Modulation of gene expression in endothelial cells in response to high LET nickel ion irradiation

  • Authors:
    • Michaël Beck
    • Charlotte Rombouts
    • Marjan Moreels
    • An Aerts
    • Roel Quintens
    • Kevin Tabury
    • Arlette Michaux
    • Ann Janssen
    • Mieke Neefs
    • Eric Ernst
    • Birger Dieriks
    • Ryonfa Lee
    • Winnok H. De Vos
    • Charles Lambert
    • Patrick Van Oostveldt
    • Sarah Baatout
  • View Affiliations

  • Published online on: August 11, 2014
  • Pages: 1124-1132
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Ionizing radiation can elicit harmful effects on the cardiovascular system at high doses. Endothelial cells are critical targets in radiation-induced cardiovascular damage. Astronauts performing a long-term deep space mission are exposed to consistently higher fluences of ionizing radiation that may accumulate to reach high effective doses. In addition, cosmic radiation contains high linear energy transfer (LET) radiation that is known to produce high values of relative biological effectiveness (RBE). The aim of this study was to broaden the understanding of the molecular response to high LET radiation by investigating the changes in gene expression in endothelial cells. For this purpose, a human endothelial cell line (EA.hy926) was irradiated with accelerated nickel ions (Ni) (LET, 183 keV/µm) at doses of 0.5, 2 and 5 Gy. DNA damage was measured 2 and 24 h following irradiation by γ-H2AX foci detection by fluorescence microscopy and gene expression changes were measured by microarrays at 8 and 24 h following irradiation. We found that exposure to accelerated nickel particles induced a persistent DNA damage response up to 24 h after treatment. This was accompanied by a downregulation in the expression of a multitude of genes involved in the regulation of the cell cycle and an upregulation in the expression of genes involved in cell cycle checkpoints. In addition, genes involved in DNA damage response, oxidative stress, apoptosis and cell-cell signaling (cytokines) were found to be upregulated. An in silico analysis of the involved genes suggested that the transcription factors, E2F and nuclear factor (NF)-κB, may be involved in these cellular responses.


Cardiovascular disease is considered to be one of the most important non-cancer long-term effects of ionizing radiation, as evidenced by the epidemiological data of atomic bomb survivors exposed to doses of 0.5 to 2 Gy (1). In the context of space exploration, high linear energy transfer (LET) radiation found in space produces high values of relative biological effectiveness (RBE), as compared to low LET radiation, such as X-rays or gamma-rays, which can increase the health risks to astronauts (2). Indeed, during long-term missions, such as a journey to Mars, astronauts are bound to be exposed to cumulative doses between 0.3 and 4 Sv, depending on the spacecraft shielding and on the intensity of solar particle events (3).

Heavy ion irradiation is also used for terrestrial applications, such as non-conventional radiotherapy (hadron therapy), which takes advantage of the depth distribution of the dose, which is maximal at the Bragg peak, and of the increased RBE, allowing the enhanced killing effect on tumor cells while sparing the healthy tissue (4,5). However, little is known of the molecular mechanisms involved in the enhanced killing properties of heavy ion irradiation. Improving our understanding of the effects of heavy ion radiation, particularly on the cardiovascular system that may be irradiated during treatment, is therefore of utmost importance for both long-term space missions and hadron therapy.

Endothelial cells are critical targets in radiation-induced cardiovascular damage (1,6,7). While high doses of low LET radiation induce pro-inflammatory responses in endothelial cells, the opposite has been observed upon exposure to low doses (810). The mechanisms involved are not yet fully understood; however, they appear to be at least partly linked to the transcription factor, nuclear factor (NF)-κB, and the nitric oxide signaling pathway, which in turn mediates various cellular responses, including the secretion of cytokines [such as transforming growth factor (TGF)-β1, interleukin (IL)-6, interferon (IFN)-γ, IFN-β and tumor necrosis factor (TNF)-α] and chemokines (911). Another possible mechanism of radiation-induced cardiovascular alteration, as shown upon low LET radiation (1216), is the endothelial retraction and the impairment of cellular adhesion. Matrix metalloproteinases (MMPs), Rho GTPases, calcium signaling and reactive oxygen species seem to be important factors that stimulate modifications in cell junctions and the cytoskeleton through adhesion molecules and actin (1216). Although high LET radiation has been shown to reduce the length of a 3D human endothelial vessel model, both developing and mature (17), only a few studies have been conducted to identify the mechanisms involved in the endothelial response to high LET radiation (18,19).

Thus, the aim of this study was to investigate the effects of moderate and high doses of high LET nickel ion (Ni) irradiation on gene expression in endothelial cells in order to elucidate the molecular mechanisms responsible for radiation-induced cardiovascular damage. For this purpose, the EA.hy926 cell line, which originates from human umbilical vein endothelial cells, was irradiated with nickel ions (LET, 183 keV/μm) at moderate (0.5 Gy) and high (2 and 5 Gy) doses after which gene expression was determined by whole-genome microarray analysis.

