expression was higher in chronic myeloid leukemia (CML) in blastic crisis than that in chronic phase (7.831 vs 1.672, P < 0.01). LYL1 expression in AML in complete remission (CR) was down-regulated as compared with that of un-remission patients (1.400 vs 9.985, P < 0.01). Down-regulation of endogenous expression of LYL1 in K562 cells by a combination of three specific siRNA could inhibit cellular growth and clonogenicity to some extent. CONCLUSION: Over-expression of LYL1 is highly associated with AML as well as ALL. RNA interference targeting specific oncogenes such as LYL1 is potentially useful in the treatment of hematological malignancies.

[Comparative proteomics research of apoptosis initiation induced by homoharringtonine in HL-60 cells].

OBJECTIVE: To study the related proteins of apoptosis initiation induced by homoharringtonine (HHT) in HL-60 cells. METHODS: After establishment of an apoptosis initiation model induced by HHT in HL-60 cells, proteins of untreated and HHT treated HL-60 cells were extracted, and the two-dimensional polyacrylamide gel electrophoresis (2-DE) maps of the extracted proteins were established by using the immobilized pH gradient (IPG) two-dimensional electrophoresis respectively. The alteration protein spots were identified with assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and database searching. RESULTS: Proteomics analysis showed that proteins including MHC class I antigen, calbindin D-28K, chloride channel protein 6, oncoprotein 18, zinc finger protein Helios and apoptosis inhibitor like protein 2 were involved in apoptosis initiation induced by HHT. CONCLUSION: The present study might conduce to the researches of HL-60 cells carcinogenesis and pave the way to exploit drug precursor related to HHT and initiation of apoptosis in HL-60 cells.