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Radiation and chemotherapy bystander effects induce early genomic instability events: Telomere shortening and bridge formation coupled with mitochondrial dysfunction
Mutation Research 669 (2009) 131–138
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Radiation and chemotherapy bystander effects induce early genomic instability events: Telomere shortening and bridge formation coupled with mitochondrial dysfunction Sheeona Gorman a , Miriam Tosetto a , Fiona Lyng b , Orla Howe b , Kieran Sheahan a , Diarmuid O’Donoghue a , John Hyland a , Hugh Mulcahy a , Jacintha O’Sullivan a,∗ a b
Centre for Colorectal Disease, St. Vincent’s University Hospital, Elm Park, Dublin 4, Ireland Radiation & Environmental Science Centre, Dublin Institute of Technology and St. Luke’s Hospital, Dublin, Ireland
a r t i c l e
i n f o
Article history: Received 20 November 2008 Received in revised form 3 June 2009 Accepted 9 June 2009 Available online 18 June 2009 Keywords: Radiation bystander Mitochondria Nuclear instability
a b s t r a c t The bridge breakage fusion cycle is a chromosomal instability mechanism responsible for genomic changes. Radiation bystander effects induce genomic instability; however, the mechanism driving this instability is unknown. We examined if radiation and chemotherapy bystander effects induce early genomic instability events such as telomere shortening and bridge formation using a human colon cancer explant model. We assessed telomere lengths, bridge formations, mitochondrial membrane potential and levels of reactive oxygen species in bystander cells exposed to medium from irradiated and chemotherapytreated explant tissues. Bystander cells exposed to media from 2 Gy, 5 Gy, FOLFOX treated tumor and matching normal tissue showed a significant reduction in telomere lengths (all p values <0.018) and an increase in bridge formations (all p values <0.017) compared to bystander cells treated with media from unirradiated tissue (0 Gy) at 24 h. There was no significant difference between 2 Gy and 5 Gy treatments, or between effects elicited by tumor versus matched normal tissue. Bystander cells exposed to media from 2 Gy irradiated tumor tissue showed significant depolarisation of the mitochondrial membrane potential (p = 0.012) and an increase in reactive oxygen species levels. We also used bystander cells overexpressing a mitochondrial antioxidant manganese superoxide dismutase (MnSOD) to examine if this antioxidant could rescue the mitochondrial changes and subsequently influence nuclear instability events. In MnSOD cells, ROS levels were reduced (p = 0.02) and mitochondrial membrane potential increased (p = 0.04). These events were coupled with a decrease in percentage of cells with anaphase bridges and a decrease in the number of cells undergoing telomere length shortening (p values 0.01 and 0.028 respectively). We demonstrate that radiation and chemotherapy bystander responses induce early genomic instability coupled with defects in mitochondrial function. Restoring mitochondrial function through overexpression of MnSOD significantly rescues nuclear instability events; anaphase bridges and telomere length shortening. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The radiation induced bystander effect is a biological phenomenon whereby cells not directly exposed to treatment exhibit cellular responses similar to those of directly irradiated cells [1,2]. Radiation bystander effects can lead to cell death [3,4], mitochondrial alterations [5,6], DNA damage [7,8] and enhanced mutagenesis [9,10], ultimately leading to oncogenic transformation [11]. Studies have examined whether chemotherapy treatment also induces a bystander effect which includes apoptosis [12], an increase in
∗ Corresponding author. Tel.: +353 1 2213464; fax: +353 1 2838123. E-mail address: [email protected] (J. O’Sullivan). 0027-5107/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2009.06.003
reactive oxygen species (ROS) [13] and cell differentiation [14]. The mechanisms driving radiation and chemotherapy bystander effects are unknown, but soluble signalling factors [15,16] and cellular communication (i.e. via gap junctions and/or release of molecular messengers into the extracellular environment) may play a pivotal role [17]. ROS have been shown to be involved in the induction of sister chromatid exchanges [15] and stress inducible signalling pathways [18] and micronuclei formation in bystander cells [19]. An important biological endpoint observed in bystander cells is genomic instability [20], however the cellular events driving this instability is unknown. Telomere shortening and bridge formations affect the integrity of chromosomes [21]. When telomeres become critically short, they can fuse together and during cell division, they can form a bridge [22] which can break, resulting in daughter cells
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with altered genomic material [21]. We have previously shown that these events are activated very early in disease progression [23]. The majority of bystander work has been performed in cell lines [18,24,25] and limited studies have been performed in vivo [19,26]. Using a human explant model, the aim of this study was to investigate if radiation and chemotherapy bystander effects induce telomere length shortening and bridge formations and if these events were coupled with mitochondrial dysfunction. Additionally, we assessed if mitochondrial function was restored, did this influence nuclear instability events: anaphase bridges and telomere length shortening. 2. Materials and methods 2.1. Ex vivo explant culture Resected tumor and matching normal adjacent mucosa was obtained from eight patients from the Centre for Colorectal Disease’s explant tissue bio-bank in St. Vincent’s University Hospital, Dublin (five male, three female, median age 69). Explant tissue was cut into 12 equal-sized pieces of approximately 5 mm3 allowing each treatment (described below) to be performed in duplicate. Tumor and matched normal explant tissue were cultured in 5 ml RPMI 1640 containing 100 U/ml Penicillin, 100 g/ml Streptomycin, 4 g/ml Fungizone and supplemented with 20% foetal bovine serum for 24 h prior to treatment in 6-well plates.
2.2. Irradiation and chemotherapy treatments There were six treatment arms to this study performed for both the tumor and matched normal tissue: (1) unirradiated controls (0 Gy), (2) 2 Gy treatment, (3) 5 Gy treatment, (4) FOLFOX chemotherapy (10 M 5-fluoruracil, 5 M oxaliplatin and 2.5 g/ml folinic acid), (5) FOLFOX + 2 Gy and (6) FOLFOX + 5 Gy. The 2 Gy and 5 Gy doses were administered at room temperature using a cobalt-60 teletherapy unit in St. Luke’s Hospital, Dublin, delivering approximately 0.8 Gy/min. The tissue was treated with the above treatments for 24 h at 37◦ C.
2.3. Media transfer Following 24 h treatment, media from tumor and matched normal tissue was removed from the untreated and treated explant tissue and filtered through a 0.2-m filter (Nalgene, Rochester, NY) to remove any floating cells and cellular debris. This ‘conditioned media’ was incubated on bystander SW480 (American Type Culture Collection) cells for 24 h at 37◦ C cultured in RPMI 1640 medium and supplemented with 10% foetal bovine serum and 50 U/ml Penicillin, 50 g/ml Streptomycin, 4 g/ml Fungizone in an atmosphere of 5% CO2 . Following 24 h incubation, these bystander cells were processed for assays outlined below.
