J Korean Med Sci.  2020 Dec;35(48):e371. 10.3346/jkms.2020.35.e371.

Cell-type-specific Modulation of Nonhomologous End Joining of Gamma Ray-induced DNA Double-strand Breaks by cAMP Signaling in Human Cancer Cells

• Noh SE
• Juhnn YS

• KMID: 2509524
• DOI: http://doi.org/10.3346/jkms.2020.35.e371

Abstract

Background
Cyclic AMP (cAMP) signaling is activated by various hormones and neurotransmitters and regulates numerous physiological phenomena, including energy metabolism, gene expression, and proliferation. cAMP signaling plays a role in the repair of DNA damage, but its specific function is inconsistent in the literature. The present study aimed to investigate the mechanism of the different roles of cAMP signaling in DNA repair by analyzing the cell-type differences in the modulation of DNA repair by cAMP signaling following γ-ray irradiation.
Methods
cAMP signaling was activated in human malignant melanoma cells (SK-MEL-2 and SK-MEL-28), human uterine cervical cancer cells (HeLa and SiHa) and human non-small cell lung cancer cells (H1299 and A549) by expressing a constitutively active mutant of the long-form stimulatory α subunit of GTP-binding protein or by treating with isoproterenol and prostaglandin E2 before γ-ray irradiation. DNA damage was quantitated by western blot analysis of γ-H2AX, and non-homologous end joining (NHEJ) was assessed by fluorescent reporter plasmid repair assay and immunofluorescence of microscopic foci of XRCC4 and DNA-ligase IV.
Results
cAMP signaling modulated DNA damage, apoptosis and the NHEJ repair following γ-ray irradiation differently depending upon the cell type. cAMP signaling regulated the phosphorylation of DNA-dependent protein kinase catalytic subunit (DNA-PKcs) at Ser2056 and Thr2609 in cell-type-specific manners following γ-ray irradiation, an activity that was mediated by protein kinase A.
Conclusion
cAMP signaling modulates the NHEJ repair of γ-ray-induced DNA damage in melanoma cells, uterine cervical cancer cells and lung cancer cells in a cell-typespecific manner, and the modulation is likely mediated by protein kinase A-dependent phosphorylation of DNA-PKcs. This study suggests that cell- and tissue-specific modulation of DNA damage repair by cAMP signaling may contribute to improve the therapeutic efficiency of radiation therapy.

Keyword

cAMP; DNA Repair; Cell-type Specificity; Non-homologous End Joining; DNA-dependent Protein Kinase; Gamma Radiation

Figure

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  Fig. 1 Cell-type-specific modulation of γ-ray-induced DNA damage and apoptosis by cAMP signaling. (A) Effects of pretreatment with isoproterenol and PGE2 on γ-H2AX formation following γ-ray irradiation in human malignant melanoma cells. The empty bars present SK-MEL-2 cells, and the filled bars present SK-MEL-28 cells. (B) Effects of PGE2 and isoproterenol on γ-H2AX formation following γ-ray irradiation in human uterine cervical cancer cells. The empty bars present HeLa cells, and the filled bars present SiHa cells. (C) Effects of PGE2 and isoproterenol on γ-H2AX formation following γ-ray irradiation of human non-small cell lung cancer cells. The empty bars present H1299 cells, and the filled bars present A549 cells. The cells pretreated with 1 μM isoproterenol (ISO) or 20 μM PGE2 for 30 minutes were irradiated with γ-rays (5 Gy) and collected with a cell scraper after 1 h for analysis by western blotting. (D, E, F) PGE2 effects on the cleavage of PARP and caspase-9 following γ-ray irradiation in SK-MEL-28 cells (D), HeLa cells (E), and A549 cells (F). The cells pretreated with PGE2 (20 μM, 30 minutes) were irradiated with γ-rays (5 Gy) and collected with a cell scraper after 24 hours (A549 and HeLa) or 48 hours (SK-MEL-28) for analysis by western blotting. The empty bars shows cleaved PARP, and the filled bars shows cleaved caspase-9. The means ± standard error calculated from the three independent experiments were presented as columns. The asterisk (*) denotes statistically significant differences compared with the respective control (P ≤ 0.05, Mann-Whitney U test).

