Bryostatin-1 and IFN-γ synergize for the expression of the inducible nitric oxide synthase gene and for nitric oxide production in murine macrophages

Lynn S. Taylor, George W. Cox, Giovanni Melillo, Maria Carla Bosco, Igor Espinoza-Delgado

Research output: Contribution to journalArticle

Abstract

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

Original languageEnglish
Pages (from-to)2468-2473
Number of pages6
JournalCancer Research
Volume57
Issue number12
Publication statusPublished - Jun 15 1997

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Nitric Oxide Synthase Type II
Nitric Oxide
Macrophages
Tumor Necrosis Factor-alpha
Genes
Messenger RNA
Gene Expression
bryostatin 1
Antibodies
Nitrites
Protein Kinase C

ASJC Scopus subject areas

  • Cancer Research
  • Oncology

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Bryostatin-1 and IFN-γ synergize for the expression of the inducible nitric oxide synthase gene and for nitric oxide production in murine macrophages. / Taylor, Lynn S.; Cox, George W.; Melillo, Giovanni; Bosco, Maria Carla; Espinoza-Delgado, Igor.

In: Cancer Research, Vol. 57, No. 12, 15.06.1997, p. 2468-2473.

Research output: Contribution to journalArticle

Taylor, Lynn S. ; Cox, George W. ; Melillo, Giovanni ; Bosco, Maria Carla ; Espinoza-Delgado, Igor. / Bryostatin-1 and IFN-γ synergize for the expression of the inducible nitric oxide synthase gene and for nitric oxide production in murine macrophages. In: Cancer Research. 1997 ; Vol. 57, No. 12. pp. 2468-2473.
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T1 - Bryostatin-1 and IFN-γ synergize for the expression of the inducible nitric oxide synthase gene and for nitric oxide production in murine macrophages

AU - Taylor, Lynn S.

AU - Cox, George W.

AU - Melillo, Giovanni

AU - Bosco, Maria Carla

AU - Espinoza-Delgado, Igor

PY - 1997/6/15

Y1 - 1997/6/15

N2 - 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-γ. Bryo, although ineffective alone, and IFN-γ 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-γ mainly regulate iNOS gene expression posttranscriptionally through stabilization of iNOS mRNA. Experiments designed to investigate the role of tumor necrosis factor α (TNF-α) in NO2- production by Bryo- and IFN-γ-activated macrophages revealed that ANA-1 macrophages expressed low levels of TNF-α mRNA constitutively that were not augmented in the presence of IFN-γ. However, Bryo alone augmented the TNF-α mRNA expression, which was only slightly increased with the addition of IFN-γ. A polyclonal antibody to TNF-α was able to completely neutralize TNF-α secreted in either medium or Bryo plus IFN-γ-treated cultures. Neutralizing concentrations of anti-TNF-α antibody suppressed the Bryo plus IFN-γ- induced NO2- production approximately by 50%, suggesting that NO2- produced by Bryo plus IFN-γ-treated ANA-1 macrophages may involve both TNF- α-dependent and TNF-α-independent mechanisms. Overall, these findings provide the first evidence that Bryo and [IFN-γ can synergize for the induction of NO2- production us well as iNOS gene expression and show the involvement of posttranscriptional mechanisms in the induction of iNOS mRNA.

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

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