2.4. Telomere length analysis Bystander cells were seeded in chamber slides (BD Falcon) at a density of 2000 cells/well and incubated conditioned media from treated (2 Gy, 5 Gy and FOLFOX alone and in combination) and untreated tumor tissue for 24 h. Following incubation, cells were fixed in methacarn buffer (3:1 methanol:acetic acid) for 8 min at room temperature and incubated in a graded ethanol series of 50%, 70% and 100% ethanol. Slides were incubated in 10 mM sodium citrate for 8 min at 88 ◦ C, rinsed in PBS, dipped through the above ethanol series and allowed to air dry. Cells were incubated with 100 M PNA TAMRA (Tetra methylu-6-Carboxyrhodamine) probe (Applied Biosytems) and hybridised in the dark overnight at room temperature. Slides were washed in 70% Formamide buffer four times for 15 min each, followed by PBS/0.05% Tween 20 washes. Cell nuclei were counterstained with TOTO-3 DNA dye (Molecular Probes) for 5 min at room temperature, briefly washed in PBS, dipped through the ethanol series and air-dried. Slides were mounted with Vectashield (Vector Laboratories). Images were obtained on a Zeiss Axiovert confocal microscope using LSM software. Telomere length analyses was performed using image analysis algorithms [27] and the mean level of telomere fluorescence was proportional to mean telomere length for each case.
2.5. Bridge assessment Bystander cells were seeded in chamber slides at a density of 1000 cells/well and incubated with conditioned media for 24 h at 37◦ C. Slides were washed with PBS, fixed in Carnoys solution for 8 min at room temperature, dehydrated through a graded ethanol series of 50%, 70% and 100% ethanol and air-dried for 10 min. Slides were stained with 1:3 Mayer’s Haematoxylin, washed in water and put through the above graded alcohol series and fixed in xylene. The number of bridges were scored and expressed as a percentage of total cell number.
2.6. Mitochondrial membrane potential Bystander cells were seeded on glass cover slips (100,000 cells) and incubated with conditioned media from treated 2 Gy treated and untreated tumor tissue for 6 h. Conditioned media was removed and cells washed twice with a buffer containing 130 mM NaCl, 5 mM KCl, 1 mM Na2 HPO4 , 1 mM CaCl2 , 1 mM MgCl2 and 25 mM Hepes (pH 7.4). Cells were loaded with 5 M rhodamine 123 (Sigma) for 30 min in the buffer at 37 ◦ C. Cells were washed three times with the above buffer and then analysed using a confocal microscope (Zeiss LSM 510 META). Rhodamine 123 was excited at 488 nm, and fluorescence emission at 525 nm was recorded. Mean fluorescence values from ten random fields for each condition were obtained using Image J software. 2.7. Measurement of reactive oxygen species Bystander cells were seeded on glass cover slips (100,000 cells) and incubated with conditioned media from 2 Gy treated and untreated tumor tissue for 6 h for the reasons stated above. Conditioned media was removed and cells washed twice with a buffer containing 130 mM NaCl, 5 mM KCl, 1 mM Na2 HPO4 , 1 mM CaCl2 , 1 mM MgCl2 and 25 mM Hepes (pH 7.4). Cells were loaded with 5 M 2,7 dichlorofluorescein diacetate (Sigma) for 30 min in the buffer at 37 ◦ C. Cells were washed three times with the above buffer and then analysed using a confocal microscope (Zeiss LSM 510 META). 2,7 dichlorofluorescein diacetate was excited at 488 nm, and fluorescence emission at 525 nm was recorded. Mean fluorescence values from ten random fields for each condition were obtained using Image J software. 2.8. Generation of a manganese superoxide dismutase (MnSOD) bystander cell line In addition to the culturing of parental SW480 cell lines, another cell line over expressing MnSOD was prepared. MnSOD cDNA in a mammalian expressing vector (pcDNA3.1) was stably transfected into SW480 colorectal cells. Briefly, cells were seeded at 2 × 106 cells/well in a 6-well plate 24 h prior to transfection with MnSOD cDNA and the empty vector DNA. Transfection was carried out following the manufacturer’s protocol (LipofectamineTM 2000 reagent, Invitrogen). Briefly, 35 ng of DNA was diluted in serum free media containing 20 l of lipofectamineTM 2000 reagent and incubated for 20 min at room temperature. This DNA/lipid mixture were added to cells in serum free media and incubated for 16 h. Transfection medium was replaced with fresh medium supplemented with 700 g/ml G418 (Geneticin, Invitrogen) to select for MnSOD overexpressing stably transfected cells for a period of 14 days. Overexpression of MnSOD was confirmed by Western blot analysis. Actin was used as the internal control. Densiometry was performed and data were presented as MnSOD levels/-actin levels in the same samples. 2.9. Statistical analysis Data are presented as medians and interquartile ranges. Data were assessed using Wilcoxon’s signed rank test. All p values are two-sided and p-values less than 0.05 were considered statistically significant in all analyses.
3. Results 3.1. Telomere lengths in bystander cells Fig. 1A and B shows representative images of telomere staining in bystander cells incubated with conditioned media from untreated, 0 Gy (A) and 2 Gy (B) treated tumor explant tissue. Lower telomere fluorescence intensity is observed in bystander cells exposed to conditioned media from 2 Gy treated explant tissue (B). Conditioned media from irradiated tumor (Fig. 1C) and normal (Fig. 1D) tissue induced significant telomere shortening in bystander cells at 24 h compared to bystander cells incubated with conditioned media from untreated tumor and matched normal tissue (p values ≤0.012 and 0.018 respectively). There was no significant difference in telomere length shortening between 2 Gy and 5 Gy treated conditions or between effects induced by the tumor versus normal treated tissue. Therefore, 2 Gy conditioned media is adequate to elicit a maximum bystander response. Table 1 shows telomere shortening induced by a chemotherapy bystander effect. Conditioned media from FOLFOX treated tumor and normal tissue induced telomere shortening in bystander cells compared to control (p ≤ 0.012 and 0.018 respectively). Combination of radio and chemotherapy treatment did not have an additive effect compared to single treatments.
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Fig. 1. Telomere length assessment using QFISH in bystander cells. Representative images of bystander cells following incubation with conditioned media from control (0 Gy) (A) and 2 Gy (B) treated tumor explant tissue. DNA is stained with TOTO-blue dye and telomeres are hybridised with telomere-specific PNA probe (red). Images were taken by confocal microscopy and analysed using image analysis algorithims. Conditioned media from irradiated tumor (C) and matched normal (D) explant tissue induced significant telomere shortening in bystander cells compared to cells incubated with conditioned media from unirradiated tumor and matched normal tissue.
3.2. Bridge formation in bystander cells Fig. 2A and B shows representative images of bridges in bystander cells. Conditioned media from irradiated tumor (Fig. 2C) and normal (Fig. 2D) explant tissue induced significantly higher level of chromatin bridges in bystander cells (p = 0.012 and ≤0.018 respectively). Bridge formation was also significantly increased in bystander cells incubated with conditioned media from FOLFOX treated tumor and normal tissue (p ≤ 0.012), however, as seen with telomere lengths, the levels of bridges did not increase with combination treatments (Table 2). 3.3. Mitochondrial dysfunction in bystander cells As the above statistical analysis showed no significant differences detected between 2 Gy and 5 Gy treatments nor between tumor and normal, mitochondrial dysfunction as measured by
reduction in mitochondrial membrane potential and an increase in ROS, was performed in bystander cells exposed to media from 2 Gy irradiated tumor explant tissue. Fig. 3A and B shows representative images of mitochondrial membrane staining with rhodamine 123 fluorescent dye in bystander cells following incubation with conditioned media from untreated tumor tissue (0 Gy) (A) and 2 Gy treated tumor tissue (B). Bystander cells exposed to conditioned media from 2 Gy irradiated tumor tissue caused significant reduction in rhodamine staining intensity indicating depolarisation of the mitochondrial membrane potential versus control (Fig. 3E) (p = 0.012). Fig. 3C and D shows representative images of ROS levels in these bystander cells following staining with 2,7 dichlorofluorescein diacetate. Bystander cells exposed to conditioned media from 2 Gy irradiated tumor tissue showed an increase in ROS levels, however this was not significant. Five out of the eight patients showed elevated levels of ROS (p = 0.02).
