Background
Radiotherapy is one of the major treatment modalities for benign and malignant diseases throughout the body. Approximately 50% of all cancer patients are treated with radiotherapy, and there is a wide inter-patient variability in tumor responses. Strategies to improve radiotherapy seek to increase the effects of radiation on the tumor or decrease the effects on normal tissues. An improved understanding of the molecular response of cells and tissues to ionizing radiation has contributed to improvements in radiotherapy[
1]. Ionizing radiation can induce single-strand breaks (SSBs) and double-strand breaks (DSBs) in the DNA double helix backbone that trigger DNA damage responses. The DNA damage response machinery delays cell cycle progression and activates cell cycle checkpoints to provide more time for lesion repair and prevent the transfer of damaged DNA to progeny. When repair fails, the damaged cells are commonly eliminated from the proliferative pool through cellular senescence or several types of cell death, including apoptosis[
2].
Together with ataxia–telangiectasia and RAD3-related (ATR) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the ataxia–telangiectasia mutated (ATM) protein kinase plays a central role in coordinating the cellular response to DNA damage[
3‐
5]. Deficiency in the ATM kinase causes ataxia-telangiectasia, a rare autosomal recessive disorder characterized by hypersensitivity to radiation and predisposition to cancer. ATM belongs to the phosphatidylinositol 3 kinase-like kinase (PIKK) family of Ser/Thr-protein kinases, which contains ATR, DNA-PKcs and mTOR (mammalian target of rapamycin)[
6]. Following DNA damage, an intermolecular autophosphorylation occurs on Ser-1981 of ATM that disrupts the inactive homodimer and enables the kinase domain to phosphorylate several target substrates and trigger downstream signaling pathways[
5]. Many ATM substrates regulate gene expression, cell-cycle checkpoints, DNA repair and apoptosis[
7]. Thus, ATM is a potential target molecule for the development of novel radiosensitizers[
8,
9].
Cyclic adenosine 3', 5'-monophosphate (cAMP) is a second messenger that is produced from ATP by adenylate cyclases and degraded into 5’-AMP by cyclic nucleotide phosphodiesterases. Adenylate cyclase is activated by stimulatory heterotrimeric GTP-binding proteins (G proteins), which are activated by G protein coupled receptor (GPCR)-agonist complexes[
10]. cAMP binds to and activates the cAMP-dependent protein kinase (PKA), the cAMP-activated guanine exchange factors (Epacs), which are the guanine nucleotide exchange factors (GEFs) for monomeric G protein Raps[
11], and the cyclic nucleotide-gated channels functioning in transduction of sensory signals (CNGs). The cAMP signaling system regulates numerous cellular responses including gene expression, growth, differentiation, proliferation, and apoptosis.
We have reported that the cAMP signaling system modulates cancer cell apoptosis by regulating the expression of Bcl-2 family proteins[
12,
13] and the inhibitor of apoptosis protein (IAP)[
14] in response to various DNA damaging agents, including ionizing radiation. Recently, the cAMP signaling system was found to inhibit the repair of γ-ray-induced DNA damage by promoting degradation of the XRCC1 protein in human lung cancer cells[
15]. The cAMP signaling system was also reported to inhibit DNA-damage induced apoptosis of leukemia cells by promoting acetylation and turnover of p53[
16,
17]. Thus, we hypothesized that the cAMP signaling system might be involved in the regulation of ATM activation, the key event triggering signaling pathways in response to DNA damage. This study aimed to investigate the mechanism through which the cAMP signaling system regulates ATM activation and cellular responses following γ-ray irradiation. We found that Gαs inhibits ATM activation via the Gαs-cAMP-PKA-PP2A pathway and augments radiation-induced apoptosis following γ-ray irradiation in non-small cell lung cancer cells.
