Biological Significance of Constitutive NF-κB Activity Levels in Human Myeloma Cell Lines

To elucidate the mechanism of constitutive NF-κB activity in human myeloma cell lines, constitutive expression of all the NF-κB family, RelA (p65), c-Rel, RelB, NF-κB1 (p105/p50) and NF-κB2 (p100/p52), genes in different human myeloma cell lines, such as IL-6-independent (AMO1, NOP2), IL-6-dependent (ILKM2, ILKM8) and IL-6-responsive cell line (U266), were first checked by RT-PCR and western blot analysis. In the electrophoretic mobility shift assay (EMSA) for κB main consensus, total DNA binding activity levels for NF-κB were higher in IL-6-dependent, IL-6-responsive and IL-6-independent human myeloma cell lines respectively. Supershift analysis with anti-NF-κB family antibodies and immunoprecipitation followed by western blotting, confirmed that p50/p50, p50/RelB and p50/p52 as the principal player responsible for constitutive activation of NF-κB in IL-6-independent cell lines. However, beside these major dimers, p50/p65 dimer was also involved in the constitutive NF-κB activation in IL-6-dependent and IL-6-responsive cell lines. Moreover, on the basis of microarray data in human myeloma cell lines, we confirmed that the expression levels of NF-κB target genes, such as NFKB2, RELB, NFKBIA, MALT1 and CD54, showed correlation with the level of DNA binding activity for NF-κB. Therefore, these data suggest that the difference of total DNA-binding activities for NF-κB partly depended on the different p50/p65 activity among the IL-6-independent, IL-6-dependent and IL-6-responsive human myeloma cell lines, and further studies in the different NF-κB-dimers activity levels might contribute to the clarification of the regulation of NF-κB target genes expression in human myeloma cell lines.