DETA-NONOate, a nitric oxide (NO) donor, induced cytostasis in the human breast cancer cells MDA-MB-231, and the cells were arrested in the G 1 phase of the cell cycle. This cytostatic effect of the NO donor was associated with the down-regulation of cyclin D1 and hypophosphorylation of the retinoblastoma protein. No changes in the levels of cyclin E or the catalytic partners of these cyclins, CDK2, CDK4, or CDK6, were observed. This NO-induced cytostasis and decrease in cyclin D1 was reversible for up to 48 h of DETA-NONOate (1 mM) treatment. DETA-NONOate (1 mM) produced a steady-state concentration of 0.5 M of NO over a 24-h period. Synchronized population of the cells exposed to DETANONOate remained arrested at the G 1 phase of the cell cycle whereas untreated control cells progressed through the cell cycle after serum stimulation. The cells arrested at the G 1 phase after exposure to the NO donor had low cyclin D1 levels compared with the control cells. The levels of cyclin E and CDK4, however, were similar to the control cells. The decline in cyclin D1 protein preceded the decrease of its mRNA. This decline of cyclin D1 was due to a decrease in its synthesis induced by the NO donor and not due to an increase in its degradation. We conclude that down-regulation of cyclin D1 protein by DETA-NONOate played an important role in the cytostasis and arrest of these tumor cells in the G 1 phase of the cell cycle.A ctivated macrophages produce large amounts of nitric oxide (NO), which induces cytostasis and cytotoxicity to tumor cells both in vitro and in vivo (1-8). Macrophages also play a significant role in host resistance to tumors (7-9). Tumor cells cocultured with activated macrophages rapidly develop cytostasis, which precedes the cytotoxic effects (5, 10).It has been reported that NO-induced early cytostasis begins with a rapid and reversible inhibition of ribonucleotide reductase (RR) (10). However, the possibility of targets other than RR being involved in the NO-induced long-lasting cytostasis was suggested by other investigators (10). Hydroxyurea, a specific inhibitor of RR (10), decreased thymidine incorporation in cultured cells and, after its removal, the thymidine incorporation rapidly returned to control values. The rate of recovery was much faster after withdrawal of hydroxyurea when compared with the rate of recovery observed in cells that initially were exposed either to activated macrophages or to a NO donor and then kept in a NO-free environment. On this basis, it was suggested that targets other than RR may be responsible for producing long-lasting cytostasis in target cells exposed to NO (5,10,11). This long-lasting cytostasis, therefore, would be associated with metabolic alterations in target cells, producing long-lasting growth inhibition and requiring a longer time for these cells to resume their normal rate of proliferation.Because the precise mechanism(s) by which NO induces this long-lasting cytostasis is not completely known, the present work was undertaken to assess this mechanism(s)...