Table 1 Telomere length analysis in bystander cells using QFISH. Tumor
Normal
Treatment
Telo fluorescence
Treatment
Telo fluorescence
p value
Treatment
Telo fluorescence
Treatment
Telo fluorescence
p value
Control Control Control 2 Gy 5 Gy
209.3 209.3 209.3 157.8 148.1
FOLFOX 2 Gy FOLFOX 5 Gy FOLFOX 2 Gy FOLFOX 5 Gy FOLFOX
143.8 166.8 153.7 166.8 153.7
0.012 0.012 0.012 0.856 0.486
Control Control Control 2 Gy 5 Gy
208.3 208.3 208.3 178.5 169.9
FOLFOX 2 Gy FOLFOX 5 Gy FOLFOX 2 Gy FOLFOX 5 Gy FOLFOX
198.9 187.3 171.4 187.3 171.4
0.018 0.017 0.015 0.695 0.974
Telomere shortening induced by a chemotherapy bystander effect. Values shown are median fluorescence for eight patients. Conditioned media from FOLFOX treated tumor and matched normal explant tissue induced significant telomere shortening in bystander cells compared to cells incubated with conditioned media from unirradiated tumor and matched normal tissue. Radiation + FOLFOX conditioned media had no additive effect on telomere lengths observed. All statistical values obtained by Wilcoxon Signed Ranks test.
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Fig. 2. (A) and (B) are representative images of bridges observed in bystander cells. Cells were incubated with conditioned media, fixed in methacarn and nuclei were stained with haemotoxyllin. Bridges were defined as unresolved chromatin strings linking two nuclei as indicated by the arrows and calculated as a percentage of total cell number. Conditioned media from irradiated tumor (C) and matched normal (D) explant tissue induced significantly higher levels of bridging in bystander cells compared to cells incubated with conditioned media from unirradiated tumor and matched normal tissue.
3.4. Restoring mitochondrial function rescues nuclear instability events Fig. 4 (panel A) shows the level of overexpression of MnSOD in the stable line generated. There is an approximate 10-fold increase in expression of MnSOD in this stable line compared to parental untransfected cells. Panel B shows levels of ROS in 0 Gy and 2 Gy parental cells (Par), empty vector cells (EV) and MnSOD overexpressing cells (MnSOD). There is a significant increase in ROS levels between 0 Gy Par and 2 Gy Par cells (p = 0.03). There is a significant decrease in the levels of ROS in 2 Gy MnSOD cells compared to 2 Gy parental cells (p = 0.02). There is no difference between empty vector and MnSOD stable cells. Panel C shows a significant reduction in mitochondrial membrane potential between 0 Gy versus 2 Gy parental cells (p = 0.04). Overexpression of MnSOD rescues this mitochondrial defect and significantly increases the mitochondrial
membrane potential (p = 0.04). While in the 0 Gy conditions there seems to be a decrease in mitochondrial membrane potential in MnSOD cells, this did not reach significance. Panel D shows percentage increase in the numbers of anaphase bridges. Levels of bridges induced by 2 Gy conditioned media are significantly decreased in MnSOD cells compared to untransfected bystander cells (p = 0.01). This was coupled with a decrease in the number of cells undergoing telomere length shortening (p = 0.028, panel E).
4. Discussion The majority of bystander studies have been studied in vitro [3,4,8,20]. Tissue explant models have investigated the formation of outgrowths [28] and apoptosis [29–31] following radiation, however, the cellular events which may drive bystander radiation
Table 2 Bridge formation in bystander cells. Tumor
Normal
Treatment
% bridging
Treatment
% bridging
p value
Treatment
% bridging
Treatment
% bridging
p value
Control Control Control 2 Gy 5 Gy
1.36 1.36 1.36 6.11 5.25
FOLFOX 2 Gy FOLFOX 5 Gy FOLFOX 2 Gy FOLFOX 5 Gy FOLFOX
4.61 4.06 4.81 4.06 4.81
0.012 0.012 0.012 0.049 0.22
Control Control Control 2 Gy 5 Gy
1.45 1.45 1.45 4.21 3.82
FOLFOX 2 Gy FOLFOX 5 Gy FOLFOX 2 Gy FOLFOX 5 Gy FOLFOX
2.73 3.37 3.18 3.37 3.18
0.012 0.012 0.025 0.11 0.77
Bridge formations induced by a chemotherapy bystander effect. Conditioned media from FOLFOX treated tumor and matched normal explant tissue induced significant bridge formations in bystander cells compared to cells incubated with conditioned media from unirradiated tumor and matched normal tissue. Radiation + FOLFOX conditioned media had no additive effect on levels of bridging observed.
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Fig. 3. Representative images of mitochondrial membrane potential in SW480 bystander cells incubated with control (0 Gy) (A) and 2 Gy (B) conditioned media from tumor tissue following staining with Rhodamine 123 dye. C and D show levels of reactive oxygen species in bystander cells incubated with control (0 Gy) and 2 Gy conditioned media respectively following staining with 2,7 dichlorofluorescein diacetate. Panel E shows a decrease in mitochondrial membrane potential in 2 Gy versus 0 Gy treated bystander cells. Panel F shows a trend of increasing ROS levels following exposure of bystander cells to condition media from 2 Gy irradiated tumors. Statistical values obtained using Wilcoxon Signed Ranks test.
