Growth of a variety of human tumor cell lines is inhibited by interferon-gamma (IFN-gamma) in vitro. This mechanism is not well understood. The present experiments identify two separate mechanisms which account for the growth inhibitory activity of IFN-gamma. Cell lines most sensitive to IFN-gamma (inhibited by 10-30 U/ml IFN-gamma in 3 d) were stimulated by IFN-gamma to oxidize tryptophan in media to kynurenine and completely eliminated tryptophan from the culture media after 48-72 h. Addition of L-tryptophan, but not other aromatic amino acids, other essential amino acids, or D-tryptophan, prevented inhibition of cell growth by IFN-gamma. The amount of IFN-gamma required to yield 50% inhibition of cell growth was directly related to the concentration of L-tryptophan in culture media and increased from approximately 3 to 600 U/ml as the concentration of tryptophan in the media was increased from 25 to 1,000 microM. By contrast, inhibition of growth of the cell lines, BT20 and HT29, was not prevented by addition of tryptophan. Inhibition by IFN-gamma (100-300 U/ml after 5-6 d) was, however, completely prevented by addition of two inhibitors of adenosine diphosphate-ribosyl transferase (ADP-RT), 3-aminobenzamide or nicotinamide. Activity of ADP-RT was increased in these cell lines after addition of IFN-gamma. ADP-RT catalyzes the incorporation of the ADP moiety of nicotinamide adenine dinucleotide (NAD) into proteins and causes depletion of intracellular NAD. All tumor cell lines tested had reduced levels of intracellular NAD after treatment with IFN-gamma and loss of NAD preceded inhibition of cell growth by 12-24 h. Inhibitors of IFN-gamma-mediated inhibition of cell growth prevented loss of levels of intracellular NAD. Generation of reactive oxygen species lead to DNA strand breaks which result in activation of ADP-RT. Increased DNA strand breaks were induced in BT20 and HT29 cells but not ME180 and A549 cells after culture with IFN-gamma. The two enzymes known to catalyze the decyclization of tryptophan to kynurenine require superoxide anion for activity. Increased amounts of superoxide anion were released from ME180 and A549 cells after culture with IFN-gamma. Reduced oxygen concentration decreased the ability of IFN-gamma to inhibit tumor cell growth in vitro. Intracellular glutathione has been shown to protect cells against oxidative damage by various agents. Elevation or reduction of intracellular glutathione concentrations lowered or raised sensitivity of cell lines to IFN-gamma, respectively. These data indicate that at least two distinct mechanisms can account for IFN-gamma-madiated inhibition of tumor cell growth. Both mechanisms appear to be sensitive to oxygen tension and to changes in intracellular glutathione concentrations, and both mechanisms lead to loss of intracellular NAD.
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