Initial radiation-induced DNA damage in human tumour cell lines: a correlation with intrinsic cellular radiosensitivity.

The role of the initial DNA double-strand breaks (dsb) as a determinant of cellular radiosensitivity was studied in human breast and bladder cancer cell lines. Cell survival was measured by monolayer colony-forming assay as appropriate and differences in radiosensitivity were seen (alpha-values ranged from 0.12 to 0.54). After pulsed-field gel electrophoresis (PFGE) the initial slopes of dose-response curves were biphasic with a flattening of the curves above 30 Gy. When the frequency of DNA dsb induction was assessed using a mathematical model based on the DNA fragment size distribution into the gel lane, we found a statistically significant relationship between the number of DNA dsb induced and the corresponding alpha-values and fraction surviving after 2Gy (P = 0.0049 and P = 0.0031 respectively). These results support the view that initial damage is a major determinant of cell radiosensitivity.     

Relationship between DNA double-strand breaks, cell killing, and fibrosis studied in confluent skin fibroblasts derived from breast cancer patients

PURPOSE: To investigate the relationship between DNA double-strand breaks (dsbs), cell killing, and fibrosis using skin fibroblasts derived from breast cancer patients who received postmastectomy radiotherapy. METHODS AND MATERIALS: Experiments were performed with 12 lines of normal skin fibroblasts derived from recurrence-free breast cancer patients. Cells were irradiated in confluence and cell survival was determined either after immediate or delayed (14 h) plating using a colony-forming assay. Dsbs were measured by constant-field gel electrophoresis. The "excess risk of fibrosis" was previously scored by Johansen et al. (IJRB 1994;66:407-412). RESULTS: The 12 cell lines showed a typical spectrum of radiosensitivity. The mean value of surviving fraction after 3.5 Gy (SF3.5) was 0.063 for immediate and 0.174 for delayed plating with a coefficient of variation (CV) of 44 and 39%, respectively. There was also a broad variation in the extent of recovery from potentially lethal damage (RPLD), which was not correlated with the immediate sensitivity. The number of initial dsbs as well as the half-times of dsb repair showed little variation, whereas there were considerable differences in the number of residual dsbs (CV = 29%). The number of residual dsbs after 100 Gy was correlated significantly only with SF3.5 after delayed (r2 = O.59; p = 0.006) but not after immediate plating (r2 = 0.21, p = 0.16). There was also no significant relationship between residual dsbs and the "excess risk of fibrosis" determined for the respective patients. CONCLUSION: It is shown that the number of residual dsbs measured in confluent human fibroblast lines can be used to predict the cellular radiosensitivity after delayed but not after immediate plating and also not to predict the excess risk of fibrosis of the respective breast cancer patients.     

Fibroblast radiosensitivity measured using the comet DNA-damage assay correlates with clonogenic survival parameters

A study was made of the neutral comet assay as a potential method for measuring normal cell radiosensitivity. Eleven fibroblast strains were studied comprising nine derived from vaginal biopsies from pretreatment cervical cancer patients and two strains from radiosensitive individuals. DNA double strand break (dsbs) dose-response curves for both initial and residual (20-h repair time) damage were obtained over the dose range 0-240 Gy, with slopes varying 3.2 and 8-fold respectively. Clonogenic cell survival parameters were available for all the cell strains following both high- and low-dose rate irradiation. There were no correlations between the dose-response slope of the initial level of DNA dsbs and parameters that mainly describe the initial portion of clonogenic radiation survival curves (SF2, alpha, D). A significant correlation (r = -0.63, P = 0.04) was found between the extent of residual DNA dsbs and clonogenicity for all 11 fibroblast strains. The parameter showing the highest correlation with fibroblast cell killing (D) for the nine normal fibroblasts alone was the ratio of initial/residual DNA dsb dose-response slope (r = 0.80, P = < 0.01). A significant correlation (r = -0.67, P = 0.03) with clonogenic radiosensitivity was also found for all 11 cell strains when using the ratio of initial/residual DNA dsb damage at a single dose of 180 Gy. This study shows that fibroblast radiosensitivity measured using the neutral comet assay correlates with clonogenic radiation survival parameters, and therefore may have potential value in predictive testing of normal tissue radiosensitivity.     

