Mechanisms of suppression of neoplastic transformation in vitro by low doses of low LET radiation

M. C. Pant1, X.-Y. Liao1, Q. Lu1, S. Molloi2, E. Elmore1 and J. L. Redpath1,3

1 Department of Radiation Oncology and 2 Department of Radiological Sciences, University of California Irvine, Irvine, CA 92697, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Suppression of neoplastic transformation of HeLa x skin fibroblast human hybrid cells in vitro following low doses of low linear energy transfer radiation has been reported previously. The present study represents an exploration of two hypothesized mechanisms that may underlie this observed suppression. These are the up-regulation of reduced glutathione (GSH), a known antioxidant, and induction of DNA repair activity. The hybrid cells were found to have a high endogenous level of GSH and no induction following low doses of 60 kVp X-rays was observed. Buthionine sulfoximine (BSO), a GSH biosynthesis inhibitor, completely suppressed GSH levels in both unirradiated and irradiated cells. Furthermore, there was no significant impact of BSO-induced suppression of GSH on the neoplastic transformation frequency of either unirradiated or low dose irradiated cells indicating that glutathione levels play no role in the low dose suppression of transformation frequency. To assess the possible role of DNA repair in the low dose suppression of transformation the effect of 3-aminobenzamide (3-AB), a poly-ADP-ribose polymerase (PARP) inhibitor was examined. In these experiments, there was no significant effect of 3-AB on the transformation frequency at a dose of Cs-137 gamma rays of 0.5 cGy, however, at a dose of 5 cGy there was a significant increase (P < 0.05) in the transformation frequency in the presence of 3-AB. These findings suggest that the influence of DNA repair on the low dose suppression of transformation is significant at a dose of 5 cGy, but not at the lower dose of 0.5 cGy.

Abbreviations: 3-AB, 3-aminobenzamide; BSO, buthionine sulfoximine; DAPI, 4,6-diamidino-2-phenylindole; DSB, double strand break; GSH, glutathione; HRS, hyper-radiosensitivity; IAP, intestinal alkaline phosphatase; IRR, induced radioresistance; LET, linear energy transfer


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Suppression of neoplastic transformation in vitro below spontaneous levels by low doses of low linear energy transfer (LET) radiation has been reported. This was initially described in C3H 10T1/2 cells in the context of an adaptive response where an initial low dose of ionizing radiation resulted in a reduction of the effectiveness of a subsequent challenge dose (1). The same research group subsequently demonstrated that exposure to low doses of ionizing radiation (0.1–10 cGy) resulted in the suppression of the transformation frequency of C3H 10T1/2 cells to levels significantly below that seen for spontaneous transformation of unirradiated cells (2). These latter studies were performed with a clone of C3H 10T1/2 cells that had an unusually high spontaneous transformation frequency, perhaps indicative of genetic instability. The HeLa x skin fibroblast human hybrid cell system, a more genetically stable hybrid cell line with a lower spontaneous transformation frequency, has also demonstrated such suppression of transformation following low doses of Cs-137 gamma radiation (3,4). Recently, we have reported a similar finding with diagnostic energy X-rays (5).

These findings have been described as a type of adaptive response induced by low radiation doses (14). Adaptive response following low dose radiation is more typically demonstrated by the reduced effect of a subsequent high challenge dose and this has been demonstrated both in vitro and in vivo using a variety of endpoints (for review see ref. 6). Mechanistic studies have demonstrated that such an adaptive response is abrogated by cyclohexamide, an inhibitor of protein synthesis, and by 3-aminobenzamide (3-AB), an inhibitor of poly ADP-ribose polymerase, an enzyme involved in DNA damage repair (e.g. ref. 7). DNA repair activity has also been directly shown to be up-regulated by low dose radiation (8). Glutathione (GSH) is a known antioxidant and radioprotective agent and recent studies in mouse splenocytes have shown that endogeneous levels can be raised by low doses of gamma rays (9). In view of the above findings, we decided to test whether GSH-induction and/or DNA repair were involved in the suppression of transformation in vitro by low doses of low LET radiation.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cell system
The non-tumorigenic HeLa x skin fibroblast cell line CGL1 (10) was maintained in Eagle's minimum essential medium supplemented with 5% heat-inactivated calf serum, 2 mM glutamine, non-essential amino acids and 100 IU/ml penicillin (growth medium). Cultures were incubated in 5% CO2 in air at 37°C and routinely tested for mycoplasma contamination by the 4,6-diamidino-2-phenylindole (DAPI) assay (11). Cells in the population doubling number range of 43–45 were used. Because of the large scale of the experiments, two separate lots of serum had to be used for the studies with 3-AB. A difference between the data obtained with each serum lot was noted, however, the relative differences between each treatment arm were similar for each lot of serum and the data were therefore pooled for analysis.

