Mutagenesis vol. 18 no. 6 pp. 549-560,
November 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Mechanisms of cell death associated with death-inducing factors from genomically unstable cell lines
1Radiation Oncology Research Laboratory, BRB-6010, 2Graduate Program in Human Genetics and 3Marlene and Stewart Greenebaum Cancer Center, University of Maryland, 655 West Baltimore Street, Baltimore, MD 21201-1559, USA
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We recently described a unique non-targeted effect of ionizing radiation whereby growth medium from two clones of GM10115 cells exhibiting radiation-induced chromosomal instability was cytotoxic to parental GM10115 cells. We termed this the death-inducing effect (DIE). The goal of the present study was to determine how DIE killed cells. Our hypothesis was that DIE medium contained either a secreted factor(s) from unstable clones or products from dead/dying cells that were cytotoxic to parental cells. First, we investigated the apoptotic characteristics of our unstable clones by Annexin V binding and TUNEL assays. Both the parental GM10115 cells and cells from the unstable clone LS12 had a low background (
2%) level of apoptosis. The unstable Fe-10-3 clone showed a high spontaneous level of apoptosis, indicating major differences in the spontaneously occurring levels of apoptosis. We then analyzed how medium from these unstable clones killed cells by investigating the induction of DNA breaks, micronucleus formation and apoptosis induction in cells exposed to medium from unstable clones. Medium from unstable clones was capable of eliciting DNA double-strand breaks and increased apoptosis. Increased micronucleus frequencies were also observed in cells exposed to medium from either unstable clone, indicating a role of mitotis-linked cell death in DIE. These data suggest that DIE most likely results from cytotoxic factors secreted into the culture medium that can cause DNA double-strand breaks in recipient cells. These breaks can then lead to mitotis-linked cell death, as measured by micronuclei, or apoptosis, which accounts for the DIE. |
Introduction |
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Recent experimental evidence supports a role for long-term, non-targeted effects occurring in cells surviving exposure to ionizing radiation that can manifest as genomic instability and/or bystander effects (reviewed in Morgan, 2003a,b
). Genomic instability may be an early cellular event in the multi-step process of radiation carcinogenesis, but the processes that initiate and perpetuate the unstable phenotype remain unclear. Altered gene expression, defects in cellular responses to DNA damage and/or interactions between persistent oxidative stress and signal transduction processes may all be involved in the observed instability (Limoli et al., 1998; Cui et al., 1999; Baverstock, 2000; Lorimore et al., 2001). Bystander effects can occur in non-irradiated cells and are thought to result from a secreted signal from irradiated cells that leads to a response in unirradiated cells as well as cell-to-cell gap junction communication between irradiated and non-irradiated cells (Mothersill and Seymour, 1997; Azzam et al., 2001). Both chromosomal instability and bystander effects are characterized by chromosomal rearrangements, micronuclei, transformation and increased cell killing, and there appears to be a mechanistic link between the two phenomena (Seymour and Mothersill, 1997; Watson et al., 2000; Lorimore et al., 2001; Morgan et al., 2002). These non-targeted effects associated with exposure to ionizing radiation have significant implications for understanding radiation-induced carcinogenesis and potential long-term health consequences in irradiated individuals and indicate that the target for radiation effects may be larger than those cells actually irradiated.
We have previously described the initial characterization of a novel non-targeted effect of ionizing radiation, the death-inducing effect (DIE) (Nagar et al., 2003). Medium from clonally expanded cells surviving exposure to ionizing radiation that manifest chromosomal instability was cytotoxic to parental non-irradiated cells. We attributed this drastic reduction in clonogenic survival to the production of DIE factor(s) by chromosomally unstable clones or to by-products of dead and dying cells released into the culture medium that were ultimately cytotoxic to parental cells.
In this report, we extend our previous studies on DIE to investigate how medium from these unstable clones kills cells. Specifically, we test the hypothesis that either lytic products produced by dead or dying cells or secreted factors produced by unstable cells induce DIE. The goal is to provide a mechanistic link between the death-inducing signal and cell death induced in cells after medium transfer.
