Abstract
N-Methyl-N'-nitro-N'-nitrosoguanidine (MNNG) is a DNA-methylating agent, and deficiency in mismatch repair (MMR) results in lack of sensitivity to this genotoxin (termed alkylation tolerance). A number of DNA damage response pathways are activated in a MMR-dependent manner following MNNG, and several also require ATM kinase activity. Here we show that activation of the transcription factor c-Jun is dependent upon both the MMR component MLH1 and ATM, but not ATR, in response to MNNG. In addition to c-Jun, the upstream MAPKs JNK and MKK4 are also activated in a MLH1- and ATM-dependent manner. We document that c-Jun activation is dependent on the MAPK kinase kinase MEKK1. Additionally, the tyrosine kinase c-Abl is required to activate this signaling cascade and forms a complex with MEKK1 and MLH1. This study indicates that an arm of DNA damage-activated MAPK signaling is activated in an MLH1- and ATM-dependent manner in response to MNNG and perhaps suggests that dysregulation of this signaling is responsible, in part, for alkylation tolerance.
Highlights
DNA damage activates a number of cellular signaling mechanisms that promote DNA repair, halt cell growth, or activate cell death mechanisms
This study indicates that an arm of DNA damage-activated mitogen-activated protein kinase (MAPK) signaling is activated in an MLH1- and ATM-dependent manner in response to MNNG and perhaps suggests that dysregulation of this signaling is responsible, in part, for alkylation tolerance
It is clear that DNA damage response mechanisms are crucial to limiting the occurrence of somatic mutations, maintaining genomic homeostasis, and limiting cancer development [2, 3]
Summary
Cell Culture and Drug Treatment—The SV40-immortalized A-T fibroblast line AT22IJE-T stably expressing full-length recombinant human ATM (designated YZ-5) or stably transfected with empty vector (designated EBS-7) were cultured as previously indicated [36]. To test if JNK is activated in response to MNNG and, if so, through a MLH1- and ATM-dependent mechanism we treated HCT116/HCT116ϩch and EBS-7/YZ-5 cells with MNNG or vehicle alone (mock treated) and subjected extracts to immunoblot analysis with anti-phospho Thr-183/Tyr-185 JNK antibody. We did not observe increased levels of phospho-Ser-271/ Thr-275 MKK7 in either HCT116 (Fig. 3D, lanes 5 and 6) or HCT116ϩch (Fig. 3D, lanes 7 and 8) following treatment with MNNG When taken together, these findings indicate that MKK4 is phosphorylated/activated in response to MNNG exposure and that this signaling event occurs in an MLH1- and ATM-dependent manner. 3 h after drug exposure cells were harvested and immunoblotted for phosphorylated c-Jun This experiment revealed that knock-down of MEKK1 reduced MNNG-induced c-Jun phosphorylation by ϳ3-fold compared with MNNG-treated cells transfected with control siRNA (Fig. 4A, bottom). 1.0 6.0 3.4 c-Abl siRNA control siRNA :MNNG phospho c-jun signal intensity total c-jun
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