The Nuclear Factor κB pathway: A link to the immune system in the radiation response

ARTICLE IN PRESS Cancer Letters ■■ (2015) ■■–■■

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Cancer Letters j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c a n l e t

Mini-review

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The nuclear factor κB pathway: A link to the immune system in the radiation response Q2 Christine E. Hellweg * German Aerospace Centre (DLR), Institute of Aerospace Medicine, Radiation Biology, Linder Höhe, Köln D-51147, Germany

A R T I C L E

Q3

I N F O

Keywords: Nuclear factor κB Innate immune system Adaptive immune system Cellular radiation response Linear energy transfer Bystander effect Chemokines

A B S T R A C T

Exposure to ionizing radiation modulates immune responses in a complex dose-dependent pattern, with possible anti-inflammatory effects in the low dose range, expression of pro-inflammatory cytokines at moderate doses and immunosuppression after exposure to higher doses due to precursor cell death together with concomitant exacerbated innate immune responses. A central regulator in the immune system is the transcription factor Nuclear Factor κB (NF-κB). NF-κB is involved in the regulation of cellular survival, immune responses and inflammation, resulting in eminent importance in cancerogenesis. After exposure to ionizing radiation, NF-κB activation is initially triggered by ATM which is activated by DNA double strand breaks. Together with the NF-κB essential modulator (NEMO), it serves as a nucleoplasmic shuttle. The pathway converges with the classical NF-κB pathway at IκB kinase (IKK) complex activation. Resulting cytokine expression can activate NF-κB in a positive feed forward loop. Danger signals released from dying cells can activate NF-κB via Toll-like receptors (TLRs). The resulting immune activation can be beneficial or detrimental. In the low dose range, pro- and anticancerogenic effects are possible. In the radiotherapy-relevant dose range, tolerogenic immune responses should be avoided, and an antitumor immune response might be supported by TLR agonists activating NF-κB. © 2015 Published by Elsevier Ireland Ltd.

36 Introduction

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Since its discovery as a nuclear protein bound to the enhancer of immunoglobulin κ light chain genes in pre-B cells stimulated with bacterial lipopolysaccharide (LPS) [1], a growing list of functions has been

attributed to the transcription factor Nuclear Factor κB (NF-κB) and it was shown to be involved in the pathogenesis of a large number of diseases. These include cancer, chronic inflammatory disorders such as rheumatoid arthritis, diabetes, transplant intolerance, cachexia, Alzheimer’s disease, and organ ischemia/reperfusion injury [2–4].

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Q1

Abbreviations: A20 (TNFAIP3), tumor necrosis factor inducible protein 3; RD, ankyrin repeat domain; ATM, ataxia telangiecta-sia mutated protein; β-TrCP, β-transducin repeat containing protein; BIRC, baculoviral IAP repeat-containing; BTG2, B-cell translocation gene 2; CCL3, chemokine (C-C motif) ligand 3; cIAP-1 (BIRC2), cIAP-2 (BIRC3), cellular inhibitor of apoptosis protein 1 and 2; CHO, Chinese hamster ovary; COX-2/PTGS2, cyclooxygenase 2/prostaglandin-endoperoxide synthase 2; CXCL, chemokine (C-X-C motif) ligand; DAMPs, damage-associated molecular patterns; DDR, DNA damage response; DNA-PK, DNA-dependent protein kinase; DSB, double strand break; DUSP, dual specificity phosphatase; EBV, Epstein–Barr virus; ELKS, protein abundant in glutamic acid (E), leucine (L), lysine (K), and serine (S); EMSA, electrophoretic mobility shift assay; EMT, epithelial mesenchymal transition; GADD45B, growth arrest & DNA-damage-inducible, β; h, hours; HEK/293, human embryonic kidney; HMBG1, highmobility-group box 1; HUVEC, human umbilical vein endothelial cells; IAP, inhibitor of apoptosis; ICAM1, intercellular adhesion molecule; IκB, inhibitor of NF-κB; IKK, IκB kinase; IKK-K, IKK kinases; IFN, interferon; IL, interleukin; iNOS, inducible nitric oxide synthase; LET, linear energy transfer; LPS, lipopolysaccharide; LRR, leucine-rich repeat; MAP3K (or MAPKKK), MAPK kinase kinase; MAPK, mitogen-activated protein kinase; MCP-1, monocyte chemotactic protein-1; MD-2, myeloid differentiation factor-2; MEF, murine embryonic fibroblasts; MEKK3 (or MAP3K3), MAPK kinase kinase 3; MCP-1, monocyte chemotactic protein-1; MHC, major histocompatibility complex; MMP, matrix metalloproteinase; NEMO, NF-κB essential modulator; NF-κB, nuclear factor κB; NIK, NF-κB-inducing kinase; NK, natural killer; NRE, NF-κB response element; PAMPs, pathogenassociated molecular patterns; PARP-1, poly(ADP-ribose)-polymerase-1; PGE2, prostaglandin E2; PI3K, phosphatidyl-inositol 3-kinase; PIASy, protein inhibitor of activated STAT 4; PIDD, p53-induced protein with a death domain; PRR, pattern recognition receptors; RHD, Rel homology domain; RIP1, receptor interacting protein 1; RNS, reactive nitrogen species; ROS, reactive oxygen species; SASP, senescence-associated secretory phenotype; SUMO-1, small ubiquitin-like modifier 1; SV40, simian virus 40; TAB, TGF-β activated kinase; TAD, transcriptional activation domain; TAK1, TGF-β-activated protein kinase 1; Th, T helper; TLR, Toll-like receptor; TNF-α, tumor necrosis factor α; TNFR, TNF receptor; TRAF, TNF-R associated factor; Tregs, regulatory T cells; Ubc13, ubiquitin-conjugating enzyme 13; VCAM-1, vascular cell adhesion molecule-1; VEGF, vascular endothelial growth factor; XIAP, X-linked IAP. Present address (September 13, 2014–March 13, 2015): Center for Radiological Research, Lab of Prof. Tom K. Hei, 630 West 168th Street, VC11-244, New York, NY 10032. Tel.: +001 212 305 9514. * Tel.: +49 2203 601 3243; fax: +49 2203 61 970. E-mail address: [email protected]. http://dx.doi.org/10.1016/j.canlet.2015.02.019 0304-3835/© 2015 Published by Elsevier Ireland Ltd.

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C.E. Hellweg/Cancer Letters ■■ (2015) ■■–■■

NF-κB is a master switch in inflammation, the central element of an acute innate immune response. Furthermore, it plays a fundamental role in the adaptive immune response and is involved in the regulation of embryonic development, lymphopoiesis and osteogenesis [5–12]. It contributes to oncogenesis as it can affect most of the hallmarks of cancer through the transcriptional activation of genes associated with cell proliferation, angiogenesis, metastasis, tumor promotion, inflammation and suppression of apoptosis [7]. NF-κB is considered as a crucial promoter of inflammation-linked cancers, with chronic inflammation being a route to carcinogenesis [13]. Aberrant NF-κB regulation, including constitutive or induced activation, has been observed in many cancers. Constitutive NF-κB activation was found e.g. in breast, thyroid, bladder and colon cancer [14–18], and NF-κB is often activated in malignant cells in response to inflammatory stimuli from the microenvironment. NF-κB can be critical for cancer progression [19] and for radio- and chemotherapy resistance in cancer [8]. This resistance can rise from constitutive NF-κB activity in the tumor cells, or by activation of NFκB in response to DNA damaging agents such as chemotherapeutics and ionizing radiation [15]. Activation of the NF-κB pathway can protect cells from apoptosis after treatment with various genotoxic agents via expression of anti-apoptotic proteins [20]. NF-κB also enhances the expression of degradative enzymes and adhesion molecules supporting the idea that it makes a major contribution to tumor progression and metastasis in various cancers [21]. Therefore, most interest for the role of NF-κB in DNA damage pathways comes from the field of cancer therapy, where resistance of tumors to chemo- and radiotherapy is a major problem [15], and NF-κB was identified quite early as a potential target of innovative cancer therapies [22]. Otherwise, radiotherapy induced NF-κB activation in the tumor microenvironment might be beneficial as it might contribute to an antitumor immune response by modulating cytokine production of tumor cells and of tumor infiltrating lymphocytes [19]. The role of cytokines in radiobiological responses was recently reviewed [23]. High radiation doses result in significant cell death with release of danger signals which could support the rise of an antitumor immune response with strong involvement of NF-κB activation [24]. Low dose radiotherapy, e.g. for different inflammatory diseases, tries to regulate and terminate inflammation by applying a dose at which the anti-inflammatory arm of the response outweighs the pro-inflammatory arm. It acts on already inflamed tissue with upregulated inflammatory responses. A suppression of NF-κB by low-dose radiation therapy was found in allergic asthma with chronic airway remodeling: Whole body irradiation of mice (0.5 Gy per day for three days) with ovalbumin induced asthma reduced NF-κB activity in mast cells [25]. In vitro, exposure to 0.5–0.7 Gy X-rays suppressed NF-κB activation in monocytes [26]. NF-κB’s role in responses to chronic low dose ionizing radiation exposure is much less investigated, but modulations in cytokine expression in response to such exposures point to the involvement of NF-κB as crucial regulator. For example, low dose (<0.2 Gy) and high dose exposure generated both pro-inflammatory responses in the thymus of irradiated mice [27]. It is suggested that chronic low dose irradiation creates an inflammatory milieu due to cytokine secretion and production of reactive oxygen and nitrogen species (ROS, RNS) with secondary genotoxic [24] and pro-tumorigenic effects [24,28,29]. On the other hand, some authors claim beneficial effects of low doses (1.2 mGy/h), such as immune activation by chronic exposure [30,31]. Shin et al. argue that lowdose rate irradiation inhibited tumor growth in mice prone to develop thymic lymphoma [32]. The natural killer (NK) cell and T cell stimulating cytokine interleukin-15 (IL-15), which expression is partially regulated by NF-κB [27], was up-regulated in the thymus after low dose rate irradiation of mice [32]. Currently, it is very difficult to judge which effects of chronic low dose ionizing radiation

