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Molecular Cancer Research 2:557-566 (2004)
© 2004 American Association for Cancer Research

DNA Damage and Cellular Stress Responses

Low-Dose Radiation Hypersensitivity Is Associated With p53-Dependent Apoptosis1

Louise Enns1, Kenneth T. Bogen2, Juanita Wizniak1, Albert D. Murtha1 and Michael Weinfeld1

1 Cross Cancer Institute, Edmonton, Alberta, Canada and 2 Environmental Science Division, Lawrence Livermore National Laboratory, Livermore, California

Requests for reprints: Michael Weinfeld, Cross Cancer Institute, 11560 University Avenue, Edmonton, Alberta, Canada T6G 1Z2. Phone: 780-432-8438; Fax: 780-432-8428. E-mail: michaelw{at}cancerboard.ab.ca


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
Exposure to environmental radiation and the application of new clinical modalities, such as radioimmunotherapy, have heightened the need to understand cellular responses to low dose and low-dose rate ionizing radiation. Many tumor cell lines have been observed to exhibit a hypersensitivity to radiation doses <50 cGy, which manifests as a significant deviation from the clonogenic survival response predicted by a linear-quadratic fit to higher doses. However, the underlying processes for this phenomenon remain unclear. Using a gel microdrop/flow cytometry assay to monitor single cell proliferation at early times postirradiation, we examined the response of human A549 lung carcinoma, T98G glioma, and MCF7 breast carcinoma cell lines exposed to {gamma} radiation doses from 0 to 200 cGy delivered at 0.18 and 22 cGy/min. The A549 and T98G cells, but not MCF7 cells, showed the marked hypersensitivity at doses <50 cGy. To further characterize the low-dose hypersensitivity, we examined the influence of low-dose radiation on cell cycle status and apoptosis by assays for active caspase-3 and phosphatidylserine translocation (Annexin V binding). We observed that caspase-3 activation and Annexin V binding mirrored the proliferation curves for the cell lines. Furthermore, the low-dose hypersensitivity and Annexin V binding to irradiated A549 and T98G cells were eliminated by treating the cells with pifithrin, an inhibitor of p53. When p53-inactive cell lines (2800T skin fibroblasts and HCT116 colorectal carcinoma cells) were examined for similar patterns, we found that there was no hyperradiosensitivity and apoptosis was not detectable by Annexin V or caspase-3 assays. Our data therefore suggest that low-dose hypersensitivity is associated with p53-dependent apoptosis.


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
The effects of low dose and low-dose rate ionizing radiation continue to be of interest because of the potential dangers posed by exposure to environmental and occupational sources of radiation (1) and because of the potential clinical benefits of radioimmunotherapy (2). Seminal studies by Joiner and colleagues (3, 4) and Wouters and colleagues (5-7) revealed that many human tumor cell lines exhibit a low-dose hypersensitivity to radiation (termed HRS for hyperradiosensitivity). This is usually manifested <0.5 Gy as a clear deviation from the standard linear-quadratic cell survival response extrapolated from higher doses back to 0 Gy. It is accompanied by an increase in radioresistance at doses between ~50 and 100 cGy (termed IRR for increased radioresistance).

Several explanations have been proposed for the HRS/IRR phenomenon (reviewed in refs. 8-10). HRS may represent a subpopulation of cells that are hypersensitive due possibly to a cell cycle factor or, more remotely, a genetic predisposition. It has been reasoned that, if there is a hypersensitive phase of the cell cycle, continuous low-dose rate irradiation should be more effective at inducing HRS, because cells would be eliminated as they moved through this phase (6). Earlier models for IRR, on the other hand, assumed that the population of cells is uniformly hypersensitive to start with and becomes resistant as a function of dose due to some protective mechanism, such as induction of DNA repair (11) or down-regulation of programmed cell death (6). Recently, it was shown that cells in G2 phase display elevated HRS in comparison with cells in G1 or S phase (12) and a model has emerged for IRR that involves the activation of a G2-phase checkpoint (13), which promotes DNA repair and cell survival (9, 10). To date, however, the mode of death for those cells that die at the low doses has still not been ascertained.

HRS/IRR has been observed in many cell lines by a variety of different assays for cell killing and cell proliferation, including conventional colony-forming assays (14), and newer microimaging methods (15). We recently reported on the application of a gel microdrop (GMD) protocol (16, 17) to a low-dose radiation response of human A549 cells (18). The principal advantage of this approach is that it allows direct assessment of proliferation of the irradiated cells. By comparison, the colony-forming assay cannot distinguish between early and late effects that may lead to a reduction in the eventual number of colonies derived from the irradiated population. Other advantages of the GMD approach are that results can be obtained within 4 to 5 days postirradiation and, because it is a flow cytometric (FC) technique, up to 2 x 104 single cells or microcolonies in GMDs can be analyzed at each dose.

