Interaction Of P65 And SMRT In The Signaling Transduction Of FLT3 And The Study Of Targeting Inhibition In Acute Myelogenous Leukemia Cells

Acute leukemia is a hematologic malignancy characterized by clonal proliferation of variably differentiated myeloid or lymphoid precursors in bone marrow. It represents the most common type of childhood cancer, accounting for about 30% of all tumours diagnosed in children. Within this population, acute myelogenous leukemia (AML) accounts for approximately 25%-30% of all childhood leukemia diagnosed. Over the past few decades, the prognosis of children with AML has greatly improved, because of advances in tailored chemotherapy, optimal recourse to hematopoietic stem cell transplantation and innovative supportive care. However, the 5-year disease free survival (DFS) still only approaches 45%, and the long-term survival remains low, highlighting the need for novel therapeutic approaches.As the progress of technology, cell biology and molecular biology in the past decades, researchers realize that the only way to improve the prognosis of AML is to uncover the pathogenesis of it. Now, the increased understanding of the biology of AML has led to the development of molecular targeted therapies, especially the over-expressed genes that play crucial roles in the pathogenesis of AML. FMS-like tyrosine kinase 3 (FLT3), a receptor tyrosine kinase (RTK) mainly expressed in normal human bone marrow selectively in CD34+ hematopoietic stem/progenitor cells, and associated with the proliferation and differentiation of normal hematopoietic stem cells and the survival of AML blasts, has been an increasing focus of research. It is aberrant activated in 70%-90% of pediatric AML, including overexpression of wild-type FLT3 and FLT3 mutations. Aberrant activation of FLT3 is closely associated with leukocytosis and poor prognosis in AML patients. Previous studies found that some FLT3 mutations were associated with overexpression of wild-type FLT3, indicating that regulating of wild-type FLT3 is more important.Now, studies of signaling pathway in the wild-type FLT3 mainly focuse on the PI3K-Akt and Ras-MAPK pathways, whereas in the ITD-type FLT3 also involved STAT5 pathway. But as to the roles of NF-κB, Silencing Medtiator for Retinoic Acid and Thyroid Hormone Receptor (SMRT) in FLT3 signaling transduction has not been determined, which may play key roles in the pathogenesis of AML. NF-κB family is a eucaryotic transcription factor playing an important role in regulating multiple biological functions including inflammation, immunity, cell proliferation, apoptosis and tumor migration through regulating transcription of cyclinDl, c-myc, and so on. SMRT has been identified as fundamental components in the regulation of eukaryotic gene transcription inhibition.In the previous study, the expression of FLT3, p65 and SMRT was identified in the THP-1 cell line, and down regulation of FLT3 could effectively induced the translocation of p65 from the nucleus to the cytoplasm and SMRT from the cytoplasm to the nucleus. But only inhibiting the expression of FLT3 could not completely block the NF-κB signaling pathway, we suppose that inhibiting the expression of FLT3 and p65 simultaneously maybe more effective. We also presume that FLT3/NF-κB p65/SMRT signaling pathway may play an important role in the pathogenesis of AML. So in this study, we will construct the recombinant expression vectors of p65 and SMRT, then use the co-immunoprecipitation and GST pull-down to detect the interaction of them, in order to confirm the FLT3/NF-κB p65/SMRT signaling pathway in THP-1 cell line. Then we use FLT3 small interfering RNA (siRNA) and p65 siRNA to specifically down-regulate the expression of FLT3 and p65 in AML cells and THP-1 xenograft tumor models. The levels of FLT3, p65, cyclin D1,IκBαand silencing mediator of retinoic and thyroid hormone receptors (SMRT) were measured by semi-quantitative Reverse Transcription Polymerase Chain Reaction (RT-PCR), flow cytometry (FCM) and Western blotting, respectively. The cell cycle was investigated by FCM, and apoptosis was analyzed by Annexin V-fluorescein isothiocyanate/propidium iodide staining and TUNEL method, respectively. Cell proliferation was assayed using