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.
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.
Rouget
Raphael
R
Department of Immunology/Oncology, Maisonneuve-Rosemont Hospital Research Center, Faculty of Medicine, University of Montreal, Montreal, Quebec H1T 2M4, Canada.
Auclair
Yannick
Y
Loignon
Martin
M
Affar
El Bachir
el B
Drobetsky
Elliot A
EA
D017209:Apoptosis / Q000187:drug effects / Q000528:radiation effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects / Q000528:radiation effects; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D018736:DNA Adducts / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects / Q000528:radiation effects*; D004260:DNA Repair / Q000187:drug effects / Q000528:radiation effects*; D005434:Flow Cytometry; D015894:Genome, Human / Q000187:drug effects / Q000528:radiation effects; D006801:Humans; D020935:MAP Kinase Signaling System / Q000187:drug effects / Q000528:radiation effects*; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048290:Mitogen-Activated Protein Kinase 11 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048055:Mitogen-Activated Protein Kinase 8 / Q000037:antagonists & inhibitors / Q000378:metabolism; D048056:Mitogen-Activated Protein Kinase 9 / Q000037:antagonists & inhibitors / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000528:radiation effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D014466:Ultraviolet Rays / Q000009:adverse effects*
D018736:DNA Adducts; D047428:Protein Kinase Inhibitors; D048056:Mitogen-Activated Protein Kinase 9; D019950:Mitogen-Activated Protein Kinase 1; D048290:Mitogen-Activated Protein Kinase 11; D048052:Mitogen-Activated Protein Kinase 3; D048055:Mitogen-Activated Protein Kinase 8