Repair of radiation induced damage in two human tumour cell lines grown as spheroids and monolayers

Growth curves of two human tumour cell lines grown as multicellular tumour spheroid (MTS) were used to obtain survival estimates by back-extrapolation after split and single dose irradiation. Neuroblastoma (NB-100) and squamous cell carcinoma (HN-1) single cells from monolayer culture were assessed for repair of sublethal and potentially lethal damage. The extent of repair was calculated on an iso-effect basis. When grown as spheroids squamous cell carcinoma cells showed a higher capacity to repair sublethal damage than neuroblastoma cells. Repair of potentially lethal damage did not contribute to this higher capacity of HN-1 cells, since this cell line was found to be deficient for this type of repair. Using the recovery ratio to estimate the beta-component of the survival curves, it was found that differences in repair capacity were determined by the alpha-component of the equation. Our results show the feasibility of back-extrapolating multicellular tumour spheroid growth curves to obtain survival estimates that can be applied to establish sublethal damage repair capacity.     

Comparison of repair and rejoining fidelity between two isogenic human ovarian carcinoma cell lines

PURPOSE: The difference in radiosensitivity between two isogenic tumour cell lines was evaluated to determine whether factors such as sublethal and potentially damage repair, DNA double-strand break repair and fidelity of repair can be related to differences in radiosensitivity. MATERIALS AND METHODS: The cell lines used were the ovarian carcinoma A2780s and a radiation-resistant derivative A2780cp. Radiation response was measured in terms of cell survival, recovery of sublethal (SLD) and potentially lethal damage (PLD), induction of and recovery of DNA strand breaks, and fidelity of DNA repair using a cell-free plasmid assay. RESULTS: While A2780cp was more resistant to radiation than A2780s, it showed less ability for recovery of SLD and PLD. DNA strand-break induction was the same for both cell lines, while only at very high doses did A2780cp show greater DNA strand-break recovery than A2780s. Fidelity of rejoining DNA was significantly higher in the A2780cp cell line. CONCLUSION: The difference in radiosensitivity between these two cell lines was not related to recovery of PLD or SLD or to the induction of DNA damage. It appears that fidelity of DNA rejoining, which was significantly higher in the resistant cell line, may be related to the difference in radiosensitivity.     

Radiation-induced DNA damage and repair in quiescent and proliferating human tumor cells in vitro

The purpose of this study was to examine radiation-induced DNA strand breakage and repair in quiescent and proliferating human tumor cells in vitro and determine their relationship to radiation sensitivity and potentially lethal damage repair (PLDR). Using centrifugal elutriation we have isolated from fed plateau-phase cultures of HEp-3 human squamous carcinoma cells, relatively pure populations of quiescent and proliferating cells. This was confirmed by both [3H]-thymidine labelling and acridine orange (AO) staining with flow cytometry. Quiescent cells were more sensitive to ionizing radiation (Do = 0.97 Gy) than were proliferating cells (Do = 1.28 Gy). However, quiescent cells showed higher repair of potentially lethal damage (PLDR) than did proliferating cells. Repair of single-strand breaks (ssb) and double-strand breaks (dsb) as measured by filter elution did not differ significantly between quiescent and proliferating cells. For both populations, ssb and dsb repair kinetics and final damage remaining were the same, suggesting that repair of DNA strand breaks is not entirely responsible for the difference in radiation sensitivity between quiescent and proliferating cells.     

DNA double-strand breaks: their repair and relationship to cell killing in yeast

Yeast is a suitable eukaryotic organism in which to study DNA double-strand breakage measured by the neutral sucrose gradient sedimentation technique and cell killing in the same range dose of sparsely ionizing radiations. Radiosensitive mutants (including temperature conditional ones) exist in which rejoining of double-strand breaks (dsb) is not detectable. In such mutants approximately one dsb per cell corresponds to a lethal event, suggesting that a dsb is a potentially lethal lesion. There are two modes by which dsb may confer cell lethality: firstly, an unrepaired dsb may be lethal on its own and secondly, two dsb may interact to form a lethal lesion (binary misrepair). The operationally defined cellular phenomena of potentially lethal damage (PLD) repair and sublethal damage (SLD) repair are both based on the repair of dsb. Induced dsb show a linear and unrejoined dsb a linear-quadratic relationship with dose. At low dose rate the quadratic component is abolished in accordance with the exponential survival curve observed. The dose-rate effect is based on dsb repair during irradiation; it is absent in dsb repair-deficient mutants.     

