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     https://doi.org/10.3892/ijmm.2014.1893
  • Pages: 1124-1132
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Abstract

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.

Introduction

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.

Results

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
CRYBB2NM_0004961,02E-03−1,800HSP90AA6PNR_0367516,55E-031,630
GNAT1NM_1444995,54E-03−1,601RFT1NM_0528596,83E-031,572
DNAJB13NM_1536143,79E-03−1,521DEFB123NM_1533248,50E-031,518
List of differentially expressed genes 24 h after 0.5 Gy nickel-ion irradiation
UPF3ANM_0230111,53E-03−1,932C1orf113 ENST000003128088,96E-032,224
E2F8aNM_0246805,38E-03−1,716SNORD53NR_0027416,78E-032,182
LIMA1NM_0011135469,63E-03−1,670SLC45A4BC0332238,10E-032,049
C16orf55AK3030247,00E-04−1,600HIST1H2BDNM_0210639,01E-032,028
HLA-DRB4AK2930203,18E-03−1,583ZNF16NM_0010299763,65E-031,952
MCM10aNM_1827519,77E-03−1,530RNF207NM_2073967,07E-031,677
PROK2NM_0011261288,29E-03−1,518 LCE1EbNM_1783534,89E-031,619
C10orf72NM_0010317466,04E-041,592
PMCHNM_0026747,49E-031,591
RUNDC3BNM_1382901,98E-031,571
ORAI3bNM_1522887,01E-031,515
List of differentially expressed genes 24 h after 5 Gy nickel-ion irradiation
FAM111BaNM_1989475,65E-03−5,894 ACTA2bNM_0011419452,01E-053,424
PCBP1aNM_0061966,05E-04−3,611TP53INP1NM_0332851,02E-042,976
DHRS2NM_1829086,48E-05−3,090 CD70bNM_0012529,56E-032,702
MCM6aNM_0059151,81E-05−3,069PHOSPHO1NM_0011438049,47E-042,629
HIST1H1TNM_0053236,99E-03−2,762CDKN1ANR_0371518,27E-032,221
ZNF367aNM_1536951,61E-04−2,669CEACAM1NM_0017125,56E-042,184
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
UPF3ANM_0230114,54E-04−2,292TUBA4ANM_0060001,36E-031,932
DLGAP5aNM_0147509,85E-03−2,264PSTPIP2NM_0244305,51E-041,924
ATAD2aNM_0141097,92E-03−2,239 ACY3bNM_0806588,81E-031,922
UNGaNM_0033622,21E-05−2,220 SLC40A1bNM_0145852,55E-031,921
HELLSaNM_0180638,16E-03−2,194TMEM150ANM_0010317385,23E-041,904
MCM3aNM_0023881,81E-03−2,189MXD4NM_0064541,09E-041,903
FIGNL1NM_0010427626,39E-03−2,184 IL-6bNM_0006002,89E-031,883
KIF11NM_0045239,41E-03−2,164NCRNA00219NR_0153708,85E-031,864
FBXO5NM_0121771,38E-05−2,150 KBTBD8bNM_0325058,57E-031,833
E2F2aNM_0040913,11E-04−2,126 NIPAL3bNM_0204481,16E-061,819
WDR76NM_0249084,11E-04−2,108RAB4BNM_0161543,30E-031,811
HIST1H2BGaNM_0035181,90E-03−2,106 SAT1bNR_0277837,52E-031,810
LOC1720NR_0334231,54E-03−2,101SLC22A4NM_0030597,67E-041,796
CAMK2N1NM_0185841,24E-03−2,091NEU1NM_0004342,68E-031,778
DLEU2aNR_0026126,87E-03−2,088 CYFIP2bNM_0010373325,48E-031,770
MCM5aNM_0067392,95E-04−2,084TNFSF9NM_0038117,09E-041,736