a Cell Counting Kit-8. So we can evaluate the antitumor activity of them.Part I Interaction between NF-κB p65 and SMRTObjectiveConstruct the recombinant expression vectors of p65 and SMRT, after restriction enzyme digestion and DNA sequencing, the interaction between them was identified in THP-1 cells and vitro.Methods1 Human SMRT-C 645bp sequences and p65 coding regions were synthesized in vitro and cloned into pUC57 vector, after restriction enzyme digestion and DNA sequenced, then amplified by PCR. The PCR products were subcloned into eukaryotic expression vectors of pCMV-HA and pCMV-Myc, then these recombined plasmids were confirmed by restriction enzyme digestion and DNA sequencing, then co-transfected in THP-1 cell line. The interaction between p65 and SMRT was detected by co-immunoprecipitation.2 The recombinant prokaryotic expression vectors of pET-32a-p65 and pGEX-6p-1-SMRT were constructed in vitro, after restriction enzymes digestion and DNA sequenced, fusion proteins were induced expression by IPTG in E.coli BL21. The interaction between p65 and SMRT was measured by GST Pull-Down after purified. Results1 Restriction enzymes digestion and DNA sequencing showed that the recombinant eukaryotic expression vectors of pCMV-HA-p65 and pCMV-Myc-SMRT were constructed correctly. After immunoprecipitated by anti-HA and anti-Myc multi-antibodies, HA-p65 and Myc-SMRT fusion proteins were identified by Western blotting with anti-Myc and anti-HA antibodies.2 Restriction enzymes digestion and DNA sequencing showed that the recombinant prokaryotic expression vectors of pET-32a-p65 and pGEX-6p-1-SMRT were constructed correctly, and the fusion proteins and GST were successfully purified. After GST pull-down, the interaction between His-p65 and GST-SMRT fusion proteins was identified by Western blotting.Conclusions The recombinant expression vectors of p65 and SMRT were constructedsuccessfully. The interaction between p65 and SMRT were identified byco-immunoprecipitation and GST pull-down.Part II Researches on the the blockade strategies of FLT3 signaling pathway in acute myelogenous leukemia cells in vitro and in vivoObjectiveInhibit the expression of FLT3 and p65 alone or simultaneously, detect the effect of them on the downstream signaling molecules and proliferation and apoptosis in THP-1 cells and THP-1 xenograft tumor models, in order to find more effective therapeutic strategies of AML.Methods1 THP-1 cells were plated at densities of 4,000 cells/well in 100μl culture medium in 96-well plates, then the FLT3 inhibitor (FLT3-I) was added to the plates at different contentions, cell viabilities were tested by CCK-8 and inhibitory concentration 50%(IC50) were calculated. 2 After THP-1 cells treated with FLT3 siRNA or FLT3-I, the expression levels of FLT3 mRNA and protein were measured by semi-quantitative RT-PCR and FCM, respectively, and the expression levels p65, cyclin D1, IκBαand SMRT were measured RT-PCR and Western blotting, respectively. The cell cycle was investigated by FCM, and apoptosis was analyzed by Annexin V-fluorescein isothiocyanate/propidium iodide staining. Cell proliferation was assayed using Cell Counting Kit-8.3 After THP-1 cells treated with p65 siRNA or PN, the expression levels of FLT3 mRNA and protein were measured by semi-quantitative RT-PCR and FCM, respectively, and the expression levels p65, cyclin D1, IκBαand SMRT were measured RT-PCR and Western blotting, respectively. The cell cycle was investigated by FCM, and apoptosis was analyzed by Annexin V-fluorescein isothiocyanate/propidium iodide staining. Cell proliferation was assayed using Cell Counting Kit-8.4 After THP-1 cells treated with FLT3 siRNA, p65 siRNA or FLT3 siRNA and p65 siRNA simultaneously, the expression levels of FLT3 mRNA and protein were measured by semi-quantitative RT-PCR and FCM, respectively, and the expression levels p65, cyclin D1,IκBαand SMRT were measured RT-PCR and Western blotting, respectively. The cell apoptosis was analyzed by Annexin V-fluorescein isothiocyanate/propidium iodide staining. Cell proliferation was assayed using Cell Counting Kit-8.5 Logarithmically growing THP-1 cells were harvested and resuspended in PBS, and 1×107 cells in 0.2 ml were injected subcutaneously into the right