A Cytoplasmic Fragment Of Gp190 Induces The Differentiation Of Leukemia Cell Differentiations By Activating The STAT3 Pathway

Background and objectiveLIF induces terminal differentiation of myeloid leukemia cells, also modulates growth/differentiation of many other types of target cells. Other cellular functions influenced by this molecule include: embryogenesis, inflammation, neural development. LIF receptor is a heterodimer consisting of a low affinity binding protein gp190 and a signal transducing unit gp130. gp190 belongs to the hematopoietin receptor family, and is characterized by a cytokine receptor homology (CRH) domain. Cytoplasmic domain of gp190 contains three homologous and functionally important motifs: Box 1, 2 and 3. Gp190 binds to the src-homology 2 (SH2) domain of STAT3 via the YXXQ consensus sequences in the C-terminal. In addition to a membrane form, gp190 also exists in a soluble form that lacks transmembrane domain. Previous reports indicated that certain motif of the cytoplasmic gp190 could activate cell proliferation and differentiation. In the current study, transfecting leukemia cells with vectors containing a sequence encoding the C-terminal fragment (CTF) of gp190 (containing BOX 3 or BOX 2+3 ) inhibited cell proliferation, and induced differentiation. Such effects were significantly attenuated by a mutation in CTF (containing Box 3), as well as by inhibiting STAT3 using a selective inhibitor. STAT3 phosphorylation and nuclear translocation were significantly enhanced in cells expressing the CTF (containg Box 3). The cells expressing the CTF had a slower rate of proliferation upon transplantation into nude mice Analysis of cell morphology and markers for maturity indicated that these cells have higher degree of differentiation. We also found that cytoplasmic region of gp190 could be cleaved byγ-secretase upon LIF stimulation. Taken together, these findings suggest that a gp190 CTF may exist in leukemia cells, and modulate the property of these cells via the STAT3 pathway. It is beneficial to explore the application value about the single motif of cytoplasmic receptor, induce leukemia cells to differentiation.PartⅠRecombination of human dissociated LIFR (gp190) cytoplasmic receptor in HL-60 cells and identification its expression Methods: (1) Enzyme BamHⅠand XbaⅠcleaving the recombined plasmid pcDNA3.0-190CT2+3,pcDNA3.0-190CT3, and checking the sequences. Mutant of 190CT3: mutagenic oligonucleotide primers were complementary to the gene sequence in the region to be mutated, except Y-to-F conversion at three YXXQ sequences. The mutagenic oligonucleotide was then allowed to prime new DNA synthesis to create a complementary full-length sequence containing the desired mutation. Desired sequences were amplified using PCR and subcloned into pcDNA3.0. (2) Transfected the eukaryotic expression plasmid pcDNA3.0-190CT2+3, pcDNA3.0-190CT3, pcDNA3.0-MUT into leukemia (HL-60/K562) cells with liposome FuGENE-6. Two control group were setup which were vector pcDNA3.0 tansfected and wild-type HL-60/K562 cells. In the culture processes, we selected the clones with different G-418 concentration. The transfected cells were survival and expanding in presence of neo-gene. We detected the protein expression with western blotting and RT-PCR to identify the leukemia cell lines with stable protein expression. (3) Hematoxylin staining showed the morphous of nuclei. Describing the growth curve. Staphylococcus aureus were added into HL-60 cells. Cell smear was stained using a Wright method, and examined under a light microscope. Percentage of the HL-60 cells containing staphylococcus was used as an index for phagocytosis. The levels of CD15/CD14/CD11b were assayed by flow cytometer. Results: (1)After BamHⅠand XbaⅠcleaving, we observed electrophoretic irradiance strip under the UV light. Sequence detection was consistent with the LIFR sequence in Genbank. After PCR reaction, there is one 300-400bp DNA strip. Blast the MUT sequence and the original LIFR sequence, we found that the original TAT were exactly changed to TTC. The MUT4 colony was expressed the higher MUT by RT-PCR. (2) After subsequent selection by G418, G418-resistent colonies were isolated, namely ,the pcDNA3.0-190CT2+3, pcDNA3.0-190CT3, pcDNA3.0-MUT and pcDNA3.0 leukemia cell lines. What western blotting and RT-PCR analyzed demonstrated that the 190CT had expressed in leukemia cells stably. Cells expressing the gp190 C-terminal fragment were significantly larger than cells transfected with pcDNA3.0. The nuclei of these cells were more leafy than the wild-type as well as the pcDNA3.0 controls. The rate of proliferation was significantly decreased by both 190CT3 and 190CT2+3. Expression of the mature granulocyte marker CD15/CD14/CD11b and the phagocytosis index were significantly increased. Conclusion: 