Cancer Therapy: Preclinical Distinct Effects of Ionizing Radiation on In vivo Murine Kidney and Brain Normal Tissue Gene Expression Weiling Zhao,1Eric Y. Chuang,2 Mark Mishra,3 Rania Awwad,3 Kheem Bisht,3 Lunching Sun,3 Phuongmai Nguyen,3 J. Daniel Pennington, 3 Tony Jau Cheng Wang,3 C. Matthew Bradbury, 3 Lei Huang,3 Zhijun Chen,3 Gil Bar-Sela,3 Michael E.C. Robbins,1and David Gius3 Abstract Purpose: There is a growing awareness that radiation-induced normal tissue injury in late- responding organs, such as the brain, kidney, and lung, involves complex and dynamic responses between multiple cell types that not only lead to targeted cell death but also acute and chronic alterations in cell function.The specific genes involved in the acute and chronic responses of these late-responding normal tissues remain ill defined; understanding these changes is critical to understanding the mechanism of organ damage. As such, the aim of the present study was to identify candidate genes involved in the development of radiation injury in the murine kidney and brain using microarray analysis. Experimental Design: A multimodality experimental approach combined with a comprehen- sive expression analysis was done to determine changes in normal murine tissue gene expression at 8 and 24 hours after irradiation. Results: A comparison of the gene expression patterns in normal mouse kidney and brain was strikingly different. This observation was surprising because it has been long assumed that the changes in irradiation-induced gene expression in normal tissues are preprogrammed genetic changes that are not affected by tissue-specific origin. Conclusions: This study shows the potential of microarray analysis to identify gene expression changes in irradiated normal tissue cells and suggests how normal cells respond to the damaging effects of ionizing radiation is complex and markedly different in cells of differing origin. The ongoing improvements in cancer therapy and health care the new areas of public health emphasis,particularly studying have resulted in an ever-growing population of long-term adverse long-term or late effects of cancer and its treatment cancer survivors: 62% of adult and 77% of pediatric cancer (National Cancer Institute’s Plans and Priorities for Cancer patients survive beyond 5 years. For these individuals,a Research).4 Given the increasing population of long-term particular concern is the late effects that arise several months survivors,the need to mitigate or treat late effects has emerged to years after irradiation. Although improvements in radiation as a primary area of radiobiological research (1–3). oncology,such as intensity-modulated radiation therapy,have Radiation-induced late effects,once viewed solely as a late led to a reduction in the volume of normal tissue irradiated, consequence of clonogenic cell loss,now seems to be due to a late effects remain a significant risk. The National Cancer complex interrelationship between cell loss and changes in Institute has identified long-term survival from cancer as one of gene expression (4,5). It has long been assumed that the damage to normal tissues from therapeutic irradiation is inevitable,progressive,and untreatable; however,it is now Authors’ Affiliations: 1Department of Radiation Oncology, BrainTumor Center of viewed in terms of dynamic interactions between multiple cell Wake ForestUniversity, Wake ForestUniversitySchool of Medicine, Winston- 2 types within a particular organ (6–9) that can be modulated Salem, North Carolina; Biomedical Engineering Group, Department of Electrical (2,10,11). Some of the important lesions include fibrosis, Engineering, National Taiwan University, Taipei, Taiwan; and 3Radiation Oncology Sciences Program, Radiation Oncology Branch, Center for Cancer Research, necrosis,atrophy,and vascular damage. Irradiated normal cells National Cancer Institute, NIH, Bethesda, Maryland are not passive bystanders,merely dying as they attempt to Received 11/7/05; revised 2/4/06; accepted 4/13/06. divide,but are active participants in an orchestrated,yet Grant support: Intramural Research Program of the NIH, National Cancer Institute, limited,response to injury. Center for Cancer Research, Radiation Oncology Branch, and grants DK51612 and CA82722 (M.E.C. Robbins). In general,it has been hypothesized that irradiating normal The costs of publication of this article were defrayed in part by the payment of page tissues leads to an acute activation of stress kinases and charges. This article must therefore be hereby marked advertisement in accordance transcription factors (12) and increased production of inflam- with 18 U.S.C. Section 1734 solely to indicate this fact. matory cytokines that may play a central role in the resulting