The Role of Radiation Quality in the Stimulation of Intercellular ...

RADIATION RESEARCH

176, 346–355 (2011)

0033-7587/11 $15.00 g 2011 by Radiation Research Society. All rights of reproduction in any form reserved. DOI: 10.1667/RR2509.1

The Role of Radiation Quality in the Stimulation of Intercellular Induction of Apoptosis in Transformed Cells at Very Low Doses Abdelrazek B. Abdelrazzak,a,b David L. Stevens,a Georg Bauer,c Peter O’Neilla and Mark A. Hilla,1 a

CRUK/MRC Gray Institute for Radiation Oncology & Biology, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, United Kingdom; b Physics Research Division, National Research Centre, Giza, Egypt; and c Department of Virology, Institute of Medical Microbiology and Hygiene, University of Freiburg, D-79104 Freiburg, Germany

particles, leading to a plateau in radiation-stimulated intercellular induction of apoptosis at higher doses. g 2011 by Radiation Research Society

Abdelrazzak, A. B., Stevens, D. L., Bauer, G., O’Neill, P. and Hill, M. A. The Role of Radiation Quality in the Stimulation of Intercellular Induction of Apoptosis in Transformed Cells at Very Low Doses. Radiat. Res. 176, 346–355 (2011).

INTRODUCTION

Tumorigenesis is a complex multistage process, with intercellular signaling from neighboring cells playing an important role in the progression of transformed cells to tumors (1). Intercellular signaling may either contribute to tumor formation (2) or cause selective elimination of transformed cells (3, 4). An important stage in this progression is the ability of a precancerous cell to escape natural anticancer signals imposed on it by its environment. Under normal conditions, cell growth within a tissue is tightly controlled by multiple intercellular signals that control proliferation and so inhibit cancer formation (5). Any external stimuli that affect this cellular signaling network may in turn have an effect on the risk of tumorigenesis. Therefore, cells do not act in isolation, and many studies have shown that after exposure to ionizing radiation, biological effects are observed not only in cells exposed to radiation but also in nonirradiated ‘‘neighboring’’ cells (6–15). Such nontargeted effects may lead to deviations from the linear no-threshold dose response for ionizing radiation based on risks measured at high doses of radiation in epidemiology studies (16). A major challenge remains characterizing the underlying mechanism for these phenomena. To address some of these issues, we have used a previously well-characterized model of intercellular induction of apoptosis (3, 17) to explore how intercellular signaling mechanisms are perturbed as a result of low doses of radiation. This model has been demonstrated in a number of cell lines, although the most detailed information has been obtained in the 208F rat fibroblast cell line and the 208Fsrc3-transformed derivative (4, 18, 19). The nontransformed 208F cells are able to selectively eliminate cocultured transformed 208Fsrc3 cells via cytokine and reactive oxygen/nitrogen species

An important stage in tumorigenesis is the ability of precancerous cells to escape natural anticancer signals. Apoptosis can be selectively induced in transformed cells by neighboring normal cells through cytokine and ROS/RNS signaling. The intercellular induction of apoptosis in transformed cells has previously been found to be enhanced after exposure of the normal cells to very low doses of both low- and high-LET ionizing radiation. Low-LET ultrasoft X rays with a range of irradiation masks were used to vary both the dose to the cells and the percentage of normal cells irradiated. The results obtained were compared with those after a-particle irradiation. The intercellular induction of apoptosis in nonirradiated src-transformed 208Fsrc3 cells observed after exposure of normal 208F cells to ultrasoft X rays was similar to that observed for c rays. Intercellular induction of apoptosis was stimulated by irradiation of greater than 1% of the nontransformed 208F cells and increased with the fraction of cells irradiated. A maximal response was observed when ,10–12% of the cells were irradiated, which gave a similar response to 100% irradiated cells. Between 1% and 10%, highLET a particles were more effective than low-LET ultrasoft X rays in stimulating intercellular induction of apoptosis for a given fraction of cells irradiated. Scavenger experiments show that the increase in intercellular induction of apoptosis results from NON and peroxidase signaling mediated by TGF-b. In the absence of radiation, intercellular induction of apoptosis was also stimulated by TGF-b treatment of the nontransformed 208F cells prior to coculture; however, no additional increase in intercellular induction of apoptosis was observed if these cells were also irradiated. These data suggest that the TGF-b-mediated ROS/ RNS production reaches a maximum at low doses or fluences of Note. The online version of this article (DOI: 10.1667/RR2509.1) contains supplementary information that is available to all authorized users. 1 Address for correspondence: Gray Institute for Radiation Oncology & Biology, University of Oxford, Old Road Campus Research Building, Oxford OX3 7DQ, UK; e-mail: mark.hill@rob. ox.ac.uk. 346

