Differential effect of thalidomide and dexamethasone on the transcription factor NF-kappa B.
Thalidomide was initially used as a sedative during pregnancy but was withdrawn from the market due to its teratogenic effects. In vitro studies have shown that thalidomide inhibits tumour necrosis factor alpha (TNF-alpha) mRNA expression and protein production by mitogen-stimulated macrophages and activated T cells. Even at the highest concentration (10-1 mM) tested, however, TNF-alpha levels are inhibited only partially and the mechanism of action is unknown. In the present investigations, we have examined the influence of thalidomide on nuclear levels of NF-kappa B in human peripheral blood mononuclear cells (PBMC) following activation with mitogen or phorbol myristate acetate (PMA)/ionophore. Dexamethasone was used as a positive control due to its well-characterised mechanism of action and NF-kappa B-mediated effects on TNF-alpha expression. PBMC from healthy human volunteers were stimulated optimally with phytohemagglutinin (PHA) or PMA/ionophore in the presence of 10(-1)-10(-5) mM thalidomide or dexamethasone, concentrations that displayed a range of inhibitory effects on TNF-alpha production. Cells were harvested at varying time points and nuclear extracts prepared. Nuclear levels of NF-kappa B were measured using electrophoretic mobility shift assays (EMSA) with a radiolabelled DNA probe specific for NF-kappa B. Results were analysed using optical densitometry. Nuclear levels of NF-kappa B were found to be unaffected by thalidomide at all concentrations tested, including concentrations (10(-1)-10(-3) mM) that exhibited significant inhibition of TNF-alpha protein and mRNA expression. In concurrent experiments, dexamethasone was found to reduce NF-kappa B expression in a dose-dependent manner with maximal inhibition at the highest dose tested (10(-1) mM). TNF-alpha gene expression is controlled by at least three separate transcription factors that are involved in binding to the promoter region. These observations suggest that thalidomide does not act directly on NF-kappa B and therefore inhibits TNF-alpha production through another independent mechanism.
Rowland
T L
TL
Glaxo Wellcome, Stevenage, SG1 2NY Hertfordshire, UK.
McHugh
S M
SM
Deighton
J
J
Ewan
P W
PW
Dearman
R J
RJ
Kimber
I
I
D000328:Adult; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism*; D010835:Phytohemagglutinins / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013792:Thalidomide / Q000494:pharmacology*; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics
D017931:DNA Primers; D016328:NF-kappa B; D010835:Phytohemagglutinins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D013792:Thalidomide; D003907:Dexamethasone; D013755:Tetradecanoylphorbol Acetate