S-phase arrest by reactive nitrogen species is bypassed by okadaic acid, an inhibitor of protein phosphatases PP1/PP2A.
In mammalian cells DNA damage activates a checkpoint that halts progression through S phase. To determine the ability of nitrating agents to induce S-phase arrest, mouse C10 cells synchronized in S phase were treated with nitrogen dioxide (NO(2)) or SIN-1, a generator of reactive nitrogen species (RNS). SIN-1 or NO(2) induced S-phase arrest in a dose- and time-dependent manner. As for the positive controls adozelesin and cisplatin, arrest was accompanied by phosphorylation of ATM kinase; dephosphorylation of pRB; decreases in RF-C, cyclin D1, Cdc25A, and Cdc6; and increases in p21. Comet assays indicated that RNS induce minimal DNA damage. Moreover, in a cell-free replication system, nuclei from cells treated with RNS were able to support control levels of DNA synthesis when incubated in cytosolic extracts from untreated cells, whereas nuclei from cells treated with cisplatin were not. Induction of phosphatase activity may represent one mechanism of RNS-induced arrest, for the PP1/PP2A phosphatase inhibitor okadaic acid inhibited dephosphorylation of pRB; prevented decreases in the levels of RF-C, cyclin D1, Cdc6, and Cdc25A; and bypassed arrest by SIN-1 or NO(2), but not cisplatin or adozelesin. Our studies suggest that RNS may induce S-phase arrest through mechanisms that differ from those elicited by classical DNA-damaging agents.
Ranjan
Priya
P
Department of Pathology and Vermont Cancer Center, University of Vermont College of Medicine, Burlington, VT 05405, USA.
Heintz
Nicholas H
NH
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D002945:Cisplatin / Q000494:pharmacology; D019938:Cyclin D1 / Q000187:drug effects / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis / Q000187:drug effects; D004249:DNA Damage / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D066298:In Vitro Techniques; D051379:Mice; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009585:Nitrogen Dioxide / Q000494:pharmacology; D009687:Nuclear Proteins / Q000187:drug effects / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000378:metabolism; D026361:Reactive Nitrogen Species / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D051818:Replication Protein C / Q000187:drug effects / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000187:drug effects / Q000378:metabolism; D016196:S Phase / Q000187:drug effects*; D013997:Time Factors; D025521:Tumor Suppressor Proteins / Q000187:drug effects / Q000378:metabolism; D020687:cdc25 Phosphatases / Q000187:drug effects / Q000378:metabolism
C502132:CDC6 protein, mouse; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D026361:Reactive Nitrogen Species; D016160:Retinoblastoma Protein; D025521:Tumor Suppressor Proteins; D019938:Cyclin D1; D019319:Okadaic Acid; C002385:linsidomine; D004247:DNA; D008981:Molsidomine; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases; D010749:Phosphoprotein Phosphatases; C517005:Cdc25a protein, mouse; D020687:cdc25 Phosphatases; D051818:Replication Protein C; D002945:Cisplatin; D009585:Nitrogen Dioxide