Here, we describe a detailed study of the low-dose response of three human cell lines, A549 and T98G cells, which have been shown previously to display HRS/IRR (6, 19), and MCF7 cells, which do not.3 Two dose rates differing by two orders of magnitude were used to examine the potential influence of low-dose rate radiation. The GMD assay confirmed the early nature of the HRS response, so we sought to further characterize the HRS response by analyzing the effects of low-dose irradiation of these cell lines, particularly the influences on cell cycle distribution and modes of cell death. An early clue that HRS may be due to an apoptotic response lay in the fact that MCF7 cells lack caspase-3 activation (20), a key caspase for a p53-dependent apoptotic pathway. By further examination of apoptosis with markers for translocation of phosphatidylserine and caspase-3 cleavage and p53 dependence using pifithrin, a specific inhibitor of p53, we found that HRS reflects a p53-dependent apoptotic pathway. This finding was strengthened by an analysis of HRS in p53+/+ and p53–/– HCT116 cells and in three human fibroblast lines including a p53-inactive fibroblast line.


   Results
Top
 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 Acknowledgements
 References
 
Cell Proliferation
We employed the GMD/FC approach to follow early growth of single A549, T98G, and MCF7 cells into GMD encapsulated microcolonies. Optimum detection of altered relative proliferative capacity of singly encapsulated cells was determined to be ~4 days post–GMD encapsulation both with and without irradiation at 22 or 0.18 cGy/min (data not shown). The analysis of A549 and T98G cells irradiated at 22 cGy/min yielded clear evidence of HRS (Fig. 1). MCF7 cells, on the other hand, did not exhibit this hypersensitivity (Fig. 1). Similar HRS/IRR responses were also apparent in the A549 and T98G cells when irradiated at 0.18 cGy/min (Fig. 1). To confirm the data obtained with the GMD/FC assay, we carried out conventional clonogenic survival assays with all three cell lines. The survival curves (Fig. 2) matched the GMD/FC data showing a HRS response for A549 and T98G cells but not for MCF7 cells.



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  		FIGURE 1. Cell proliferation determined by the GMD/FC assay for A549, T98G, and MCF7 cells irradiated with doses up to 2 Gy delivered at 22 or 0.18 cGy/min. Points, mean of n independent dose-specific determinations (each from 104 cells) of cell growth in exposed relative to unexposed GMDs, ~95% of which contained a single cell at the time of exposure; bars, SE. Values of n (from lowest to highest dose) are as follows. For A549 cells, n = {17, 11, 11, 14, 13, 11, 11, 10, 2} (22 cGy/min) and n = {111, 24, 22, 46, 19, 30, 42, 33, 33, 3} (0.18 cGy/min); for T98G cells, n = {15, 9, 9, 10, 13, 13, 10, 11} (22 cGy/min) and n = {21, 12, 12, 12, 9, 12, 6, 13, 11} (0.18 cGy/min); for MCF7 cells, n = {17, 14, 15, 15, 13, 10, 11} (22 cGy/min). Solid circles, points statistically inconsistent (P < 10–7) with (constrained) linear-quadratic fits to all data in each plot; similar fits (dashed curves) are all statistically consistent (P > 0.01) with the remaining data (open circles) shown in each plot.

 


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  		FIGURE 2. Comparison of the cell proliferation, as determined by the GMD/FC assay (GMDS; solid symbols), to cell survival, as determined by the colony-forming assay (CFA; open symbols), for A549 cells irradiated at 22 cGy/min, T98G cells irradiated at 0.18 cGy/min, and MCF7 cells irradiated at 22 cGy/min. Points, means of at least three independent assays for the colony-forming assays; bars, SE.

 
Cell Cycle Analysis
To begin to identify possible mechanisms for the hypersensitivity exhibited in the GMD/FC assay, we examined the relative numbers of A549 cells in various phases of the cell cycle at times up to 24 hours postirradiation at 5, 10, and 20 cGy in comparison with unirradiated cells (Fig. 3). We were specifically interested in differences in 5, 10 (a dose at which A549 cells display HRS), and 20 cGy in irradiated cells. After irradiation at 22 cGy/min, no statistically significant differences were observed between the three doses in any phase of the cell cycle. A small but statistically significant difference was observed for cells in G2-M post–10 cGy irradiation at 0.18 cGy/min (i.e., more cells appeared in G2-M at 6 and 12 hours postirradiation). However, because this result was not observed with the higher dose rate, it is unlikely to be specifically related to HRS.



