Carcinogen-induced S-phase arrest is Chk1 mediated and caffeine sensitive.

Abstract	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.
Authors	Ning Guo, Douglas V Faller, Cyrus Vaziri
Journal	Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research (Cell Growth Differ) Vol. 13 Issue 2 Pg. 77-86 (Feb 2002) ISSN: 1044-9523 [Print] United States
PMID	11864911 (Publication Type: Journal Article, Research Support, U.S. Gov't, P.H.S.)
Chemical References	Alkaloids Antineoplastic Agents Carcinogens Cell Cycle Proteins DNA-Binding Proteins Enzyme Inhibitors Protein Kinase Inhibitors Tumor Suppressor Proteins Caffeine 7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide 7-hydroxystaurosporine DNA Protein Kinases ATM protein, human Ataxia Telangiectasia Mutated Proteins CHEK1 protein, human Checkpoint Kinase 1 Protein Serine-Threonine Kinases Protein Kinase C CDC2-CDC28 Kinases CDK2 protein, human Cyclin-Dependent Kinase 2 Cyclin-Dependent Kinases Staurosporine
Topics	7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide (toxicity) Adenoviridae (genetics) Alkaloids (pharmacology) Antineoplastic Agents (pharmacology) Ataxia Telangiectasia (metabolism) Ataxia Telangiectasia Mutated Proteins CDC2-CDC28 Kinases Caffeine (pharmacology) Carcinogens (toxicity) Cell Cycle Proteins Checkpoint Kinase 1 Cyclin-Dependent Kinase 2 Cyclin-Dependent Kinases (metabolism) DNA (biosynthesis) DNA Replication (drug effects, radiation effects) DNA-Binding Proteins Drug Resistance Enzyme Activation Enzyme Inhibitors (pharmacology) Genes, Dominant Humans Immunoblotting Phosphorylation Protein Kinase C (antagonists & inhibitors) Protein Kinase Inhibitors Protein Kinases (metabolism) Protein Serine-Threonine Kinases (metabolism) S Phase (drug effects) Staurosporine (analogs & derivatives) Tumor Cells, Cultured (drug effects, enzymology, radiation effects) Tumor Suppressor Proteins

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