Materials and methods

Cell culture

The human EA.hy926 endothelial cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). They were cultured (37°C-5% CO2) in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (all from N.V. Invitrogen S.A., Merelbeke, Belgium). The cells were regularly examined for the absence of mycoplasma using the LookOut® Mycoplasma PCR Detection kit (Sigma-Aldrich, St. Louis, MO, USA).

Nickel irradiation

The cells were seeded at a density of 105 cells in 12.5 cm2 flasks. Twenty-four hours after plating, the flasks were placed in a transportable incubator (37°C) and moved from the resident laboratory (Mol, Belgium) to the GSI Helmholtzzentrum für Schwerionenforschung GmbH (Darmstadt, Germany). Forty-eight hours after plating, the subconfluent cells were irradiated in flasks completely filled with culture medium with a 1 GeV/u Ni beam at the SIS facility at GSI with the intensity controlled raster scanning technique as described by Haberer et al (20). The ion energy at the sample position was approximately 930 MeV/u with a LET of 183 keV/μm (calculated with the program code ATIMA). The culture flasks were placed vertically and exposed perpendicularly to the nickel ion beam at the following doses: 0.5, 2 and 5 Gy. Non-irradiated control samples were treated similarly to the irradiated samples, but placed out of the beam. Following irradiation, the cells were incubated (37°C, 5% CO2) in 2 ml of conditioned medium until fixation time points (2, 8 and 24 h).

DNA double-strand break detection (detection of γ-H2AX foci)

The cells were fixed in 4% paraformaldehyde (Merck KGaA, Darmstadt, Germany) 2 and 24 h after irradiation. They were then treated with 0.25% Triton X for 5 min, blocked with 3% bovine serum albumin (both from Sigma-Aldrich) for 30 min and incubated overnight with mouse anti-γ-H2AX antibody (Abcam, Cambridge, MA, USA) at 4°C. After a second blocking of 10 min, the cells were incubated for 1 h with anti-mouse secondary antibody coupled to FITC (Sigma-Aldrich) at 37°C and then mounted in Vectashield mounting medium (Vector Laboratories, Burlingame, CA, USA) with DAPI. Between each of the previous steps, the slides were washed with phosphate-buffered saline (PBS).

An automated inverted fluorescence microscope (TE2000-E; Nikon, Tokyo, Japan), equipped with a motorized XYZ stage, emission and excitation filter wheels, shutters and a triple dichroic mirror (436/514/604) was used for the image acquisition of the immunostained slides. Images were acquired with a 40X Plan Fluor oil objective (NA 1.3) and an Andor iXon EMCCD camera (Andor Technology, South Windsor, CT, USA). For each sample, at least 12 fields were acquired on 5 z-stack focusses (1 μm). The γ-H2AX spot number and spot occupancy were analyzed with the INSCYDE plugin for ImageJ as previously described (21). Spot occupancy was defined for each nucleus as the sum of the spot areas divided by the nucleus area (spot_occupancy = sum (spot_area)/nuclear_area). A minimum number of 100 cells was analyzed in 2 biological replicates per condition. For statistical analyses, the data were analyzed using the Mann-Whitney U test with SPSS version 17.0 software (IBM Corp., Chicago, IL, USA) and box plots were generated using the same software. P-values <0.05 were considered to indicate statistically significant differences.

RNA extraction

At 2 time points after irradiation (8 and 24 h), the adherent cells were washed in PBS, lysed in 350 ml of AllPrep DNA/RNA/Protein Mini kit lysis buffer (Qiagen, Hilden, Germany) and frozen at −80°C. RNA was extracted using the same kit and its concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), while its quality (RNA integrity number, RIN) was determined using Agilent’s lab-on-chip Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA). All RNA samples had a RIN value >9.0.

Affymetrix microarrays and data analysis

RNA was processed using the GeneChip WT cDNA Synthesis and Amplification kit (Affymetrix, Santa Clara, CA, USA) according to the manufacturer’s instructions. The resulting RNA was hybridized to Affymetrix Human Gene 1.0 ST arrays which contain an estimated number of 28,869 genes based on the March 2006 [UCSC Hg 18; National Center for Biotechnology Information (NCBI) build 36] human genome assembly. Biological triplicates were collected for each condition.