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  		FIGURE 3. Cell cycle analysis of A549 cells. The cells were irradiated at either 22 cGy/min (left) or 0.18 cGy/min (right) for times up to 24 hours postirradiation with 5 cGy ({blacksquare}), 10 cGy ({square}), or 20 cGy (). Points, mean of 3-7 (22 cGy/min) and 5-10 (0.18 cGy/min) determinations; bars, SE. P values were calculated by ANOVA.

 
In addition to FC cell cycle analysis, we also examined cyclin B1 (p62) and cdk1 (p34) protein levels in irradiated A549 cells (0.18 cGy/min) to ascertain whether they were affected at these low doses. Badie et al. (21) have shown that both cdk1 and cyclin B1 protein levels are down-regulated in response to DNA damage in cells with functional p53 at higher radiation doses. Figure 4 shows accumulated data from densitometry done on five immunoblots all corrected for protein concentration relative to actin (p43). The results indicate that both proteins are down-regulated at these low doses relative to controls, but the differences seen in 5, 10, and 20 cGy were not statistically significant (P > 0.05).



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  		FIGURE 4. Analysis of cyclin B1 (p62) and cdk1 (p34) protein levels in A549 cells in response to irradiation at 0.18 cGy/min to doses of 0, 5, 10, and 20 cGy. Top, a representative Western blot; bottom, data accumulated from densitometry done on cyclin B1 (p62) and cdk1 (p34) bands relative to actin (p43) on five (cyclin B1) and four (cdk1) separate blots. There was no significant difference found in 5, 10, and 20 cGy for cyclin B1 (P = 0.57) or cdk1 (P = 0.88). The cdk1 untreated control was significantly different from the irradiated samples (P = 0.0096). Bars, SE. P values were calculated by t test.

 
Apoptotic Responses
We chose to examine two independent variables of initiation of apoptosis following irradiation of A549, T98G, and MCF7 cells: caspase-3 activation and phosphatidylserine translocation. During apoptosis, caspase-3 is cleaved into its active form, a heterodimer of 17 and 12 kDa subunits, which can proteolytically cleave other cellular enzymes, including poly(ADP-ribose) polymerase (22). As a marker of apoptosis, caspase-3 activation is typically measured up to 24 hours and later after the cellular damage. Figure 5 shows the dose responses we obtained with a FC assay for active caspase-3. A549 and T98G cells showed a discernable activation of caspase-3 at the doses that induce HRS. MCF7 cells, which displayed no significant caspase-3 activation, in agreement with Jaenicke et al. (20), served as a negative control.



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  		FIGURE 5. Activation of caspase-3 24 hours postirradiation of A549 cells ({blacksquare}), T98G cells ({circ}), and MCF7 cells ({square}) at a dose rate of 22 cGy/min. In A549 cells, there was a significant difference (P < 0.005) between the unirradiated control and each of the 10, 15, and 100 cGy dose responses. For T98G cells, there was a significant difference between the control and the 10 and 20 cGy doses (P < 0.05). For both A549 and T98G cells, the 10 and 20 cGy dose responses are significantly different from the dose response of MCF7 cells (P < 0.0001). Points, data accumulated from 7 to 10 independent repeats; bars, SE. P values were calculated by t test or ANOVA as appropriate. See Materials and Methods for details of the assay.

 
A second marker of apoptosis is the translocation of phosphatidylserine to the exterior of the cell membrane, which can be detected by binding of fluorescently tagged Annexin V (23). Transfer of phosphatidylserine is considered an early-to-intermediate event in the apoptotic process (24); consequently, the Annexin V assay has been applied at times after cellular insult ranging from 2 to 24 hours. We therefore examined Annexin V binding to A549 cells at 0, 5, 15, and 25 cGy postirradiation for times up to 24 hours. The response (Fig. 6A and B) indicated optimum binding at 4 hours. Similarly, we observed optimum binding to T98G cells at 6 hours postirradiation (data not shown). No significant Annexin V binding was seen with MCF7 cells at any time up to 24 hours. A more complete dose response at 4 hours (A549 and MCF7) and 6 hours (T98G) is shown in Fig. 6C. In the same assay, the cells were also stained with propidium iodide (PI), which is used to gauge cell permeability as a measure of necrosis. The irradiated cells (A549, T98G, and MCF7) showed no significant increase in uptake of PI in comparison with unirradiated cells at the doses at which HRS was observed (data not shown). We also examined the cell cycle status of the A549 cells binding Annexin V 4 hours post–10 cGy irradiation (~4% of the total irradiated cell population) in comparison with the cell cycle status of cells in the total irradiated population (Fig. 6D). The data indicate that the cell cycle distribution of irradiated cells that bound Annexin V was fairly similar to the distribution of the total irradiated cell population. The most significant difference between the two populations was the proportionally low level of G2-M cells binding Annexin V (6% versus 12%) and a concomitant increase in the percentage of cells in G1 binding Annexin V (43% versus 38%).



