Objective To explore the effect of sodium butyrate(NaB) on phosphorylation/ acetylation of histone H3(ph/acH3) at G?-globin gene and A?-globin gene promoter regions in K562 cells.Methods K562 cells were devided into 2 groups:K562 cells were grown in the presence or absence of 0.5 mmol?L-1NaB for 48 h [K562(NaB) group] and untreated K562 cells group(K562 group).Semi-quantitative RT-PCR was employed to measure the levels of G?-globin mRNA and A?-globin mRNA.The real time PCR-based chromatin immunoprecipitation(ChIP) was used to detect the levels of ph/acH3 at G?-globin gene and A?-globin gene promoter regions.Results Compared with the K562 group,there was a 1.4-fold(t=-149.022,P=0.000) and 1.2-fold(t=-13.363,P=0.000) increase in G?-globin mRNA and A?-globin mRNA,respectively,in K562(NaB) group.The level of ph/acH3 at G?-globin gene and A?-globin gene promoter region increased by 2.9-fold(t=-12.833,P=0.006) and 3.2-fold(t=-10.484,P=0.000),respectively,in K562(NaB) group,compared with the K562 group.The %Input value of G?-globin and A?-globin promoter fragment was 10.0-fold(P=0.000) and 9.5-fold(P=0.000) higher than that value of Necdin gene promoter fragment in the K562(NaB) group,while the %Input value of G?-globin and A?-globin promoter fragment was 3.2-fold(P=0.000) and 2.7-fold(P=0.000) higher than that value of necdin gene promoter fragment in K562 group.Conclusions NaB improves the phosphorylation and acetylation of H3 at ?-globin gene promoter regions,and this may be one of the mechanisms of expression of ?-globin genes induced by NaB.

Cri-du-Chat Syndrome ; complications ; Humans ; Infant, Newborn ; Male ; XYY Karyotype

AIM To investigate the effect of allicin on the regulation of VEGF mRNA expression in human hepatocellular carcinoma cells. METHODS Hepatocellular carcinoma cells were treated with the concentration 10 ?g?L -1 allicin in culture medium,and then the relative VEGF mRNA level at 8 h in human hepatocellular carcinoma cells was evaluated by reverse transcriptase polymerase chain reaction using HPRT(hypoxanthine phosphoribosyltransferase)as an internal control standard. RESULTS The expression of VEGF gene mRNA was inhibited obviously by allicin. Compared with control group, the relative expression level of VEGF gene mRNA was decreased by about 66 36%( P

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

Objective To explore the effects of astragalus polysaccharides(APS) on fetal hemoglobin(HbF) synthesis and cell proli-feration in K562 cells.Methods K562 cells were chosen as the cell model and cells treated with Na-butyrate(NaB) were taken as the po-sitive control.Western blot was applied to study the level of HbF expression in K562 cells and Trypan blue dye exclusion test was employed to analyze the influence of APS(150 mg/L,300 mg/L,450 mg/L)on K562 cells proliferation.Results 1.Dosage effect:when compared with untreated K562 cells,the HbF expression level increased to(1.56?0.03),(1.78?0.04) and(1.51?0.32) fold,respectively after 48 h treated with different concentrations of APS(150 mg/L,300 mg/L,450 mg/L,F=310.476 P=0).The best inducing concentration was 300 mg/L(P=0.005).2.Time course: HbF levels raised up gradually and the maximum was(2.88?0.27) fold over baseline(P=0) at 48-60 h in the presence of 300 mg/L APS.Then it went to decline.There was statistical significance of HbF expression between K562 cells treated with 300 mg/L APS or NaB [(2.88?0.27) folds,P=0].3.Effects of APS on K562 cells proliferation:the highest reduction of the cell proliferative was obtained in K562 cells cultured in the presence of 0.5 mmol/L NaB.As detected by Trypan blue exclusion met-hod,growth rate of cells stimulated by APS was affect in a dose dependent manner,and significantly higher than NaB.For example,the inhibition rate at 48 hours was 20.45% for 300 mg/L APS but 79.55% for 0.5 mmol/L NaB(P=0).Conclusion APS has ability to induce HbF synthesis in K562 cells and revealed less cells reduction than that of NaB.

