A survivin-associated adaptive response in radiation therapy

Abstract

Adaptive responses can be induced in cells by very low doses of ionizing radiation resulting in an enhanced resistance to much larger exposures. The inhibitor of apoptosis protein, survivin, has been implicated in many adaptive responses to cellular stress. Computerized axial tomography used in image-guided radiotherapy to position and monitor tumor response uses very low radiation doses ranging from 0.5 to 100 mGy. We investigated the ability of these very low radiation doses administered along with two 2 Gy doses separated by 24 hours, a standard conventional radiotherapy dosing schedule, to initiate adaptive responses resulting in the elevation of radiation resistance in exposed cells. Human colon carcinoma (RKO36), mouse sarcoma (SA-NH), along with transformed mouse embryo fibroblasts, wild type or cells lacking functional tumor necrosis factor receptors 1 and 2 were used to assess their relative ability to express an adaptive response when grown either to confluence in vitro or as tumors in the flank of C57BL/6 mice. The survival of each of these cells was elevated from 5% to 20% (P ≤ 0.05) as compared to cells not receiving a 100 mGy or lesser dose. In addition, the cells exposed to 100 mGy exhibited elevations in survivin levels, reductions in apoptosis frequencies, and loss of an adaptive response if transfected with survivin siRNA. This survivin-mediated adaptive response has the potential for affecting outcomes if regularly induced throughout a course of image guided radiation therapy.

Figure 1

The percentage of surviving RKO36 human colon carcinoma cells grown to confluence as a function of the timing of very low radiation doses of 5, 10, 20 and 100 mGy, administered 30 min or 6 h following the first of two 2 Gy doses separated by 24 h, are presented. 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 various very low mGy doses using a two-tailed Student’s t test with values ≤ 0.05 identified as significant. Each experiment was repeated 3x and error bars represent the standard error of the mean (SEM).

Figure 2

Representative Western blots from 3x experiments describe changes in survivin protein levels in RKO36 cells over 24 h as a function of exposure to a single 2 Gy dose of ionizing radiation only (A), or a 2 Gy dose followed 30 min later by a 100 mGy exposure (B). Relative survivin levels were determined through densitometry measurements of survivin and corresponding α-Tubulin loading control band densities and normalized to unirradiated control values.

Figure 3

Cell survival as a function of the timing of a 100 mGy dose given prior to or following the first of two 2 Gy doses separated by 24 h is presented for: transformed mouse embryo fibroblast (MEF) wild type (WT) cells (A); transformed MEF cells lacking tumor necrosis factor receptors 1 and 2 (TNFR1⁻R2⁻) (B); human colon carcinoma RKO36 cells (C), and SA-NH mouse sarcoma cells (D). Western blots monitor the relative change in survivin band densities as a function of treatment as compared to cells exposed to a 2 Gy dose only. Times indicate when survivin analysis was performed following the 100 mGy dose and represent survivin levels at 24 h after the first 2 Gy dose. P values were determined by comparing the survival of cells following two 2 Gy doses with those also exposed to an additional 100 mGy dose using a two-tailed Student’s t test with values ≤ 0.05 identified as significant. Each experiment was repeated 3x and error bars represent the SEM.

Figure 4

Cell survival of MEF grown in the flanks of C57BL/6 mice as solid tumor growths, as a function of timing of a 100 mGy dose given 30 min or 3 h following the first of two 2 Gy doses separated by 24 h are presented for MEF WT (A) and MEF TNFR1⁻R2⁻ (B). Both the first 2 Gy and subsequent 100 mGy doses were delivered to MEF growing in C57BL/6 mice. Tumors were removed just prior to the second 2 Gy dose, made into single cell suspension, and irradiated under in vitro conditions and surviving fraction assessed using an in vitro colony forming assay. P values were determined by comparing the survival of cells following two 2 Gy doses with those also exposed to a 100 mGy dose delivered 30 min or 3 h following the first 2 Gy dose using a Student’s two-tailed t test with values ≤ 0.05 identified as significant. Experiments were repeated 2x and error bars represent the SEM.

Figure 5

The effects of transfection of RKO36 cells with negative control (NC) and survivin siRNA sequences, 5′-UAGCAAAAGGGACACUGCCtt-3′ (A, C) or 5′-UGUAGAGAUGCGGUGGUCCtt-3′ (B, D) on survival and apoptosis responses, respectively, to two 2 Gy doses or 2 Gy followed 30 min or 3 h later with an additional 100 mGy exposure and relative survivin protein levels in control cells as determined by Western blotting. Survival and apoptosis experiments were repeated 3x and P values were determined by comparing the survival of each non-transfected cohort of cells following either two 2 Gy doses only or two 2 Gy doses with an additional 100 mGy exposure with their respective survivin siRNA transfected cohorts using a Student’s two-tailed t test. Error bars represent the SEM.

Figure 6

Effects of transfection of MEF WT (A), MEF TNFR1⁻R2⁻ (B), and SA-NH cells (C) with negative control (NC) or survivin siRNA, 5′-UAGCAAAAGGGACACUGCCtt-3′, on respective cell survival after two 2 Gy doses or 2 Gy followed 30 min or 3 h later with an additional 100 mGy exposure. Survivin protein levels in control cells were determined by Western blotting. Survival experiments were repeated 3x and P values were determined by comparing the survival of each non-transfected cohort of cells following either two 2 Gy doses only or two 2 Gy doses with an additional 100 mGy exposure with their respective survivin siRNA transfected cohorts using a Student’s two-tailed t test. Error bars represent the SEM.

Figure 7

The percentage of apoptotic cells as determined by the TUNEL assay are plotted as a function of radiation treatment and transfection with survivin siRNA, 5′-UAGCAAAAGGGACACUGCCtt-3′, for MEF WT (A), MEF TNFR1⁻ R2⁻ (B), and SA-NH cells (C). Comparisons were made in each cell system between the percentage of apoptotic cells counted 24 h after the first 2 Gy dose in the non-transfected group and its negative control (NC) or survivin siRNA transfected counterpart. Apoptosis was significantly elevated only in cells transfected with survivin siRNA and exposed to 100 mGy (P ≤ 0.019) as compared to no effect in transfected cells only exposed to 2 Gy (P > 0.05). Each experiment was repeated 3x and error bars represent the SEM.