Detecting normal cell radiosensitivity via assay of DNA damage in lymphocytes for individualizing radiotherapy in head and neck cancer patients

PURPOSE: The purpose of this study was to determine whether the distribution of radiosensitivities in normal tissues of head and neck cancer patients, measured using a DNA damage assay on lymphocytes, is likely to provide sufficient discrimination to enable reliable identification of patients with abnormal sensitivities. MATERIAL AND METHODS: Radiosensitivity was assessed in 307 lymphocyte samples from unselected head and neck cancer patients and was quantified as the initial number of DNA double-strand breaks (dsb) induced per Gray and per DNA unit (200 Mbp). RESULTS: The existence of an inter-individual variation in the radiosensitivity parameter is described by the range (0.41--9.38 dsb/Gy/DNA unit) of the values found. We detected 37 patients who developed severe skin reactions during radiotherapy treatment and we compared their radiosensitivity values with the remaining patients treated. Radiosensitivity values of >7.20 dsb/Gy/DNA unit should theoretically correspond to highly radiosensitive patients. CONCLUSIONS: Our results suggest that initial DNA damage measured on lymphocytes offers an approach to predict the acute response of human normal tissues prior to radiotherapy. .     

The ratio of initial/residual DNA damage predicts intrinsic radiosensitivity in seven cervix carcinoma cell lines.

The single-cell gel electrophoresis (comet) assay was used to measure radiation-produced DNA double-strand breaks (dsbs) in a series of seven cervical tumour cell lines (ME180, HT3, C33A, C41, SiHa, MS751 and CaSki). The proportion of DNA dsbs was measured immediately after radiation treatment (initial damage) and 16 h later after incubation at 37 degrees C (residual damage). Linear dose-response curves were seen for initial (slopes 0.23-0.66) and residual (slopes 0.16-0.87) DNA dsbs. Neither of the slopes of the linear regression analysis on the initial and on the residual DNA dsbs dose-response curves (range 0-80 Gy) correlated with SF2 (surviving fraction at 2 Gy) measured after high- (HDR) or low-dose-rate (LDR) irradiation. An association was evident between SF2 after HDR and LDR irradiation and the ratio of the absolute level of initial and residual damage after a single dose of 60 Gy. However, a significant correlation was found between HDR (r= -0.78, P = 0.04) and LDR (r = -0.86, P = 0.03) SF2 values and the ratio of the slopes of the initial and residual DNA dsbs dose-response curves (range 0.47-0.99), representing the fraction of DNA damage remaining. These results indicate that the neutral comet assay can be used to predict radiosensitivity of cervical tumour cell lines by assessing the ratio of initial and residual DNA dsbs.     

Potential use of the alkaline comet assay as a predictor of bladder tumour response to radiation

Bladder tumours show a variable response to radiotherapy with only about 50% showing good local control; currently there is no test to predict outcome prior to treatment. We have used five bladder tumour cell lines (T24, UM-UC-3, TCC-SUP, RT112, HT1376) to investigate the potential of the alkaline comet assay (ACA) to predict radiosensitivity. Radiation-induced DNA damage and repair were compared to clonogenic survival. When the five cell lines were irradiated and initial DNA damage was plotted against cell survival, at all doses (0-6 Gy), a significant correlation was found (r2=0.9514). Following 4 Gy X-irradiation, all cell lines, except T24, showed a correlation between SF2 vs half-time for repair and SF2 vs residual damage at 5, 10, 20 and 30 min. The T24 cell line showed radioresistance at low doses (0-2 Gy) and radiosensitivity at higher doses (4-6 Gy) using both cell survival and ACA end points, explaining the lack of correlation observed for this cell line. These data indicate that initial DNA damage and residual damage can be used to predict for radiosensitivity. Our data suggest that predictive tests of radiosensitivity, appropriate to the clinical situation, may require the use of test doses in the clinical range.     

