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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.
Highlights
The 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
Stimulatory α subunit of G protein (Gαs) inhibited radiation-induced ATM activation in lung cancer cells To investigate the effects of cAMP signaling on radiationinduced DNA damage responses, an EE-tagged constitutively active mutant long form of the α subunit of stimulatory heterotrimeric GTP binding protein (GαsQL) was transiently expressed in H1299 human lung cancer cells
Irradiation of H1299 cells with γ-rays induced a biphasic phosphorylation of ATM: ATM phosphorylation started at 15 min after irradiation and reached peak levels at 30 min, followed by a second peak at 120 min
Summary
The 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. The 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. 50% of all cancer patients are treated with radiotherapy, and there is a wide inter-patient variability in tumor responses. 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 damaged cells are commonly eliminated from the proliferative pool through cellular senescence or several types of cell death, including apoptosis [2]
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