125I-induced DNA Double Strand Breaks: Use in Calibration of the Neutral Filter Elution Technique and Comparison with X-ray Induced Breaks

Summary

The neutral filter elution assay, for measurement of DNA double strand breakage, has been calibrated using mouse L cells and Chinese hamster V79 cells labelled with [¹²⁵I]dUrd and then held at liquid nitrogen temperature to accumulate decays. The basis of the calibration is the observation that each ¹²⁵I decay, occurring the DNA, produces a DNA double strand break. Linear relationships between ¹²⁵I decays per cell and lethal lesions per cell (minus natural logarithm survival) and the level of elution, were found. Using the calibration data, it was calculated that the yield of DNA double strand breaks after X-irradiation of both cell types was from 6 to 9 × 10^?12 DNA double strand breaks per Gy per dalton of DNA, for doses greater than 6 Gy. Neutral filter elution and survival data for X-irradiated and ¹²⁵I-labelled cells suggested that the relationships between lethal lesions and DNA double strand breakage were significantly different for both cell types. An attempt was made to study the repair kinetics for ¹²⁵I-induced DNA double strand breaks, but was frustrated by the rapid DNA degradation which occurs in cells that have been killed by the freezing-thawing process.     

Effect of cisplatin on the recovery from sublethal and potentially lethal radiation damage in cell culture

The effect of Cisplatin upon the recovery from sublethal damage during fractionated irradiation and upon the recovery from potentially lethal damage after a single dose has been investigated. In three mammalian cell lines, Cisplatin did not influence the recovery from sublethal and potentially lethal damage. There were differences among the three cell lines in their ability to recover from radiation damage. However, a significant quantitative difference between the recovery from sublethal and potentially lethal damage in any one cell line could not be found.     

Production and repair of chromosome damage in an X-ray sensitive CHO mutant visualized and analysed in interphase using the technique of premature chromosome condensation

Production and repair of chromosome damage were studied in interphase xrs-5 cells by means of premature chromosome condensation (PCC). The results obtained were compared with those previously reported for CHO cells. Production of chromosome damage per unit of absorbed radiation dose was in xrs-5 cells larger by a factor of 2.6 than in CHO cells (5.2 breaks per cell per Gy). Changes in chromatin structure, associated with the radiation-sensitive phenotype of xrs-5 cells, that increase the probability of conversion of a DNA double-stand break (dsb) to a chromosome break are involved to explain this effect. Repair of chromosome breaks as measured in plateau-phase G1 cells was deficient in xrs-5 cells and the number of residual chromosome breaks was practically identical to the number of lethal lesions calculated from survival data. This observation suggests that non-repaired chromosome breaks are likely to be manifestations of lethal events in the cell. The yield of ring chromosomes scored after a few hours of repair was higher by a factor of three in xrs-5 compared with CHO cells. This increase in ring formation suggests an increase in the probability of misrepair of chromosome damage that may stem either from the reduced ability of xrs-5 cells to repair dsb, or from the higher production of chromosome fragments observed per cell and per Gy.     

Comparison of DNA damage and repair following radiation challenge in buccal cells and lymphocytes using single‐cell gel electrophoresis

Purpose: To develop a reproducible single‐cell gel electrophoresis assay for DNA damage and repair in buccal mucosa and sublingual exfoliated cells.

Materials and methods: Buccal mucosa and sublingual cells and lymphocytes from six individuals (three males, three females, aged 34–45 years) were challenged with increasing doses of gamma‐rays. DNA strand breaks and DNA repair were measured using the single‐cell gel electrophoresis assay.

Results: Baseline DNA strand breaks were significantly greater in buccal mucosa and sublingual cells compared with lymphocytes. Buccal mucosa and sublingual cells did not differ from each other with respect to induction of DNA strand breaks by 2 or 4?Gy gamma‐rays. However, they showed a smaller increase in gamma‐ray‐induced DNA strand breaks compared with lymphocytes (32–53% less than lymphocytes; ANOVA p<0.0001). Unlike lymphocytes, which repaired 83% of DNA strand breaks, buccal mucosa and sublingual cells exhibited only a minimal capacity for DNA repair (approximately 0–14% of the level in lymphocytes).

Conclusions: Buccal mucosa and sublingual cells exhibit an apparent resistance to the expression of radiation‐induced DNA strand breaks in vitro and an apparent lack of DNA strand break repair in the single‐cell gel electrophoresis assay.     