Raw data (.cel-files) were imported at exon level in Partek Genomics Suite version 6.5 (Partek, Inc., St. Louis, MO, USA). Briefly, robust multi-array average (RMA) background correction was applied, data were normalized by quantile normalization and probeset summarization was performed by the median polish method. Gene summarization was performed using one-step Tukey’s biweight method. The obtained data were analyzed with Partek Genomics Suite for single gene analysis. One- or two-way ANOVA, taking into consideration the scan date (where applicable) and the dose as factors, were performed for each time point. In order to determine statistical significance, thresholds were set on the p-value <0.001 and on the fold-change >1.5.

The enrichment of the transcription factor binding motifs was analyzed using Pscan Ver. 1.2 (22) and the JASPAR database, scanning in a region from −950 to +50 base pairs from the transcription start site.


DNA damage

To assess DNA damage induction by nickel ion irradiation and evaluate the cell capacity required to repair this damage, we performed a high content cytometric assay of γ-H2AX, 2 and 24 h after exposure. As measured by the number of γ-H2AX foci, DNA damage was significantly increased 2 h after nickel ion irradiation (2 Gy), with an average number of 15 foci per nucleus vs. 2 foci per nucleus in the control samples (Fig. 1A). Twenty-four hours after irradiation, the number of foci decreased to 9 per nucleus, which was significantly higher than the values of controls, indicating that part of the DNA damage persisted for at least 24 h. Similar trends were observed for the spot occupancy, which is the fraction of the projected area of the nucleus occupied by the signal from the γ-H2AX foci (Fig. 1B).

Effects of nickel irradiation on gene expression

In order to evaluate gene expression, we performed microarrays 8 and 24 h after irradiation. A 0.5 Gy irradiation, both after 8 and 24 h, elicited a subtle effect on gene expression in the EA.hy926 cells. Six annotated genes were differentially regulated with fold changes (FC) between 1.5 and 1.8 after 8 h, and 18 genes were differentially regulated with an FC between 1.5 and 2.3 after 24 h. A more drastic effect was observed at 5 Gy, 24 h after irradiation. At this time point, we detected the upregulation of 77 annotated genes (Fig. 2 and Table I; maximum FC, 3.4). Among these genes, cytokines and chemokines (CXCL5, TGFA, TRIM22, TNFSF9, EBI3, IL-6, IL-11 and CD70) were identified, as well as genes involved in DNA damage response (SPATA18, POLL, APOBEC3H and SESN1), cell cycle arrest (ZMAT3, MXD4, TP53INP1, HSPB8, TGFA, SESN2, BTG2, DTX3, TOB1, HBP1, CDKN1A and PLK3) and apoptosis (TP53INP1, HSPB8, TGFA, TP53I3, MOAP1, CYFIP2, TRADD, DTX3 and FAS). In addition, we observed the upregulation of genes coding for ion channels (SLC22A4, KCNJ2, ORAI3 and CLIC3), cell adhesion (CEACAM1 and NEU1) and oxidative stress response proteins (FMO4, FDXR, SIRT2 and SESN1).

Table I

List of the differentially expressed genes at 8 and 24 h after 0.5 and 5 Gy of nickel ion irradiation.

Table I

List of the differentially expressed genes at 8 and 24 h after 0.5 and 5 Gy of nickel ion irradiation.

