cAMP signaling inhibits radiation-induced ATM phosphorylation leading to the augmentation of apoptosis in human lung cancer cells

Abstract

BackgroundThe ataxia–telangiectasia mutated (ATM) protein kinase plays a central role in coordinating the cellular response to radiation-induced DNA damage. cAMP signaling regulates various cellular responses including metabolism and gene expression. This study aimed to investigate the mechanism through which cAMP signaling regulates ATM activation and cellular responses to ionizing radiation in lung cancer cells.MethodsLung cancer cells were transfected with constitutively active stimulatory G protein (GαsQL), and irradiated with γ-rays. The phosphorylation of ATM and protein phosphatase 2A was analyzed by western blotting, and apoptosis was assessed by western blotting, flow cytometry, and TUNNEL staining. The promoter activity of NF-κB was determined by dual luciferase reporter assay. BALB/c mice were treated with forskolin to assess the effect in the lung tissue.ResultsTransient expression of GαsQL significantly inhibited radiation-induced ATM phosphorylation in H1299 human lung cancer cells. Treatment with okadaic acid or knock down of PP2A B56δ subunit abolished the inhibitory effect of Gαs on radiation-induced ATM phosphorylation. Expression of GαsQL increased phosphorylation of the B56δ and PP2A activity, and inhibition of PKA blocked Gαs-induced PP2A activation. GαsQL enhanced radiation-induced cleavage of caspase-3 and PARP and increased the number of early apoptotic cells. The radiation-induced apoptosis was increased by inhibition of NF-κB using PDTC or inhibition of ATM using KU55933 or siRNA against ATM. Pretreatment of BALB/c mice with forskolin stimulated phosphorylation of PP2A B56δ, inhibited the activation of ATM and NF-κB, and augmented radiation-induced apoptosis in the lung tissue. GαsQL expression decreased the nuclear levels of the p50 and p65 subunits and NF-κB-dependent activity after γ-ray irradiation in H1299 cells. Pretreatment with prostaglandin E2 or isoproterenol increased B56δ phosphorylation, decreased radiation-induced ATM phosphorylation and increased apoptosis.ConclusionscAMP signaling inhibits radiation-induced ATM activation by PKA-dependent activation of PP2A, and this signaling mechanism augments radiation-induced apoptosis by reducing ATM-dependent activation of NF-κB in lung cancer cells.

Cyclic AMP Signaling Reduces Sirtuin 6 Expression in Non-small Cell Lung Cancer Cells by Promoting Ubiquitin-Proteasomal Degradation via Inhibition of the Raf-MEK-ERK (Raf/Mitogen-activated Extracellular Signal-regulated Kinase/Extracellular Signal-regulated Kinase) Pathway

“…Experiments showed that 1) cAMP signaling augmented radiation-induced apoptosis in lung cancer cells, 2) that cAMP signaling reduced SIRT6 expression, 3) that exogenous expression of SIRT6 abolished the cAMP signaling-mediated effects on apoptosis, and 4) that exogenous expression of SIRT6 reduced the level of radiation-induced apoptosis. We previously showed that cAMP signaling regulates apoptosis in various cancer cells, including lung cancer cells, by modulating the expression of Bcl-2 family proteins (8), the expression of X-linked inhibitor of apoptosis protein (22), and the activity of ataxia-telangiectasia mutated (ATM) protein kinase (21). Here, we describe a novel mechanism through which cAMP signaling augments apoptosis, reducing SIRT6 expression.…”

“…Western Blot Analysis-Western blotting was performed as previously described (21). Antibodies against SIRT1, -2, and -7, Skp2, ubiquitin, and p-CREB were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and antibodies against SIRT6, CHIP, Mdm2, p-c-Raf, c-Raf, p-MEK, MEK, p-ERK, ERK, CREB, p-p38, p38, p-JNK, JNK, poly(ADP-ribose) polymerases (PARP), caspase-3, and FLAG were purchased from Cell Signaling Technology (Beverly, MA).…”

Impact of p38 mitogen-activated protein kinase inhibition on immunostimulatory properties of human 6-sulfo LacNAc dendritic cells

“…Interestingly, cAMP signaling inhibits radiation induced phosphorylation of ATM in lung cancer [42]. Addition of forskolin In summary we show that although IR inhibits IL-23 secretion from DC mono-cultures, the inclusion of FB provides a positive feedback loop that serves to maintain IL-23 secretion by DC.…”

“…However PGE 2 also up-regulates IL-23 through activation of the cAMP-PKA (Protein Kinase-A) signaling pathway [40,41]. Interestingly, in lung cancer cAMP signaling inhibits IR-induced phosphorylation of ATM [42]. Therefore, we sought to dissect the molecular mechanism responsible for IL-23 secretion in irradiated DC-FB co-cultures.…”

Analyses of PDE-regulated phosphoproteomes reveal unique and specific cAMP-signaling modules in T cells

“…A number of the biological processes identified by GO analysis have been previously reported to be regulated by cAMP. These include mRNA splicing (64)(65)(66), spindle organization (67,68), fibroblast migration (69,70), lamellipodium assembly (71,72), apoptosis (73)(74)(75)(76), ATM signaling (77,78), gene silencing via microRNA (79)(80)(81)(82), T-cell selection (83), and cytoskeletal reorganization (84). This study suggests which molecular substrates might participate in the regulation of these processes.…”