Evidence that de novo protein synthesis is dispensable for anti-apoptotic effects of NF-κB

Abstract

The transcription factor NF-kB is a positive transcription factor for a number of genes and has been recognized as an anti-apoptotic regulator. However, the mechanism by which NF-kB blocks apoptosis is still controversial. Here, we demonstrate the evidence that NF-kB could attenuate the TNF-a-induced apoptosis without de novo protein synthesis using human pancreatic cancer cell lines, MIA PaCa-2 and Capan-2. The TNF-a-induced apoptosis was blocked by IL-1b, a potent inducer of NF-kB activation. This inhibitory eect of IL-1b was evident when cells were treated with protein synthesis inhibitors such as cycloheximide (CHX). Moreover, NF-kB decoy oligonucleotides could not block the anti-apoptotic eect of IL-1b at doses sucient to block the NF-kB-dependent transcription induced by IL-1b. To con®rm the role of NF-kB in blocking apoptosis, we generated stable cell lines expressing IkBDN, a highly stable form of IkBa, a cytoplasmic inhibitor of NF-kB. In these stable transfectants, the antiapoptotic eect of IL-1b was totally abolished, indicating that the anti-apoptotic action of IL-1b could be ascribed to the NF-kB action. These ®ndings show that de novo protein synthesis is dispensable for anti-apoptotic eects of NF-kB and support the possibility that NF-kB could exert its anti-apoptotic action through protein-protein interaction.

Nuclear Factor-κB Activation: A Question of Life or Death

“…NF-κB can attenuate TNF-α-induced apoptosis without de novo protein synthesis in the human pancreatic cancer cell lines, MIA PaCa-2 and Capan-2. TNF-α-induced apoptosis was blocked by IL-1β, a potent inducer of NF-κB activation (Kajino et al, 2000). These findings suggest that de novo protein synthesis is dispensable for anti-apoptotic effects of NF-κB, and support the possibility that NF-κB exerts its anti-apoptotic action through protein-protein interaction.…”

“…A large number of reports have demonstrated the anti-apoptotic effect of NF-κB in a wide variety of cell types. The protective role of NF-κB is shown in a large variety of cell types, including the human breast carcinoma (Liu et al, 1996) , T cells (Van Antwerp et al, 1996;Chu et al, 1997;Khoshnan et al, 2000), fibroblasts and macrophages (Beg and Baltimore, 1996), endothelial cells (Stehlik et al, 1998), EBV-infected lymphoblastoid cells (Asso-bonnet et al, 1998), non-small lung cancer cells , glomerular mesangial cells (Sugiyama et al, 1999), human ovarian cancer cells (Shao et al, 1997), human pancreatic cancer cell lines (Kajino et al, 2000), Ewing sarcoma cells (Javelaud and Besancon, 2001), cardiomyocytes (Bergmann et al, 2001), mouse embryos , and HT1080 fibrosarcoma . Treatment of RelA-deficient (the transcriptionally active subunit of NF-κB) mouse fibroblasts and macrophages with TNF significantly reduced cell viability, whereas RelA +/+ cells were unaffected.…”

Induction of Notch signaling by tumor necrosis factor in rheumatoid synovial fibroblasts

“…4 0 ,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was used for the nuclear counter staining. Primary and secondary antibodies were diluted at 1 : 400 and 1 : 50, respectively, in PBS containing 3% bovine serum albumin (Kajino et al, 2000).…”

Cdc25C interacts with PCNA at G2/M transition

“…The cell cycle pattern was examined by¯ow cytometry using FACScan (Beckton Dickinson) and CELL Quest (Beckton Dickinson) software as described (Suganuma et al, 1999;Kajino et al, 2000). Brie¯y, 2610 6 cells were washed twice with cold PBS, resuspended in 300 ml Krishan's solution (0.1% Sodium Citrate, 50 mg/ml propidium iodide (PI), 20 mg/ml RNase A, 0.5% NP-40).…”

“…To detect the hyperphosphorylated Cdc25C, Cdc25C was immunoprecipitated, separated by 12% SDS ± PAGE, and immunoblotted with anti-Cdc25C antibody (a generous gift from H Piwnica-Worms; Peng et al, 1997) and horseradish peroxidase-labeled anti-mouse IgG (Amersham Pharmacia Biotech). To detect the interaction between PCNA and Cdc25C, PCNA was immunoprecipitated with 5 ml of anti-PCNA antibody (Santa Cruz Biotechnology), separated by 10% SDS ± PAGE, and immunoblotted with anti-Cdc25C (Santa Cruz Biotechnology) and horseradish peroxidase-labeled anti-rabbit IgG (Amersham Pharmacia Biotech) as described previously (Kajino et al, 2000;Uranishi et al, 2001). The immunoprecipitated PCNA was detected with anti-PCNA (Santa Cruz Biotechnology) and horseradish peroxidase labeled anti-mouse IgG (Amersham Pharmacia Biotech).…”