Downregulated genesUpregulated genes

Gene symbolGenBankp-valueFCGene symbolGenBankp-valueFC
List of differentially expressed genes 8 h after 0.5 Gy nickel ion irradiation
List of differentially expressed genes 24 h after 0.5 Gy nickel-ion irradiation
UPF3ANM_0230111,53E-03−1,932C1orf113 ENST000003128088,96E-032,224
PROK2NM_0011261288,29E-03−1,518 LCE1EbNM_1783534,89E-031,619
List of differentially expressed genes 24 h after 5 Gy nickel-ion irradiation
FAM111BaNM_1989475,65E-03−5,894 ACTA2bNM_0011419452,01E-053,424
DHRS2NM_1829086,48E-05−3,090 CD70bNM_0012529,56E-032,702
KIF20ANM_0057334,17E-04−2,595 BTG2bNM_0067631,08E-032,170
LMNB1NM_0055739,94E-04−2,561 APOBEC3HbNM_0011660034,32E-032,070
HIST1H1DNM_0053204,45E-03−2,478 TRIM22bNM_0060744,27E-042,063
E2F8aNM_0246803,88E-04−2,469 KCNJ2bNM_0008911,88E-032,055
HAUS8NM_0334171,71E-05−2,444 SPATA18bNM_1452631,16E-062,026
MYBL2NM_0024667,56E-04−2,369 ZNF223bNM_0133614,56E-031,997
UHRF1aNM_0010482015,93E-03−2,363 LCE1EbNM_1783538,26E-041,986
SRP19 ENST000005127906,66E-03−2,337FDXRNM_0244171,72E-031,972
ATAD2aNM_0141097,92E-03−2,239 ACY3bNM_0806588,81E-031,922
UNGaNM_0033622,21E-05−2,220 SLC40A1bNM_0145852,55E-031,921
FIGNL1NM_0010427626,39E-03−2,184 IL-6bNM_0006002,89E-031,883
FBXO5NM_0121771,38E-05−2,150 KBTBD8bNM_0325058,57E-031,833
E2F2aNM_0040913,11E-04−2,126 NIPAL3bNM_0204481,16E-061,819
HIST1H2BGaNM_0035181,90E-03−2,106 SAT1bNR_0277837,52E-031,810
DLEU2aNR_0026126,87E-03−2,088 CYFIP2bNM_0010373325,48E-031,770
ANKRD36BNM_0251903,63E-03−2,082 TMEM217bNM_1453167,60E-031,730
MCM2aNM_0045262,05E-04−1,927 CLIC3bNM_0046698,25E-031,666
EMP2NM_0014241,87E-03−1,915 NCF2bNM_0004336,46E-031,656
LHX2NM_0047898,33E-03−1,883 CXCL5bNM_0029942,65E-051,643
NCAPG2NM_0177601,59E-03−1,881 SESN2bNM_0314598,22E-041,642
PER3aNM_0168318,02E-03−1,867 FAM84AbNM_1451753,91E-041,623
SEMA3DNM_1527547,14E-03−1,867 ORAI3bNM_1522883,54E-031,616
KIFC1NM_0022631,96E-04−1,860 C9orf150bNM_2034032,10E-031,614
CCNE2NM_0577491,34E-03−1,841 MAGED4bNM_0010988001,62E-031,611
FAM111AaNM_0220745,61E-04−1,798 RDH10bNM_1720378,25E-031,556
STX11aNM_0037641,61E-03−1,795 BHLHE40bNM_0036709,87E-031,553
RFC4NM_0029163,92E-03−1,789 TP53I3bNM_0048811,71E-041,543
MLF1IPaNM_0246292,81E-04−1,785 NIPSNAP1bNM_0036343,88E-041,541
CEP55NM_0181314,87E-04−1,782 HHATbNM_0011705802,41E-031,536
BUB1NM_0043366,08E-04−1,780 SIRT2bNM_0122374,30E-031,533
CHAF1BNM_0054411,22E-03−1,773 C15orf33bNM_1526471,43E-031,526
EZH2aNM_0044565,38E-04−1,772 RBKSbNM_0221285,82E-031,523
PLK4NM_0142642,16E-04−1,757 DTX3bNM_1785024,95E-031,519
H1F0aNM_0053181,51E-04−1,740 ADCY4bNM_0011985925,24E-031,514
NUSAP1aNM_0163598,15E-05−1,730 TRADDbNM_0037892,41E-031,509
ESPL1NM_0122919,12E-03−1,724 ZMAT3bNM_0224705,71E-051,502
KIAA1524NM_0208901,99E-04−1,680 USP37aNM_0209353,15E-04−1,566
WHSC1NM_1333307,07E-04−1,677 KCTD12aNM_1384447,18E-03−1,555
MMS22LNM_1984682,44E-04−1,675 ZNF749aNM_0010235613,89E-03−1,553
FAM72DAB0966832,28E-03−1,674 CENPHaNM_0229091,88E-03−1,547
GINS2aNM_0160957,86E-03−1,657 MTBPaNM_0220454,20E-03−1,539
YAP1NM_0011301451,60E-03−1,655 CCNFaNM_0017618,64E-04−1,529
PKP4NM_0036284,89E-03−1,653 PBKaNM_0184923,92E-03−1,528
NFKBIL2NM_0134322,44E-03−1,632 EXO1aNM_1303983,35E-03−1,521
DTLaNM_0164482,55E-04−1,591 DUX4L4aNM_0011773768,97E-03−1,514

[i] The genes containing a potential binding motif for E2F1 or NF-κB are respectively marked by ‘a’ and ‘b’. The score of all marked genes was calculated by Pscan to be higher than the average matching score for all the promoters of the genome.

A total of 145 annotated genes was downregulated 24 h after nickel ion irradiation (5 Gy) (Fig. 3 and Table I). The majority (62 genes) is known to be involved in various aspects of cell division, such as DNA replication, replication forks and chromosome assembly and segregation (Table II and Fig. 4). Other downregulated genes found have been implicated in post-replicative DNA repair (UNG, UPF3A, MSH2 and MSH6), nucleotide biosynthesis (DHFR and RRM2), DNA repair (FANCA, MMS22L, NFKBIL2, RAD51, EXO1 and HMGB2), positive (YAP1) and negative regulation of apoptosis (DHRS2, DHCR24 and MTBP), Rho signaling (ARHGAP19, ARHGAP11B and RACGAP1) and cell adhesion (PVRL1 and DLGAP5).

