Characterization of NF-κB expression in Hodgkin's disease: Inhibition of constitutively expressed NF-κB results in spontaneous caspase-independent apoptosis in Hodgkin and Reed-Sternberg cells

Abstract

Although the neoplastic cells of classical Hodgkin's disease (CHD) demonstrate high levels of constitutively active nuclear NF-κB, the precise physiologic and clinical significance of NF-κB expression is currently undefined. Expression of active NF-κB p65(Rel A) was evaluated in patient samples of CHD and nodular lymphocyte predominance Hodgkin's disease. The action of the chemical NF-κB inhibitors gliotoxin and MG132 and the effect of NF-κB inhibition utilizing an adenovirus vector carrying a dominant-negative IκBα mutant (Ad5IκB) were then demonstrated in CHD cell lines (LA28, 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-κB inhibition, p65(Rel A) was significantly reduced in nuclear extracts from cultured HRS cells as revealed by electrophoretic mobility shift assays. Furthermore, chemical NF-κB inhibition resulted in time- and concentration-dependent apoptosis in HRS cells. With the exception of MG132-induced apoptosis in HS445, apoptosis by chemical NF-κB 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 Ad5IκB also showed a marked increase in spontaneous apoptosis compared with wild type adenovirus-infected and control cells. Overall, the inhibition of active NF-κB in HRS cells resulting in spontaneous caspase-independent apoptosis demonstrates a critical role for NF-κB in HRS cell survival and resistance to apoptosis.