Note: W. Zhao and E.Y.Chuang contributed equally to this work. damaging late effects (13). This is followed by an aberrant Requests for reprints: David Gius, Radiation Oncology Sciences Program, Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, chronic inflammatory/wound-healing response,in which NIH, Building 10, B3B69, Bethesda, MD 20892. Phone: 301-496-5457; Fax: 301- vascular and parenchymal cell dysfunction and loss,associated 480-5439; E-mail. [email protected]. F 2006 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-05-2418 4 http://plan.cancer.gov/public/survivor.htm. www.aacrjournals.org 3823 Clin Cancer Res 2006;12(12) June 15, 2006 Downloaded from clincancerres.aacrjournals.org on September 30, 2021. © 2006 American Association for Cancer Research. Cancer Therapy: Preclinical with chronic overproduction of particular cytokines and growth whole-body irradiation using a 12,000 Ci self-shielded 137Cs irradiator factors,result in fibrosis and/or necrosis,depending on the and a dose rate of 4 Gy/min. Sham-irradiated mice were placed 137 particular organ involved (9). More recently,chronic oxidative similarly in the Perspex box and positioned in the Cs irradiator for n stress has been suggested to contribute to the progression of 2.5 minutes without g-ray exposure. Groups of mice ( = 3) were euthanized using an overdose of pentobarbital (Nembutal,90 mg/kg radiation-induced late effects (14). body weight,given i.p.) at 8 and 24 hours after irradiation. Kidneys and Despite this paradigm shift,details of the specific genes and/ brains were removed,frozen in liquid nitrogen,and stored at À70jC. or mechanisms involved in the pathogenesis of radiation- RNA extraction. Total RNA of the kidneys and brains was isolated induced late effects remain unclear. The development of tech- using TRIzol according to the protocol supplied by the manufacturer nological advances to determine changes in gene expression (Invitrogen,Carlsbad,CA); the concentration of RNA was determined using cDNA and oligonucleotide microarray analysis presents spectrophotometrically at 260 nm. RNA was further purified using the potential to increase our understanding of the complex Rneasy Mini kit according to the manufacturer’s recommendations nature of normal tissue responses to radiation (15). It has also (Qiagen,Valencia,CA) with the addition of DNase digestion with enabled investigators to obtain a global view of gene expression RNase-free DNase set (Qiagen). patterns in both normal and tumor cells to profile how these Microarray fabrication. The microarrays used for this study contained 7,680 human cDNA clones and were prepared from the cells may respond to cellular stress,including that induced by Research Genetics Named Genes set (Huntsville,AL). These cDNA anticancer agents. clones are enriched for known genes. All 7,680 cDNAs were spotted A genomic approach has been used to identify candidate onto poly-L-lysine-coated slides (National Cancer Institute ROSP 8k genes of interest in the irradiated mouse kidney and rectum at Human Array) using an OmniGrid arrayer (GeneMachines,San Carlos, 10 and 20 weeks after irradiation with a single dose of 16-Gy, CA) as described in Eisen and Brown (19). 250-kV X rays (16,17),as well as in the mouse brain after Probe labeling, microarray hybridization, image, and data analysis. exposure to low dose (0.1 or 2.0 Gy 137Cs g-rays) ionizing Methodologies for the probe labeling reaction and microarray hybrid- radiation (18). Here,we report microarray results obtained ization were the same as described previously (20). Microarrays were A 8 and 24 hours after irradiating the mouse brain and kidney scanned at 10- m resolution on a GenePix 4000A scanner (Axon with a single dose of 10 Gy 137Cs g-rays. These data show not Instruments,Inc.,Foster City,CA). The Cy5- and the Cy3-labeled cDNA samples were scanned at 635 and 532 nm,respectively. The resulting TIFF only the potential of microarray analysis to identify gene images were analyzed by GenePix Pro 3.0 software (Axon Instruments). expression changes in irradiated normal tissues but also that the Each sample was tested in duplicate with alternating of the dyes. Thus, radiation-induced changes in irradiated normal cells vary a total of four microarrays for each sample of every brain and kidney markedly between cells from different tissue types. sample was done. This was necessary to have enough data points for statistical significance. The ratios of the sample intensity to the reference Materials and Methods intensity (green [Cy3]/red [Cy5]) for all targets were determined. Because a normal distribution could not be applied to all components of the data Irradiation of murine kidney and brain. A total of 12 adult male set,a Mann-Whitney