Table II

List of the downregulated genes involved in cell cycle progression 24 h after 5 Gy of nickel ion irradiation.

Table II

List of the downregulated genes involved in cell cycle progression 24 h after 5 Gy of nickel ion irradiation.

DNA replicationReplication forksSpindleKinetochoreCentromeresChromosome formation/stability

[i] The genes containing a potential binding motif for E2F1 or NF-κB are marked by ‘a’. The score of all marked genes was calculated by Pscan to be higher than the average matching score for all the promoters of the genome.

Enrichment of transcription factor binding motifs

In order to identify the transcription factors potentially responsible for the differential gene expression upon irradiation, we scanned sequences close to the transcription start sites of these genes using Pscan (22). We found motifs for E2F1 among the transcription factor binding motifs enriched in the downregulated gene list, (p-value <10–19). On the other hand, we found two members of the REL family (RelA and NF-κB) with significantly enriched binding motifs in the list of upregulated genes (p-values <0.05).


DNA damage persists 24 h after irradiation

We measured a significant increase in the number of γ-H2AX foci 2 h following nickel ion irradiation. This number was lower than the 30 spots per nucleus that we measured on average upon X-irradiation with the same dose (data not shown). However, it is not so surprising since high LET irradiation deposits high amounts of energy along well-separated tracks. For nickel ions with a LET of 183 keV/μm and at a dose of 2 Gy, we calculated an average of 6.8 direct particle hits per nucleus (100 μm2), which follows a Poisson distribution. However, we observed an average of 15 spots per nucleus. This may be due to the secondary radiation from the ion track and the basal level of endogenous γ-H2AX foci as observed in the controls.

Considering that the imaging of γ-H2AX foci was performed at the same angle as ion tracks produced by the irradiation beam, the complexity of the damage along these tracks could not be taken into account. However, the DNA damage complexity is known to be important in high LET irradiation (2326). Although significantly increased, the γ-H2AX spot occupancy did not seem to be able to account for the complexity of DNA damage and showed similar results to the spot number measurement. This complex DNA damage is associated with slower repair (27) and therefore leads to a more pronounced delayed cellular damage (26). Our results revealed a significant level of γ-H2AX foci 24 h following nickel ion irradiation, as compared to controls; this suggests the presence of complex DNA damage.

Effects of high LET irradiation on the cell cycle

Nickel ion irradiation at a dose of 0.5 Gy elicited a lower gene expression response as compared to a dose of 5 Gy, in terms of the number of regulated genes and FC. At 24 h post-irradiation (5 Gy), we observed an upregulation of 12 genes involved in cell cycle arrest and a downregulation of 62 genes involved in cell cycle progression, among which were 3 members of the E2F transcription factor family (E2F1, E2F2 and E2F8). Moreover, the transcription factor binding motifs for E2F1 were found to be highly enriched in the list of downregulated genes. E2F is a family of transcription factors known to control G1- to S-phase transition (28), and to regulate the expression of a large variety of genes involved in DNA replication, DNA repair and apoptosis (29). Among the E2F transcription factors, E2F1 is known to be stabilized upon DNA damage through its phosphorylation by ataxia telangiectasia-mutated (ATM) kinase, ATM and Rad3-related (ATR) kinase and checkpoint kinase 2 (CHK2), as well as through its acetylation (29). Our results suggest a major role of E2F transcription factors in the response of EA.hy926 cells to high LET irradiation.

Six components of the minichromosome maintenance (MCM) complex, a heterohexamer helicase essential for the initiation and elongation step of DNA replication (30), were downregulated. This helicase may be a target for replication checkpoints (31), and is thought to be regulated mostly through post-transcriptional modifications (32). However, our results indicate a possible transcriptional regulation of several members of the MCM complex. Apart from MCM, many of the observed downregulated genes are involved in DNA replication and in chromosome formation, maintenance and segregation, indicating their key role in cell cycle regulation in response to high LET radiation. Of note, we also reported the downregulation of 4 genes involved in post-replication DNA repair (UNG, UPF3A, MSH2 and MSH6), which may be silenced in the absence of active replication.

During this study, irradiation was performed on proliferating endothelial cells. The results gathered on cell cycle gene expression are therefore of moderate interest for mature blood vessels where proliferation is limited. However, as far as hadron therapy is concerned, our data indicate that high LET radiation may have a significant impact on the cellular proliferation of newly formed vascular vessels in the vicinity of the targeted tumor.

DNA damage response, oxidative stress and apoptosis

The expression of several genes involved in the DNA damage response, oxidative stress response and apoptosis was induced 24 h after 5 Gy of nickel ion irradiation, with a concomitant reduction of genes involved in DNA repair. However, these effects were not significant at a dose of 0.5 Gy, at either time points (8 and 24 h). These results suggest that a high dose of nickel ion irradiation induces a global DNA damage response, accompanied by cell cycle arrest and an increase in pro-apoptotic gene expression 24 h after irradiation.

