Abstract

The accurate joining of DNA double-strand breaks by homologous recombination repair (HRR) is critical to the long-term survival of the cell. The three major mitogen-activated protein (MAP) kinase (MAPK) signaling pathways, extracellular signal-regulated kinase (ERK), p38, and c-Jun-NH(2)-kinase (JNK), regulate cell growth, survival, and apoptosis. To determine the role of MAPK signaling in HRR, we used a human in vivo I-SceI-based repair system. First, we verified that this repair platform is amenable to pharmacologic manipulation and show that the ataxia telangiectasia mutated (ATM) kinase is critical for HRR. The ATM-specific inhibitor KU-55933 compromised HRR up to 90% in growth-arrested cells, whereas this effect was less pronounced in cycling cells. Then, using well-characterized MAPK small-molecule inhibitors, we show that ERK1/2 and JNK signaling are important positive regulators of HRR in growth-arrested cells. On the other hand, inhibition of the p38 MAPK pathway generated an almost 2-fold stimulation of HRR. When ERK1/2 signaling was stimulated by oncogenic RAF-1, an approximately 2-fold increase in HRR was observed. KU-55933 partly blocked radiation-induced ERK1/2 phosphorylation, suggesting that ATM regulates ERK1/2 signaling. Furthermore, inhibition of MAP/ERK kinase (MEK)/ERK signaling resulted in severely reduced levels of phosphorylated (S1981) ATM foci but not gamma-H2AX foci, and suppressed ATM phosphorylation levels >85% throughout the cell cycle. Collectively, these results show that MAPK signaling positively and negatively regulates HRR in human cells. More specifically, ATM-dependent signaling through the RAF/MEK/ERK pathway is critical for efficient HRR and for radiation-induced ATM activation, suggestive of a regulatory feedback loop between ERK and ATM.

Highlights

  • Mitogen-activated protein (MAP) kinases (MAPK) play an evolutionarily conserved role in mediating and amplifying growth factor–mediated and mitogenic signals from the cytoplasm to the nucleus [1]

  • To first determine whether the I-SceI repair system would be suitable for assessing the effects of pharmacologic inhibitors on double-strand break repair, we investigated the effect of a small-molecule inhibitor specific for the ataxia telangiectasia mutated (ATM) kinase, KU-55933, on homologous recombination repair (HRR) [13]

  • U87/DR-green fluorescent protein (GFP) cells were growth-arrested in G0-G1 by serum starvation before and during the course of the experiment [6], facilitating a closer investigation of double-strand break repair by minimizing the interference of cell cycle effects

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Summary

Introduction

Mitogen-activated protein (MAP) kinases (MAPK) play an evolutionarily conserved role in mediating and amplifying growth factor–mediated and mitogenic signals from the cytoplasm to the nucleus [1]. Three major MAPK pathways have been described; the extracellular signal-related kinase (ERK), the c-Jun NH2-terminal. Doi:10.1158/0008-5472.CAN-06-2371 kinase (JNK), and the p38 MAPK pathways. In addition to their roles in regulating normal cell growth, all three classes are known to be activated in response to genotoxic stresses such as ionizing radiation (IR) and UV light. The JNK and p38 MAPKs are largely associated with apoptosis, whereas signaling via the ERK pathway is primarily linked with cell proliferation and survival, and seems to prevent apoptosis. Deregulation of the MAPK pathways is associated with genomic instability and cancer

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