Abstract
In response to diverse genotoxic stimuli (e.g. UV and cisplatin), the mitogen-activated protein kinases ERK1/2, JNK1/2, and p38alpha/beta become rapidly phosphorylated and in turn activate multiple downstream effectors that modulate apoptosis and/or growth arrest. Furthermore, previous lines of evidence have strongly suggested that ERK1/2 and JNK1/2 participate in global-genomic nucleotide excision repair, a critical antineoplastic pathway that removes helix-distorting DNA adducts induced by a variety of mutagenic agents, including UV. To rigorously evaluate the potential role of mitogen-activated protein kinases in global-genomic nucleotide excision repair, various human cell strains (primary skin fibroblasts, primary lung fibroblasts, and HCT116 colon carcinoma cells) were treated with highly specific chemical inhibitors, which, following UV exposure, (i) abrogated the capacities of ERK1/2, JNK1/2, or p38alpha/beta to phosphorylate specific downstream effectors and (ii) characteristically modulated cellular proliferation, clonogenic survival, and/or apoptosis. A highly sensitive flow cytometry-based nucleotide excision repair assay recently optimized and validated in our laboratory was then employed to directly demonstrate that the kinetics of UV DNA photoadduct repair are highly similar in mock-treated versus mitogen-activated protein kinase inhibitor-treated cells. These data on primary and tumor cells treated with pharmacological inhibitors were fully corroborated by repair studies using (i) short hairpin RNA-mediated knockdown of ERK1/2 or JNK1/2 in human U2OS osteosarcoma cells and (ii) expression of a dominant negative p38alpha mutant in human primary lung fibroblasts. Our results provide solid evidence for the first time, in disaccord with a burgeoning perception, that mitogen-activated protein kinase signaling does not influence the efficiency of human global-genomic nucleotide excision repair.
Highlights
Nucleotide excision repair (NER) is composed of two distinct subpathways (i.e. global genomic-NER (GG-NER) removes bulky adducts from the genome overall, whereas transcription-coupled NER (TCNER) removes such adducts exclusively from the transcribed strands of active genes)
GG-NER is triggered when the UV-DDB1/UV-DDB2 heterodimer recognizes and binds the helical distortion created by bulky adducts, which is followed by recruitment of the XPChHR23B complex
A sensitive flow cytometry-based NER assay recently optimized and validated in our laboratory was used to directly determine, in each UV-irradiated human strain, whether or not individual mitogen-activated protein kinases (MAPKs) modulate the efficiency of DNA photoproduct removal via GG-NER
Summary
Human U2OS osteosarcoma cells were grown in Dulbecco’s minimal essential medium containing 10% fetal bovine serum, L-glutamine, and antibiotics. Cells were washed with 0.1 M Na2B4O7 (pH 9.0) antibodies (BIOSOURCE International) and/or (ii) by meas- and with PBS and resuspended in 300 l of RNase (100 uring levels of phosphorylated downstream MAPK effectors, g/ml in PBS) for 1 h at 37 °C followed by washing with PBS-TB where solid-phase kinase assay kits (Cell Signaling Technology, (1% bovine serum albumin plus 0.25% Tween 20 in PBS). This was followed by phosphoryl- antibody against either CPDs or 6-4PPs (Kamiya Biomedical ation reactions in vitro and detection of activated substrates (i.e. Company) for 1 h at room temperature, washed with PBS-TB, phospho-Elk-1, -c-Jun, or -ATF-2) using specific anti-phos- and resuspended in 300 l of fluorescein isothiocyanatephoantibodies. Pellets were washed twice with PBS-TB and rylated forms of the specific p38␣/ substrate MAPKAPK2 resuspended in 300 l of PBS containing 5 g/ml propidium
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
