Bryostatin-1 and IFN-gamma synergize for the expression of the inducible nitric oxide synthase gene and for nitric oxide production in murine macrophages.Cancer Res 1997; 57(12):2468-73CR
Bryostatin-1 (Bryo) is a nontumor-promoting protein kinase C modulator that has been shown to have both in vitro and in vivo activity against several murine and human tumors. In this study, we investigated the effects of Bryo on nitric oxide production, measured as accumulated nitrite (NO2-) in culture supernatant, and inducible nitric oxide synthase (iNOS) gene expression in the murine macrophage cell line ANA-1. ANA-1 macrophages did not produce NO2- or iNOS mRNA constitutively, and very little or no NO2- or iNOS mRNA were detectable upon exposure to IFN-gamma. Bryo, although ineffective alone, and IFN-gamma synergized to produce high levels of NO2- and iNOS mRNA. The activity of Bryo was evident at a concentration of 0.1 ng/ml and reached its maximum at 1 ng/ml. The effects of Bryo were time dependent because expression of iNOS mRNA was detectable as early as 6 h and increased through 24 h. Analyses of the molecular mechanisms involved indicate that Bryo and IFN-gamma mainly regulate iNOS gene expression posttranscriptionally through stabilization of iNOS mRNA. Experiments designed to investigate the role of tumor necrosis factor alpha (TNF-alpha) in NO2- production by Bryo- and IFN-gamma-activated macrophages revealed that ANA-1 macrophages expressed low levels of TNF-alpha mRNA constitutively that were not augmented in the presence of IFN-gamma. However, Bryo alone augmented the TNF-alpha mRNA expression, which was only slightly increased with the addition of IFN-gamma. A polyclonal antibody to TNF-alpha was able to completely neutralize TNF-alpha secreted in either medium or Bryo plus IFN-gamma-treated cultures. Neutralizing concentrations of anti-TNF-alpha antibody suppressed the Bryo plus IFN-gamma-induced NO2- production approximately by 50%, suggesting that NO2- produced by Bryo plus IFN-gamma-treated ANA-1 macrophages may involve both TNF-alpha-dependent and TNF-alpha-independent mechanisms. Overall, these findings provide the first evidence that Bryo and IFN-gamma can synergize for the induction of NO2- production as well as iNOS gene expression and show the involvement of posttranscriptional mechanisms in the induction of iNOS mRNA.