Gene transfection was done using Nucleofector II (Amaxa Biosystems, Cologne, Germany) with a protocol specifically designed for the transfection of U266 by Amaxa Biosystems. The plasmid used for transfection was the expression vector pCMV6SOX4GFP (purchased from Origene Technologies Inc, MD, USA), or empty vector as
the control. U266 cells transfected with the gene encoding green fluorescent protein (EGFP) served as a transfection control in which the efficiency of transfection was between 30 and 40%. U266 cells (approximately 2 9 106 cells) were transfected with 5 lg of the vector. After transfection,
U266 cells were kept for 48 h at 37C in 10% FBS supplemented
RPMI1640.
CD56 is frequently detected on primary myeloma cells from more than 80% patients with overt myeloma. In order to clarify the possible mechanisms of CD56 expression in human myeloma, we underwent screening for potential targets by microarray analysis, where the CD56(+) myeloma cell lines showed markedly increased expressions of transcription factors involved in the neuronal cell lineage compared to the CD56(-) myeloma cell lines. Here, we show that among the SOX family of transcription factors, SOX4 was highly up-regulated and SOX1 was down-regulated in the CD56(+) myeloma cell lines as well as in primary myeloma cases as confirmed by the RT-PCR. ChIP analysis of the CD56 promoter region showed specific bindings of SOX4 in the CD56(+) and SOX1 in the CD56(-) myeloma cell lines, respectively. shRNA against SOX1 failed to induce CD56 expression in CD56(-) myeloma cell line, U266. On the contrary, over-expression of SOX4 in the CD56(-) myeloma cell line could induce the CD56 expression. Silencing of SOX4 by shRNA transfection down-regulated CD56 expression and induced apoptosis to CD56(+) human myeloma cell line, AMO1. Thus, induction of SOX4 gene expression might be responsible for the CD56 expression in human myeloma cells.