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Cell culture
All studies were carried out using hamster–human hybrid GM10115 cells (Human Genetic Mutant Cell Repository, Camden, NJ), which contain one copy of human chromosome 4 in a background of 20–24 Chinese hamster ovary chromosomes. Cells were grown as monolayers in Dulbecco’s minimal essential medium containing 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.2 mM L-proline. All cultures were grown at 34°C in humidified incubators containing 5% CO2. Cells were routinely monitored for mycoplasma (MycoFluorTM Mycoplasma Detection Kit; Molecular Probes, Eugene, OR) and showed no evidence of infection.
Clones were isolated from parental GM10115 cells surviving exposure to ionizing radiation and characterized cytogenetically for chromosomal instability. Chromosomally unstable clones were defined as those having at least three distinct aberrant metaphase subpopulations involving rearrangements of human chromosome 4, of which the total number constituted 
5% of the 200 metaphases scored (Limoli et al., 1997). The Fe-10-3 unstable clone was isolated after exposure to 10 Gy of iron ions from the alternating gradient synchrotron facility at Brookhaven National Laboratory (Upton, NY) and shows 18 unique cytogenetic subpopulations of cells having rearrangements involving human chromosome 4. Clone LS12 was isolated following exposure to 10 Gy X-rays and shows 13 subpopulations of unique cytogenetic rearrangements. Maintenance of the stable or unstable phenotype was monitored regularly using fluorescence in situ hybridization (FISH) (Marder and Morgan, 1993).
Characterization of apoptosis in unstable clones
All experiments involved seeding 5 x 104 cells into chambered glass microscope slides (Nalge Nunc International, Naperville, IL) and incubating cells overnight to facilitate cell attachment and subsequent growth. Dead and/or dying, floating cells were discarded and not taken into account for these studies.
Annexin V–GFP staining. To detect early apoptotic events, cells were stained for Annexin V according to the manufacturer’s instructions (ApoAlert® Annexin V kit; Clontech Laboratories, Palo Alto, CA). Briefly, culture medium was discarded and cells were stained with Annexin V–GFP (1 µg/ml), fixed in 4% formaldehyde, counterstained with propidium iodide (0.5 µg/ml) and observed under a fluorescence microscope using a dual filter set for fluorescein at 520 ± 20 nm and rhodamine at >620 nm. A total of 500 cells per time point per coded slide were scored for Annexin V–GFP staining.
TUNEL assay. Nuclear DNA fragmentation of apoptotic cells was measured by the TUNEL assay (DeadEnd Fluorometric TUNEL System; Promega, Madison, WI). Briefly, culture medium was discarded, cells were fixed in 4% formaldehyde, permeabilized in 0.2% Triton X-100 and incubated with TdT incubation buffer for 60 min in a 37°C humidified incubator for 3'-OH labeling. Cells were counterstained with 1 µg/ml propidium iodide and observed under a fluorescence microscope (Nikon Eclipse E600). A total of 500 cells per coded slide per time point were scored for TUNEL labeling.
Mechanisms of cell death after transfer of medium from unstable clones
Cells were seeded at a cell density of 5 x 104 cells in fresh medium into chambered glass microscope slides and incubated overnight to facilitate cell attachment. After 24 h fresh medium was replaced with either fresh medium (control) or filtered medium taken from either GM10115 cells (conditioned medium) or Fe-10-3 or LS12 cells after 48 h of culture. We had previously determined that medium that had been on unstable clones for 48 h was optimal for maximal DIE (Nagar et al., 2003). Medium from irradiated but chromosomally stable clones was not included, as we have previously found that this has no cytotoxic effect on GM10115 cells.
Cell killing in medium from unstable clones
To determine the kinetics of cell killing by DIE, 5 x 104 cells were seeded into chambered glass microscope slides. At 24 h intervals cells were removed from the slide by trypzinization and counted using a Coulter Counter. This was repeated for 6 days, after which time no cells were detected in slides cultured with medium from LS12 or Fe-10-3 cells.