Radiation-induced DNA double-strand break rejoining in human tumour cells.

Five established human breast cancer cell lines and one established human bladder cancer cell line of varying radiosensitivity have been used to determine whether the rejoining of DNA double-strand breaks (dsbs) shows a correlation with radiosensitivity. The kinetics of dsb rejoining was biphasic and both components proceeded exponentially with time. The half-time (t1/2) of rejoining ranged from 18.0 +/- 1.4 to 36.4 +/- 3.2 min (fast rejoining process) and from 1.5 +/- 0.2 to 5.1 +/- 0.2 h (slow rejoining process). We found a statistically significant relationship between the survival fraction at 2 Gy (SF2) and the t1/2 of the fast rejoining component (r = 0.949, P = 0.0039). Our results suggest that cell lines which show rapid rejoining are more radioresistant. These results support the view that, as well as the level of damage induction that we have reported previously, the repair process is a major determinant of cellular radiosensitivity. It is possible that the differences found in DNA dsb rejoining and the differences in DNA dsb induction are related by a common mechanism, e.g. conformation of chromatin in the cell.     

DNA double strand breaks in fibroblast cell lines, from non-Hodgkin's lymphoma patients, showing increased sensitivity to chronic gamma irradiation

Cultured skin fibroblast cell lines from two non-Hodgkin's lymphoma patients (NHL) and a normal subject were studied for cell killing, chromosomal aberrations (breaks, translocations, dicentrics and rings) and DNA double strand breaks (dsbs) following chronic gamma irradiation. Compared to the cell line from the normal donor, the NHL patients' fibroblasts showed enhanced radiosensitivity for both cell survival and chromosomal aberrations. While spontaneous breaks were observed in both normal and patients' cells, spontaneous translocations and radiation-induced dicentrics and rings were found only in the latter. Radiation-induced DNA double-strand breaks (dsb) were determined by CHEF electrophoresis. After chronic irradiation with gamma rays the fraction of residual dsb was significantly increased from 1.4% in controls to 1.9% in the NHL cell lines. These data, thus, suggest that the cellular and chromosomal sensitivity to chronic irradiation observed in NHL patients may be due to a deficiency in the repair of a small fraction of DNA double strand breaks.     

Individualization of radiotherapy in breast cancer patients: possible usefulness of a DNA damage assay to measure normal cell radiosensitivity.

PURPOSE: The purpose of this study was to determine whether the distribution of sensitivities in breast cancer patients, measured using a DNA damage assay on lymphocytes, is likely to provide sufficient discrimination to enable the reliable identification of patients with abnormal sensitivities.MATERIAL AND METHODS: Radiosensitivity (x) was assessed in 226 samples of lymphocytes from unselected women with breast cancer and was quantified as the initial number of DNA double-strand breaks (dsb) induced per Gy and per DNA unit (200 Mbp).RESULTS: The existence of an inter-individual variation in the parameter (x) is described through the range (0.40-4.72 dsb/Gy/DNA unit) of values found, which have been fitted to the mathematical model defined by the log-normal distribution (mu = 0.42+/-0.03; sigma = 0.52+/-0.03; R(2)=0.9475). A total of 189 patients received radiotherapy after surgical treatment. Among them, we have detected 15 patients who developed severe skin reactions and we have compared their radiosensitivity values with the rest of patients treated.CONCLUSIONS: Our results suggest that DNA initial damage measured on lymphocytes offers an approach to predict the acute response of human normal tissues prior to radiotherapy. Values of x higher than 3.20 dsb/Gy/DNA unit theoretically should correspond to the highly radio-sensitive patients. Using the experimental results, we have calculated the strength of the test by means of the area under the receiver operator characteristic curves (A(Z)) to determine whether the radiosensitivity assay can discriminate between patients according to their radiation response. The value found (A(Z)=0.675+/-0.072) is indicative of a fair-poor discriminating capacity of the test to identify the patients with higher risk of developing a severe acute reaction during the radiotherapy treatment.     