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  		FIGURE 6. Translocation of phosphatidylserine as determined by the Annexin V-FITC binding assay after irradiation at 22 cGy/min (see Materials and Methods for experimental details). A. Representative FC plots of A549 cells stained 2, 4, 8, or 24 hours postirradiation with 0, 15, or 25 cGy. Annexin V quantitation is based on the percentage of cells in the bottom right quadrant. B. Plot of the time course for Annexin V binding based on five independent assays. Significant differences (see below) are indicated. P values were calculated by t test. C. Dose response for Annexin V-FITC binding to A549 cells ({blacksquare}), MCF7 cells ({square}), and T98G cells ({circ}). A549 and MCF7 cells were examined 4 hours postirradiation and T98G cells 6 hours postirradiation. For the A549 dose-response curve, P ≤ 0.002 at 10 and 20 cGy when compared with the unirradiated control. Values at 50 and 100 cGy are also different from control (P < 0.02). T98G cells had significantly more Annexin V bound after 10 and 20 cGy doses (P < 0.01) than they did prior to irradiation. By ANOVA, radiation doses of 10 and 20 cGy to A549 and T98G cells caused significantly more Annexin V binding than in MCF7 cells (P < 0.005). Points, means of the values obtained from 9-14 (A549), 5 (MCF7), and 7-11 (T98G) independent experiments; bars, SE. P values were calculated using a t test. Significant differences are marked. D. Cell cycle status 4 hours postirradiation (10 cGy) of the total population of A549 cells compared with the status of the subpopulation binding Annexin V-FITC. Significant differences between total cells and Annexin V binding cells are marked. Bars, SE. P values were calculated by t test.

 
Role of p53 in the HRS Apoptotic Response
The tumor suppressor p53 is considered a key regulator of the apoptotic pathway (25). All three tumor cell lines used in this study express p53, although p53 in T98G cells is mutated at Met-237. We therefore examined the potential participation of p53 in the initiation of the apoptotic response by irradiation of the cells in the presence of pifithrin, an inhibitor of p53-mediated apoptosis (26), and then measured Annexin V binding 4 hours (A549 and MCF7) and 6 hours (T98G) postirradiation. The data (Fig. 7) show that 3 µmol/L pifithrin eliminated the Annexin V-FITC signal observed previously at the HRS doses with A549 and T98G cells. We followed up this experiment by testing the influence of pifithrin on HRS itself. Figure 8 reveals that treating A549 and T98G cells with pifithrin completely ablated HRS. Finally, we compared the low-dose hypersensitivity of HCT116 p53+/+ cells and p53–/– cells generated by targeted disruption of the p53 alleles in HCT116 cells (27). The survival curves (Fig. 9) confirm the association of active p53 with HRS.



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  		FIGURE 7. Influence of the p53 inhibitor pifithrin at 3 µmol/L ({square}) compared with no pifithrin ({blacksquare}) as determined by the Annexin V binding assay. *, P < 0.05, significantly different from controls. Points, mean of three to four independent determinations; bars, SE. Top, A549 cells; middle, T98G cells; bottom, MCF7 cells.

 


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  		FIGURE 8. Effect of 30 µmol/L pifithrin (PFT) on A549 and T98G cells as measured by a low-dose radiation dose-response colony-forming assay. n is at least three separate experiments for each point. Bars, SE.

 


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  		FIGURE 9. Survival of p53+/+ (#8 p53wt) and p53–/– (#2 p53null) HCT116 cells to low-dose radiation measured by colony-forming assay. n is at least three separate experiments for each point. Bars, SE.

 
HRS in Human Fibroblasts
Because tumor cell lines by their nature often possess more than one mutation that may affect apoptotic pathways, we sought to further confirm our results in noncancerous and nontransformed human cells. To date, such cells have not been extensively examined for HRS, the only report being that of a study of L132 human epithelial cells (28). Consequently, we analyzed the HRS and apoptotic responses of three human fibroblast strains, CRL2522, GM38, and 2800T. The first two possess wild-type p53 activity, whereas 2800T cells lack active p53 (29). The results, shown in Fig. 10, indicate that the two normal cell lines displayed a typical HRS response as well as Annexin V binding and elevated caspase-3, but the 2800T cells showed no indication of HRS or apoptosis.



