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Keywords:

  • DNA double-strand breaks;
  • ionizing radiation;
  • γ-H2AX analysis;
  • radiosensitivity;
  • radiotherapy

Abstract

  1. Top of page
  2. Abstract
  3. Immunofluorescence detection of radiation induced DNA DSB
  4. Assessing DSB and their repair in patients after CT examinations
  5. Discussion
  6. References

Ionizing radiation is a powerful tool to treat cancer. The curing effect is mainly based on the efficiency of ionizing radiation to kill the cancer cells and it is believed that DNA double-strand breaks (DSBs) represent the most significant genetic lesion introduced by radiation that causes cell killing. One limitation in radiotherapy is the unavoidable damage delivered to the normal, noncancer cells that can give rise to side effects. The ultimate goal in treatment planning is to maximize cell killing in the tumor by minimizing damage induction in the normal tissue surrounding the tumor. The biological response to the induction of DSBs is largely affected by DSB repair processes and it has, therefore, been a long-standing goal to determine a patient's DSB repair capacity to “individualize” treatment planning. A recently developed DSB repair assay that allows the assessment of patients' repair capacity under in vivo conditions may provide a new approach to predict individuals' responses to radiotherapy and may be able to contribute to improvements in treatment planning. © 2006 Wiley-Liss, Inc.

Radiation therapy, together with surgery and chemotherapy, constitutes an important treatment of malignant diseases. In clinical practice, it follows highly standardized schedules based on long standing experience. These are tailored to the “average patient” and normally do not take into account individuals with an increased radiosensitivity, who are more likely than others to react to radiation exposure with severe, sometimes life threatening, side effects.1, 2 These radiosensitive individuals may amount to about 5–10% of all patients3, 4 and cannot be identified by conventional clinical anamnesis and it has, therefore, been suggested to use “predictive assays”5, 6, 7, 8 to single out those patients who may overreact to radiation exposure. A number of methods have been proposed, e.g. measuring clonogenic survival of cultured fibroblasts or lymphocytes,9, 10 detection of repair deficiencies by applying the comet assay in lymphocytes11, 12, 13 or determination of chromosomal aberrations or micronuclei in lymphocytes.2, 4, 10, 14 The studies displayed variable correlations of the measured parameters with the clinical observations: poor results were reported for clonogenic survival,9, 10 a better trend was observed for chromosomal alterations2, 10, 14 and a reasonably acceptable prognostic power was suggested for the comet assay.3, 11, 12 In general, the analysis of lymphocytes appears to be more promising than studies using fibroblasts.2, 10 However, the most promising approach, the comet assay, requires special laboratory equipment and relies heavily on the experience of the experimenter. Lymphocytes have to be cultured and irradiated in vitro to doses in the range of a few gray.

Although ionizing radiation can lead to many alterations in DNA, there is no doubt that DNA double-strand breaks (DSBs) play an eminent role. This is demonstrated by the fact that cell lines deficient in DSB repair are drastically more radiosensitive in terms of cell killing or mutation induction.15 Moreover, patients carrying a mutation in the ATM gene, which is mutated in the hereditary disorder ataxia telangtiectasia (A-T), exhibit defects in the cellular response to DSBs and are also prone to develop severe side effects in radiotherapy.16 Another link between a defect in DSB repair and cellular as well as clinical radiosensitivity has recently been demonstrated by the identification of mutations in DNA ligase IV in patients with developmental delay and immunodeficiency.17 However, the frequency with which these diseases occur is low and the clinical relevance is limited.18

The classical methods to measure DSBs, e.g. pulsed-field gel electrophoresis, sedimentation analysis or neutral filter elution, suffer from the necessity to require comparatively high doses. The comet assay is more sensitive but still requires doses around 1 Gy, which is higher than the doses delivered to most of the normal tissue during radiotherapy. Moreover, this dose limit restricts the comet assay to in vitro measurements that may only partly reflect the in vivo response to radiation damage. A recently established method based on immunofluorescence detection of individual DSBs19 has the potential of significant improvements for the development of predictive assays.20, 21 The technique is able to quantify DSBs after doses as low as a few milligray22 and can be applied to quantitatively assess DSB induction and repair in lymphocytes of patients after computer tomography (CT) scans,23 which are routinely being performed during the preparation for radiotherapy. There is no need for external irradiation, and it can be investigated whether and how far radiation damage is repaired in vivo, i.e. before blood samples are taken.

