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Gene Expression Analysis of and Apoptosis in Proton-irradiated Rat Retina

XIAO W. MAO, LORA M. GREEN, TSEHAY MEKONNEN, NATHAN LINDSEY and DAILA S. GRIDLEY

Department of Radiation Medicine, Radiation Research Laboratories, Loma Linda University and Medical Center, Loma Linda, CA 92354, U.S.A.

Abstract. The aim of this study was to examine the induction studies have shown that oxidative stress is involved in the of oxidative stress and apoptosis-associated expression pathogenesis of radiation-induced retinal damage (5). In profiles in retina after proton irradiation exposure at 0.5 to 4 Gy. many cases, cell death induced by ionizing radiation has Materials and methods: One eye of each Sprague-Dawley rat (6 been identified as occurring via the process of apoptosis (6, per group) was irradiated with a conformal proton beam to total 7). Photoreceptor death by apoptosis is central in the doses of 0, 0.5, 1 and 4 Gy. Retinal tissues were isolated for pathology of most forms of retinal degeneration (8). characterization of profiles 6 hours after proton Photoreceptor loss causes irreversible blindness in many radiation. Results: For oxidative stress, many responsible retinal diseases (9). Radiation-induced photoreceptor loss for regulating the production of reactive oxygen species (ROS) was also evident in our previous study (10). However, as were significantly up-regulated (Fmo2, Gpx2, Noxa1 and Sod3) increased ROS and reduced activity are linked compared to controls. Several important genes involved in the with retinal changes, the underlying mechanisms invoked by initiation or activation of apoptotic signaling pathways were retinal cells to regulate radiation-induced oxidative stress are significantly up-regulated following irradiation (Fas, Faslg, poorly understood; To date, few reports are available on Trp63 and Trp73). TUNEL assay and caspase-3 immuno- proton radiation-induced changes in gene expression relating cytochemical analysis revealed increased apoptotic immuno- to oxidative stress and apoptosis in retinal cells. Thus, the reactivity following irradiation. Conclusion: The data revealed details of cellular mechanisms of radiation-induced retinal that exposure to proton radiation induced oxidative stress- cell apoptosis remain unclear. Characterization of radiation- associated apoptosis. In response to ionizing radiation, the induced marker genes for oxidative stress and apoptosis may expression of genes involved in pathways mediating apoptosis provide insight into pathways governing the outcome of may be differentially regulated in different dose regimens. differential doses of radiation on the retina. The purpose of the present study was to investigate the The eye is a unique organ because it is relatively unprotected effect of proton irradiation on apoptotic and oxidative stress- and is constantly exposed to radiation, atmospheric oxygen, associated gene expression profiles in a rat retinal model. environmental chemicals, and physical abrasion. Concern exists, therefore, about possible radiation damage from Materials and Methods occupational exposure and medical procedures. Radiation-induced oxidative stress occurs via reactive Animals and experimental design. Male Sprague-Dawley rats (n=24, oxygen species (ROS), produced as a consequence of 6 per group), each weighing approximately 250 g, were purchased locally (Charles River Breeding Lab, Hollister, CA, USA) and irradiation of biological water (1). Oxidative stress-induced maintained, three per cage, under constant ambient temperature of ocular tissue damage resulting from ROS has been associated 18.3˚C with a 12-hour day/night cycle. Commercial pellet chow and with a variety of pathological conditions (2-4). Previous water were available ad libitum. The animals were observed post- irradiation for signs of radiation-related toxicity. Euthanasia was performed 6 hours after irradiation for analyses. The Loma Linda University Animal Care Facility is fully accredited by the American Correspondence to: Xiao Wen Mao, MD, Chan Shun Pavilion, Room Association for Accreditation of Laboratory Animal Care and the A1010, 11175 Campus Street, Loma Linda University and Medical protocol was approved by the Institutional Animal Care and Use Center, Loma Linda, CA 92350, U.S.A. Tel: +1 9095588373, Fax: Committee. +1 9095580825, e-mail: [email protected] Proton radiation. Animals were lightly anesthetized with isoflurane Key Words: Gene expression profile, apoptosis, oxidative stress, (Halocarbon Laboratories, NJ, USA; 4% induction, 1.5% maintenance). proton radiation, rat retina. One eye of each rat in the appropriate groups was irradiated with a

