Signal transducer and activator of transcription 5 (STAT5) is an important transcription factor existing in the cytoplasm of various types of cells. Once activated, STAT5 dimers translocate into nucleus and bind to the corresponding DNA sequence to regulate the transcription of its target genes. There are two isoforms of STAT5: STAT5A and STAT5B with 96% sequence homology and are encoded by two closely related but different genes. Studies have shown that STAT5 can regulate the survival, proliferation, differentiation and death of hematopoietic cells. Furthermore, elevated activation of STAT5 was found in many malignant hematologic diseases and therefore raised the possibility that STAT5 may be used as a new therapeutic target for blood related diseases. This review discusses the regulatory role of STAT5 in hematopoietic cells and its effect on the occurrence and development of blood diseases.
Animals ; Hematologic Diseases ; genetics ; metabolism ; Hematopoietic System ; metabolism ; Humans ; STAT5 Transcription Factor ; genetics ; metabolism

OBJECTIVETo investigate the frequency of JAK2 V617F mutation in 145 myeloproliferative disorders (MPDs) patients, analyze the correlation between JAK2 V617F mutation and clinical features.

METHODSThe JAK2 V617F mutation was detected by direct DNA sequencing of PCR product and allele-specific PCR respectively. The expression of JAK2, phospho-JAK2 and phospho-STAT5 proteins was determined by Western blot. The clinical data of MPDs patients with or without JAK2 V617F mutation was collected and analyzed for evaluating the clinical significance of JAK2 V617F mutation.

RESULTS1) The frequency of JAK2 V617F mutation for PV, IMF, ET was 92%, 58%, 50% respectively. Compared with conventional DNA sequencing (PV 84%, IMF 44%, ET 39%, respectively), allele-specific PCR exhibited a higher sensitivity in JAK2 V617F mutation detection. 2) The expression levels of phospho-JAK2 and phospho-STAT5 in peripheral blood mononuclear cells (PBMNCs) were upregulated significantly in JAK2 V617F-positive patients than in JAK2 V617F negative patients. 3) Compared with the patients with no JAK2 V617F mutation, the JAK2 V 617F-positive patients' features were as follows: older age of onset, higher mean leukocyte counts, lower platelet counts and smaller spleen volume. Frequency of thrombosis events in PT, ET, IMF was 17%, 32%, 16% respectively for JAK2 V617F positive group, and 0% (PV), 16% (ET), 5% (IMF) for JAK2 V617F negative group.

CONCLUSIONSMPDs patients display higher frequency of JAK2 V617F mutation. JAK2 V617F mutation positive patients predispose to a thrombosis tendency.

Female ; Humans ; Janus Kinase 2 ; genetics ; metabolism ; Male ; Mutation ; Myeloproliferative Disorders ; genetics ; metabolism ; Phosphorylation ; STAT5 Transcription Factor ; metabolism ; Sequence Analysis, DNA

OBJECTIVETo investigate the effect of curcumin on STAT 5 signaling molecule in K562 cells and its molecular mechanism of antileukemia.

METHODSCell proliferation was studied by tetrazolium dye assay. The expressions of STAT5 mRNA and protein were assayed by in situ hybridization, and Western blotting respectively.

RESULTSCurcumin could inhibit K562 cell proliferation in a time- and dose-dependent manner. The percentage of STAT5-positive cells was 19% in curcunin group, significantly less than 31% of that in K562 cell group (P < 0.01). The A value of the expression level of STAT5 protein in curcumin group was 15 266 +/- 769, significantly less than 25 781 +/- 1240 of that in K562 cell group (P < 0.01).

CONCLUSIONThe expressions of STAT5 mRNA and protein in K562 cells were inhibited by curcumin and curcumin could inhibit K562 cell proliferation.

Cell Proliferation ; drug effects ; Curcumin ; pharmacology ; Gene Expression Regulation, Leukemic ; drug effects ; Humans ; K562 Cells ; Leukemia ; genetics ; metabolism ; physiopathology ; STAT5 Transcription Factor ; genetics ; metabolism ; Signal Transduction ; drug effects