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  		FIGURE 10. Responses of human fibroblasts to low-dose radiation. Top, colony-forming assay; middle, Annexin V-FITC binding; bottom, anti–active caspase-3 binding. There were at least three determinations for each point. Bars, SE.

 

   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 Acknowledgements
 References
 
The GMD/FC assay was found to be an efficient method to follow initial proliferation of single A549, T98G, and MCF7 cells into GMD encapsulated microcolonies, the evolution of which could be followed with or without prior exposure to radiation. The advantage of this technique, as with other FC-based assays, is the capacity to analyze a relatively large number of individual cells or, in this case, GMDs. (We were unable to apply this approach to normal human fibroblasts because of a tendency for these cells to escape the microdrops.)

In A549 cells exposed to 0 to 100 cGy at 22 cGy/min, the GMD/FC data indicate a nonlinear, nonmonotonic dose survival response at the lower doses (i.e., there was significant hypersensitivity at 10 to 17.5 cGy). If combined data for the 10, 15, and 17.5 cGy exposure groups are excluded, goodness-of-fit analysis indicates that all other data points obtained are consistent with a linear-quadratic fit (P = 0.28; i.e., no significant departure from the pattern predicted by a linear-quadratic fit), whereas that fit is rejected if the 10 to 17.5 cGy data are included (P {approx} 0; Fig. 1). Similar results were also obtained for T98G cells in GMDs, but no radiation hypersensitivity was observed in similarly exposed MCF7 cells (Fig. 1). These findings are consistent with recent evidence of low-dose hypersensitivity in many tumor cell lines (but not in MCF7 cells) exposed chronically to low-dose low linear energy transfer radiation reported by Joiner et al. (8) and by M.C. Joiner (personal communication), who have applied automated imaging techniques to increase the ability of the classic colony-forming assay to detect small reductions in clonogenic survival at low-level irradiation. The GMD/FC data as well as the apoptosis results show that hypersensitivity in A549 and T98G cell lines occurs during the first cell cycle after low-dose irradiation and argue against the possibility that HRS is the result of a longer-term consequence, such as accrual of chromosome damage in descendents of the irradiated cells seen after high-dose irradiation (30).

In addition to examining cell proliferation at 22 cGy/min, we also looked at the response to radiation delivered at a 100-fold lower dose rate (0.18 cGy/min). This dose rate is equivalent to ~10 cGy/h, which is the dose rate estimated by Fowler (31) to be the maximum dose intensity to tumor cells that can be achieved with radioimmunotherapy. As mentioned in Introduction, a lower dose rate would be expected to be more effective at inducing HRS if a phase of the cell cycle was associated with HRS, because more cells would have the opportunity to move through this phase of the cell cycle during irradiation (6). This approach to analyzing HRS dependence on cell cycle status has been proven inconclusive, because neither the A549 nor the T98G cells showed such an enhancement of HRS at the lower dose rate (Fig. 1).

Our examination of the effects of low doses of radiation on cell cycle checkpoints (Figs. 3 and 4), including expression of key proteins, failed to reveal any indication that the doses inducing maximal HRS had a different effect on the cell cycle than the other doses <50 cGy. This is in general agreement with others (14, 32), although Hendrikse et al. (33) reported a modest arrest in G2-M, but no change in cyclin B1, after irradiation of a lymphoblast cell line (TK6) with 10 and 30 cGy.

To date, there has been no clear identification of the mode of death—necrosis, apoptosis, or senescence—of the cells dying at low dose. Part of the problem has been identifying the fate of a small component of the cell population. We chose to use two well-characterized and independent markers of apoptosis, caspase-3 activation and Annexin V binding to translocated phosphatidylserine, because both could be monitored by FC, a technique that measures cell characteristics on a cell by cell basis. Our data indicate that apoptosis plays a role in the death of A549 and T98G cells after low-dose radiation (Figs. 5 and 6). Although the percentage of apoptotic cells identified by FC was small, the data were consistent over the many times that each assay was repeated. Further credence can be given to the data because (a) neither assay identified an increase in apoptotic MCF7 cells, in line with its lack of HRS response noted above, and (b) the apoptotic response seen with A549 and T98G cells closely correlated with the dose response seen for HRS. We are aware that others have used a similar approach to look for apoptosis in cells displaying HRS without success (14). We are not sure why these assays failed to reveal apoptosis, but one possibility may lie in the timing of the assays after irradiation. For example, we observed that the difference in Annexin V binding between irradiated A549 cells and control cells reached a maximum at 4 hours and was negligible by 24 hours (Fig. 6B), the latter unfortunately being the time at which others (14) looked for binding.