ROLE OF RADIATION QUALITY IN INTERCELLULAR APOPTOSIS SIGNALING

(ROS/RNS) signaling. Transformed cells may also induce apoptosis in the transformed cells via autocrine2 destruction (AD). The proposed mechanisms for the induction of apoptosis in transformed cells and the role of intercellular signaling have been discussed in detail elsewhere (3, 20). The transformed cells rather than the nontransformed cells are susceptible to apoptosis induction due to the superoxide produced by membranebound NADPH oxidase expressed constitutively in the transformed cells (20). Essentially, the transformed 208Fsrc3 cells secrete TGF-b into the medium, which can ultimately stimulate nontransformed cells to produce peroxidase (POD) and nitric oxide (NON). The NADPH oxidase-derived O2N2 surrounding the membranes of the transformed 208Fsrc3 cells leads to the production of hydrogen peroxide (H2O2). POD produced by the TGF-b-stimulated cells interacts with the H2O2 in the presence of chlorine ions to generate hypochlorous acid (HOCl). HOCl can subsequently interact with O2N2 surrounding the transformed 208Fsrc3 cells to form the highly reactive hydroxyl radical (NOH), which can ultimately induce apoptosis in the transformed cells. Likewise the secreted NON from stimulated cells interacts with the O2N2 located around the membranes of the transformed cells to produce peroxynitrite (ONOO2), which is highly reactive and can also induce apoptosis in the transformed 208Fsrc3 cells. We have previously shown (20) that irradiation of these nontransformed cells with low doses of either highlinear energy transfer (LET) a particles or low-LET c rays leads to stimulation of intercellular induction of apoptosis, resulting in an increase in the rate of apoptosis in the transformed cells. The use of scavengers and inhibitors confirmed the involvement of ROS/RNS signaling and the importance of transformed cell NADPH oxidase in the selectivity of the system. The use of TGF-b neutralizing antibody confirmed a role for the cytokine in the radiation-induced signaling. Doses of 2 mGy c rays or 0.29 mGy a particles are sufficient to produce an observable increase in apoptosis of transformed cells, with radiation-stimulated effects saturating at low doses (50 mGy for c rays and 25 mGy for a particles). The system may represent a natural anticancer mechanism that could potentially be stimulated by extremely low doses of ionizing radiation. The general shape of the dose response in the low-dose region was similar for a particles and c rays, although the variation in the energy deposition profiles within the cell population is very different. Essentially, for c-ray exposure over the dose range studied, all cells will be traversed by multiple electron tracks. As the dose to the cell population is reduced, there is a corresponding reduction in the number of electron tracks and therefore 2 In the autocrine signaling process, molecules act on the same cells that produce them.

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a reduction in the dose to each cell. Reducing the exposure of the cell population to a particles essentially reduces the fraction of cells irradiated, while the few that are traversed by a single a particle will receive a significant dose, similar to the dose to the cell for those traversed by single a particles at higher doses to the cell population. To study the interplay between dose delivered to individual cells and the fraction of the cell population irradiated, experiments were performed using low-LET ultrasoft X rays in conjunction with radiation masks. This enabled both the dose delivered in the irradiated cells and the percentage of cells irradiated to be varied independently. The aim of the study is to identify factors that may limit the radiation stimulation of intercellular induction of apoptosis, to understand how differences in radiation quality affect the kinetics of intercellular induction of apoptosis in the unirradiated transformed cells, and to explore the relative importance of the various intercellular signaling pathways using scavengers and inhibitors. MATERIALS AND METHODS Cell Lines and Cell Culture The 208F and v-src-transformed 208Fsrc3 rat fibroblast cell lines were used between passage number 18 and 30 (19). Cells were kept in Eagle’s MEM (Gibco Invitrogen, UK) supplemented with 5% FBS (Sigma), 2 mM L-glutamine and 50 mg/ml penicillin-streptomycin in a penicillin-streptomycin-glutamine solution (Gibco, UK) and incubated at 37uC in a 5% CO2/air gassed incubator. Cell Seeding and Coculture For coculture, 2 ml of 208Fsrc3 cells at a density of 5 3 104 cells/ml were seeded into each well of a six-well plate (Becton Dickinson Labware Europe) and 1 ml of 208F cells at a density of 1 3 105 cells/ml was seeded into each custom insert. The custom inserts consist of a 2.5mm-thick Hostaphan (polyethylene terephthalate; Hoechst, Weisbaden, Germany) base of glass ring (internal diameter of 20 mm, wall thickness 1.8 mm) designed to be located 1 mm above the base of a well from the six-well plate. These inserts contain three 1-mm-wide slits, 1 mm above the base, to allow medium-borne signaling between the two cell lines in the insert and well after coculture. These slits extend to ,55% of the circumference on the inner wall of the insert. After cell seeding, both cell populations were incubated independently to allow cells to attach to the bases of both the wells and the inserts. After 5 h, the 208F cells cultured in custom inserts were irradiated as described later, then immediately placed into the six-well plates containing 208Fsrc3 cells. After addition of 1 ml of medium to the well, the cells were incubated for 65 h. Determination of Apoptosis in Transformed 208Fsrc3 Cells Apoptosis in live 208Fsrc3 cells was measured quantitatively using well-established criteria (4, 17, 20) in which scoring is dependent on the morphological characteristics of the cells undergoing apoptosis, namely, nuclear condensation and fragmentation and membrane blebbing. Cellular imaging was carried out using a Nikon TS-100F (Nikon, UK) inverted phase-contrast microscope with the percentage of apoptotic cells determined from at least 200 cells per assay, with at least three samples taken for each point and samples scored blind.

