Influence Of Biological Character Of Jurkat After Overexpression Of C/EBPα And PU.1

Background and objective:Leukemia is a group of heterogeneous hematopoietic system malignant tumor which results from differentiation block in differentiation process, apoptosis disorders and malignant proliferation of hematopoietic stem or progenitor cells. More and more study considered that most acute leukemias are the consequence of the propose of second-hit hypothesis. Most of acute leukemia is resulted from mutations of different kinds of genes. Class I mutations give hematopoietic stem cells with proliferation and survival advantage, it sustain activates tyrosine kinase or its downstream molecules; Class II mutations involve transcription factors which play an important role in regulation of normal hematopoietic differentiation. These two mutations all give leukemia cells proliferation and survival advantages as well as block of differentiation, then causes acute leukemia. Transcription factors plays a crucial role in development of leukemia. In most of the transcription factors, the CCATT/enhancer binding protein alpha(C/EBPa) and CCATT/enhancer binding protein(CEBPA) gene promote hematopoietic stem/progenitor cells differentiate into granulates and inhibits proliferation of cells, C/EBPa gene is a key transcription factor in hematopoietic system. C/EBPa plays highly adjustment in proferlition、 apoptosis、cell cycle and cell transfection. C/EBPa plays as proto-oncogene in acute myeloid leukemia, while tumor suppressor gene in acute lymphoblastic leukemia. Mutations of CEBPA and abnormal control of transcription、translation and protein level after translation may lead to abnormal of C/EBPa protein.Transcription factor PU.1 is one member of Ets family. Kueh considers that mature of B cells dependent on low expression of PU.1, but formation of macrophage dependent on highly expression of PU.1. Then PU.1 and Then PU.1 and C/EBPa all play important role in formation of hematopoietic stern cells and differentiation of granulates or monocytes.Transcription to grain cells or mononuclear cells dependents on the ratio between expression of PU.1 and C/EBPa. Recent reports have demonstrated that overexpression of C/EBPa and PU.1 in 3T3 cell can force it transform into macrophages with functions of chemotactic. Overexpression of C/EBPa can increase the ability of oxidative stress,then inhibit formation of fat, PU.1 also plays an important role in adipose cell lineage. Overexpression of C/EBPa and PU.1 in acute B lymphocytic leukemia also can transform it into macrophages with functions of chemotactic and phagocytosis. In the clinical work, conversion between ALL and AML or conversion from SAA to AML and MDS occasionally occurred, apart from the influence of radiation and chemotherapy drugs,Whether interaction with transcription factor C/EBPa and PU.1 can partly explain this factor is still unknown.In this paper, we establish new jurkat cell lines with stable expression of C/EBPa or and PU.1 by retrovirus vectors in order to explore the influence of C/EBPa and PU.1 gene. We inject the cells into the immunodeficiency mouse through the tail vein, then observe the changes of biological behaviour of mice, survival times and leukemia in organs. We study whether acute lymphoblastic leukemia cell line-jurkat can transform into macrophages through the overexpression of transcription factors and reduce the carcinogenicity and prolong survival of the mouse. At the same time, this can partly explain the mechanism of translation between ALL and AML in clinical work and let us better understand leukemia. We use plasmids which is correct to package vectors through 293T cells, correcting the virus supernatant and inject the jurkat cells to establish cell line which can express C/EBPa and PU.1 stably, QRT-PCR extraim the expression of C/EBPa and PU.1. Detection of cell proliferation、 apoptosis and changes of surface antigen were done to know the influence of C/EBPa and PU.1. Through the help of cytokine (GM-CSF、MCSF、IL-3、FLT-3L),we observe whether jurkat can transform into macrophages with chemotaxis consuming yeast. RT-PCR ananlysis was done to detect the expression of myeloid related gene. BCIP/NBT kit was used to detect alkaline phosphatase of macrophage. Cells which overexpress C/EBPa and PU.1 were injected into immunodeficiency mice, biolobical beheviour and survival time were observed. Leukemia violations were