induced genomic instability is unknown. Using a human colorectal cancer explant tissue model, this study has demonstrated that radiation and chemotherapy bystander responses induce early genomic instability events such as telomere shortening and bridge formations coupled with mitochondrial dysfunction. Telomere length shortening is an early genomic instability event which can drive malignant progression [23,32–34]. We demonstrated that telomere lengths were significantly shorter in bystander cells incubated with conditioned media from irradiated and chemotherapy-treated tissue. The frequency of bridges is commonly used as an indicator of telomere-mediated chromosomal instability [23,35,36] which can lead to whole-arm translocations and loss of whole chromosomes through defects in mitotic spindle machinery [37]. These bystander cells with short telomeres also display significantly higher levels of bridges. This is a novel finding and has not previously been linked with radiation and chemotherapy bystander effects. Interestingly, conditioned media from treated tumor and matched normal tissue induce similar levels of telomere length shortening and bridge formations. If the accumulation of this
instability in the normal mucosa is not repaired or the cells do not undergo apoptosis, this may initiate further disease progression. These early instability events we detect may be influenced through mitochondrial dysfunction [38,39]. Mitochondria are a rich source of reactive oxygen species [3,5,40] and have been implicated in bystander effects in vitro [5,41], however, a role for mitochondria in bystander signalling using ex vivo models has not yet been established nor has it been linked with nuclear instability events (telomere shortening and bridge formations) during the bystander response. We have shown that bystander cells exhibited a significant depolarisation of the mitochondrial membrane at 6 h following incubation with conditioned media from 2 Gy treated tumor explant tissue. Caspase activity following membrane depolarisation of the outer membrane may cause a release of reactive oxygen species further influencing mitochondria function [42] which in turn may influence treatment response. The levels of reactive oxygen species were higher overall in the 2 Gy conditioned media compared to media from unirradiated tumor tissue. Reactive oxygen species have been well documented
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Fig. 4. Panel (A) shows approximately 10-fold over expression of MnSOD in the stable line. Panel (B) shows levels of ROS in 0 Gy and 2 Gy parental cells (Par), empty vector cells (EV) and MnSOD overexpressing cells (MnSOD). There is a significant decrease in the levels of ROS in 2 Gy MnSOD cells compared to 2 Gy parental cells (p = 0.02). Panel (C) shows a significant reduction in mitochondrial membrane potential between 0 Gy versus 2 Gy parental cells (p = 0.04). Overexpression of MnSOD rescues this mitochondrial defect and significantly increases the mitochondrial membrane potential (p = 0.04). Panel (D) shows percentage increase in the numbers of anaphase bridges. Levels of bridges induced by 2 Gy conditioned media are significantly decreased in MnSOD cells compared to untransfected bystander cells (p = 0.01). This was coupled with a decrease in the number of cells undergoing telomere length shortening (p = 0.028, panel E). Statistical values obtained using Wilcoxon Signed Ranks test.
as important mediators of bystander signalling. Previous studies have shown the induction of reactive oxygen species in culture media following ␣ particle and ␥ irradiation [3,43]. In vitro studies have shown that ROS can mediate sister chromatid exchanges and signalling pathways in bystander cells and these events can be inhibited with manipulation at the mitochondrial level [44,45]. In this study, we have shown that restoring mitochondrial function
through overexpression of an antioxidant, manganese superoxide dismutase significantly decreases nuclear instability events: anaphase bridges and telomere length shortening. While the radiation bystander effect has been well studied in cell lines, there are very few examples of chemotherapy bystander effects in the literature. In a recent study, an enhanced cell killing effect was observed in an untreated MCF-7 breast cancer cell line
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using an in vitro co-culture system. Extracellular release of ROS was seen to mediate bystander growth inhibition in thymidine labelled bystander cells co-cultured with chemotherapy-treated cells [13]. In a similar study, conditioned media from chemotherapy-treated cells induced growth inhibition and cytoskeleton disorders along with altered protein secretion activity in vivo [14]. Our study is the first to illustrate that conditioned media from chemotherapytreated tissue can induce early nuclear instability events. These results should not be attributed to residual FOLFOX as its half life in culture is approximately 60 min and the conditioned media was removed after 24 h, placed on bystander cells and incubated for a further 24 h. This study has demonstrated using an ex vivo explant model that bystander signalling in colorectal cancer initiates telomere shortening and bridge formations which are early instability events which maybe important in driving malignant transformation [23,46,47]. These events are coupled with dysfunctional mitochondrial which we have shown have an important mechanistic role in driving nuclear instability events. Conflicts of interest All authors have nothing to declare. Acknowledgements The authors would like to thank Peter McLoone and Gemma McConnell for assisting with the Cobalt 60 irradiations in St. Luke’s Hospital. This work was supported by the Irish Health Foundation. References [1] C. Mothersill, C. Seymour, Radiation-induced bystander effects: past history and future directions, Radiat. Res. 155 (2001) 759–767. [2] K.L. Chapman, J.W. Kelly, R. Lee, E.H. Goodwin, M.A. Kadhim, Tracking genomic instability within irradiated and bystander populations, J. Pharm. Pharmacol. 60 (2008) 959–968. [3] F.M. Lyng, C.B. Seymour, C. Mothersill, Production of a signal by irradiated cells which leads to a response in unirradiated cells characteristic of initiation of apoptosis, Br. J. Cancer 83 (2000) 1223–1230. [4] K.M. Prise, O.V. Belyakov, M. Folkard, B.D. Michael, Studies of bystander effects in human fibroblasts using a charged particle microbeam, Int. J. Radiat. Biol. 74 (1998) 793–798. [5] J.E. Murphy, S. Nugent, C. Seymour, C. Mothersill, Mitochondrial DNA point mutations and a novel deletion induced by direct low-LET radiation and by medium from irradiated cells, Mutat. Res. 585 (2005) 127–136. [6] P. Maguire, C. Mothersill, C. Seymour, F.M. Lyng, Medium from irradiated cells induces dose-dependent mitochondrial changes and BCL2 responses in unirradiated human keratinocytes, Radiat. Res. 163 (2005) 384–390. [7] L. Huo, H. Nagasawa, J.B. Little, HPRT mutants induced in bystander cells by very low fluences of alpha particles result primarily from point mutations, Radiat. Res. 156 (2001) 521–525. [8] H. Nagasawa, J.B. Little, Induction of sister chromatid exchanges by extremely low doses of alpha-particles, Cancer Res. 52 (1992) 6394–6396. [9] W.F. Morgan, A. Hartmann, C.L. Limoli, S. Nagar, B. Ponnaiya, Bystander effects in radiation-induced genomic instability, Mutat. Res. 504 (2002) 91–100. [10] E.J. Hall, T.K. Hei, Genomic instability and bystander effects induced by high-LET radiation, Oncogene 22 (2003) 7034–7042. [11] D.A. Lewis, B.M. Mayhugh, Y. Qin, K. Trott, M.S. Mendonca, Production of delayed death and neoplastic transformation in CGL1 cells by radiation-induced bystander effects, Radiat. Res. 156 (2001) 251–258. [12] R.R. Chhipa, M.K. Bhat, Bystander killing of breast cancer MCF-7 cells by MDAMB-231 cells exposed to 5-fluorouracil is mediated via Fas, J. Cell. Biochem. 101 (2007) 68–79. [13] J. Alexandre, Y. Hu, W. Lu, H. Pelicano, P. Huang, Novel action of paclitaxel against cancer cells: bystander effect mediated by reactive oxygen species, Cancer Res. 67 (2007) 3512–3517. [14] A. Demidem, D. Morvan, J.C. Madelmont, Bystander effects are induced by CENU treatment and associated with altered protein secretory activity of treated tumor cells: a relay for chemotherapy? Int. J. Cancer. 119 (2006) 992–1004. [15] P.K. Narayanan, E.H. Goodwin, B.E. Lehnert, Alpha particles initiate biological production of superoxide anions and hydrogen peroxide in human cells, Cancer Res. 57 (1997) 3963–3971. [16] C. Shao, M. Folkard, K.M. Prise, Role of TGF-beta1 and nitric oxide in the bystander response of irradiated glioma cells, Oncogene 27 (2008) 434–440.