Impact of radiation on genes related to cell adhesion

The impermeability of the endothelium is essential for the vasculature integrity and is determined by the cooperation of cell junctions and the cytoskeleton (33,34). In turn, adhesion molecules regulate cell homeostasis, growth and apoptosis (33). A number of cellular pathways are known to regulate cell adhesion in endothelial cells. These include growth factors, Rho GTPases, protein kinases and calcium signaling (34,35). The alteration of these pathways or of adhesion molecules may trigger the radiation-induced retraction observed by others in endothelial cells (13,14). Our study identified the differential expression of a number of genes known to be involved in cell adhesion (CEACAM1 and NEU1), cytoskeleton architecture (TUBA4A, LIMA1 and PLS1), Rho signaling (ARHGAP19, ARHGAP11B and RACGAP1) and calcium metabolism (ORAI3, CAMK2N1 and CALML4) 24 h after 5 Gy of nickel ion irradiation, which are potentially involved in endothelial cell retraction.

Expression of cytokines and chemokines

Inflammatory responses mediated by endothelial cells are believed to be involved in radiation-induced cardiovascular disease (7). Our study revealed the upregulation of 8 cytokines or chemokines that may be linked to inflammation (CXCL5, TGFA, TRIM22, TNFSF9, EBI3, IL-6, IL-11 and CD70). Of note, a search for transcription factor binding motifs that are significantly enriched in the list of upregulated genes upon 5 Gy of irradiation, revealed 2 members of the REL family (RelA and NF-κB). This family of transcription factors induces the expression of a multitude of genes, such as cytokines, proliferation, pro-survival and anti-apoptotic genes (36). For instance, we found IL-6 to be upregulated after 5 Gy of nickel ion irradiation. IL-6 expression was also shown to be upregulated by low-dose radiation therapy (10). IL-6 is known to be activated by NF-κB (36,37) and is thought to play a role in radiation-induced cardiovascular disease (1,7). The secretion of cytokines may also affect non-irradiated cells by a bystander effect. Indeed, in human fibroblasts, the external addition of IL-6 has been shown to increase γH2AX spot occupancy (38). The activation of NF-κB may be linked to the transcription factor, signal transducer and activator of transcription 3 (STAT3) (37), of which we also found significant binding motif enrichment.

In conclusion, we observed a downregulation of multiple genes involved in cell division, particularly at 24 h after nickel ion irradiation. Our results suggest an important role for E2F transcription factors in this process. The endothelial function being based on a plethora of intercellular interactions within a dynamic structure involving cell movements and turnover, cell cycle arrest may play a role in the radiation-induced cardiovascular disease. On the other hand, we observed an upregulation of various cytokines which may be induced by NF-κB. Other studies have also suggested that these cytokines may be linked to radiation-induced cardiovascular disease (10). The effects on gene expression were observed upon high doses of acute irradiation and are less relevant to space exploration. However, during hadron therapy, healthy tissues surrounding tumors, such as endothelial cells, may be subjected to high doses, which may lead to complications. In this study, we identified a multitude of potential molecular targets for further mechanistic studies out of which the gene expression changes upon high doses of nickel ion irradiation may be important for patients treated with hadron-therapy.


This study was supported by 4 PRODEX/BELSPO/ESA contracts (C90-303, C90-380, C90-391 and 42-000-90-380) and the ESA IBER-2 program. The authors wish to thank Professor Marco Durante for providing access to the GSI irradiation facilities.



Schultz-Hector S and Trott KR: Radiation-induced cardiovascular diseases: is the epidemiologic evidence compatible with the radiobiologic data? Int J Radiat Oncol Biol Phys. 67:10–18. 2007. View Article : Google Scholar : PubMed/NCBI


Blaber E, Marçal H and Burns BP: Bioastronautics: the influence of microgravity on astronaut health. Astrobiology. 10:463–473. 2010. View Article : Google Scholar : PubMed/NCBI


Brinckmann E: Biology in Space and Life on Earth: Effects of Spaceflight on Biological Systems. WILEY-VCH Verlag GmbH & Co. KGaA; Weinheim: 2007, View Article : Google Scholar


Rong Y and Welsh J: Basics of particle therapy II biologic and dosimetric aspects of clinical hadron therapy. Am J Clin Oncol. 33:646–649. 2010. View Article : Google Scholar : PubMed/NCBI


Blakely EA and Chang PY: Biology of charged particles. Cancer J. 15:271–284. 2009. View Article : Google Scholar : PubMed/NCBI


Halle M, Hall P and Tornvall P: Cardiovascular disease associated with radiotherapy: Activation of nuclear factor kappa-B. J Intern Med. 269:469–477. 2011. View Article : Google Scholar : PubMed/NCBI