Induction and repair of DNA double strand breaks in radiation-resistant cells obtained by transformation of primary rat embryo cells with the oncogenes H-ras and v-myc

Rat embryo cells (REC) transformed by the H-ras oncogene plus the cooperating oncogene v-myc are highly resistant to ionizing radiation as compared with the nontransformed parent cells, REC, or immortalized REC. In an attempt to understand the potential mechanism of resistance in these cells, the induction and repair of double strand breaks (dsb) in DNA were measured in a H-ras plus v-myc transformed (3.7) and an immortalized REC (mycREC) line using pulsed field gel electrophoresis. Cells were irradiated in the exponential phase of growth, and the amount of DNA dsb present was quantified by measuring the fraction of DNA activity released from the agarose plugs in which cells were embedded. Similar values of the fraction of DNA activity released were measured for both cell lines at equal X-ray doses, after correction for differences in cell cycle distribution, suggesting a similar induction of DNA dsb per Gy. Repair of DNA dsb measured after exposure to 40 Gy of X-rays was similar in both cell lines and displayed a fast and a slow component. The fast component had a 50% repair time of approximately 12 min, and the slow component, 50% repair time of about 3 h. These results suggest that the relative radioresistance of 3.7 cells is not conferred by a decrease in the amount of DNA dsb induced per Gy per dalton or by alterations in the capacity of the cells to repair DNA dsb. It is hypothesized that alterations in the expression of potentially lethal damage underlie this phenomenon.     

DNA damage and prediction of radiation response in lymphocytes and epidermal skin human cells

The success of radiotherapy in eradicating tumours depends on the total radiation dose, but what limits this dose is the tolerance of the normal tissues within the treatment volume. Studies involving fibroblast survival have demonstrated the theoretical feasibility of a predictive assay of radiation sensitivity, but such an assay is still far from clinical application. Using pulsed-field gel electrophoresis (PFGE), we have quantified the initial “apparent” number of DNA double-strand breaks (dsb) induced by the radiation as an alternative measure of sensitivity in 2 different normal cell types from the same patients, epidermal skin cells and lymphocytes. We found significant inter-individual variation in the measured dsb (1–5 dsb/Gy/DNA unit). We also found a linear correlation between molecular damage in lymphocytes and skin samples from the same patient (slope = 0.83; r = 0.694; p = 0.0001). These results suggest that the initial number of dsb could be used as an indicator of the in vivo response to radiation. Int. J. Cancer 76:354–361, 1998.© 1998 Wiley-Liss, Inc.     

Relationship between clonogenic radiosensitivity, radiation-induced apoptosis and DNA damage/repair in human colon cancer cells

The intrinsic radiation sensitivity of normal and tumour tissue is a major determinant of the outcome of radiotherapy. There is currently no established test that can be used routinely to measure the radiosensitivity of the cells in an individual patient's cancer in a manner that can inform treatment planning. The purpose of this study was to evaluate, in four human colorectal adenocarcinoma cell lines, two possible end points as surrogate markers of radiation response--apoptosis and induction of DNA single-strand breaks--and to compare the results with those of a conventional clonogenic assay. Cell lines (SW707 SW480, SW48 and HT29) known to differ in radiosensitivity were exposed to single doses of X-rays ranging from 0.5 to 5 Gy and cell survival was measured using the clonogenic assay. Apoptosis was determined on the basis of morphology under fluorescent microscopy and DNA damage/repair was measured, as tail moment, using an adaptation of the alkaline comet assay. The relationship between surviving fraction at 2 Gy (SF2) and the percentage of apoptotic cells 24 h after the same dose was complex, but apoptosis accurately predicted the order of radiosensitivities as measured by SF2. Initial damage measured after 2 Gy using the alkaline comet assay gave a close correlation with SF2 (r2=0.95), whereas there was no correlation between initial DNA damage repair rate and SF2.     

A comparison of heat sensitivity, radiosensitivity and PLDR in four human melanoma cell lines.

