LINE-1 Retrotransposition Events Regulate Gene Expression After X-Ray Irradiation

Abstract

Long interspersed nuclear element-1 (LINE-1) retrotransposons are mobile elements that insert into new genomic locations via reverse transcription of an RNA intermediate. The mechanism of retrotransposition is not entirely understood. The integration of these elements occurs by target-primed reverse transcription (TPRT), which initiates double-strand breaks (DSBs) during the LINE-1 integration. Also, X-ray is known to induce DNA damage. The aim of this study was to evaluate the potential effects of LINE-1 de novo retrotransposition on the expression of different genes after X-ray irradiation in human endothelial cells. After stable transfection of the human hybrid endothelial cell line EA.hy926 with the human LINE-1 element, we analyzed the expression of different genes after irradiation with 5 Gy X-rays by reverse transcription–polymerase chain reaction (RT-PCR). We determine the expression level of phosphorylated p53 and γ-histone H2AX protein levels upon X-ray irradiation with 5 Gy for 24 h. Our results showed that EA.hy926 LINE-1 cell clones react with a strong upregulation of phosphorylated p53 protein, already 15 min after irradiation compared to the wild type (WT) cells. Also, the expression of γ-histone H2AX protein was elevated in the cell clones with retrotransposition events 15 min after irradiation, whereas the WT cells have a delayed expression of phosphorylated histone H2AX protein. Taken together, our findings provide that LINE-1 retrotransposition events regulate different gene expression after irradiation in the EA.hy926 cell line.

Introduction

L ong interspersed nuclear element-1 (LINE-1, L1) is the most active mobile element in the human genome and is considered to be one of the major driving forces in the evolution of eukaryotic genomes (Lander et al., 2001). It comprises about 17% of the human genome. Due to truncations and mutations, only 80–100 of the more than 500,000 copies are still capable of retrotransposition (Sassaman et al., 1997; Kazazian, 2004). A full-length LINE-1 element is 6 kb in length and contains an internal promoter at the 5′-end that generates a genomic transcript that also serves as an mRNA (Schulz, 2006). LINE-1 encodes two open reading frames (ORF1 and ORF2), which are both required for LINE-1 retrotransposition (Belancio et al., 2009). ORF1 protein (ORF1p) is approximately 40 kDa in size and is further a high-affinity, nonsequence-specific RNA-binding protein with nucleic acid chaperone activity (Callahan et al., 2012). ORF2 encodes the endonuclease (EN) and reverse transcriptase activities. The mechanism of retrotransposition is not entirely understood, but it is known that LINE-1 mobilization can generate reinsertions containing LINE-1 sequence, transduced sequences, and deletion of adjacent genomic sequences (Moran et al., 1996; Meyer et al., 2010). The LINE-1 retrotransposition occurs by target-primed reverse transcription (TPRT) (Luan and Eickbush, 1996). Via this mechanism, LINE-1 cleavage of both strands of genomic DNA is required, which implies that an intermediate equivalent to a double-strand break (DSB) is present during the integration process (Belgnaoui et al., 2006). DNA DSBs are probably the most dangerous among different types of DNA damages that occur within the cell. A very early step in the cellular response to DSBs is the phosphorylation of the histone H2A variant, H2AX, at the sites of DNA damage (Burma et al., 2001). It has been reported that H2AX is rapidly phosphorylated at serine 139 when DSBs were induced within mammalian cells, resulting in H2AX focus formation at the DNA-damaged site (Rogakou et al., 1998, 1999).

