Interferon γ promotes terminal erythroid differentiation / 基础医学与临床

Objective To study the effects of interferon γ(IFN-γ) on terminal erythroid differentiation.Methods RT-qPCR was used to detect the expression of IFN-γ receptors during erythroid differentiation of K562 induced by hemin.Both hemin-induced K562 cells and human umbilical cord CD34+ cell derived primary erythroid cells were treated with IFN-γ.Erythroid differentiation of the cells was evaluated using RT-qPCR to detect the mRNA level of erythroid specific surface markers CD71 and CD235a,and benzidine staining assay was applied to explore the change of hemoglobin expression.Results The expression of IFN-γ receptors in K562 cells decreased and climbed up again after reaching the lowest point at 48 h of hemin induction.IFN-γ treatment increased CD71 and CD235a expression in both hemin-induced K562 cells and the later stage (E15D) primary erythroid cells.Benzidine staining showing increased globin protein expression in hemin-induced K562 cells after IFN-γstimulation.Furthermore,our results indicated that IFN-γ promoted hemin-induced K562 erythroid differentiation in a time dependent manner.Mechanistically,the results showed that IFN-γ treatment stimulated the expression of erythroid transcription factors NFE2,which was critically for erythroid maturation.Conclusions IFN-γaccelerates terminal erythroid differentiation in hemin-induced K562 cells and human umbilical cord CD34+ derived primary erythroid cells.

TET2 is upregulated during erythroid differentiation of CD34+ cells from healthy donors and myelodysplastic syndrome patients

Background: Tet methylcytosine dioxygenase 2 (TET2) is frequently mutated and/or downregulated in myeloid neoplasm, including myelodysplastic syndromes. Despite the extensive studies, the specific contribution of TET2 in disease phenotype of myeloid neoplasms is not fully elucidated. Recent findings have grown attention on the role of TET2 in normal and malignant erythropoiesis. Methods: In the present study, we investigated TET2 mRNA levels by quantitative PCR during erythropoietin-induced erythroid differentiation CD34+ cells from healthy donor and myelodysplastic syndrome patients. Statistical analyses were performed using the ANOVA and Bonferroni post hoc test and a p-value <0.05 was considered statically significant. Results: TET2 expression is upregulated during erythroid differentiation of CD34+ cells from healthy donor and myelodysplastic syndrome patients. Conclusions: Our findings corroborate that TET2 is involved in the erythrocyte differentiation (AU)

miR-34a-5p inhibits the erythroid differentiation of K562 cells / 基础医学与临床

Objective To study the effects of microRNA-34a-5p on erythroid differentiation of K562 cells.Methods K562 cells were transfected with the microRNA-34a-5p mimics and antisense inhibitors specifically targeting mi-croRNA-34a-5p, respectively.The effects of over-expression or knocking-down of microRNA-34a-5p were exam-ined by Quantitative RT-PCR.Flow cytometry was performed to detect specific surface marker of erythroid cells . The benzidine staining assay was used to access the differentiation of K 562 cells.Western blot was performed to de-tect miRNA targets.Results microRNA-34a-5p was down-regulated at the early stage of K562 erythroid differenti-ation.Over-expression of microRNA-34a-5p in K562 cells attenuates erythroid differentiation , in contrast, inhibi-tion of microRNA-34a-5p accelerates erythroid pheotypes in K562 cells.c-MYB was found to be the direct target of microRNA-34 a-5 p in erythroid cells .Conclusions microRNA-34 a-5 p regulates early erythroid differentiation of K562 cells via repressing c-MYB.

ZNF330 promotes erythroid differentiation / 基础医学与临床

Objective To explore the effects of ZNF330 on erythroid differentiation of K562 cells and underlying mechanism .Methods Realtime PCR was performed to detect the expression of ZNF 330 in K562 cells induced by hemin .After CD34 +cells being infected by the recombination lentivirus ZNF 330-RNAi, Realtime PCR was applied to detect the expression of CD235a and γ-globin.The luciferase report assay was performed to examine if ZNF 330 could act as a trans-acting factor in 293T/17 cells.Co-Immunoprecipitation (Co-IP) was applied in 293T/17 cells to detect the interaction between ZNF 330 and ZNF408 which was involved in mRNA degradation .Results The ex-pression of ZNF330 was up-regulated after hemin treatment .The expression of CD235a andγ-globin decreased after inhibition expression of ZNF 330 had no effect on report gene .Co-Ip in two ways confirmed the direct binding be-tween ZNF330 and ZNF408 .Conclusions ZNF330 can promote erythroid differentiation , and a possible mecha-nism is that ZNF330 inhibits the function of ZNF 408 , a factor that is involved in mRNA degradation , through the interaction between the two proteins .

