[Corrigendum] Fully human VEGFR2 monoclonal antibody BC001 attenuates tumor angiogenesis and inhibits tumor growth.

Subsequently to the publication of the above paper, an interested reader drew to the authors' attention that, for the scratch wound assay experiments shown in Fig. 1 on p. 2413, the panels showing the '0 h' experiments for the respective incubations with VEGF or BC001 were apparently identical. The authors were able to re­examine their original data files, and realized that this figure had been inadverently assembled incorrectly. The revised version of Fig. 1, containing the correct data for the '0 h / BC001' panel, is shown below. Note that the revisions made to this figure do not affect the overall conclusions reported in the paper. The authors are grateful to the Editor of International Journal of Oncology for allowing them the opportunity to publish this Corrigendum, and apologize to the readership for any inconvenience caused. [International Journal of Oncology 45: 2411­2420, 2014; DOI: 10.3892/ijo.2014.2690].

EDAG mediates Hsp70 nuclear localization in erythroblasts and rescues dyserythropoiesis in myelodysplastic syndrome.

During human erythroid maturation, Hsp70 translocates into the nucleus and protects GATA-1 from caspase-3 cleavage. Failure of Hsp70 to localize to the nucleus was found in Myelodysplastic syndrome (MDS) erythroblasts and can induce dyserythropoiesis, with arrest of maturation and death of erythroblasts. However, the mechanism of the nuclear trafficking of Hsp70 in erythroblasts remains unknown. Here, we found the hematopoietic transcriptional regulator, EDAG, to be a novel binding partner of Hsp70 that forms a protein complex with Hsp70 and GATA-1 during human normal erythroid differentiation. EDAG overexpression blocked the cytoplasmic translocation of Hsp70 induced by EPO deprivation, inhibited GATA-1 degradation, thereby promoting erythroid maturation in an Hsp70-dependent manner. Furthermore, in myelodysplastic syndrome (MDS) patients with dyserythropoiesis, EDAG is dramatically down-regulated, and forced expression of EDAG has been found to restore the localization of Hsp70 in the nucleus and elevate the protein level of GATA-1 to a significant extent. In addition, EDAG rescued the dyserythropoiesis of MDS patients by increasing erythroid differentiation and decreasing cell apoptosis. This study demonstrates the molecular mechanism of Hsp70 nuclear sustaining during erythroid maturation and establishes that EDAG might be a suitable therapeutic target for dyserythropoiesis in MDS patients.

Fully human VEGFR2 monoclonal antibody BC001 attenuates tumor angiogenesis and inhibits tumor growth.

The critical role of VEGFR2 in tumor neovascularization and progression has allowed the design of clinically beneficial therapies based on it. Here we show that BC001, a new fully human anti-VEGFR2 monoclonal antibody, inhibits VEGF-stimulated endothelial cell migration, tube formation, and effectively suppressed the transdifferentiation of cancer stem cells into endothelial cells in vitro. Since BC001 exhibited no activity against the mouse VEGFR2 and mouse based study was required to confirm its efficacy in vivo, BC101, the mouse analogue of BC001, was developed. BC101 significantly attenuated angiogenesis according to Matrigel plug assay and resulted in ~80% growth inhibition of mouse B16F10 homograft tumors relative to vehicle control. Similarly, human analogue BC001 suppressed the growth of human xenograft tumors HCT116 and BGC823. Furthermore, immunohistochemical results showed reduced expression of CD31, VEGFR2 and Ki-67, as well as increased expression of Caspase 3 in BC001-treated tumor, which indicated BC001 was able to significantly decrease microvessel density, suppress proliferation and promote apoptosis. These results demonstrate the fully human VEGFR2 monoclonal antibody BC001 can work as an effective inhibitor of tumor angiogenesis and tumor growth both in vitro and in vivo.

EDAG positively regulates erythroid differentiation and modifies GATA1 acetylation through recruiting p300.

Erythroid differentiation-associated gene (EDAG) has been considered to be a transcriptional regulator that controls hematopoietic cell differentiation, proliferation, and apoptosis. The role of EDAG in erythroid differentiation of primary erythroid progenitor cells and in vivo remains unknown. In this study, we found that EDAG is highly expressed in CMPs and MEPs and upregulated during the erythroid differentiation of CD34(+) cells following erythropoietin (EPO) treatment. Overexpression of EDAG induced erythroid differentiation of CD34(+) cells in vitro and in vivo using immunodeficient mice. Conversely, EDAG knockdown reduced erythroid differentiation in EPO-treated CD34(+) cells. Detailed mechanistic analysis suggested that EDAG forms complex with GATA1 and p300 and increases GATA1 acetylation and transcriptional activity by facilitating the interaction between GATA1 and p300. EDAG deletion mutants lacking the binding domain with GATA1 or p300 failed to enhance erythroid differentiation, suggesting that EDAG regulates erythroid differentiation partly through forming EDAG/GATA1/p300 complex. In the presence of the specific inhibitor of p300 acetyltransferase activity, C646, EDAG was unable to accelerate erythroid differentiation, indicating an involvement of p300 acetyltransferase activity in EDAG-induced erythroid differentiation. ChIP-PCR experiments confirmed that GATA1 and EDAG co-occupy GATA1-targeted genes in primary erythroid cells and in vivo. ChIP-seq was further performed to examine the global occupancy of EDAG during erythroid differentiation and a total of 7,133 enrichment peaks corresponding to 3,847 genes were identified. Merging EDAG ChIP-Seq and GATA1 ChIP-Seq datasets revealed that 782 genes overlapped. Microarray analysis suggested that EDAG knockdown selectively inhibits GATA1-activated target genes. These data provide novel insights into EDAG in regulation of erythroid differentiation.