Cellular stress can further induce the activation of the most important tumor suppressor gene p53. Typical stresses that activate p53 are exposure to chemicals, exposure to radiation, and reactive oxygen species (Harris et al., 2009). The activation of p53 can trigger several responses like cell cycle arrest, apoptosis, or cellular senescence, so the cell can prevent and propagate the genome alterations (Lane, 1992). As LINE-1 is known as a genome modifier, the LINE-1-encoded proteins ORF1p und ORF2p have been shown to also be present in endothelial cells (Ergun et al., 2004). Recently, we could show that LINE-1 de novo retrotransposition takes place in endothelial cells (Banaz-Yasar et al., 2010). In this study, we analyzed the potential role of LINE-1 in activation of p53 and H2AX in the endothelial cell line EA.hy926 after irradiation with 5-Gy X-rays. To this aim, the EA.hy926 cells were transfected with a LINE-1 expression vector (pJM101/L1_RP) before using them for analyses. This vector allows a high frequency of retrotransposition in cultured cells. We used X-ray irradiation to determine whether the cell clones with retrotransposition events go through the process of programmed cell death or activate cellular DNA repair mechanisms. Our results demonstrate that LINE-1 de novo retrotransposition results in an increased level of phosphorylated p53 and H2AX, already 15 min after irradiation with 5-Gy X-rays. In contrast in EA.hy926 wild type (WT) cells, this activation of p53 and H2AX started 1 h after irradiation. We could also show that the endogenous expression of LINE-1 is significantly increased 2 h after irradiation in the nontransfected EA.hy926 WT cells, whereas the LINE-1 protein expression level was not changed in the analyzed cell clones. The analysis of different genes of the ATM-signaling pathway on the mRNA level demonstrated that in cell clones with LINE-1 de novo retrotransposition, the expression levels of different genes are decreased after irradiation. Taken together, our data show that LINE-1 de novo retrotransposition may activate the expression of p53 and initiate DSB after irradiation.

Materials and Methods

Cell line and cell culture

The human hybrid EA.hy926 cell line was derived by fusing human umbilical vein endothelial cells with the permanent human lung epithelial cell line A549 (kindly provided by Dr. Cora-Jean S. Edgell, University of North Carolina) (Edgell et al., 1983). EA.hy926 cells were grown in the Dulbecco's modified Eagle's medium (DMEM; Invitrogen) with 4.5 g/L glucose and supplemented with 10% fetal calf serum.

Plasmid construction and transfection

The LINE-1 retrotransposition reporter plasmid pJM101/L1_RP and the episomal expression vector pCEP4 were kindly provided by John Moran (University of Michigan). The plasmid pJM101/L1_RP contains a hygromycin gene, which facilitates the selection for the presence of LINE-1 reporter plasmid. In addition, the plasmid contains an antisense copy of the neo gene, which is disrupted by an intron in the opposite transcriptional orientation. This system ensures that G418-resistant cells will only arise when a transcript initiated from the promoter and neo expression is spliced, reverse transcribed, and reintegrated into chromosomal DNA (Moran et al., 1996). To create the pCEP4/neo control vector, neomycin was amplified by polymerase chain reaction (PCR; GenBank no. EF550208) and cloned into NotI/HindIII sites of pCEP4 (Invitrogen). The sequence and orientation of the inserts were analyzed by sequencing (Eurofins MWG Operon). The new generated pCEP4/neo vector contains both a gene encoding hygromycin and a gene encoding G418 resistance.

To establish LINE-1 EA.hy926 cell clones, EA.hy926 cells were transfected using Lipofectamine (Invitrogen), according to the recommended protocol. After 24 h, cells were selected by the DMEM, containing 100 μg/mL hygromycin (Roth). Seven days after hygromycin selection, the cells were additionally selected with 150 μg/mL G418 (Biochrome) for 14 days.

Plasmid DNA isolation

The plasmid DNA was isolated with the Plasmid Purification Kit^® (Qiagen), according to the user manual. After the DNA pellet was dissolved in the Tris EDTA buffer, the concentration was determined photometrically. Finally, the isolated DNA was characterized by restriction and sequencing analysis.

Cell irradiation

Confluent cell cultures were irradiated at room temperature using a conventional Pantak X-ray machine (Pantak) operated at 310 kV, 10 mA, with a 2-mm AI filter (effective photon energy ∼90 kV), at a distance of 75 cm and a dose rate from 2.7 Gy/min with 5-Gy X-rays. Dosimetry was performed with a Victoreen dosimeter that was used to calibrate an in-field ionizing monitor. Culture flasks were returned immediately to the incubator after irradiation. We used 5-Gy X-rays for our experiments, because it is the clinically relevant irradiation dose used for diagnosis and therapy (Banaz-Yasar et al., 2008).

RNA preparation and cDNA synthesis

Total RNA was isolated from confluent cell monolayers using a Qiagen RNeasy Kit (Qiagen), according to the protocol. Total RNA (1 μg) was converted to cDNA by using a QuantiTect^® Reverse Transcription Kit (Qiagen).

Preparation of genomic DNA from eukaryotic cells

Genomic DNA was isolated from ∼10⁷ cells by using a DNazol^® Genomic DNA Isolation Reagent (Invitrogen), according to producer's instructions. The preparations were incubated with RNAse A at 37°C for at least 3 h to degrade the co-isolated RNA.