0258-851X/2010 $2.00+.40 425 in vivo 24: 425-430 (2010) modulated and shaped 100-MeV conformal proton beam using a 100. Retinal tissues were subjected to terminal previously reported technique (11). Rats were positioned in the path deoxynucleotidyltransferase dUTP nick-end labeling (TUNEL) and field localization was achieved using a 0.8 cm diameter light using DeadEnd™ Fluorometric TUNEL system kit (Promega, Corp., field projected to the (iso) center. The left eye of each rat received a Madison, WI, USA). As an additional way to confirm the apoptotic single dose of either 0 (control), 0.5, 1, or 4 Gy, delivered at an process in the retina, sections were immunocytochemically labeled average dose rate of 7 Gy/min. A polycarbonate range shifter with Anti-ACTIVE® Caspase-3 polyclonal antibody (Promega) reduced the range of protons to 0.9 cm in tissue using a stepped specific for activated caspase-3. Sections were then incubated with propeller. The beam yielded a uniform dose distribution across the anti-active caspase-3 primary antibody at room temperature for 2 irradiated eye at the 90% isodose line. hours followed by a donkey anti-rabbit IgG fluorescent-conjugated secondary antibody (Invitrogen Corp. Carlsbad, CA, USA) for 2 Eye and retina collection. At euthanasia, three retinas of left eyes hours at room temperature and counterstained with propidium from each group were isolated for RNA microarray analysis. The iodide (PI). Five random fields on one tissue section of the retina retinal tissue from each animal was placed individually in sterile (horizontal, central sections, passing through the optic nerve head) cryovials, snap frozen in liquid nitrogen and kept at –80˚C prior to were examined at 300 μm nasally and temporally of the optic nerve use. Three left eyes from each subset of group were used for using an Olympus IX81 microscope (Olympus America Inc., Center immunohistochemistry. Isolated eyes were fixed in 4% Valley, PA, USA). TUNEL-positive cells were identified by green paraformaldehyde in phosphate-buffered saline (PBS) overnight, fluorescence; the nuclei of photoreceptor were counterstained with rinsed with PBS, infiltrated overnight with 30% sucrose in PBS at PI (red). For each field, the numbers of TUNEL-positive cells and 4oC, and then embedded in Optimal Cutting Temperature (OCT) total nuclei in the outer nuclear (ONL) and inner nuclear layer (Sakura, Torrance, CA) and frozen at –80˚C. (INL) were counted. Percentages of apoptotic-to-total nuclei were averaged across three sections per animal (separated by 50 μm) to RNA purification microarrays. RNA extraction and polymerase provide a single value for the entire retina. chain reaction (PCR) array services were performed at To obtain the resulting activated caspase-3 immunoreactivity, SABiosciences service core (Frederick, MD, USA). RNA was fluorescence intensity was measured on 5 randomly selected fields extracted from each frozen retina by first disrupting tissues using a on each section and calculated using Image J 1.41 software ceramic bead-based tissue disruption method on a FastPrep FP120, (National Institutes of Health, Bethesda, MD, USA; Bio101 homogenizer (Savant Instruments, Inc., Nassau-Suffolk, NY, http://rsb.info.nih.gov/ij/Java). Once the green channel was USA), followed by phenol/chloroform extraction combined with separated from the red channel in an image, fluorescence intensities isopropanol precipitation. from the areas of interest were measured using the integral/density For PCR array service, RNA was quantitated with NanoDrop feature in the Image J program and data were extracted and spectrophotometer, and RNA quality analyzed using Agilent averaged within the group. Bioanalyzer with RNA Nano Chip 6000 (Agilent Technologies, Santa Ana, CA, USA). Reverse transcription was performed with Statistical analyses. Results were subjected to statistical analyses SABiosciences’ RT2 First Strand Kit (C-03) which contained an including one-way analysis of variance (ANOVA) and Tukey’s HSD effective genomic DNA elimination step and a built-in external (honestly significant difference) test using SigmaStat™ version 3.5 RNA control. RNA input was 1000 ng per RT reaction. cDNA software (SPSS Inc., Chicago, IL, USA). A p-value of <0.05 was template was then run on appropriate PCR arrays in an ABI 7900HT selected to indicate significant differences among groups. sequence detection system. PCR reactions were performed to evaluate expression of 84 genes using RT2 Prolifer™ PCR array Results PARN-12 (Rat Apoptosis SuperArray) and PARN-065 (Rat Oxidative Stress SuperArray). Data analysis was performed using Gene expression. The genes that were statistically SABiosciences’ PCR array data analysis software. Relative changes significantly (p<0.05) altered between irradiated and control in gene expression were calculated using the comparative threshold groups by >1.5-fold are presented in a tabular format: Table cycle (Delta Delta Ct, DDCt) method. This method first subtracts I lists genes of the oxidative stress-related pathway. Table II the Ct (threshold cycle number) of the gene-average Ct of the 5 contains the list of apoptosis-associated genes. For the 84 housekeeping genes on the array (Rplp1, Hprt, Prl13a Ldha and oxidative stress genes that were evaluated, significant dose- Actb) to normalize for the amount of RNA per sample. Finally the DDCt was calculated as the difference between the normalized dependent changes in irradiated retina were noted when compared with the 0 Gy-treated group (p<0.05): 5 genes average Ct of the irradiated group and the normalized average Ct of the non-irradiated control group. This DDCt value was raised to the were up-regulated and 2 were down-regulated at 0.5 Gy; 8 power of 2 to calculate the degree of change. The genes of interest were up-regulated and none were down-regulated at 1 Gy; 3 included those involved in oxidative stress and apoptosis. Three rat were up-regulated and 1 was down-regulated at 4 Gy. Several retinas per group were used for this analysis. genes playing a central role in regulating ROS production under conditions of oxidative stress were up-regulated. These Photoreceptor cell apoptosis detection by TUNEL assay and include: flavin-containing monooxygenase 2 (Fmo2), activated caspase-3 immunohistochemical labeling. In the present study, apoptosis was evaluated 6 hours post irradiation. Sections of glutathione peroxidase 2 (Gpx2), NADPH oxidase activator 1 5 μm bisecting the optic disc superiorly to inferiorly were cut on a (Noxa1), and dismutase 3 (Sod3). microtome (Leica Inc., Deerfield, IL, USA). Paraffin sections were For the apoptotic gene profile based on the 84 evaluated deparaffinized in Histo-clear, then permeabilized in 0.3% TritonX- genes, the data revealed that 7 genes were up-regulated and

