Calibration of MTT assay in proton beams using radiochromic films.

PURPOSE: This study provides methodology of calibrating as well as controlling the output for an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) colorimetric assay irradiated in a low energy proton beam using EBT3-model GAFCHROMICTM film, without correcting for quenching effect. METHODS: A calibrated Markus ionization chamber was used to measure the depth dose and beam output for 26.5 MeV protons produced by a CS30 cyclotron. A time-controlled aluminum cylinder was added in front of the horizontal beam-exit serving as a radiation shutter. Following the TRS-398 reference dosimetry protocol for proton beams, the output was calibrated in water at a reference depth of 3 mm. EBT3 film was calibrated for doses up to 8 Gy at the same depth. To verify the dose distribution for each 96-well MTT assay plate, EBT3 film was placed at the reference depth during irradiation and cell doses were scaled by measured percent depth dose (PDD) data. RESULTS: The radiochromic film dosimetry system in this study provides dose measurements with an uncertainty better than 3.3% for doses higher than 1 Gy. From a single exposure and utilizing the Gaussian shape of the beam, multiple dose points can be obtained within different wells of the same plate ranging from 6.9 Gy (sigma ∼4%) in the central well, and 2 Gy (sigma ∼8%) for wells positioned closer to the periphery. CONCLUSIONS: We described a methodology for radiochromic film-based dose monitoring system, using low-energy protons, which can be used for the MTT assay in any proton beam, except within Bragg peak region.

Dosimetry of biological irradiations using radiochromic films.

Delivering accurate radiation dose to blood specimens during biological irradiations is essential in quantifying damage of ionizing radiation. To estimate dose to blood samples as accurately as possible, pieces of EBT2 model GAFCHROMIC™ film were placed within an approximately 10 mm finely ground rice layer that was used to simulate test specimens inside 40 mL plastic flasks. Irradiations of flasks were carried out using an X-RAD 320 irradiator with a beam quality of 320 kVp and a measured half value layer of 1.12 mm Cu, in air and in a full scattering setup which consisted of either rice or Solid Water™ (SW) surrounding flasks, filled to the same level at top of the flasks, together with a 5 cm thick SW slab beneath them. Outputs, per cent depth doses and beam profiles at different depths were measured and compared between setups. For the same setting, the dose delivered to the middle flask under the full scattering setup is 22% larger than with the in-air setup at the depth of the specimen and 9.2% more homogeneous across the specimen thickness of 10 mm (2.3% variation in comparison to the surface). Rice showed a fairly similar performance to SW within 1% at the same depth of 10 mm. Experimental setup based on full scattering conditions was shown to provide faster, more homogenous and fairly uniform dose delivery to biological specimens in comparison to conventionally used in-air setups.

Association between normal tissue complications after radiotherapy and polymorphic variations in TGFB1 and XRCC1 genes.

Genetic predictive biomarkers of radiosensitivity are being sought to individualize radiation treatment of cancer patients. In this pilot case-control study, we tested the association between TGFB1 T869C codon 10 Leu/Pro (rs1982073), XRCC1 G28152A codon 399 Arg/Gln (rs25487), and XRCC3 C18067T codon 241 Thr/Met (rs861539) single-nucleotide polymorphisms (SNPs) and late reaction to radiotherapy in 60 nasopharyngeal cancer patients. Subcutaneous and deep tissue fibrosis was scored using the RTOG/EORTC grading system. Patients with moderate to severe fibrosis (radiosensitive cases, G2-3, n = 30) were matched and compared to those with little or no reaction (controls, G0-1, n = 30). The three nonsynonymous SNPs were genotyped by direct DNA sequencing. Significant association was observed for TGFB1 T869C and XRCC1 G28152A genotypes (P < or = 0.05). Both variant alleles, TGFB1 869C and XRCC1 28152A, were associated with a lower grade of fibrosis (odds ratios were 0.41, 95% CI: 0.20-0.86, P = 0.02 and 0.30, 95% CI: 0.10-0.89, P = 0.02, respectively), and therefore the wild-types were the risk alleles. Interestingly, there was a significant difference in the median number of risk alleles between the radiosensitive and the control groups (P = 0.006). We conclude that radiotherapy complications are associated with genetic variations in our nasopharynx cancer patients. Our findings support the assumption of the combined effects of multiple SNPs. Large-scale studies are required to confirm these findings before polymorphisms can be used as predictive markers to individualize radiation therapy on genetic bases.