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Radiation and chemotherapy bystander effects induce early genomic instability events: Telomere shortening and bridge formation coupled with mitochondrial dysfunction Sheeona Gorman a , Miriam Tosetto a , Fiona Lyng b , Orla Howe b , Kieran Sheahan a , Diarmuid O’Donoghue a , John Hyland a , Hugh Mulcahy a , Jacintha O’Sullivan a,∗ a b
Centre for Colorectal Disease, St. Vincent’s University Hospital, Elm Park, Dublin 4, Ireland Radiation & Environmental Science Centre, Dublin Institute of Technology and St. Luke’s Hospital, Dublin, Ireland
a r t i c l e
i n f o
Article history: Received 20 November 2008 Received in revised form 3 June 2009 Accepted 9 June 2009 Available online 18 June 2009 Keywords: Radiation bystander Mitochondria Nuclear instability
a b s t r a c t The bridge breakage fusion cycle is a chromosomal instability mechanism responsible for genomic changes. Radiation bystander effects induce genomic instability; however, the mechanism driving this instability is unknown. We examined if radiation and chemotherapy bystander effects induce early genomic instability events such as telomere shortening and bridge formation using a human colon cancer explant model. We assessed telomere lengths, bridge formations, mitochondrial membrane potential and levels of reactive oxygen species in bystander cells exposed to medium from irradiated and chemotherapytreated explant tissues. Bystander cells exposed to media from 2 Gy, 5 Gy, FOLFOX treated tumor and matching normal tissue showed a significant reduction in telomere lengths (all p values <0.018) and an increase in bridge formations (all p values <0.017) compared to bystander cells treated with media from unirradiated tissue (0 Gy) at 24 h. There was no significant difference between 2 Gy and 5 Gy treatments, or between effects elicited by tumor versus matched normal tissue. Bystander cells exposed to media from 2 Gy irradiated tumor tissue showed significant depolarisation of the mitochondrial membrane potential (p = 0.012) and an increase in reactive oxygen species levels. We also used bystander cells overexpressing a mitochondrial antioxidant manganese superoxide dismutase (MnSOD) to examine if this antioxidant could rescue the mitochondrial changes and subsequently influence nuclear instability events. In MnSOD cells, ROS levels were reduced (p = 0.02) and mitochondrial membrane potential increased (p = 0.04). These events were coupled with a decrease in percentage of cells with anaphase bridges and a decrease in the number of cells undergoing telomere length shortening (p values 0.01 and 0.028 respectively). We demonstrate that radiation and chemotherapy bystander responses induce early genomic instability coupled with defects in mitochondrial function. Restoring mitochondrial function through overexpression of MnSOD significantly rescues nuclear instability events; anaphase bridges and telomere length shortening. © 2009 Elsevier B.V. All rights reserved.
1. Introduction The radiation induced bystander effect is a biological phenomenon whereby cells not directly exposed to treatment exhibit cellular responses similar to those of directly irradiated cells [1,2]. Radiation bystander effects can lead to cell death [3,4], mitochondrial alterations [5,6], DNA damage [7,8] and enhanced mutagenesis [9,10], ultimately leading to oncogenic transformation [11]. Studies have examined whether chemotherapy treatment also induces a bystander effect which includes apoptosis [12], an increase in
∗ Corresponding author. Tel.: +353 1 2213464; fax: +353 1 2838123. E-mail address: [email protected] (J. O’Sullivan). 0027-5107/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.mrfmmm.2009.06.003
reactive oxygen species (ROS) [13] and cell differentiation [14]. The mechanisms driving radiation and chemotherapy bystander effects are unknown, but soluble signalling factors [15,16] and cellular communication (i.e. via gap junctions and/or release of molecular messengers into the extracellular environment) may play a pivotal role [17]. ROS have been shown to be involved in the induction of sister chromatid exchanges [15] and stress inducible signalling pathways [18] and micronuclei formation in bystander cells [19]. An important biological endpoint observed in bystander cells is genomic instability [20], however the cellular events driving this instability is unknown. Telomere shortening and bridge formations affect the integrity of chromosomes [21]. When telomeres become critically short, they can fuse together and during cell division, they can form a bridge [22] which can break, resulting in daughter cells
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with altered genomic material [21]. We have previously shown that these events are activated very early in disease progression [23]. The majority of bystander work has been performed in cell lines [18,24,25] and limited studies have been performed in vivo [19,26]. Using a human explant model, the aim of this study was to investigate if radiation and chemotherapy bystander effects induce telomere length shortening and bridge formations and if these events were coupled with mitochondrial dysfunction. Additionally, we assessed if mitochondrial function was restored, did this influence nuclear instability events: anaphase bridges and telomere length shortening. 2. Materials and methods 2.1. Ex vivo explant culture Resected tumor and matching normal adjacent mucosa was obtained from eight patients from the Centre for Colorectal Disease’s explant tissue bio-bank in St. Vincent’s University Hospital, Dublin (five male, three female, median age 69). Explant tissue was cut into 12 equal-sized pieces of approximately 5 mm3 allowing each treatment (described below) to be performed in duplicate. Tumor and matched normal explant tissue were cultured in 5 ml RPMI 1640 containing 100 U/ml Penicillin, 100 g/ml Streptomycin, 4 g/ml Fungizone and supplemented with 20% foetal bovine serum for 24 h prior to treatment in 6-well plates.
2.2. Irradiation and chemotherapy treatments There were six treatment arms to this study performed for both the tumor and matched normal tissue: (1) unirradiated controls (0 Gy), (2) 2 Gy treatment, (3) 5 Gy treatment, (4) FOLFOX chemotherapy (10 M 5-fluoruracil, 5 M oxaliplatin and 2.5 g/ml folinic acid), (5) FOLFOX + 2 Gy and (6) FOLFOX + 5 Gy. The 2 Gy and 5 Gy doses were administered at room temperature using a cobalt-60 teletherapy unit in St. Luke’s Hospital, Dublin, delivering approximately 0.8 Gy/min. The tissue was treated with the above treatments for 24 h at 37◦ C.
2.3. Media transfer Following 24 h treatment, media from tumor and matched normal tissue was removed from the untreated and treated explant tissue and filtered through a 0.2-m filter (Nalgene, Rochester, NY) to remove any floating cells and cellular debris. This ‘conditioned media’ was incubated on bystander SW480 (American Type Culture Collection) cells for 24 h at 37◦ C cultured in RPMI 1640 medium and supplemented with 10% foetal bovine serum and 50 U/ml Penicillin, 50 g/ml Streptomycin, 4 g/ml Fungizone in an atmosphere of 5% CO2 . Following 24 h incubation, these bystander cells were processed for assays outlined below.