Discussion
This study aimed to investigate the mechanism through which the cAMP signaling system might regulate the activation of ATM and apoptosis following γ-ray irradiation. We found that cAMP signaling inhibits radiation-induced activation of ATM by PKA-dependent activation of PP2A, and the cAMP signaling system augments radiation-induced apoptosis partially by reducing the ATM-dependent activation of NF-κB in human lung cancer cells and mouse lung.
Our finding that the cAMP signaling system inhibits radiation-induced activation of ATM by PKA-dependent activation of PP2A is supported by several results. First, radiation-induced phosphorylation of ATM was inhibited by expression of constitutively active Gαs and by treatment with Gαs-coupled receptor agonists or an adenylate cyclase activator, forskolin. Second, treatment with a PP2A inhibitor or knock down of PP2A B56δ subunit abolished the ATM-inhibitory effect of Gαs. Third, expression of the active Gαs increased the phosphorylation of the PP2A B56δ subunit and enhanced PP2A activity. In addition, inhibition of PKA abolished the PP2A activation induced by Gαs, thereby restoring ATM phosphorylation. Moreover, inhibition of radiation-induced ATM phosphorylation by the cAMP signaling system was observed in human lung cancer cells, murine melanoma cells, and murine lung tissue, suggesting that the inhibition occurs in many tissues.
ATM is primarily recruited to double-strand DNA breaks and activated through interactions with the MRE11–RAD50–NBS1 (MRN) complex[
19]. ATM protein undergoes autophosphorylation at Ser-1981 and forms monomers from an inactive dimer following double-strand DNA breaks; ATM autophosphorylation is considered a hallmark of ATM activation[
20]. Recently, ATM was found to be activated independently from DNA damage through redox-dependent mechanisms and to participate in diverse signaling pathways involved in metabolic regulation and cancer[
5]. However, no previous reports show that the cAMP signaling system regulates radiation-induced activation of ATM. Caffeine is known to inhibit ATM activation and has been studied as a potential radioenhancer[
8]. Caffeine is also known to inhibit cAMP phosphodiesterase, which may increase the cAMP level[
21], suggesting the involvement of the cAMP signaling system in ATM activation. However, caffeine was reported to inhibit the enzymatic activity of ATM immunoprecipitates in vitro, which was interpreted as direct inhibition of ATM by caffeine[
22], independent of the cAMP signaling system. Thus, to the best of our knowledge, this paper presents the first evidence that the cAMP signaling system can regulate radiation-induced ATM activation.
PP2A-mediated inhibition of ATM activation in a PKA-dependent pathway is supported by the previous report that PKA phosphorylates Ser-566 of the PP2A B56δ subunit to stimulate PP2A activity[
23]. PP2A forms complexes with ATM and dephosphorylates the autophosphorylated Ser-1981 in undamaged cells to suppress the intrinsic ATM activity[
24].
This study shows that the cAMP signaling system augments radiation-induced apoptosis by inhibiting ATM activation. This finding is based on the result that radiation-induced apoptosis was augmented by the activation of the cAMP signaling system and by inhibition of ATM with a specific inhibitor, KU55933, and siRNA against ATM in cancer cells and mouse lung. In addition, the cAMP signaling system inhibits radiation-induced activation of ATM. This finding is supported by the fact that ATM is a master regulator of cellular responses to DNA damage caused by ionizing radiation and activates downstream signaling pathways to regulate various DNA damage responses including cell cycle, DNA repair, and apoptosis[
25,
26]. This finding suggests that cAMP signaling can modulate radiation-induced apoptosis by regulating radiation-induced ATM activation. This finding also implies that drugs targeting the cAMP signaling pathway could be possibly used to modulate radiation-induced apoptosis, thereby increasing the radiosensitivity of cancer cells or protecting normal cells from radiation. The cAMP signaling system can stimulate or inhibit apoptosis depending on cell types[
27] through diverse molecular mechanisms involving Bcl-2 family proteins, p53, and histone deacetylase[
16,
28,
29]. Thus, this study presents a novel mechanism for the cAMP signaling system to regulate cancer cell apoptosis. It is also plausible that the cAMP signaling system modulates other cellular responses to DNA damage mediated by ATM, such as DNA damage repair and cell cycle arrest.