Diagnostic PCR analysis to demonstrate LINE-1 retrotransposition

To confirm that retrotransposition events had occurred in transfected cells, PCR was performed on genomic DNA isolated from G418-selected EA.hy926 cells. Each PCR was performed in a 25-μL volume containing 1 U Taq DNA polymerase, 1.5 mM MgCl₂, 10×PCR buffer, 1 mM of each dNTP, 5 μM of primers Neo sense (5′-GAA GAA CTC GTC AAG AAG GCG ATA GAA GG-3′) and Neo antisense (5′-GCC ATT GAA CAA GAT GGA TTG CAC GCA GG-3′), which are specific for the neo gene. PCR was performed in a BioRad Thermocycler using the following cycling conditions: initial denaturation at 94°C for 3 min, followed by 35 cycles of amplification (20 s at 94°C, 15 s at 60°C, and 2 min at 72°C), and a final elongation step at 72°C for 5 min (Kirilyuk et al., 2008).

Reverse transcription–polymerase chain reaction

PCR amplifications were obtained using gene-specific primers designed by the Primer3 software program (Table 1). The primers were purchased from Eurofins MWG Operon.

Table 1.

List of Primer Sequences used for RT-PCR Studies

Primer	Sequence	Product size (bp)
ATM	3′-GCAAACGAACCTGGAGAGAG; 5′-GGTGGAGGGATTTGGTAGGT	226
ATR	3′-GTGGTCATGAGCCGATTTTT; 5′-GCTGGTAGTCCTCCAAGCTG	272
DNA-PK	3′-AGTGGTGTGCCCACACAAAA; 5′-CTTGCAGCGCTGAATGAGC	280
p53	3′-CCTCACCATCATCACACTGG; 5′-CCTCATTCAGCTCTCGGAAC	287
MDM2	3′-CTGTGTGTCGGAAAGATGGA; 5′-TATTGCACATTTGCCTGCTC	274
p21^CIP1	3′-GGATTCGCCGAGGCACCG; 5′-GCATCACAGTCGCGGCTCA	260
TopoIIα	3′-CGCGTCTGTGGAGAAGCGG; 5′-CCATGGTGACGGTCGTGAA	210
TopoIIβ	3′-TGCTGCCATTACCTTGGCA; 5′-CTTTCCGCTGGCCAGGTTT	282
p27^KIP1	3′-CTCTCCGCTTGCCTGGTCC; 5′-CTTTCTCCCGGGTCTGCAC	270
RAD50	3′-CTTGGATATGCGAGGACGAT; 5′-CCAGAAGCTGGAAGTTACGC	215
RAD51	3′-CTCAGCCTCCCGAGTAGTTG; 5′-CATCACTGCCAGAGAGACCA	239
H2AX	3′-GGCCTCCAGTTCCCAGTG; 5′-AGCTTGTTGAGCTCCTCGTC	224
GAPDH	3′-TCCATGGCACCGTCAAGGC; 5′-CCACGACGTACTCAGCGCC	280

The reaction mixture consisted of 0.5 μL cDNA, 0.2 μL dNTP-mixture (10 mM each), 1.2 μL 10×PCR buffer, 5 μmol/μL of the respective sense and antisense primers (Table 1), 1 U Taq-Polymerase (Genecraft), and sterile water in a total volume of 12 μL. The amplification program consisted of the following steps: 5 min at 95°C, 40 cycles of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C. The amplification program ended with a final extension step of 7 min at 72°C. The generated PCR products were analyzed on a 1.5% agarose gel.

Cell proliferation assay

The effect of LINE-1 retrotransposition events on EA.hy926 cell proliferation before and after irradiation was measured using a Biotrak cell proliferation ELISA system (GE Healthcare), following the manufacturer's instructions. The cells were seeded at subconfluent levels into 96-well plates (10,000 cells/well) and incubated for 24 h. The cells were cultivated in a basal medium containing 2% fetal calf serum (PAA) and 2 ng fibroblast growth factor-2 (Immunotools) at 37°C. After 24 h, the medium was removed and replaced with a medium containing bromodeoxyuridine-(BrdU)-labeling reagent (final concentration 10 μM BrdU). Cells were incubated for an additional 24 h before fixing. Incorporation of BrdU was assessed by an enzyme-linked immunosorbent assay (ELISA). Data were analyzed by 450/620 nm using an ELISA reader (TECAN).

