title: Early Chk1 phosphorylation is driven by temozolomide-induced, DNA double strand break- and mismatch repair-independent DNA damage.
abstract: Temozolomide (TMZ) is a DNA methylating agent used to treat brain cancer. TMZ-induced O6-methylguanine adducts, in the absence of repair by O6-methylguanine DNA methyltransferase (MGMT), mispair during DNA replication and trigger cycles of futile mismatch repair (MMR). Futile MMR in turn leads to the formation of DNA single and double strand breaks, Chk1 and Chk2 phosphorylation/activation, cell cycle arrest, and ultimately cell death. Although both pChk1 and pChk2 are considered to be biomarkers of TMZ-induced DNA damage, cell-cycle arrest, and TMZ induced cytotoxicity, we found that levels of pChk1 (ser345), its downstream target pCdc25C (ser216), and the activity of its upstream activator ATR, were elevated within 3 hours of TMZ exposure, long before the onset of TMZ-induced DNA double strand breaks, Chk2 phosphorylation/activation, and cell cycle arrest. Furthermore, TMZ-induced early phosphorylation of Chk1 was noted in glioma cells regardless of whether they were MGMT-proficient or MGMT-deficient, and regardless of their MMR status. Early Chk1 phosphorylation was not associated with TMZ-induced reactive oxygen species, but was temporally associated with TMZ-induced alkalai-labile DNA damage produced by the non-O6-methylguanine DNA adducts and which, like Chk1 phosphorylation, was transient in MGMT-proficient cells but persistent in MGMT-deficient cells. These results re-define the TMZ-induced DNA damage response, and show that Chk1 phosphorylation is driven by TMZ-induced mismatch repair-independent DNA damage independently of DNA double strand breaks, Chk2 activation, and cell cycle arrest, and as such is a suboptimal biomarker of TMZ-induced drug action.
authors.affiliation: Department of Neurological Surgery, University of California-San Francisco, San Francisco, California, United States of America.
authors.forename: Motokazu
authors.initials: M
authors.lastname: Ito
authors.forename: Shigeo
authors.initials: S
authors.lastname: Ohba
authors.forename: Karin
authors.initials: K
authors.lastname: Gaensler
authors.forename: Sabrina M
authors.initials: SM
authors.lastname: Ronen
authors.forename: Joydeep
authors.initials: J
authors.lastname: Mukherjee
authors.forename: Russell O
authors.initials: RO
authors.lastname: Pieper
mesh_terms: D000970:Antineoplastic Agents / Q000494:pharmacology*; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects*; D053904:DNA Breaks, Single-Stranded / Q000187:drug effects; D019175:DNA Methylation / Q000187:drug effects; D053843:DNA Mismatch Repair / Q000187:drug effects*; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism*; D000077204:Temozolomide; D013997:Time Factors
chemical_list: D000970:Antineoplastic Agents; D003606:Dacarbazine; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D000077204:Temozolomide