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J. Biol. Chem., Vol. 283, Issue 9, 5533-5541, February 29, 2008

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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^*

From the Department of Immunology/Oncology, Maisonneuve-Rosemont Hospital Research Center, Faculty of Medicine, University of Montreal, Montreal, Quebec H1T 2M4, Canada

In response to diverse genotoxic stimuli (e.g. UV and cisplatin), the mitogen-activated protein kinases ERK1/2, JNK1/2, and p38/β 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 p38/β 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 p38 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.

Received for publication, July 30, 2007 , and in revised form, December 17, 2007.

^* This study was supported by the National Cancer Institute of Canada with funds from the Canadian Cancer Society and by the Canadian Institutes for Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence should be addressed: Maisonneuve-Rosemont Hospital Research Center, 5415 Boul. de l'Assomption, Montreal, Quebec H1T 2M4, Canada. Tel.: 514-252-3400 (ext. 4665); Fax: 514-252-3430; E-mail: elliot.drobetsky{at}umontreal.ca.

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