*4.4. COX-2*/*Prostaglandin E2 Signaling*

The inflammatory cyclooxygenase-2 (COX-2) signaling pathway that is required for PGE2 synthesis has been implicated in promoting bystander effects [149–152,157]. Most bystander studies involve the transfer of conditioned medium from irradiated cells to non-irradiated cells in vitro. Zhou et al. used a novel mylar strip culture dish to shield a portion of human fibroblasts from irradiation while remaining in the same culture vessel as irradiated cells [150]. Using this technique, bystander human fibroblasts showed overexpression of COX-2 [150]. Inhibition of COX-2 with NS-398 decreased the bystander effect in non-irradiated cells, as determined by the frequency of mutations in the hypoxanthine-guanine phosphoribosyltransferase (HPRT- ) locus and the percent of surviving cells [150]. They also demonstrated that inhibition of ERK1/2, which lies upstream of COX-2, could diminish the bystander effects. More recently, this same group reported that NF-κB was important for mediating bystander effects in human fibroblasts, which could be attributed to NF-κB-mediated inducible NO synthase (iNOS) and COX-2 expression [149]. The NF-κB inhibitor, Bay 11-7082, and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a scavenger of NO, decreased mutation frequency in bystander cells [149].

PGE2 has also been suggested to mediate the bystander effect [147,157]. We have previously reported that PGE2 levels are increased in parotid acinar cell culture supernatant 24–72 h post-IR [48]. We also reported reduced levels of PGE2 release in mice lacking P2X7R [48]. In addition to these findings, P2X7Rs and P2Y2Rs lie upstream of many of the signaling pathways resulting in expression of COX-2 (Figure 3). Together, these findings suggest that COX-2/PGE2-mediated bystander effects are dependent on signaling through P2X7Rs or P2Y2Rs.

#### *4.5. DNA Damage Response*

Elevated levels of γH2AX are an indicator of DNA damage, a well-documented response to ionizing radiation [59]. In irradiated A549 human lung cancer cells, elevated γH2AX levels were abrogated by pretreatment with the ectonucleotidase apyrase [158], a response that could be eliminated by the ectonucleotidase inhibitor ARL67156 or addition of ATP or UTP. Similar to our findings in primary murine parotid epithelial cells [48], ATP is released from A549 cells following γ-irradiation (2 Gy) in a P2X7R-dependent manner. The finding that antagonism of P2Y6R, P2Y12R or P2X7R blocked activation of DNA repair mechanisms led to the overall hypothesis that P2X7R-dependent release of ATP and subsequent autocrine activation of G protein-coupled P2Y receptors was responsible for the observed DNA damage response [158]. Additionally, downstream P2R signaling molecules, such as nitric oxide (NO), are important mediators of radiation-induced DNA damage in bystander cells [128,159].

#### *4.6. Reactive Oxygen Species and TGF-*β*1*

ROS and TGF-β1 are well-appreciated mediators of the bystander effect in multiple IR models [160], serving as second messengers in an autocrine or paracrine fashion. In response to ATP, P2X7Rs regulate production of ROS by NLRP3 inflammasome activation [161] and ROS promote TGF-β1 release [162]. P2Y2Rs may also contribute to the bystander effect by promoting latent TGF-β signaling. TGF-β, latency activated protein (LAP) and latent TGF-β binding proteins (LTBP) form a covalently-bound large latent complex (LLC) that is secreted from cells, whereupon it binds to integrins (e.g., αvβ5, αvβ6, αvβ<sup>3</sup> and αvβ8) in the extracellular matrix (ECM) through LAP interaction with RGD sequences. Latent activation of TGF-β signaling can occur through metalloprotease-dependent cleavage of LAP or LTBP or disruption of LAP-RGD interaction with integrins [163–166]. P2Y2R activation also induces metalloprotease activity [167,168] and the P2Y2R contains an extracellularly-oriented RGD sequence

that enables its direct binding and activation of the αvβ<sup>5</sup> integrin [167]. Through these mechanisms, activation of P2Y2R or P2X7R could promote the TGF-β1 and ROS-mediated bystander effects.