Mechano-growth factor (MGF) is one of IGF-1 isoforms. MGF is mechanosensitive and has important functions in muscle hypertrophy, regeneration and nerve injury recovery. In this study, MGF cDNA (330 bp) was cloned from stretched osteoblasts by RT-PCR. In order to avoid prolin residue inhibiting enterokinase cleavage, 9bp of MGF cDNA 5' end sequence was truncated by primer, then the obtained truncated MGF (des(1-3)MGF) cDNA (321 bp) was subcloned in pET32a(+) vector to construct a prokaryotic recombination expression plasmid. Trx/des(1-3)MGF fusion protein, existing in forms of solution, was expressed in transformed Escherichia coli strain BL21(DE3) by IPTG induction at 30 degres C. The supernatant of cell lysates was subjected to ion exchange chromatography and Ni2+ metal affinity chromatography, and the fusion protein was obtained with the purity over 95%. After the fusion protein was cleaved by enterokinase, Trx and des(1-3)MGF was isolated by reverse-phase HPLC. Through these procedures, des(1-3) MGF was obtained with the purity of 98%. The protein molecular mass was conformity to the theoretical value by SDS-PAGE and mass spectrometry analysis. The purified des(1-3)MGF was incubated with MC3T3-E1 for cell proliferation and migration assays. The results show that des(1-3)MGF exhibited more facilitative effects on proliferation and migration of MC3T3-E1 than that of des(1-3)IGF-1.
Cloning, Molecular ; DNA, Complementary ; genetics ; Escherichia coli ; genetics ; metabolism ; Humans ; Insulin-Like Growth Factor I ; Osteoblasts ; metabolism ; Protein Isoforms ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; STAT5 Transcription Factor ; biosynthesis ; genetics ; Tumor Suppressor Proteins ; biosynthesis ; genetics

OBJECTIVETo study the effects of hermap gene on kinases in erythroid signal transduction pathway and investigate the mechanism of hermap on erythroid differentiation.

METHODSThe K562 cells expressing hermap and hermap-siRNA respectively were established for up- and down-regulating the expression of hermap gene. These K562 cells were then induced by Ara-C to erythroid differentiation and analyzed at 0, 24, 48, 72 and 96 h, respectively, for cell morphology and biphenylamine staining positive cells, determination of CD235a, CD36, kinases p-STAT5, p-Akt, p-MAPK and p-c-JUN by FCM; and quantification of hermap gene and γ (Aγ,Gγ) globin gene by FQ-PCR.

RESULTSWith up-regulating hermap gene and inducing by Ara-C, K562 cells were changing to low ratio of nucleus to cytoplasm, cytoplasm colour from basophilic to pinkish or amethyst tinge, increase of number of biphenylamine positive cells and expression of CD235a, CD36, γ (Aγ,Gγ) globin gene, hermap gene and p-STAT5 from 0 to 96 h. At 0, 24, 48, 72 and 96 h of culture, the positive rates of p-STAT5 cells were detected of 0.46%, 4.54%, 20.01%, 23.65% and 33.08%, respectively. This results demonstrated that there was a positive correlation between expression of p-STAT5 and hermap gene expression (P < 0.05).

CONCLUSIONhermap gene can stimulate erythroid differentiation of Ara-C induced K562 cells mainly through JAK/STAT5 signal transduction pathway.

Cell Differentiation ; Erythrocyte Membrane ; Erythrocytes ; cytology ; Erythropoiesis ; Gene Expression ; Humans ; K562 Cells ; Receptors, Erythropoietin ; genetics ; STAT5 Transcription Factor ; metabolism ; Signal Transduction

Entamoeba histolytica is a tissue-invasive protozoan parasite causing dysentery in humans. During infection of colonic tissues, amoebic trophozoites are able to kill host cells via apoptosis or necrosis, both of which trigger IL-8-mediated acute inflammatory responses. However, the signaling pathways involved in host cell death induced by E. histolytica have not yet been fully defined. In this study, we examined whether calpain plays a role in the cleavage of pro-survival transcription factors during cell death of colonic epithelial cells, induced by live E. histolytica trophozoites. Incubation with amoebic trophozoites induced activation of m-calpain in a time- and dose-dependent manner. Moreover, incubation with amoebae resulted in marked degradation of STAT proteins (STAT3 and STAT5) and NF-kappaB (p65) in Caco-2 cells. However, IkappaB, an inhibitor of NF-kappaB, was not cleaved in Caco-2 cells following adherence of E. histolytica. Entamoeba-induced cleavage of STAT proteins and NF-kappaB was partially inhibited by pretreatment of cells with a cell-permeable calpain inhibitor, calpeptin. In contrast, E. histolytica did not induce cleavage of caspase-3 in Caco-2 cells. Furthermore, pretreatment of Caco-2 cells with a calpain inhibitor, calpeptin (but not the pan-caspase inhibitor, z-VAD-fmk) or m-calpain siRNA partially reduced Entamoeba-induced DNA fragmentation in Caco-2 cells. These results suggest that calpain plays an important role in E. histolytica-induced degradation of NF-kappaB and STATs in colonic epithelial cells, which ultimately accelerates cell death.
Caco-2 Cells ; Calcium-Binding Proteins ; Calpain/genetics/metabolism ; Caspase 3/genetics/metabolism ; Caspases ; *Cell Death ; Colon/cytology ; Entamoeba histolytica/*physiology ; Epithelial Cells/cytology/parasitology ; Humans ; I-kappa B Proteins/metabolism ; Intestinal Mucosa/cytology ; NF-kappa B/genetics/*metabolism ; RNA Interference ; RNA, Small Interfering ; STAT3 Transcription Factor/genetics/*metabolism ; STAT5 Transcription Factor/genetics/*metabolism ; Signal Transduction