Immunostaining for anti-phospho-H2AX foci
To investigate whether transfer of medium from unstable cells causes DNA double-strand breaks in recipient cells, immunostaining for anti-phospho-H2AX (
-H2AX) foci was performed. -H2AX focus formation is a rapid and sensitive cellular response to the presence of DNA double-strand breaks (Rogakou et al., 1998; Sedelnikova et al., 2002). A total of 5 x 106 cells were seeded into chambered glass slides and exposed to medium from unstable cells. After 10, 30 and 60 min and 4, 8 and 24 h, cells were stained for -H2AX focus formation. Briefly, cells were fixed in cold methanol for 20 min, permeabilized in cold methanol:acetone 1:1 and blocked with 10% fetal bovine serum in phosphate-buffered saline (PBS) for 1 h at 37°C. Subsequently, cells were incubated with -H2AX antibody (1:200) (Upstate Cell Signaling, Lake Placid, NY) for 1 h at 37°C and FITC-labeled goat anti-rabbit antibody (1:50) (Santa Cruz Biotechnology, Santa Cruz, CA) for an additional 1 h at 37°C. Nuclei were counterstained with DAPI (0.5 µg/ml) and observed under a fluorescence microscope (Nikon Eclipse E600). Cells irradiated with 1 Gy X-rays and incubated for 10 min prior to fixation served as positive controls. Five independent slides per condition were analyzed and a total of 500 cells per time point per coded slide were scored for the presence of -H2AX foci and the number of foci per cell.
Analysis of DIE cell death
Cell killing by apoptosis was monitored by Annexin V–GFP staining and TUNEL assays as described above. In order to be able to score 500 cells after 6 days of exposure to medium from Fe-10-3 or LS12 cells, the initial number of GM10115 cells seeded on chambered slides was increased. In addition, apoptotic DNA fragmentation was confirmed by Cell Death Detection ELISA (Roche Molecular Biochemicals, Mannheim, Germany). Cells were diluted to 1 x 105 cells/ml, lysed, centrifuged and the resulting supernatant incubated in a microtiter plate coated with mouse monoclonal anti-histone antibody. After 1.5 h at 25°C the supernatant was incubated with mouse monoclonal anti-DNA antibody conjugated with horseradish peroxidase for 1.5 h at room temperature. Subsequently, ABTS substrate [2,2'-azino-di-(3-ethylbenzthiazoline sulfonate)] was added to the samples and incubated until sufficient color developed for photometric analysis measured as absorbance at 405 nm. Positive controls included cells irradiated with 10 Gy X-rays and incubated for 48 h.
Detection of micronuclei and morphological cellular abnormalities
To investigate the presence of micronuclei in GM10115 cells exposed to unstable medium, slides were analyzed at daily intervals for 6 days after staining with 100 µl of Cell Tracker CM-Dil (Molecular Probes), rinsed with PBS, fixed using 4% formaldehyde, permeabilized with 0.1% Triton-X and counterstained with DAPI. Micronuclei were identified using a triple bandpass filter on a fluorescence microscope (Nikon Eclipse E600). Five hundred cells were examined per time point per coded slide. In addition to the frequency of micronuclei, cellular morphological abnormalities, such as the presence of giant cells and bridges between cells, were also recorded.
Immunostaining for the centromere
To determine if the micronuclei observed in GM10115 cells exposed to unstable medium contained intact chromosomes, cells with micronuclei were analyzed for the presence of a centromere. Cells on chambered microscope slides were permeabilized in PBS containing 0.01% Tween-20, washed with PNM (0.1 M phosphate buffer, pH 8.0, containing 0.5% IGEPAL and 5% non-fat dry milk) and incubated with human anti-centromere antibody (Antibodies Inc., Davis, CA) (1:1 dilution of antibody in PBS containing 0.2% Tween-20) in a humidified chamber at 37°C for 1 h. Slides were then washed twice with 0.1% Tween-20 in PBS and incubated with FITC-goat anti-human IgG (1:120 dilution of antibody in PBS containing 0.5% Tween-20) in a humidified chamber at 37°C for 1 h. Nuclear material was counterstained with 2.5 µg/ml DAPI in antifade and centromeric labeling analyzed in 200 cells containing micronuclei.
Fluorescence in situ hybridization using a mitochondrial DNA probe
LS12 cells and GM10115 cells cultured in medium from LS12 cells displayed a high frequency of cells with what appeared to be multiple micronuclei. To investigate the possibility that these ‘multi-micronuclei’ were aggregates of mitochondria, FISH was performed using a biotin-labeled probe for the total mitochondrial genome. The probe was generated by PCR amplification of the HeLa mitochondrial genome and consisted of nine fragments of 2 kb as described by Taylor et al. (2001). The resulting nine fragments were mixed and 1 µg of DNA was nick translated using the Bionick DNA labeling system (Invitrogen, Carlsbad, CA) and labeled probe cleaned with the ConcertTM Rapid PCR Purification System (Invitrogen). Cells were seeded into chambered slides at a density of 2 x 105 cells/ml and incubated for 3 days, after which time cells were rinsed with 1x PBS and cell membranes stained with 100 µl of Cell Tracker CM-Dil (at a final concentration of 0.05 µM). Cells were rinsed with PBS, fixed using 4% formaldehyde, permeabilized with 0.1% Triton-X, air dried and hybridized with 20 ng of labeled mitochondrial probe in 50% formamide, 2x SSC, 10% dextran sulfate with 1 µg herring sperm DNA. After overnight incubation at 37°C in a humidified chamber, slides were stained, coded and scored for the presence or absence of mitochondrial DNA in the multiple micronuclei.
