Redox reagents and staurosporine inhibit stimulation of the transcription regulator NF-κB following tumour necrosis factor treatment of chronic B-leukaemia cells

Abstract

B-chronic lymphocytic leukaemia (B-CLL) and hairy cell leukaemia cells (HCL) are refractory to stimulation by several cytokines which activate normal B-cells. However, tumour necrosis factor (TNF) promotes the proliferation of these cells. TNF regulates some of its cellular responses via the transcription factor NF-κB. Using an electrophoretic mobility shift assay, we demonstrate that TNF treatment of B-CLL and HCL cells in vitro resulted in the augmentation of NF-κB levels. In haemopoietic cell lines. TNF induction of NF-ϰB is mediated via the generation of reactive oxygen intermediates and by the activation of protein kinase C (PKC). We have used activators and inhibitors of these pathways to unravel TNF signalling in the cells of ten patients with B-CLL and two with HCL, using the increase in NF-ϰB levels following TNF treatment as an end point. Raising glutathione levels with N-acetyl cysteine substantially reduced NF-ϰB induction by TNF in two of four samples, as did treatment of cells with the antioxidant butylated-hydroxytoluene in all three samples tested. These data suggest that redox mechanisms are involved in TNF signalling in these cells. Treatment with the P KC activator phorbol myristate acetate failed to activate NF-ϰB suggesting that this enzyme does not mediate the induction of NF-ϰB in these cells. However, the protein kinase inhibitor staurosporine inhibited TNF induction of NF-ϰB in four of five samples, suggesting that staurosporine-sensitive protein kinases (other than P KC) are involved in the signalling pathway. Our results suggest that P KC-independent pathways, including pathways sensitive to redox reagents, mediate the induction of NF- KB by TNF in chronic B-leukaemia cells. Additionally, these data suggest that defects in P KC-mediated pathways may contribute to the general reluctance of B-CLL and HCL cells to respond to mitogenic signals.