Quenching of the tyrosyl free radical of ribonucleotide reductase by nitric oxide. Relationship to cytostasis induced in tumor cells by cytotoxic macrophages.

Abstract

Nitric oxide (NO) synthesized by macrophages inhibits tumor cell replication. NO also inhibits ribonucleotide reductase, an enzyme essential for DNA synthesis, probably by quenching the catalytically active tyrosyl free radical of its R2 subunit. The role of this inhibition in NO-mediated cytostasis was thus evaluated. After a 4-h coculture with macrophages, quenching of the radical was demonstrated by electron paramagnetic resonance spectroscopy in transfected L1210-R2 cells over-expressing the R2 protein. Pronounced cytostasis was simultaneously observed. A NO synthase inhibitor greatly reduced both phenomena. Target cells withdrawn from macrophages partially recovered from cytostasis and radical loss within 90 min. Deoxyribonucleosides added to by-pass ribonucleotide reductase inhibition efficiently reversed cytostasis of K-562 cells. After a 24-h coculture, the quenched tyrosyl radical still reappeared in L1210-R2 cells withdrawn from macrophages, but DNA synthesis did not resume. Moreover, deoxyribonucleosides marginally reversed overnight cytostasis of K-562 cells mediated by macrophages but were efficient against cytostasis induced by hydroxyurea, a ribonucleotide reductase inhibitor. Autocrine cytostasis observed early in TA3-H2 cells committed to produce NO was closely correlated with quenching of the tyrosyl radical but not with formation of dinitrosyl-iron complexes. We thus propose that NO-dependent cytostasis begins with a rapid and reversible inhibition of ribonucleotide reductase, progressively reinforced by other, long-lasting antiproliferative effects.