A Sensitive Flow Cytometry-based Nucleotide Excision Repair Assay Unexpectedly Reveals That Mitogen-activated Protein Kinase Signaling Does Not Regulate the Removal of UV-induced DNA Damage in Human Cells
| Autor: | El Bachir Affar, Martin Loignon, Raphael Rouget, Yannick Auclair, Elliot Drobetsky |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
DNA Repair
Cell Survival MAP Kinase Signaling System Ultraviolet Rays Mitogen-Activated Protein Kinase 3 DNA repair DNA damage Apoptosis Biochemistry DNA Adducts Mitogen-Activated Protein Kinase 11 Cell Line Tumor medicine Humans Mitogen-Activated Protein Kinase 9 Mitogen-Activated Protein Kinase 8 Phosphorylation Protein kinase A Protein Kinase Inhibitors Molecular Biology Cell Proliferation Mitogen-Activated Protein Kinase 1 Cisplatin biology Genome Human Kinase Cell Biology Flow Cytometry Molecular biology Mitogen-activated protein kinase biology.protein DNA Damage Nucleotide excision repair medicine.drug |
| Zdroj: | Journal of Biological Chemistry. 283:5533-5541 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m706257200 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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