Summary. Nuclear factor-kB (NF-kB) is an important transcription factor that regulates survival in many cells. Activated NF-kB has been shown to protect some haematopoietic neoplastic cells from apoptosis. In the present study, we analysed NF-kB 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-kB 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-kB was present in all MM patient samples as well as in all four myeloma cell lines. Inhibition of constitutively active NF-kB, by either proteasome inhibitors (MG132, gliotoxin) or inhibitors of IkB 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-kB as determined by the electrophoretic mobility shift assay. In addition, adenovirus vector with dominant negative IkBa (Ad5IkB) was used for inhibition of NF-kB 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-kB plays an important role in plasma cell survival in MM.

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Although the neoplastic cells of classical Hodgkin's disease (CHD) demonstrate high levels of constitutively active nuclear NF-B, the precise physiologic and clinical significance of NF-B expression is currently undefined. Expression of active NF-B p65(Rel A) was evaluated in patient samples of CHD and nodular lymphocyte predominance Hodgkin's disease. The action of the chemical NF-B inhibitors gliotoxin and MG132 and the effect of NF-B inhibition utilizing an adenovirus vector carrying a dominant-negative IB␣ mutant (Ad5IB) were then demonstrated in CHD cell lines (L428, KMH2, and HS445). Hodgkin and Reed-Sternberg (HRS) cells from all patient and cell line specimens showed strong immunopositivity for active p65(Rel A). Expression was also seen in lymphocytic/histiocytic cells from all cases of nodular lymphocyte predominance Hodgkin's disease. After chemical NF-B inhibition, p65(Rel A) was significantly reduced in nuclear extracts from cultured HRS cells as revealed by electrophoretic mobility shift assays. Furthermore, chemical NF-B inhibition resulted in time-and concentration-dependent apoptosis in HRS cells. With the exception of MG132-induced apoptosis in HS445, apoptosis by chemical NF-B inhibition was not significantly altered by preincubation with various caspase inhibitors (z-DQMD- FMK, z-DEVD-FMK, z-VAD-FMK, z-VEID-FMK, and z-IETD-FMK). Regardless of the chemical inhibitor used, no significant change in caspase-3 functional activity was found in CHD cell lines. HRS cells infected with Ad5IB also showed a marked increase in spontaneous apoptosis compared with wild type adenovirus-infected and control cells. Overall, the inhibition of active NF-B in HRS cells resulting in spontaneous caspase-independent apoptosis demonstrates a critical role for NF-B in HRS cell survival and resistance to apoptosis.

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