60 kVp X-irradiation
For X-irradiation, the cells were irradiated with 60 kVp X-rays delivered using a conventional X-ray tube (Dynamax 79-45/120, Machlett Laboratories, Stamford, CT) and a constant X-ray generator (Optimus M200, Philips Medical Systems, Shelton, CT) operated at 60 kVp. The current used was 100 mA with varying pulse widths, depending on the dose to be delivered. Dosimetry was performed using a 60 cc ionization chamber (Model 20 x 6-6, Radcal Co., Monrovia, CA).

Cs-137 gamma irradiation
For gamma irradiation the cells were irradiated in a J.L.Shepherd and Associates Mark I self-shielded 137-cesium {gamma}-irradiator. The dose rate can be adjusted by the use of attenuators. For the 3-AB study, doses of 0.5 and 5 cGy were used with dose rates of 0.29 and 1.05 cGy/min, respectively. The impact of this variation in dose rate on the response of the cells would not be expected to be great over this low dose range.

GSH measurement
Intracellular glutathione (GSH) was quantified by the glutathione reductase-DTNB recycling assay as described by Vandeputte et al. (12) with modifications as described by Cen et al. (13). Four hours following completion of treatment (radiation ± BSO), the cells were trypsinized, counted and washed twice with PBS. Cell lysates were prepared by adding 200 µl of 10 mM HCl to the pellet. The sample was freeze-thawed three times using liquid nitrogen and then stored at -80°C until analyzed. Prior to analysis, 40 µl was removed for protein measurement using the microwell protocol with the Pierce BCA Protein Assay Kit, Pierce Technology, Rockford, IL. The remaining lysate (160 µl) was mixed with 40 µl of 5-sulfosalicylic acid (6.5%) for 10 min in microfuge tubes to precipitate the proteins. The tubes were centrifuged at 200 g for 15 min at 4°C. The supernatant was stored at -80°C in microfuge tubes until tested. The GSH assay was conducted in 96-well culture plates using a microtiter plate assay. All reagents, including the GSH standard, were prepared as described previously (12). The assay was conducted in a Spectramax micro-plate reader (Molecular Devices, Sunnyvale, CA). The rate of OD change was measured at 415 nm and the amount of GSH determined from the standard curve. The amount of GSH was expressed as nanomoles per milligram protein.

Buthionine sulfoximine treatment protocol
Since the buthionine sulfoximine (BSO) treatment was for 24 h before and after irradiation, we needed to determine a concentration of BSO that would be non-toxic over a 48-h period. This was done by exposing cultures at the density used in the transformation protocol to BSO concentrations of 0.01, 0.1 and 1.0 mM for 48 h and then plating for colony forming assay. The results indicated that a concentration of 0.1 mM had minimal toxicity under these conditions and this was the concentration used for all of the BSO experiments in this study.

For BSO treatment, standard medium was replaced by BSO (0.1 mM) contained in fresh medium 24 h prior to irradiation. At the same time, the control flasks had their media replaced by fresh medium. The flasks were then irradiated with Cs-137 gamma radiation to a dose of either 0.1 or 4 cGy, or sham-irradiated. The cells were placed at 37°C for a 24 h holding period before seeding for the transformation assay.

3-AB treatment protocol
For all studies, preparation of 3-AB solutions was done under yellow light conditions due to its light sensitivity. Flasks containing cell cultures were also wrapped in aluminum foil until treatment was completed.

Since the transformation protocol involves a 24-h post-irradiation holding period, we needed to determine a concentration of 3-AB that would be non-toxic over such a period. This was done by exposing cultures at the density used in the transformation protocol to 3-AB concentrations of 1 and 5 mM for 24 h and then plating for colony forming assay. The results indicated that a concentration of 1 mM had minimal toxicity under these conditions and this was the concentration used for all of the 3-AB experiments in this study.

For 3-AB treatment, standard medium was replaced by 3-AB (1 mM) contained in fresh medium 30 min prior to irradiation. At the same time, the control flasks had their media replaced by fresh medium. The flasks were then irradiated with Cs-137 gamma radiation to a dose of either 0.5 or 5 cGy, or sham-irradiated. The irradiator room was darkened and the aluminum foil was removed only for irradiation, and replaced after irradiation was completed. The cells were placed at 37°C for a 24 h holding period before seeding for the transformation assay.