Comparison of DNA damage and repair following radiation challenge in buccal cells and lymphocytes using single-cell gel electrophoresis

PURPOSE: To develop a reproducible single-cell gel electrophoresis assay for DNA damage and repair in buccal mucosa and sublingual exfoliated cells. MATERIALS AND METHODS: Buccal mucosa and sublingual cells and lymphocytes from six individuals (three males, three females, aged 34-45 years) were challenged with increasing doses of gamma-rays. DNA strand breaks and DNA repair were measured using the single-cell gel electrophoresis assay. RESULTS: Baseline DNA strand breaks were significantly greater in buccal mucosa and sublingual cells compared with lymphocytes. Buccal mucosa and sublingual cells did not differ from each other with respect to induction of DNA strand breaks by 2 or 4 Gy gamma-rays. However, they showed a smaller increase in gamma-ray-induced DNA strand breaks compared with lymphocytes (32-53% less than lymphocytes; ANOVA p<0.0001). Unlike lymphocytes, which repaired 83% of DNA strand breaks, buccal mucosa and sublingual cells exhibited only a minimal capacity for DNA repair (approximately 0-14% of the level in lymphocytes). CONCLUSIONS: Buccal mucosa and sublingual cells exhibit an apparent resistance to the expression of radiation-induced DNA strand breaks in vitro and an apparent lack of DNA strand break repair in the single-cell gel electrophoresis assay.     

Determination of the initial DNA damage and residual DNA damage remaining after 12 hours of repair in eleven cell lines at low doses of irradiation

Purpose : To determine the relationship between DNA damage and radiosensitivity at low doses (1-10Gy) for the initial DNA damage and residual DNA damage remaining after 12-h repair. Materials and methods : Eleven cell lines, normal human lung epithelial L132, HT29 human colon carcinoma, ATs4 human ataxia telangiectasia, normal CHO-K1 hamster, repair-deficient xrs1 and xrs5 mutants, repair-deficient SCID rodent cell line, the human normal fibroblast 1BR.3, human ataxia telangiectasia fibroblast AT1BR and the repair-deficient fibroblasts 180BR.B and 46BR.1 were irradiated with 60 Co γ-rays. Radiosensitivity was measured by clonogenic survival assay. DNA damage was measured by fluorometric analysis of DNA unwinding (FADU). Results : The radiosensitivity in the 11 cell lines ranged from SF2 of 0.02-0.61. By FADU assay, the undamaged DNA at 5-Gy ranged from 56 to 93%. The initial DNA damage and radiosensitivity were highly correlated (r 2 =0.81). After 5-Gy irradiation and 12-h repair, two groups of cell lines emerged. Group 1 restored undamaged DNA to a level ranging from 94 to 98%. Group 2 restored the undamaged DNA to a level ranging from 77 to 82%. No correlation was seen between residual DNA damage remaining after 12-h repair and radiosensitivity. Conclusion : It is shown that the initial DNA damage correlates with radiosensitivity at low doses of irradiation. This suggests that the initial DNA damage must be considered as a determinant for radiosensitivity.     

Determination of the initial DNA damage and residual DNA damage remaining after 12 hours of repair in eleven cell lines at low doses of irradiation

PURPOSE: To determine the relationship between DNA damage and radiosensitivity at low doses (1-10 Gy) for the initial DNA damage and residual DNA damage remaining after 12-h repair. MATERIALS AND METHODS: Eleven cell lines, normal human lung epithelial L132, HT29 human colon carcinoma, ATs4 human ataxia telangiectasia, normal CHO-K1 hamster, repair-deficient xrs1 and xrs5 mutants, repair-deficient SCID rodent cell line, the human normal fibroblast 1BR.3, human ataxia telangiectasia fibroblast AT1BR and the repair-deficient fibroblasts 180BR.B and 46BR.1 were irradiated with 60Co gamma-rays. Radiosensitivity was measured by clonogenic survival assay. DNA damage was measured by fluorometric analysis of DNA unwinding (FADU). RESULTS: The radiosensitivity in the 11 cell lines ranged from SF2 of 0.02-0.61. By FADU assay, the undamaged DNA at 5-Gy ranged from 56 to 93%. The initial DNA damage and radiosensitivity were highly correlated (r2 = 0.81). After 5-Gy irradiation and 12-h repair, two groups of cell lines emerged. Group 1 restored undamaged DNA to a level ranging from 94 to 98%. Group 2 restored the undamaged DNA to a level ranging from 77 to 82%. No correlation was seen between residual DNA damage remaining after 12-h repair and radiosensitivity. CONCLUSION: It is shown that the initial DNA damage correlates with radiosensitivity at low doses of irradiation. This suggests that the initial DNA damage must be considered as a determinant for radiosensitivity.     

