Macrophage-derived
nitric oxide (NO) is
cytostatic to
tumor cells and microbial pathogens. We tested whether one molecular target for the
cytostatic action of NO may be
ribonucleotide reductase (RR), a rate-limiting
enzyme in
DNA synthesis. In a concentration-dependent manner, NO gas and lysates of activated macrophages that generated comparable amounts of NO led to the same degree of inhibition of partially purified RR from L1210 mouse
lymphoma cells. Lysates from nonactivated macrophages, which do not produce NO, were noninhibitory. With lysates from activated macrophages, RR was protected by omitting
L-arginine or by adding the
NO synthase inhibitors
diphenyleneiodonium, N omega-methyl-
L-arginine, or N omega-amino-
L-arginine.
L-Arginine, but not D-
arginine, abolished the protective effect of N omega-amino-
L-arginine. The prototypic pharmacologic inhibitor of RR is
hydroxyurea. Its structural resemblance to
N omega-hydroxy-L-arginine, a reaction intermediate of
NO synthase, prompted us to test if
hydroxyurea can generate NO. In the presence of H2O2 and CuSO4,
hydroxyurea produced NO2-/NO3-, aerobic reaction products of NO. Addition of
morpholine blocked NO2-/NO3- generation from
hydroxyurea and led to formation of nitrosomorpholine, as detected by gas chromatography/mass spectrometry. Thus,
hydroxyurea can produce an NO-like, nitrosating rectant. L1210 cell
DNA synthesis was inhibited completely by activated macrophages or by
hydroxyurea, and was partially restored to the same degree in both settings by providing
deoxyribonucleosides to bypass the block in RR. Thus, both NO gas and NO generated by activated macrophage lysates inhibit
tumor cell RR. The RR inhibitor
hydroxyurea can also generate an NO-like species. Similar, partial restoration of
tumor cell
DNA synthesis by
deoxyribonucleosides in the presence of activated macrophages or
hydroxyurea suggests that cytostasis by activated macrophages and by
hydroxyurea has comparable mechanisms, including, but probably not limited to, inhibition of RR.