extraimed through flow cytometry, the transcription of cells were observed by cytology dyeing, chemical dyeing and immunohistochemical to further verify the influence of C/EBPa and PU.1 on acute lymphoblastic leukemia cell line-jurkat.Materials and methods:1. Expand the retrovirus vectors which has been built and extracted the plasmid, then confirm it by enzyme. The retrovirus vectors pwpxld-gfp, pwpxld-PU.1-gfp, pwpxld-C/EBPa-PU.1-gfp were transformed into DH5a, then coated in solid medium containing ampicillin LB 37 degrees inversion training for the night. The next day, pick a larger single colony and put it into 2 ml contains ampicillin LB liquid medium 37 degrees,210r/s cultivating 12-16h. Extracted the plasmid and confirm it by enzyme again, in order to make it correct and no matution.2、Retrovirus packaging, jurkat infection and indentification of gene. Using three plasmid packaging system to package retrovirus by 293T cells, then observe gfp expression of 293T cells by fluorescence microscope and collect the virus supernatant after 24h,48h,72h to infect jurkat cells. Observe the gfp expression of jurkat cells after 48h by fluorescence microscope and sorting gfp+ cells after 72h.Extract RNA of jurkat after sorting and identify the amount of expression of transcription factors by QRT-PCR.3、To observe the influences of biology activity of jurkat by overexpression of C/EBPa and PU.1:To study the influences of biology activity by detection cells proliferation, apoptosis, changes of cell surface marker and cellular morphology.4、To observe the influences of biology activity of jurkat by overexpression of C/EBPa and PU.1 through help of myeloid cell factors. Cell surface markers were observed by flow cytometry, cell morphology and cell staining,cytochemical staining were observed after sorting cells jurkat-gfp, jurkat-PU.1-gfp, jurkat-C/EBPa-gfp, jurkat-C/EBPa-PU.1-gfp by coculture with myeloid cell factors(100ng/ul).5、Switzerland’s dyeing yeast, and observe the ability of chemotaxis and consuming of macrophage. Coated yeast in YPD 37 degrees inversion training for the night. The next day, pick a larger single colony and put it into YPD liquid medium 37 degrees and cultivating 12-16h, then heating yeast to inactivate. To centrifugal it and stain with wright stain for night. The next day, centrifugal yeast, and wash it with PBS, adjust the concentration of 1*10^6 to 1*10^7. Plate the macrophages in the six well plates and add 400ul yeast. After coculture with 12h, then observe the ability of chemotaxis and consuming of macrophage by fluorescence microscope. Throw the media and wash 3 times with PBS, observe the same vision.6、To study whether jurkat can transform into myeloid cells in vivo by stable expression of C/EBPa and PU.1. Sorting gfp+cells which stably express C/EBPa or/and PU.1, then inject them into NSI mouse by tail vein and observe the changes of biological behavior (gait, spirit, hair, eyes and appetite) and the disease,survival time as well as invasion of leukemia cells in peripheral blood, liver, spleen and bone marrow. Transcription of cells was observed by wright stain, peroxidase staining and immunohistochemical.7、Statistical analysis:The statistical analysis is performed by SPSS20.0, significant difference was defined by P<0.05.Results:1、Expand the retrovirus vectors which has been built and extracted the plasmid then confirm it by enzyme,all the plasmid are correct and no mutation after compare with plasmid profiles.2、Using three plasmid packaging system to transform plasmids (pwpxld-gfp, pwpxld-PU.1-gfp, pwpxld-C/EBPa-PU.1-gfp) into 293T cells, expression of gfp can be seen after 24h. Gfp positive cells in jurkat-gfp is almost 100%, gfp positive cells in jurkat-PU.l-gfp is 80%, gfp positive cells in jurkat-C/EBPa-gfp is 60%, gfp positive cells in jurkat-C/EBPa-pu.l-gfp is 20% after infection. Sorting gfp+ cells by flow cytometry, then extract RNA and confirm the stable expression of transcription factors by Q-RT PCR. The expression of C/EBPa and PU.1 in jurkat-C/EBPa-pu.1-gfp is 4 times and 1.5 times than jurkat-gfp respectively.The expression of PU.1 in jurkat-PU.1-gfp is 3.5 times than jurkat-gfp.The expression of PU.1 is 6 times than C/EBPa in jurkat-C/EBPa-pu.l-gfp.3、Results of flow-cytometry:CD3 positive cells in gfp-jurkat is 90%, almost no expression of CDllb, CD3 positive cells in jurkat-pu.1-gfp is 40%, CD11b positive cells is highly as 20%, CD3 positive