exposure are pro- or anti-inflammatory, pro- or anticancerogenic, or might even cross the border to autoimmune effects. Seemingly, in both cases, exposure to radiotherapy relevant doses Q4 or chronic low dose exposure, the outcome of the inflammatory response involving NF-κB can be desirable or undesirable [24], and requires further investigation. In this review, first, the multitude of pathways leading to NF-κB activation after exposure to ionizing radiation will be highlighted. In the second part, the involvement of NF-κB target genes in survival, inflammatory, immune and bystander responses will be discussed. NF-κB and IκB families: the basics NF-κB consists of homo- or hetereodimeric complexes made up of members of the NF-κB/Rel family. These carry the characteristic 300-amino-acid long N-terminal stretch, called the Rel homology domain (RHD), which is responsible for binding of DNA and inhibitory factors and for homo- and heterodimerization: RelA (p65), RelB, c-Rel, p50/p105 (NF-κB1) and p52/p100 (NF-κB2) [33–36], whereby p105 and p100 are precursor proteins. RelA, RelB and c-Rel possess a transcriptional activation domain (TAD), rendering NF-κB a potent transcription factor, which lacks in p50 and p52 [35]. Under resting conditions, retention of NF-κB in the cytoplasm is achieved by the inhibitor of NF-κB (IκB) proteins, which bind through their ankyrin repeat domain (ARD) to NF-κB. Thereby the nuclear localization sequences are masked [35,37]. In their free state, IκB proteins are unstable and rapidly degraded, while binding to NF-κB strongly increases their stability [35]. The three canonical IκB proteins, IκBα, IκBβ, and IκBε, are encoded by the NFΚBIA, NFΚBIB, and NFΚBIE genes [35]. The prototypical p50:p65 heterodimer is mainly bound by IκBα. p105 and p100 proteins, which are involved in the alternative NF-κB pathway, contain the inhibitory part in their C-terminal region in addition to the NF-κB part in the N-terminal half. Two novel IκBs (IκBζ and BCL-3) were described. BCL-3 is a non-inhibiting IκB family member that acts as transcriptional co-activator for p50:p50 and p52:p52 homodimers [2]. Canonical, alternative and atypical NF-κB pathways The list of NF-κB activators is long and comprises diverse endogenous and exogenous ligands and physical and chemical stresses [4,38,39]. Frequent ligands are inflammatory cytokines or bacteria, viruses and pathogen-derived agents (e.g. LPS). Among the activating cellular stress factors are phorbol esters, ROS, necrotic cell products, growth factor depletion, hypoxia, heat shock, and ultraviolet as well as ionizing radiation [4,40–42]. These activators trigger pathway run-through via activation by membrane receptors (cytokine receptors, toll-like receptors, TLRs), as seen in the canonical (or classical) and alternative (or noncanonical) pathway, or from intracellular sites such as the nucleus in the case of atypical pathways. Ligands bind to their receptors resulting in recruitment of distinct proximal signaling molecules. These use common intermediates (receptor interacting protein 1, RIP1; TNF-R associated factor, TRAF) to activate IκB kinase (IKK) complex, which is as the proteasome a central element of all sub-pathways [43]. Depending on the sub-pathway, the IKK complex is composed by different members of the IKK family. In the canonical and the atypical pathway, the IKK complex is composed of the two catalytic subunits, IKK-α (IKK1) and IKK-β (IKK2), and the regulatory subunit, IKK-γ/NF-κB essential modulator (NEMO) [6,35]. ELKS associates as another regulatory subunit within the IKK complex [44]. It is abundant in glutamic acid (E), leucine (L), lysine (K), and serine (S) [45] and is suggested to recruit IκBα to the IKK complex [44]. The activated IKK phosphorylates IκB in the signal responsive domain at the serine residues 32 and 36 and thereby targets IκB for

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ubiquitination [34,46–48]. Phosphorylated IκB is polyubiquitinylated by the E3 ubiquitin ligase containing β-transducin repeat containing protein (β-TrCP) and subsequently degraded by the 26S proteasome [33,35,49]. The canonical pathway plays a major role in inflammation. It is activated by proinflammatory cytokines, e.g. tumor necrosis factor α (TNF-α) or interleukin 1 (IL-1) [7], growth factors, pathogenassociated molecular patterns (PAMPs), which are ligands of TLR, or antigens, which bind to the T- or B-cell receptor. They induce a rapid and strong NF-κB-activating signal, which is needed for a fast response in acute stress situations, such as viral infection [36]. Activation of IKK is achieved via a complex pathway involving several adaptor proteins, ubiquitin ligases, binding proteins and kinases such as RIP1 and TRAF2, 5 or 6 [2,33,35,50], resulting in activation of IKK kinases (IKK-K). These kinases are responsible for phosphorylation of IKK and might be TGF-β-activated protein kinase 1 (TAK1) or MAPK kinase kinase 3 (MEKK3) after stimulation with TNF-α [2]. TRAF proteins act as E3 ligases together with the ubiquitinconjugating enzyme Ubc13/Uev1A to catalyze the formation of K63linked polyubiquitin. This activates the TAK1/TAB1/TAB2 complex. IKK is activated by phosphorylation via TAK1 or autophosphorylation [34,47,48]. The exact contribution of different kinases to IKK activation is not completely known, and redundancy in function may occur. The NF-κB is mostly composed of p50:p65 and p50:c-Rel heterodimers. The alternative pathway is involved in non-inflammatory signaling, e.g. in control of development, organization and function of secondary lymphoid organs such as lymph nodes, in B-cell maturation and survival, and in osteoclastogenesis [35]. After ligand binding to membrane receptors of the TNF-R superfamily, NF-κBinducing kinase (NIK) activates the IKKα homodimer [2,35]. IKKα phosphorylates p100 in the p100:RelB complex, resulting in polyubiquitination of p100 and proteolytic degradation of p100’s C-terminal region, releasing p52 [7,35]. The released NF-κB translocates to the nucleus and binds to κB DNA motifs (NF-κB response elements, NREs) initiating gene transcription. Finally, phosphorylation and acetylation of key residues in NF-κB, mainly in the cell nucleus, is required for full activation and effects on transcription [36]. NF-κB activation after ionizing radiation exposure: many roads lead to Rome Activation of NF-κB by ionizing radiation in cell cultures was first shown by Brach et al. [51]. An overview of NF-κB activation in different cell lines, organs and tissues by different radiation qualities is given in Table 1. Different cell types and tissues show different sensitivity toward NF-κB activation by ionizing radiation. A strong activation is observed in cells of lymphoid or myeloid origin and in some epithelial cells. In vivo, NF-κB was activated in spleen, mesenteric lymph nodes and bone marrow at 8.5 Gy as measured by electrophoretic mobility shift assay (EMSA) [80], or already after 1 Gy in the bone marrow as detected by an oligonucleotide-ELISA [81]. NF-κB activation was also elicited in bone marrow cells of mice exposed to 1 Gy whole body proton irradiation [81]. A strong nuclear translocation of p65 and p50 was observed in kidney homogenates after whole body γ-irradiation of mice [83], and in the liver and kidney in response to 20 Gy whole-body irradiation of mice [82]. In the rodent brain, NF-κB activation occurred after whole body irradiation with 5 Gy [85]. In cells of the connective tissue as fibroblasts, the response is lower or absent [86]. An extremely high dose of ionizing radiation (80 Gy) was required in murine fibroblasts to elicit DNA-binding activity of NF-κB [52], while 5 Gy was sufficient in human embryonic lung fibroblasts [54]. The higher sensitivity for radiation-induced NF-κB activation seems to be retained in

3

lymphoblastoid cells with activation already at 0.25 Gy [76]. In solid tumors, mostly doses above 5 Gy are required (Table 1). In many publications, only one dose was examined and dose–effect curves for NF-κB activation are not available. Furthermore, different time points were analyzed, and the sensitivity of the detection method also strongly affects at which dose NF-κB activation can be detected. For example, depending on the reporter system, the lowest dose at which X-rays induced NF-κB activation in human embryonic kidney (HEK/293) cells varied between 3 and 8 Gy (Table 1). Luciferase reporter assays containing NREs are expected to have the highest sensitivity due to enzymatic signal amplification. Furthermore, the radiation quality affects the degree of NF-κB activation. α-Particles with an LET of 120 keV/μm activated NF-kB in human skin fibroblasts at a dose as low as 0.5 Gy [53]. Exposure of human epithelial cells to accelerated heavy ions (36Ar, linear energy transfer (LET) 272 keV/μm) resulted in strong activation of the NF-κB, starting at a dose of 0.7 Gy [59]. Other groups reported NF-κB translocation after exposure of normal human monocytes (MM6 cells) to 0.7 Gy of 56Fe ions [66]. Heavy ions with an LET of 100–300 keV/μm had a nine times higher potential to activate the NF-κB pathway compared with X-rays [62]. High-LET radiation such as heavy ions is known to induce complex DNA damage which can persist several times. This might be a reason for the strong NF-κB activation in response to high-LET radiation. The pathway leading to this activation was unknown for a long time, and ROS were suspected as important intermediates because NF-κB is known as redox-sensitive transcription factor [87–90], and oxidative stress arising from exposure to ionizing radiation might contribute to NF-κB activation. However, in the last two decades, DNA double strand breaks (DSBs) as initial trigger were spotlighted. Early studies supposed DNA-dependent protein kinase (DNA-PK) [69], phosphatidylinositol 3-kinase (PI3K) and mitogenactivated protein kinase (MAPK) [91] as mediators of radiationinduced NF-κB activation. In a cell culture model, it has been found that ataxia telangiectasia mutated protein (ATM) plays a role in sustained activation of NF-κB in response to DNA DSBs [92–94]. As this atypical sub-pathway is activated not only by ionizing radiation, but also by other genotoxic agents such as DNA DSB inducing chemicals (e.g. topoisomerase-targeting drugs), it is called genotoxic stress induced NF-κB pathway [95]. After transmittal of the signal from the cell nucleus to the cytoplasm, this sub-pathway follows more or less the canonical NF-κB activation scheme. Signals from the cell nucleus The genotoxic stress induced NF-κB sub-pathway is initiated by DNA DSBs in the cell nucleus (Fig. 1) [95]. Sensing of DNA DSBs in the cell is part of the DNA damage response (DDR) [96–100], and ATM is recognized as a dominant sensor [99,101]. In the presence of DNA DSBs, ATM homodimers dissociate after autophosphorylation of serine 1981 to activated monomers [102]. The ATM-dependent network expanded as ATM was shown to be involved in NF-κB activation in response to DNA damage [103]. NF-κB is now regarded as second arm of the ATM signaling pathway besides p53, providing survival signals [36,43,52,103–107]. ATM was shown to be required for NF-κB activation after DNA damage [92,93,106,108], and NF-κB/RelA translocation and Ser-276 phosphorylation was reduced in ATM(−/−) murine embryonic fibroblasts (MEF) [109]. ATM that was activated after DNA DSB formation phosphorylates NEMO on serine 85 (S85) followed by mono-ubiquitination and thereby promotes NEMO’s nuclear export [103,106]. Mutation of S85 in NEMO resulted in higher sensitivity to ionizing radiation, revealing its role in the NF-κB induced survival response [103]. However, ATM activation alone is not sufficient to achieve genotoxic stress induced NF-κB activation. A nucleoplasmic signalosome has to form that initiates extensive posttranslational

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Table 1 Activation of the NF-κB pathway by sparsely and densely ionizing radiation. Experimental model

Radiation quality

Dose (Gy)

Effect

Methods

Reference

Cell lines or primary cells Murine fibroblasts

Ionizing radiation

80

ions (LET 120 keV/ μm):

0.5

Electrophoretic mobility shift assay (EMSA) EMSA (NF-κB) Western blot (IκBα)

[52]

4He

DNA-binding activity of NF-κB starting 3 hours (h) after exposure, maximum after 5–7 h Nuclear p65-p50 activity ⇑ after 1 h Degradation of IκBα Both in directly hit and in bystander cells

γ-Radiation (137Cs irradiator, 4.3 Gy/min)

5

DNA-binding activity of NF-κB 15 min to 4 h after irradiation

EMSA

[54]

X-rays

10

NF-κB binding activity 30–60 min after exposure

EMSA with supershift (p65), reporter assay with human growth hormone

[55]

6 MV photons (137Cs irradiator) 12C ions (LET not published)

8

NF-κB p65 translocation to the cell nucleus

EMSA, Immunofluorescence

[56]

0.1 1

NF-κB p65 expression ⇑ after 8 h NF-κB p65 expression ⇑ after 3 h

Western Blot

[57,58]

X-rays (1 Gy/min)

8

NF-κB dependent reporter gene expression ⇑

[59,60]

X-rays (1 Gy/min)

8

Argon ions (95 MeV/n, LET 272 keV/μm, 1 Gy/ min) Carbon ions (75 Mev/n, LET 33 keV/μm, 1 Gy/ min) X-rays (1 Gy/min)

0.7

Nuclear p65 content ⇑ indicating nuclear translocation of NF-κB NF-κB dependent reporter gene expression ⇑

Stably transfected green fluorescent reporter system: HEK-pNF-κB-d2EGFP/ Neo Oligonucleotide ELISA for p65 with nuclear extracts Stably transfected green fluorescent reporter system: HEK-pNF-κB-d2EGFP/ Neo Oligonucleotide ELISA for p65 with nuclear extracts Stably transfected red fluorescent reporter system: HEK-pNFκB-DDtdTomato-C8 HEK-pNFκB-DD-tdTomatoC8 + Shield-1 Luciferase reporter assay

[63]

EMSA with supershift (p65 and p50)

[65]

DNA binding assay: binding of NF-κB to its consensus sequence of 5′GGGGACTTTCC-3 EMSA

[66]

Human skin fibroblasts immortalized by SV40 T antigen Human embryonic lung fibroblasts (FH109) Human umbilical vein endothelial cells (HUVEC) HUVEC Chinese hamster ovary (CHO) V79 cells Human embryonic kidney (HEK/293) cells HEK/293 HEK/293

HEK/293

HEK/293

Mouse epidermal cell line (JB6P+) Rat primary astrocytes Normal human monocytes (MM6 cells) Murine macrophages (RAW 264.7) Tumor and Leukemia Cell Lines Human cervix adenocarcinoma cell line (HeLa) HeLa Human breast cancer cell line (MCF-7) Human oral squamous cell carcinoma cell lines (B88, BHY, HNt) ECV 304 cells, derivative of human urinary bladder carcinoma cell line T-24 Human colon cancer cell line (HT29) Human malignant glioma cell line (MO54) Human glioma cell lines M059J and M059K Human melanoma cells (U1-Mel, radioresistant)

1.3

Nuclear p65 content ⇑ indicating nuclear translocation of NF-κB

~6

NF-κB dependent reporter gene expression ⇑

X-rays (1 Gy/min)

~3

NF-κB dependent reporter gene expression ⇑

X-rays (0.028 Gy/min) X-rays (1.8 Gy/min)

0.05, 0.1

Increased luciferase activity 24 h after 0.1 Gy

3.8–15

56

0.7

γ-Radiation (137Cs irradiator, 5.5 Gy/min)

8, 20

NF-κB binding activity increased dosedependently, maximum 2–4 h after exposure Rapid and persistent NF-κB translocation, phosphorylation of IκBα and subsequent proteasome-dependent degradation Dose-dependent repression of NF-κB 3 h after irradiation

γ-Radiation (137Cs irradiator)

20–100

Ionizing radiation γ-Radiation (137Cs irradiator, 4.4 Gy/min) X-rays

10 5

γ-Radiation (137Cs irradiator, 5.5 Gy/min)

Fe ions (high LET)

[53]

[61] [62]

[61]

[64]

[67]

NF-κB activation 1–5 h after exposure with a peak after 2–3 h, maximum after 20 Gy, IκBα degradation 1–5 h after irradiation NF-κB activation 1–4 h after irradiation Increased luciferase activity 24 h after irradiation Nuclear translocation of p65, transcriptional activation

EMSA with supershift (p65), Western Blot, p65 immunofluorescence

[68]

EMSA, luciferase reporter assay Luciferase reporter assay, EMSA

[69,70] [71]

EMSA, p65 immunofluorescence staining, NF-κB luciferase reporter assay

[72]

8, 20

IκBα levels mostly unchanged NF-κB activation 3 h after irradiation

Western Blot EMSA

[67]

X-rays (1 Gy/min)

3

Weak NF-κB activation 2 h after exposure

EMSA

[73]

X-rays (1 Gy/min)

8

Nuclear translocation of p65 containing NF-κB dimers 2 h after exposure

Western blot (p65) of nuclear extracts

[46]

Ionizing radiation

10

EMSA

[69]

60

4.5

M059K: NF-κB activation 1–4 h after irradiation M059J: no NF-κB activation NF-κB activation 4 h after irradiation

EMSA

[74]

Co γ-rays (0.95 Gy/ min)

15, 30

(continued on next page)

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Table 1 (continued) Experimental model

Radiation quality

Dose (Gy)

Effect

Methods

Reference

Human melanoma cells (U1-Mel, radioresistant)

60

1.5–7.5

EMSA

[75]

Epstein–Barr virus (EBV)-transformed human lymphoblastoid cells (244B) Human myeloid leukemia cells (KG1) Human myeloblastic leukemia cell line ML-1 (wild-type p53) Human B lymphoma cells (Ramos)

137

Cs γ-rays (1.17 Gy/ min)

0.25–1 0.1–2

Long-lasting NF-κB activation starting 30 min after irradiation, peaking at 12–24 h, reaching control levels again after 48 h. Maximal response 12 h after exposure to 3 Gy Maximal response after exposure to 0.5 Gy Maximal response 8 h after exposure to 0.5 Gy, suppressed b N-acetylcysteine

EMSA

[76] [77]

137Cs

2–50

NF-κB activation 15 min to 6 h after 20 Gy irradiation with a maximum after 3–4 h; maximum after 5–20 Gy Activation of NF-κB (0.5 Gy had no effect) 0.5 and 2 h after exposure

EMSA

[51]

EMSA

[78]

Organs and Tissues Bone marrow, lymph nodes, and spleen of male BALB/c mice Bone marrow of male BALB/cJ mice

Bone marrow of male BALB/cJ mice

Murine liver and kidney Kidney homogenates of mice Small intestine of C57BL/6J mice Cerebral cortex of male SpragueDawley rats

Co γ-rays (6 Gy/min)

γ-rays (14.3 Gy/

min) X-rays (0.7 Gy/min)

20

137Cs

1–20

Dose-dependent increase of nuclear translocation and transcriptional activity of NF-κB

EMSA

[79]

Whole body γ-irradiation (137Cs irradiator, 2.4 Gy/min)

8.5

NF-κB activation 1–10 h after irradiation with a maximum after 1–2.5 h

EMSA with supershift (p65 and p50)

[80]

Whole body γ-irradiation (137Cs irradiator, LET 0.66 keV/μm, 10 mGy/ min) Whole body irradiation with protons (100 MeV, LET 0.7 keV/μm)

1.0

NF-κB activation 1.5 h and 1 month postirradiation

Oligonucleotide enzyme-linked immunosorbent assay (ELISA) for p65 with nuclear extracts

[81]

1.0

Oligonucleotide ELISA for p65 with nuclear extracts

[81]

Whole body irradiation

20

NF-κB activation 3 h, 24 h and 1 month postirradiation (5 mGy/min) NF-κB activation 1.5 h, 3 h, 24 h and 1 month postirradiation (10 mGy/min) NF-κB activation

EMSA

[82]

γ-rays (5.7 Gy/

min)

Whole-body γ-irradiation Whole-body γ-irradiation (137Cs irradiator, 2.4 Gy/min) Whole body γ-irradiation (137Cs irradiator, 3.8 Gy/min)

Strong nuclear translocation of p65 and p50 0.5–12

5–30

NF-κB DNA-binding activity detectable after all doses, maximal response after 12 Gy, peak after 2–4 h NF-κB DNA-binding activity 2 h after exposure increased dose-dependently

[83] EMSA

[84]

EMSA

[85]

⇑ increase, ⇓ decrease. The dose rate is indicated when it was given in the original publication.

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modification of NEMO, especially its SUMOylation1 [106,110,111] and shuttling of parts of the complex from the nucleus to the cytoplasm is required. Ionizing radiation induces both signals, ATM activation and NEMO SUMOylation. The composition of the nucleoplasmic signalosome is not fully elucidated. It has to be considered that the pathway analyses were partially performed with extremely high doses of ionizing radiation (e.g. 30 or 80 Gy). p53-induced protein with a death domain (PIDD), RIP1, Poly(ADP-ribose)-polymerase-1 (PARP-1) and NEMO were suggested to represent the link between nuclear DNA damage and cytoplasmic IKK activation [36]. While NEMO and ATM are currently considered essential, participation of PIDD, RIP1 and PARP-1 was not observed in all test systems and they are suggested to have supporting roles in this pathway [52,110,112]. The direct DNA damage sensor PARP-1 is proposed to assemble the nucleoplasmic signalosome consisting of NEMO, protein inhibitor of activated STAT 4 (PIASy) and activated ATM [43]. In this complex, NEMO is phosphorylated by ATM and SUMOylated by the SUMO ligase PIASy on the lysine residues K277 and K309 and also monoubiquitinated at K285 [43,112]. Monoubiquitinated and

1

Modification with small ubiquitin-like modifier 1 (SUMO-1).

SUMOylated NEMO shuttles back to the cytoplasm [35,43]. SUMOylation of ATM promotes its export from the nucleus. PARP-1 might also act as transcriptional cofactor for p65 [112]. PIDD contains a leucine-rich repeat (LRR)-domain and three isoforms were identified. PIDD is suggested to act upstream of RIP1 [110], recruiting it after its translocation to the nucleus [36,113]. In MEF, 30 Gy γ-irradiation resulted in phosphorylation of IKKα/β and IκBα, which was less pronounced in MEF lacking PIDD [114]. The loss of NF-κB activation (e.g. due to loss of PIDD) did not alter the survival of MEF and hematopoietic stem/progenitor cells after exposure to ionizing radiation, but impaired cytokine synthesis and release and thereby the induction of a sterile inflammation [114]. The serine/threonine death domain kinase RIP1 is suggested to be upstream of IKK in the genotoxic stress induced NF-κB subpathway [52]. RIP1 was shown to be essential for NF-κB activation in response to TNF-α, interacting with the TNFR1 via its death domain [115–117]. RIP1 was not involved in UVC, LPS or IL-1β induced NF-κB activation, but in NF-κB activation by the DNA damaging agents adriamycin and camptothecin [52]. RIP1 kinase activity was also required for CXL8/IL-8 secretion in response to DNA DSB induction by etoposide [118], which is a known stimulator of NF-κB-dependent gene expression [36]. The NEMO-RIP1 complex assembled in response to low and high level damage and

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Fig. 1. The genotoxic stress induced NF-κB sub-pathway. This pathway starts in the cell nucleus with activation of ATM and PARP-1 by ionizing radiation induced DNA DSB. A nucleoplasmic signalosome forms, which contains ATM and NEMO; facultative binding partners are PIDD and RIP1. PIASy SUMOylates NEMO in the complex. SUMOylated NEMO and ATM shuttle to the cytoplasm, where they assemble the IKK complex including ELKS. Phosphorylation of IκB by IKK results in IκB ubiquitination and subsequent proteasomal degradation. The liberated NF-κB, in case of ionizing radiation mostly a p50:p65 heterodimer, translocates to the nucleus and activates target gene expression. For more details, see text.

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remained active after high level damage [118]. RIP1 was necessary for a second wave of NF-κB activation in response to strong DNA damage [118]. In the signalosome, both PIDD and RIP1 might foster NEMO’s localization in the nucleus and facilitate the SUMOylation of NEMO [35,114]. SUMOylated NEMO in complex with ATM therefore represents the long searched nuclear to cytoplasmic shuttle of the NF-κB activating signal [95]. Hinz et al. showed that ATM activates TRAF6 in the cytoplasm, leading to TRAF6 polyubiquitination and cellular inhibitor of apoptosis protein 1 (cIAP1) recruitment in HepG2 cells 40–60 min after ionizing radiation exposure [43]. The resulting ATM-TRAF6-cIAP-1 complex was responsible for stimulation of TAB2-dependent TAK1 phosphorylation, which is required for NF-κB activation by genotoxic stress [43]. PARP-1 and ATM-TRAF6cIAP1 signaling pathways converge at NEMO monoubiquitination at K285 [43]. In the cytoplasm, ELKS binds to the ATM-NEMO complex, enabling ATM-dependent activation of the canonical IKK complex containing IKKα/β [35,103]. The active kinase complex phosphorylates IκBα on serines 32 and 36, resulting in its subsequent ubiquitination and its proteasomal degradation [36,43,68]. The liberated NF-κB translocates to the nucleus where it binds to NRE containing promoters and enhancers [107]. In the radiation response, the heterodimer p50:p65 is most often found to be activated [95], but also other NF-κB subunits such as RelB might be relevant.

Signals from the outside In addition to this direct NF-κB activation in response to DNA DSBs after ionizing radiation exposure via the genotoxic stress induced subpathway, other pathway input ports using membrane receptors can be frequented. The initial direct NF-κB activation can induce cytokine expression which sustains NF-κB activation in a positive feed forward loop via the canonical pathway. After excessive DNA damage, ATM stimulates cytokine secretion to admonish neighboring cells of danger [118] and two waves of NF-κB activation were observed: In the first phase, NF-κB induced TNF-α secretion resulted in its binding to TNFR1 and thereby autocrine feed forward signaling (~6 h after etoposide exposure in HeLa cells), leading to a second wave of TNF-α secretion. In the second phase (after 26 h), CXCL8 secretion depended on the feed forward signaling of the first phase [118]. Such chemokines expressed after initial NF-κB activation might also be responsible for perpetuation of the NF-κB activation. Chemokines are small secreted proteins and promote cell trafficking (chemoattraction of leukocytes, invasion of tumor cells) and have local effects on cell activation [19]. Chemokines can thereby contribute to the clearance of dying cells by recruit-ment of macrophages and/or neutrophils [119–121] and are involved in regulation of angiogenesis, fibrosis, wound healing, proliferation, apoptosis susceptibility and survival [19,122,123].

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At high radiation doses, products released from damaged or dying cells represent damage-associated molecular patterns (DAMPs) and bind to pattern recognition receptors (PRR) such as TLR [13], resulting in NF-κB activation and production of proinflammatory cytokines and type I interferon (IFN)2 [124]. These receptors recognize non-self molecular patterns (e.g. PAMPs) and altered molecular patterns (such as DAMPs). Depending on the immune cell, they mediate activation of innate and acquired immunity, e.g. dendritic cells as professional antigen presenting cells can be activated and cross present tumor antigens and thereby activate T-cells [124]. Activation of these receptors has an important impact on the interaction of an irradiated tumor with the immune system. On the one hand, signals from DAMPs may promote carcinogenesis [125], while on the other hand, stimulation of selected TLRs is tested as anti-tumor strategy. Currently, TLR4 and TLR7 are in the focus of adjuvant immunotherapy combined with chemo- or radiotherapy of a tumor [126]. TLR4, together with its coreceptor, myeloid differentiation factor-2 (MD-2), is activated after binding of LPS [127], resulting in activation of MAPK and NF-κB which trigger the proinflammatory cascade and oxidative stress [128]. The role of TLR4 in the antitumor immune response is highlighted by the observation that a TLR4-loss-of-function-allele in cancer patients seems to worsen the outcome after radiotherapy [126]. TLR4 also promotes fibrosis development in the liver [127,129,130] and is regarded as largely tumor-promoting [13], as chronic low level stimulation of TLRs is suggested to support tumor survival and promotion in general [13]. Stimulation of TLR7 by imiquimod results in NF-κB activation and production of proinflammatory cytokines by dendritic cells [124].The alarmin high-mobility group box 1 (HMGB1) protein [126,131,132] and hyaluronan breakdown products [133] are endogenous ligands for TLR. The nonhistone chromatin binding protein HMGB1 is passively released by dying cells (necrotic and apoptotic cells) after exposure to high radiation doses (10 Gy) (immunogenic cell death) [19,24,126,134,135], but also actively secreted by inflammatory antigen presenting cells and acts then as potent inflammatory mediator [134,135]. HMGB1 and hyaluronan can activate NF-κB after binding to TLR2/TLR6 and TLR4 on dendritic cells, resulting in release of proinflammatory cytokines and prevention of apoptosis [13]. Together with an “eat me” signal on the cell surface (calreticulin) and release of ATP, HMBG1 can activate dendritic cells after binding to TLR4 [24] and stimulate an anti-tumor immune response [19,126,136,137]. Calreticulin surface translocation and ATP release can also be triggered by ionizing radiation exposure [138]. This allows dendritic cells to cross-present tumor antigens from dying tumor cells after processing and maturation to naïve CD4 T helper (Th) cells and cytotoxic CD8 T cells, which eliminate tumor cells [126]. Stimulation of TLRs on antigen-presenting cells by exogenous ligands in combination with radiotherapy might help to overcome the immunosuppressive microenvironment in a tumor that is generated by immunosuppressive cytokines, M2 macrophages and regulatory T cells (Tregs) [137]. The adjuvant action will be transmitted through a TLR. In addition to endogenous ligands liberated from dying cells (HMBG1 binding to TLR4) after localized radiation therapy, synthetic ligands such as imiquimod for TLR7 and synthetic C-G rich oligonucleotides (CpG) for TLR9 might be used in cancer immunotherapy. In fibrosarcoma and lung carcinoma, CpG activated dendritic cells after binding to TLR9, resulting in improved effectiveness of the anticancer therapy [137]. Injection of CpG in low-grade B-cell lymphoma of 15 patients induced tumorreactive memory CD8 T cells and resulted in one complete and three partial responses at tumor locations outside the irradiation field [139]. The challenge of TLR stimulation to act as immunoadjuvant

2 IFN-γ promotes effective cytotoxic T cell response, higher trafficking, better tumor cell recognition and then lysis by increased MHC class I expression [168].

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lies in the appropriate dose and timing with radiotherapy, to avoid tolerogenic effects, achieve highest immunogenicity of tumor antigens while avoiding severe side effects or chronic low level stimulation. In summary, in vitro and in vivo studies have shown that irradiated tumor cells can be a source of tumor antigen with immunoadjuvant properties [140,141], and more studies are required to assess possible therapeutic implications of TLRs in cancer immunotherapy. Role of NF-κB in the radiation response Activation of the NF-κB pathway results in modulation of NFκB target gene expression. NREs have been identified in the promoter or enhancer regions of a number of growth factors, cytokines, chemokines and adhesion molecules and other immunoregulatory proteins involved in fibrosis and inflammation [40,142]. In addition, NF-κB regulates the expression of many genes whose products are involved in the control of cell proliferation and cell death [143]. The role of NF-κB in the radiation response depends on the set of target genes that are up- or downregulated and the duration of its activation. An overview of NF-κB target genes that were shown to be upregulated after ionizing radiation exposure is given in Table 2. As shown in the section “NF-κB activation after ionizing radiation exposure”, several pathways exists that could sustain NF-κB activation after its initial radiation-induced activation via ATM. Antiapoptotic and antioxidative NF-κB target genes as survival factor NF-κB activation, especially RelA [177], in response to different stimuli, including genotoxic agents, can protect cells from apoptosis triggered by the mitochondrial pathway or by death receptors [178,179] via expression of anti-apoptotic proteins [20]. Possible pro-survival genes that might be transcribed at a higher rate are the inhibitor of apoptosis protein (IAP) genes, such as cIAP1/ BIRC2, cIAP2/BIRC3 and X-linked IAP (XIAP). XIAP is suggested to preclude apoptosis by interaction with caspase-9 [172]. cIAP1 was upregulated in spontaneously immortalized human breast epithelial cells (MCF-10F) after exposure to 0.5 and 1 Gy X-rays and Fe ions (1 GeV/n, LET 150 keV/μm) [144]. In the human breast cancer cell line CAL51, the RelA dependent antiapoptotic target genes BIRC3 and TNF-α induced protein 3 (TNFAIP3/A20) were upregulated after exposure to 5 Gy ionizing radiation [101]. BIRC3 encodes an ubiquitin ligase that targets caspases 3 and 7 and TRAF1 and 2. After whole body irradiation (15 Gy), BIRC2 and TNFAIP3 were upregulated in murine lymph nodes [104]. Other anti-apoptotic genes are bcl-2, bcl-XL, survivin, FLIP and A1. Preliminary experiments by Gong et al. have shown that high-LET radiation has a higher potential to activate survivin expression compared with low-LET radiation [173,174]. Upregulation of the NF-κB regulated gene GADD45B was found in HEK/293 cells after exposure to α-particles (0.5 MeV/n, LET 160 keV/ μm) [176]. GADD45β was shown to block cell death induced by DNA damaging agents [179]. As activation of the NF-κB pathway is supposed to play a role in the negative regulation of apoptosis, survival of cells with residual DNA damage might thus be favored. Even if this happens only in a small fraction of irradiated cells, it might be important for the initiation or promotion of cancer [180]. A protective mechanism against cancer can be the survival of a cell with persisting DNA damage as senescent cell, preventing the damaged cell to be transformed. However, senescent fibroblasts, which secretory profile comprises NF-κB controlled cytokines, can promote growth of preneoplastic and malignant cells and angiogenesis [161,181]. They can accumulate in premalignant lesions [119,182]. However, under certain circumstances, NF-κB may facilitate proapoptotic signals, through repression of prosurvival genes or upregulation of proapoptotic factors [4,7,34,118,183].

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1 Table 2 2 Q10 NF-κB target genes involved in the radiation response. 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67

Functional group

Genes with evidence for regulation after ionizing radiation exposure

Proinflammatory molecules Cytokines Tumor necrosis factor α (TNF-α)

Interleukin 1α and 1β (IL-1α, IL-1β) Interleukin 6 (IL-6)

Enzymes

Interleukin 33 (IL-33) Cyclooxygenase (COX-2/PTGS2) Inducible nitric oxide synthase (iNOS) Manganese superoxide dismutase (MnSOD/SOD2)

Chemokines

CXCL1, CXCL2, CXCL8

Chemokine (CXC motif) ligand 1 (CXCL1) Chemokine (CXC motif) ligand 2 (CXCL2) Chemokine (CXC motif) ligand 8 (CXCL8/IL-8)

Adhesion molecules

Chemokine (CXC motif) ligand 10 (CXCL10) Chemokine (C-C motif) ligand 3 (CCL3) CD83 Vascular cell adhesion molecule-1 (VCAM1) Intercellular adhesion molecule (ICAM1) E-selectin Major histocompatibility complex class I (MHC class I)

Tissue modulation Antiapoptotic proteins

Growth factors Cell cycle control NF-κB pathway components

Matrix metalloproteinases (MMP1, MMP3) TNF-α-induced protein 3 (TNFAIP3/A20) Baculoviral IAP repeat containing 2 (BIRC2/cIAP1) Baculoviral IAP repeat containing 3 (BIRC3/cIAP2) X-linked inhibitor of apoptosis (XIAP) BCL2 related protein1 (BCL2A1) Survivin Vascular endothelial growth factor (VEGF) Growth arrest and DNA damage-inducible 45β (GADD45B/MyD118) Nuclear factor of kappa light polypeptide gene enhancer in B-cells 2 (NFKB2 (p52/p100)), v-rel avian reticuloendotheliosis viral oncogene homolog B (RelB) NF-κB inhibitor, alpha & epsilon (NFKBIA, NFKBIE)

Possible role in radiation response and in early, late and chronic radiation sequelae

Reference

Immunity, inflammation, control of cell proliferation, differentiation, apoptosis, acute radiation syndrome (whole body exposure), acute radiation syndrome (whole body exposure), acute local inflammatory response, central nervous system injury, radiation enteritis, skin eythema and desquamation Central nervous system injury, radiation enteritis, skin eythema and desquamation Inflammatory response, bystander effect, senescence, fibrosis, radiation enteritis, skin eythema and desquamation Bystander effect Inflammatory response (Prostaglandin E2, PGE2) Bystander effect Inflammatory response (NO production) Bystander effect Control of superoxide prodution in mitochondria, survival advantage for tumor cells (radioresistance), protective effect in normal tissues such as bone marrow stroma cells, adaptive response Intercellular communication, modulation of cellular radiation response including persistent DNA damage response, growth arrest, chemoattraction of neutrophils, angiogenesis Attraction of neutrophils, angiogenesis, senescence Bystander effect, late radiation enteritis Chemoattractant for neutrophils, angiogenesis, tumorigenicity, metastasis, senescence, bystander effect Recruitment o CD8 and CD4 T cells to the tumor Leukocyte chemotaxis Increased recognition of tumor cells by the immune system Expression on tumor vascular endothelium facilitates T cell infiltration Bystander effect; improved recognition and killing of tumor cells by CD8 T cells Adhesion molecule guides immune cells into the tumor Anti-tumor immune response of MHC class I restricted cytotoxic T cells resulting in increased recognition of tumor cells by the immune system Tissue remodeling, migration of cells and angiogenesis., late radiation enteritis Inhibition of radiation-induced apoptosis Inhibition of apoptosis, radioresistance of tumor cells Inhibition of apoptosis by interaction with caspases 3 &7 Inhibition of radiation-induced apoptosis Inhibition of radiation-induced apoptosis Inhibition of radiation-induced apoptosis Tumor vasculature becomes more permeable

[23,144–150]

Cell cycle regulation, survival of tumor cells or normal tissue cells, radioresistance of tumor cells Alternative NF-κB pathway

[71,176]

Negative feedback loop resulting in transient or longlasting termination of NF-κB activation

[101,104,163,176]

[23,146–148,150–153] [23,54,56,146–148,150,151,154]

[53,151] [151,153,155,156] [23,153,155,156] [23,71,151,157,158]

[118,147,148,159,160]

[161,162] [151,163,164] [118,119,121,148,151,153,161,165,166]

[137,153] [153,163] [167] [55,137,163,167–169] [55,137,150,151,163,167–169] [55] [137,170]

[151,164] [101,104,151] [104,144,171] [101] [172] [151] [173,174] [175]

[101,104]

68 69 70 71 72 73 74

α-Particle exposure resulted among others in upregulation of MnSOD in human lung fibroblasts [151]. MnSOD scavenges superoxide radicals in the mitochondrial matrix and is thought to be an important determinant of sensitivity to ROS-induced cytotoxicity. Its gene contains NF-κB binding sites in the promoter and the first intron [184,185].

Radiation induced production of proinflammatory molecules: NF-κB as master regulator NF-κB controls the expression of more than 100 proinflammatory genes, including TNF-α, IL-1β, IL-6, CXCL8, cyclooxygenase (COX2), vascular cell adhesion molecule-1 (VCAM1) [186] and intercellular

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adhesion molecule (ICAM1) [38], which ensure NF-κB a central role in acute inflammatory responses, but are also known to promote cancer in different organs [13]. Besides the survival signals, alerting neighboring or even distant cells might be a major role of ionizing radiation induced NF-κB activation. Recently, it was shown that ionizing radiation activated NF-κB predominantly upregulates genes involved in intercellular communication processes, especially genes coding for cytokines and chemokines, and the gene expression profiles depended on radiation quality and dose. Cytokines such as IL-1α and β, IL-6 and TNF-α were shown to modulate the cellular radiation response [187]. In vitro, a single dose of 20 Gy rapidly induced IL-1β expression in isolated normal mouse spleen cells and leukemia cells [152]. In human alveolar macrophages, IL-1β was induced by a single dose of 2 Gy [188]. X-irradiation increased IL-6 synthesis of human embryonic lung fibroblasts FH109 with a maximum 9 hours after exposure to 5 Gy [54]. Human sarcoma cells produced TNF-α after exposure to a single dose of 5 Gy X-rays [145]. In mice, after irradiation of the thorax with 12 Gy X-rays (8 MV), NF-κB activation and expression of IL-1β, TNF-α and IL-6 were observed [146]. The soy isoflavone 5,7,4′-genistein suppressed these reactions in the lung and thereby radiation-induced pneumonitis [146]. Whole-body irradiation leads to NF-κB induced cytokine expression at least after exposure to high doses. After lethal irradiation of mice with 15 Gy, serum levels of TNF-α, IL-6, IL-10 and IL-1β increased [114]. IL-6 in murine plasma increased also after exposure to lower whole body doses with a clear dose dependence (2–7 Gy) [154]. Lethal γ-irradiation (8.5 Gy) of BALB/c mice activated NF-κB in the spleen, mesenteric lymph nodes, and bone marrow, and resulted in upregulation of TNF-α, IL-1α, IL-1β, and IL-6-mRNA in these organs [147]. Expression of TNF-α, IL-6 and IL-1β increased also in the ileal muscularis layer 3 and 6 h after γ-irradiation (10 Gy) of rats [148]. The anti-inflammatory cytokine IL-10 increased transiently, but was repressed on day 3 [148]. High dose whole body exposure induced inflammation in the liver with NF-κB as a central regulating factor, increasing the expression of inducible nitric oxide synthase (iNOS), COX-2, IL-6, and TNF-α. The enzymes iNOS and COX-2 produce secondary inflammatory mediators [10]. IL-6 boosts cancer cell invasion and controls many other cytokines in a selffeeding loop [159]. CCL3, CXCL10, CXCL8, IL1B and iNOS expression was upregulated in rat skin one day after exposure of rats to 1.1 GeV/n Fe ions, with a skin dose of 3 Gy [153]. CXCL10 is a chemoattractant for T cells [189]. NF-κB regulated genes upregulated in response to irradiation encompassed the chemokines CXCL13 and CXCL24 [104]. The chemokine CXCL2 was upregulated in murine tumor models after carbon ion irradiation (290 MeV/n, LET 50 keV/ μm) or γ-irradiation (30 Gy) [163]. Its expression was recently shown to depend on NF-κB [190]. Whether such upregulation results in immune cell chemotaxis and infiltration into tumor has still to be determined. Also the expression of adhesion molecules such as E-Selectin, ICAM1 and VCAM1 which are involved in adhesion of leukocytes to endothelia prior to their extravasation can increase in response to ionizing radiation exposure. The glycoprotein E-selectin which mediates leukocyte rolling on the endothelium was induced after X-ray exposure in human endothelial cells in a NF-κB dependent fashion [55]. ICAM1 is expressed e.g. on endothelial cells upon inflammatory stimulation and mediates strong attachment of leukocytes by binding to integrins (CD11a or b/CD18), initiating

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3 CXCL1 is also designated as GRO1 oncogene, GROα, KC, neutrophil-activating protein 3 (NAP-3) and melanoma growth stimulating activity, α (MSGA-α). 4 CXCL2 is also called macrophage inflammatory protein 2-α (MIP2-α), growthregulated protein β (Gro-β) and Gro oncogene-2 (Gro-2).

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transmigration. For example, ICAM1 expression increased 3 days after 67 carbon ion exposure in murine tumor models [163] and in oral 68 69 mucosa of head and neck cancer patients after radiotherapy (2 Gy 70 fractions with a total dose 60 Gy) [169]. ICAM1 expression also in71 creased in human adenocarcinoma cells 4 days after X-irradiation 72 (5, 10 and 12 Gy) [191], in human breast cancer cells (MDA-MB73 231) 3 days after γ-irradiation [141] and in human lens epithelial 74 cells 6 h after exposure to 55 MeV protons (4, 8 or 12 Gy) [167]. VCAM1 usually appears on endothelial cells in response to cytokines, Q5 75 76 mediating the adhesion of monocytes and T cells, and its promot77 er contains two NREs [186]. In a mouse model, VCAM1 expression 78 was induced on tumor vasculature after exposure to 15 Gy [168]. 79 Increased expression of ICAM1 and VCAM1 on tumor and endo80 thelial cells could promote immune cell infiltration into the tumor [140]. Furthermore, carbon ion irradiation induced the expression 81 of vascular endothelial growth factor (VEGF) in lung carcinoma 82 cells at high doses (15 Gy, LET 13.3–90 keV/μm) [175]. As a conse83 quence, tumor vasculature becomes more permeable and adhesion 84 molecules guide immune cells into the tumor [19]. 85 86 87 Gene expression profile in radiation-induced senescence: 88 role of NF-κB 89 90 Recently, it was shown that human umbilical vain endothelial 91 cells (HUVEC) gain a senescent phenotype after ionizing radiation 92 exposure (2–8 Gy) through the genotoxic stress induced NF-κB 93 subpathway, possibly mediated by increased IL-6 production [56]. Persistent DNA damage was identified to promote secretion of 94 IL-6 and CXCL8 with NF-κB as controlling transcription factor 95 [118,147,148,159,160]. Such persisting NF-κB activation was ob96 served in bone marrow cells of mice one month after whole body 97 γ- or proton irradiation [81]. A persistent DDR can lead to cellular 98 senescence which is associated with secretion of several proteins 99 (senescence-associated secretory phenotype, SASP) leading to main100 tenance of senescence and innate immune response for tumor 101 suppression and clearance [159,192]. Radiation-induced SASP 102 depends among others on ATM and NF-κB. Senescent cells produce 103 higher amounts of extracellular proteases such as matrix 104 metalloproteinases (MMP), matrix components and cyto- and 105 chemokines (monocyte chemotactic protein-1 (MCP-1), CXCL1, IL106 15) than early passage cells [193,194]. Some secreted molecules are 107 tumor promoting by supporting invasion, angiogenesis, tumor 108 growth, epithelial mesenchymal transition (EMT) (IL-6, CXCL8) and 109 altered differentiation (MMP-3) [159]. Chemokines such as CXCL8 110 and CXCL1 bind to the chemokine receptor CXCR25 and can main111 tain the growth arrest [119–121,159]. CXCL8 is a chemoattractant 112 for neutrophils and promotes their degranulation [119]. It modu113 lates the migration of endothelial cells and cancer cells and thereby 114 promotes angiogenesis, tumor growth and metastasis in a variety 115 of human cancers [121,162,195,196]. CXCL1 may also promote an116 giogenesis [162]. Because of this paracrine action on other cell types, 117 CXCL8 and CXCL1 are regarded as positive mediators of tumor 118 progression [161]. For both genes, the involvement of NF-κB in their 119 expression was shown, as RelA binds to the promoter of the CXCL8 120 gene [119], and the NF-κB binding site in the CXCL1 gene promot121 er was essential for induction of its expression [162]. Also IL-15 122 expression can be upregulated by NF-κB activation [194]. 123 124 Radiation induced regulatory loops in the NF-κB pathway 125 126 NF-κB dependent genes upregulated in response to irradiation 127 encompassed NFKB2, RelB, and NFKBIA [104]. Upregulation of 128 129 130 5 CXCL1, 2, 3 (GROα, β and γ) and CXCL8 (IL-8) bind to CXCR2; CXCL8 also binds 131 to CXCR1. 132

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NFKBIA was also found in HEK/293 cells after exposure to α-particles (0.5 MeV/n, LET 160 keV/μm) [176]. In the human breast cancer cell line CAL51, 37 RelA dependent genes were upregulated after exposure to 5 Gy ionizing radiation, including the NF-κB subunits NFKB2 and RelB, and the NF-κB inhibitors NFKBIA and NFKBIE [101]. NFKBIA expression increased weakly 1 and 3 h after exposure of human melanoma cell lines to carbon ions (2 Gy, 290 MeV/n, LET 50 keV/μm) [197]. A negative feedback regulation via IκB upregulation was also found after carbon ion and γ-irradiation of murine tumors – the inhibitor NFKBIE was upregulated 3 days after exposure [163]. NF-κB in the bystander effect A radiation-induced bystander effect implicates that cells which were not exposed to ionizing radiation respond to a stress signal from nearby irradiated cells [198]. Bystander effects were observed in monolayer cell cultures, 3D human tissues [199] and whole organisms [200]. They include sister chromatid exchanges [201,202], micronucleus formation [203], chromosome aberrations, mutation induction [204], oncogenic transformation [205], apoptosis [206] and terminal differentiation [207]. In the low dose range, the number of non-hit cells, which are potential bystander cells, is high in the neighborhood of the irradiated cells. In the high dose range, when all cells are hit, no bystander effect might be observed. The bystander effect is mediated by exchange of small molecules such as ROS and NO via gap junctions and by cytokines and chemokines that are released by the irradiated cells [19]. In a bystander model with sister chromatid exchanges as endpoint, ATM was necessary for generation of the bystander signal [202]. NF-κB was shown to play a role in the bystander effect as well as the adaptive response [151,208]. While p53 activation was minimal in bystander cells, NF-κB activation was observed in irradiated and in bystander cells [151]. In normal human fibroblasts, exposure to 4He ions (LET 120 keV/μm, 0.5 Gy) resulted in NF-κB activation and threefold increased expression of COX-2 which exerts proinflammatory effects by production of prostaglandin E2 (PGE2) [155,156]. The same irradiation conditions also resulted in up-regulation of CXCL8, IL1B and IL33 in directly irradiated cells and, by a paracrine loop, in bystander cells [53]. Later on, expression of IL-6 and SOD2 and TNF-α also increased. NF-κB activation in this setting was ATM-dependent [53]. In the expression profile of bystander cells, genes involved in proliferation, cell death and cell cycle progression were less important, while upregulation of stress signaling, cytokine and matrix metalloproteinase genes preponderated [151]. For the interpretation of risks arising from exposure to low doses of ionizing radiation, it has to be evaluated in future whether the bystander effect is pro- or anticancerogenic and whether it induces an innate and/or acquired immune response. Role of NF-κB in cancer patients subjected to radiotherapy Constitutive NF-κB activity in tumors and in many lymphoid malignancies [209] can represent an obstacle to successful radiotherapy because of antiapoptotic effects. NF-κB might be activated in a tumor prior to radiotherapy by other stress factors such as hypoxia. In an irradiated tumor, NF-κB can be activated in different cell types, the tumor cells and the stroma cells including fibroblasts, endothelial cells of the tumor vasculature and immune cells. As inducer of cytoproctective responses such as antiapoptosis and senescence, NF-κB activation can modulate the outcome of exposure to radiation and genotoxic drugs. Radiation-induced NF-kB activation may promote development of radiation or chemotherapy resistance. Therapy resistance was observed in some cases such as multiple myeloma due to enhanced expression of antiapoptotic target genes [209]. In MCF-7 breast carcinoma cells, induced

radioresistance was attributed to increased MnSOD expression after radiation induced NF-κB activation [157]. Radiation-induced NF-κB activation could also enhance the survival of tumor cells which already show constitutive NF-κB activity. Inhibition of NF-κB can sensitize tumor cells for radiotherapy [210]. For example, indomethacin increased the radiosensitivity of HeLa cells by inhibiting NF-κB-DNA binding [70]. It has to be considered that for example IKK inhibitors have severe side effects and that more specific targeting is required, especially because immune suppression will not be beneficial for an effective antitumor immune response. Heat shock pretreatment transiently reduced X-ray induced NF-κB activation via inhibition of IKK activity in HeLa cells [211]. Suppression of the NF-κB target gene CXCL8 was favorable for therapy sensitivity of cancer cells [121]. In the irradiated normal tissues, activated NF-κB contributes via increased cytokine expression to an inflammatory response, as observed in skin and oral mucosa of head and neck cancer patients, and to systemic and abscopal effects after radiotherapy [140]. Proinflammatory effects are seen with doses as low as 2 Gy [140]. Production of inflammatory cytokines (IL-1 β, TNF-α, IL-6) is induced by radiation in the tumor and in the surrounding normal tissue and contributes to side effects in blood, peripheral lymphoid tissues, lungs [212–214]. Prolonged secretion of TNF-α from epithelial, endothelial and connective tissue cells has an important role in the development of cutaneous radiation syndrome [215]. In addition, increased expression of TNF-α, IL-1α, IL-1β, IL-6, and TGF-β was observed outside of the irradiated field in the lung of rats exposed to partial lung irradiation [12–14], revealing the involvement of the innate immune system in the non-targeted effects of radiotherapy [24]. Depending on set of target genes that is expressed, NF-κB might support tissue repair or participate in the pathogenesis of radiation-induced tissue damage. A persisting inflammatory response after initial radiation injury might pave the way to late side effects. It is unclear whether NF-κB activation in the brain after ionizing radiation exposure is neuroprotective or contributes to neurodegeneration [85]. While inappropriate continued recruitment of innate immune responses can contribute to acute and chronic side effects of radiotherapy including the development of secondary cancers and non-targeted effects, appropriate signaling to immune cells could support tumor clearance [192,216]. The expression of adhesion molecules and cyto- and chemokines resulting from radiation induced NF-κB activation changes the tumor microenvironment and sends signals to the immune system. Together with up-regulated cell surface receptors and adhesion molecules, the cytokines and chemokines modulate the interaction with the immune cells. A negative outcome would be an immunosuppressive reaction with attraction of Tregs. The best outcome would be the conversion of a tumor by radiotherapy into a personalized in situ vaccine by recovering immunogenicity [124], resulting in an enhanced antitumor immune response with infiltration of cytotoxic T cells. For effective tumor antigen cross presentation by dendritic cells to T cells and subsequent lysis by cytotoxic T cells, immunogenic tumor cell death, MHC class I expression by the tumor cells and upregulation of cytokines/ chemokines with immune cell recruitment to the tumor are required [168]. MHC class I expression can also be upregulated by radiationinduced NF-κB activation. Hereby, the relative ability of a radiotherapy regimen to induce cross-priming without increasing the numbers of regulatory T cells might determine the immunogenic effect [217]. In general, NF-κB activation is strong in the range of 7–10 Gy and combined radiation and immunotherapy is also most effective with fractions in this dose range [23,137].

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Conclusions Aside from the changes occurring within the cell, radiationinduced DNA damage promotes cytokine and chemokine release via

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activation of NF-κB [118,218,219], allowing for altered communication between cells organized into tissues and microenvironments [91,220]. NF-κB is a central factor in modification of the tumor microenvironment after localized radiation therapy and elicits complex responses which can be immunosuppressive or enhance the antitumor immune response. According to the current understanding, a better immune response can be expected at higher doses (7–10 Gy) because of the induction of immunogenic cell death and stronger NFκB activation in this dose range. To optimally use NF-κB modulation for cancer therapy, optimal regimens of radiotherapy have to be defined (dose, fractionation, sequencing with immunotherapy) and a dissociation of its immunregulatory function from its antiapoptotic effects would be desirable. In addition, a better understanding of the underlying pathways and inhibition of main players that do not affect the killing effect of radiation on the tumor might help to avoid inflammation of surrounding normal issues as side effect of tumor radiotherapy. For example, the plant flavonoid ferulic acid reduced radiation-induced inflammation after high dose exposure (10 Gy) of mice via diminished NF-κB activation and COX-2 and iNOS-2 expression, and the increase of TNF-α and IL-6 in serum was prevented [221]. In the low dose range, NF-κB has to be considered as a major player in radiation-induced bystander effects. Furthermore, complex DNA damage which is caused to a higher extent by high LET than by low LET radiation might be difficult to repair, resulting in persistent NF-κB activation. A possible outcome is senescence of the cell, which prohibits this cell to become a cancer cell, but the changed gene expression profile might be procancerogenic. As pathway analyses were performed with high or extremely high doses, the role of different components in low dose range should be reassessed. In normal skin tissue, low doses (0.01 and 1 Gy) of ionizing radiation resulted in transient alterations in the expression of genes involved in DNA and tissue remodeling, cell-cycle transition, and inflammation [222], suggesting an involvement of the NF-κB pathway. Also, curcumin which can inhibit NF-κB activation was shown to reduce radiation induced initiation of mammary tumorigenesis in rats [223]. Currently, it is very difficult to judge whether the induced cytokine signaling has beneficial or detrimental effects. Further analysis of the NF-κB pathway and its involvement in cell and tissue outcome after low dose exposures is necessary to provide information for risk assessment of occupational and other low dose exposures. Funding The Helmholtz Association and the German Aerospace Center are acknowledged for funding SpaceLife doctoral students inQ6 volved in the accelerator experiments. Acknowledgements The DLR Cellular Biodiagnostics team and the beam operator team are acknowledged for help given beam times at the French Heavy Ion Accelerator GANIL which provided some of the results cited in this review. Conflict of interest

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