Aberrant CDKN1A transcriptional response associates with abnormal sensitivity to radiation treatment.

Normal tissue reactions to radiation therapy vary in severity among patients and cannot be accurately predicted, limiting treatment doses. The existence of heritable radiosensitivity syndromes suggests that normal tissue reaction severity is determined, at least in part, by genetic factors and these may be revealed by differences in gene expression. To test this hypothesis, peripheral blood lymphocyte cultures from 22 breast cancer patients with either minimal (11) or very severe acute skin reactions (11) have been used to analyse gene expression. Basal and post-irradiation expression of four radiation-responsive genes (CDKN1A, GADD45A, CCNB1, and BBC3) was determined by quantitative real-time PCR in T-cell cultures established from the two patient groups before radiotherapy. Relative expression levels of BBC3, CCNB1, and GADD45A 2 h following 2 Gy X-rays did not discriminate between groups. However, post-irradiation expression response was significantly reduced for CDKN1A (P<0.002) in severe reactors compared to normal. Prediction of reaction severity of approximately 91% of individuals sampled was achieved using this end point. Analysis of TP53 Arg72Pro and CDKN1A Ser31Arg single nucleotide polymorphisms did not show any significant association with reaction sensitivity. Although these results require confirmation and extension, this study demonstrates the possibility of predicting the severity of acute skin radiation toxicity in simple tests.

Association between TP53 codon 72 single-nucleotide polymorphism and radiation sensitivity of human fibroblasts.

Inherent radiosensitivity varies widely between individuals. We hypothesized that amino acid substitution variants in two highly radiation-responsive proteins, TP53 (p53) and CDKN1A (p21, Waf1, Cip1), are associated with and could explain individual variations in radiosensitivity. The two non-synonymous single-nucleotide polymorphisms (SNPs) TP53 codon 72 Arg/Pro G>C and CDKN1A codon 31 Ser/Arg C>A were genotyped in 92 normal fibroblast cell strains of different radiosensitivity. The clonogenic surviving fraction at 2 Gy (SF2) ranged between 0.15 and 0.50 (mean = 0.34, SD = 0.08). The mean SF2 was used to divide the cell strains into radiosensitive (45) and normal groups (47). A significant association was observed between SF2 and the TP53 codon 72 haplotype (C compared to G, P = 0.01). No association was observed between CDKN1A codon 31 haplotype and radiosensitivity (P = 0.86). The variant TP53 Arg72 allele was associated with a decrease in radiosensitivity, presumably due to suboptimal function leading to less stringent control of cell division. We conclude that certain SNPs in susceptible genes can influence cellular radiation response. Such risk alleles could ultimately be used as predictive markers for radiosensitivity to help stratifying individuals during assessment of risk of radiation exposure.

Evidence that individual variations in TP53 and CDKN1A protein responsiveness are related to inherent radiation sensitivity.