Comparison of the heat sensitivity and radiosensitivity of four human melanoma cell lines in culture revealed a large variation in sensitivity amongst the four cell lines. Three of the four cell lines had large shoulders on the survival curves when exposed to hyperthermia (44 degrees C or 45 degrees C). These three cell lines also had demonstrable shoulders on the acute radiation dose response curves. The most radiosensitive cell line did not show a shoulder region in the heat or radiation survival curves (HT-144, Dq = 0.2 Gy). Despite this consistency in the presence or absence of shoulder, there was no correlation between heat and radiation sensitivity in the four melanoma cell lines. Furthermore, regardless of radiosensitivity, all four lines studied showed competent repair of potentially lethal damage. The recovery ratios at a surviving fraction of 0.001 ranged from 5.7 to 7.6. All four lines had a similar cell cycle distribution at the time of treatment, hence the variation observed in the response of these four lines to radiation and heat was not due to differences in cell cycle kinetics. Preliminary results of DNA polymerase-alpha and -beta activities do not demonstrate a clear correlation between cellular levels of these two enzymes and radiosensitivity.     

Ku70/80 gene expression and DNA-dependent protein kinase (DNA-PK) activity do not correlate with double-strand break (dsb) repair capacity and cellular radiosensitivity in normal human fibroblasts

The expression of the Ku70 and Ku80 genes as well as the activity of the DNA-dependent protein kinase (DNA-PK) were studied in 11 normal human fibroblast lines. The proteins studied are known to be part of a double-strand break (dsb) repair complex involved in non-homologous recombination, as was demonstrated for the radiosensitive rodent mutant cell lines of the complementation groups 5-7. The 11 fibroblast lines used in this study represent a typical spectrum of normal human radiosensitivity with the surviving fraction measured for a dose of 3.5 Gy, SF3.5 GY, ranging from 0.03 to 0.28. These differences in cell survival were previously shown to correlate with the number of non-repaired dsbs. We found that the mRNA signal intensities of both Ku70 and Ku80 genes were fairly similar for the 11 cell lines investigated. In addition, the DNA-PK activity determined by the pulldown assay was fairly constant in these fibroblast lines. Despite the correlation between cell survival and dsb repair capacity, there was no correlation between dsb repair capacity and DNA-PK activity in the tested normal human fibroblast lines. Obviously, in this respect, other proteins/pathways appear to be more relevant.     

辐射所致残存染色体易位与放射敏感性关系研究

目的 :应用荧光原位杂交 (FISH)方法研究人肿瘤细胞放射敏感性与染色体残存易位关系及临床应用的可行性。方法 :采用 3种放射敏感性不同的人肿瘤细胞系 :鼻咽鳞癌 (CNE)、肺腺癌 (SPC)和乳腺腺癌 (MCF 7)。通过克隆形成方法测定 2Gy、4Gy、6Gy和 8GyX线照射剂量下肿瘤细胞的存活率。采用常规染色体制片过程和 2号染色体涂染探针及FISH方法 ,测定 2Gy、4Gy和 6GyX线照射2 4h后 ,肿瘤细胞 2号染色体内在和诱导的畸变量。结果 :未照射的对照细胞 2号染色体存在不同程度的内在畸变。 2Gy、4Gy和 6Gy照射后 2 4h ,CNE、SPC和MCF 7细胞诱导生成的残存染色体畸变与剂量关系一致 ,能够反映细胞的放射敏感性 ,所有细胞系诱导 2号染色体生成的畸变与细胞存活率均存在良好相关性 (rs=0 96)。结论 :诱导的残存染色体畸变与照射剂量呈线性关系 ,采用FISH方法计数照射诱导的残存染色体畸变 ,可以预测肿瘤细胞间的放射敏感性差异并具有重要的临床意义     

DNA repair and tumour radiosensitivity: focus on ATM gene

Numerous parameters influenced tumour radiosensitivity. The number of clonogenic cells, growth fraction, hypoxia and intrinsic radiosensitivity are among the most important determinants of radiocurability. Detection of DNA damage and repair pathways are important components of intrinsic radiosensitivity. ATM plays a major role in the cellular response to ionizing radiation: it induced DNA repair, cell cycle arrest, and apoptosis via induction of p53. Analysis of single nucleotide polymorphisms could help us to predict normal tissue sensitivity on an individual basis. Mutations of ATM is probably involved in some cases of severe radiation-induced late effects. Measure of residual double-strand breaks by immunochemistry of H2AX, but also ATM or MRE11, is another way to evaluate tumour radiosensitivity. Integration of genomics and functional approach are needed to better predict what the best candidates for a curative radiotherapy are.     