OBJECTIVETo study the expression of signal transducer and activator of transcription 5b (STAT5b) mRNA and interleukin-4 (IL-4) mRNA in blood mononuclearcells in a rat model of asthma and the effect of montelukast (MK) and BCG-polysaccharide and nucleic acid injection (BCG-PSN) on STAT5b mRNA and IL-4 mRNA expression.

METHODSFifty-two male Sprague-Dawley rats (weight:140-200 g) were randomly divided into four groups: asthma, MK treated and BCG-PSN-treated and control groups. Rat model of asthma was prepared by ovalbumin (OVA) sensitization. The rats were sacrificed 24 hrs after the last sensitization. Blood eosinophils (EOS) were counted. Plasma contens of IL-4 and interferon-gamma (IFN-gamma) were measured using ELISA. Expression of STAT5b mRNA and IL-4 mRNA in blood mononuclearcells was detected with SYBR GREEN I fluorescent quantitation PCR method.

RESULTSBlood contents of STAT5b mRNA and IL-4 mRNA in the untreated asthma group were significantly higher than those in the other three groups (<0.01). Blood EOS count and plasma IL-4 contents in the untreated asthma group significantly increased, while plasma IFN-gamma contents significantly decreased compared with the other three groups (<0.01). There were no significant differences in the parameters measured among the MK-treated, the BCG-PSNjtreated and the control groups. STAT5b mRNA expression was positively correlated to IL-4 mRNA expression, IL-4 content and EOS count (r=0.730,0.650, 0.664, respectively; <0.01), but negatively correlated to IFN-gamma content (r=-0.798; <0.01).

CONCLUSIONSSTAT5b mRNA and IL-4 mRNA were strongly expressed in blood mononuclearcells in rats with asthma, and there was a positive correlation between them. MK and BCG-PSN had inhibitory effects on the expression of STAT5b mRNA and IL-4 mRNA, which might be contributed to suppression of airway inflammation in asthma.

Acetates ; pharmacology ; Animals ; Asthma ; blood ; drug therapy ; pathology ; BCG Vaccine ; pharmacology ; Interferon-gamma ; biosynthesis ; Interleukin-4 ; genetics ; Leukocytes, Mononuclear ; metabolism ; Male ; Quinolines ; pharmacology ; RNA, Messenger ; blood ; Rats ; Rats, Sprague-Dawley ; STAT5 Transcription Factor ; genetics

OBJECTIVETo investigate the effect of meisoindigo on bcr-abl signaling pathway and to explore the mechanism of meisoindigo inducing apoptosis in K562 cells.

METHODSApoptosis and mitochondria membrane potential (MMP) were evaluated by flow cytometry. In K562 cells, the expression level of Bcl-2 family members, cleaved caspase members, bcr-abl, STAT5 and CRKL were determined by Western blot and bcr-abl mRNA expression level was measured by RT-PCR before and after meisoindigo treatment. The DNA binding potential of STAT3 and STAT5 was checked by electronic mobility shift assay (EMSA).

RESULTSDown-regulation of total and phosphorylated bcr-abl protein level in K562 cells was observed when treated with 20 micromol/L meisoindigo, but its mRNA level was not changed. The expression level of phosphorylated STAT5 and CRKL was decreased and the DNA binding potential of STAT3 and STAT5 were inhibited in K562 cell after exposure to meisoindigo. Exposure to 5 - 20 micromol/L meisoindigo induced apoptosis accompanied with activating of caspase 3, 8, 9 and decreasing of MMP in K562 cells in a dose-dependent manner. The apoptosis was blocked by 50 micromol/L z-DEVD-fmk, z-IETD-cho, z-LETD-fmk, the specific inhibitors of caspase 3, 8, 9, respectively. No change in Bcl-2, Bax and Bid protein expression levels were observed before and after meisoindigo inducing apoptosis.

CONCLUSIONMeisoindigo can inhibit the proliferation of K562 cells by affecting the bcr-abl signaling transduction pathway. Meisoindigo induces K562 cell apoptosis through a novel caspase dependent pathway in addition to the contribution of mitochondria. The Bcl-2 family members are not involved in the apoptosis induction by meisoindigo in K562 cells.