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Inhibitors The HOCl scavenger taurine (TAU) (Sigma-Aldrich Company Ltd, UK) was used to inhibit the POD/HOCl pathway while the NON/ONOO2 pathway was inhibited using the peroxynitrite decomposition catalyst FeTPPS [4-sulfonatophenyl phorphyirinatoiron (III) chloride, Calbiochem UK]. These inhibitors were added either individually or in combination to the cell culture medium at the time of coculture to give final concentrations of 25 mM and 10 mM for taurine and FeTTPS, respectively. Taurine inhibits the PODmediated apoptosis through scavenging HOCl, thus preventing production of NOH, which induces apoptosis in the cells (3, 20). FeTPPS inhibits the NON-mediated apoptosis through scavenging ONOO2, formed on interaction of NON with O2N2 produced by the 208Fsrc3 cells. Pretreatment of 208F Cells with TGF-b The effect of pretreatment of 208F cells with TGF-b was studied by adding 10 ng/ml TGF-b (Sigma-Aldrich Company Ltd, UK) for 48 h prior to coculture with 208Fsrc3 cells. Just before coculture, the medium of 208F cells was replaced by fresh medium prior to either the sham or 500-mGy c irradiation. Irradiations 1. Low-LET ultrasoft X rays Ultrasoft X irradiations were performed using a cold-cathode discharge tube (3 kV, 3 mA) with a thin aluminum foil transmission target (1.27 mg cm22) to produce 1.49 keV AlK characteristic X rays (21). The custom insert was placed inside a brass ring dish, so the Hostaphan base of the insert, on which the 208F cells were grown, was in contact with the 0.9 mm Mylar [Dupont (UK) Ltd, Stevenage, UK] base on the bottom of the brass ring, through which the cells are irradiated. Irradiations were performed at room temperature with a surface dose rate of ,0.5 Gy/min to give incident surface doses in the range 0.5–500 mGy, with exposure time controlled using a fast Uniblitz shutter (Vincent Associates, NY) controlled using a programmable IMO timer (RS Components Ltd. Corby, UK). Due to the high attenuation of the ultrasoft X rays while traversing the cells, measurements of the cell monolayer thickness were performed to calculate the mean dose to the cell population. For a cell thickness, d, the mean dose (Dm) was calculated using Eq. (1). Dm ~Ds 1{ exp { ðm=rÞcell rcell d ðm=rÞcell rcell d ,ð1Þ where Ds is the dose on the incident surface of the cells, rcell is the density of the cell (assumed to be 1.08 g cm23), and (m/r) is the mass absorption coefficient of ultrasoft X rays in the cell (assumed to be 1319 cm2 g21) (21). A confocal laser-scanning microscope (BioRadLasersharp MRC600) was used to obtain cell thickness measurements on live attached cells as described previously (22). Fluorescein isothiocyanate (FITC) dextran (0.1%) of a high molecular weight was added to make the medium fluorescent and leave the cells unstained. Random fields were examined by recording a series of sections in 1-mm steps and the height of individual cells was determined by counting the number of steps from the base to the top of the cell. The resulting cell height distribution (mean cell height 6.8 ± 1.7 mm) was used to determine the average mean to surface dose ratio of 0.657. Characteristic AlK X rays are highly attenuated (half-value layer of 0.2 mm in Cu) and so are easily shielded. They interact via the photoelectric effect with negligible scatter due to the low fluorescence yield, and energy is deposited locally by short-range electrons (combined range of electron pair is ,70 nm). These properties make ultrasoft X rays an ideal candidate for partially shielding irradiations, as illustrated in Fig. 1. The use of patterned radiation masks positioned directly beneath the irradiated dishes enables the percentage of cells irradiated with ultrasoft X rays to be varied (23).

