Analysis of expression of nuclear factor κB (NF-κB) in multiple myeloma:: downregulation of NF-κB induces apoptosis

Nuclear factor-kappaB (NF-kappaB) is an important transcription factor that regulates survival in many cells. Activated NF-kappaB has been shown to protect some haematopoietic neoplastic cells from apoptosis. In the present study, we analysed NF-kappaB status in 13 primary samples from patients with multiple myeloma (MM) and in four myeloma cell lines including U266, RPMI 8226, HS-Sultan and K620. Constitutive activation of NF-kappaB was evaluated by either immunohistochemistry or immunofluorescence using a monoclonal mouse anti-human p65 (Rel A) antibody, which recognizes the unbound, active form of p65 (Rel A). Constitutively active NF-kappaB was present in all MM patient samples as well as in all four myeloma cell lines. Inhibition of constitutively active NF-kappaB. by either proteasome inhibitors (MG132, gliotoxin) or inhibitors of I kappaB phosphorylation (Bay117082, and Bay117085), induced apoptosis as demonstrated by both flow cytometric analysis and light microscopic morphological evaluation. This chemically induced apoptosis was associated with decreased DNA binding of nuclear NF-kappaB as determined by the electrophoretic mobility shift assay. In addition, adenovirus vector with dominant negative I kappaB alpha (Ad51 kappaB) was used for inhibition of NF-kappaB in the U266 cell line. Compared with wild-type, super-repressor-treated cells showed an increased level of apoptosis. These results suggest that constitutive expression of NF-kappaB plays an important role in plasma cell survival in MM.