Western blot analysis

Protein extracts were prepared with a modified radioimmunoprecipitation assay buffer lysis buffer (50 mM Tris/HCl, 150 mM NaCl, 1% NP-40, 0.25% Na-deoxycholate, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% sodium dodecyl sulfate) supplemented with EDTA-free complete protease inhibitors (Roche).

Protein samples were separated on a 9% tricine gel and electrophoretically transferred to a nitrocellulose membrane (Amersham Biosciences). Membranes were blocked with 5% nonfat dried milk in Tris-buffered saline with 0.15% Tween-20 and incubated with the primary antibody. The following primary antibodies were used: rabbit anti-p53 (1:1000; Cell Signaling), mouse anti-LINE-1 (1:1000), and monoclonal mouse anti-β-actin peroxidase antibody (clone AC-15) (1:10,000; Sigma-Aldrich) for normalization of protein expression. Primary antibody binding was detected using the horseradish peroxidase-coupled secondary goat anti-rabbit immunoglobulin G obtained from Jackson ImmunoResearch.

Phospho-H2AX activation

For detection and quantification of phosphorylated H2AX (Ser139) in the cells, the Cellomics^® Phospho-H2AX Activation Kit was used according to the manufacturer's instructions. The assay was performed in 96-well microplates. The cells were plated at a density of 10,000 cells per well. After 24 h, the cells were irradiated with 5 Gy. The cells were fixed 15 min up to 24 h after irradiation. The primary monoclonal antibody detects the phosphorylated form of human H2AX. Cells were visualized applying a DyLight 549 secondary antibody using a confocal laser-scanning microscope (Leica).

Statistical analysis

Results are reported as the mean±standard deviation of the mean. Levels of significance were determined at the 0.05 level by the Student's t-test.

Results

Characterization of the generated EA.hy926 cell clones with LINE-1 de novo retrotransposition events

To investigate the effect of irradiation on LINE-1 de novo retrotransposition, we transfected the human hybrid endothelial cell line EA.hy926 with the episomal expression vector pJM101/L1_RP. The transfection with this vector allowed the selection of G418-resistant cells that had successfully undergone transcription, splicing, and reverse transcription and genomic reintegration of the LINE-1 sequence (Rangwala and Kazazian, 2009). EA.hy926 cells were selected for de novo retrotransposition by growth in a G418-containing medium for up to 21 days. To verify that the observed G418 resistance of pJM101/L1_RP-transfected cells was a consequence of retrotransposition events caused by this plasmid, genomic DNA was isolated from G418-resistant cell clones and used as a template for PCR analysis (Fig. 1A). EA.hy926 pCEP4/neo was used as control. The PCR was performed with neo gene-specific primers flanking the intron in the neo gene to distinguish the spliced neo cassette in genomic LINE-1 de-novo insertions from the unspliced neo gene in the LINE-1 reporter plasmid (Kirilyuk et al., 2008). The 870-bp PCR product clearly indicates the presence of genomic LINE-1 de-novo retrotransposition events that were launched from the LINE-1 reporter plasmid pJM101/L1_RP (Fig. 1A). Generation of a 1773-bp PCR product demonstrates the presence of the unspliced neo gene, which is localized in the LINE-1 reporter plasmid.

FIG. 1.

Characterization of the EA.hy926 LINE-1 cell clones. (A) Genomic DNA was extracted from the stably transfected EA.hy926 cells after G418 selection and used as template for diagnostic PCR analysis of the neo gene after retrotransposition from the reporter plasmid pJM101/L1_RP. This PCR allows to determine if the γ-globulin intron had been removed by splicing of the neo gene (870-bp product). The spliced form was expressed from the reporter construct and confirmed integration into the genome via retrotransposition. The original unspliced form of neo (1703-bp product) is expressed when the pJM101/L1_RP plasmid DNA is mixed with genomic EA.hy926 WT DNA (pJM101/L1_RP). PCR was performed on pCEP4/neo mixed with genomic EA.hy926 WT DNA (pCEP4/neo), genomic DNA from pJM101/L1_RP-transfected EA.hy926 cells, clone1–clone5, and WT cells expressed no neomycin, H₂O-minus-template control; M, 1-kb DNA ladder (Fermentas). (B) Representative Western blot and densitometric analysis of LINE-1 expression in the generated cell clones. The LINE-1 protein expression was increased in the clones detected with an antibody specific for LINE-1. The detection of beta-actin served as a loading control. LINE-1, long interspersed nuclear element-1; PCR, polymerase chain reaction; WT, wild type.