190CT3 and 190CT2+3 could promote the differentiation and inhibit the proliferation of leukemia cells. PartⅡ: Signaling pathway investigation for human dissociated LIFR (gp190) cytoplasmic receptorMethod: (1)Western blotting analyzed the levels of related signal molecular as P -STAT3(T705), P44/42-MAPK. Immunofluorescence detected the expression of STAT3 and P -STAT3(T705). (2) pEGFP-PIAS3: Total RNA was extracted and reversely transcribed using RT-PCR with the designed primers. PIAS3 coding sequences were then subcloned into the eukaryotic expression vector pEGFP-N1. All constructs were verified with gene sequencing. Western blotting analyzed STAT3 dimer after PIAS3 transfection. (3) After human LIF (20 ng/ml) and theγ-secretase inhibitor dropped in HL-60 cells, detected the expression of gp190-CTF and PS1-NTF. Resultes: (1) Transfection with pcDNA3.0 containing the CTF of the gp190 increased the level of phosphorylated STAT3. Immunofluorescence microscopy revealed that P-STAT3 was significantly increased in both the cytoplasm and nuclei. Cells expressing either 190CT3 or 190CT2+3 had significantly increased level of P–STAT3 in comparison to the wild-type or pcDNA3.0 controls, and decreased leval of P-MAPK. In comparison to cells expressing the gp190 CTF, cell expressing mutant C-terminal had higher rate of proliferatio, lower degree of differentiation. Cells expressing mutant C-terminal gp190 had lower P-STAT3 level and lower P-MAPK. (2) Co-transfection with PIAS3 significantly reduced the level of dimeric STAT3. (3) LIF significantly increased the level of PS1-NTF as well as the amount of the 26-kD cytoplasmic gp190. Theγ-secretase inhibitor significantly decreased the 26-kD cytoplasmic gp190 fragment and PS1-NTF and increased PS1-FL. Conclusion: The gp190 C–terminal fragment could enhance STAT3 phosphorylation and nuclear translocation. After YXXQ mutation or PIAS3 transfection ,activated STAT3 decreased. Gp190 could be cleaved byγ–secretase.PartⅢBALB/c nude mouse experimentMethod: (1) Animal models: Male BALB/c nude mice (4 weeks of age) were kept under clean condition. At the first time, Mice (7 in each group) received 2×105 HL-60 cells transfected with pcDNA3.0-190CT3 or pcDNA3.0-Mutant in 200μl via the tail vein. A PBS control, a HL-60 wild-type control, and a pcDNA3.0 control were also included. After 30 days, mice were injected 2×106 cells at the second time. (2) Peripheric blood was collected at days 7, 14, 21 and 28. Granulocytes and lymphoid cells were counted using Wright staining under a microscope. Bone marrow was obtained from the femur after sacrifice at days 28. Flow cytometric analysis the CD15 and HLA-1 expression in bone marrow cells. After 60d, the mice were killed and organs were harvest and were in the processing of H.E. staining. Resultes: (1) Lymphoid cell count in the mice receiving HL-60 cells expressing 190CT3 was significantly lower than that in mice receiving the widetype or pcDNA3.0 control. In contrast, granulocyte count was considerably higher in mice receiving cells expressing 190CT3. In mice receiving HL-60 cells expressing mutant 190CT3, count for lymphoid cells and granulocytes was somewhere between that in the wild-type and cells expressing the 190CT3. (2) Cytometric analysis revealed that CD15-positive cells in the bone marrow were significantly higher in mice receiving HL-60 cells expressing the 190CT3 on day 28. CD15-positive cell count in the mice receiving the mutant was higher than the wild-type control, but lower than the 190CT3 group. HLA-1 positive cells in the bone marrow was also significantly higher in mice receiving HL-60 cells expressing the 190CT3 on day 28. (3) Lung pathology: the necrosis configuration was showed in wild-type, pcDNA3.0 and MUT- HL60 transplanted mice, but not in the 190CT3-HL60 mice. Conclusion: gp190 C-terminal fragment trended to differentiation and had low infestation and metastasis in vivo.ConclusionsIn conclusion, this study provided strong, albeit not conclusive evidence that the C-terminal of the gp190 could be released via enzymatic cleavage byγ-secretase. The released fragment could inhibit the proliferation of leukemia cells, induce their differentiation into granulocytes by enhancing STAT3 phosphorylation and nuclear translocation. Results from our in vivo experiments suggest that targeting the gp190 C-terminal and related pathways may be a promising novel approach in the treatment of leukemia. Effects of 190CT3 on the proliferation of HL-60 cells could be partially blocked by eitherboth mutation of the YXXQ sequence in the 190CT3 and or PIAS3 transfection partially blocked the effects of gp190 Box 3 on the proliferation of HL-60 cells. In vivo, 190CT3 in HL-60 had a low malignancy. Gp190 C-terminal fragment prevented tumor growth in animal models of human acute myeloid leukemia.