Reduced pH increases recovery from radiation damage potentially leading to cell death and to in vitro transformation

C3H-10T1/2 cells were grown to plateau phase and assessed for recovery from radiation damage under acidic and normal pH conditions. Repair of potentially lethal damage (PLD) was observed after X-ray doses of 5.0, 6.0 and 12.0 Gy and repair increased with dose. The repair at an acidic pH of 6.8 was greater than at the normal pH of 7.4. Repair of potentially transforming damage (PTD) was also observed when cells were held in plateau phase after irradiation. Repair of PTD was greater at the acidic pH than at the normal pH. When PTD recovery was plotted vs PLD recovery the results showed a good linear correlation with approximately twice as much PTDR. The experimental conditions allowing PLDR and PTDR indicate that the repair process may be error-free, since transformation was reduced in all experiments which showed recovery from PLD.     

DNA base and strand damage in X-irradiated monkey CV-1 cells: influence of pretreatment using small doses of radiation

Base damage in alpha DNA from irradiated monkey CV-1 cells was determined by measuring release of 5'-32P-end labelled DNA fragments after digestion with endonuclease III of E. coli. The frequency and base sequence locations of the enzyme-sensitive sites were determined. Fragments were released from irradiated DNA at sequence sites of pyrimidines and guanines. The time for repair of half the single strand breaks was approximately 1.5 h. Repair of base damage as judged from loss of enzyme-sensitive sites in DNA was slower, with more than half of the damaged bases still detectable after 4 h of repair. Two important changes in the pattern of fragment release from DNA were produced when small radiation doses preceded the large ones needed to produce measurable DNA strand breaks and base damage. 5 Gy to cells incubated several hours before 320 Gy increased by five-fold the abundance of small DNA fragments with 3'-phosphoryl termini detected in high-resolution denaturing gels. These increases were detectable with doses as small as 0.2 Gy and were accompanied by the appearance of new species of DNA fragments of intermediate mobility at specific locations in the base sequence. The patterns resemble those produced by digesting DNA from heavily irradiated cells with endonuclease III.     

Underestimation of the small residual damage when measuring DNA double-strand breaks (DSB): is the repair of radiation-induced DSB complete?

PURPOSE: To overcome the underestimation of the small residual damage when measuring DNA double-strand breaks (DSB) as fraction of activity released (FAR) by pulsed-field gel electrophoresis. MATERIALS AND METHODS: The techniques used to assess DNA damage (e.g. pulsed-field gel electrophoresis, neutral elution, comet assay) do not directly measure the number of DSB. The Bl?cher model can be used to express data as DSB after irradiation at 4 degrees C by calculating the distribution of all radiation-induced DNA fragments as a function of their size. We have used this model to measure the residual DSB (irradiation at 4 degrees C followed by incubation at 37 degrees C) in untransformed human fibroblasts. RESULTS: The DSB induction rate after irradiation at 4 degrees C was 39.1+/-2.0 Gy(-1). The DSB repair rate obtained after doses of 10 to 80 Gy followed by repair times of 0 to 24 h was expressed as unrepaired DSB calculated from the Bl?cher formula. All the damage appeared to be repaired at 24h when the data were expressed as FAR, whereas 15% of DSB remained unrepaired. The DSB repair rate and the chromosome break repair rate assessed by premature condensation chromosome (PCC) techniques were similar. CONCLUSION: The expression of repair data in terms of FAR dramatically underestimates the amount of unrepaired DNA damage. The Bl?cher model that takes into account the size distribution of radiation-induced DNA fragments should therefore be used to avoid this bias. Applied to a normal human fibroblast cell line, this model shows that DSB repair is never complete.     

Cytotoxicity of 125I decay in the DNA double strand break repair deficient mutant cell line, xrs-5.

The survival of parental Chinese hamster ovary (CHO) K1 cells and the DNA double strand break (DSB) repair deficient mutant, xrs-5 was determined after accumulation of 125I decays. Both CHO and xrs-5 cells were extremely sensitive to accumulated 125I decays. The D0 values for CHO and xrs-5 cells were 40 and approximately 7 decays per cell, respectively. The difference in cell survival between CHO and xrs-5 cells was not due to differences in overall 125IUdR incorporation, differences in labelling index (LI) or differences in plating efficiency (PE). Relative biological effectiveness (RBE) values calculated relative to 137Cs gamma radiation survival values (D0 and D10) were higher in xrs-5 cells compared with CHO cells. Although both CHO and xrs-5 cells have high RBE values that correspond to a high sensitivity of CHO and xrs-5 cells to 125I decay. The higher RBE observed for xrs-5 cells in combination with the known repair defect in xrs-5 cells support the idea that unrepaired DNA double strand breaks are lethal to the cell.     