cells in jurkat-C/EBPa-gfp is 60%, almost no expression of CD11b, CD3 positive cells in jurkat-C/EBPa-pu.l-gfp is 20%,CD11b positive cells is as highly as 10% after sorting gfp positive cells. There is significant difference of expression of CD3 and CD11b between four groups(P<0.001,P=0.005).4、After adding cytokine (GM-CSF、M-CSF、IL-3、FLT-3L,100ng/ml) to the cells,jurkat-gfp is still suspension cells,while jurkat-PU.1-gfp, jurkat-C/EBPa-gfp,jurkat-C/EBPa-PU.1-gfp all transform into adherent cells. Positive rate of CD3 in jurkat-PU.1-gfp is 30%, positive rate of CD11b is 4%, positive rate of CD3 is 50% in jurkat-C/EBPa-gfp, positive rate of CD11b is 1%. positive rate of CD3 is 20% in jurkat-C/EBPa-PU.l-gfp, positive rate of CDllb is 2%.After adding cytokine(GM-CSF and M-CSF or IL-3 and FLT-3L,100ng/ml),they are still suspension cells and the positive rate of CD3 and CD11b has no significant difference.5、Results of proliferation exprement by resazurin:Each cell has trend to proliferation within 72h after cultivation, significant difference was observed in proliferation at different times(P<0.001), At the time of 0h、24h、48h、72h, significant difference was observed in proliferation between jurkat-gfp、 jurkat-PU.1-gfp、jurkat-C/EBPa-gfp、jurkat-C/EBPa-PU.1-gfp cell. OD value in proliferation of jurkat-C/EBPa-gfp group is the lowest. It was prove to be true that C/EBPa inhibit the proliferation of cells.6、Results of apoptosis:Apoptosis of jurkat-gfp and jurkat-PU.1-gfp is low, while apoptosis of jurkat-C/EBPa-gfp is significantly higher after one day of infection, apoptosis rate increased after 7 days of infection in jurkat-C/EBPa-PU.l-gfp. Ther is a significant difference of apoptosis rate in different groups. Overexpression of C/EBPa significantly increase apoptosis rate of cells, while PU.1 has no influence to apoptosis, apoptosis rate drops in jurkat-C/EBPa-PU.l-gfp then jurkat-C/EBPa-gfp group.7、Results of the cell morphology:Jurkat cells have abundant cytoplasm with irregular nodular more bumps and clear nucleoli.Jurkat-gfp has similar cell morphology as jurkat. Jurkat cells with overexpression of C/EBPa and PU.1 has inner and outside cytoplasm with a few red granules and misty nucleoli. Adherent cells have abundant and rough cytoplasm, large cell body, misty and multiple nucleoli. Results of POX staining:All the cells are POX negative.8、Results of the chemotaxis and phagocytosis of macrophages:After co-culture 12h with yeast, macrophages have the ability of chemotaxis and phagocytosis. Yeast which has not yet been swallowed can be washed by PBS. Analysis of BCIP/NBT kit:Within the cell cytoplasm are red of jurkat-gfp, Blue particles precipitate in cytoplasm are observed in macrophage of jurkat-C/EBPa-gfp、 jurkat-PU.1-gfp、jurkat-C/EBPa-PU.1-gfp group. It is proved the presence of alkaline phosphatase in macrophage.9、Results of myeloid related genes:jurkat-PU.l-gfp and jurkat-C/EBPa-PU.1-gfp all express CD64 and Mar-1,jurkat-C/EBPa-PU.l-gfp express CD14,expression of CD 16 has no significantly increased. Significantly decrease were observed of BCL-11B and Gata-3. Macrophages have significantly decrease of BCL-11B.10、Results of NSI mouse:NSI mice all develop leukemia with depression, hair disorder, light weight, paralyzed hind legs and depraved appetite in 1-2 months after injection with cells.They The mean survival time of jurkat-gfp and jurkat-PU.1-gfp is shortly compared to the jurkat- C/EBPa-gfp, it showed significant difference. CD3 positive rate of peripheral blood, liver, spleen and BM of jurkat-gfp group is about 90%, CD11b positive rate is about 0%. CD3 positive rate of peripheral blood, liver, spleen and BM of jurkat-PU.1-gfp and jurkat-C/EBPa-gfp group is about 70%, CD11b positive rate has significantly increase.Conclusion:There is a drop of CD3 expression and increase of CD11b expression in acute lymphoblastic leukemia cell line jurkat after overexpression of C/EBPa and PU.1. Expression of CD3 is lowest in jurkat-C/EBPa-PU.1-gfp, while expression of CD11b is highest in jurkat-PU.1-gfp. Apoptosis increase and proliferation inhibit after overexpression of C/EBPa. After adding GM-CSF、M-CSF、IL-3 and FLT-3L, cells all transform into macrophages with function chemotaxis and devour yeast. Macrophage is the presence of alkaline phosphatase. Jurkat-C/EBPa-gfp and jurkat-C/EBPa-PU.1 can significantly reduce carcinogenicity of jurkat cells and prolong the survival time of mice.