ANKRD36BNM_0251903,63E-03−2,082 TMEM217bNM_1453167,60E-031,730
POLA1aNM_0169372,54E-03−2,073IL-11NM_0006413,38E-031,721
BUB1BNM_0012111,55E-03−1,995ATP6V0A4NM_0206327,57E-031,712
GPSM2NM_0132962,43E-04−1,988FMO4NM_0020221,05E-031,707
HMGB2NM_0011306883,20E-03−1,979WIPI1NM_0179837,41E-041,705
DHCR24NM_0147623,24E-04−1,970HBP1NM_0122577,81E-031,684
MCM7aNM_0059162,60E-05−1,956UCN2NM_0331994,81E-031,683
ANLNNM_0186857,33E-03−1,953bEBI3NM_0057551,99E-031,676
NDC80NM_0061012,04E-04−1,953bFASNM_0000439,18E-031,673
MCM2aNM_0045262,05E-04−1,927 CLIC3bNM_0046698,25E-031,666
CDKN3NM_0051929,25E-03−1,917TGFANM_0032361,70E-041,662
EMP2NM_0014241,87E-03−1,915 NCF2bNM_0004336,46E-031,656
TACC3NM_0063422,72E-03−1,906NADSYN1NM_0181614,99E-041,651
LHX2NM_0047898,33E-03−1,883 CXCL5bNM_0029942,65E-051,643
NCAPG2NM_0177601,59E-03−1,881 SESN2bNM_0314598,22E-041,642
DHFRNM_0007911,91E-05−1,878HSPB8NM_0143651,53E-041,634
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
DEPDC1BNM_0183695,71E-03−1,853C2orf80NM_0010993345,67E-041,613
USP1NM_0033681,74E-03−1,848PLK3NM_0040738,79E-031,611
CCNE2NM_0577491,34E-03−1,841 MAGED4bNM_0010988001,62E-031,611
PRC1NM_0039811,06E-03−1,838LRRC29NM_0121632,25E-061,589
DEPDC1NM_0011141201,89E-04−1,819POLLNM_0011740842,96E-031,583
ORC1NM_0041532,50E-03−1,811DFNA5NM_0044036,35E-041,578
CDCA7aNM_0319421,42E-04−1,807CRYABNM_0018853,49E-041,577
MCM10aNM_1827512,11E-03−1,801WBP5NM_0163032,50E-041,569
CDT1aNM_0309285,55E-03−1,800SESN1NM_0144546,34E-031,565
FAM111AaNM_0220745,61E-04−1,798 RDH10bNM_1720378,25E-031,556
STX11aNM_0037641,61E-03−1,795 BHLHE40bNM_0036709,87E-031,553
MSH2aNM_0002517,76E-04−1,794FAM113AAK2936387,81E-041,550
MKKSNM_0188486,55E-04−1,794LOC100130581NR_0274136,57E-031,546
CEP78NM_0010988029,07E-03−1,790MOAP1NM_0221512,89E-041,543
RFC4NM_0029163,92E-03−1,789 TP53I3bNM_0048811,71E-041,543
KIF23NM_1385553,79E-03−1,789OR51B6NM_0010047505,49E-031,542
MLF1IPaNM_0246292,81E-04−1,785 NIPSNAP1bNM_0036343,88E-041,541
CEP55NM_0181314,87E-04−1,782 HHATbNM_0011705802,41E-031,536
TCF19aNM_0071096,78E-04−1,781ARR3NM_0043127,01E-041,535
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
E2F1aNM_0052251,61E-03−1,755TOB1NM_0057496,18E-031,517
H1F0aNM_0053181,51E-04−1,740 ADCY4bNM_0011985925,24E-031,514
CEP57L1NM_0010835355,12E-03−1,739ARL15NM_0190871,98E-031,512
NUSAP1aNM_0163598,15E-05−1,730 TRADDbNM_0037892,41E-031,509
ESPL1NM_0122919,12E-03−1,724 ZMAT3bNM_0224705,71E-051,502
KIF2CNM_0068457,54E-04−1,718
DEPDC4NM_1523173,25E-03−1,714
MSH6NM_0001796,14E-03−1,712
CDC6aNM_0012542,07E-03−1,710
PM20D2aNM_0010108537,65E-04−1,706
PVRL1NM_0028559,24E-03−1,693
C16orf55AK3030243,86E-04−1,691
RRM2aNM_0011659317,98E-03−1,687