2.4. Telomere length analysis Bystander cells were seeded in chamber slides (BD Falcon) at a density of 2000 cells/well and incubated conditioned media from treated (2 Gy, 5 Gy and FOLFOX alone and in combination) and untreated tumor tissue for 24 h. Following incubation, cells were fixed in methacarn buffer (3:1 methanol:acetic acid) for 8 min at room temperature and incubated in a graded ethanol series of 50%, 70% and 100% ethanol. Slides were incubated in 10 mM sodium citrate for 8 min at 88 ◦ C, rinsed in PBS, dipped through the above ethanol series and allowed to air dry. Cells were incubated with 100 M PNA TAMRA (Tetra methylu-6-Carboxyrhodamine) probe (Applied Biosytems) and hybridised in the dark overnight at room temperature. Slides were washed in 70% Formamide buffer four times for 15 min each, followed by PBS/0.05% Tween 20 washes. Cell nuclei were counterstained with TOTO-3 DNA dye (Molecular Probes) for 5 min at room temperature, briefly washed in PBS, dipped through the ethanol series and air-dried. Slides were mounted with Vectashield (Vector Laboratories). Images were obtained on a Zeiss Axiovert confocal microscope using LSM software. Telomere length analyses was performed using image analysis algorithms [27] and the mean level of telomere fluorescence was proportional to mean telomere length for each case.
2.5. Bridge assessment Bystander cells were seeded in chamber slides at a density of 1000 cells/well and incubated with conditioned media for 24 h at 37◦ C. Slides were washed with PBS, fixed in Carnoys solution for 8 min at room temperature, dehydrated through a graded ethanol series of 50%, 70% and 100% ethanol and air-dried for 10 min. Slides were stained with 1:3 Mayer’s Haematoxylin, washed in water and put through the above graded alcohol series and fixed in xylene. The number of bridges were scored and expressed as a percentage of total cell number.
2.6. Mitochondrial membrane potential Bystander cells were seeded on glass cover slips (100,000 cells) and incubated with conditioned media from treated 2 Gy treated and untreated tumor tissue for 6 h. Conditioned media was removed and cells washed twice with a buffer containing 130 mM NaCl, 5 mM KCl, 1 mM Na2 HPO4 , 1 mM CaCl2 , 1 mM MgCl2 and 25 mM Hepes (pH 7.4). Cells were loaded with 5 M rhodamine 123 (Sigma) for 30 min in the buffer at 37 ◦ C. Cells were washed three times with the above buffer and then analysed using a confocal microscope (Zeiss LSM 510 META). Rhodamine 123 was excited at 488 nm, and fluorescence emission at 525 nm was recorded. Mean fluorescence values from ten random fields for each condition were obtained using Image J software. 2.7. Measurement of reactive oxygen species Bystander cells were seeded on glass cover slips (100,000 cells) and incubated with conditioned media from 2 Gy treated and untreated tumor tissue for 6 h for the reasons stated above. Conditioned media was removed and cells washed twice with a buffer containing 130 mM NaCl, 5 mM KCl, 1 mM Na2 HPO4 , 1 mM CaCl2 , 1 mM MgCl2 and 25 mM Hepes (pH 7.4). Cells were loaded with 5 M 2,7 dichlorofluorescein diacetate (Sigma) for 30 min in the buffer at 37 ◦ C. Cells were washed three times with the above buffer and then analysed using a confocal microscope (Zeiss LSM 510 META). 2,7 dichlorofluorescein diacetate was excited at 488 nm, and fluorescence emission at 525 nm was recorded. Mean fluorescence values from ten random fields for each condition were obtained using Image J software. 2.8. Generation of a manganese superoxide dismutase (MnSOD) bystander cell line In addition to the culturing of parental SW480 cell lines, another cell line over expressing MnSOD was prepared. MnSOD cDNA in a mammalian expressing vector (pcDNA3.1) was stably transfected into SW480 colorectal cells. Briefly, cells were seeded at 2 × 106 cells/well in a 6-well plate 24 h prior to transfection with MnSOD cDNA and the empty vector DNA. Transfection was carried out following the manufacturer’s protocol (LipofectamineTM 2000 reagent, Invitrogen). Briefly, 35 ng of DNA was diluted in serum free media containing 20 l of lipofectamineTM 2000 reagent and incubated for 20 min at room temperature. This DNA/lipid mixture were added to cells in serum free media and incubated for 16 h. Transfection medium was replaced with fresh medium supplemented with 700 g/ml G418 (Geneticin, Invitrogen) to select for MnSOD overexpressing stably transfected cells for a period of 14 days. Overexpression of MnSOD was confirmed by Western blot analysis. Actin was used as the internal control. Densiometry was performed and data were presented as MnSOD levels/-actin levels in the same samples. 2.9. Statistical analysis Data are presented as medians and interquartile ranges. Data were assessed using Wilcoxon’s signed rank test. All p values are two-sided and p-values less than 0.05 were considered statistically significant in all analyses.
3. Results 3.1. Telomere lengths in bystander cells Fig. 1A and B shows representative images of telomere staining in bystander cells incubated with conditioned media from untreated, 0 Gy (A) and 2 Gy (B) treated tumor explant tissue. Lower telomere fluorescence intensity is observed in bystander cells exposed to conditioned media from 2 Gy treated explant tissue (B). Conditioned media from irradiated tumor (Fig. 1C) and normal (Fig. 1D) tissue induced significant telomere shortening in bystander cells at 24 h compared to bystander cells incubated with conditioned media from untreated tumor and matched normal tissue (p values ≤0.012 and 0.018 respectively). There was no significant difference in telomere length shortening between 2 Gy and 5 Gy treated conditions or between effects induced by the tumor versus normal treated tissue. Therefore, 2 Gy conditioned media is adequate to elicit a maximum bystander response. Table 1 shows telomere shortening induced by a chemotherapy bystander effect. Conditioned media from FOLFOX treated tumor and normal tissue induced telomere shortening in bystander cells compared to control (p ≤ 0.012 and 0.018 respectively). Combination of radio and chemotherapy treatment did not have an additive effect compared to single treatments.
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Fig. 1. Telomere length assessment using QFISH in bystander cells. Representative images of bystander cells following incubation with conditioned media from control (0 Gy) (A) and 2 Gy (B) treated tumor explant tissue. DNA is stained with TOTO-blue dye and telomeres are hybridised with telomere-specific PNA probe (red). Images were taken by confocal microscopy and analysed using image analysis algorithims. Conditioned media from irradiated tumor (C) and matched normal (D) explant tissue induced significant telomere shortening in bystander cells compared to cells incubated with conditioned media from unirradiated tumor and matched normal tissue.
3.2. Bridge formation in bystander cells Fig. 2A and B shows representative images of bridges in bystander cells. Conditioned media from irradiated tumor (Fig. 2C) and normal (Fig. 2D) explant tissue induced significantly higher level of chromatin bridges in bystander cells (p = 0.012 and ≤0.018 respectively). Bridge formation was also significantly increased in bystander cells incubated with conditioned media from FOLFOX treated tumor and normal tissue (p ≤ 0.012), however, as seen with telomere lengths, the levels of bridges did not increase with combination treatments (Table 2). 3.3. Mitochondrial dysfunction in bystander cells As the above statistical analysis showed no significant differences detected between 2 Gy and 5 Gy treatments nor between tumor and normal, mitochondrial dysfunction as measured by
reduction in mitochondrial membrane potential and an increase in ROS, was performed in bystander cells exposed to media from 2 Gy irradiated tumor explant tissue. Fig. 3A and B shows representative images of mitochondrial membrane staining with rhodamine 123 fluorescent dye in bystander cells following incubation with conditioned media from untreated tumor tissue (0 Gy) (A) and 2 Gy treated tumor tissue (B). Bystander cells exposed to conditioned media from 2 Gy irradiated tumor tissue caused significant reduction in rhodamine staining intensity indicating depolarisation of the mitochondrial membrane potential versus control (Fig. 3E) (p = 0.012). Fig. 3C and D shows representative images of ROS levels in these bystander cells following staining with 2,7 dichlorofluorescein diacetate. Bystander cells exposed to conditioned media from 2 Gy irradiated tumor tissue showed an increase in ROS levels, however this was not significant. Five out of the eight patients showed elevated levels of ROS (p = 0.02).
