The Study On The Role Of Eukaryotic Translation Initiation Factor 4E In Chronic Myelocytic Leukemia

Part I The expression of eukaryotic initiation factor 4E in human leukemia and lymphoma cell linesObjective To explore the expression of eukaryotic initiation factor 4E (EIF4E) in human leukemia and lymphoma cell lines.Methods Peripheral blood mononuclear cells (PBMCs) collected from healthy persons and 6 human leukemia and lymphoma cell lines including K562, Molt-4, HL60, Raji, Daudi and U937 were studied by using RT-PCR and Western blot.Results Expression of EIF4E can be detected in human leukemia and lymphoma cell lines and PBMCs. EIF4E was noticed to higher express in 6 human leukemia and lymphoma cell lines than in normal human PBMCs. The mean relative OD values of EIF4E mRNA were HL-60:(1.12 ±0.03), Molt-4:(3.20 ± 0.08), K562:(4.11 ±0.03), Raji:(4.92±0.03), Daudi:(3.73±0.02), U937:(2.06±0.03), PBMCs:(0.30±0.02); while the mean relative OD values of EIF4E protein were HL-60:(0.72±0.11), Molt-4:(1.30± 0.08), K562:(1.86±0.03), Raji:(3.42±0.23), Daudi:(3.02± 0.17), U937:(1.39±0.03), PBMCs:(0.39±0.06). There was a strong direct correlation between the level of EIF4E mRNA expression and the EIF4E protein expression. Expression of EIF4E in transcription and translation level showed the same tendency in 6 human leukemia cell lines and lymphoma cell lines.Conclusion Expression of EIF4E may play an important role in leukemia and lymphoma development and progression.Part II Construction and identification of eukaryotic expression plasmid of sense and antisense EIF4EObjective To construct the eukaryotic expression vector of sense and antisense eukaryotic initiation factor 4E(EIF4E) gene and to provide a tool for studying EIF4E gene function.Methods PCR primers were designed according to the coding sequence of human EIF4E gene, and Apa I and Kpn I recognition sequence and cutting sites were added to the 5'end of the sense and antisense primer respectively. The 760bp specific PCR fragment was obtained from the cDNA of chronic myelocytic leukemia cell line K562 by using RT-PCR, the purified PCR fragment was then inserted reversedly into the multiple cloning site of eukaryotic expression vector pEGFP-C1. The constructed recombinant plasmid was identified by PCR confirmation, Kpn I and Apa I double enzyme digestion and DNA sequencing.Results A 512bp specific DNA band was observed by PCR, Xba I and Kpn I double digestion produced a 760bp and a 4.7kb DNA fragments which represented the inserted DNA fragment and the vector respectively. The sequencing result confirmed that the sequence of inserted DNA fragment was correct.Conclusion The eukaryotic expression plasmid of sense and antisense EIF4E gene were constructed successfully by using gene-cloning technique. Part III The role of EIF4E on the proliferation of chronic myelocytic leukemiaObjective Translational control plays a major role in early development, different-tiation and the cell cycle in cancer. The translation initiation factor EIF4E is elevated in the most chronic myelocytic leukemia patients and chronic myelocytic leukemia cell line K562. In this part, the aim of study is to research the effects of EIF4E gene on proliferation of K562 cells.Methods The sense and antisense recombinant EIF4E plasmids were induced in K562 by electroporation. Selections were performed using G418 and flow cytometry, respectively. The integration of the transferred gene and its expression were determined by flow cytometry, fluorescence microscope, polymerase chain reaction and Western blot, respectively. The expression change of trasfected K562 cells was invested, including cell proliferation rate by MTT, cell cycle by flow cytometry and cyclinD1 expression by Immunocytochemistry and confocol.Results After a stable transfection, we obtained positive cell clones that induced sense EIF4E gene. RT-PCR and western-blot assay showed EIF4E gene had been integrated into K562 cells and expressed highly. Proliferation of pEIF4E-transfected K562 cells was increased compared to K562 and plasmid-transfected K562 cells. Conversely, freshly isolated antisense recombinant EIF4E plasmid-transfected K562 cells by FCM, the study revealed that blocking EIF-4E result in G1 cell cycle arrested and decreasing cyclinDl protein expression.Conclusion These data provide evidence that EIF-4E plays a pivotal role in the proliferation of chronic myelocytic leukemia cells.Part IV The role of EIF4E on the differentiation of chronic myelocytic leukemiaObjective Many researchers have reported that EIF4E has closed related not only to proliferation of tumor cells but also to cell differentiation of many kinds of tumors. In most cases, increasing expression of EIF4E in tumor cells may block the differentiation of cells. The loss of differentiation and the malignant proliferation are the common characteristics of leukemia cells. So we study the role of EIF4E on the differentiation of chronic myelocytic leukemia.Methods We used pEIF4E/K562, pEGFP-C1 /K562 and K562 cells as experimental objects. Three differentiation models of leukemia cell line K562 were established. HMBA(Hexamethylene Biscaetamide),TPA(0-Tetra decanoylphorbol 13-acetate)and Hu(Hydroxyurea)were selected to induce pEIF4E/K562, pEGFP-C1/K562 and K562 cells directional differentiation to monocytia series , megacaryocyte series and erythrocyte series determined by benzidine staining, CD41 and CD14, respectively.Results After treatment, pEGFP-C1/K562 and K562 cells tend into erythro-progen-itor cells, mononuclear cell and megacaryocyte, While the OD value of benzidine staining, the percentage of CD14 and CD41 were increased. However, pEIF4E/K562 cells hadn't obvious change after treatment, without obvious change of the OD value of benzidine staining and the percentage of CD14 and CD41.Conclusion The differentiation of K562 cells to monocytia series, megacaryocyte series and erythrocyte series induced by HMBA, TPA and Hu can be blocked by increased expression of EIF4E. EIF4E is significantly involved in differentiation of chronic myelocytic leukemia.