Immunofluorescence detection of radiation induced DNA DSB

  1. Top of page
  2. Abstract
  3. Immunofluorescence detection of radiation induced DNA DSB
  4. Assessing DSB and their repair in patients after CT examinations
  5. Discussion
  6. References

The method is based on the detection of a specific histone modification occurring upon induction of DSBs. An early event in the DNA damage response after ionizing irradiation is phosphorylation of serine 139 of the histone H2A variant, H2AX. Phosphorylated H2AX, designated γ-H2AX, extends to megabase regions of DNA around the lesions and can be visualized by using immunofluororescence microscopy as discrete nuclear foci at the site of DSBs, either induced by exogenous agents such as radiation19, 24 or generated endogenously during programmed DNA rearrangements.25, 26, 27 The precise biological function of the foci is still unclear but H2AX phosphorylation is required for the retention of several damage response proteins at the break site.28, 29, 30 Initial studies observed a close correlation between the number of foci and the number of DSBs produced by decay of 125I incorporated into cellular DNA,31 suggesting that each focus may represent an individual DSB and that each break may form a focus. A recent study in our laboratory has compared the number of γ-H2AX foci formed after low doses of ionizing radiation (1 mGy–2 Gy) with the number of DSBs introduced by much higher doses (10–80 Gy) and analyzed by pulsed-field gel electrophoresis, a standard assay for quantifying DSBs (Fig. 1). The observed correlation is close to a 1:1 ratio, strongly suggesting that the number of foci formed after ionzing radiation at low doses is similar to the predicted number of DSBs introduced.22 Because γ-H2AX can also form at sites of single-stranded DNA generated during replication fork stalling or nucleotide excision repair following UV irradiation,32 it is important to note that these studies investigating induction and repair of DSBs after ionizing radiation were performed with nonreplicating cells. This avoids complications from replication and provides conditions under which γ-H2AX foci are specific for DSBs.33

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Figure 1. DSBs are induced linearly with dose between 1 mGy and 100 Gy. Noncycling primary human fibroblasts, MRC-5 cells, were irradiated in vitro (i.e. in cell culture vessels) and analyzed by γ-H2AX immunofluorescence microscopy (IFM) or pulsed-field gel electrophoresis (PFGE) immediately after irradiation. The linear correlation between γ-H2AX foci and DSBs measured by PFGE provides evidence for a 1:1 relationship between γ-H2AX foci and DSBs. Radiation doses between 1 mGy and 100 Gy introduce ˜0.03 to 3,000 DSBs per cell. Doses typically encountered during X-ray examinations (X), CT examinations (CT) or radiotherapy (RT) are indicated above the figure. The range of damage levels that can be examined by IFM, PFGE or chromosomal analyses (CA) are indicated on the right. Figure was modified from Rothkamm and Löbrich.22

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We have recently shown that the rate of loss of γ-H2AX foci at physiological radiation doses correlates with repair kinetics for DSBs as analyzed by pulsed-field gel electrophoresis after high, nonphysiological doses in primary human fibroblasts that are either proficient or deficient for DSB rejoining,22, 34, 35 as well as in rodent cell lines with various deficiencies in DSB repair, including Chinese hamster ovary cells and mouse embryo fibroblasts.35, 36 In all these studies, the degree and nature of the DSB repair defect was remarkably similar between these 2 techniques,37, 38 providing strong evidence that the dephosphorylation of γ-H2AX at the break site coincides with the physical rejoining of the DSB and establishing γ-H2AX foci analysis as a sensitive tool to analyze the repair of DSBs after low radiation doses. A remarkable finding provided by the analysis of cells exposed to different doses is the similarity in the rate of γ-H2AX foci loss irrespective of the initial number of DSBs introduced. For example, primary human fibroblasts proficient or deficient for DSB rejoining show kinetics for γ-H2AX foci loss nearly identical for doses between 20 mGy, corresponding to an average of less than 1 DSB per cell, and 2 Gy, a dose that introduces about 70 DSBs per cell (Fig. 2). Indeed, the doses typically used for pulsed-field gel electrophoresis studies introduce several thousand DSBs per cell (see Fig. 1); yet the breaks are rejoined at a rate that is similar to the rate of repair for an individual DSB.22 This finding is consistent with the model that DSB repair enzymes are in abundant supply such that the rejoining process can start simultaneously at all damaged sites. However, although many DSBs per cell can be efficiently rejoined by the DSB repair machinery, the fidelity of the rejoining process is likely to be compromized after higher radiation doses.40, 41

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Figure 2. The rate of DSB repair is similar for doses between 20 mGy and 2 Gy. Noncycling primary human fibroblasts from a normal person (MRC-5 cells) or from patients with ataxia telangiectasia (AT1BR cells) or the LIG4 syndrome (180BR cells), two hereditary diseases associated with a DSB repair defect and clinical radiosensitivity,17, 34, 39 were irradiated in vitro with doses of 20 mGy, 200 mGy or 2 Gy and analyzed for γ-H2AX foci at the indicated times postirradiation. The similarity in the kinetics of repair suggests that cells process and repair a DSB at a rate that is independent of the presence of additional breaks (i.e. the average repair time for 1 DSB per cell is the same as for 100 DSBs per cell). Data were taken from Kühne et al.34