Hildebrandt G: Non-cancer diseases and non-targeted effects. Mutat Res. 687:73–77. 2010. View Article : Google Scholar : PubMed/NCBI


Rödel F, Frey B, Capalbo G, et al: Discontinuous induction of x-linked inhibitor of apoptosis in ea. Hy926 endothelial cells is linked to NF-κB activation and mediates the anti-inflammatory properties of low-dose ionising-radiation. Radiother Oncol. 97:346–351. 2011.PubMed/NCBI


Rödel F, Hofmann D, Auer J, et al: The anti-inflammatory effect of low-dose radiation therapy involves a diminished CCL20 chemokine expression and granulocyte/endothelial cell adhesion. Strahlenther Onkol. 184:41–47. 2008.


Rödel F, Keilholz L, Herrmann M, Sauer R and Hildebrandt G: Radiobiological mechanisms in inflammatory diseases of low-dose radiation therapy. Int J Radiat Biol. 83:357–366. 2007.PubMed/NCBI


Rödel F, Schaller U, Schultze-Mosgau S, et al: The induction of TGF-beta(1) and NF-kappaB parallels a biphasic time course of leukocyte/endothelial cell adhesion following low-dose X-irradiation. Strahlenther Onkol. 180:194–200. 2004.PubMed/NCBI


Ando K, Ishibashi T, Ohkawara H, et al: Crucial role of membrane type 1 matrix metalloproteinase (MT1-MMP) in Rhoa/Rac1-dependent signaling pathways in thrombin-stimulated endothelial cells. J Atheroscler Thromb. 18:762–773. 2011. View Article : Google Scholar


Kantak SS, Diglio CA and Onoda JM: Low dose radiation-induced endothelial cell retraction. Int J Radiat Biol. 64:319–328. 1993. View Article : Google Scholar : PubMed/NCBI


Onoda JM, Kantak SS and Diglio CA: Radiation induced endothelial cell retraction in vitro: correlation with acute pulmonary edema. Pathol Oncol Res. 5:49–55. 1999. View Article : Google Scholar : PubMed/NCBI


Gabrys D, Greco O, Patel G, Prise KM, Tozer GM and Kanthou C: Radiation effects on the cytoskeleton of endothelial cells and endothelial monolayer permeability. Int J Radiat Oncol Biol Phys. 69:1553–1562. 2007. View Article : Google Scholar : PubMed/NCBI


Pluder F, Barjaktarovic Z, Azimzadeh O, et al: Low-dose irradiation causes rapid alterations to the proteome of the human endothelial cell line EA. hy926. Radiat Environ Biophys. 50:155–166. 2011. View Article : Google Scholar : PubMed/NCBI


Grabham P, Hu B, Sharma P and Geard C: Effects of ionizing radiation on three-dimensional human vessel models: differential effects according to radiation quality and cellular development. Radiat Res. 175:21–28. 2011. View Article : Google Scholar


Takahashi Y, Teshima T, Kawaguchi N, Hamada Y, Mori S, Madachi A, Ikeda S, Mizuno H, Ogata T, Nojima K, Furusawa Y and Matsuura N: Heavy ion irradiation inhibits in vitro angiogenesis even at sublethal dose. Cancer Res. 63:4253–4257. 2003.PubMed/NCBI


Kiyohara H, Ishizaki Y, Suzuki Y, Katoh H, Hamada N, Ohno T, Takahashi T, Kobayashi Y and Nakano T: Radiation-induced ICAM-1 expression via TGF-β1 pathway on human umbilical vein endothelial cells; comparison between X-ray and carbon-ion beam irradiation. J Radiat Res. 52:287–292. 2011.PubMed/NCBI


Haberer T, Becher W, Schardt D and Kraft G: Magnetic scanning system for heavy ion therapy. Nucl Instrum Methods Phys Res A. 330:296–305. 1993. View Article : Google Scholar


De Vos WH, Van Neste L, Dieriks B, Joss GH and Van Oostveldt P: High content image cytometry in the context of subnuclear organization. Cytometry A. 77:64–75. 2010.PubMed/NCBI


Zambelli F, Pesole G and Pavesi G: Pscan: finding over-represented transcription factor binding site motifs in sequences from co-regulated or co-expressed genes. Nucleic Acids Res. 37:W247–W252. 2009. View Article : Google Scholar


Fokas E, Kraft G, An H and Engenhart-Cabillic R: Ion beam radiobiology and cancer: time to update ourselves. Biochim Biophys Acta. 1796:216–229. 2009.PubMed/NCBI


Held KD: Effects of low fluences of radiations found in space on cellular systems. Int J Radiat Biol. 85:379–390. 2009. View Article : Google Scholar : PubMed/NCBI


