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Bryostatin-1 and IFN- Synergize for the Expression of the Inducible Nitric Oxide Synthase Gene and for Nitric Oxide Production in Murine Macrophages

Laboratory of Experimental Immunology, Division of Basic Sciences [L. S. T., G. W. C., G. M.], Intramural Research Support Program [M. C. B., I. E-D.], Science Applications International Corp., National Cancer Institute-Frederick Cancer Research and Development Center, NIH, Frederick, Maryland 21702-1201, and Hematology/Oncology, Department of Medicine and Stanley S. Scott Cancer Center, Louisiana State University Medical Center, New Orleans, Louisiana [I. E-D.] 70112

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-. Bryo, although ineffective alone, and IFN- 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- mainly regulate iNOS gene expression posttranscriptionally through stabilization of iNOS mRNA. Experiments designed to investigate the role of tumor necrosis factor (TNF-) in NO₂^- production by Bryo- and IFN--activated macrophages revealed that ANA-1 macrophages expressed low levels of TNF- mRNA constitutively that were not augmented in the presence of IFN-. However, Bryo alone augmented the TNF- mRNA expression, which was only slightly increased with the addition of IFN-. A polyclonal antibody to TNF- was able to completely neutralize TNF- secreted in either medium or Bryo plus IFN--treated cultures. Neutralizing concentrations of anti-TNF- antibody suppressed the Bryo plus IFN--induced NO₂^- production approximately by 50%, suggesting that NO₂^- produced by Bryo plus IFN--treated ANA-1 macrophages may involve both TNF--dependent and TNF--independent mechanisms. Overall, these findings provide the first evidence that Bryo and IFN- 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.

¹ To whom requests for reprints should be addressed, at Hematology/Oncology, Department of Medicine and Stanley S. Scott Cancer Center, Louisiana State University Medical Center, 1542 Tulane Avenue, New Orleans, LA 70112. Fax: (504) 568-3694.

Received 1/14/97. Accepted 4/22/97.

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