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Assessing DSB and their repair in patients after CT examinations

  1. Top of page
  2. Abstract
  3. Immunofluorescence detection of radiation induced DNA DSB
  4. Assessing DSB and their repair in patients after CT examinations
  5. Discussion
  6. References

Whilst DSB repair studies had hitherto been restricted to human cells in culture, γ-H2AX foci analysis offered the intriguing possibility to investigate the induction and repair of DSBs in humans. In an ongoing study with the Radiological Clinic in Homburg, we have extended the methodological approach of γ-H2AX foci analysis to cells of the peripheral blood system and were able to detect DSBs in humans that were exposed to diagnostic X-ray doses during CT examinations.23 The analysis of lymphocytes has the advantage that access to the cells is easy and fast and does not require massive medical intervention. CT examinations were chosen to establish the γ-H2AX in vivo approach because this diagnostic procedure is frequently performed, involves a well defined radiation protocol and covers a reasonably broad dose range. This allowed the investigation of normal individuals who were exposed to a biologically relevant dose range. Figure 3 shows the relationship observed between the number of γ-H2AX foci at 30 min after CT and the dose length product, DLP, delivered during CT of the thorax or the abdomen (the DLP is defined as the product of the dose deposited within the exposure field and the length of the body examined). Despite some interindividual variation, a linear relationship is evident, suggesting that the amount of damage introduced is proportional to both the local dose delivered and the length of the body exposed. By comparing these in vivo γ-H2AX foci levels with those obtained by irradiating human lymphocytes in vitro, the DLP values could be transformed into dose index values. This transformation showed that the average damage level in lymphocytes from individuals exposed in vivo with a DLP of 1,000 mGy times cm is similar to that for lymphocytes irradiated in vitro with a dose of about 20 mGy (see Fig. 3). However, the lymphocytes exposed in vivo represent a broader range of cells, including those within the exposure field and unexposed cell. Cells irradiated in vitro, in contrast, will have all received the same dose. These results show that γ-H2AX foci formation can be used to measure the in vivo induction of DSBs after low radiation doses.

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Figure 3. In vivo γ-H2AX foci analysis in lymphocytes from irradiated individuals. Blood samples were taken immediately before and at 30 min after a CT examination and the excess number of foci is shown as a function of the DLP delivered by the CT scan. The linear relationship between γ-H2AX foci and DLP suggests that the amount of DSBs introduced by this diagnostic irradiation procedure is proportional to both the local dose delivered and the length of the body exposed. A comparison between foci levels after in vivo and in vitro exposure provided lymphocyte dose indices for the CT scans (shown on the x-axis above the figure). Figure was modified from Löbrich et al.23

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Analysis of lymphocytes sampled up to 1 day post-CT provided kinetics for the in vivo loss of γ-H2AX foci (Fig. 4a). Kinetics similar to those of Figure 4a were obtained for several normal individuals exposed to DLP values from 150 to 1,500 mGy times cm and the rate of loss of γ-H2AX foci in lymphocytes in vivo was found to be similar to that observed when fibroblasts were irradiated in vitro with similar doses.23 Because foci loss in fibroblasts in vitro has been shown to monitor DSB repair,22, 34, 35, 36 it is suggested that the assay monitors DSB repair in vivo. Importantly, we observed that normal individuals repair the CT-induced DSBs to background levels within 24 hr (Fig. 4b). The conclusion that the γ-H2AX foci loss in vivo monitors DSB repair was further supported by the investigation of a patient with a compromized DSB repair capacity. Fibroblasts established from this patient were analyzed by using pulsed-field gel electrophoresis, γ-H2AX foci analysis and cellular radiosensitivity measurements and were found to exhibit a significant DSB repair defect.23 Strikingly, this patient showed a strongly impaired rate of γ-H2AX foci loss and exhibits a significant level of unrejoined foci at 24 hr post-CT (Fig. 4b). This patient had previously shown exceptionally severe side effects after radiotherapy, which were likely caused by this DSB repair defect. Taken together, these studies establish γ-H2AX foci loss in lymphocytes from exposed individuals as a sensitive technique to quantify the repair of radiation-induced DSBs in vivo.