We tested the hypothesis that individual variations in the induction of the TP53 tumor suppressor protein by radiation are related to inherent radiosensitivity. Thirty-two fibroblast cell strains were examined. Radiosensitivity was measured by a clonogenic survival assay. The induction of TP53 and its transcriptionally activated CDKN1A (p21) protein were studied by Western blotting 3 h after a single dose of 5 Gy. The relative cell culture age, as determined by the colony size distribution, was studied as a confounding factor. Survival curves showed wide range of radiosensitivity. The surviving fraction at 2 Gy (SF2) ranged between 0.02 and 0.49 (mean = 0.29, SD = 0.13). TP53 induction ranged between 1.28 and 2.34 (mean = 1.80, SD = 0.31). CDKN1A showed a wider induction (1.09-4.05, mean = 2.33, SD = 0.78). Positive correlations were observed between SF2 and TP53 induction (R(2) = 0.62, P < 0.001) and CDKN1A (R(2) = 0.64, P < 0.001). No correlation with the colony size distribution was observed. In conclusion, these results suggest that the individual variations in radiosensitivity and in the level of induction of TP53 (and consequently CDKN1A) are congruent, irrespective of the genetic background of these nontransformed fibroblasts. It is postulated that underlying mechanisms culminating in a stronger TP53 induction lead to higher survival, presumably due to more efficient repair of radiation-induced damage.

Loss of wild-type Trp53 protein in mouse fibroblasts leads to increased radioresistance with consequent decrease in repair of potentially lethal damage.

It has been reported that the loss of function of Trp53 protein is associated with a reduction in the expression of radiation-induced potentially lethal damage (PLD). These studies, however, were carried out using either transformed or transfected cell lines, and other factors may have existed that could interfere with PLD repair. In this study, we used isogenic fibroblasts derived from Trp53 knockout mice to study radiation sensitivity, PLD repair, and repair of DNA double-strand breaks (DSBs). Experiments were carried out using wild-type (Trp53(+/+)), heterozygous (Trp53(+/-)) and homozygous mutant (Trp53(-/-)) cells. This is an ideal system because the only difference in the three cell strains is the status of the Trp53 protein. DSB repair was measured by pulsed-field-gel electrophoresis (PFGE), while radiosensitivity and PLD repair were studied using the clonogenic survival assay. Cells were irradiated in plateau phase and then trypsinized and plated either immediately or 24 h later to allow for PLD repair. The results of Western blot analyses showed that Trp53(-/-) cells expressed a putative mutant form of Trp53 that was unable to transcriptionally activate Cdkn1a (p21) protein in response to irradiation. The Trp53(-/-) cells were significantly more radioresistant than the Trp53(+/+) cells, and this was associated with a moderate reduction in PLD repair. DNA repair experiments showed no difference in DSB rejoining capability between the two cell lines. In conclusion, our results show that loss of wild-type Trp53 leads to increased radioresistance with consequent reduction in PLD repair but with no effect on DNA DSB repair.

Analysis of radiation-induced DNA double-strand breaks misrepair is not compromized by broken DNA in human fibroblasts.

It has been suggested that the technique for measuring repair fidelity of radiation-induced DNA double-strand breaks (DSBs) using Southern blotting and hybridization to defined regions of the genome could be compromised by broken or poorly-digested DNA. Since misrepair of DNA DSBs is an important aspect of radiation-induced chromosome aberrations, mutations, and cell killing, we checked for such a supposition in non-transformed human fibroblasts. DSB misrepair was assessed in a NotI-cleavable DNA fragment of 3.2 Mbp located on the long arm of chromosome 21 and detected by D21S1 probe. We hypothesized that the suggested DNA degradation, whether spurious in nature or the results of irradiation-induced phenomena such as apoptosis and/or necrosis, should be detectable with or without NotI restriction enzyme treatment. When the DNA embedded in agarose plugs was separated by electrophoresis without prior NotI restriction, no significant difference was observed in the relative amount of migrating DNA between the control (no irradiation) and 24 h of repair following 80 Gy irradiation. Furthermore, only about 10% of the total signal was located below the 3.2 Mbp band. This suggests that the amount of DNA fragmentation due to biological (apoptosis or necrosis) or technical processes was negligible. The Tunel assay supported these results, as there was little to no apoptosis detectable in these fibroblasts up to 24 h after irradiation. We conclude that in primary human fibroblasts, the NotI method for measuring radiation-induced misrepair is not compromised by DNA degradation.

Equivalence of pulsed-dose-rate to low-dose-rate irradiation in tumor and normal cell lines.