HIST1H3FaNM_0210188,20E-03−1,682DDX12NR_0333993,55E-03−1,570
ZNF716NM_0011592792,70E-04−1,682FLJ30064AK0546262,40E-03−1,562
KIAA1524NM_0208901,99E-04−1,680 USP37aNM_0209353,15E-04−1,566
FANCAaNM_0001356,54E-06−1,680PLS1NM_0011723129,23E-04−1,559
KLHL23NM_1447118,07E-03−1,679MT4NM_0329357,81E-03−1,558
CDCA2NM_1525623,22E-03−1,677GTSE1NM_0164266,65E-03−1,556
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
KIAA0101aNM_0147362,64E-03−1,663DDX11NM_0306537,35E-04−1,545
AREGNM_0016578,49E-03−1,658SNX5NM_1522274,96E-03−1,543
GINS2aNM_0160957,86E-03−1,657 MTBPaNM_0220454,20E-03−1,539
ARHGAP11BaNM_0010398411,80E-03−1,657GAR1NM_0189838,34E-03−1,539
LYARNM_0178169,45E-03−1,656NUF2NM_1456972,74E-04−1,531
YAP1NM_0011301451,60E-03−1,655 CCNFaNM_0017618,64E-04−1,529
PKP4NM_0036284,89E-03−1,653 PBKaNM_0184923,92E-03−1,528
FGF12NM_0210322,85E-03−1,649NCAPHNM_0153413,03E-05−1,521
NFKBIL2NM_0134322,44E-03−1,632 EXO1aNM_1303983,35E-03−1,521
FOXD4L3aNM_1991353,14E-03−1,631NOS1APNM_0146977,34E-03−1,520
CALML4NM_0334296,12E-03−1,609RACGAP1NM_0132776,33E-03−1,517
DSCC1NM_0240942,26E-03−1,602CLCNKANM_0040702,68E-03−1,517
PRIM1NM_0009462,41E-05−1,593FAM133BNM_0010400577,16E-03−1,515
DTLaNM_0164482,55E-04−1,591 DUX4L4aNM_0011773768,97E-03−1,514
WDHD1NM_0070865,97E-04−1,590GABRA6NM_0008113,58E-03−1,513
SUN2NM_0153742,49E-03−1,586L2HGDHNM_0248843,44E-03−1,512
PHF10aNM_0182882,15E-03−1,583CDKN2CNM_0012621,50E-03−1,511
SKA1NM_0010395354,42E-04−1,576ARHGAP19NM_0329001,78E-03−1,510
CNTNAP3aNM_0336552,76E-04−1,576SLFN11NM_0011045875,13E-03−1,508
RAD51aNM_0028752,80E-03−1,575C14orf80NM_0011348751,09E-03−1,506
CDCA8NM_0181014,13E-04−1,573NCAPGNM_0223463,17E-03−1,501

[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
PRIM1 MCM6aHAUS8NDC80PLK4NCAPH
CDC6a MCM7aKIFC1NUF2MLF1IPDDX11
DSCC1 MCM2a NUSAP1aSKA1CEP55 PHF10a
ORC1 MCM5aCDCA8 CENPHaNCAPG2
POLA1a MCM3aKIF20ACEP57L1NCAPG
GINS2a MCM10aSKA1CEP78CDCA2
CDT1aNFKBIL2BUB1
RFC4KIF2C
PRC1
BUB1B
KIF23
ESPL1
KIF11

[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).

Discussion

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.

Acknowledgements

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.

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October 2014
Volume 34 Issue 4

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Spandidos Publications style
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.
APA
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. https://doi.org/10.3892/ijmm.2014.1893
MLA
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.
Chicago
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. https://doi.org/10.3892/ijmm.2014.1893