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

Background: 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. Methods: Lung 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. Results: Transient 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. Conclusions: cAMP 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.This study was supported by a National Research Foundation (NRF) grant funded by the Korea government (MEST) (No. 2007–2001258), by Basic Science Research Program through the NRF funded by the Ministry of Education, Science and Technology (2012R1A1A2044374), and a grant from the National R&D Program for Cancer Control, Ministry of Health and Welfare, Republic of Korea (0720540).Peer Reviewe

인체 태반조직 Glutathione S-transferase π의 정제 및 특성구명

Human placental glutathione S-transferase(GST - π ) was purified to the apparent homgeneity through salting-out with ammonium sulfate and the consecutive chromatography on carboxymethyI(CM)-, diethylaminoethyI(DEAE)-cellulose and S-hexylglutathione sepharose 6B affinity column. For the characterization of the apparently purified enzyme, sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS PAGE), kinetic studies, neuraminidase digestion, and isoelectrofocusing were performed. The yield of the enzyme was 11 percent with the 1107 fold purification and the respective specific activity to l-chloro-2,4-dinitrobenzene(CDNB), 1,2-dichloro-4-nitrobenzene(DCNB) and p-nitrophenyl chloride was 62 JU/mg, 0.12 IU/mg, and almost non-detectable. The Km of the enzyme for reduced glutathione(GSH) was 0.085 mM at the concentration of 2 mM CDNB, while its Km for CDNB was 0.46 mM at the fixed concentration of 5 mM GSH. Calcium, magnesium, zinc, ethylenediamintertraacetic acid(EDTA) and ethylenedioxydiethylenedinitrilotetraacetic acid (EGTA) did not show any significant effect on enzyme activity. The subunit of the enzyme with a molecular weight of 25,000 did not reveal the molecular weight change after neuraminidase treatment. Isoelectrofocusing of the enzyme showed two bands, of which the pi of the major band was 4.48, while that of the minor band, 4.55. The specific antibody, raised against the purified GST- π in the rabbit serum indicated the immunologic cross-reactivity to the acidic GST from the human granulocyte

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

Background 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. Methods Lung 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. Results Transient 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. Conclusions cAMP 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

A novel balanced-lethal host-vector system based on glmS.

During the last decade, an increasing number of papers have described the use of various genera of bacteria, including E. coli and S. typhimurium, in the treatment of cancer. This is primarily due to the facts that not only are these bacteria capable of accumulating in the tumor mass, but they can also be engineered to deliver specific therapeutic proteins directly to the tumor site. However, a major obstacle exists in that bacteria because the plasmid carrying the therapeutic gene is not needed for bacterial survival, these plasmids are often lost from the bacteria. Here, we report the development of a balanced-lethal host-vector system based on deletion of the glmS gene in E. coli and S. typhimurium. This system takes advantage of the phenotype of the GlmS(-) mutant, which undergoes lysis in animal systems that lack the nutrients required for proliferation of the mutant bacteria, D-glucosamine (GlcN) or N-acetyl-D-glucosamine (GlcNAc), components necessary for peptidoglycan synthesis. We demonstrate that plasmids carrying a glmS gene (GlmS(+)p) complemented the phenotype of the GlmS(-) mutant, and that GlmS(+) p was maintained faithfully both in vitro and in an animal system in the absence of selection pressure. This was further verified by bioluminescent signals from GlmS (+)pLux carried in bacteria that accumulated in grafted tumor tissue in a mouse model. The signal was up to several hundred-fold stronger than that from the control plasmid, pLux, due to faithful maintenance of the plasmid. We believe this system will allow to package a therapeutic gene onto an expression plasmid for bacterial delivery to the tumor site without subsequent loss of plasmid expression as well as to quantify bioluminescent bacteria using in vivo imaging by providing a direct correlation between photon flux and bacterial number

Maintenance of <i>^S.tGlmS⁺p</i> in <i>S. typhimurium</i> proliferating in tumor tissues.

<p><i>SMR2130</i> (GlmS⁻) and <i>SKS1002</i> (WT) strains carrying <i>^S.tGlmS⁺pLux</i> (3×10⁷ CFU), were injected intravenously into mouse bearing 4T-1 (mouse breast cancer) or ASPC-1 (human pancreatic cancer). Tumor tissue were sampled at 7 days after the injection, homogenized, spread on LB plates containing kanamycin and chloramphenicol and/or amphicilin, and enumerated total number of bacteria (Km^R Cm^R) and those carrying plasmid(Amp^R).</p