Objective To investigate the role of directly constitutive activation of p38 mitogen-activated protein kinases(p38MAPKs)signaling in ?-globin gene expression and fetal hemoglobin(HbF)induction,and provide direct data for the relationship between phosphorylation of p38 and erythroid differentiation of human K562 erythroleukemia cells.Methods The human K562 erythroleukemia cells were transfected with pCDNA 3.1-MKK3(Glu)and pCDNA 3.1-MKK3(Ala)recombinant plasmids by lipofectamineTM 2000.Then,the stable cell lines overexpressing constitutively active p38 and constitutively inhibitive p38 activation were established by the addition of G418 to select single cell G418-resistant clones and identification with reverse transcriptase-polymerase chain reaction(RT-RCR)and Western blot assays,named K562-MKK3(Glu)and K562-MKK3(Ala)cells,respectively.Furthermore,the direct effects of constitutively active p38 on the ?-globin gene expression and HbF induction were analyzed by RT-PCR and benzidine staining,respectively.Results The results of RT-PCR and Western blot showed that there were no evident changes in the mRNA and protein levels of p38 for various cell models,but compared with K562,K562-vect,and K562-MKK3(Ala)cells,the phosphorylation of p38 and expression of ?-globin levels in K562-MKK3(Glu)cells were significantly up-regulated.The results of benzidine staining displayed that the mean percentages of positive cells stained by benzidine in K562,K562-vect,K562-MKK3(Ala),K562-MKK3(Glu)cells,and K562-MKK3(Glu)cells treated with SB203580 were(3.2?1.4)%,(3.7?1.2)%,(2.8?0.9)%,(32.6?5.3)%,and(7.8? 2.3)%(q = 7.56 P

OBJECTIVETo develop a real-time PCR-based chromatin immunoprecipitation (ChIP) assay for determining the effect of sodium butyrate on acetylation of histone in gamma-globin gene promoter regions in K562 cells.

METHODSK562 cells were grown in the presence or absence of 0.5 mmol/L sodium butyrate for 48 h, and 1=10(7) cells per group were used for real-time PCR-based ChIP with anti-acetylated histone H3 or H4 antibodies. The levels of acetylated histone H3 and H4 (acH3 and acH4) in Ggamma- and Agamma-globin gene promoter regions were measured.

RESULTSIn the K562 cells with sodium butyrate treatment or without any treatment, the levels of acH3 or acH4 in Ggamma- or Agamma-globin gene promoter were higher than that in the necdin gene (negative control). Compared with the untreated K562 cells, the cells treated with 0.5 mmol/L sodium butyrate showed a 3.1-fold or 2.6-fold increase in acH3 or acH4 in Ggamma-globin gene promoter region, with also a 3.7-fold or 3.2-fold increase in acH3 or acH4 in Agamma-globin gene promoter region, respectively (P<0.01).

CONCLUSIONWe have successfully developed a real-time PCR-based ChIP assay for analyzing the acetylation of histone H3 and H4 in gamma-globin gene promoter regions. Our results support the role of sodium butyrate in increasing the level of acetylated histone in gamma-globin gene promoter regions.

Acetylation ; Butyrates ; pharmacology ; Chromatin Immunoprecipitation ; methods ; Histones ; chemistry ; Humans ; K562 Cells ; Promoter Regions, Genetic ; genetics ; Real-Time Polymerase Chain Reaction ; methods ; gamma-Globins ; genetics

Abstracting and Indexing as Topic ; Neoplasms ; Periodicals as Topic

Objective To investigate the effects of Epigallocatechin gallate(EGCG)on IL-1βinduced MIN6 cells apoptosis. M.Methods The experiment group was divided into control group,IL-1β group,IL-1β+ EGCG low concentration group and IL-1β+EGCG high concentration group.Cell activity was detected by CCK8.Insulin secretion was detected by ELISA.cell apoptosis was detected by flow cytometry.The mitochondrial membrane potential was detected by flow cytometry.ATP content and cell ac-tivity of ROS were detected by colorimetry and chemiluminescence method.Results Compared with normal group,IL-1β group showed much lower cell activity,insulin secretion,cell mitochondrial membrane potential and ATP content,and at the same time IL-1βgroup had significantly higher cell apoptosis and ROS activities.After given EGCG,both low concentration group and high con-centration group had higher cell activity,insulin secretion,cell mitochondrial membrane potential and ATP content,at the same time lower cell apoptosis and ROS activities was showed.And the IL-1β+EGCG high concentration group worked more powerful.Con-clusion EGCG has protective effects on IL-1βinduced MIN6 cells apoptosis.Its mechanism may be related to increasing the content of the ATP and mitochondrial membrane potential and protecting mitochondrial function as well reducing the activity of ROS.