The cAMP signaling system was found to augment radiation-induced apoptosis partly by inhibiting ATM-mediated NF-κB activation in this study. This finding is substantiated by the result that activation of the cAMP signaling system or inhibition of ATM resulted in a reduction of radiation-induced NF-κB activation and augmentation of apoptosis. In addition, inhibition of NF-κB activation by treatment with several NF-κB specific inhibitors augmented radiation-induced apoptosis, but activation of NF-κB signaling by expression of constitutively active IKKs abolished apoptosis-augmenting effect of cAMP signaling system. ATM can stimulate NF-κB activation, which induces the expression of anti-apoptotic proteins to protect cells from apoptosis. Thus, inhibition of ATM may compel the cells to undergo apoptosis as observed in this study[
30,
31]. However, ATM can play contrasting roles in DNA damage-induced apoptosis, and ATM induces apoptosis by phosphorylating downstream target substrates such as p53, TRF1[
32] and NBS1[
33]. Therefore, ATM seems to show different apoptotic effects depending on the cell type, DNA damage-inducing agent, the severity of DNA damage, and the presence of functional p53[
34].
NF-κB is activated in response to various immune and inflammatory stimuli, and it is also activated by ionizing radiation to protect damaged cells from apoptotic cell death[
35,
36]. The signal transduction mechanisms that link DNA damage to NF-κB activation are relatively unknown, but signaling pathways involving ATM and NF-κB essential modulator (NEMO) are reported to cooperate to directly link DNA damage in the nucleus to NF-κB activation in the cytosol[
37]. ATM is involved in the sequential post-translational modification of NEMO, and ATM translocates in a calcium-dependent manner to the cytosol and membrane[
38]. Cytosolic ATM activates TGFβ-activated kinase (TAK1), which phosphorylates IKKβ to trigger ubiquitin-proteasome dependent degradation of IκB and NF-κB activation[
18]. In agreement with these findings, the cAMP signaling system was observed to reduce the cytosolic translocation of phosphorylated ATM accompanied with increased IκB level following γ-ray irradiation in this study, which may have resulted from inhibition of radiation-induced ATM phosphorylation and could cause reduced NF-κB activation and augmented apoptosis.
In this study, the role of the cAMP signaling system in ATM, PP2A and NF-κB activation, as well as in apoptosis, following γ-ray irradiation was assessed by activating the signaling system using various mechanisms: expression of constitutively active Gαs, treatment with Gαs-coupled receptor agonists such as isoproterenol for β-adrenergic receptors and prostaglandin E2 for prostanoid receptors, or treatment with the adenylate cyclase activator forskolin. Furthermore, similar effects were observed in A549 and p53-null H1299 human lung cancer cells, murine melanoma cells, and murine lung tissue, suggesting comparable effects of the cAMP signaling system in various cells and tissues. These results reinforce the inhibitory role of the cAMP pathway in radiation-induced activation of ATM by PKA-dependent activation of PP2A. These findings also suggest the augmentation of radiation-induced apoptosis potentially through a reduction of ATM-dependent NF-κB activation.