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Results |
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Background apoptosis in parental and chromosomally unstable clones
The average background level of apoptotic cells in parental GM10115 cells was 2–7% over 1–6 days in culture as measured by TUNEL-positive cells and Annexin V fluorescence, respectively (Fig. 1a and b) and there was only a small increase after X irradiation (data not shown). A similar profile was observed in the unstable clone LS12 (Fig. 1a and b), indicating that this clone does not show an elevated spontaneous level of apoptosis. Furthermore, this background level was only slightly increased by X-irradiation (data not shown). In contrast, elevated levels of apoptotic cells were observed for the unstable clone Fe-10-3 as measured by both assays (Fig. 1a and b). This difference between parental GM10115 cells, LS12 cells and Fe-10-3 cells is striking and immediately apparent when cells were observed by fluorescence microscopy (Fig. 2a–f).
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Kinetics of cell killing by DIE
As an alternative to clonogenic survival and to determine how rapidly medium transfer from unstable clones led to cell death, cell numbers were determined as a function of time after exposure to medium from either unstable clone. The data in Figure 3 indicate that while GM10115 cells can attach and divide in fresh or conditioned medium, medium from either unstable clone prevents population doubling and after 6 days in culture very few cells can be detected in chambered slides.
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Kinetics of induction of DNA breaks by medium from unstable cells
Exposure of GM10115 cells to medium from either Fe-10-3 or LS12 cells results in a rapid increase in the number of cells showing
-H2AX foci (Fig. 4a). There is a gradual increase in the percentage of cells with foci with increasing exposure time to medium from Fe-10-3 cells. In contrast, the percentage of foci-containing cells upon exposure to medium from LS12 cells peaks at 4 h (9.5%) and declines at the 8 and 24 h exposure times (Fig. 4a).
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In addition to the percentage of cells with foci, the number of foci per cell was also determined. Data in Figure 4b indicate that after exposure to conditioned medium background levels of cells with 1–5 foci/cell is 1.25%. No cells with >10 foci/cell were detected. An increase in the percentage of cells with 11–15 foci/cell occurs after 24 h exposure to medium from Fe-10-3 cells, but only 4 h exposure to medium from LS12 cells is sufficient to cause a similar increase (Fig. 4b). Exposure to medium from Fe-10-3 cells for 8 and 24 h also causes some cells to produce >20 foci/cell (Fig. 2i). Interestingly, after 8 and 24 h exposure to medium from Fe-10-3 cells foci appeared predominantly at the nuclear periphery (Fig. 2j). These observations were made consistently from five independent slides and only in GM10115 cells exposed to medium from Fe-10-3 cells. This argues against them being immuno-artifacts, which should have been observed in all cell types, including GM10115 and LS12 cells. Why this occurs is unclear. The percentage of cells with foci after 1 Gy X-rays was 12%, and after 10 Gy X- irradiation it rose to
70% (data not shown).
Mode of cell killing by DIE
Apoptosis. Transferring medium from either unstable clone resulted in a significant increase in apoptosis in recipient GM10115 cells as measured by both Annexin V- and TUNEL-positive cells (Fig. 5a and b). While medium from Fe-10-3 cells was clearly more efficient at inducing apoptosis as detected by both assays, medium from LS12 cells also increased the percentage of apoptosis in the recipient cells.
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Apoptosis as a result of DIE was confirmed by ELISA using anti-histone antibody as a function of time after transfer of unstable medium (Fig. 6). Notable differences in the kinetics of detection of apoptotic DNA fragmentation were observed between the two clones.