Apoptosis ; drug effects ; Caspases ; metabolism ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Humans ; Indoles ; pharmacology ; K562 Cells ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; RNA, Messenger ; genetics ; STAT3 Transcription Factor ; metabolism ; STAT5 Transcription Factor ; metabolism ; Signal Transduction ; drug effects ; bcl-2-Associated X Protein ; metabolism

To study the effect of curcumin on signaling pathway of signal transducers and activators of transcription (STAT5) in primary newly-diagnosed chronic myelocytic leukemia (CML) cells, and to explore the clinical significance of curcumin in the treatment of primary CML cells, the cells were randomly divided into 3 groups: normal control group, CML cells group, and curcumin group; the cellular proliferation was assayed by MTT test; the expression of cellular STAT5 mRNA in CML cells was detected by RT-PCR; the activation of STAT5 in CML cell was detected by electrophoretic mobility shift assay (EMSA). The results showed that the cellular proliferation of curcumin group (OD value 0.640 +/- 0.073) was decreased, as compared with that of the CML cells group (OD value 0.856 +/- 0.083, P <0.01). The expression levels of STAT5 mRNA in CML cells group (integral ratio of OD 1.782 +/- 0.156) were significantly greater than that in the normal control group (integral ratio of OD 0.289 +/- 0.025, P <0.01). The expression of STAT5 mRNA in curcumin group (integral ratio of OD 1.398 +/- 0.126) was significantly decreased as compared with that in the CML cells group (P <0.01). The activation of STAT5 was significantly increased in CML cells group (gray value 5323.375 +/- 515.640) as compared with that in the normal control group (gray value 2943.000 +/- 273.377, P <0.01). The activation of STAT5 of curcumin group (gray value 4331.750 +/- 398.035) was significantly decreased as compared with that of CML cells group (P <0.01). It is concluded that the cellular proliferation and the expression of STAT5 mRNA are increased in the primary CML cells. The activation of STAT5 in primary CML cells is markedly enhanced. STAT5 signaling pathway may be involved in the proliferation of primary CML cells. Curcumin can inhibit the cellular proliferation and the expression of STAT5 mRNA, and down-regulate the activation of STAT5 in primary CML cells. Curcumin may be used in treatment of leukemia.
Adult ; Antineoplastic Agents ; pharmacology ; Cell Proliferation ; drug effects ; Curcumin ; pharmacology ; DNA ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Female ; Humans ; Leukemia, Myelogenous, Chronic, BCR-ABL Positive ; drug therapy ; metabolism ; pathology ; Male ; Milk Proteins ; genetics ; metabolism ; RNA, Messenger ; analysis ; STAT5 Transcription Factor ; Signal Transduction ; drug effects ; Trans-Activators ; genetics ; metabolism

OBJECTIVESTo investigate targeted blockage of BCR/ABL oncoprotein mediated cell transformation by STAT5 decoy oligodeoxynucleotide (ODN), its effect on the growth and proliferation inhibition of K562 cells and the related molecular mechanisms.

METHODSSTAT5 decoy ODN, designed and synthesized in vitro, was transfected into K562 cells by cationic lipid. The cell growth curve and colony formation assay were used to reflect the growth and proliferation capacity of K562 cells, RT-PCR to detect the expression of three genes downstream STAT5.

RESULTSConfocal microscopy demonstrated that STAT5 decoy ODN was successfully transfected into K562 cells (95.2% positive cells). STAT5 decoy ODN inhibited the growth of K562 cells (inhibition rate 77.7%) and their colony formation capacity (Decoy ODN treated group 8.3% vs control group 35.7%, P < 0.05) after the treatment with STAT5 decoy ODN, the expressions of c-myc, bcl-X(L), cyclin D1 mRNA were down-regulated by 15.4%, 30.8%, 29.1%, respectively in the K562 cells.

CONCLUSIONSSTAT5 decoy ODN inhibits the growth and proliferation of K562 cells. The mechanisms may be that decoy ODN blocks the transcriptional activation potent of STAT5 and down-regulates the expression of these tumor related genes downstream STAT5.

Cell Proliferation ; Cyclin D1 ; genetics ; Fusion Proteins, bcr-abl ; genetics ; metabolism ; Gene Expression ; Humans ; K562 Cells ; Liposomes ; Microscopy, Confocal ; Oligodeoxyribonucleotides, Antisense ; genetics ; Proto-Oncogene Proteins c-myc ; genetics ; Reverse Transcriptase Polymerase Chain Reaction ; STAT5 Transcription Factor ; genetics ; physiology ; Transfection ; bcl-X Protein ; genetics