Quantitative assay of neoplastic transformation
Cells were seeded at 75,000 cells per T-75 flask. Three days later the culture was subconfluent. At this time, the flasks containing the cultures were either irradiated or sham-irradiated. Following treatment, the cultures were placed at 37°C for a 24-h holding period. Following holding, the cells were subcultured and plated at low cell density into T-75 flasks for assay of clonogenic survival at 8 days post-irradiation, and at higher cell density into T-75 flasks for assay of neoplastic transformation at 25 days post-irradiation. The cultures were re-fed twice weekly after the first week. Neoplastic transformation was assessed by staining for foci expressing cell surface intestinal alkaline phosphatase (IAP) using the Western Blue staining method that we developed (14). IAP is a marker for neoplastic transformation in this system (15). Our previous studies have shown that the transformation frequency is dependent upon the density of viable cells plated after irradiation (16), and we attempt to control this parameter carefully and aim to plate at a viable cell density of 50 cells/cm2. However, when necessary, we are able to correct for variation in viable cell density as described previously (16). A minimum of three and a maximum of seven separate experiments were performed at each dose using cells of comparable passage number and the same two independent persons scoring IAP-positive foci.

Calculation of transformation frequency
The null method (17,18) was used to calculate transformation frequencies expressed as transformants per surviving cell as well as associated 95% confidence intervals. This method is used to avoid possible errors from satellite colony formation as a consequence of the multiple re-feedings, which cultures must receive during the 25-day post-irradiation expression period.


    Results
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 Abstract
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 Materials and methods
 Results
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 References
 
Lack of induction of GSH by low doses of 60 kVp X-rays in HeLa x skin fibroblast hybrid cells
No induction of GSH following low dose X-ray irradiation was observed (Figure 1). When BSO, an inhibitor of GSH biosynthesis (19) was added to the control and the 0.1 cGy group, GSH levels were, as expected, almost totally suppressed (Figure 1).



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Fig. 1. Glutathione (GSH) levels in nanomoles per milligram of protein in unirradiated and irradiated HeLa x skin fibroblast human hybrid cells (CGL1) with and without treatment with 0.1 mM BSO. Errors are ±1 standard deviation.

 
Lack of effect of suppression of GSH by BSO on transformation frequencies at low doses of 60 kVp X-rays
There was no impact of BSO-induced suppression of GSH on the transformation frequency at low doses (Table I and Figure 2). Importantly, the previously observed suppression of transformation at 0.1 cGy and lack of suppression at 4 cGy of 60 kVp X-rays (5) was seen.


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Table I. Effect of BSO (0.1 mM) on neoplastic transformation of HeLa x skin fibroblast hybrid cells induced by low doses of 60 kVp X-raysa

 


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Fig. 2. The effect of BSO treatment on radiation-induced neoplastic transformation of unirradiated and irradiated HeLa x skin fibroblast human hybrid cells (CGL1). Errors are ± standard errors of the mean.

 
Effect of 3-AB on transformation frequency at low doses of Cs-137 gamma rays
As we have observed previously (3,4), low doses of Cs-137 gamma rays showed a trend, albeit not significant in the current experiments, towards suppression of transformation frequency compared with the unirradiated control (Table II and Figure 3). There was no significant effect of 3-AB on the transformation frequencies with the exception of the 5 cGy dose level where there was a significant increase (P < 0.05) in the transformation frequency.


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Table II. Effect of 3-AB (1.0 mM) on neoplastic transformation of HeLa x skin fibroblast hybrid cells induced by low doses of Cs-137 gamma raysa

 


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Fig. 3. The effect of 3-AB treatment on radiation-induced neoplastic transformation of unirradiated and irradiated HeLa x skin fibroblast human hybrid cells (CGL1). Errors are 95% confidence intervals.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The present study indicates that up-regulation of GSH levels is not involved in the previously reported suppression of neoplastic transformation of HeLa x skin fibroblast human hybrid cells following low doses of low LET radiation (35). On the other hand, the present study does indicate a possible role for up-regulation of DNA repair, since 3-AB was able to abrogate the suppression seen at a dose of 5 cGy. Interestingly, this was not the case at a dose of 0.5 cGy.

The lack of induction of GSH following low dose X-ray irradiation seen in the present study (Figure 1) stands in contrast to a report in the literature (9) but may be explicable by the very high endogenous GSH levels in our unirradiated cells. This finding implies that in these cells modulation of GSH levels by low dose radiation is not involved in the suppression of transformation. The lack of any effect of suppression of GSH levels by BSO on the transformation frequencies seen at low doses (Table I and Figure 2) further indicates that GSH levels do not play a role in the low dose response.

