The Potentials of Constitutive NF-κB Activation in U266 Cell Lines.

NF-κB has been described as a family of ubiquitously expressed transcription factors, composed of a dimer of distinct members of the Rel protein family (RelA/p65, cRel, RelB, p50, p52), and after stimulation, NF-κB dimer translocate into the nucleus to activate the transcription of various target genes. In the present study, we aimed to elucidate the mechanism of constitutive activation of NF-κB in human myeloma cells. For this purpose here, we used a human myeloma cell line, U266 cells, and performed gel shift assay and also examined the expression of different target genes of NF-κB by RT-PCR and determined the up-regulation of some of the anti-apoptopic genes (IAP-1, IAP-2, Survivin, Bcl-2, Bfl-1) and cell cycle regulators (Cox-2, Cyclin-D1, Cyclin-D2). This NF-κB-mediated gene expression provided an approach for investigating the protein expression of NF-κB family in U266. Our data revealed that the expression of p65, p50, RelB and cRel was strongly detected in the whole lysate of U266, while weak expression of p52 was found in U266. Furthermore, in the nuclear fractions of U266 cells, we strongly detected the expression of p65, RelB and p50, but not the cRel, for cRel was present in the cytoplasm. It has been recognized that dimerization is essential for NF-κB activity. Then, we performed immunoprecipitation-immunoblot analysis to demonstrate the dimer that might present in the U266, and we detected the possible dimerization such as p65-RelB in the nucleus. Therefore, these results suggest that U266 shows high constitutive activation of NF-kB, and possible hetero- and homodimer of NF-kB except for p52 may be responsible for the constitutive activation of NF-kB in U266 cells.