Mechanism of radiation-induced bystander effect: role of the cyclooxygenase-2 signaling pathway - PubMed (original) (raw)

Comparative Study

. 2005 Oct 11;102(41):14641-6.

doi: 10.1073/pnas.0505473102. Epub 2005 Oct 3.

Affiliations

Comparative Study

Mechanism of radiation-induced bystander effect: role of the cyclooxygenase-2 signaling pathway

Hongning Zhou et al. Proc Natl Acad Sci U S A. 2005.

Abstract

The radiation-induced bystander effect is defined as "the induction of biological effects in cells that are not directly traversed by a charged particle but are in close proximity to cells that are." Although these bystander effects have been demonstrated with a variety of biological endpoints in both human and rodent cell lines (as well as in 3D tissue samples), the mechanism of the phenomenon is not known. Although gap junction communication and the presence of soluble mediator(s) are both known to play important roles in the bystander response, the precise signaling molecules have yet to be identified. By using the Columbia University charged particle beam in conjunction with a strip dish design, we show here that the cyclooxygenase-2 (COX-2, also known as prostaglandin endoperoxide synthase-2) signaling cascade plays an essential role in the bystander process. Treatment of bystander cells with NS-398, which suppresses COX-2 activity, significantly reduced the bystander effect. Because the critical event of the COX-2 signaling is the activation of the mitogen-activated protein kinase pathways, our finding that inhibition of the extracellular signal-related kinase phosphorylation suppressed bystander response further confirmed the important role of mitogen-activated protein kinase signaling cascade in the bystander process. These results provide evidence that the COX-2-related pathway, which is essential in mediating cellular inflammatory response, is the critical signaling link for the bystander phenomenon.

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Figures

Fig. 1.

Fig. 1.

A schematic diagram of the stripped dish used in the present study. A6-μm-thick mylar sheet was epoxied to the outer stainless ring to provide a bottom for the dish. The mylar of the inner rings (38 μm thick) was cut as strips. Exponentially growing NHLF cells were plated into the concentric rings 2 days before irradiation at a density to ensure a confluent state. (See Materials and Methods for further details.)

Fig. 2.

Fig. 2.

Survival fraction and _HPRT_- mutations in bystander and directly irradiated cells. Cultures were irradiated with a 0.5-Gy dose of α particle radiation by using the stripped dishes. Data are pooled from six to eight independent experiments. Bar represents ± SD.

Fig. 3.

Fig. 3.

Identification of differentially expressed genes in radiation-induced bystander cells. (A) Differentially expressed signaling genes in bystander and control normal human lung fibroblasts using the cDNA signal transduction pathway finder array (SuperArray). (B) RT-PCR analysis and confirmation of the array data showing down-regulated IGFBP-3 and up-regulated COX-2.(C) Western blotting of COX-2 protein expression (72 kD) in bystander and control cells with or without treatment with NS 398 (50 μM), a specific COX-2 inhibitor.

Fig. 4.

Fig. 4.

Effect of NS 398 treatment (50 μM, 24 h before and maintained overnight after irradiation), a specific COX-2 inhibitor, on _HPRT_- mutant fractions of NHLFs. Data are from three to four independent experiments. Bar represents ± SD.

Fig. 5.

Fig. 5.

Effect of IGFBP-3 treatment (1 μg/ml, 24 h before and maintained overnight after irradiation) on survival fraction (Left) and _HPRT_- mutation fraction (Right) in control and bystander NHLF cells in the vicinity of cells irradiated with 0.5-Gy α particle plated in strip dishes. Data are pooled from three independent experiments. Bar represents ± SD.

Fig. 6.

Fig. 6.

Activation of the MAPK signaling pathways is involved in the radiation-induced bystander effects. Western blot analysis of phospho-ERK1/2, total ERK1/2, phospho-p38, and total p38 expression in control, α-irradiated, and bystander NHLF cells 4 hr after irradiation with a 0.5-Gy dose of α particle plated in stripped dishes (A). β-actin was used as a loading control. Phospho-ERK/ERK ratio is indicated. PD 98059 (50 μM), an inhibitor of MEK-ERK, was added to the cultures immediately after irradiation (B). Anti-TNF-α mAb (2 μg/ml) was added to the cultures immediately after irradiation (C), PD98059 (50 μM) suppressed expression of COX-2 in bystander cells (D).

Fig. 7.

Fig. 7.

Effect of PD 98059 treatment (50 μM, immediately after and maintained overnight after irradiation), an inhibitor of MEK-ERK, on surviving fractions of control, directly irradiated, and bystander NHLF cells. Data are from three independent experiments. Bar represents ± SD.

Fig. 8.

Fig. 8.

Working model of the signaling pathways involved in the radiation-induced bystander effect. Binding of TNF-α, TGFβ, IGF, IL-1, and IL-8 ligands to the corresponding receptors activate signaling pathways including MEK1/2; mitogen-activated protein kinase kinase (MKK3/6), and p38 kinase (all in blue lines), which lead to COX-2 gene expression. Specific inhibitors of the various signaling pathways, such as Wortmannin (wt) for phosphatidylinositol 3-kinase and _N_-(α)-tosyl-

l

-lysine chloromethylketone hydrochloride for the NF-κB can be used to delineate specific signaling routes involved in the bystander phenomenon.

References

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