Mammalian S-phase checkpoint integrity is dependent on transformation status and purine deoxyribonucleosides

In eukaryotic cells arrested in S-phase, checkpoint controls normally restr ain mitosis until after replication. We have identified an array of previou sly unsuspected factors that modulate this restraint, using transformed ham ster cells in which cycle controls are known to be altered in S-phase arres t. Arrested cells accumulate cyclin B, the regulatory partner of the mitoti c p34(cdc2) kinase, which is normally not abundant until late G(2) phase; t reatment of arrested cells with caffeine produces rapid S-phase condensatio n. We show here that such S-phase checkpoint slippage, as visualised throug h caffeine-dependent S-phase condensation, correlates with rodent origin an d transformed status, is opposed by reverse transformation, and is favoured by c-src and opposed by wnt1 overexpression, Slippage is also dependent on a prolonged replicative arrest, and is favoured by arrest with hydroxyurea , which inhibits ribonucleotide reductase, This last is a key enzyme in deo xyribonucleotide synthesis, recently identified as a determinant of maligna ncy. Addition of deoxyribonucleosides shows that rapid S-phase condensation is suppressed by a novel checkpoint mechanism: purine (but not pyrimidine) deoxyribonucleosides, like reverse transformation, suppress cyclin B/p34(c dc2) activation by caffeine, but not cyclin B accumulation. Thus, ribonucle otide reductase has an unexpectedly complex role in mammalian cell cycle re gulation: not only is it regulated in response to cycle progression, but it s products can also reciprocally influence cell cycle control kinase activa tion.