FIG. 1. Schematic of partial irradiation of a cell population with ultrasoft X rays using irradiation masks. Also shown are images of the transmitted X rays through the 50% and 1% transmission masks detected using HD810 Gafchromic film (ISP). These masks consisted of a ,50-mm copper disc with a square array of circular holes, at 3-mm intervals, with an appropriate diameter to produced the required transmission (1.0, 2.5, 4.9, 7.7, 9.6, 12.6 and 14.7%). The 17% transmission mask consisted of a square array of circular holes 0.54 mm in diameter at 1.17-mm intervals and the 50% transmission mask a square array of 0.94-mm-wide square holes with rounded corners repeated at 1.17-mm intervals. In addition to the patterned array of holes, irradiations were also performed by shielding half of the dish. 2. Low-LET c radiation 208F cells were irradiated in custom inserts supported in empty sixwell plates at room temperature on a 10-mm Perspex buildup sheet with 60Co c rays at the Medical Research Council, Harwell, UK as described previously (20), with all exposure times being ,5 min. 3. High-LET a-particle radiation 208F cells on custom inserts supported in 0.9-mm Mylar-based brass rings were irradiated with 3.0 MeV a particles (LET of 127 keV/mm) from a 238Pu source at room temperature. The details of the irradiator and associated dosimetry have been documented previously by Goodhead et al. (24).

RESULTS

Comparison of the Dose-Dependent Induction of Apoptosis by Ultrasoft X Rays and c Rays The increase in the level of apoptosis observed in transformed 208Fsrc3 cells cocultured for 65 h with irradiated nontransformed 208F cells compared to sham-irradiated controls after exposure to both c rays and ultrasoft X rays is shown in Fig. 2. The observed c-ray dose response and plateau level are similar to those reported previously by Portess et al (20). A similar dose response was observed for both ultrasoft X rays and c


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FIG. 2. Percentage apoptosis scored in nonirradiated transformed 208Fsrc3 cells after 65 h coculture with nontransformed 208F cells irradiated with either ultrasoft X rays (&) or c radiation (%) as a function of mean dose to the cell. Each data point is a mean value derived from three independent experiments, with the sham control (0 Gy) value (ultrasoft X rays: 27.4 ± 0.3%; c rays: 27.3 ± 0.2%) from experiments carried out in parallel subtracted from each data point. The error bars represent the standard deviation derived from three independent experiments.

rays, with the level of apoptosis increasing from ,0.5 mGy and reaching a plateau at .,100 mGy. Variation in the Level of Apoptosis as a Function of the Fraction of Cells Irradiated 1. Masked ultrasoft X irradiations The effect of varying the fraction of irradiated nontransformed 208F cells on the level of apoptosis in the nonirradiated transformed 208Fsrc3 cells after coculture for 65 h was investigated. No difference was observed in the level of apoptosis induced in 208Fsrc3 cells for the same dose of 100 mGy to irradiated 208F cells when either 100% of the cells were irradiated (45 ± 2.5%), 50% were irradiated by shielding half of the dish (43 ± 2.4%), or 50% were irradiated using a patterned array of holes (42 ± 2.7%). The errors presented represent the standard deviations from three independent experiments. All configurations resulted in a significant increase in apoptosis compared to unirradiated controls (29 ± 1.0%). The variation in the increase in apoptosis induced in nonirradiated transformed cells above the level observed in sham-irradiated controls was next investigated as a function of the fraction of nontransformed 208F cells exposed to either 66 or 330 mGy of ultrasoft X rays using a range of radiation masks (average dose to the whole cell population will be reduced as the percentage of irradiated cells decreases). Figure 3 shows that the radiation-induced stimulation of apoptosis in the transformed cells is independent of the percentage of nontransformed cells irradiated from ,12% up to 100%. Below 12%, the level of apoptosis decreases with minimal stimulation observed when only 1% of the 208F cells have been irradiated.

Alpha Particles To determine the variation in the level of apoptosis induced by high-LET a particles, it was necessary to determine the mean number of a-particle traversals for different doses. The mean cellular and nuclear area of 208F cells grown on the base of the custom inserts have previously (20) been measured as 746 mm2 and 176 mm2, respectively. These areas have been used to calculate the mean number of a-particle traversals, n, using Eqs. (2) and (3).

FIG. 3. Percentage apoptosis scored in nonirradiated transformed 208Fsrc3 cells after 65 h coculture with nontransformed 208F cells irradiated with 66 or 330 mGy (100 and 500 mGy surface doses) ultrasoft X rays or different doses of a particles as a function of the percentage of cells irradiated. Each data point represents the mean value from at least three different experiments carried out independently, with the sham control (0 Gy) value (330 mGy ultrasoft X rays: 27.3 ± 0.8%; 66 mGy ultrasoft X rays: 30 ± 3%; a particles: 29 ± 2%) from experiments carried out in parallel subtracted from each data point. Error bars represent the standard deviation from at least three independent experiments.