To determine whether different fractionation schemes could simulate low-dose-rate irradiation, ovarian cells of the carcinoma cell lines A2780s (radiosensitive) and A2780cp (radioresistant) and AG1522 normal human fibroblasts were irradiated in vitro using different fraction sizes and intervals between fractions with an overall average dose rate of 0.53 Gy/h. For the resistant cell line, the three fractionation schemes, 0.53 Gy given every hour, 1.1 Gy every 2 h, and 1.6 Gy every 3 h, were equivalent to low dose rate (0.53 Gy/h). Two larger fraction sizes, 2.1 Gy every 4 h and 3.2 Gy every 6 h, resulted in lower survival than that after low-dose-rate irradiation for the resistant cell line, suggesting incomplete repair of radiation damage due to the larger fraction sizes. The survival for the sensitive cell line was lower at small doses, but then it increased until it was equivalent to that after low-dose-rate irradiation for some fractionation schemes. The sensitive cell line showed equivalence only with the 1.6-Gy fraction every 3 h, although 0.53 Gy every 1 h and 1.1 Gy every 2 h showed equivalence at lower doses. This cell line also showed an adaptive response. The normal cell line showed a sensitization to the pulsed-dose-rate schemes compared to low-dose-rate irradiation. These data indicate that the response to pulsed-dose-rate irradiation is dependent on the cell line and that compared to the response to low-dose-rate irradiation, it shows some equivalence with the resistant carcinoma cell line, an adaptive response with the parental carcinoma cell line, and sensitization with the normal cells. Therefore, further evaluation is required before implementing pulsed-dose-rate irradiation in the clinic.

Enhanced in vitro radiosensitivity of skin fibroblasts in two patients developing brain necrosis following AVM radiosurgery: a new risk factor with potential for a predictive assay.

PURPOSE: Radiosurgery is an effective treatment for arteriovenous malformations (AVM) with a low risk of developing brain necrosis. Models have been developed to predict the risk of complications. We postulated that genetic differences in radiosensitivity may also be a risk factor. METHODS AND MATERIALS: Fibroblast cultures were established from skin biopsies in two AVM patients developing radiation necrosis. The results of clonogenic survival assays were compared to a parallel study with two groups of cancer patients treated with radiation: 1) patients without late side effects; 2) patients experiencing severe late sequelae. RESULTS: The survival fraction at 2 Gy (SF2) of the 2 AVM patients was 0.17 (0.14-0.19) and 0.18 (0.14-0.22). The SF2's of the cancer patients ranged between 0.25-0.38 (mean = 0.31) for the control group, and between 0. 10-0.20 (mean = 0.17) for the hypersensitive group. The SF2's of the AVM patients who developed brain necrosis were comparable to that of the hypersensitive group (p = 0.85) but significantly lower than the control group (p = 0.05). CONCLUSION: The two patients who developed radiation necrosis demonstrate increased fibroblast radiosensitivity. The SF2 of skin fibroblasts may potentially be used as a predictive assay to detect patients at risk for brain necrosis.

Correlation between normal tissue complications and in vitro radiosensitivity of skin fibroblasts derived from radiotherapy patients treated for variety of tumors.

PURPOSE: To assess the relationship between fibroblast intrinsic radiosensitivity in vitro and late reactions of normal tissues in patients treated by definitive radiotherapy for variety of tumors. PATIENTS AND METHODS: Ten patients were selected for this study. They were treated by radical radiotherapy for variety of tumors, including non-Hodgkin's lymphoma, prostate, glottic larynx, anal canal, cervix, bladder, thyroid gland, and tonsil pillar. Five patients did not develop any significant late reactions (normally sensitive group, NS). The other five developed late complications in different normal tissues and organs that proved to be fatal in one patient (clinically hyper-sensitive group, HS). Fibroblast cultures were established from punch skin biopsy and radiosensitivity in vitro was measured. The survival fraction at 2 Gy (SF2) was calculated and compared between the two groups. RESULTS: SF2 ranged between 0.10 and 0.38 with a mean of 0.24. The mean SF2 for each of the NS and the HS groups were 0.31 and 0.17, respectively. The non-parametric rank test of Mann-Whitney shows that the difference between the two groups is statistically significant (p = 0.01). CONCLUSION: This study indicates that the in vitro radiosensitivity of skin fibroblasts is correlated with late complications in different organs and normal tissues following radiotherapy for variety of tumors. It also lends support to the existence of a common genetic component determining the radiosensitivity of cells targeted by the late effects of ionizing radiation.