SHP1 gene induces apoptosis and erythroid differentiation in human erythromyeloblastoid leukemia cell line K562 / 第二军医大学学报

Objective: To investigate the role of SHP1 gene in inducing apoptosis and erythroid differentiation in human erythromyeloblastoid leukemia cell line K562. Methods: The full length cDNA of SHP1 gene was cloned by RT-PCR and was subcloned into mammalian expression vector pcDNA3.0. The cDNA sequence of the cloned gene was validated by enzyme digestion and DNA sequencing. Then the recombinant plasmid was used to transfect K562 cells via lipofectin. The apoptosis of K562 cells was examined by Hoechst 33258 staining assay and Annexin V/PI double-labeled assay; the differentiation of K562 cells was observed by benzidine staining and expression of glycophorin A (GPA). Results: RT-PCR and Western blotting analysis showed expression of SHP1 in K562 cells after transfection with pcDAN3-SHP1 plasmid. Apoptotic cells were detected in the K562 cells 48 h after treatment with pcDNA3-SHP1, with the apoptosis rate being 16.84%, which was significantly higher than that in cells transfected with pcDNA3.0 (6.23% , P=0.000). The positive rate of benzidine staining was 14.67% and the positive rate of GPA expression was 19.38% in cells treated with pcNDA3-SHP1, both were significantly different from those in the cells transfected with pcDNA3.0 (P=0.005). Conclusion: Over-expression of SHP1 can effectively induce apoptosis and erythroid differentiation in K562 cells.

Enforced Expression of BMI-1 in Postnatal Human CD34+ Cells Promotes Erythroid Differentiation

BACKGROUND: The Polycomb-group gene Bmi-1 is known to be a molecular regulator of self-renewal of normal and leukemic stem cells and be involved in various aspects of cellular proliferation, differentiation, and survival. METHODS: This study evaluated the effects of overexpression of Bmi-1 on human cord blood CD34+ cells. Bmi-1 was introduced into CD34+ cells through lentivirus transduction. Bmi-1 expressing CD34+ cells were applied to colony forming assay, stromal co-culture, and cytokine-stimulatied culture. RESULTS: Ectopic expression of Bmi-1 resulted in the increased number of erythroid colonies in primary and secondary colony forming assay in an erythropoietin dependent manner. In stromal co-culture, Bmi-1-expressing postnatal hematopoietic stem cells seemed to lose the ability of self-renewal, as determined by week 5 cobblestone area-forming cell assay and by week 5 secondary colony assay. In cytokine-stimulated suspension culture of Bmi-1-transduced CD34+ cells, we observed increased erythropoiesis marked by Glycophorin A expression. CONCLUSION: Our data suggest that ectopic expression of Bmi-1 in human hematopoietic stem/progenitor cells may result in the differentiation to the erythroid lineage rather than promoting self-renewal.

DNA-Protein Interaction of gamma-Globin Gene Promoter by Differentiation Inducers / 대한혈액학회지