The tumor suppressor protein p53 is involved in ionizing radiation–induced caspase-3 activation and apoptosis at 4 Gy (34). To examine whether p53 played a role in the low-dose apoptosis, we employed a p53 inhibitor, pifithrin, which was originally identified by screening a chemical library for inhibitors of p53 transactivation and then for suppression of p53-dependent apoptosis (26). A549 cells express wild-type active p53 (35) and T98G cells express p53 with a point mutation in codon 237 (36). Although this point mutation severely reduces the transactivation of several genes, including CDKN1A (p21WAF1,Cip1), it does not seem to suppress p53-dependent apoptosis in response to agents such as camptothecin (37). The loss of Annexin V binding to cells irradiated in the presence of pifithrin (Fig. 7) provided evidence for p53 involvement in the low-dose apoptosis. This was confirmed by showing (a) that pifithrin ablated HRS in A549 and T98G cells (Fig. 8) and (b) that p53-inactive human cell lines did not display HRS, whereas p53 wild-type controls had normal HRS responses (Figs. 9 and 10).

We have provided evidence that low-dose irradiation of A549 and T98G cells giving rise to HRS does not lead to readily discernable cell cycle arrest but induces p53-dependent apoptosis. When we compared the cell cycle status of the subpopulation of A549 cells binding Annexin V with the total 10 cGy irradiated cell population (Fig. 6D), we observed that the overall difference between the two populations was not substantial and was primarily confined to a decreased proportion of cells in G2-M binding Annexin V and a concomitant increase in Annexin V binding cells in G1. These measurements were taken 4 hours postirradiation. Because this time is too short for the cells to traverse more than one phase of the cell cycle (e.g., from G2-M to S), this would suggest that the apoptosis responsible for HRS can be triggered by irradiation in any phase of the cell cycle. At first sight, this would seem to contradict the observation by Short et al. (12) of a more pronounced HRS/IRR response in cell populations enriched for G2 at the time of irradiation in comparison with populations enriched for G1 or S phase. However, both sets of observations can be reconciled if the ATM-dependent early G2 checkpoint (13), proposed by Marples and colleagues (9, 10) to mediate the switch from HRS to IRR, regulates p53-dependent apoptosis. If cells pass through this checkpoint relatively soon after irradiation and if their DNA damage is repairable, the checkpoint would serve to prevent the apoptosis. On the other hand, cells reaching the checkpoint at later times would be further along the apoptotic pathway and less responsive to stop signals. Thus, a population of cells enriched for G2 when irradiated would have to traverse the complete cell cycle before encountering the checkpoint and therefore undergo higher levels of apoptosis than a population enriched for G1 or S. This explanation would also account for the comparatively low level of G2 cells binding Annexin V seen in Fig. 6D because a high percentage of these cells would have been in S phase 4 hours earlier when the cells were irradiated.

The dose relationship between HRS and IRR would thus reflect a combination of (a) a stochastic process giving rise to a percentage of cells that receive sufficient damage to elicit p53-dependent apoptosis before passing through the early G2 checkpoint and (b) a potential dose-dependent activation of the checkpoint. In their description of the early G2 checkpoint found in HeLa cells, Xu et al. (13) noted a diminution in the activity of the checkpoint at doses <40 cGy; thereafter, activity seems to be dose independent. Such a "threshold" dose would probably vary from cell line to cell line, which may explain, at least in part, the variation in HRS/IRR responses seen among different cell lines.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
Acknowledgements
 References
 
Reagents
Cell culture reagents included DMEM:Ham's F12 nutrient mix, Ham's balanced salt solution, L-glutamine, trypsin, and FCS (all from Invitrogen, Grand Island, NY). General cell culture–grade chemicals were obtained from Sigma Chemical Co. (St. Louis, MO). Anti-mouse and anti-rabbit secondary antibodies (horseradish peroxidase–conjugated) were supplied by Santa Cruz Biotechnology (Santa Cruz, CA) and cyclin B1 and cdk1 were from BD Biosciences (Mississauga, Ontario, Canada). An enhanced chemiluminescence detection kit was purchased from Amersham (Baie d'Urfe, Quebec, Canada) as was the Kodak X-OMAT AR autoradiographic film. Annexin V-FITC and a caspase-3 detection kit were obtained from BD Biosciences. Pifithrin was purchased from Biomol (Plymouth Meeting, PA).