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  Fig. 2 Cell-type-specific modulation of NHEJ repair by cAMP signaling. (A, B, C) Effects of cAMP signaling upon the disappearance of γ-H2AX formed by γ-ray irradiation in SK-MEL-28 cells (A), HeLa cells (B), and A549 cells (C). The cells were irradiated with γ-ray, then incubated for 10 min before PGE2 treatment and collected at the presented time points for analysis by western blotting. The empty circles present vehicle-stimulated control cells, and the filled circles present PGE2- stimulated cells. (D) cAMP signaling effects upon NHEJ repair efficiency. The cells were cotransfected with GαsQL and SceI-linearized GFP fluorescent reporter plasmids using Lipofectamine 3000 and harvested after 24 hours for flow cytometric analysis. The NHEJ repair efficiencies were determined as the ratios of green fluorescence emitted from the repaired reporter plasmid to the red fluorescence emitted from the DsRed control plasmid. (E, F, G) PGE2 effects upon the recruitment of XRCC4 and DNA-ligase IV after exposure to γ-rays of SK-MEL-28 cells (E), HeLa cells (F), and A549 cells (G). The empty bars present XRCC4, and the filled bars present DNA-ligase IV. After pretreatment with PGE2, the cells were irradiated with γ-rays. The irradiated cells were reacted with antibodies against XRCC4 or DNA-ligase IV, followed by DAPI staining. The number of γ-H2AX foci per cell was derived by analyzing the confocal images of stained cells. The means ± standard error calculated from the three independent experiments were presented as columns. The asterisk (*) denotes statistically significant differences compared with the respective control (P ≤ 0.05, Mann-Whitney U test).

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  Fig. 3 Cell-type-specific modulation of DNA-PKcs phosphorylation at T2609 and S2056 following γ-ray irradiation by cAMP signaling. (A, B, C) PGE2 effects upon the phosphorylation of DNA-PKcs on T2609 and ATM on S1981 following γ-ray irradiation in SK-MEL-28 cells (A), HeLa cells (B), and A549 cells (C). The cells were treated with PGE2 with/without pretreatment with okadaic acid (100 nM, 30 minutes). The treated cells were exposed to γ-rays and collected after 1 hour for analysis by western blotting. The empty bars present the phosphorylation of DNA-PKcs at T2609, and the filled bars present the phosphorylation at ATM on S1981. (D) PGE2 effects upon the phosphorylation of DNA-PKcs at S2056 following exposure to γ-rays. (E) Bar graph obtained by densitometric analysis of images in (D). The cells were irradiated with γ-rays after pretreatment with PGE2, and the irradiated cells were collected for analysis by western blotting. The empty bars present SK-MEL-28 cells, the filled bars present HeLa cells, and the slant bars present A549 cells. The means ± standard error calculated from the three independent experiments were presented as columns. An asterisk (*) denotes statistically significant differences compared with the irradiated control, and double asterisk (**) denotes differences indicating statistical significance compared with the PGE2- stimulated cells (P ≤ 0.05, Mann-Whitney U test).

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  Fig. 4 Mediation of cell-type-specific effects of cAMP by PKA on the phosphorylation of DNA-PKcs at T2609. (A, B, C) Effects of PGE2, H-89 and 6-Phe-cAMP on T2609 phosphorylation resulting from exposure to γ-rays in SK-MEL-28 cells (A), HeLa cells (B), and A549 cells (C). (D) Confocal images of DNA-ligase IV recruited to DSBs following γ-ray irradiation in SK-MEL-28 cells, HeLa cells and A549 cells. (E, F, G) Effects of PGE2 and H-89 on DNA-ligase IV foci per cell, obtained from the images in D of SK-MEL-28 cells (E), HeLa cells (F) and A549 cells (G). The cells were incubated with PGE2 after pretreatment with 20 μM H-89 for 30 minutes or were treated with 50 μM 6-Phe-cAMP for 30 minutes. Next, the cells were exposed to γ-rays, and collected after 1 hour for analysis by western blotting or confocal microscopy. Green color represents XRCC4 and DNA-Ligase IV, and blue color represents DAPI stained nucleus (100-fold amplification). The means ± standard error calculated from the three independent experiments were presented as columns. An asterisk (*) denotes statistically significant differences compared with the irradiated control, and a double asterisk (**) denotes statistically significant differences compared with the PGE2- stimulated cells (P ≤ 0.05, Mann-Whitney U test).

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