BACKGROUND: The K562 erythroleukemia cell line was used to study the molecular mechanisms regulating lineage commitment of hematopoietic cells. There are numerous similarities between the erythroid or megakaryocytic lineages. In this study, we examined role of the region -269~-240 of gamma-globin gene promoter in fetal hemoglobin expression during either erythroid or megakaryocytic differentiation. METHODS: K562 cells were cultured and treated with differentiation inducers. Hemoglobin content was scored by benzidine staining, and hemoglobin F was stained by acid elution technique. To determine whether transcription factor binding to the gamma-globin gene promoter is critical to lineage determination, DNA-protein interaction of gamma-globin gene promoter was examined under both uninduced and induced conditions of K562 cells using gel mobility shift assay and southwestern blot analysis. RESULTS: Phorbol 12-myristate 13-acetate (PMA) induced a megakaryocytic differentiation, but suppressed erythroid differentiation. On the other hand, hydroxyurea (HU), hemin, n-butanol, and sodium butyrate (NaB) induced the expression of erythroid phenotypes. Parallel to hemoglobinization, increase in gamma-globin mRNA was observed in HU- and hemin-treated K562 cells. Gel mobility shift assay and southwestern blot analysis revealed that binding of a erythroid-specific protein (p120) to the region -269~-240 of gamma-globin gene promoter occurred with treatment of erythroid differentiation inducers and did not occur with treatment of PMA. CONCLUSION: These results suggest that erythroid differentiation inducers may act via DNA- protein interaction at the gamma-globin gene promoter region to induce erythroid differentiation.

Role of Ras/ERK-dependent pathway in the erythroid differentiation of K562 cells

The chronic myelogenous leukemic K562 cell line carrying Bcr-Abl tyrosine kinase is considered as pluripotent hematopoietic progenitor cells expressing markers for erythroid, granulocytic, monocytic, and megakaryocytic lineages. Here we investigated the signaling modulations required for induction of erythroid differentiation of K562 cells. When the K562 cells were treated with herbimycin A (an inhibitor of protein tyrosine kinase), ras antisense oligonucleotide, and PD98059 (a specific inhibitor of MEK), inhibition of ERK/MAPK activity and cell growth, and induction of erythroid differentiation were observed. The ras mutant, pZIPRas61leu-transfected cells, K562-Ras61leu, have shown a markedly decreased cell proliferation rate with approximately 2-fold doubling time, compared with the parental K562 cells, and about 60% of these cells have shown the phenotype of erythroid differentiation. In addition, herbimycin A inhibited the growth rate and increased the erythroid differentiation, but did not affect the elevated activity of ERK/MAPK in the K562-Ras61leu cells. On the other hand, effects of PD98059 on the growth and differentiation of K562-Ras61leu cells were biphasic. At low concentration of PD98059, which inhibited the elevated activity of ERK/MAPK to the level of parental cells, the growth rate increased and the erythroid differentiation decreased slightly, and at high concentration of PD98059, which inhibited the elevated activity of ERK/MAPK below that of the parental cells, the growth rate turned down and the erythroid differentiation was restored to the untreated control level. Taken together, these results suggest that an appropriate activity of ERK/MAPK is required to maintain the rapid growth and transformed phenotype of K562 cells.

Effect of Sodium Butyrate on Erythroid Lineage by Expressional Profiles Microarray in K562 Cells / 实用儿科临床杂志

1)which represented 340 genes and 171 down-regulated(SLR

Accumulation of ? Globin mRNA and Induction of Erythroid Differentiation after Treatment of Chronic Myelocytic Leukemia Cell Line K562 with Matrine / 实用儿科临床杂志

Objective To study the effects of matrine on accumulation of ? globin mRNA and induction of erythroid differentiation in K562 cells in vitro.Methods K562 cells were cultured for 6 days with different concentration of matrine,viable cell counts were determined by trypan-blue dye exdusion test. Erythroid differentiation was evaluated by percentage of benzidine-positive cells at different days after culture. Morphological changes were observed under microscope after Wright-Gimesa staining; ? globin mRNA was quantitative by real time quantitative reverse transcript polymerase chain reaction(RT-PCR).Results Different concentrations of matrine inhibited proliferation of K562 cells in dose-dependent manner; otherwise, K562 cells were successfully induced by erythroid differentiation with matrine. After treatment with matrine, percentage of benzidine-positive cells significantly increased from 0.7% to 15.7% and characteristic changes of erythroid differentiation in the cell morphology were observed, G? globin mRNA had a preferential increase (2.7 fold)in K562 cells. Conclusions Matrine accumulation G? globin mRNA and induced erythroid differentiation of K562 cells. The results provides an experimental evidence for the pharmacological therapy of hematological diseases associated with a failure in the expression of normal ? globin genes.