flank of the BALB/c-nude mice. When the tumor volume reached 100mm3, the tumor-bearing nude mice were divided into 4 groups randomly, with five mice in each group. Intratumoral injections of the FLT3 siRNA, p65siRNA or the same volume PBS were initiated in mice of each group, and the treatment was repeated every 3 or 2 days for a total of seven times. Tumor size was measured using calipers at the indicated times. The expression levels of FLT3, p65, cyclin D1, IκBαand SMRT in tumor tissues were detected by immunohistochemistry, RT-PCR, and Western blotting, respectively, and the cell apoptosis was measured by TUNEL.Results1 Growth inhibition was observed in THP-1 cells treated with FLT3-I in a concentration-dependant manner. The IC50 concentration of FLT3-I was at 100nM-150nM.2 After THP-1 cells treated with FLT3 siRNA or FLT3-I, the expression levels of FLT3 and cyclin D1 were strongly inhibited (P<0.05). The expression levels of p65, IκBαand SMRT mRNA, protein in the whole cell extracts weren't affected (P>0.05), but p65 protein in the nuclear extracts decreased and SMRT protein in the nuclear extracts decreased (P<0.05). The cell cycle was blocked from G0/G1 phase to S phase (P< 0.05), and early apoptosis rate increased dramatically (P<0.05). Cell proliferation was strongly inhibited, and the growth curves become low, flat which lacked the typical character of exponential growth. All the results in FLT3 siRNA treated cells were more obvious than those in FLT3-I treated cells (P<0.05), but there is no significant difference between the groups of control and control siRNA (P>0.05)3 After THP-1 cells treated with p65 siRNA or PN, the expression levels of p65, FLT3 and cyclin D1 decreased significantly (P<0.05), but the expression levels of IκBα, and SMRT increased obviously (P<0.05). The cell cycle was blocked from G0/G1 phase to S phase (P<0.05), and early apoptosis rate increased dramatically (P< 0.05). Cell proliferation was strongly inhibited, and the growth curves become low, flat which lacked the typical character of exponential growth. All the results in p65 siRNA treated cells were more obvious than those in PN treated cells (P<0.05), but there is no significant difference between the groups of control and control siRNA (P>0.05)4 After THP-1 cells treated with FLT3 siRNA, p65 siRNA or FLT3 siRNA and p65 siRNA simultaneously, the expression levels of FLT3, p65 and cyclin D1 decreased significantly (P<0.05), but the expression levels of IκBαand SMRT increased dramatically (P<0.05). The cell early apoptosis rate increased dramatically (P<0.05). Cell proliferation was strongly inhibited, and the growth curves become low, flat which lacked the typical character of exponential growth. All the results in FLT3 siRNA and p65 siRNA simultaneously treated cells were more obvious than those in FLT3 siRNA or p65 siRNA treated cells alone (P< 0.05), but there is no significant difference between the groups of control and control siRNA (P>0.05)5 THP-1 xenograft tumor models were constructed successfully, and treatments with FLT3 siRNA, p65 siRNA or FLT3 siRNA and p65 siRNA all led to tumor growth inhibition in BALB/c-nude mice bearing THP-1 xenograft tumor. RT-PCR, immunohistochemistry and Western blotting results show that the expression levels of FLT3, p65 and cyclin D1 decreased significantly (P<0.05), but the expression levels of IκBαand SMRT increased dramatically in tumor tissues (P<0.05), especially in the FLT3 siRNA and p65 siRNA simultaneously group (P<0.05). TUNEL results suggest that more apoptotic cells in tumor tissues were observed in all the treated groups (P<0.05), especially in the FLT3 siRNA and p65 siRNA simultaneously group (P<0.05). But all of results are less significant in vivo than those in vitro.Conclusion1 Both FLT3 and p65 inhibition can effectively block the FLT3 signaling pathway, and inhibit the proliferation and induce the apoptosis of THP-1 cells.2 Comparing effect of FLT3/p65 siRNA and their targeting inhibitor FLT3-I/PN results indicate that antileukemic efficacy of FLT3/p65 siRNA is more strong, and they may be worthy of studied for clinical usage.3 Inhibiting the expression of FLT3 and p65 together in FLT3 signaling pathway may be an effective therapy strategy for AML.