Methods
Cell culture and reagents
Human non-small cell lung cancer cell lines H1299 and A549 (Korea Cell Line Bank, Seoul, Korea) and B16-F10 mouse melanoma cells (ATCC, Manassas, VA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (JBI, Korea) and 100 units/ml penicillin/streptomycin. The cells were incubated in a 5% CO2 incubator at 37°C. H89, isoproterenol, dimethyl sulfoxide (DMSO), and 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) were purchased from Sigma (St. Louis, MO, USA). Forskolin, pyrrolidine dithiocarbamate (PDTC), IKK inhibitor VII, BAY 11–7082 and isobutylmethylxanthine (IBMX) were purchased from Calbiochem (La Jolla, CA, USA). The FITC Annexin V apoptosis detection kit was purchased from BD Biosciences (San Diego, CA, USA). Prostaglandin E2 (PGE2) and okadaic acid were purchased from Cayman Chemical (Ann Arbor, MI, USA). KU-55933 was purchased from Selleck Chemicals (Houston, TX, USA). Bovine serum albumin (BSA) and goat anti-rabbit IgG-FITC were purchased from Santa Cruz Biotechnology (CA USA). Phenylmethanesulfonyl fluoride (PMSF), sodium orthovanadate, sodium fluoride, and a protease inhibitor mixture were purchased from Roche Molecular Biochemicals (Indianapolis, IN, USA).
Animal experiment
Care, use, and treatment of animals were done in agreement with the guidelines established by the Seoul National University Institutional Animal Care and Use Committee (SNU- 110415–2). Male BALB/c mice (4 week-old) were housed for 1 week before the experiments and maintained on a 12-h light/dark cycle, with food and water freely available. The mice were divided into the control (n = 6) and the treatment (n = 6) group. The treatment group mice were injected intraperitoneally with forskolin (20 μg/g), and the control mice received an equal volume of Dulbecco's Phosphate-Buffered Saline. After 6 h, the mice were exposed to whole body γ-ray irradiation (10 Gy).
Expression constructs and transient transfection
H1299 cells were transfected with a EE-tagged constitutively active mutant (GαsQ227L, GαsQL) of long form stimulatory α subunit of G protein (Gαs) in a pcDNA3 vector (Invitrogen, Paisley, UK) using the calcium phosphate method[
39]. A glutamine residue that is essential for the intrinsic GTPase activity is replaced with leucine in GαsQL[
40]. A dominant negative mutant of PKA (dnPKA) was a gift from Dr. G. Stanley McKnight (University of Washington, WA, USA)[
41]. Constitutively active mutant of I-kappa B kinase alpha S176E/S180E (IKKαSE) and beta S177E/181E (IKKβSE) were gifts from Dr. Dae-Myung Jue (The Catholic University of Korea)[
42]. Small interfering RNAs (siRNAs) against ATM (cat. sc-29761) were purchased from Santa Cruz Biotechnology (CA, USA), and siRNA against PP2A B56δ (FlexiTube no. SI2653350) from Qiagen (Hilden, Germany). Control siRNA (5’-AATTCTCCGAACGTGTCACGT-3’) were purchased from Bioneer (Daejeon, Korea). siRNAs were transfected using Lipofectaimine (Invitrogen, Paisley, UK), and the cells were treated with other reagents at 48 h after transfection.
Preparation of cytosolic and nuclear fractions
The cultured cells were harvested and then disrupted in lysis buffer A (0.33 M sucrose, 10 mM Hepes (pH 7.4), 1 mM MgCl2, 0.1% Triton X-100, protease inhibitor cocktail (PIC), and PMSF). The cell lysates were centrifuged for 5 min at 800 g, and the supernatants were collected to use as the cytosolic fractions. The resulting pellets were resuspended in lysis buffer B (0.45 M NaCl, 10 mM Hepes (pH 7.4), PIC, and PMSF) and centrifuged for 5 min at 20,000 g. The supernatants were collected to use as the nuclear fractions.