Cell Culture
Human A549 lung adenocarcinoma cells, T98G glioma cells, and MCF7 breast carcinoma cells were obtained from American Type Culture Collection (Rockland, MD); normal human fibroblasts, CRL2522 and GM38, were purchased from the National Institute of General Medical Sciences Human Genetic Cell Repository (Camden, NJ); the p53–/– human colorectal carcinoma cell line, HCT116 #2, and its p53+/+ parent cell line, HCT116 #8, were kindly supplied by Dr. Bert Vogelstein (Howard Hughes Medical Institute, Johns Hopkins Medical Institutions, Baltimore, MD); and the p53-inactive 2800T human fibroblasts were kindly provided by Dr. Razmik Mirzayans (Cross Cancer Institute, Edmonton, Alberta, Canada). All cells were grown in DMEM:Ham's F12 with 10% FCS at 37°C and 5% CO2.

GMD Production
Cells in GMDs were prepared for each assay using a microdrop maker (One Cell Systems, Cambridge, MA) as described by Bogen et al. (18). Briefly, cells were trypsinized, centrifuged, and resuspended in PBS with 0.5% FCS at 0.6 x 106 to 1.0 x 106 cells per 100 µL. After filtering the cells through a 20 µm nylon mesh (Small Parts, Miami Lakes, FL), 100 µL were added to 0.5 mL CelGel Encapsulation Matrix (One Cell Systems) together with 100 µL FCS and 10 µL Pluronic F68 (Sigma Chemical). This suspension was added to 15 mL CelMix Emulsion Matrix (One Cell Systems) and then emulsified using the GMD maker (1,200 x g at room temperature for 1 minute, 1,200 x g at 4°C for 1 minute, and 600 x g at 4°C for 8 minutes). Resulting GMDs were washed three times with Ham's balanced salt solution, filtered through a 74 µm mesh, and resuspended in culture medium and either incubated or irradiated and incubated.

Radiation Exposures
We used two different radiation dose rates for these experiments to give doses from 0 to 200 cGy. Encapsulated cells were irradiated at 0.18 cGy/min using a 137Cs source (Picker International, Cleveland, OH) while being incubated at 37°C with 5% CO2 or at 22 cGy/min in a Mark I 137Cs irradiator (J.L. Shepherd, San Fernando, CA) for relatively short times under ambient conditions.

GMD/FC Assay of Colony Formation
At 96 hours postencapsulation, exposed and control GMDs were washed and resuspended in PBS and live/dead stain (2.5 µmol/L ethidium homodimer, 75 nmol/L calcein AM, Molecular Probes, Eugene OR). In each GMD/FC assay, 104 cell-bearing GMDs were analyzed by FACSort FC using CellQuest Software (Becton Dickinson, San Jose, CA). Linear forward scatter versus log-scale side scatter plots were obtained as well as FL1 (log) versus FL2 (log) plots (calcein AM versus ethidium homodimer, respectively) for live/dead–stained GMDs. Dot-plot regions defining GMDs containing single (S) versus multiple (M) cells were assigned based on corresponding S/M ratios estimated by microscopic analysis of control and treated GMDs at 96 hours postencapsulation. GMDs with single or multiple cells inside can be distinguished on a forward scatter-side scatter dot-plot because they define different GMD populations based on the amount of scatter caused by the varying number of cells constituting the microdrop. From each GMD/FC analysis, the estimated fraction, FM = [M / (S + M)], of occupied GMDs that each contain a live microcolony was normalized to reflect a corresponding fraction relative to untreated FM measured using unexposed concurrent control GMDs. Typically, FM of unirradiated controls was 85% to 90% after 4 days in culture.

Conventional Colony-Forming Assay
Tumor cells were plated at 100 cells per 60 mm tissue culture dish, irradiated 4 hours postseeding, and incubated for 8 to 10 days. Fibroblasts were plated at 300 cells per 100 mm culture dish and irradiated after 18 hours of incubation. For colony-forming assays requiring pifithrin, cells were incubated with 30 µmol/L pifithrin for 16 to 20 hours prior to irradiation and incubated a further 24 hours in the presence of the drug. After 2 to 3 weeks, surviving cells forming visible colonies (containing >50 cells) were counted after staining with crystal violet in 60% methanol.

Data Analysis
Analysis of growth data plotted in Fig. 1 was done using constrained least squares linear regression and associated F tests for fit; all these calculations were done using Mathematica 4.2 software (Wolfram, Champaign, IL; refs. 38, 39). To facilitate these analyses, regressions were done using untransformed (rather than log transformed) y-axis (fractional) data values, because log transformation is very nearly linear in the range of the y-axis data obtained. Mean and SE were calculated with reference to untreated controls to analyze data shown in Figs. 2 to 10. To test statistical significance of response differences, we did two-tailed t tests or ANOVA after in each case confirming variance homogeneity by F test using the Prism version 3.03 graphics and statistics software package (GraphPad, San Diego, CA). Results were considered significant if P ≤ 0.05.