426 Mao et al: Proton Radiation-induced Gene Expression in Retina

Table I. Oxidative stress gene expression in retina after proton Table II. Apoptotic gene expression in retina after proton irradiation irradiation. exposures.

Gene Description Fold Gene Description Fold change change

0.5 Gy 0.5 Gy Up-regulated Up-regulated Dhcr24 24-dehydrocholesterol reductase 1.58 Apaf Apoptotic peptidase-activating factor 1 2.33 Ehd2 EH-domain-containing 2 1.52 Casp 8 Caspase 8 1.50 Fmo2 Flavin-containing monooxygenase 2 2.83 Dffb DNA fragmentation factor, beta subunit 2.0 Gpx2 Glutathione peroxidase 2 1.53 Gadd45a Growth arrest and DNA-damage- –2.09 Mb Myoglobin 2.25 inducible 45 alpha 1.50 Down-regulated Prlr Prolactin receptor 2.14 Noxa1 NADPH oxidase activator 1 –2.10 Fas TNF receptor superfamily, member 6 2.01 Vcp3 Uncoupling protein 3 (mitochondrial) –5.35 Faslg Fas ligand (TNF superfamily, member 6) 1.6 Down-regulated 1 Gy Birc1b Baculoviral IAP repeat-containing 1b –3.37 Up-regulated Als2 Amyotrophic lateral sclerosis 2 2.17 1 Gy Dnm2 Dynamin 2 2.29 Up-regulated Gpx2 Glutathione peroxidase 2 1.53 Apaf Apoptotic peptidase-activating factor 1 3.80 Mpo Myeloperoxidase 3.86 Bax Bcl2-associated X protein 2.38 Prdx4 Peroxiredoxin 4 3.45 Casp3 Caspase-3 1.90 Sod3 3, extracellular 3.36 Casp 8 Caspase-8 2.18 Tpo Thyroid peroxidase 1.86 Casp14 Caspase-14 13.22 Txnrd1 Thioredoxin reductase 1 1.50 Dapk1 Death-associated protein kinase 1 1.54 Gadd45a Growth arrest and DNA-damage- 1.54 4 Gy inducible 45 alpha Up-regulated Prlr Prolactin receptor 2.00 Mpo Myeloperoxidase 3.80 Traf3 Tnf receptor-associated factor 3 1.50 Nqo1 NAD(P)H dehydrogenase 1.52 Faslg Fas ligand (TNF superfamily, member 6) 3.20 Prdx4 Peroxiredoxin 4 3.53 Downregulated Down-regulated Prok2 Prokineticin 2 –2.24 Idh1 Isocitrate dehydrogenase 1 –2.09 Dad1 Defender against cell death 1 –1.53 (NADP+), soluble 4 Gy Up-regulated Casp 3 Caspase 3 2.16 IL10 Interleukin 10 4.17 CD40Ig CD40 ligand 1.64 Trp63 Transformation-related protein 63 2.40 1 was down-regulated at 0.5 Gy; at 1 Gy, 10 were up- Trp73 Transformation-related protein 73 2.11 regulated and 2 were down-regulated; whereas, 5 were up- regulated and none were down-regulated at 4 Gy (p<0.05 vs.0 Gy; Table II). Several important genes involved in the initiation or activation of apoptotic signaling pathways were significantly up-regulated. They included: TNF receptor superfamily, member 6 (Fas), Fas ligand (Faslg), in the INL and ONL after 1 Gy (Figure 1c) and 4 Gy transformation related protein 63 (Trp63), and Trp73. Genes (Figure 1d) exposure compared to controls (Figure 1a) or participating in the caspase cascade were also up-regulated: retinas that received 0.5 Gy (Figure 1b). Only sparse apoptotic peptidase activating factor 1 (Apaf), Bcl2- TUNEL-positive cells were found in the photoreceptor layer associated X protein (Bax), caspases 3, 8 and 14 (Casp3, of eyes that received 0.5 Gy. 35.5±7.2% and 21.8± 4.3% of Casp8 and Casp14). the cells that received 4 Gy and 1 Gy were labeled TUNEL positively in ONL and INL compared to age-matched Apoptosis assessment following radiation. To investigate controls (p<0.05) respectively. Our assessment (Figure 2) whether radiation induced retinal cell loss via apoptosis, a also revealed that the immunoreactivity for activated TUNEL assay and caspases-3 immunohistochemical caspase-3 after 1 and 4 Gy of proton irradiation exposure at labeling of retinal sections were performed. At 6 hours’ post 6 hours were significantly higher than that of controls and irradiation, greater numbers of apoptotic cells were observed the 0.5 Gy irradiated group (p<0.01).

