Nitrosative stress induces DNA strand breaks but not caspase mediated apoptosis in a lung cancer cell line

BACKGROUND: Key steps crucial to the process of tumor progression are genomic instability and escape from apoptosis. Nitric oxide and its interrelated reactive intermediates (collectively denoted as NO(X)) have been implicated in DNA damage and mutational events leading to cancer development, while also being implicated in the inhibition of apoptosis through S-nitrosation of key apoptotic enzymes. The purpose of this study was to explore the interrelationship between NO(X)-mediated DNA strand breaks (DSBs) and apoptosis in cultured tumor cell lines. METHODS: Two well-characterized cell lines were exposed to increasing concentrations of exogenous NO(X )via donor compounds. Production of NO(X )was quantified by the Greiss reaction and spectrophotometery, and confirmed by nitrotyrosine immunostaining. DSBs were measured by the alkaline single-cell gel electrophoresis assay (the COMET assay), and correlated with cell viability by the MTT assay. Apoptosis was analyzed both by TUNEL staining and Annexin V/propidium iodine FACS. Finally, caspase enzymatic activity was measured using an in-vitro fluorogenic caspase assay. RESULTS: Increases in DNA strand breaks in our tumor cells, but not in control fibroblasts, correlated with the concentration as well as rate of release of exogenously administered NO(X). This increase in DSBs did not correlate with an increase in cell death or apoptosis in our tumor cell line. Finally, this lack of apoptosis was found to correlate with inhibition of caspase activity upon exposure to thiol- but not NONOate-based NO(X )donor compounds. CONCLUSIONS: Genotoxicity appears to be highly interrelated with both the concentration and kinetic delivery of NO(X). Moreover, alterations in cell apoptosis can be seen as a consequence of the explicit mechanisms of NO(X )delivery. These findings lend credence to the hypothesis that NO(X )may play an important role in tumor progression, and underscores potential pitfalls which should be considered when developing NO(X)-based chemotherapeutic agents.