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

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.

We 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.

1. 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.

C/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.

No relevant conflicts of interest to declare.