Erythroid differentiation-associated gene interacts with NPM1 (nucleophosmin/B23) and increases its protein stability, resisting cell apoptosis.

Erythroid differentiation-associated gene (EDAG) is a haematopoietic tissue-specific transcription regulator that plays a key role in maintaining the homeostasis of haematopoietic lineage commitment. In acute myeloid leukaemia (AML) patients, the high expression level of EDAG is associated with poor prognosis. NPM1 (nucleophosmin/B23), a ubiquitous nucleolar phosphoprotein, comprises a multifunctional protein that is involved in several cellular processes, including ribosome biogenesis, centrosome duplication, cell cycle progression, cell growth and transformation. Various studies have implicated NPM1 overexpression in promoting tumour cell proliferation, blocking the differentiation of leukaemia cells and resisting apoptosis. In the present study, using co-immunoprecipitation, we characterized EDAG as a physiological binding partner of NPM1; The N-terminal (amino acids 1-124) region of EDAG interacts with the N-terminal (amino acids 118-187) of NPM1. Under cycloheximide treatment, the stability of NPM1 protein was enhanced by EDAG overexpression, whereas knockdown of EDAG by lentivirus-mediated small interfering RNA resulted in an increased degradation rate of NPM1 in K562 cells. During 4ß-phorbol l2-myristate 13-acetate-induced K562 megakaryocytic differentiation, overexpression of EDAG prevented the down-regulation of NPM1 proteins, whereas knockdown of EDAG accelerated the down-regulation of NPM1. EDAG deletion mutant lacking the binding domain with NPM1 lost the ability to stabilize NPM1 protein. Furthermore, knockdown of EDAG in K562 cells led to increased cell apoptosis induced by imatinib, and re-expression of NPM1 attenuated the increased apoptosis. These results suggest that EDAG enhances the protein stability of NPM1 via binding to NPM1, which plays a critical role in the anti-apoptosis of leukaemia cells.

[Targeted regulation of mll-af4 fusion gene by miR-142-3p].

In order to analyze the possible epigenetic regulation mechanism of mll-af4 gene expression and find the possible microRNA regulating mll-af4 gene expression, targetscan software was used to analyze potential microRNA target sites in 3'-UTR of mll-af4. 3'-UTR fragment of mll-af4 was amplified by PCR. PCR products were cloned into EcoR I/Pst I-digested pGL3-M reporter vector, placing the 3'-UTR with potential microRNA binding site downstream of coding sequence of luciferase. The construct was cotransfected in 293T cells with control plasmid or plasmids expressing microRNAs regulating mll-af4 potentially. Western blot and RT-PCR were used to detect the expression level of mll-af4 protein and mRNA in RS4; 11 cells after transfection of miR-142-3p, respectively. The results showed that the pGL3-AF4-3'UTR of luciferase reporter recombinant plasmid contain the 3'UTR sequence of mll-af4 gene with 1935 bp, 2104 bp and 1371 bp was constructed successfully and was confirmed by enzyme digestion and gene sequencing. The luciferase assay revealed that overexpression of miR-142 could reduce the luciferase activity from the reporter construct containing the mll-af4 3'-UTR significantly. Protein and mRNA expressions of mll-af4 were found to be downregulated by miR-142-3p. It is concluded that miR-142-3p regulates the expression of mll-af4 through target binding the mll-af4 gene 3'UTR site.

Over-expression of EDAG in the myeloid cell line 32D: induction of GATA-1 expression and erythroid/megakaryocytic phenotype.

Erythroid differentiation-associated gene (EDAG), a hematopoietic tissue-specific transcription regulator, plays a key role in maintaining the homeostasis of hematopoietic lineage commitment. However, the mechanism and genes regulated by EDAG remain unknown. In this study, we showed that overexpression of EDAG in a myeloid cell line 32D led to an erythroid phenotype with increased number of benzidine-positive cells and up-regulation of erythroid specific surface marker TER119. The megakaryocytic specific marker CD61 was also induced significantly. Using a genome-wide microarray analysis and a twofold change cutoff, we identified 332 genes with reduced expression and 288 genes with increased expression. Among up-regulation genes, transcription factor GATA-1 and its target genes including EKLF, NF-E2, Gfi-1b, hemogen, SCL, hemoglobin alpha, beta and megakaryocytic gene GPIX were increased. Silencing of EDAG by RNA interference in K562 cells resulted in down-regulation of these genes. Taken together, EDAG functions as a positive regulator of erythroid/megakaryocytic differentiation in 32D cells associated with the induction of GATA-1 and its target genes.