Next, we isolated proteins from EA.hy926 WT and LINE-1 cell clones to characterize the LINE-1 expression pattern in the generated cell clones (Fig. 1B). Densitometric analysis revealed that the LINE-1 protein levels were increased in all generated cell clones.

LINE-1 de novo retrotransposition events exhibited an increase in cell proliferation

To clarify whether LINE-1 de novo retrotransposition events are functionally implicated in control of cell growth, we analyzed the cell proliferation rate in EA.hy926 WT cells, in control vector pCEP4/neo- and in pJM101/L1_RP-transfected EA.hy926 cell clones (Fig. 2A). Five different G418-resistant EA.hy926 LINE-1 cell clones were analyzed. Nontransfected parental WT cells were set as 0%. Our results demonstrated that there was no difference between the WT and the pCEP4/neo-transfected EA.hy926 cells. Therefore, we used the EA.hy926 WT cells as control in further experiments. All analyzed that LINE-1 cell clones harboring de novo retrotransposition revealed a significant increase in cell proliferation. The proliferation was increased up to 18–45% in the clones compared to EA.hy926 WT cells.

FIG. 2.

LINE-1 de novo retrotransposition events increase cell proliferation. Cell proliferation assays with EA.hy926 WT and EA.hy926 pJM101/L1_RP cell clones exhibited a significant increase in cell proliferation in all analyzed cell clones (A), whereas treatment with 5-Gy irradiation showed no effect on cell proliferation in clones 1, 3, and 4. Cell clones 2 and 5 demonstrate a decrease in proliferation activity after irradiation (B) (WT=0%). Data represent means±SD (n=4). *p≤0.05. SD, standard deviation.

To investigate further the effect of irradiation on the proliferation activity of cell clones with de-novo retrotransposition events, we performed cell proliferation assays. In Figure 2B, the effect of ionizing radiation on cell proliferation is shown. While treatment with 5-Gy X-rays did not result in a significant alteration in cell proliferation in clones 1, 3, and 4, the proliferation rate of clones 2 and 5 was reduced compared to the nonirradiated cell clones. Taken together, LINE-1 de novo retrotransposition can reduce cell proliferation in irradiated cell clones harboring LINE-1 de novo retrotransposition.

Reverse transcription–polymerase chain reaction analysis in EA.hy926 cell clones harboring LINE-1 de novo retrotransposition events after irradiation with 5-Gy X-rays

Using reverse transcription–polymerase chain reaction (RT-PCR), we analyzed genes of the ATM/ATR signalling pathway to explore whether they were regulated by LINE-1 de novo retrotransposition events. The expression levels of eight known cell cycle control and DNA damage response genes were determined (Fig. 3). p21 ^Cip1 and p27 ^Kip1 were analyzed as cell cycle control genes, whereas RAD50 and RAD51 were used as DNA repair factors. Furthermore, we evaluated the expression of p53, which acts as a transcriptional activator of p21^Cip1, and finally, we determined the H2AX mRNA level as a marker for DNA DSBs. GAPDH was used as a housekeeping gene. Two hours and 24 h after irradiation the expression of following genes was downregulated in all analyzed EA.hy926 LINE-1 cell clones harboring LINE-1 de novo retrotransposition: ATM, ATR, p53, p21 ^Cip1, p27 ^Kip1, γ - H2AX, and RAD51. The expression of RAD50 was not altered in these cell clones. In contrast in EA.hy926 WT and pCEP4/neo cells, there was no change of the expression of the mentioned genes after irradiation.

FIG. 3.

LINE-1 de novo retrotransposition events regulated some genes after irradiation. Expression levels of different genes in reverse transcription–polymerase chain reaction analysis of total RNA extracted from EA.hy926 cells before and after irradiation. EA.hy926 LINE-1 cell clones showed no expression or weak expression of the analyzed genes 2 and 24 h after irradiation. RAD50 was not regulated after irradiation. The studies have been repeated with three different RNAs.

