Roles Of Stat3α　and Stat3β In Mediating The Growth Arrest And Differentiation Of HL-60Cells Through The190CT3

Leukaemia is a heterogeneous clonal disorder of haemopoietic progenitor cellsand the most common malignant myeloid disorder in adults. Dysregulation ofhematopoietic cellular differentiation contributes to leukemogenesis. Despite thatvisual masses might not be observed throughout the whole course of leukemia, thisdisease has plenty shared characteristics with other cancer, such as uncontrolledproliferation, differential disorder, hindered apoptosis, etc. As the research on thebiological characters of leukemic cells,it is showed that acute leukemic cells can beinduced to differentiation under some certain conditions．So inducing differention ofleukemic cells is a new way in the treatment of leukemia.Looking for inductive drugswith high-effect and low-toxicity can become a new focus in the field of leukemictreatment．Many cytokines and growth factors are involved in regulating the proliferationand differentiation of hematopoietic cells. Leukemia inhibitory factor (LIF),as amember of the IL-6family of cytokines, involved in a variety of biological responses,including the immune response, inflammation, hematopoiesis, and oncogenesis byregulating cell growth, survival, and differentiation. LIF produces biological effectsvia binding to its receptor, which consists of a specific ligand-binding α-chain(gp190)and a shared signal-transducing β-chain(gp130). Cytoplasmic domain of gp190contains three homologous and functionally important motifs: Box1,2and3. Gp190binds to the src-homology2(SH2) domain of STAT3via the YXXQ consensussequences in the C-terminal. In the present study, transfection was performed tooverxpress190CT3(containing the structurally conserved triple YXXQ motifs ofLIFR α, termed190CT3) in HL-60cells. Our findings clearly demonstrate that theenrichment of the190CT3can inhibit proliferation in human promyeloid leukemiaHL-60cells, However, so far the mechanism of action of190CT3in the treatment ofHL-60cells has not been completely clarified.Therefore,in this work,we will conductan In-depth investigation in the molecular mechanisms during HL-60cellsdifferentiation and growth inhibit through the190CT3.In the first part, We Constructed and packaged gene over expression lentivimsparticle of190CT3and green fluorescent protein．Use lentivirus particle expressingGFP to infect HL-60acute myeloid leukemia (AML) cells,observe fluorescenceefficiency under the inverted microscope to get the best infecting condition and multiplicity of infection．Use target gene over-expression lentivirus particle of190CT3to infect HL-60cells,observe the morphological changes to get the bestlaboratory conditon．GFP positive clones were selected by fluorescence microscopy.Western blot was used to detect the protein level of190CT3in this positive clons. Theresult reveal that190CT3protein was high expressed in this positive clons.The cellclones with stably expression of190CT3and blank vectors were picked out. To studythe effects of190CT3on the proliferation and differentiation of HL-60acute myeloidleukemia (AML) cells. The proliferation of HL-60cells were detected by cellcounting method. The morphology and ultrastructure of HL-60cells were observe by1ight microscope and transmission electron microscope,measure cell viability bytrypan blue assay. Describing the growth curve. The morphology characteristic of celltreated by190CT3was investigated by phase contrast microsope and stained byWright's-Gimsa.Flow cytometry was used to examine the differentiation antigens on190CT3treated cells, the cell cycle distribution of190CT3treated cells.Thedifferentiation of HL-60cells were examined by immunocytochemical staining ofNBT reduction reaction and α-NAE method. We found that the growth of HL-60cellswas inhibited after treated by190CT3, but their Vitality was not affected. HL-60cellswere arrested at G0/G1phase after treated by190CT3. The morphological andultrastructure studies showed that differentiated HL-60cells tended to be more maturein treated group than in control group. In brief, HL-60cells treated by190CT3demonstrated smaller Volume, decreased nuclei/cytoplasm ratio, more cytoplasm,reduced and smaller of nucleolus, and increased cell organs. NBT positive cellscovered significantly more in treated group than in controI group． The α-NAEstaining was weakly positive,and its positive intensity could be inhibited by NaF intreated group．The expressions of mature granulocyte differentiation antigen of CD15,CD11b were significantly increased in treated group. All the above suggested that theproliferation of HL60cells was obviously inhibited and the differentiation wasinduced in HL60cells by the190CT3.However, so far the mechanism of action of190CT3in the treatment of HL-60cells has not been completely clarified．Therefore,we will conduct an in-depthinvestigation in the signal transduction of STAT3during HL-60cells differentiation．Signal Transducers and Activators of Transcription (STAT)family are potentialcytoplasmic transcription factors. STAT3plays important roles in embryodevelopment and cell proliferation．The activation of STAT3is instantaneous and strictly regulated in normal tissue．However, it was constitutively activated in manyhematopoietic and solid tumors.The activated STAT3could regulate the expression ofmany genes including negative factors for immune response and genes associatedwith proliferation,apoptosis and cell cycle. It could enhance proliferation,inhibitapoptosis,induce angiogenesis and invade immune surveillance．STAT3was thereforebeing considered as oncogene．