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Figure 4. (a) Kinetics for the in vivo γ-H2AX foci loss in lymphocytes from 2 representative individuals undergoing CT examinations. Blood samples were taken immediately before (control) and at the indicated times after CT. The patients received different doses and hence showed a different level of induced DSBs; despite this, the rate of foci loss appears to be similar and the amount of damage has returned to background levels after 24 hr post exposure. (b) A series of patients were analyzed for their repair capacity following CT exposure. Except for 1 patient (see the right-most column in this figure), all individuals have returned their γ-H2AX foci to background levels within 24 hr of repair. Intriguingly, this patient showed severe clinical radiosensitivity (see text for a further discussion of this patient). The exposure conditions are given inside the figure. Data were taken from Löbrich et al.23

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Discussion

  1. Top of page
  2. Abstract
  3. Immunofluorescence detection of radiation induced DNA DSB
  4. Assessing DSB and their repair in patients after CT examinations
  5. Discussion
  6. References

Crompton et al.7 discussed in some detail theory and practice of predictive assays and listed important requirements for new approaches to be useful for clinical applications. Although they addressed mainly tumor sensitivity, their arguments are equally valid for the assessment of side effects. The first point to be checked is “clinical relevance,” which has to be seen together with “predictor error.” Both parameters can only be estimated by larger trials that are not yet available for the method described in this report. The number of patients is too small to allow a reasonable answer but the results show nevertheless a correlation between the experimental data and the clinical experience. This justifies more extended tests. Compared to other predictive assays discussed in the literature (see Introduction), the approach suggested in this paper appears to have some advantages:

  • 1
    The assay is based on in vivo irradiation and repair. Because of its exceptional sensitivity, repair can be assessed after CT examinations that are performed prior to radiotherapy. All other methods require that cells (either peripheral blood lymphocytes or fibroblasts) are irradiated and cultivated outside the body. The outcome may thus depend on the specific experimental conditions and may vary from laboratory to laboratory. With the possible exception of the comet assay, the doses employed are comparatively high and do not necessarily reflect those to which normal tissues outside the tumor are usually subjected.
  • 2
    The test can be performed as part of the clinical diagnostic routine. Tumor patients always have to undergo CT examinations and blood counts. If the time schedule is appropriately adjusted, the samples for the assay are “automatically” provided.
  • 3
    The assay is fast, results can be obtained within less than 1 day. It can be incorporated into the clinical routine if the necessary equipment is available and the laboratory staff trained in the experimental method. If laboratories for radiobiology or molecular biology are on-site, as is the case in most major establishments, the evaluation may be performed in cooperation.
  • 4
    The assay is based on the assessment of an individual's capacity to repair a specific DNA lesion, namely a DSB. Although it is not clear whether all (or even the majority of) side effects seen in the clinic after radiotherapy can be attributed to reduced DSB repair, this lesion is probably more significant for most of the radiation effects than any other lesion introduced by ionizing radiation.
  • 5
    There is very little variation in the number of initial foci between different individuals if the values are adjusted according to the DLP of the CT scan. This is not the case with the comet assay where marked differences are seen, particularly at low doses (see, e.g., ref11). These differences may suggest that the comet assay is not specific for the detection of DSBs but is also affected by other factors including changes in chromatin structure. Such radiation-induced alterations, however, may be less important for biological effects that may limit the comet assay's predictable power.

In summary, one may state that the γ-H2AX in vivo assay can be considered to have some potential for predicting severe side effects in radiotherapy. The main shortcoming is that clinical data are presently very limited; the parallelism between clinical complications and measured repair deficiencies is based on only 1 patient. Clearly, more studies are needed before definite conclusions can be drawn. Irrespective of the assay's applicability as a predictive test for patients' responses to radiotherapy, the possibility to assess quantitatively radiation effects in patients who have undergone normal CT scans may open up also other avenues of clinical applications. An example could be the assessments of the level of radiation damage acquired during radioimmuno tumor treatments where physical dose measurements are difficult to obtain. The biodosimetric assessement of extended angiographic procedures may also be helpful in the clinic. Further applications are seen in the detection of alterations in DSB repair genes that are associated with subtle repair deficiencies, including heterozygous mutations and hypomorphic gene variants. These could be uncovered by screening apparently normal persons for their in vivo repair capacity after CT scans followed by a sequence analysis of the relevant DSB repair genes in those persons who exhibit unusual repair kinetics. Alterations in DSB repair genes may render these people sensitive for radiotherapy treatments but could also predispose them to a potentially increased risk for cancer development as it is known that mutations in some DSB repair genes (e.g. in the beast cancer susceptibility genes, BRCA1 and BRCA2, in ATM, or in the gene defective in patients with the Nijmegen breakage syndrome, Nbs1) are related to increased cancer risk.42, 43, 44, 45

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  2. Abstract
  3. Immunofluorescence detection of radiation induced DNA DSB
  4. Assessing DSB and their repair in patients after CT examinations
  5. Discussion
  6. References
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