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.

Keywords: Adaptive response; Emodin; Radiation response; Reactive oxygen species; Survivin; p53.

Fig. 1.

Demonstration of very low radiation dose induced adaptive responses in two mouse tumor cell lines differing in p53 mutational status. P values were determined by comparing the survival of cells following two 2 Gy doses with those exposed to two 2 Gy doses along with the 5 mGy doses using a Student’s two-tailed t-test with values ≤0.05 identified as significant. Exposure to 5 mGy induced a significant elevation in SA-NH p53 WT (P=0.001) and reduction in FSa p53 Mut (P<0.003) cell survival. The presence of NAC during the 5 mGy irradiation inhibited these effects in both SA-NH (P=0.572) and FSa (P=0.841). Each experiment was repeated three times and error bars represent the standard error of the mean (SEM).

Fig. 2.

Survivin protein in cytoplasmic and nuclear fractions measured by ELISA as a function of treatment protocol in SA-NH and FSa tumor cells. P values were determined by comparing the protein content in the unirradiated control cytoplasmic (C) and nuclear (N) fractions with their corresponding groups from the three treatment protocols: 2 Gy only (SA-NH, P = 0.416 and 0.686, C and N, respectively and FSa, P = 0.814 and 0.469, C and N, respectively; 5 mGy+ 2 Gy ( SA-NH, P = 0.020 and 0.133, C and N, respectively; FSa, P = 0.597 and 0.009, C and N, respectively; and 5 mGy+NAC +2 Gy (SA-NH, P = 0.773 and 0.700, C and N, respectively; FSa, P = 0.016 and 0.996, C and N, respectively. Comparisons were performed using a Student’s two-tailed t-test with P values 0.05 identi ed as signi cant. Each experiment was repeated three times and error bars represent the SEM.

Fig. 3.

Demonstration of Emodin induced adaptive responses in SA-NH and FSa tumor cell lines. P values were determined by comparing the survival of cells following two 2 Gy doses with those exposed to two 2 Gy doses along with Emodin using a Student’s two-tailed t-test with values ≤0.05 identified as significant. Exposure to 50 μM Emodin induced a significant elevation in SA-NH (P=0.002) and reduction in FSa (P <0.001) cell survival. The presence of NAC during the Emodin treatment inhibited these effects in both SA-NH (P = 0.546) and FSa (P = 0.649). Each experiment was repeated three times and error bars represent the SEM.

Fig. 4.

Survivin protein in cytoplasmic and nuclear fractions measured by ELISA as a function of Emodin treatment protocol in SA-NH and FSa tumor cell lines. P values were determined by comparing the protein content in the unirradiated control cytoplasmic (C) and nuclear (N) fractions with their corresponding groups from the three treatment protocols: 2 Gy only (SA-NH, P = 0.407 and 0.045, C and N, respectively and FSa, P = 0.377 and 0.173, C and N, respectively; Emodin + 2 Gy ( SA-NH, P = 0.012 and 0.004, C and N, respectively; FSa, P = 0.186 and 0.002, C and N, respectively; and Emodin+ NAC +2 Gy (SA-NH, P = 0.827 and 0.192, C and N, respectively; FSa, P = 0.186 and 0.122, C and N, respectively. Comparisons were performed using a Student’s two-tailed t-test with P values ≤0.05 identi ed as signi cant. Each experiment was repeated three times and error bars represent the SEM.

Fig. 5.

The effect of p53 siRNA transfection on the expression of the survivin-associated adaptive response in SA-NH tumor cells. The cell survival of untransformed (UT) and mock transformed (MT) SA-NH control cells were contrasted to negative control (NC) and p53 siRNA transfected cells following two 2 Gy doses separated by 24 h and 5 mGy administered 15 min prior to each of two 2 Gy doses. P values were determined by comparing the surviving fractions of the two 2 Gy treatment with that of the 5 mGy+ 2 Gy exposures × 2 of nontransfected and p53 siRNA transfected SA-NH. 5 mGy induced a cytoprotective adaptive response in nontransfected cells (P < 0.001) and a sensitizing response in p53 siRNA transfected SA-NH cells (P = 0.003). Experiments were repeated three times and error bars represent the SEM.

Fig. 6.

CRM-1 and phosphorylated survivin protein levels measured by Western Blotting as a function of radiation treatment in SA-NH and FSa tumor cells. Representative blots from three experiments.

Fig. 7.

Tumor growth delay studies demonstrating the effects of the very low radiation dose induced adaptive responses in SA-NH and FSa tumors. Each tumor type is compared to their respective unirradiated control tumor growth curve. Irradiations, designated by (↓), were performed after tumors reached a volume of approximately 250 mm³ and tumor growth was monitored daily until they reached a volume of about 1000 mm³. Mean growth times and SEM were determined. Mean tumor volumes over time were compared among groups using repeated measures analysis of covariance (ANOVA) assuming a quadratic model and accounting for separate variances in each group. A post-hoc pairwise comparison among groups was performed using the Tukey-Kramer method.

Fig. 8.

Model of the redox sensitive, survivin-associated adaptive response as a function of very low radiation dose-induced translocation of the bi-functional inhibitor of apoptosis protein survivin in tumor cells differing in p53 mutational status.