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FIG. 4. Percentage apoptosis scored in nonirradiated transformed 208Fsrc3 cells after 65 h of coculture with sham-irradiated or irradiated (33 or 330 mGy ultrasoft X rays) nontransformed 208F cells in the presence or absence of taurine (TAU), FeTPPS or both. Error bars represent the standard deviation from at least three independent experiments.

n~DA=0:16L,

ð2Þ

where D is the dose (Gy), L the LET (keV/mm) and A the nuclear or cellular area (mm2) and assuming a cell density of 1 g cm23. The particle distribution is Poisson across the dish. The fraction of cells traversed, f, is given by Eq. (3). f ~1{ expð{nÞ:

ð3Þ

Figure 3 shows the dose-dependence for the induction of apoptosis in nontransformed 208Fsrc3 cells after coculturing with a-particle-irradiated nontransformed 208F cells as a function of the percentage of cells irradiated. A dose of 2.5 mGy to the cell population results in essentially full stimulation of apoptosis and corresponds to ,9.6% of cells and 2.3% of nuclei being traversed. It should be noted that varying the dose varies the fraction of cells traversed, but those cells traversed by a single a particle receive on average ,26 mGy. The results show that for a given fraction of cells irradiated in the range 1– ,10%, high-LET a particles are more effective at stimulating apoptosis in the cocultured, unirradiated transformed cells than low-LET ultrasoft X rays, although the maximum plateau level of apoptosis at 65 h is similar for both types of radiation. Relative Importance of Signaling Pathways in Intercellular Induction of Apoptosis after Irradiation with Ultrasoft X Rays 1. The effect of the ultrasoft X-ray dose Figure 4 shows the reduction in the percentage of 208Fsrc3 cells undergoing apoptosis after coculture for 65h with sham-irradiated and X-irradiated (33 and 330 mGy) nontransformed 208F cells in the presence of taurine to inhibit the POD/HOCl pathway, FeTPPS to inhibit the NON/ONOO2 pathway or both inhibitors combined. The two radiation doses were chosen to represent the stimulation of intercellular induction of

apoptosis at the plateau level (dose-independent level) and a dose where the stimulation of apoptosis is approximately half its maximal level. At both doses, taurine and FeTPPS when added independently lead to a reduction in the level of apoptosis, implying that both signaling pathways are involved in the induction of apoptosis. When both inhibitors were used in combination, a further reduction in the level of apoptosis was observed. At all doses, though, the levels of apoptosis were higher than the corresponding levels seen for the sham irradiation. 2. The effect of the a-particle dose To examine the effect of the percentage of cells irradiated on the signaling pathways involved in intercellular induction of apoptosis, the cells were exposed to either 1.43 mGy or 460 mGy of a particles. These doses correspond to either 5.1% or 100%, respectively, of the cell population being traversed by at least one a particle. Figure 5 shows the reduction in the percentage of cells undergoing apoptosis in 208Fsrc3 cells when cocultured for 65 h with sham-irradiated or a-particle-irradiated nontransformed 208F cells in the presence or absence of taurine, FeTPPs or taurine/ FeTPPS. Again, at both doses, taurine and FeTPPS, when they were added independently, led to a reduction in the level of apoptosis, with an additional reduction observed when used together. At both doses, the level of apoptosis approached that of the sham-irradiated cells but only when the inhibitors were used in combination. The Effect of TGF-b Pretreatment of 208F Cells on Apoptosis Induced in 208Fsrc3 Cells Figure 6 shows that pretreatment of 208F cells with TGF-b prior to coculture results in significant stimulation in apoptosis of 208Fsrc3 cells in sham-irradiated samples compared to that observed with no pretreat-


FIG. 5. Percentage apoptosis scored in nonirradiated transformed 208Fsrc3 cells after 65 h of coculture with sham-irradiated or 1.43 or 460 mGy a-particle-irradiated nontransformed 208F cells in the presence or absence of taurine (TAU), FeTPPS or both. Error bars represent the standard deviation from at least three independent experiments.

ment of the 208F cells. The increase in levels of apoptosis is the same as observed after irradiation of the 208F cells with 500 mGy c radiation. If the 208F cells were pretreated with TGF-b, subsequent irradiation of these cells did not lead to additional stimulation of apoptosis in the transformed 208Fsrc3 cells compared to sham-irradiated pretreated cells. Addition of the HOCl scavenger taurine (to block POD/HOCl pathway) to the TGF-b-pretreated cells, either irradiated or sham-irradiated, reduced the level of apoptosis in 208Fsrc3 cells to that observed for shamirradiated controls with no pretreatment (,27%). A similar reduction was observed with TGF-b-pretreated 208F cells, either sham- or 500 mGy c-irradiated after the addition of FeTPPS (to block NON/ONOO2 pathway). DISCUSSION

The exposure of nontransformed 208F cells to ionizing radiation leads to an increase in the levels of apoptosis in cocultured nonirradiated transformed 208Fsrc3 cells through intercellular induction of apoptosis. In this paper, the levels of apoptosis stimulated by radiation after 65 h of coculture are reported and are representative of a single time in a continuous kinetic process. The level of apoptosis continues to increase as the cells remain in coculture, and eventually the vast majority of transformed cells are removed by intercellular induction of apoptosis. The kinetics is dependent on the relative cell densities of the two cell lines. Ionizing radiation stimulates the signaling involved in intercellular induction of apoptosis and therefore increases the rate at which transformed cells are removed from coculture, with the onset of apoptosis occurring at earlier times (20). An example of the kinetics for the induction of apoptosis in 208Fsrc3 cells when culture in