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Micronucleus formation. Analysis of Fe-10-3 and LS12 cells revealed an increased spontaneous frequency of micronuclei compared to parental GM10115 cells (
2%) when cultured in fresh medium (Figs 7a and 2k). Interestingly, cellular abnormalities, including micronuclei and/or apoptotic cells, were observed in GM10115 cells (Fig. 2l). Not only is a higher micronucleus frequency observed in LS12 cells, but cells with multiple micronuclei or DNA bridges between cell nuclei were also frequently observed (Fig. 2l and m, respectively). Fe-10-3 cells were found to have a higher frequency of DNA bridges than LS12 or GM10115 cells (Table I). We then investigated whether DIE observed after transferring medium from the unstable clones was due to chromosomal fragmentation or mitotic abnormalities that could lead to chromatin loss via micronuclei and whether the kinetics of micronucleus formation were similar in the two unstable clones. Conditioned medium from parental cells did not increase the baseline micronucleus frequency from 2% over the time of the study (Fig. 7b). However, medium from both unstable clones increased the micronucleus frequency in GM10115 cells, although the kinetics of induction differed. GM10115 cells exposed to Fe-10-3 medium had a peak micronucleus frequency of 17% by day four (Fig. 7b), while the micronucleus frequency in cells exposed to LS12 medium peaked at 16% on day two (Fig. 7b). Interestingly, GM10115 cells cultured in medium from clone LS12 had cells with multiple micronuclei similar to those observed in LS12 cells.
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Micronuclei can consist of chromosomal fragments lacking a functional centromere induced by DNA double-strand breaks and/or reflect whole chromosomes harboring a centromere resulting from failed cytokinesis. Since aneuploidy and DNA double-strand breaks are both characteristics of genomic instability, we determined whether the micronuclei observed in individual clones represented chromosome fragments or whole chromosomes by immunostaining micronuclei with an antibody that recognizes centromeres. Although the antibody used is for human centromeres, it is not specific for the human centromere alone but is also able to bind to hamster centromeres. Centromere-positive micronuclei in GM10115 and LS12 cells were rarely found (0 and 4.6%, respectively; Table I) while Fe-10-3 cells showed a higher frequency of centromere-positive cells (17%; Table I). Although not a common observation, during analysis of DNA double-strand break induction by
-H2AX analysis after medium transfer we observed a micronucleus containing a strong -H2AX signal (Fig. 2h). This may support the contention that the observed micronuclei may be the result of failed segregation of broken chromatids. Furthermore, the multiple micronuclei observed in LS12 or GM10115 cells cultured in medium from clone LS12 showed no evidence of centromeric regions (Fig. 2p), suggesting that the multiple micronuclei are unlikely to be the result of failed cytokinesis.
Micronuclei do not contain mitochondrial DNA
One characteristic of our genomically unstable clones is increased reactive oxygen species (Limoli et al., 1998, 2001), which may be due to increased levels of dysfunctional mitochondria (Limoli et al., 2003). To determine whether the multiple micronuclei observed in unstable clone LS12 were actually aggregates of mitochondria, cells with multiple micronuclei were analyzed after hybridization with a mitochondrial DNA probe. Figure 2q and r demonstrates a micronuclei positive for mitochondrial DNA signals and a giant cell, respectively. The data in Table I indicate that neither the micronuclei nor multiple micronuclei exhibited preferential hybridization to the mitochondrial DNA probe. These data further support the idea that the observed micronuclei result from preferential exclusion of acentric DNA fragments.
Gross morphological abnormalities associated with the DIE
Following transfer of unstable medium, grossly altered cellular morphology was frequently observed in the recipient cells. These abnormalities include increased giant cell formation and bridges between dividing cells (Fig. 2m and Table I).