Modulation of LINE-1 protein expression after irradiation with 5-Gy X-rays in EA.hy926 cells

Since UV light and ionizing radiation regulate and activate the LINE-1 element (Servomaa and Rytomaa, 1990; Asakawa et al., 2004; Farkash et al., 2006), we analyzed the protein expression level of LINE-1 in EA.hy926 WT and LINE-1 cell clones 1, 2, and 5 in time-course experiments by Western blot analysis (Fig. 4). Cells were harvested 15 min up to 24 h after irradiation with 5-Gy X-rays with subsequent protein extraction. These studies revealed that endogenous expression of LINE-1 proteins is significantly elevated already 2 h after irradiation of EA.hy926 WT cells (Fig. 4A). At later timepoints, the endogenous LINE-1 protein expression is declined up to 24 h. In contrast, we could not detect any significant changes in LINE-1 protein expression levels in the LINE-1 cell clones 1, 2, and 5 after exposure to 5-Gy X-rays (Fig. 4B–D).

FIG. 4.

Expression of LINE- 1 protein after exposure of EA.hy926 cells to X-ray. Western blot analysis of LINE-1 protein expression in EA.hy926 WT cells (A) and EA.hy926 cell clones with de novo retrotransposition events (B–D) exposed to 5-Gy X-rays. Proteins were harvested 15 min up to 24 h after irradiation. Densitometric analyses of three independent experiments are shown. The endogenous LINE-1 proteins were significantly upregulated 2 h after irradiation with 5 Gy in EA.hy926 WT cells (A). Cell clones with retrotransposition events exhibited no modulation of LINE-1 protein after irradiation with 5 Gy for 24 h (B–D). Data represent means±SD (n=3). *p≤0.05.

Analysis of phosphorylated p53 expression levels in EA.hy926 WT and LINE-1 cell clones after irradiation with 5-Gy X-rays

The level of the activated tumor suppressor protein p53 has been investigated after irradiation with 5-Gy X-rays in time-course experiments in EA.hy926 cells (Fig. 5). For Western blot analysis, the cellular proteins were extracted 15 min up to 24 h after irradiation. One hour after irradiation, we detected in EA.hy926 WT cells a significantly elevated expression of phosphorylated p53, which was diminished after 4 h (Fig. 5A). In contrast, the analyzed that cell clones with LINE-1 de novo retrotransposition events showed already 15 min after irradiation a significant increase of phosphorylated p53 protein expression (Fig. 5B–D). The phosphorylated p53 protein levels decline up to 24 h. Our results demonstrated that in the analyzed LINE-1 cell clones, the phosphorylation of p53 starts earlier than in EA.hy926 WT cells.

FIG. 5.

Expression of p53 in EA.hy926 WT and cell clones with LINE-1 de novo retrotransposition events after irradiation. Western blot and densitometric analyses of phosphorylated p53 expression in EA.hy926 WT cells (A) and EA.hy926 cell clones with retrotransposition events (B–D) exposed to 5-Gy X-rays. Phosphorylated p53 was significantly upregulated 1 h after irradiation with 5 Gy in EA.hy926 WT cells (A), whereas in cell clones with retrotransposition events, p53 was significantly upregulated already 15 min after irradiation (B–D). Data represent means±SD (n=3). *p≤0.05.

Activation of γ-H2AX in EA.hy926 cell clones harboring LINE-1 de novo retrotransposition events after exposure to 5-Gy X-rays

Next, we wanted to determine whether LINE-1 de novo retrotransposition events create DNA DSBs in endothelial cells. To this aim, we analyzed the expression of γ-H2AX in EA.hy926 cells stably transfected with pJM101/L1_RP. We used the Cellomics Phospho-H2AX Activation Kit for the detection of phosphorylated H2AX at Ser139 in the cell nuclei. The activation of H2AX was determined 15 min up to 24 h after irradiation in EA.hy926 WT cells and in EA.hy926 LINE-1 clones 1, 2, and 5. Figure 5 exemplarily shows the results for 15 min and 1 h after irradiation. The analysis of the cell nuclei by confocal microscopy demonstrated that the expression of γ-H2AX in WT cells was elevated 1 h after irradiation (Fig. 6A). In contrast, the analyzed LINE-1 cell clones exhibited already 15 min after irradiation a high γ-H2AX protein expression (Fig. 6B).

FIG. 6.

Phospho-H2AX expression in EA.hy926 cells harboring LINE-1 de novo retrotransposition. EA.hy926 WT cells exhibited 1 h after irradiation the strongest phospho-H2AX expression (A), whereas all analyzed LINE-1 clones exhibited the highest protein expression already 15 min after irradiation (B).