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FIG. 6. The effect of apoptosis induced in transformed 208Fsrc3 cells after pretreatment of nontransformed 208F cells with 10 ng/ml TGF-b for 48 h, prior to coculture. Experiments were done in the presence or absence of either 25 mM of HOCl scavenger taurine or 10 mM of peroxinitrite decomposition catalyst FeTPPS to investigate the pathways involved in intercellular induction of apoptosis signaling in the presence or absence of 500 mGy c radiation. The error bars represent the standard deviation derived from three independent experiments.

isolation or coculture with 208F cells (sham-irradiated or irradiated with 0.5 Gy c rays) from a related study is shown in Supplementary Fig. 1 (http://dx.doi.org/10. 1667/RR2509.1.S1). A very similar response was seen for the induction of apoptosis in transformed cells after irradiation of nontransformed cells with either c radiation or ultrasoft X rays (Fig. 2). This similarity supports the use of ultrasoft X rays to investigate the effects of low-LET radiation with the added advantage that they can be used in combination with irradiation masks, enabling a known fraction of cells to be irradiated. The low-energy electrons produced by ultrasoft X rays are very similar to the low-energy secondary electrons (0.1–5.0 keV) produced by c rays that contribute ,30% of the absorbed dose and show all the characteristic features of other low-LET radiations such as c rays (25). Exposure to 60Co c rays corresponds to approximately 12,000 electron tracks per Gy interacting with a cell (assumed to have a mean cross-sectional area of 746 mm2 and height of 5 mm) compared to approximately 17,000 electron tracks per Gy for AlK ultrasoft X rays. Therefore, for both c rays and ultrasoft X rays, as the dose to the irradiated cell population is reduced to 1 mGy, the dose to all individual cells is correspondingly reduced, with all cells being irradiated. These results show that the amount of energy deposited within a cell (dose to individual cell) is important in stimulating intercellular induction of apoptosis in the 208Fsrc3 cells above the control level. This may be due either to a minimum amount of energy being required to be deposited within a ‘‘target’’ volume (or volumes) or to a reduced frequency of hitting the ‘‘targets’’ within the cell population. The response saturates at a low dose (,200 mGy) above which increasing the dose further

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TABLE 1 Percentage Apoptosis in Nonirradiated 208Fsrc3 Cells Cocultured with 208F Cells that were either Sham-Irradiated or Irradiated with 33 mGy or 330 mGy Ultrasoft X Rays due to either POD/ HOCl or NON/ONOO2 Pathway (inhibited using taurine and FeTPPS, respectively)

TABLE 2 Percentage Apoptosis in Nonirradiated 208Fsrc3 Cells Cocultured with 208F Cells that were either Sham-Irradiated or Irradiated with 1.43 mGy or 460 mGy a Particles due to either POD/HOCl or NON/ONOO2 Pathway (inhibited using taurine and FeTPPS, respectively)

Pathway

Sham control

33 mGy

330 mGy

Pathway

Sham control

1.43 mGy

460 mGy

POD/HOCl NON/ONOO2

8.9% (±0.7) 9.0% (±0.6)

5.9% (±1.3) 13.5% (±0.9)

16% (±5) 21% (±11)

POD/HOCl NON/ONOO2

7.0% (±0.8) 11% (±3)

7.5% (±2.6) 13.3% (±2.0)

16.4% (±0.3) 19.6% (±1.0)

Note. All percentages are ±SD.

does not increase the level of response. This is consistent with previously observed nontargeted effects that have been shown to saturate for many end points at very low doses (26). The saturation observed in intercellular induction of apoptosis at higher doses/percentage cells irradiated for all types of radiation may be due to a number of reasons, for instance, a negative feedback loop in TGF-b signaling pathway (27), saturation of TGF-b receptors in the nontransformed cells, or saturation of signaling molecule release, such as POD and NON. No difference was seen in the response when two different masks were used to irradiate 50% of the cell population but with very different patterns and average distances between irradiated and unirradiated cells. This implies that the intercellular signals (TGF-b signals) between irradiated and nonirradiated cells that stimulate the nonirradiated transformed 208Fsrc3 cells to undergo intercellular induction of apoptosis can travel many millimeters within this model system. The results presented in Fig. 3 show the importance of the percentage of cells irradiated as well as the energy deposited (related to radiation dose to the exposed cell) in stimulating apoptosis in transformed cells. The overall shapes of the curves for a particles and ultrasoft X rays as a function of cell population irradiated are similar, with maximal stimulation observed from 100% down to ,10–12% of the population irradiated. Below this, the frequency of apoptosis decreased, with minimal stimulation observed when only 1% of the population was irradiated. However, when between 1% and 10% of the cell population is irradiated, high-LET a particles are more effective than low-LET ultrasoft X rays for a given fraction of cells irradiated even though the mean dose to the cells traversed by an a particle is of the order of 26 mGy compared to ,330 mGy for ultrasoft X rays. It is inferred that the difference in the radiation track structure between the different radiation qualities is also important in influencing the kinetics of intercellular induction of apoptosis. Therefore, the pattern of irradiated cells across the cell population is not critical, but the pattern of energy deposition within the cell is important in influencing the kinetics of intercellular induction of apoptosis. While low-LET radiation deposits