Costes SV, Boissière A, Ravani S, Romano R, Parvin B and Barcellos-Hoff MH: Imaging features that discriminate between foci induced by high- and low-let radiation in human fibroblasts. Radiat Res. 165:505–515. 2006. View Article : Google Scholar : PubMed/NCBI


Blakely EA and Kronenberg A: Heavy-ion radiobiology: new approaches to delineate mechanisms underlying enhanced biological effectiveness. Radiat Res. 150:S126–S145. 1998. View Article : Google Scholar


Chappell LJ, Whalen MK, Gurai S, Ponomarev A, Cucinotta FA and Pluth JM: Analysis of flow cytometry DNA damage response protein activation kinetics after exposure to x rays and high-energy iron nuclei. Radiat Res. 174:691–702. 2010. View Article : Google Scholar : PubMed/NCBI


Dyson N: The regulation of E2F by pRB-family proteins. Genes Dev. 12:2245–2262. 1998. View Article : Google Scholar : PubMed/NCBI


Biswas AK and Johnson DG: Transcriptional and nontranscriptional functions of E2F1 in response to DNA damage. Cancer Res. 72:13–17. 2012. View Article : Google Scholar : PubMed/NCBI


Costa A and Onesti S: The MCM complex: (just) a replicative helicase? Biochem Soc Trans. 36:136–140. 2008. View Article : Google Scholar : PubMed/NCBI


Forsburg SL: The MCM helicase: Linking checkpoints to the replication fork. Biochem Soc Trans. 36:114–119. 2008. View Article : Google Scholar : PubMed/NCBI


Chuang CH, Yang D, Bai G, Freeland A, Pruitt SC and Schimenti JC: Post-transcriptional homeostasis and regulation of MCM2-7 in mammalian cells. Nucleic Acids Res. 40:4914–4924. 2012. View Article : Google Scholar : PubMed/NCBI


Dejana E, Orsenigo F, Molendini C, Baluk P and McDonald DM: Organization and signaling of endothelial cell-to-cell junctions in various regions of the blood and lymphatic vascular trees. Cell Tissue Res. 335:17–25. 2009. View Article : Google Scholar : PubMed/NCBI


Prasain N and Stevens T: The actin cytoskeleton in endothelial cell phenotypes. Microvasc Res. 77:53–63. 2009. View Article : Google Scholar


Bogatcheva NV and Verin AD: The role of cytoskeleton in the regulation of vascular endothelial barrier function. Microvasc Res. 76:202–207. 2008. View Article : Google Scholar : PubMed/NCBI


Oeckinghaus A and Ghosh S: The NF-kappaB family of transcription factors and its regulation. Cold Spring Harb Perspect Biol. 1:a0000342009. View Article : Google Scholar : PubMed/NCBI


Karin M: NF-kappaB as a critical link between inflammation and cancer. Cold Spring Harb Perspect Biol. 1:a0001412009. View Article : Google Scholar : PubMed/NCBI


Dieriks B, De Vos WH, Derradji H, Baatout S and Van Oostveldt P: Medium-mediated DNA repair response after ionizing radiation is correlated with the increase of specific cytokines in human fibroblasts. Mutat Res. 687:40–48. 2010. View Article : Google Scholar

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October 2014
Volume 34 Issue 4

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Beck M, Rombouts C, Moreels M, Aerts A, Quintens R, Tabury K, Michaux A, Janssen A, Neefs M, Ernst E, Ernst E, et al: Modulation of gene expression in endothelial cells in response to high LET nickel ion irradiation. Int J Mol Med 34: 1124-1132, 2014
Beck, M., Rombouts, C., Moreels, M., Aerts, A., Quintens, R., Tabury, K. ... Baatout, S. (2014). Modulation of gene expression in endothelial cells in response to high LET nickel ion irradiation. International Journal of Molecular Medicine, 34, 1124-1132.
Beck, M., Rombouts, C., Moreels, M., Aerts, A., Quintens, R., Tabury, K., Michaux, A., Janssen, A., Neefs, M., Ernst, E., Dieriks, B., Lee, R., De Vos, W. H., Lambert, C., Van Oostveldt, P., Baatout, S."Modulation of gene expression in endothelial cells in response to high LET nickel ion irradiation". International Journal of Molecular Medicine 34.4 (2014): 1124-1132.
Beck, M., Rombouts, C., Moreels, M., Aerts, A., Quintens, R., Tabury, K., Michaux, A., Janssen, A., Neefs, M., Ernst, E., Dieriks, B., Lee, R., De Vos, W. H., Lambert, C., Van Oostveldt, P., Baatout, S."Modulation of gene expression in endothelial cells in response to high LET nickel ion irradiation". International Journal of Molecular Medicine 34, no. 4 (2014): 1124-1132.