Reactive nitrogen species block cell cycle re-entry through sustained production of hydrogen peroxide

Endogenous sources of reactive nitrogen species (RNS) act as second messengers in a variety of cell signaling events, whereas environmental sources of RNS like nitrogen dioxide (NO₂) inhibit cell survival and growth through covalent modification of cellular macromolecules. To examine the effects of RNS on cell cycle progression, murine type II alveolar C10 cells arrested in GO by serum deprivation were exposed to either NO₂ or SIN-1, a generator of RNS, during cell cycle re-entry. In serum-stimulated cells, RNS did not prevent the immediate early gene response by AP-1, but rather blocked cyclin D1 gene expression, resulting cell cycle arrest at the boundary between GO and G1. Dichlorofluorescin diacetate (DCF) fluorescence indicated that RNS induced sustained production of intracellular hydrogen peroxide (H₂O₂), which normally is produced only transiently in response to serum growth factors. Loading cells with catalase did not diminish the formation of 3-nitrotyrosine on the cell surface, but rather prevented enhanced DCF fluorescence and rescued cyclin D1 expression and S phase entry. These studies indicate environmental RNS interfere with cell cycle re-entry through an H₂O₂-dependent mechanism that influences expression of cyclin D1 and progression from GO to the G1 phase of the cell cycle.