N-methyl-N'-nitro-N-nitrosoguanidine activates cell-cycle arrest through distinct mechanisms activated in a dose-dependent manner.
S(N)1-alkylating agents, such as the mutagenic and cytotoxic drug N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), robustly activate the DNA damage-responsive G(2) checkpoint. Establishment of this checkpoint is dependent on a functional mismatch repair (MMR) system; however, exposure to high doses of MNNG overrides the requirement for MMR to trigger G(2) arrest. In addition, unlike moderate-dose exposure, in which the G(2) checkpoint is attenuated in ataxia-telangiectasia, mutated (ATM)-deficient cells, high-dose MNNG treatment activates G(2) arrest through an ATM-independent mechanism. We document that this arrest is sensitive to the pharmacological agents caffeine and 7-hydroxystaurosporine (UCN-01) that inhibit the checkpoint kinases ATM/ATM and Rad-3-related (ATR) and Chk1/Chk2, respectively. Furthermore, these agents block inactivation of the cell-cycle regulatory molecules Cdc25C and Cdc2, establishing the downstream mechanism through which high-dose MNNG establishes G(2) arrest. Activation of both Chk2 and Chk1 was independent of ATM and MMR in response to high-dose MNNG, unlike the response to moderate doses of this drug. Chk2 was found to be dispensable for cell-cycle arrest in response to high-dose MNNG treatment; however, ATR deficiency and decreased Chk1 expression forced by RNA interference resulted in diminished checkpoint response. These results indicate that MNNG activates the G(2) checkpoint through different mechanisms activated in a dose-dependent fashion.
Beardsley
Dillon I
DI
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL 32611, USA.
Kim
Wan-Ju
WJ
Brown
Kevin D
KD
D002453:Cell Cycle / Q000187:drug effects*; D002461:Cell Line, Transformed; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism
D008769:Methylnitronitrosoguanidine; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases