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Cellular Monitoring of the Nuclear Factor κB Pathway for Assessment of SpaceEnvironmental RadiationAuthor(s): Christa Baumstark-Khan, Christine E. Hellweg, Andrea Arenz, and Matthias M. MeierSource: Radiation Research, 164(4):527-530. 2005.Published By: Radiation Research SocietyDOI: http://dx.doi.org/10.1667/RR3397.1URL: http://www.bioone.org/doi/full/10.1667/RR3397.1

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RADIATION RESEARCH 164, 527–530 (2005)0033-7587/05 $15.00q 2005 by Radiation Research Society.All rights of reproduction in any form reserved.

Cellular Monitoring of the Nuclear Factor kB Pathway for Assessment ofSpace Environmental Radiation

Christa Baumstark-Khan,1 Christine E. Hellweg, Andrea Arenz and Matthias M. Meier

German Aerospace Centre (DLR), Institute of Aerospace Medicine, Linder Hohe, D-51147 Koln, Germany

Baumstark-Khan, C., Hellweg, C. E., Arenz, A. and Meier,M. M. Cellular Monitoring of the Nuclear Factor kB Pathwayfor Assessment of Space Environmental Radiation. Radiat.Res. 164, 527–530 (2005).

A screening assay for the detection of NF-kB-dependentgene induction using the destabilized variant of the reporterprotein enhanced green fluorescent protein (d2EGFP) is usedfor assessing the biological effects of accelerated heavy ions asa model of space environmental radiation conditions. The timecourse of d2EGFP expression and therefore of activation ofNF-kB-dependent gene expression was measured after treat-ment with TNFA or after heavy-ion exposure using flow cy-tometry. The reported experiments clearly show that accel-erated argon ions (95 MeV/nucleon, LET 230 keV/mm) inducethe NF-kB pathway at low particle densities (1–2 particle hitsper nucleus), which result in as few as 5–50 induced DSBs percell. q 2005 by Radiation Research Society

INTRODUCTION

Astronauts are exposed not only to greater amounts ofnatural radiation in space than they receive on Earth butalso to a different radiation quality, which can result inimmediate and long-term risks. It has been estimated (1)that on a 3-year mission to Mars, even if it is shielded by4 g/cm2 of aluminum, a mammalian cell nucleus (area 100mm2) would be traversed on average by 400 protons, 0.6carbon nuclei, and 0.03 iron nuclei. This means that about3% of the body’s cell nuclei would have 2000 primary ion-izations deposited in them (1). The probability of such acell surviving unharmed is small. Accordingly, radiationprotection issues are concerned mainly with late effects,especially cancers, which are stochastic in nature.

The cellular response to radiation begins with the sensingof DNA damage and ends with DNA repair or cell death,and it involves numerous steps in multiple signal transduc-tion pathways (2, 3). A general scheme is that several sen-sors detect the induced DNA damage and trigger signaltransduction pathways. This then leads to the activation of

1 Address for correspondence: German Aerospace Centre (DLR), In-stitute of Aerospace Medicine, Linder Hohe, D-51147 Koln, Germany;e-mail: christa.baumstark-khan@dlr.de.

various transcription factors, resulting in the expression ofcertain genes whose protein products are involved in pro-ducing several possible outcomes, such as cell cycle arrest,allowing repair of the damaged DNA, or apoptosis, if thedamage is too severe and cannot be repaired.

The NF-kB (NFKB)/REL is a family of transcription fac-tors [NF-kB1 (p50/p105), NF-kB2 (p52/p100), RelA (p65),RelB and c-Rel] that can be activated in response to oxi-dative stress. It is involved in proliferation, inflammationand apoptosis (4–7). In the inactive state, NF-kB is boundto its inhibitor IkB (IKB), mainly to IkBa, which controlsnuclear uptake of NF-kB by masking the nuclear localiza-tion sequence of p65 and p50 (8). Upon activation, IkBacan be degraded by several proteases (9), and the releasedNF-kB translocates to the nucleus and binds to kB or kB-like DNA motifs (NREs), initiating gene transcription. NF-kB sites have been identified in the promoter or enhancerregions of a number of growth factors, cytokines and ad-hesion molecules involved in fibrosis and inflammation(10). In addition, NF-kB also regulates the expression ofmany genes whose products are involved in the control ofcell proliferation and cell death (11). In a cell culture mod-el, it has been found that ataxia telangiectasia mutated pro-tein (ATM) plays a role in sustained activation of NF-kBin response to DNA double-strand breaks (12, 13), probablyby its PI3 kinase-like activity (3). Activation of the NF-kBpathway not only protects cells from apoptosis after treat-ment with various genotoxic agents through expression ofanti-apoptosis proteins such as TRAF1, TRAF2, cIAP1 andcIAP2 (14) but also gives transformed cells a growth andsurvival advantage and further renders tumor cells resistantto treatment (15). NF-kB also enhances the expression ofdegradative enzymes, supporting the idea that it makes amajor contribution to tumor progression and metastasis invarious cancers (16).

In view of its tumor-promoting capacity, NF-kB is animportant factor involved in the modulation of environ-ment-induced gene expression. Many investigations wereperformed on the activation of the NF-kB pathway usinghundreds of inducers of NF-kB DNA binding (17). Besidesimmune-modulating agents (lipopolysaccharides), a varietyof other cellular stress factors are able to induce this path-way, including cytokines, phorbol esters, viruses, ultraviolet

528 BAUMSTARK-KHAN ET AL.

FIG. 1. NF-kB activation of stably transfected HEK cells expressingd2EGFP controlled by the KB4-TK promoter (pNF-kB-d2EGFP/Neoclone L2) by various stimuli after 24 h. Treatment at 378C with 0.6 nmol/liter TNF-a and 16 nM PMA. X-ray experiments with various doses wereperformed at 08C and at 378C. Means and standard deviations of at leasttwo independent experiments with three samples per dose.

radiation, reactive oxygen species, growth factor depletion,hypoxia, heat shock and ionizing radiation (10, 18).

Recently, we developed a recombinant mammalianscreening assay for quick and reliable measurements of cel-lular NF-kB response (19). It is based on a mammalian cellsystem harboring a plasmid controlling the expression ofthe destabilized variant (20) of enhanced green fluorescentprotein (d2EGFP) as a reporter molecule by a syntheticpromoter containing four tandem copies of kB elements. Inthis cell system (HEK-pNF-kB-d2EGFP/Neo cells), acti-vation of the cellular NF-kB pathway leads to binding ofliberated NF-kB not only to its target genes but also to thesynthetic promoter. Thus the cells respond with increasedtranscription of d2EGFP, followed by translation and mat-uration of the protein. It is then possible to quantify NF-kB-dependent transcriptional activation in response to stim-uli simply by measuring the levels of green fluorescence ofthe reporter protein. The kinetics of NF-kB activation byTNFA could thus be followed (19). To determine whetherheavy-ion exposure of human cells can lead to alterationsin molecular regulation, we have investigated the time- anddose-dependent response of NF-kB activation after expos-ing human embryonic kidney cells to accelerated heavyions (95 MeV/nucleon argon). Here we show that radiationqualities that are relevant for space missions are capable ofactivating the NF-kB pathway.

MATERIALS AND METHODS

Cell Culture

The cell line HEK 293 was established by Graham et al. (21) fromhuman embryonic kidney cells immortalized by transformation withsheared fragments of adenovirus type 5 DNA. The cells contain only thestably integrated transforming region (E1a and E1b genes) of the ade-novirus genome and do not produce viral particles. Cells were obtainedfrom the American Type Culture Collection (Manassas, VA, ATCC CRL-1573). Cloning of the plasmid pNF-kB-d2EGFP/Neo and stable transfec-tion and selection of an appropriate cell clone (HEK-pNF-kB-d2EGFP/Neo clone L2) have been described (19). Cells were maintained in amedium (Biochrom KG, Berlin, Germany) containing 1.5 mg/ml G418and 10% fetal bovine serum (FBS) under standard conditions (378C, 95%air and 5% CO2 atmosphere).

Treatment Modalities

For chemical treatment or irradiation experiments, 3 3 104 cells/cm2

were seeded into suitable culture vessels (3-cm dishes, Nunc; 96-wellstrip plate, Costar) freshly coated with poly-L-lysine (10 mg/cm2) andgrown for 4 days.

Chemical treatment. Human recombinant TNFA, bovine serum albumin(BSA), and phorbol-12-myristate-13-acetate (PMA) were obtained fromSigma-Aldrich Chemie, Steinheim, Germany. TNFA (final concentration0.6 nM) dissolved in PBS containing 1% BSA or PMA (final concentra-tion 16 nM) dissolved in dimethyl sulfoxide (DMSO, final concentration0.1%) was added to medium containing serum. Cells were harvested forFACS analysis after different times.

X Irradiation. Cells in early exponential growth phase were irradiatedon ice or at 378C with 150 kV X rays generated by an X-ray unit (MullerTyp MG 150, Germany) yielding a dose rate of 2 Gy min21.

Heavy-ion exposure. Cells were exposed to high-LET heavy-ion par-ticles (95 MeV/nucleon argon, LET 232.2 keV/mm) at the French heavy-

ion accelerator GANIL (Caen, France). Irradiation was performed at roomtemperature with dose rates adjusted to achieve irradiation within 2 min.Dosimetry was performed by Dr. Isabelle Testard, GANIL, and her do-simetry group.

Flow Cytometry

Cells were harvested at different times after radiation or chemical treat-ment by trypsinization and fixed with 3 ml cold 3.5% paraformaldehydein PBS. For FACS analysis, cells were centrifuged and resuspended inPBS. Forward and scatter and green fluorescence (FL1) of 20,000 cellsfrom the samples were measured in a FACScan (Becton Dickinson, SanJose, CA) with an argon laser (488 nm) as the excitation source. Themarkers M1 (EGFP2) and M2 (EGFP1) were set by means of untreated(M1) and TNFA-treated (M2) cells.

RESULTS AND DISCUSSION

NF-kB Activation by Various Stimuli

The stably transfected cell line HEK-pNF-kB-d2EGFP/Neo clone L2 developed by Hellweg et al. (19) allows mon-itoring of the transcription-activating potential of NF-kB insingle cells or populations by fluorometric methods. An in-crease in d2EGFP fluorescence after stimulation with dif-ferent agents results from completion of the followingevents: activation of specific kinases, liberation of NF-kBfrom its inhibitor after kinase-mediated phosphorylation, itstranslocation to the nucleus and binding to promoters con-taining NF-kB response elements, start of transcription ofthe reporter gene as an artificial NF-kB target gene, trans-lation, modification and proper folding of d2EGFP.

To test NF-kB-dependent gene transcription by d2EGFPexpression by a known NF-kB activator before performingexperiments with accelerated heavy ions, HEK-pNF-kB-d2EGFP/Neo cells were treated with known activators ofNF-kB (Fig. 1). In the case of NF-kB activation by TNFA(0.6 nM), the cells quickly responded with EGFP fluores-

529HEAVY-ION-INDUCED NUCLEAR FACTOR kB ACTIVATION

FIG. 2. Kinetics of NF-kB activation of stably transfected HEK cellsexpressing d2EGFP controlled by the KB4-TK promoter (pNF-kB-d2EGFP/Neo clone L2) after exposure to various fluences of 95 MeV/nucleon argon ions. Means and standard deviations of two independentexperiments with three samples per dose.

cence (60–80% positive cells at about 24–28 h). UsingPMA (16 nmol/liter), which has been reported to activateNF-kB in different cell types such as pre-B cells, T lym-phocytes and HeLa cells (22), cells show NF-kB-dependentd2EGFP fluorescence. Measurements 24 h after PMA treat-ment reveal a smaller fraction of the cell population(;30%) in which peak values are being reached after 48h. Experiments with low-LET radiation (150 kV X rays)using the standard DNA repair-inhibiting conditions (08C)show a slight but nonsignificant increase of NF-kB-depen-dent d2EGFP fluorescence after 5 Gy. For X irradiationapplied at 378C, a significant dose-dependent increase ind2EGFP fluorescence can be verified. This may reflect thedependence of NF-kB activation on either free-floating re-ceptors (23) or DNA repair processes already actively run-ning during irradiation.

Kinetics of NF-kB Activation

After exposure to 95 MeV/nucleon argon ions (Fig. 2),using a particle fluence from 6 3 105 (0.23 Gy) to 2 3 107

(7.4 Gy) cm22, d2EGFP fluorescence can be seen 6 h afterexposure. It reaches its maximum after 12 to 24 h with upto about 40% (for 2 3 107 particles cm22, equivalent to 7.4Gy) fluorescent cells. d2EGFP fluorescence stays at a highlevel up to 48 h, while only about 2–3% of nonirradiatedcells fall in the gate for d2EGFP1 cells. NF-kB activationcould be verified for fluences as low as 6 3 105 particlescm22 (0.23 Gy). Similar results were reported for normalhuman monocytes (MM6 cells) after exposure to 0.7 Gy of56Fe ions (24) using a DNA binding assay, clearly indicat-ing that high-LET iron-particle exposure induces rapid andpersistent NF-kB activation. This activation of NF-kB wasshown to be mediated through phosphorylation of IkBaand subsequent proteasome-dependent degradation path-way. While the iron-particle study revealed only binding of