The experiments with 3-AB using gamma radiation were carried out prior to our knowledge of the more powerful suppressive effect of 60 kVp X-rays. This is unfortunate in that the effects of modifiers of any suppressive effect would clearly be better done in the presence of a large suppressive effect. Nonetheless, we believe that the data from experiments of the effect of 3-AB are informative. First, the data points in the absence of 3-AB confirm our previous observation of a trend toward suppression of transformation frequency at multiple low doses in the range 0.1–10 cGy. Indeed, the factor by which the transformation frequency is suppressed is almost identical (1.4–1.5). This confirmation of previous work validates our conclusion that suppression of transformation is operative in the current experiments.

The observation that 3-AB, a potent poly-ADP-ribose polymerase (PARP) inhibitor, had no significant effect on the spontaneous transformation frequency is of interest. This finding implies either that DNA repair has no effect in modulating the background frequency or that 3-AB does not inhibit the specific repair process that does. At the present time we have no way of definitively discerning between these two possibilities.

The finding that 3-AB, a potent PARP inhibitor that is known to abrogate low dose (4 cGy) induced adaptive response using mutation induction as an endpoint (7), significantly abrogated the suppression of transformation at 5 cGy, but not 0.5 cGy (Table II and Figure 3), is indicative of a possible role for DNA repair in the suppression of transformation at the higher dose. It is also of some interest in view of a recent article where evidence for the lack of putative DNA double strand break (DSB) repair in human cells exposed to very low doses (0.05 and 0.12 cGy) of 90 kVp X-radiation was reported (20). On the other hand, at higher doses (>2 cGy), they concluded DNA DSB repair proceeded with kinetics similar to those seen at higher doses (20 cGy). The method used was the appearance and disappearance of nuclear foci of {gamma}-H2AX, a phosphorylated form of histone H2AX formed early on in response to DSB induction (21,22). The authors implied that the lack of foci removal at very low doses (and subsequent proliferation-dependent cell death) represented a lack of repair and suggested that this may represent a protective biological mechanism in terms of carcinogenic risk. The authors indicated that this idea must remain speculative without an evaluation of the mutagenic potential of low doses of ionizing radiation. We, and others, have shown previously such a low dose protective effect for the endpoint of neoplastic transformation in vitro (25), and the present study would support, at least in a qualitative sense, that the role of DNA repair in this process depends on radiation dose, in agreement with the speculation of Rothkamm and Lobrich (20).

This conclusion is reminiscent of low dose studies using cell survival as an endpoint where there is evidence across many cell lines and types for the existence of low dose hyper-radiosensitivity (HRS) followed by an induced radioresistance (IRR) at somewhat higher doses (23). IRR has been correlated with the induction of DNA repair (24). It has been proposed previously (25) that the observed suppression of transformation at low doses could be explained possibly by HRS-related low dose cell kill of a transformation sensitive subcomponent of the overall population. We tested recently this proposal and found that G2 cells are uniquely sensitive to cell kill at low radiation doses (26). This finding, together with our earlier finding that G2 cells represent a subpopulation that is more prone to spontaneous transformation than the remainder of the population (27), was shown to be able to explain the suppression of transformation we have seen at a dose of 5 cGy of Cs-137 gamma radiation.

In view of the fact that the low dose suppression of transformation is seen over a wide dose range (0.1–10 cGy), it is, in our opinion, highly likely there are multiple mechanisms operative and that they assume different importance at different doses. For example, at doses 0.1 cGy and less, where a significant number of cells will not even experience an ionizing event, there is still suppression of transformation (4,5). It is possible under such circumstances that bystander effects (28,29) may assume more importance. Bystander effects can potentially be of a dual nature when it comes to impacting low dose radiation risk (30). If they result in the killing of cells destined to become neoplastically transformed then this could reduce the yield of transformants. On the other hand, if they result in additional mutations in surviving cells (31), then this could increase the yield of transformants. Clearly, the balance of these opposing effects will determine the outcome. On the other hand at somewhat higher doses, it may be that induced repair becomes a more dominant mechanism contributing to the suppression of transformation frequency. Finally, as the dose gets higher (e.g. >10 cGy) induced transforming damage will begin to outweigh any potential suppressive effects and transformation frequencies greater than the spontaneous level will be observed.

In summary, this paper presents evidence that up-regulation of DNA repair is an important mechanism for the suppression of neoplastic transformation in vitro by low doses of low LET radiation, while induction of GSH is not. Inhibition of DNA repair abrogated the suppression of transformation, and this abrogation was more potent at higher (5 cGy) rather than lower (0.5 cGy) doses. In our opinion, it is highly likely that different mechanisms of suppression are dominant at different doses.


    Notes
 
3 To whom correspondence should be addressed Email: jlredpat{at}uci.edu Back


    Acknowledgments
 
This research was supported by DOE Grant No. DE-FG03-02ER63309 and the Phi Beta Psi Sorority.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received June 5, 2003; revised August 1, 2003; accepted September 10, 2003.





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