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Discussion |
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We previously demonstrated that medium from chromosomally unstable GM10115 clones causes cell death in non-irradiated parental GM10115 cells (Nagar et al., 2003
). The goal of the present study was to determine the mode of cell death in GM10115 cells exposed to medium from these unstable clones. Data from Annexin V binding indicates that GM10115, Fe-10-3 and LS12 cells are capable of undergoing early stages of the apoptotic cascade. However, TUNEL analysis demonstrated that while GM10115 and Fe-10-3 cells undergo complete cleavage of nuclear DNA, LS12 cells appear to be defective in completing this process. This conclusion is supported by the observation that TUNEL-positive Fe-10-3 cells have a homogeneous appearance of fluorescent TdT label, indicating significant cleavage of nuclear DNA. In contrast, LS12 cells have a distinct, punctate appearance of the fluorescent signal consistent with incomplete DNA cleavage (compare Fig. 2e and f). These results were also reflected in the ELISA analysis, suggesting that the two unstable clones differ in their ability to undergo apoptosis. While Fe-10-3 cells undergo extensive spontaneous apoptosis, LS12 cells show a spontaneous level of apoptosis similar to that of parental GM10115 cells, but significantly increased spontaneous micronucleus formation. This correlates with the observation that LS12 cells may be defective in apoptotic cleavage of nuclear DNA. While the multiple micronuclei observed in LS12 cells may indicate late stage apoptotic cells, they may also be manifestations of incomplete cleavage of DNA and failed apoptosis in cells that are defective in apoptosis. These micronuclei consist of fragments of chromosomes that lack a centromere and therefore did not arise as a result of failed cytokinesis during mitosis. In contrast, a larger percentage of micronuclei present in Fe-10-3 cells were centromere-positive. Fe-10-3 cells also displayed higher numbers of DNA bridges between daughter cell nuclei. These bridges presumably arise at anaphase when dicentric chromosomes are pulled towards opposite poles.
Mitochondria play a dynamic role in apoptosis. De Vos et al. (1998) have demonstrated that TNF induces clustering of mitochondria in murine fibrosarcoma cells and human leukemia cells. Desagher and Martinou (2000) showed that mitochondria aggregate around the nucleus of HeLa cells when pro-apoptotic Bax protein is overexpressed. It has recently been demonstrated that mitochondrial aggregation precedes cytochrome c release from mitochondria during apoptosis (Haga et al., 2003). Since mitochondria are clearly involved in stress-induced apoptosis we decided to rule out the possibility that the multiple micronuclei observed in the cytoplasm may actually contain mitochondrial DNA as a result of mitochondrial clustering at late stage apoptosis. Our data provide evidence that observed micronuclei in GM10115 cells exposed to unstable medium are not the result of stress-induced mitochondrial aggregates.
To explain DIE, we hypothesized that the cytotoxicity observed was a consequence of chromosomally unstable cells undergoing apoptosis and subsequently releasing lytic factors into the culture medium. Alternatively, DIE resulted from the secretion of cytotoxic factors by unstable cells. LS12 cells, unlike Fe-10-3 cells, are defective in completing apoptosis, however, medium from either clone is capable of inducing DIE in GM10115 cells. This result supports the possibility that DIE is more likely to result from secreted factors but does not definitively rule out the possibility that dead/dying cells may also contribute to the overall effect.
We then investigated the mode of DIE cytotoxicity. Data from -H2AX foci studies indicate that either cytotoxic factors and/or dead/dying cells present in the medium from either unstable clone are capable of inducing DNA breaks in unirradiated parental cells within 30 min of exposure. Although the kinetics of foci induction differed, these data clearly establish that a DIE factor(s) from either clone targets the DNA of parental cells and results in DNA strand cleavage.
Having established that medium from unstable cells can cleave the double helix, we then examined the cellular consequence of induced DNA breakage. Results from Annexin V and TUNEL assays clearly demonstrate that stable cells exposed to medium from either unstable cell line undergo increased apoptotic cell death. Likewise, analysis of micronucleus formation revealed significant increases in chromosome breakage. We have previously demonstrated by medium transfer experiments that medium from the two unstable clones differs in the kinetics of induction of cell killing in parental GM10115 cells (Nagar et al., 2003). Data from our present studies indicate an additional difference in kinetics of apoptosis and micronucleus formation. These observations support the contention that medium from either unstable clone is capable of inducing DNA breaks and these breaks can ultimately lead to lethality by chromosome breakage, resulting in micronucleus formation and/or apoptosis in parental cells.
We have proposed that DIE factors drive the chromosomal instability observed in some of our clonally expanded cells that survive irradiation (Nagar et al., 2003). While a particular clone is refractory to cell killing by the factors it secretes, using plating efficiency as an end point, we now demonstrate that DIE factors from two separate unstable clones can produce DNA strand breaks as measured by the induction of -H2AX foci after medium transfer. A recent study by Rogakou et al. suggests that phosphorylation of H2AX histone may also be induced by DNA fragmentation during apoptosis (Rogakou et al., 2000). Increased -H2AX foci observed in Fe-10-3 and LS12 cells may be the result of increased DNA breaks, which in turn may induce increased levels of apoptosis. Apoptotic DNA fragmentation may further increase the levels of -H2AX foci observed in these unstable cells. It is reasonable to assume that these same secreted factors can cause breakage in the unstable clone and thus perpetuate novel chromosomal rearrangements that characterize genomic instability in our model system.
