The Initial Research on Relationship of Transcription Factor C/EBPα and Raji Cell Line

Abstract

Abstract 4650 Introduction:Leukemia is the common term for a diverse group of malignancies, defined by accumulation of abnormal hematopoietic progenitor cells, which fail to undergo terminal differentiation. The CCATT/enhancer binding protein alpha, C/EBPα, is a key transcription factor involved in normal hematopoietic system and leukemia. C/EBPα acts as an inhibitor of growth and apoptosis, and induces myeloid progenitors to differentiate. It is also reported that enforced expression of C/EBPα in B cells leads to their rapid and efficient reprogramming into macrophages. According to the phenomenon of cell-type transformation in clinical practice, we explore the role of C/EBPα in transformation of Raji cell line. Materials and MethodsWe detected expression of C/EBPα gene in Raji cell line by RT-PCR.The retroviral vector was inducted into the packaging cell Phoenix 293 by Lipofectamine™ 2000 (Invitrogen). Retroviruses encoding full-lengh cDNAs of murine C/EBPα were cloned by PCR into the BglII/XhoI sites of the pMIG retrovirus vector by creating a BglII site on the 5 end and a XhoI site at the 3 end. The supernatant of retrovirus was used to transfect Raji cells. To study the biological differences in C/EBPα positive-Raji cells, we used multiple methods such as cytomorphology, flow cytometry, MTT and RT-PCR. Results1. Raji, 6T-CEM, Molt-4 and K562 cell line were negative for C/EBPα gene.However, HL60 and NB4 cells were positive for C/EBPα gene. Sequencing results showed that there is no genetic mutation.2. The retrovirus pMIG and pMIG-C/EBPα were used to transfect Raji cell.The 72h transfection rates were 28.5% and 8.4% respectively. We used FACS to sort the GFP+ cells and amplification these cells. The GFP+ cells were taken account above 80%. By RT-PCR, we confirmed that C/EBPα gene was stably expressed.3. Cytomorphology showed that the nuclein of Raji+pMIG-C/EBPα cells was loose compared to the other two cells. All the three cells were PAS and POX negative. FACS results showed that CD19 positive rate was (97.76±1.48)% A(97.93±0.64)% A(96.98±1.80)% in Raji cells, Raji+pMIG cells and Raji+pMIG-C/EBPα cells separately(P=0.688), and their mean value was 7003.5±276.48, 5803.5±159.10, 4808.5±327.39 (P=0.008). However, they were all negative for CD33 and CD14.RT-QPCR results showed that Raji cells, Raji+pMIG cells and Raji+pMIG-C/EBPα cells were Pax5 and PU.1 positive(P value was 0.450 and 0.186), and negative for MPO, G-CSFR and GM-CSFR gene.4. We knew that C/EBPα gene may decrease proliferation of the cell. And this was maybe the reason for why we didn't find a cell-type tansformation we expected. We did FACS 72h after transfection and found that CD19 positive rate of the Raji+pMIG-C/EBPα cell was decreased, but CD14 and CD33 were still nagetive. By sorting the CD19 negative cells which including all the GFP positive cells, we detected that apart from β-actin and C/EBPα gene were positive, the other five genes were all nagetive including Pax5 and PU.1. ConclusionC/EBPα acts as an inhibitor of growth in Raji cell line. B lymphoid cell related antigen CD19 and Pax5 and PU.1 genes were decreased after C/EBPα gene transfection.We conclude that more than one transcription factor were involved in this complex progress, and PU.1 may act an equal important role in it. Disclosures:No relevant conflicts of interest to declare.