Discussion

In this article, we demonstrate for the first time the interaction between LINE-1 de novo retrotransposition events and X-ray irradiation in endothelial cells using the endothelial cell line EA.hy926. Briefly, our results show (1) changed proliferation activity of LINE-1 EA.hy926 cell line clones, (2) a differential regulation of several genes in EA.hy926 LINE-1 cell clones after irradiation, (3) alteration of the phosphorylation of the tumor suppressor p53 and H2AX proteins after X-ray irradiation of EA.hy926 LINE-1 cell clones, and (4) finally, an increased LINE-1 protein level in WT EA.hy926 cells, but not in EA.hy926 LINE-1 cell clones after X-ray irradiation.

We previously showed the presence of LINE-1-encoded proteins ORF1p and ORF2p in endothelial cells of normal blood vessels and demonstrated later that these proteins are not detectable in endothelial cells of tumor blood vessels (Ergun et al., 2004; Banaz-Yasar et al., 2010). Ionizing radiation is still an effective modality for the treatment of many tumors. Tumor growth and metastasis require angiogenesis, which is provided by proliferation and migration of mature endothelial cells and/or endothelial progenitor cells. The cultivation conditions of cell clones that harbor LINE-1 de novo retrotransposition did not allow the survival of cultivated human primary dermal microvascular endothelial cells up to now. Therefore, we used the EA.hy926 cell line.

It was shown that LINE-1 retrotransposition plays a significant role in controlling tumor cell proliferation and differentiation (Sciamanna et al., 2005; Oricchio et al., 2007), as stable inactivation of LINE-1 expression reduced proliferation rate, induced typical signs of morphological differentiation, and reprogrammed the expression pattern of key genes encoding differentiation markers analyzed in A-375 cells (Oricchio et al., 2007). So far, little is known about the effect of LINE-1 retrotransposition events on endothelial cells and their angiogenic properties like cell proliferation, and whether X-ray irradiation influences cell proliferation after LINE-1 de novo retrotransposition. Our present study revealed a significant increase of cell proliferation in all analyzed EA.hy926 LINE-1 clones after de novo retrotransposition events compared to the WT and to the vector control. After irradiation with 5-Gy X-rays, only a part of EA.hy926 LINE-1 cell clones (clone 2 and clone 5) showed a reduced cell proliferation, suggesting that these clones were sensitive to irradiation. In previous studies, we used a porcine aortic endothelial cell line (PAE) to analyze whether LINE-1 retrotransposition events influence cell proliferation (Banaz-Yasar et al., 2010). In contrast to our current results, the proliferation activity of PAE cells was suppressed after LINE-1 de novo retrotransposition. Furthermore, PAE cell clones delayed also in the G0/G1 phase of the cell cycle measured by flow cytometric studies. A possible explanation for this discrepancy could be the different response to LINE-1 retrotransposition events in the used cell lines. Each cell line could have an own mechanism to reduce the consequence of LINE-1 activity. Moreover, the reinsertion of these elements into the genome could occur randomly, and it additionally can depend on different other factors influencing the genomic structure.

Since it has been shown that treatment with low-linear energy transfer (X-ray) irradiation can influence LINE-1 retrotransposition, and LINE-1 expression can further contribute to DNA damage not only through insertional mutagenesis but also via generation of DSBs, we analyzed the effect of irradiation on EA.hy926 cells with LINE-1 de novo retrotransposition. For this purpose, we used the clinically relevant radiation dose for diagnosis and therapy of 5 Gy (Dale and Carabe-Fernandez, 2005). It is known that genotoxins such as γ-radiation and heavy metals lead to increased levels of retrotransposition events (El-Sawy et al., 2005; Kale et al., 2005; Rangwala and Kazazian, 2009). To this aim, we analyzed the LINE-1 protein expression after irradiation by Western blot analysis. Our results demonstrate that in the generated cell clones harboring LINE-1 de novo retrotransposition, the expression of LINE-1 was not changed, whereas the endogenous expression of LINE-1 proteins in WT cells or cells transfected with the control vector was significantly increased 2 h after irradiation. UV light and ionizing radiation are known as DNA-damaging agents. Also, it is known that UV-light and ionizing radiation may activate mobile genetic elements, including retrotransposons, suggesting that retrotransposition may cause somatic mutations (Servomaa and Rytomaa, 1990). It is also discussed in the literature that the radiation-induced activation of LINE-1 is associated with direct DNA damage and must not necessarily followed by lethal consequences to the cells (Farkash et al., 2006). Both chemotherapeutic agents and irradiation can induce tumor cell death primarily by causing DNA damage. The EA.hy926 LINE-1 cell clones did not undergo apoptosis observed by the fluorescence-activated cell sorting analysis (data not shown). One possible explanation for the missing of apoptosis could be cellular DNA repair mechanisms, which might be activated during the retrotransposition process.