Although DIE and bystander effects share a common end point, namely cytotoxicity (Lyng et al., 2000, 2002), these two non-targeted effects associated with exposure to ionizing radiation are separate. Our GM10115 cells do not display a bystander effect as measured by increased cell kill after transfer of medium from irradiated cells (Nagar et al., 2003). We presume that this is because GM10115 cells either do not produce a bystander signal upon irradiation or are not responsive to that particular secreted factor. While the factors responsible for DIE and the bystander effects are likely different in GM10115 cells, the induction of secreted factors has been described after human exposure to ionizing radiation in vivo. These include accidentally (Goh and Sumner, 1968) or therapeutically (Hollowell and Littlefield, 1968; Littlefield et al., 1969) irradiated individuals, as well as some A-bomb survivors (Pant and Kamada, 1977), Chernobyl salvage personnel (Emerit et al., 1994) and children living in areas contaminated by Chernobyl (Emerit et al., 1997; Gemignani et al., 1999). It should be stressed that secreted factors are not unique to radiation exposures, but have been associated with exposure to asbestos (Emerit et al., 1991) as well as ischemia–reperfusion injury (Emerit et al., 1995a) and occur spontaneously in patients with the cancer-prone chromosome breakage syndromes ataxia telangiectasia (Shaham et al., 1980), Bloom syndrome (Emerit et al., 1982) and Fanconi anemia (Emerit et al., 1995b). The role of factors with chromosome breaking activity in cancer has recently been reviewed by Huang et al. (2003).
Why cells produce clastogenic and/or cytotoxic factors, either spontaneously or after carcinogenic insult, is an intriguing question. We speculate that the factor(s) is likely to be a cytokine(s) (Hallahan et al., 1989; Narayanan et al., 1999; Iyer and Lehnert, 2000). While cytokine expression usually has significantly less dramatic effects, aberrant expression or inappropriate cellular/tissue responses can lead to increased production of reactive oxygen species. For example, in response to ionizing radiation transforming growth factor ß1 (TGF-ß1) is rapidly secreted (Barcellos-Hoff et al., 1994). TGF-ß1 can activate cell surface membrane-associated NADH oxidase, releasing a variety of reactive oxygen species as a consequence (Thannickal et al., 1993, 1998; Thannickal and Fanburg, 1995). This altered redox metabolism can in turn stimulate TGF-ß1 (Barcellos-Hoff and Dix, 1996; Barcellos-Hoff, 1998). Consequently, persistent cytokine expression might create a hostile cellular environment that may initiate and perpetuate dynamic changes in the genome (Iyer and Lehnert, 2000; Morgan et al., 2002), reminiscent of inflammatory type responses occurring after radiation insult (Lorimore et al., 2001). Increased levels of reactive oxygen species have been described as a delayed effect of radiation exposure (Rugo et al., 2002) and in a variety of different experimental systems showing radiation-induced genomic instability (Clutton et al., 1996; Limoli et al., 1998, 2001, 2003; Roy et al., 2000) and bystander effects (Wu et al., 1999; Mothersill et al., 2000).
Clearly identifying those factors leading to DIE and/or bystander effects will provide important insights into delayed cellular responses to DNA-damaging agents and how cells exposed to such insults may acquire the multiple mutations associated with the carcinogenic process that ultimately results in the neoplastic phenotype.
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Acknowledgements |
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This work was supported by the Biological and Environmental Research Program (BER), US Department of Energy grant no. DE-FG02-01ER63230 and National Institutes of Health Awards CA73924 and CA 83872.
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4To whom correspondence should be addressed at: Radiation Oncology Research Laboratory, BRB-6010, University of Maryland, 655 West Baltimore Street, Baltimore, MD 21201-1559, USA. Tel: +1 410 706 0254; Fax: +1 410 706 6138; Email: snaga001{at}umaryland.edu
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Received on July 14, 2003; accepted on September 11, 2003.
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