[In vitro radiosensitivity of skin fibroblasts can identify a group of patients with complications in various health tissues after radiotherapy]. / La radiosensibilité in vitro des fibroblastes de la peau peut identifier un groupe de patients ayant des complications dans différents tissus sains après radiothérapie.

PURPOSE: A retrospective study of the in vitro radiosensitivity of skin fibroblasts derived from two groups of patients treated by definitive radiotherapy for a variety of tumors who either displayed or did not display severe complications. PATIENTS AND METHODS: Seven radiotherapy patients were selected: three were treated for head and neck, prostate and non-Hodgkin lymphoma tumors, and did not develop any significant complications (control group); four patients were treated for bladder, thyroid, head and neck and anal canal tumors and developed serious acute and especially late reactions (hypersensitive group). Primary cell cultures of skin fibroblasts were established and their radiosensitivity studied by the clonogenic assay after exposing to single radiation doses ranging between 1 and 8 Gy. RESULTS: The survival fraction at 2 Gy (SF2) ranged from 0.27 to 0.38, with a mean of 0.33 for the control group, and from 0.10 to 0.20 with a mean of 0.17 for the hypersensitive group. The Mann-Whitney non-parametric test showed that the difference between the two means was statistically significant (p = 0.03). CONCLUSION: The data are in favor of a correlation between the radiosensitivity of patients' fibroblasts and the reactions of different normal tissues to radiotherapy. This association supports the use of the clonogenic survival, or a surrogate test, as a predictive assay. The multiplicity of normal tissues and organs implicated in this association suggests the existence of genetic factors that determine, at least in part, the radiosensitivity of target cells involved in the expression of normal tissues complications following radiotherapy.

Differential induction of premature chromosome condensation by calyculin A in human fibroblast and tumor cell lines.

The feasibility of chemically-induced premature chromosome condensation (PCC) was tested in two human fibroblast and two human malignant melanoma cell lines. To induce PCCs, calyculin A, a potent protein phosphatase inhibitor, was used at a final concentration of 80 nM. Attached cells and cells put into suspension by trypsinization were incubated with calyculin A at 37 degrees C for 1 hour. Calyculin A was able to induce PCCs in all the phases of the cell cycle with the tumour cell lines giving the highest PCC frequency. No systematic differences were observed between attached cells and cells put into suspension by trypsinization. However, a cytotoxic effect that led to the loss of 50% to 80% of the treated cells was observed. The cytotoxic effect was more severe in the fibroblast than in the tumour cell lines. The appearance of deformed, fragile and fragmented nuclei with no particular chromatin condensation would explain to some extent this cytotoxic effect. A better understanding of the mechanism of action of calyculin A would help generalizing its use to study interphase chromosome aberrations.

Hypersensitivity to low single doses and split-dose recovery: two manifestations of induced resistance that might be related.

