Endogenously generated or exogenously supplied nitric oxide (NO)-induced apoptotic cell death in the mouse macrophage cell line RAW 264.7. Apoptotic signaling caused an early accumulation of the tumor suppressor p53 prior to DNA fragmentation. Contrary to the notion of specific activating signals, inhibitory transduction mechanisms largely remain unknown. Therefore, RAW 264.7 macrophages were stably transfected with human Bcl-2, an anti-apoptotic protein. Bcl-2 transfectants showed substantial protection from cell death induced following the exposure to NO donors such as S-nitrosoglutathione (GSNO) and spermine-NO. In contrast, in RAW 264.7 parent or in neomycin control-transformed cells, these NO donors induced internucleosomal DNA cleavage in a dose-dependent manner. Similarly, expression of the inducible NO synthase in response to lipopolysaccharide and interferon-␥ also caused apoptosis in RAW macrophages and neo controls within 24 h. In contrast, Bcl-2 transfectants appeared highly resistant, although inducible NO synthase levels increased along with concomitant nitrite production similar to control cells. The expression of p53 and Bax was also explored in controls and Bcl-2 transfectants after GSNO addition. GSNO induced p53 expression in Bcl-2 transfectants at levels comparable with nontransfected RAW macrophages. Moreover, GSNO induced increases in the steady-state levels of Bax protein in parental and Bcl-2-transfected cells. We conclude therefore, that Bcl-2 acts downstream of p53, presumably nullifying the NO-mediated increase in Bax protein in RAW 264.7 cells.
Nitric oxide (NO)1 is generated from L-arginine by a family of constitutive or cytokine inducible NO synthase (NOS) isoenzymes (1). Signaling of NO via rich redox and additive chemistry can be broadly discriminated as cGMP-dependent versus -independent. Activation of soluble guanylyl cyclase is considered a main physiological event of NO production. A pathological role for NO has been suggested for several diseases, including immune-mediated cytotoxicity, septic shock, neurotoxicity, -cell destruction, autoimmune diseases, and chronic inflammation (see Ref. 2 for references). Besides activation of NOS, a chemically heterogeneous group of NO-releasing compounds preserve NO in their molecular structure and exhibit biological activity after NO liberation (3). These drugs are valuable biochemical tools because they permit the investigation of NO's role in signaling pathways without interfering with second messenger systems involved in NOS activation or NOS induction.In several systems, the progressive intra-or extracellular generation of NO results in cytostasis and/or cytotoxicity. Multiple target interactions, with protein thiol groups, iron-sulfur proteins, or a direct DNA damage, with either NO, ONOO Ϫ