Actinomycin D (ActD) enhances the potency of tumor necrosis factor-α (TNF-α) in killing cancer cells. However, it is determined in this study that murine L929 fibrosarcoma cells, when pretreated with bovine testicular hyaluronidase for 12–24h, became resistant to the cytotoxic effect of TNF-α in the presence of DNA interacalators, such as ActD, doxorubicin, and daunorubicin. Monoclonal anti-Fas antibody-mediated apoptosis in the presence of ActD was also blocked in hyaluronidase-pretreated L929 cells. Hyaluronidase failed to up-or downregulate the expression of apoptosis regulatory proteins, including Bcl-2, Bcl-xL, ICH-1, and TIAR, suggesting that these proteins were not involved in the hyaluronidase-induced resistance to TNF/ActD. A semisynthetic polysulfated hyaluronic acid (HA) inhibited the increased TNF/ActD resistance, whereas unmodified HA, dextran sulfate, and naturally polysulfated glycosaminoglycans had no effect. Evidence is provided here that the induced resistance is related to serum fetuin and a novel intracellular 35-kDa TNF-binding protein (intra TBP). Under serum-free conditions, L929 became refractory to TNF/ActD cytotoxicity and hyaluronidase reversed the resistance. Exogenous fetuin increased L929 cell spreading and proliferation, and restored hyaluronidase-induction of TNF/ActD resistance in these serum-starved cells. Hyaluronidase failed to reduce the expression of TNF-receptors and their binding of TNF-α. However, binding and Western-blotting analyses revealed that hyaluronidase downregulated the intra-TBP. Overall, these observations suggest that serum fetuin and intra TBP are involved in the hyaluronidase induction of TNF/ActD resistance.
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