PURPOSE: To study retrospectively the relationship between intrinsic radiosensitivity (SF2), and both the low-dose inducible response (alpha(s)/alpha(r)) and the amount of split-dose recovery (betaRR). MATERIALS AND METHODS: A total of 53 sets of experimental data obtained with 44 human cell lines were collected from the literature and the above relationships were studied. RESULTS: Analysis showed a statistically significant correlation between alpha(s)/alpha(r) and SF2 (p = 0.0023, 10 sets of data), and a statistically significant inverse correlation between betaRR and SF2 (p = 0.0005, 36 sets of data, AT excluded). Furthermore, the analysis of the relationship between the challenge dose SF2 (after a clinical-sized priming dose) and that of the single-dose SF2 (27 sets of data, AT excluded) showed a statistically significant correlation (p<0.0001), which deviates from, and becomes higher than, the one-to-one relationship for single-dose SF2<0.30, suggesting that the final response to fractionated irradiation in radiosensitive cells might not be predictable on the basis of simple reconstitution of survival from the single-dose treatment. CONCLUSION: The comparison between the two relationships: SF2/(alpha(s)/alpha(r)) and SF2/betaRR, suggests some parallelism indicating that these two phenomena may be inversely correlated and could be attributed to induced resistance mechanisms that might be triggered differently in sensitive and resistant cell lines.

Hypersensitivity of ataxia telangiectasia fibroblasts to ionizing radiation is associated with a repair deficiency of DNA double-strand breaks.

We have studied the intrinsic radiosensitivity, repair of potentially lethal damage (PLD) and the repair rate of radiation-induced DNA double-strand breaks (DSB) in 11 non-transformed human fibroblast cell lines, four of which were homozygous for the A-T mutation and two that were heterozygous (A-TH). All the experiments were done on cells in plateau phase of growth (97-99% of cells in G0/G1). With a dose of 30 Gy delivered at 4 degrees C, the A-T cell lines had faster repair rates of up to 6 h, after which the repair curve crossed that of the control so that the residual damage at 24 h was higher in the A-T cells. Irradiation at 37 degrees C at low dose rate 1 cGy.min-1) produced even more marked differences between the A-T cells and controls: the residual DSB level was always higher in A-T cells than controls at doses of 5-40 Gy, due to defective repair of a small fraction of DSB in A-T cells. The two protocols showed DSB repair rates for the A-TH cell lines that were intermediate between those of the A-T and control cells. There was a quantitative relationship between the residual DSB after irradiation at 37 degrees C and the intrinsic radiosensitivity, and with the extent of PLD repair. There were very few apoptotic cells in the non-transformed control and A-T cell line, both before and after irradiation. In combination, these result support the contention that the defective repair of DSB is a mechanism of the hypersensitivity linked to the A-T mutation.

Dose-rate effects on the survival of irradiated hypersensitive and normal human fibroblasts.

The linear-quadratic model describes cell killing by radiation as due to two processes defined by the linear (alpha) component and by the quadratic (beta) component. As alpha and beta are interdependent, it is difficult to evaluate accurately the alpha component (which characterizes the intrinsic radiosensitivity). It has been suggested that irradiation at low dose-rate (around 1 cGy/min) allows the disappearance of the beta component and thus gives a direct measure of alpha. The present results verify this hypothesis with plateau phase cells. The survival of five human fibroblast cell lines in exponentially growing and density-inhibited, confluent cultures maintained at 37 degrees C following exposure to 60Co gamma-rays at dose-rates of 0.33-100 cGy/min followed by delayed plating (only for plateau phase cells) was monitored. Three of these cell lines are considered to be 'normal' and two are derived from hypersensitive individuals. The mean inactivation doses (D) of the five cell lines for acute doses with immediate plating were 173, 163, 136, 107 and 67 cGy. (D) increased with delayed plating recovery for 4 of the 5 cell lines and the survival of the 5 cell lines increased after low dose-rate exposure (1 cGy/min) without altering the ranking. The differences between cell lines (absolute values of (D) increased with decreasing the dose-rate. Analysis of the survival curves with the General Linear Quadratic (GLQ) model gave repair half-times for each cell line which were not correlated with the intrinsic radiosensitivities. Surprisingly, the alpha component decreased with decreasing dose-rate for all 5 cell lines (only in plateau phase). Thus low dose-rates do not allow direct measurement of the alpha component; the decrease in alpha could be interpreted as adaptive radioresistance.

A new model describing the curves for repair of both DNA double-strand breaks and chromosome damage.