Dose-rate effect between 1 and 10 Gy/min in mammalian cell culture

A dose-rate effect is observed between 1 and 10 Gy/min for cultured Chinese hamster V-79 fibroblast cells irradiated under pO2 levels of less than 10 ppm, 100 ppm, 300 ppm and 21%. The dose for a cellular surviving fraction of 0.1% increases by 10-15% as the dose rate is changed from 10 to 1 Gy/min. This observed dose-rate effect is consistent with the repair of sublethal damage taking place during the radiation delivery. A related finding is that acute hypoxia in cell culture does not inhibit sublethal damage repair.     

Production and Repair of Chromosome Damage in an X-ray Sensitive CHO Mutant Visualized and Analysed in Interphase Using the Technique of Premature Chromosome Condensation

Summary

Production and repair of chromosome damage were studied in interphase xrs-5 cells by means of premature chromosome condensation (PCC). The results obtained were compared with those previously reported for CHO cells. Production of chromosome damage per unit of absorbed radiation dose was in xrs-5 cells larger by a factor of 2·6 than in CHO cells (5·2 breaks per cell per Gy). Changes in chromatin structure, associated with the radiation-sensitive phenotype of xrs-5 cells, that increase the probability of conversion of a DNA double-strand break (dsb) to a chromosome break are invoked to explain this effect. Repair of chromosome breaks as measured in plateau-phase G₁ cells was deficient in xrs-5 cells and the number of residual chromosome breaks was practically identical to the number of lethal lesions calculated from survival data. This observation suggests that non-repaired chromosome breaks are likely to be manifestations of lethal events in the cell. The yield of ring chromosomes scored after a few hours of repair was higher by a factor of three in xrs-5 compared with CHO cells. This increase in ring formation suggests an increase in the probability of misrepair of chromosome damage that may stem either from the reduced ability of xrs-5 cells to repair dsb, or from the higher production of chromosome fragments observed per cell and per Gy.     

Sublethal damage repair and radiosensitivity of human squamous cell carcinoma cells grown with different culture techniques

In this study, cells of a human squamous cell carcinoma line, HN-1, were grown in monolayers and as multicellular tumour spheroids (MTS). Repair of radiation-induced damage was studied by irradiation with single and split doses of X rays (4-8 Gy). It was shown that the amount of sublethal damage that was repaired in this dose range was equal in cells growing in monolayers and as MTS. The radiosensitivity of spheroids, as expressed by spheroid "cure" dose, increased with increasing MTS diameter. It is postulated that, in MTS with no signs of hypoxia, radioresistance diminishes when MTS increase in diameter.     

Dose-rate effects in normal and malignant cells of human origin

Human cell lines derived from normal tissue and malignant tumours were irradiated in plateau phase under acute (81 Gy/h) or protracted (0.1-0.7 Gy/h) exposure to determine initial survival curve slopes, assess sublethal damage repair (SLDR) capability, and establish differences in survival due to SLDR over a range of dose rates. No correlation was found between clinical resistance of the tumor types and initial slope or survival at 2 Gy. Sublethal damage repair was assessed by comparing survival curves for acute and continuous irradiation. All cell lines showed significant SLDR, with the more clinically resistant tumour types demonstrating the best recovery rates. The survival data also demonstrated little difference in cell killing over the 0.1-0.7 Gy/h range. Using a modified linear-quadratic model with provisions for SLDR, it was found that for these cell lines the repair half-time was less than 1 h, with no significant change in the amount of recovery from sublethal damage at dose rates below 1 Gy/h.     

The effect of post-irradiation anisotonic treatment on cell survival and repair of DNA damage

V79 379A cells were irradiated and then exposed to anisotonic PBS for 20 min. This enhanced the radiation effect resulting from the fixation of potentially lethal damage. The induction of DNA single- and double-strand breaks is not increased by this treatment. Anisotonic treatment delayed the onset of repair of DNA damage. However when cells were returned to normal medium, they repaired the damage to a similar extent as cells not exposed to the anisotonic treatment. We suggest that the fixation of damage by post-irradiation anisotonic treatment is mediated through an increased probability of misrepair of DNA damage due to the delay in the onset of repair. This is supported by the observation that there is a reduced effect of post-irradiation anisotonic treatment on cells that have a markedly reduced ability to repair double-strand breaks.