Table 1 Telomere length analysis in bystander cells using QFISH. Tumor
Normal
Treatment
Telo fluorescence
Treatment
Telo fluorescence
p value
Treatment
Telo fluorescence
Treatment
Telo fluorescence
p value
Control Control Control 2 Gy 5 Gy
209.3 209.3 209.3 157.8 148.1
FOLFOX 2 Gy FOLFOX 5 Gy FOLFOX 2 Gy FOLFOX 5 Gy FOLFOX
143.8 166.8 153.7 166.8 153.7
0.012 0.012 0.012 0.856 0.486
Control Control Control 2 Gy 5 Gy
208.3 208.3 208.3 178.5 169.9
FOLFOX 2 Gy FOLFOX 5 Gy FOLFOX 2 Gy FOLFOX 5 Gy FOLFOX
198.9 187.3 171.4 187.3 171.4
0.018 0.017 0.015 0.695 0.974
Telomere shortening induced by a chemotherapy bystander effect. Values shown are median fluorescence for eight patients. Conditioned media from FOLFOX treated tumor and matched normal explant tissue induced significant telomere shortening in bystander cells compared to cells incubated with conditioned media from unirradiated tumor and matched normal tissue. Radiation + FOLFOX conditioned media had no additive effect on telomere lengths observed. All statistical values obtained by Wilcoxon Signed Ranks test.
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Fig. 2. (A) and (B) are representative images of bridges observed in bystander cells. Cells were incubated with conditioned media, fixed in methacarn and nuclei were stained with haemotoxyllin. Bridges were defined as unresolved chromatin strings linking two nuclei as indicated by the arrows and calculated as a percentage of total cell number. Conditioned media from irradiated tumor (C) and matched normal (D) explant tissue induced significantly higher levels of bridging in bystander cells compared to cells incubated with conditioned media from unirradiated tumor and matched normal tissue.
3.4. Restoring mitochondrial function rescues nuclear instability events Fig. 4 (panel A) shows the level of overexpression of MnSOD in the stable line generated. There is an approximate 10-fold increase in expression of MnSOD in this stable line compared to parental untransfected cells. Panel B shows levels of ROS in 0 Gy and 2 Gy parental cells (Par), empty vector cells (EV) and MnSOD overexpressing cells (MnSOD). There is a significant increase in ROS levels between 0 Gy Par and 2 Gy Par cells (p = 0.03). There is a significant decrease in the levels of ROS in 2 Gy MnSOD cells compared to 2 Gy parental cells (p = 0.02). There is no difference between empty vector and MnSOD stable cells. Panel C shows a significant reduction in mitochondrial membrane potential between 0 Gy versus 2 Gy parental cells (p = 0.04). Overexpression of MnSOD rescues this mitochondrial defect and significantly increases the mitochondrial
membrane potential (p = 0.04). While in the 0 Gy conditions there seems to be a decrease in mitochondrial membrane potential in MnSOD cells, this did not reach significance. Panel D shows percentage increase in the numbers of anaphase bridges. Levels of bridges induced by 2 Gy conditioned media are significantly decreased in MnSOD cells compared to untransfected bystander cells (p = 0.01). This was coupled with a decrease in the number of cells undergoing telomere length shortening (p = 0.028, panel E).
4. Discussion The majority of bystander studies have been studied in vitro [3,4,8,20]. Tissue explant models have investigated the formation of outgrowths [28] and apoptosis [29–31] following radiation, however, the cellular events which may drive bystander radiation
Table 2 Bridge formation in bystander cells. Tumor
Normal
Treatment
% bridging
Treatment
% bridging
p value
Treatment
% bridging
Treatment
% bridging
p value
Control Control Control 2 Gy 5 Gy
1.36 1.36 1.36 6.11 5.25
FOLFOX 2 Gy FOLFOX 5 Gy FOLFOX 2 Gy FOLFOX 5 Gy FOLFOX
4.61 4.06 4.81 4.06 4.81
0.012 0.012 0.012 0.049 0.22
Control Control Control 2 Gy 5 Gy
1.45 1.45 1.45 4.21 3.82
FOLFOX 2 Gy FOLFOX 5 Gy FOLFOX 2 Gy FOLFOX 5 Gy FOLFOX
2.73 3.37 3.18 3.37 3.18
0.012 0.012 0.025 0.11 0.77
Bridge formations induced by a chemotherapy bystander effect. Conditioned media from FOLFOX treated tumor and matched normal explant tissue induced significant bridge formations in bystander cells compared to cells incubated with conditioned media from unirradiated tumor and matched normal tissue. Radiation + FOLFOX conditioned media had no additive effect on levels of bridging observed.
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Fig. 3. Representative images of mitochondrial membrane potential in SW480 bystander cells incubated with control (0 Gy) (A) and 2 Gy (B) conditioned media from tumor tissue following staining with Rhodamine 123 dye. C and D show levels of reactive oxygen species in bystander cells incubated with control (0 Gy) and 2 Gy conditioned media respectively following staining with 2,7 dichlorofluorescein diacetate. Panel E shows a decrease in mitochondrial membrane potential in 2 Gy versus 0 Gy treated bystander cells. Panel F shows a trend of increasing ROS levels following exposure of bystander cells to condition media from 2 Gy irradiated tumors. Statistical values obtained using Wilcoxon Signed Ranks test.
