The cAMP signaling system inhibits the repair of γ-ray-induced DNA damage by promoting Epac1-mediated proteasomal degradation of XRCC1 protein in human lung cancer cells

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Abstract

Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on γ-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (GαsQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of GαsQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after γ-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2′-O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2′-O-Me-cAMP and restored XRCC1 protein level following γ-ray irradiation. From these results, we conclude that the cAMP signaling system inhibits the repair of γ-ray-induced DNA damage by promoting the ubiquitin–proteasome dependent degradation of XRCC1 in an Epac-dependent pathway in lung cancer cells.

Highlights

► cAMP signaling system inhibits repair of γ-ray-induced DNA damage. ► cAMP signaling system inhibits DNA damage repair by decreasing XRCC1 expression. ► cAMP signaling system decreases XRCC1 expression by promoting its proteasomal degradation. ► The promotion of XRCC1 degradation by cAMP signaling system is mediated by Epac1.

Introduction

Mammalian cells are constantly subjected to their DNA damage from exogenous DNA-damaging agents, such as ionizing radiation and chemical agents, and endogenous processes, such as replication and programmed genome rearrangements [1]. The resulting DNA damage may induce mutations that cause the loss or incorrect transmission of genetic information, which in turn can cause developmental abnormalities, cell death and tumorigenesis. Thus, eukaryotic cells have evolved several mechanisms to monitor the integrity of their genome and to repair the damaged DNA [2].

The X-ray repair cross-complementing protein 1 (XRCC1) gene was the first mammalian gene isolated that affects cellular sensitivity to ionizing radiation [3]. Human XRCC1 is a 70-kDa protein that contains 633 amino acid residues. XRCC1 does not have any enzyme activity, but it has at least three discrete domains that can interact specifically with enzymes involved in DNA repair [4]. Thus, XRCC1 plays an essential role in base excision repair and single strand break repair [5]. Epidemiological association of XRCC1 polymorphisms and carcinogenesis have been studied extensively in incidences of breast cancer and lung cancer [6], and XRCC1 was reported to have potential as a predictive marker in lung, head and neck cancer patients treated with radiation and chemotherapy [7].

The cyclic AMP (cAMP) signaling system is activated by cAMP, a second messenger molecule formed from ATP by adenylyl cyclase. The activity of adenylyl cyclase is stimulated by stimulatory heterotrimeric GTP-binding proteins (G proteins), which are activated by various external signals bound to G protein-coupled receptors [8]. cAMP activates cAMP-dependent protein kinase (PKA), exchange proteins directly activated by cAMP (Epac), and cyclic nucleotide-gated ion channels, and thereby regulates a variety of cellular responses. The cAMP signaling system is involved in sensory perception, metabolic control, and the regulation of gene expression, cellular growth, differentiation and proliferation [9], [10]. In previous studies, we found that the cAMP signaling system modulates DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis (IAPs) [11], [12], [13]. Thus, we hypothesized that the cAMP signaling system may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on radiation-induced DNA damage repair in H1299 lung cancer cells. We found that the cAMP signaling pathway inhibits the repair of γ-ray induced DNA damage by promoting the proteasomal degradation of XRCC1 protein, and it is mediated by EPAC1.

Section snippets

Cell culture and reagents

Human non-small cell lung cancer cells, H1299 and A549 cells (Korea Cell Line Bank, Seoul, Korea), were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (JBI, Korea) and 100 units/ml penicillin/streptomycin. Cells were maintained in a 5% CO2 incubator at 37 °C. H89, cycloheximide (CHX), dimethyl sulfoxide (DMSO), 4,6-diamidino-2-phenylindole dihydrochloride (DAPI), 8-pCPT-2′-O-Me-cAMP, and avidin-labeled 8-oxo-7,8-dihydro-2′-deoxyguanosine fluorescein

The cAMP signaling system inhibited the repair of radiation-induced DNA damage in H1299 lung cancer cells

We assessed the effect of cAMP signaling system on DNA damage repair by expressing a constitutively active Gαs or treating with forskolin, which activates the cAMP signaling system by stimulating adenylyl cyclase. Transient expression of the constitutively active GαsQL augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of GαsQL increased the green fluorescence of 8-oxo-deoxyguanosine (8-oxo-dG) by 3.27-fold over vector-transfected

Discussion

This study was performed to determine whether the cAMP signaling system can modulate the repair of γ-ray-induced DNA damage in lung cancer cells and, if so, to elucidate the underlying molecular mechanism. This study shows that the cAMP signaling system inhibits the repair of γ-ray-induced DNA damage by decreasing the expression of XRCC1 protein in lung cancer cells and that it decreases XRCC1 expression by promoting EPAC-mediated ubiquitin–proteasome dependent degradation of XRCC1.

The finding

Acknowledgments

This study was supported by the National Research Foundation of Korea(NRF) Grant funded by the Korea government (MEST) (No. 2007-2001258), and a grant from the National R&D Program for Cancer Control, Ministry of Health and Welfare, Republic of Korea (0720540). We wish to thank Dr. Keith W. Caldecott (University of Sussex, UK) for XRCC1 expression plasmids, and Dr. Stanley McKnight (University of Washington) for a PKA expression plasmid.

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