A review of reports dealing with fittings of the data for repair of DNA double-strand breaks (DSBs) and excess chromosome fragments (ECFs) shows that several models are used to fit the repair curves. Since DSBs and ECFs are correlated, it is worth developing a model describing both phenomena. The curve-fitting models used most extensively, the two repair half-times model for DSBs and the monoexponential plus residual model for ECFs, appear to be too inflexible to describe the repair curves for both DSBs and ECFs. We have therefore developed a new concept based on a variable repair half-time. According to this concept, the repair curve is continuously bending and dependent on time and probably reflects a continuous spectrum of damage repairability. The fits of the curves for DSB repair to the variable repair half-time and the variable repair half-time plus residual models were compared to those obtained with the two half-times plus residual and two half-times models. Similarly, the fits of the curves for ECF repair to the variable repair half-time and variable half-time plus residual models were compared to that obtained with the monoexponential plus residual model. The quality of fit and the dependence of adjustable parameters on the portion of the curve fitted were used as comparison criteria. We found that: (a) It is useful to postulate the existence of a residual term for unrepairable lesions, regardless of the model adopted. (b) With the two cell lines tested (a normal and a hypersensitive one), data for both DSBs and ECFs are best fitted to the variable repair half-time plus residual model, whatever the repair time range.

Induction and rejoining of DNA double-strand breaks and interphase chromosome breaks after exposure to X rays in one normal and two hypersensitive human fibroblast cell lines.

The aim of this work was to measure simultaneously and in a quantitative manner double-strand breaks (DSBs), interphase chromosome breaks and cell lethality either immediately after irradiation, or at various times thereafter (up to 24 h), in cells of three nontransformed human fibroblast cell lines of widely different intrinsic radiosensitivity. We wished to assess initial damage, repair kinetics and residual damage at the DNA and the chromosome level, and to correlate these parameters with cell killing. We employed HF19 cells, a normal fibroblast cell line, AT2 cells, a radiosensitive cell line from a patient suffering from ataxia telangiectasia (AT), and 180BR cells, a radiosensitive cell line from a patient with no clinical symptoms of AT. AT2 and 180BR cells, in addition to being radiosensitive, also display a reduced ability to repair potentially lethal damage compared to HF19 cells. The yield of DSBs, as measured by pulsed-field gel electrophoresis, is similar in all three cell lines (slopes correspond to 1.6-1.7% Gy-1 of DNA-associated radioactivity released from the gel well into the lane). In contrast, residual DSBs measured 24 h after irradiation are almost zero for HF19 cells (0.1% confidence interval = 0-1.4%), but are 12.5% (+/- 2.3%) and 43.8% (+/- 1.2%) of those measured immediately after irradiation in AT2 and 180BR cells, respectively. Residual interphase chromosome breaks are 11.6% (+/- 1.6%), 29.7% (+/- 5.7%) and 41.4% (+/- 2.2%) of those measured immediately after irradiation in HF19, AT2 and 180BR cells, respectively. Neither the initial yield of DSBs nor that of excess interphase chromosome breaks can explain the differences in radiosensitivity between the three cell lines; however, there is a correlation between residual DSBs, rate of DSB rejoining at 24 h, residual interphase chromosome breaks on the one hand and cell survival on the other hand.

Defective repair of DNA double-strand breaks and chromosome damage in fibroblasts from a radiosensitive leukemia patient.

A radiation-sensitive fibroblast culture (180BR) established from an acute lymphoblastic leukemia patient who died following radiotherapy is defective in the repair of radiation-induced DNA double-strand breaks. The cells also show a reduced capacity to repair interphase chromosome damage visualized by means of premature chromosome condensation and metaphase chromosome aberrations measured by fluorescence in situ hybridization on chromosome 4. This case represents the first example in humans where hypersensitivity to ionizing radiation can be ascribed directly to a defect in DNA and chromosome repair, and the defect may underlie the cancerous phenotype observed.