Suppression of EDAG gene expression by phorbol 12-myristate 13-acetate is mediated through down-regulation of GATA-1.

EDAG, a hematopoietic tissue-specific protein, is involved in the regulation of proliferation, differentiation and apoptosis of hematopoietic cells. In this study, a dose-dependent inhibition of EDAG expression by PMA was observed in K562 cells. The responsive element for the PMA-induced inhibition was contained in the region between -211 and +32bp of the EDAG gene promoter. By oligonucleotide-directed mutagenesis, EMSA, ChIP and transient transfection assays, we found that two tandem repeat GATA-1 sites in the promoter of EDAG gene played an important role in the PMA-mediated down-regulation of the EDAG gene expression in K562 cells. The kinetics of EDAG expression during PMA induction showed that the levels of EDAG expression were down-regulated concomitantly with GATA-1 down-expression. Decreased GATA-1 expression by siRNA reduced expression of EDAG in K562 cells, and restored expression of GATA-1 significantly rescued EDAG expression from PMA-mediated suppression. Overexpression of EDAG in K562 cells inhibited the megakaryocytic differentiation induced by PMA which raised the interesting possibility that PMA induced K562 cells differentiation toward megakaryocytic phenotype through, at least in part, the inhibition of EDAG expression. In vivo analysis confirmed that EDAG was highly expressed in primitive progenitor cells and down-regulated in megakaryocytes which was consistent with the expression pattern of GATA-1. Furthermore, PKC and MAPK specific inhibitors treatment attenuated the down-regulation of EDAG induced by PMA. Taken together, these results suggest that the inhibition of the EDAG gene by PMA is mediated through down-regulation of transcription factor GATA-1 and involved the PKC/MAPK signaling pathway.

[Expression of EDAG-1 gene in human leukemia and lymphoma cell lines].

BACKGROUND & OBJECTIVE: Embryonic development associated gene 1 (EDAG-1), located at chromosome 9q22, is specially expressed in hematopoietic cells, and related to the regulation of hematopoietic system. This study was designed to explore relationship between pathogenesis of leukemia, lymphoma and EDAG-1 through analyzing the structure of EDAG-1 coding region, and its expression in these cell lines. METHODS: Fifteen leukemia and lymphoma cell lines, HEL, K562, HL-60, Namalwa, Raji, J111, Jurkat, HuT 78, MEG-01, U937, 6T-CEM, HPB-ALL, KG-1a, THP-1, and DAMI, were selected to observe the expression of EDAG-1 by reverse transcriptase-polymerase chain reaction (RT-PCR), EDAG-1 cDNA coding fragments (1.5 kb) were purified to construct the corresponding recombinant plasmid. Then, the plasmid was sequenced to analyze mutation of the coding region. The expression of EDAG-1 protein, and mRNA in these cell lines were detected by Western blot, and Northern blot; the rearrangement and amplification of EDAG-1 genome in these cell lines were detected by Southern blot. RESULTS: EDAG-1 mRNA and protein were highly expressed in erythroleukemia cell lines (K-562, HEL), megakaryoblast leukemia cell lines (DAMI, MEG-01), and T cell leukemia cell line (Jurkat), while no gene mutation was found in coding region, no amplification and rearrangement of genome was detected in these cell lines. EDAG-1 was absent in HL-60 cell line, and rearranged in HuT 78 cells. CONCLUSION: EDAG-1 may relate with pathogenesis of erythroleukemia and megakaryoblast leukemia; its coding region may have no relation with the mechanism of its activation.

[UV-vis spectrometric characterization of the composition of active complex in the Ni-P plating solution].

A strong complex agent is normally used in the alkaline chemical plating solution for Ni-P plating in order to prevent Ni ion from precipitation by hydrolysis. To keep a stable pH condition, an NH3-NH4Cl buffer system is used. Traditionally, it is considered that NH3 does not participate in the complex because of the relatively low Ni-NH3 complexing constant, but some experimental results cannot be explained reasonably. Touhami etc. have proposed a ternary Ni-citrate-NH3 complex involved in the discharge process, however they cannot give the direct support on the presence of this complex in solution. In this paper, a UV-Vis spectrometric study was carried out to identify the nickel complex in the Ni-P plating solution, and the results indicated the presence of both Ni-cit binary complex and Ni-cit-ammonia ternary complex. After the systematic investigation of the dependence of UV-Vis spectra on the two ligands (cit and ammonia), the composition of this Ni-cit-ammonia ternary complex was supposed to be Ni(II)(C6H5O(7)3-)(NH3)3.