Stat3is activated by a number of receptors expressedon the surface of myeloid lineage cells. Binding of ligand to receptor initiates aphosphorylation cascade, which includes phosphorylation of Stat3on tyrosine (Y)705.Tyrosine-phosphorylated Stat3proteins dimerize tail-to-tail and translocate to thenucleus, where they regulate transcription. Stat3exists in two isoforms. Thefull-length protein is termed Stat3α. A shorter, Stat3β variant is generated by alternatesplicing near the carboxy terminus, resulting in replacement of the55amino acidtransactivation domain with seven unique amino acids. This isoform retains the keyY705residue required for activation and induction of DNA binding capacity but doesnot contain a serine residue (S727), which is also required for maximal activation ofSTAT3.Whereas STAT3β was reported to exert a dominant-negative inhibition onSTAT3α-mediated transcriptional activity in certain cell models, STAT3β can beactivated independently and modulate the transcription of a distinct set of genes toSTAT3α. However, distinctive additional STAT3β properties have recently beendescribed indicating that STAT3β is not simply a truncated version of STAT3α withdominant-negative properties but a protein whose unique biological functionscontribute to the complex biology of STAT3. In fact, STAT3β over-expression caninhibit tumor growth.The ratio of S Stat3α:Stat3β protein varies within myeloidcells,and the ratio is reported to decrease with cell maturation and activation. Theselective expression of STAT3isoforms and their activation is a major determinant ofgranulocytic cell development. The balance between α and β isoforms may alter acell's capacity to differentiate, with a predominance of STAT3β activation favoringdifferentiation.The specific functions of Stat3remain unclear, in part, because twoisoforms, Stat3α and Stat3β, are expressed in myeloid cells. To elucidate themolecular mechanism of CT3anti-leukemia, This study will aim to observe whetherthe balance between Stat3α and β isoforms in CT3-treated HL-60cells involved in theproliferation inhibition and differentiation of HL-60cells. Western blotting was usedto detect the expression and activity of STAT3in HL-60cells.The transcripts ofSTAT3α and β genes were detected by RT-PCR in HL-60cells which treated with 190CT3or not. the subcellular distribution of STAT3α and STAT3β will be detectedwith indirect immunofluorescence technique. The results shew that STAT3βexpression and phosphorylation levels were significantly increased and performancegathered for the nuclear in HL-60cells which treated with190CT3. STAT3αexpression and phosphorylation levels were down-regulation. STAT3α and β mRNAlevels did not change significantly. Western blotting indicated that JAK1protein andphosphorylation levels did not change significantly. These results suggest that Theexogenous190CT3can influence the expression and phosphorylation status ofSTAT3α and β in HL-60cells. the up-regulation of the expression and Y705phosphorylation of STAT3β protein, the down-regulation of the S727phosphorylationof STAT3β protein. STAT3β dimerization into the nucleus, which can produce moreintense DNA binding activity than the STAT3α dimer, The Ser727residue is theMAPK phosphorylation sites. ERK1/2phosphorylation levels lower to promoteSTAT3α Ser727sites of phosphorylation levels decreased,thereby reducing theSTAT3α itself with the DNA binding capacity, to inhibit STAT3α downstream geneexpression. The process without the participation of JAK1protein.To know the change of P-STAT3and identify the interaction between STAT3and190CT3,immunoprecipitation and coimmunoprecipitation were used. Homogenatewas prepared from HL-60cells, from which the STAT3complexes with190CT3wereimmunoprecipitated by antibody against190CT3precoupled to Protein A Agarose.The immunoprecipitates were boiled at95℃and subjected to polyacrylamide gelelectrophoresis,followed by western-blotting analysis using antibodies,such asanti-190CT3, STAT3, and P-STAT3, to detect the homologous190CT3proteins ofSTAT3．This study indicates that190CT3coimmunoprecipitated with STAT3βand P-STAT3β.Together, two combinatorial190CT3complexes,190CT3/STAT3βand190CT3/P-STAT3βcould be formed. Results lead to the increase in the ratio of Stat3βto Stat3α. Overexpression of STAT3β dimerization into the nucleus and bind to DNA,and P705-STAT3α is stranded in the cytoplasm, thereby STAT3β inhibit STAT3αfunctionality, performance to promote their differentiation to inhibit the proliferationof HL-60cells.To further explore the role of the Stat3α and Stat3β in mediating the growtharrest and differentiation of HL-60cells through the190CT3. The expression ofSTAT3target genes related differentiation and anti-proliferation were detected by reverse transcription polymerase chain reaction (RT-PCR). The expression of PU.1,C/EBPε, cyclinE,CDK2mRNA was detected by reverse transcription polymerasechain reaction (RT-PCR). The expression of c-myc was measured by Western blotmethod. we found that, compared with the control, the expression of oncogene c-mycincreased, RT-PCR results demonstrated that the mRNA levels of cyclinE and CDK2were down regulated, the mRNA levels of PU.1and C/EBPε were up regulated aftertransfected with190CT3compared with control. The results show that theup-regulation of the expression of c-myc protein promote cell differentiation, thedown-regulation of mRNA of cyclinE and CDK2arrested cell cycle at G0/G1phase,PU.1and C/EBPε play an important role in the190CT3induced granulocyticdifferentiation.In conclusion, in this study,we found that190CT3can inhibit HL-60cellproliferation and promote their differentiation to granulocyte. The molecularmechanisms we could made were listed as follows①190CT3recruited the STAT3andinduced the up-regulation of the d Y705phosphorylation of STAT3β protein in HL-60cells. Results lead to the increase in the ratio of Stat3β to Stat3α. Overexpression ofSTAT3β dimerization into the nucleus and bind to DNA, and P705-STAT3α isstranded in the cytoplasm, thereby STAT3β inhibit STAT3α functionality, performanceto promote their differentiation to inhibit the proliferation of HL-60cells.②In thisprocess, The phosphorylation levels of ERK44/42protein down regulated,overexpression of P-ERK44/42induces STAT3ser727phosphorylation. Thereby,reducing the DNA binding activity of STAT3α, Oncogene expression were downregulated.