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Figure 1. TUNEL staining of retinal tissue. Representative micrographs of retina sections were evaluated for apoptosis by TUNEL assay at 6 hours following 0.5 Gy to 4 Gy of proton irradiation. a: 0 Gy age-matched control; b: 0.5 Gy; c: 1 Gy; and d: 4 Gy. TUNEL-positive cells were identified with green fluorescence; the nuclei of photoreceptors were counterstained with propidium iodide (red).

Discussion radiation; these included Fmo2, Duox1, Mpo, Prdx4, and Tpo. Interestingly, a recent study showed thyroid peroxidase- Ionizing radiation induces the production of ROS, which induced Bcl-xL gene expression, an anti-apoptotic member of play an important causative role in apoptotic-mediated cell the Bcl-2 family (18). The data suggest possible associations death. Many studies have documented oxidative stress between oxidative stress and apoptosis. Txnrd1, the gene for associated with radiation-induced apoptosis (12, 13). Our thioredoxin reductase was also up-regulated at 1 Gy. This is aim was to characterize proton radiation-induced modulation an important selenoprotein that aids in the maintenance of of gene expression in rat retina. This study demonstrated that cellular redox balance and regulates several redox-dependent radiation significantly induced changes in oxidative stress processes in the apoptosis pathway, cell proliferation and and apoptosis-associated gene expression in the rat retina at differentiation (19). Our data indicated that Sod3 (superoxide doses as low as 0.5 Gy. There was a significant increase in dismutase 3, extracellular) was significantly up-regulated apoptotic cell death measurable at 1 Gy. Our data are in following irradiation at 1 Gy. Superoxide dismutases are a agreement with other studies of the acute effects of ionizing ubiquitous family of that function to efficiently radiation on the rat retina (14, 15). catalyze the dismutation of superoxide anions. Sod3 has been The induction of oxygen radical formation is a major shown to play an important role in scavenging ROS. A mechanism by which radiation induces DNA damage (16). previous study reported that increased levels of SOD3 protein Free radicals can also initiate a variety of cellular signal prevented the degradation of NO by oxygen radicals (20). transduction pathways that lead to damage beyond the repair The presence of oxidative stress in the irradiated retina was capabilities of the cell (17). In this study, many genes in the evident at a dose as low as 0.5 Gy. We saw the most significant oxidase or peroxidase families were up-regulated following regulation of genes in retinal samples that received 1 Gy of