NF-kB to its consensus sequence of 59-GGG GAC TTTCC-39, our study with high-LET argon ions (232 keV/mm)showed the completed sequence of NF-kB pathway-depen-dent events from DNA binding by transcription to proteintranslation and maturation. The kinetics for that completechain of events revealed that d2EGFP fluorescence oc-curred as early as 6 h after exposure, with a maximal re-sponse at about 12 to 24 h. Taking into account that thetime required for EGFP to express its fluorescence is about3 h, NF-kB is activated at about 3 h after irradiation. Asimilar time delay was shown for TNFA-induced NF-kBactivation (19). The down-regulation of NF-kB after pro-longed incubation after exposure can also be shown usingthe fluorescence screening assay, since the destabilizedEGFP variant d2EGFP has a half-life of about 3 h in pNF-kB-d2EGFP/Neo clone L2 cells. While for the higher dosesof 2 3 107 particles cm22 (7.4 Gy) and 5 3 106 particlescm22 (1.85 Gy) NF-kB is shown to be down-regulated after12 to 24 h, it is remarkable that for lower doses NF-kBactivation persists for longer postirradiation incubationtimes (Fig. 2). After exposure to 6 3 105 particles cm22

(0.23 Gy), NF-kB-dependent fluorescence persists up to 48h. Such effects, which may be related to better survival ofexposed cells through transient induction of genes involvedin maintaining DNA fidelity and in modulating cell cycleprogression and cell death, would influence astronauts’ ra-diation risk, especially at low doses (25).

Dose–Response Relationship for NF-kB Activation by 95MeV/nucleon Argon Ions

Figure 3 shows the dose dependence of the NF-kB ac-tivation capacity of accelerated argon ions as measured 24h after irradiation. Activation of the NF-kB pathway is sig-nificant for doses over 1 3 106 particles cm22 (about 0.5Gy), reaching its maximal activation at about 2 3 107 par-ticles cm22 with 30–40% of the cells activated. MaximalNF-kB activation takes place when about 50% of cells sur-vived (relative growth of ;0.5 at 1.5 3 107 particles cm22

measured 168 h after irradiation; results not shown).At a dose of 2 3 107 particles cm22, a mammalian cell

nucleus with a mean surface of 100 mm2 will receive 20particle hits on average. Since high-energy charged parti-cles deposit highly localized energy along the trajectory ofeach particle, the number of induced DSBs may well bemore than one per particle. From the first measurements ofoverall DNA strand breaks after 95 MeV/nucleon argon-ion irradiation (results not shown), it is assumed that thenumber of double-strand breaks varies from ;90 (for thelow-LET DSB/SSB ratio of 1:20) to ;1,000 (for a hypo-thetical DSB/SSB ratio of 1:1). At the lowest fluence ca-pable of activating NF-kB (about 1 3 106 particles cm22),nearly every cell nucleus is hit once by a heavy-ion particle,resulting in the induction of about 5 to 50 DSBs. For Xrays, it is known that most of the DNA damage induced isrepaired within a short time. After exposure to accelerated

530 BAUMSTARK-KHAN ET AL.

FIG. 3. Dose–effect relationship for NF-kB activation of stably trans-fected HEK cells expressing d2EGFP controlled by the KB4-TK promoter(pNF-kB-d2EGFP/Neo clone L2) after exposure to various fluences of 95MeV/nucleon argon ions. Doses (Gy) were calculated from fluences (par-ticles/cm2) according to Dose 5 Fluence 3 LET 3 1.6 3 1029 using anLET of 232.2 keV/mm for 95 MeV/nucleon argon ions. Means and standarddeviations of two independent experiments with three samples per dose.

heavy ions, DNA repair is possible, especially for the‘‘lighter’’ heavy ions. The quality of DNA repair afterheavy-ion exposure, however, may be impaired. Since ac-tivation of the NF-kB pathway is supposed to play a rolein the negative regulation of apoptosis, survival of cellswith residual DNA damage might thus be favored.

ACKNOWLEDGMENTS

We thank Dr. Isabelle Testard and Dr. Hermann Rothard (Centre Inter-disciplinaire de Recherche Ions Laser, Caen, France) for providing uswith laboratory space, equipment, valuable advice and much help givenduring numerous night shifts at the French Heavy Ion Accelerator GAN-IL. This work was supported in part by the German Academy of Flight-and Travel-Medicine (Deutsche Akademie fur Flug- und Reisemedizin).

Received: July 28, 2004; accepted: December 7, 2004

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