The adenylate cyclase activator forskolin partially protects L929 cells against tumour necrosis factor-alpha-mediated cytotoxicity via a cAMP-independent mechanism.
Recent reports indicate that cAMP-elevating agents can protect against cell death induced by many stimuli, including tumour necrosis factor-alpha (TNF-alpha). We investigated the ability of cAMP-elevating agents to modulate TNF-alpha-mediated cytotoxicity in L929 cells. Using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) reduction assay and a DNA fragmentation assay as indicators of cell survival, we have shown that forskolin confers partial protection against TNF-alpha-mediated cytotoxicity and inhibits TNF-alpha-induced internucleosomal DNA fragmentation in L929 cells. The protection conferred by forskolin is cAMP-independent since 1,9-dideoxyforskolin (an adenylate cyclase-inactive analog) also protected against TNF-alpha, while both dibutyryl-cAMP and the cAMP-phosphodiesterase inhibitor theophylline were not protective. This is the first example (that we know of) of cAMP-independent cytoprotection by forskolin. We conclude that forskolin acts in a cAMP-independent manner, potentially at a site upstream of caspase-3 activation, to protect against TNF-alpha-mediated cytotoxicity in L929 cells, and that cAMP elevation, in general, does not confer protection against TNF-alpha-induced death in L929 cells. In addition, we observed that Cyclosporin A, a mitochondrial permeability transition (MPT) inhibitor, protected L929 cells against TNF-alpha, underlining the importance of mitochondria in the cytotoxic process induced by TNF-alpha in L929 cells.
Uboldi
Alessandro D
AD
Department of Molecular Medicine and Haematology, University of the Witwatersrand Medical School, 7 York Road, Parktown, Johannesburg 2193, South Africa. uboldi@wehi.edu.au
Savage
Nerina
N
D000262:Adenylyl Cyclases / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D005576:Colforsin / Q000494:pharmacology*; D000242:Cyclic AMP / Q000378:metabolism*; D016572:Cyclosporine / Q000494:pharmacology; D053938:DNA Fragmentation / Q000187:drug effects; D006801:Humans; D010431:Pentoxifylline / Q000494:pharmacology; D013806:Theophylline / Q000494:pharmacology; D013997:Time Factors; D014409:Tumor Necrosis Factor-alpha / Q000037:antagonists & inhibitors* / Q000494:pharmacology
D014409:Tumor Necrosis Factor-alpha; D005576:Colforsin; D016572:Cyclosporine; D013806:Theophylline; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; D010431:Pentoxifylline