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The cytokine scatter factor/hepatocyte growth factor (HGF/SF) protects epithelial, carcinoma, and other cell types against cytotoxicity and apoptosis induced by DNA-damaging agents such as ionizing radiation and adriamycin (ADR, a topoisomerase IIainhibitor). We investigated the role of nuclear factor kappa B (NF-?B) signaling in HGF/SF-mediated protection of human prostate cancer (DU-145) and Madin-Darby canine kidney (MDCK) epithelial cells against ADR. HGF/SF caused the rapid nuclear translocation of the p65 (RelA) subunit of NF-?B associated with the transient loss of the inhibitory subunit I?B-a. Exposure to HGF/SF caused the activation of an NF-?B luciferase reporter that was blocked or attenuated by the expression of a mutant‘super-repressor’I?B-a. Electrophoretic mobility shift assay supershift assays revealed that HGF/SF treatment induced the transient binding of various NF-?B family proteins (p65, p50, c-Rel, and RelB) with radiolabeled NF-?B-binding oligonucleotides. The HGF/SF-mediated protection of DU-145 and MDCK cells against ADR (demonstrated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays) was abrogated by the I?B-asuper-repressor. The ability of HGF/SF to activate NF-?B signaling was dependent on c-Akt?Pak1 (p21-associated kinase-1) signaling (with Pak1 downstream of c-Akt) and was inhibited by the tumor suppressor PTEN (phosphatase and tensin homolog). Inhibitors of phosphatidylinositol-3'-kinase and Src family kinases significantly inhibited HGF/SF-mediated activation of NF-?B, while inhibitors of MEK, protein kinase C, and p70 S6 kinase had a modest effect or no effect on NF-?B activity. HGF/SF induced the expression of several known NF-?B target genes (cIAP-1 (cellular inhibitor of apoptosis-1), cIAP-2, and TRAF-2 (TNF receptor-associated factor-2)) in an NF-?B-dependent manner; HGF/SF blocked the inhibition of expression of these genes by ADR. Experimental manipulation of expression of these genes suggests that they (particularly TRAF-2 and cIAP-2) contribute to the protection against ADR by HGF/SF. These findings suggest that HGF/SF activates NF-?B through a c-Akt?Pak1 signaling pathway that is also dependent on Src, and that NF-?B contributes to HGF/SF-mediated protection against ADR.Oncogene (2005) 24, 1749-1766. doi:10.1038/sj.onc.1208327 Published online 31 January 2005 [ABSTRACT FROM AUTHOR]