[Effect of C/EBPalpha on the monocytic differentiation of HL60 cells induced by NSC67657].
OBJECTIVE
To figure out the function of C/EBPalpha in the monocytic differentiation of HL60 cells induced by a new steroidal drug NSC67657.


METHODS
The differentiation of HL60 cells was induced by NSC67657, and the cell surface antigen CD14 expression was detected by flow cytometry. The gene and protein expressions of CCAAT enhancer binding protein alpha (C/EBPalpha) before and after the induction of cell differentiation were determined by RT-PCR and Western blot. Eukaryotic expressing vector pDsRed-ICAT was constructed and transfected into HL60 cells, and its expression was verified. The effect of C/EBPalpha overexpression in HL60 cells was assessed by MTT assay, Wright's staining and flow cytometry before and after NSC67657 transfection.


RESULTS
HL60 cells could be induced into monocytes by 10 micromol/L ATRA within 5 days, and the coverage of CD14 positive cells reached 93.9% after 5 days of drug treatment. The eukaryotic expressing vector was successfully constructed, and over 90% positive clones were obtained after screening by G418 and electrotransfection. The results of proliferative analysis, chemical staining, ultrastructural observation, and CD11b detection confirmed that HL60 cells could be induced into granulocytic differentiation by overexpression of C/EBPalpha protein. Moreover, in the drug treatment group, transfected cells could not be induced into monocytic differentiation, and their granulocytic differentiation was also inhibited.


CONCLUSIONS
The monocytic differentiation of HL60 cells induced by NSC67657 may not be via the regulation by C/EBPalpha protein-mediated signal transduction. However, the overexpression of CEBPalpha may inhibit the process of NSC67657-induced monocytic differentiation in HL60 cells.
Wang
Wei-jia
WJ
Department of Laboratory Medicine, Zhongshan People's Hospital, The Affiliated Hospital of Zhongshan University, Zhongshan 528402, China.
Zhang
Xiu-ming
XM
Wang
Qian
Q
Wen
Dong-mei
DM
Qiu
Zong-yin
ZY
D022763:CCAAT-Enhancer-Binding Protein-alpha / Q000235:genetics / Q000378:metabolism*; D039481:CD11b Antigen / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects*; D005822:Genetic Vectors; D006098:Granulocytes / Q000166:cytology; D018922:HL-60 Cells; D006801:Humans; D018950:Lipopolysaccharide Receptors / Q000378:metabolism*; D008698:Mesylates / Q000494:pharmacology*; D009000:Monocytes / Q000166:cytology*; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013256:Steroids / Q000494:pharmacology*; D014162:Transfection
C544140:(4-(10, 13-dimethyl-3-methylsulfonyloxy-2,3,4,5,6,7,8,9,11,12,14,15,16, 17-tetradecahydro-1H-cyclopenta(a)phenanthren-17-yl)pentyl) methanesulfonate; D022763:CCAAT-Enhancer-Binding Protein-alpha; D039481:CD11b Antigen; C509295:ITGAM protein, human; D018950:Lipopolysaccharide Receptors; D008698:Mesylates; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013256:Steroids