title: Very low doses of ionizing radiation and redox associated modifiers affect survivin-associated changes in radiation sensitivity.
abstract: Exposure of cells to a dose of ionizing radiation as low as 5mGy can induce changes in radiation sensitivity expressed by cells exposed to subsequent higher doses at later times. This is referred to as an adaptive effect. We describe a unique survivin-associated adaptive response in which increased radiation resistance or sensitization of cells can be induced by exposure to 5mGy or to the reactive oxygen species (ROS) generating drug Emodin (1,3,8-trihydroxy-6-methylanthraquinone), a naturally occurring anthraquinone. The purpose of this study was to determine the role of ROS generating processes in affecting both the intracellular localization of the inhibitor of apoptosis protein survivin and its subsequent effect on radiation response in the presence or absence of the anti-oxidant N-acetyl-L-cysteine (NAC). Experiments were performed using two well characterized murine sarcomas: SA-NH p53 wild-type (WT) and FSa p53 mutant (Mut), grown either in culture or as solid tumors in the right hind legs of C3H mice. Doses of 5mGy or 50μM Emodin were used to induce changes in the response of these tumor cells to higher radiation exposures using a multi-dosing paradigm. Effects on radiation sensitivity were determined for SA-NH and FSa cells as a function of survivin translocation either to the cytoplasm or nucleus in the presence or absence of 10mM NAC treatment. In vitro survival assays (2Gy per fraction, two once daily fractions) and tumor growth delay (TGD) (5Gy per fraction, five once daily fractions) studies were performed. Intracellular localization of survivin was determined by enzyme-linked immunosorbent assay (ELISA) and correlated to survival response and treatment conditions. 2Gy alone had no effect on intracellular translocation of survivin. When preceded 15min earlier by 5mGy or Emodin exposures, survivin became elevated in the cytoplasm of p53 WT SA-NH as compared to the nuclei of p53 Mut FSa cells. SA-NH cells transfected with p53 small interfering RNA (siRNA), in contrast, responded similarly to p53 Mut FSa cells by becoming more radiation sensitive if exposed to 5mGy prior to each 2Gy irradiation. In contrast to their respective responses to five once daily 5Gy fractions, SA-NH tumors were protected by 5mGy exposures administered 15min prior to each daily 5Gy dose as evidenced by a more rapid growth (1.9 day decrease in TGD, P=0.032), while FSa tumors were sensitized, growing at a much slower rate (4.5 day increase in TGD, P<0.001). Exposure of SA-NH and FSa tumor cells to 10mM NAC inhibited the ability of 5mGy and Emodin to induce intracellular translocation of survivin and the corresponding altered adaptive survival response. The survivin-associated adaptive response can be induced following a multi-dosing scheme in which very low radiation doses are followed shortly thereafter by higher doses consistent with a standard image guided radiotherapy protocol that is currently widely used in the treatment of cancer. While induced by exposure to ROS generating stresses, the ultimate expression of changes in radiation response is dependent upon the bi-functionality of the tumor associated protein survivin and its intracellular translocation.
authors.affiliation: Department of Radiation and Cellular Oncology, The University of Chicago, Chicago, IL, United States.
authors.forename: Richard C
authors.initials: RC
authors.lastname: Miller
authors.affiliation: Department of Radiation and Cellular Oncology, The University of Chicago, Chicago, IL, United States.
authors.forename: Jeffrey S
authors.initials: JS
authors.lastname: Murley
authors.affiliation: Department of Preventive Medicine, Northwestern University, Feinberg School of Medicine, Chicago, IL, United States.
authors.forename: Alfred W
authors.initials: AW
authors.lastname: Rademaker
authors.affiliation: Department of Radiation Oncology, Northwestern University, Feinberg School of Medicine, Chicago, Illinois, United States.
authors.forename: Gayle E
authors.initials: GE
authors.lastname: Woloschak
authors.affiliation: Department of Radiation Oncology, The University of California Davis, Sacramento, CA, United States.
authors.forename: Jian Jian
authors.initials: JJ
authors.lastname: Li
authors.affiliation: Department of Radiation and Cellular Oncology, The University of Chicago, Chicago, IL, United States.
authors.forename: Ralph R
authors.initials: RR
authors.lastname: Weichselbaum
authors.affiliation: Department of Radiation and Cellular Oncology, The University of Chicago, Chicago, IL, United States. Electronic address: dgrdina@uchicago.edu.
authors.forename: David J
authors.initials: DJ
authors.lastname: Grdina
mesh_terms: D000111:Acetylcysteine / Q000494:pharmacology; D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D004307:Dose-Response Relationship, Radiation; D004642:Emodin / Q000494:pharmacology*; D005260:Female; D005354:Fibrosarcoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D005720:Gamma Rays; D015972:Gene Expression Regulation, Neoplastic; D006614:Hindlimb; D051034:Inhibitor of Apoptosis Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D009368:Neoplasm Transplantation; D016877:Oxidants / Q000494:pharmacology*; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011836:Radiation Tolerance / Q000187:drug effects*; D017382:Reactive Oxygen Species / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D000077022:Survivin; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D000975:Antioxidants; C117749:Birc5 protein, mouse; D051034:Inhibitor of Apoptosis Proteins; D016877:Oxidants; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; D012097:Repressor Proteins; D000077022:Survivin; D016159:Tumor Suppressor Protein p53; D004642:Emodin; D000111:Acetylcysteine
keywords: Adaptive response; Emodin; Radiation response; Reactive oxygen species; Survivin; p53