Hypoxic inducible factor 1α, extracellular signal-regulated kinase, and p53 are regulated by distinct threshold concentrations of nitric oxide

NO produced in tumors can either positively or negatively regulate growth. To examine this dichotomy, effects of NO concentration and duration on the posttranslational regulation of several key proteins were examined in human breast MCF7 cells under aerobic conditions. We found that different concentration thresholds of NO appear to elicit a discrete set of signal transduction pathways. At low steady-state concentrations of NO (<50 nM), extracellular signal-regulated kinase (ERK) phosphorylation was induced via a guanylate cyclase-dependent mechanism. Hypoxic inducible factor 1alpha (HIF-1alpha) accumulation was associated with an intermediate amount of NO (>100 nM), whereas p53 serine 15 phosphorylation occurred at considerably higher levels (>300 nM). ERK phosphorylation was transient during NO exposure. HIF-1alpha stabilization paralleled the presence of NO, whereas p53 serine 15 phosphorylation was detected during, and persisted after, NO exposure. The dose-dependent effects of synthetic NO donors were mimicked by activated macrophages cocultured with MCF7 cells at varying ratios. ERK and HIF-1alpha activation was similar in breast cancer cell lines either mutant (MB231) or null (MB157) in p53. The stabilization of HIF-1alpha by NO was not observed with increased MCF7 cell density, demonstrating the interrelationship between NO and O-2 consumption. The findings show that concentration and duration of NO exposure are critical determinants in the regulation of tumor-related proteins.