Western blot analysis
Western blotting was performed as previously described[
28]. Antibodies against Gαs, Ku70, ATM, COX-1, phosphorylated cAMP response element binding protein (p-CREB, Ser-133), PP2A B56δ, IκBα, p50 and p65 of NF-κB were obtained from Santa Cruz Biotechnology (CA, USA). Antibodies against Rad50, p-ATM (Ser-1981), γ-H2AX, Ku80, CREB, DNA-PKcs, poly (ADP-ribose) polymerase (PARP), cleaved caspase-3 (Asp-175), p-AKT (Ser-473), AKT, p-IκBα, and Myc-tag were obtained from Cell Signaling Technology (Beverly, MA, USA). An antibody against β-actin was purchased from Sigma (St. Louis, MO, USA), and an antibody against EE-tag was purchased from Covance (Princeton, NJ, USA). An antibody against phosphorylated B56δ (Ser-566) of protein phosphatase 2A (PP2A) was kindly provided by Dr. Paul Greengard (The Rockefeller University, New York)[
23]. The proteins were visualized using the Enhanced Chemiluminescence (ECL) reagent (Thermo scientific, Waltham, MA) and detected using an LAS-3000 (R&D Systems, Inc. Minneapolis, MN, USA). The densities of the protein bands were quantified using the Multi Gauge v2.3 software (Fuji, Tokyo, Japan), and the relative band densities were expressed as ratios of the corresponding control densities.
Immunofluorescence microscopy
H1299 cells were plated in 60 mm dishes and incubated until they became 60% confluent. The cells were transfected with vector or GαsQL plasmids, and after 24 h, they were irradiated with γ-rays (5 Gy) from a cesium (Cs) irradiator[
15]. After 30 min, the cells were fixed with 4% paraformaldehyde for 20 min and permeated with 0.5% Triton X-100 for 10 min. After blocking with 2% BSA for 1 h, the cells were incubated overnight with an antibody against p-ATM (1:200) in 2% BSA, followed by incubation with goat anti-rabbit IgG-FITC (1:100) and DAPI (0.5 μl/ ml) for 1 h. The stained cells were observed with a confocal microscope (LSM 501 META, Carl Zeiss, Inc. USA).
TUNEL assay
Extracted lung tissues from BALB/c mice were deparaffinized and hydrated. The tissues were stained using the ApopTag fluorescein in situ apoptosis detection kit (Chemicon International, Temecula, CA, USA), and apoptosis was observed using confocal laser scanning microscopy (TCS SP2, Leica, Wetzler, Germany).
PP2A activity assay
Cells were prepared and lysed following the protocol of the PP2A activity assay kit (R&D Systems, Inc. Minneapolis, MN, USA). In brief, the cell lysates were incubated with Serine/Threonine Phosphatase substrate I for 30 min, and then, 10 μl of Malachite Green Reagent A was added and incubated for 10 min. Then, 10 μl of Malachite Green Reagent B was added and incubated for 20 min, and the absorbance at 620 nm was measured with the Benchmark Plus™ microplate reader (Bio-Rad, PA, USA).
Flow cytometry
The cells were exposed to γ-rays (10 Gy) and incubated for 24 h. Then, the cells were washed twice with phosphate-buffered saline, harvested, and spun at 3,500 g for 5 min at 4°C. The cells were incubated in 1X Annexin V buffer containing Annexin V and PI for 15 min. Stained cells were quantified with a FacsCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) using 10,000 cells per measurement.
Dual luciferase reporter assay
H1299 cells were transfected with plasmids containing luciferase reporter genes (NF-κB-pLuc and Renilla-pLuc) together with vector or GαsQL plasmids using the calcium phosphate method. Luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega Corp., Madison, WI, USA) according to the manufacturer’s protocol. At least four independent experiments were performed in duplicate, and promoter activities were normalized using Renilla luciferase activity.
Data analysis
At least three or more independent experiments were conducted for all the analyses, and the data were presented as the means ± standard errors (SE). The non-parametric Mann–Whitney U test was used to analyze the mean values, and a p value of less than 0.05 was considered statistically significant.
Competing interests
All authors declare that they have no competing interests.
Authors’ contributions
EC and EK equally contributed in designing and performing experiments and wrote the manuscript together, and SK performed some of the experiments. YJ conceived experiments, participated in its design and coordination, edited manuscript, provided funding and resources required for conducting all experiments. All authors read and approved the final manuscript.