Note. All percentages are ±SD.

its energy through multiple sparsely ionizing electron tracks scattered randomly through the cell, a particles deposit their energy along individual densely ionizing narrow tracks [,90% of energy deposition occurring within 10 nm of the track (28)], resulting in a very nonhomogeneous distribution of correlated damage through the cell. In addition, these densely ionizing aparticle tracks produce significantly more clustering of ionization and excitation events within nanometer-sized volumes compared with low-LET radiation. We have previously shown that in the absence of nontransformed cells, the induction of apoptosis in transformed 208Fsrc3 cells occurs via AD and is dominated by the POD/HOCl signaling pathway at high cell densities (29) with little or no contribution from the NON/ONOO2 pathway unless the transformed cells have been irradiated (data not shown). However, when transformed cells are cocultured with nontransformed 208F cells, the level of apoptosis increases as a result of intercellular signaling between the nontransformed and transformed cells predominantly due to the production of NON, which initiates the NON/ONOO2 pathway (29). The effects of inhibitors on the different signaling pathways (Figs. 4 and 5) in sham-irradiated samples are consistent with these previous results with the overall level of apoptosis in the transformed cell population (,25%) comprising of ,10% from AD (as a result of the POD/HOCl pathway), ,10% as a result of intercellular signaling between the nontransformed and transformed cells (as a result of the NON/ONOO2 pathway), leaving a background level of apoptosis of ,5%. Although adding the two scavengers should reduce the percentage of apoptosis scored in transformed 208Fsrc3 cells to this basal level, the increased background level of apoptosis observed might be due to another pathway that is not elucidated here. The level of apoptosis observed in the nonirradiated transformed cells increases after irradiation of the nontransformed cells. Based on the data for inhibitors of the different signaling pathways (Figs. 4 and 5), the contributions of the various pathways to the observed levels of apoptosis are detailed in Tables 1 and 2 and calculated from the reduction in apoptosis resulting from the addition of the inhibitors. The results for the


sham-irradiated controls are consistent with the previously reported contribution of ,10% from AD as a result of the POD/HOCl pathway and ,10% as a result of intercellular signaling between nontransformed and transformed cells (29). Table 1 shows that irradiating nontransformed cells with 33 mGy ultrasoft X rays (50 mGy surface dose) did not result in any increase in the contribution of POD/HOCl pathway in signaling, although the data indicate an increase in the contribution of the NON/ONOO2 pathway, marked by an increase in the level of apoptosis scored in the transformed cells from ,9.0% to ,13.5%. After irradiation of the nontransformed cells with 330 mGy ultrasoft X rays (500 mGy surface dose), the data suggest an increase in the contribution of both the POD/HOCl and NON/ONOO2 signaling pathways. These changes in the contributions of the signaling pathways would then imply that a dose of 33 mGy ultrasoft X rays is not sufficient to stimulate the POD/HOCl pathway, whereas it is sufficient to stimulate the NON/ONOO2 pathway (see Fig. 7). When the nontransformed cells were irradiated with a dose of either 1.43 or 460 mGy of a particles, a similar pattern of response for the contributions of the signaling pathways was seen. A dose of 1.43 mGy was not sufficient to stimulate the POD/HOCl pathway, whereas there is an indication that the NON/ONOO2 pathway is stimulated. At 460 mGy, the response is also similar to the response seen with 330 mGy ultrasoft X rays, with this dose now sufficient to stimulate both pathways (Table 2). As mentioned previously, the POD/HOCl pathway contributes not only to intercellular induction of apoptosis but also to AD (,10%). Previously, Bauer and colleagues identified the inhibitory effect of TGF-b-treated normal cells against transformed cells (30–33). Further investigations showed that TGF-b plays three distinct roles in intercellular induction of apoptosis. First, TGF-b triggers nontransformed cells to release a short-lived apoptosis-inducing factor. Second, TGF-b is involved in the maintenance of the transformed state, which is required for expression of sensitivity to intercellular induction of apoptosis. Third, TGF-b sensitizes transformed cells through down-modulating their endogenous survival factors (34). Recently it was shown that TGF-b triggers the release of POD from TGF-b-treated as well as c-irradiated nontransformed and transformed cells (35). Figure 6 shows that TGF-b pretreatment of 208F cells prior to coculture leads to a significant stimulation in apoptosis observed in 208Fsrc3 cells to the same level observed after irradiation with 500 mGy in c-irradiated 208F cells (,47%, which is ,20% above the sham control in the TGF-b-untreated samples). In addition, no additional increase in apoptosis was observed when pretreated 208F cells were also irradiated.