LINE-1 affects genome stability (Cordaux and Batzer, 2009) and can generate genomic rearrangements, such as deletions, duplications, and inversions. Although the mechanism of retrotransposition is not entirely understood, it is known that TPRT occurs during LINE-1 retrotransposition. Via TPRT, LINE-1 creates DNA strand breaks using its own EN domain to create nicks in the insertion sites, and these events might be recognized by the cell as unrepaired DNA damage, but it enables newly transposed LINE-1 copies to integrate into the genome (Feng et al., 1996; Belgnaoui et al., 2006).

Since retrotransposons dramatically influenced human evolution at the DNA (Cordaux and Batzer, 2009) and RNA levels and shaped human evolution by modulation of gene expression and RNA editing, we analyzed the expression of several genes from the ATM pathway by RT-PCR in EA.hy926 LINE-1 cell clones. ATM plays a central role in the DNA damage response, and its phosphorylation is crucial for the cellular response to DNA DSBs (Gasior et al., 2006). ATM is required as a DSB repair protein during LINE-1 integration (Gasior et al., 2008). Our data show that in cell clones with LINE-1 de novo retrotransposition, the expression of the analyzed genes was changed after irradiation. LINE-1 sequences can provide new splicing sites that might promote exonization and alternative splicing (Belancio et al., 2006). The functional promoter sequences of the LINE-1 element can also initiate sense or antisense transcription through other genes (Cordaux and Batzer, 2009). Therefore, retrotransposons are also known as controlling elements of neighboring genes (McClintock, 1956; Faulkner et al., 2009).

A previous study reported that human cancer cells that contain a functional p53 underwent apoptosis after LINE-1 retrotransposition, whereas human cancer cells mutant for p53 did not (Haoudi et al., 2004). p53 is one of the most important tumor suppressor genes, as indicated by the fact that it is mutant in about half of the solid tumors (Harris et al., 2009). Furthermore, active retrotransposition by LINE-1 induces p53-dependent cell killing. Our results, however, revealed that the activation of p53 was not associated with the upregulation of endogenous LINE-1 analyzed in the WT cells, and surprisingly, the p53 activation did not result in decreased cell proliferation of EA.hy926 cells harboring LINE-1 de novo retrotransposition. We speculate that the rapid decline of phosphorylated p53 up to 24 h after irradiation could probably not affect cell proliferation. The cells could regenerate very fast and compensate the potential decline in proliferation activity. In the literature, it is described that the p53 protein binds to LINE-1 promoters, but it does not repress LINE-1 transcription (Harris et al., 2009). It is mentioned that LINE-1 transcription becomes activated by p53 protein. The consequence to assume is that the cells with LINE-1 activation would create more DSBs and recruit DNA repair pathways (Gasior et al., 2008). However, our EA.hy926 cells were not apoptotic during LINE-1 retrotransposition, suggesting that the cells use mechanisms without activating apoptosis.

Histone H2AX is phosphorylated in response to ionizing radiation (γ-H2AX) and is detectable as foci in response to DSBs (Rogakou et al., 1999; Rogakou and Sekeri-Pataryas, 1999). In several experiments, it could be shown that a correlation between γ-H2AX foci and DSB exists. Our data demonstrated that the detection of phospho-H2AX in EA.hy926 LINE-1 cell clones starts earlier than in WT cells after irradiation (Fig. 6). The cell nuclei expressed more frequent phospho-H2AX protein. The interpretation of this finding could be that in LINE-1 cell clones, the retrotransposition frequency is increased after irradiation. It is also possible that the cells use retrotransposition as a surviving mechanism. LINE-1 mobilization via ionizing radiation may have a protective function for the cells.

Taken together, we showed in this article for the first time that LINE-1 de novo retrotransposition activated the ATM pathway as a response to exposure to X-ray irradiation.

Footnotes

Acknowledgment

The authors thank Ms. Ewa Kusch for excellent technical assistance.

Disclosure Statement

No competing financial interests exist.

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