Cell Cycle Analysis
Cell cycle analysis was done on A549 cells after exposure to 0, 5, 10, and 20 cGy. Cells were seeded at 0.5 x 106 cells per dish, grown overnight, irradiated, and harvested at appropriate times up to 24 hours. This consisted of trypsinization, fixation in 95% ethanol, and treatment with 10 µg/mL RNase and 5 µg/mL PI. FC was used to analyze the resulting stained DNA and the relative numbers of cells in each phase of the cell cycle were ascertained by ModFit software (Verity Software House, Topsham, ME).

Preparation of Cell Lysates
Cells were trypsinized and washed in PBS at 4°C. The pellet was resuspended in a modified radioimmunoprecipitation assay buffer [150 mmol/L NaCl, 50 mmol/L Tris-HCl (pH 7.4), 1% NP40, 0.25% sodium deoxycholate, 1 mmol/L EGTA, 1 mmol/L sodium orthovanadate, 1 mmol/L NaF, 1 mmol/L phenylmethylsulfonyl fluoride, and protease inhibitor cocktail diluted 1:100]. Cells were sheared with a 22 gauge needle, incubated on ice for 30 minutes, and centrifuged at 13,000 x g for 20 minutes at 4°C. The supernatant lysate was decanted into fresh tubes and stored frozen at –80°C.

Western Blots
Immunoblotting was done on lysates of A549 cells that had been irradiated at 0, 5, 10, and 20 cGy and either harvested immediately or incubated for appropriate times. After blocking with PBS-Tween 20 with 5% nonfat milk, the nitrocellulose blots were probed with various monoclonal and polyclonal antibodies, and after washing, reactions were visualized by enhanced chemiluminescent detection of horseradish peroxidase–conjugated secondary antibodies. Bands on the autoradiographs were quantified with a digitized image analyzer (40).

Apoptosis Assays
Caspase-3. Cells were seeded in 60 mm culture dishes, grown overnight, and irradiated at 0 to 100 cGy at 22 cGy/min. Active caspase-3 levels were determined using an antibody kit developed by BD Biosciences. Briefly, cells are washed, fixed, permeabilized, and incubated with a FITC-conjugated caspase-3 antibody for 30 minutes. Relative amounts of active caspase-3 were quantified by FC following the protocol of Belloc et al. (41).

Annexin V. Cells were seeded in 60 mm culture dishes, grown overnight, and irradiated at 0 to 100 cGy at 22 cGy/min. Trypsinized and washed cells were treated with PI and Annexin V-FITC, which binds to phosphatidylserine translocated to the exterior of the cell membrane early in the apoptosis pathway as well as during necrosis. Determinations were made 4 hours after radiation doses to A549 and MCF7 cells and 6 hours postirradiation of T98G, 2800T, GM38, and CRL2522 cells. The assay was done as outlined in the Annexin V-FITC kit protocol of the manufacturer (BD Biosciences) and cells were counterstained with PI to distinguish apoptosis from necrosis. FC was employed to visualize the bound FITC, and necrotic cells (which were stained with both PI and FITC) were gated out so that an accurate determination of the percentage of apoptotic cells could be made (23).

Inhibition of p53 With Pifithrin. Cells were treated with 3 or 30 µmol/L pifithrin for 2 hours prior to irradiation and the Annexin V assay was done as described above.

Cell Cycle Status of Annexin V-FITC Binding Cells. A549 cells were treated as above for the Annexin V binding assay and then fixed in 75% ethanol for 30 minutes at –20°C. The fixed cells were washed twice with PBS and then stained with 5 µmol/L PI in PBS with 2 µg/mL RNase for 30 minutes in preparation for FC. During FC, we gated on the fraction of cells that had bound Annexin V-FITC and determined their cell cycle status (by examining their content of DNA stained with PI), comparing them with the cell cycle status of cells in the total cell population.


   Acknowledgements
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
References
 
We thank Drs. Brian Marples (Wayne State University, Detroit, MI) and David Murray (Cross Cancer Institute) for their helpful discussion.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 
1 U.S. Department of Energy, University of California Lawrence Livermore National Laboratory contract W-7405-Eng-48 (K.T. Bogen), National Cancer Institute (Canada) grant 013104 (M. Weinfeld), Alberta Cancer Board Bridge and Pilot grant R-418 (A.D. Murtha), and U.S. Department of Energy Low-Dose Radiation Research Program (K.T. Bogen). Back

3 M.C. Joiner, personal communication. Back

Received April 19, 2004; revised August 31, 2004; accepted September 1, 2004.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 Acknowledgements
 References
 

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