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FIG. 7. Proposed mechanisms of enhancement of apoptosis in transformed 208Fsrc3 cells by either very low doses of radiation to nontransformed 208F cells (panel A) or higher doses (panel B). The targeted nontransformed 208F cells increase the amount of active TGF-b in the medium either by increasing the activation of the latent TGF-b present in the medium or by secreting more latent TGF-b, which is subsequently activated. TGF-b acts in an autocrine fashion to stimulate the 208F cells to produce NON, which in turn leads to ROS/RNS signaling, culminating in transformed cell apoptosis. In addition, transformed cells may also induce apoptosis in transformed cells via autocrine destruction (AD) after the production of POD. In the case where 208F cells are irradiated with a very low dose (e.g. 33 mGy ultrasoft X rays or 1.43 mGy a particles) (panel A), only the NON/ONOO2 pathway is enhanced. When 208F cells are irradiated with a relatively higher dose (e.g. 330 mGy ultrasoft X rays or 460 mGy a particles) (panel B), the NON/ONOO2 pathway is enhanced and the POD/HOCl pathway is stimulated. Modified from ref. (20).

Both taurine (used to block the POD/HOCl pathway) and FeTPPS (used to block the NON/ONOO2 pathway) resulted in a similar reduction in apoptosis in transformed 208Fsrc3 cells after coculture with TGF-b pretreated 208F cells that were either sham-irradiated or c-irradiated with 500 mGy. Both the POD/HOCl and NON/ONOO2 pathways are involved with signaling between the TGF-b-pretreated nontransformed 208F cells and the transformed 208Fsrc3 cells, with the POD/ HOCl also contributing to apoptosis via AD. The reduction of ,20% is similar to that observed with

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taurine and FeTPPS when 208F cells not treated with TGF-b were irradiated (at doses in the plateau region) with either a particles [(20) and Fig. 6], ultrasoft X rays (Fig. 5) or c rays [(20) and data not shown], where a further reduction was observed when the inhibitors were used in combination. The data presented indicate that pretreatment with TGF-b acts in an equivalent manner to irradiation of the cells in stimulating the production of POD and NON by nontransformed cells. Furthermore, it suggests that rather than irradiation of the 208F cells leading directly to the production of POD and NON, their increase may ultimately result from an increase in TGFb after irradiation, which subsequently stimulates the 208F cells. This also fits into the idea that when only a fraction of the 208F cells are irradiated then a full response is observed due to stimulation of the unirradiated cells with TGF-b. Summary Irradiation of nontransformed 208F cells with very low doses of either high-LET a particles, low-LET c rays or low-LET ultrasoft X rays leads to an increase in the rate of apoptosis in the nonirradiated transformed 208Fsrc3 cells above sham-irradiated controls. This radiation-induced stimulation requires sufficient dose to be deposited in irradiated cells and a sufficient fraction of cells to be irradiated. Treatment of the 208F cells with TGF-b prior to coculture produced a similar increase in the rates of apoptosis to that observed with radiation. However, no additional increase in apoptosis was observed after irradiation of TGF-b-treated cells compared to sham-irradiated TGF-b-treated controls. Although the pattern of irradiated cells across the cell population is not critical, when a low fraction of cells were irradiated (between 1% and 10%), a particles were observed to be more effective than ultrasoft X rays at stimulating the apoptotic response for a given fraction of cells irradiated even though the energy in the irradiated cells (dose) was significantly lower. This increase in effectiveness of the a particles is likely to be due to their track structure. Not only do these a particles produce greater clustering of damage on the nanometer scale, due to their high LET, but also correlation of damage along its path through the cell results in a very nonhomogeneous dose within the cell. In the absence of radiation (or pretreatment with TGF-b), when transformed 208Fsrc3 cells are cocultured with nontransformed 208F cells, the NON/ONOO2 pathway dominates the intercellular signaling between the two cell lines, with little or no contribution of the POD/HOCl pathway. However, the POD/HOCl pathway does contribute to apoptosis of the transformed cells through autocrine destruction (signaling between the transformed cells) with little or no contribution of the NON/ONOO2 pathway. Irradiation of the nontransformed

cells or pretreatment with TGF-b leads to an increase in apoptosis observed in the nonirradiated transformed cells as a result of an enhancement of both the NON/ONOO2 and the POD/HOCl pathway, until the plateau level is reached. The data suggest that at low dose, or low fraction of cells irradiated, the NON/ONOO2 pathway may be preferentially stimulated over the POD/HOCl pathway. The data also suggest that radiation stimulation is the result of an increase in TGF-b production (or activation) by the irradiated cells. ACKNOWLEDGMENTS This work was supported by The National Research Centre, Egypt, the Medical Research Council and Cancer Research UK. We are grateful for intellectual support by the COST consortium ChemBioRadical (COST Action CM0603). Received: November 19, 2010; accepted: April 29, 2011; published online: June 10, 2011

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