Carcinogen-induced S-phase arrest is Chk1 mediated and caffeine sensitive.
We have investigated the mechanism of S-phase arrest elicited by the carcinogen benzo(a)pyrene dihydrodiol epoxide (BPDE) in p53-deficient cells. Inhibition of DNA synthesis after BPDE treatment was rapid and dose dependent (approximately 50% inhibition after 2 h with 50 nM BPDE). Cells treated with low doses (50-100 nM) of BPDE resumed DNA synthesis after a delay of approximately 4-8 h, whereas cells that received high doses of BPDE (600 nM) failed to recover from S-phase arrest. The checkpoint kinase Chk1 (but not Chk2) was phosphorylated after treatment with low doses of BPDE. High concentrations of BPDE elicited phosphorylation of both Chk1 and Chk2. Adenovirus-mediated expression of "dominant-negative" Chk1 (but not dominant-negative Chk2) and the Chk1 inhibitor UCN-01 abrogated the S-phase delay elicited by low doses of BPDE. Consistent with a role for the caffeine-sensitive ATM or ATR protein kinase in low-dose BPDE-induced S-phase arrest, both Chk1 phosphorylation and S-phase arrest were abrogated by caffeine. However, low doses of BPDE elicited Chk1 phosphorylation and S-phase arrest in AT cells (from ataxia telangiectasia patients), demonstrating that ATM is dispensable for S-phase checkpoint responses to this genotoxin. BPDE-induced Chk1 phosphorylation and S-phase arrest were abrogated by caffeine treatment in AT cells, suggesting that a caffeine-sensitive kinase other than ATM is an important mediator of responses to BPDE-adducted DNA. Overall, our data demonstrate the existence of a caffeine-sensitive, Chk1-mediated, S-phase checkpoint that is operational in response to BPDE.
Guo
Ning
N
Cancer Research Center, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Faller
Douglas V
DV
Vaziri
Cyrus
C
D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide / Q000633:toxicity*; D000256:Adenoviridae / Q000235:genetics; D000470:Alkaloids / Q000494:pharmacology; D000970:Antineoplastic Agents / Q000494:pharmacology; D001260:Ataxia Telangiectasia / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D042846:CDC2-CDC28 Kinases; D002110:Caffeine / Q000494:pharmacology*; D002273:Carcinogens / Q000633:toxicity*; D018797:Cell Cycle Proteins; D000071877:Checkpoint Kinase 1; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis; D004261:DNA Replication / Q000187:drug effects / Q000528:radiation effects; D004268:DNA-Binding Proteins; D004351:Drug Resistance; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005799:Genes, Dominant; D006801:Humans; D015151:Immunoblotting; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D016196:S Phase / Q000187:drug effects*; D019311:Staurosporine / Q000031:analogs & derivatives; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000201:enzymology / Q000528:radiation effects; D025521:Tumor Suppressor Proteins
D000470:Alkaloids; D000970:Antineoplastic Agents; D002273:Carcinogens; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D047428:Protein Kinase Inhibitors; D025521:Tumor Suppressor Proteins; D002110:Caffeine; D015123:7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide; C054852:7-hydroxystaurosporine; D004247:DNA; D011494:Protein Kinases; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases; D019311:Staurosporine