Intrinsic radiosensitivity of human cell lines is correlated with radioresponsiveness of human tumors: analysis of 101 published survival curves

One hundred and one published survival curves for 92 human cell lines (including 64 tumor lines) have been analyzed in terms of several parameters that are supposed to characterize cell radiosensitivity. Values for n, Do, alpha and beta (from the linear quadratic model), D (Mean Inactivation Dose), and survivals at 2 Gy and 8 Gy have been obtained for each curve. It was found that: I. the initial part of the survival curve is specific to the corresponding cell line; II. this initial part is well characterized by the parameters alpha and D, the values of which can be used to compare intrinsic radiosensitivity among human cell lines; III. human tumor cell line radiosensitivity (expressed in terms of alpha, D and survival at 2 Gy) reflects the clinical radioresponsiveness of the tumors from which the cell lines are derived. Thus, cells from tumors with low radioresponsiveness (melanomas and glioblastomas) are the less radiosensitive. Furthermore, the range of survival at a dose of 2 Gy is broad enough to account, in large measure, for observed differences in clinical tumor radioresponsiveness.     

Molecular assays of radiation-induced DNA damage

There is a need for assays of DNA damage in many areas of laboratory research applied to radiation therapy, in order to understand the molecular processes involved in cell killing by ionising radiation and to predict in vivo response. Assays exist which measure many types of DNA damage following ionising radiation. From studies of the dose-response relationships for different types of damage, the double-strand break (dsb) has been shown to be the most significant lesion. Assays for DNA dsb have been of low sensitivity, such that supralethal doses of radiation had to be used in order to study dsb induction or repair. New assays, such as pulsed-field gel electrophoresis, are sensitive to dsb in a dose range relevant to cell survival. In addition, these assays can assess the distribution of dsb in different parts of the genome and determine heterogeneity of damage induction and repair. Assays which measure the effects of strand breaks on DNA complexed with nuclear matrix can reveal features of chromatin organisation and their influence on cellular radiosensitivity.     

Effects of hyperthermia on the repair of radiation-induced DNA single- and double-strand breaks in DNA double-strand break repair-deficient and repair-proficient cell lines

The effect of heat on the induction and repair of DNA single (ssb) and double (dsb) strand breaks was studied in irradiated exponentially growing or plateau-phase CHO cells and their DNA dsb repair-deficient, radiation-sensitive counterpart, the xrs-5 cells. Induction and repair of DNA ssb was measured by the alkaline unwinding technique, whereas induction and repair of DNA dsb was measured by the non-unwinding filter elution technique. The results indicated that pre-exposure of cells to heat (45 x 5 degrees C) for 8-30 min did not affect the induction of DNA ssb or DNA dsb per Gy and dalton of DNA in CHO or xrs-5 cells, tested either in the exponential or in the plateau-phase of growth. On the other hand, pre-exposure to heat inhibited DNA repair processes and increased the fraction of unrepaired radiation-induced damage measured 2 h after irradiation. Repair of DNA dsb was more heat-sensitive than repair of DNA ssb in both cell lines. Repair of radiation-induced ssb or dsb was inhibited in xrs-5 cells to a larger extent than in CHO cells after a similar exposure to heat. These results complement those previously reported on heat-induced radiosensitization in these cell lines, and suggest that the reduction in heat-induced radiosensitization observed in xrs-5 cells is largely due to their deficiency in repairing DNA dsb, rather than to a reduction in the ability of heat to inhibit DNA repair processes in general. The data presented here provide further support to the hypothesis that DNA dsb repair proficiency is a prerequisite for heat-induced radiosensitization.