induced genomic instability is unknown. Using a human colorectal cancer explant tissue model, this study has demonstrated that radiation and chemotherapy bystander responses induce early genomic instability events such as telomere shortening and bridge formations coupled with mitochondrial dysfunction. Telomere length shortening is an early genomic instability event which can drive malignant progression [23,32–34]. We demonstrated that telomere lengths were significantly shorter in bystander cells incubated with conditioned media from irradiated and chemotherapy-treated tissue. The frequency of bridges is commonly used as an indicator of telomere-mediated chromosomal instability [23,35,36] which can lead to whole-arm translocations and loss of whole chromosomes through defects in mitotic spindle machinery [37]. These bystander cells with short telomeres also display significantly higher levels of bridges. This is a novel finding and has not previously been linked with radiation and chemotherapy bystander effects. Interestingly, conditioned media from treated tumor and matched normal tissue induce similar levels of telomere length shortening and bridge formations. If the accumulation of this
instability in the normal mucosa is not repaired or the cells do not undergo apoptosis, this may initiate further disease progression. These early instability events we detect may be influenced through mitochondrial dysfunction [38,39]. Mitochondria are a rich source of reactive oxygen species [3,5,40] and have been implicated in bystander effects in vitro [5,41], however, a role for mitochondria in bystander signalling using ex vivo models has not yet been established nor has it been linked with nuclear instability events (telomere shortening and bridge formations) during the bystander response. We have shown that bystander cells exhibited a significant depolarisation of the mitochondrial membrane at 6 h following incubation with conditioned media from 2 Gy treated tumor explant tissue. Caspase activity following membrane depolarisation of the outer membrane may cause a release of reactive oxygen species further influencing mitochondria function [42] which in turn may influence treatment response. The levels of reactive oxygen species were higher overall in the 2 Gy conditioned media compared to media from unirradiated tumor tissue. Reactive oxygen species have been well documented
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Fig. 4. Panel (A) shows approximately 10-fold over expression of MnSOD in the stable line. Panel (B) shows levels of ROS in 0 Gy and 2 Gy parental cells (Par), empty vector cells (EV) and MnSOD overexpressing cells (MnSOD). There is a significant decrease in the levels of ROS in 2 Gy MnSOD cells compared to 2 Gy parental cells (p = 0.02). Panel (C) shows a significant reduction in mitochondrial membrane potential between 0 Gy versus 2 Gy parental cells (p = 0.04). Overexpression of MnSOD rescues this mitochondrial defect and significantly increases the mitochondrial membrane potential (p = 0.04). Panel (D) shows percentage increase in the numbers of anaphase bridges. Levels of bridges induced by 2 Gy conditioned media are significantly decreased in MnSOD cells compared to untransfected bystander cells (p = 0.01). This was coupled with a decrease in the number of cells undergoing telomere length shortening (p = 0.028, panel E). Statistical values obtained using Wilcoxon Signed Ranks test.
as important mediators of bystander signalling. Previous studies have shown the induction of reactive oxygen species in culture media following ␣ particle and ␥ irradiation [3,43]. In vitro studies have shown that ROS can mediate sister chromatid exchanges and signalling pathways in bystander cells and these events can be inhibited with manipulation at the mitochondrial level [44,45]. In this study, we have shown that restoring mitochondrial function
through overexpression of an antioxidant, manganese superoxide dismutase significantly decreases nuclear instability events: anaphase bridges and telomere length shortening. While the radiation bystander effect has been well studied in cell lines, there are very few examples of chemotherapy bystander effects in the literature. In a recent study, an enhanced cell killing effect was observed in an untreated MCF-7 breast cancer cell line
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using an in vitro co-culture system. Extracellular release of ROS was seen to mediate bystander growth inhibition in thymidine labelled bystander cells co-cultured with chemotherapy-treated cells [13]. In a similar study, conditioned media from chemotherapy-treated cells induced growth inhibition and cytoskeleton disorders along with altered protein secretion activity in vivo [14]. Our study is the first to illustrate that conditioned media from chemotherapytreated tissue can induce early nuclear instability events. These results should not be attributed to residual FOLFOX as its half life in culture is approximately 60 min and the conditioned media was removed after 24 h, placed on bystander cells and incubated for a further 24 h. This study has demonstrated using an ex vivo explant model that bystander signalling in colorectal cancer initiates telomere shortening and bridge formations which are early instability events which maybe important in driving malignant transformation [23,46,47]. These events are coupled with dysfunctional mitochondrial which we have shown have an important mechanistic role in driving nuclear instability events. Conflicts of interest All authors have nothing to declare. Acknowledgements The authors would like to thank Peter McLoone and Gemma McConnell for assisting with the Cobalt 60 irradiations in St. Luke’s Hospital. This work was supported by the Irish Health Foundation. References [1] C. Mothersill, C. Seymour, Radiation-induced bystander effects: past history and future directions, Radiat. Res. 155 (2001) 759–767. [2] K.L. Chapman, J.W. Kelly, R. Lee, E.H. Goodwin, M.A. Kadhim, Tracking genomic instability within irradiated and bystander populations, J. Pharm. Pharmacol. 60 (2008) 959–968. [3] F.M. Lyng, C.B. Seymour, C. Mothersill, Production of a signal by irradiated cells which leads to a response in unirradiated cells characteristic of initiation of apoptosis, Br. J. Cancer 83 (2000) 1223–1230. [4] K.M. Prise, O.V. Belyakov, M. Folkard, B.D. Michael, Studies of bystander effects in human fibroblasts using a charged particle microbeam, Int. J. Radiat. Biol. 74 (1998) 793–798. [5] J.E. Murphy, S. Nugent, C. Seymour, C. Mothersill, Mitochondrial DNA point mutations and a novel deletion induced by direct low-LET radiation and by medium from irradiated cells, Mutat. Res. 585 (2005) 127–136. [6] P. Maguire, C. Mothersill, C. Seymour, F.M. Lyng, Medium from irradiated cells induces dose-dependent mitochondrial changes and BCL2 responses in unirradiated human keratinocytes, Radiat. Res. 163 (2005) 384–390. [7] L. Huo, H. Nagasawa, J.B. Little, HPRT mutants induced in bystander cells by very low fluences of alpha particles result primarily from point mutations, Radiat. Res. 156 (2001) 521–525. [8] H. Nagasawa, J.B. Little, Induction of sister chromatid exchanges by extremely low doses of alpha-particles, Cancer Res. 52 (1992) 6394–6396. [9] W.F. Morgan, A. Hartmann, C.L. Limoli, S. Nagar, B. Ponnaiya, Bystander effects in radiation-induced genomic instability, Mutat. Res. 504 (2002) 91–100. [10] E.J. Hall, T.K. Hei, Genomic instability and bystander effects induced by high-LET radiation, Oncogene 22 (2003) 7034–7042. [11] D.A. Lewis, B.M. Mayhugh, Y. Qin, K. Trott, M.S. Mendonca, Production of delayed death and neoplastic transformation in CGL1 cells by radiation-induced bystander effects, Radiat. Res. 156 (2001) 251–258. [12] R.R. Chhipa, M.K. Bhat, Bystander killing of breast cancer MCF-7 cells by MDAMB-231 cells exposed to 5-fluorouracil is mediated via Fas, J. Cell. Biochem. 101 (2007) 68–79. [13] J. Alexandre, Y. Hu, W. Lu, H. Pelicano, P. Huang, Novel action of paclitaxel against cancer cells: bystander effect mediated by reactive oxygen species, Cancer Res. 67 (2007) 3512–3517. [14] A. Demidem, D. Morvan, J.C. Madelmont, Bystander effects are induced by CENU treatment and associated with altered protein secretory activity of treated tumor cells: a relay for chemotherapy? Int. J. Cancer. 119 (2006) 992–1004. [15] P.K. Narayanan, E.H. Goodwin, B.E. Lehnert, Alpha particles initiate biological production of superoxide anions and hydrogen peroxide in human cells, Cancer Res. 57 (1997) 3963–3971. [16] C. Shao, M. Folkard, K.M. Prise, Role of TGF-beta1 and nitric oxide in the bystander response of irradiated glioma cells, Oncogene 27 (2008) 434–440.
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