428 Mao et al: Proton Radiation-induced Gene Expression in Retina

exposure to ionizing radiation (25). Recent studies reported that over expression of Trp73 can activate the transcription of p53-responsive genes and can induce apoptosis (27). At 0.5 Gy, baculoviral inhibitor of apoptosis repeat-containing 6 (Birc 6) gene was significantly down-regulated. The Birc6 gene encodes an inhibitor of apoptosis; this gene has been shown to regulate mitochondrial pathway of apoptosis (28). Down-regulation of Birc6 gene further demonstrated that radiation can induce retinal apoptosis at a dose as low as Figure 2. Immunoreactivity of caspase-3 measured in retina. The averages of fluorescence intensity for caspase-3 activity were measured 0.5Gy. These data indicate that in response to physiological and calculated using the Image J program. Fluorescence intensities stress induced by ionizing radiation, the expression of genes were averaged across three retinas per group. Each value represents involved in pathways regulating apoptosis may be mean±SEM. aSignificantly different compared to 0 or 0.5 Gy (p<0.01). differentially regulated at different doses. The universal biochemical hallmark of apoptotic death is the activation of caspases, which are cysteine proteases characterized by their ability to cleave specific proteins (29). proton radiation compared to samples that received lower (0.5 Previously published findings reported that Fas-mediated Gy) or higher doses (4 Gy). The considerable reduction of gene apoptosis involves caspase-8 upon activation of downstream expression following the 4 Gy of proton radiation was probably caspases (30). The findings of our gene expression data are due to the increased cell death at this high dose, or alternatively consistent with this. We found that caspase-8 and other caspase the inability of lethally irradiated cells to regulate their genes were significantly up-regulated following radiation response to injury. Overall, the gene expression patterns in both exposure. Among them, we measured caspase-14 as being the arrays showed a striking dependence on the total dose that was most highly up-regulated gene following 1 Gy of radiation (an delivered to the retinal tissue. approximate 13-fold increase). Caspase-14 is expressed in a The up-regulation of oxidative stress genes is an early broad range of epithelial cell types including skin, breast, marker of retinal degeneration. The presence of markers for prostate, and stomach (31). Caspase-14 protein was also present apoptosis in this study supports the hypothesis that apoptotic at lower levels in the brain, and testis of and mice (32). mechanisms are involved in retinal cell death. Radiation- To our knowledge, our study is the first to suggest the presence induced apoptosis can take place through several routes. of caspase-14 in rat retina. Caspase-14 may be involved in There are two major mechanisms for apoptosis in the retina. engagement of the death receptor in apoptotic pathways. It They involve the Fas/Fas-L interaction system and the p53 functions as a downstream signal transducer of cell death. cascade (21-23). In our study, the genes encoding Fas and Fas Caspase-14 is processed and activated by caspase-8 and ligand proteins were significantly up-regulated by 0.5 and 1 caspase-9 in mice (33). This is supported by our observations Gy of proton irradiation compared to control samples. The that expression changes were detected in both caspase-14 and data support the possible involvement of Fas in this form of caspase-8 in response to exposure to 1 Gy protons. Caspase-3 apoptosis occurring in the retina. Thus, Fas/FasL interactions acts as an effector caspase of apoptosis by cleaving cellular may play an important role in identifying and/or eliminating substrates and precipitating apoptotic death. Our immuno- damaged cells after low-dose irradiation and possibly other chemical data further support caspase 3 involvement in forms of injury. In contrast, at 4 Gy, p53 family members apoptosis of the retina measured 6 hours’ post proton irradiation. Trp63 and Trp73 genes were significantly up-regulated, Radiation-induced ROS-meditation of apoptosis is a major suggesting that p53, not Fas/Fas-L, plays a major role at oxidative stress pathway in the retina. Our study provides relative high doses of radiation-induced retinal cell apoptosis. some insight into the cellular mechanisms critical for P53 is activated and up-regulated following a range of insults responsiveness to acute oxidative injury of the retina and including DNA damage, hypoxia, oxidative stress, and viral may prompt the development of new strategies against ocular infections (24). The p53 gene product acts as a transcription damage caused by oxidative stress associated apoptosis. factor, inducing apoptosis by modulating specific downstream target genes (25). Surprisingly, in our present study, although Acknowledgements p53 family members Trp63 and Trp73 were up-regulated, Trp53 was not significantly up-regulated. The failure of The Authors thank Steve Rightnar for his support on the radiation detection of this gene at 6 hours post irradiation could point physics aspects. The study was supported by the National to its early involvement in apoptosis following irradiation. Aeronautics and Space Administration (grant No. NNX08AP21G), Previous study demonstrated that expression of p53 mRNA the National Medical Technology Testbed (NMTB), and the in RPE cells increased significantly within 2 hours after LLUMC Department of Radiation Medicine.

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