A non-radioactive, PFGE-based assay for low levels of DNA double-strand breaks in mammalian cells

A PFGE method was adapted to measure DNA double-strand breaks (DSBs) in mammalian cells after low (0-25 Gy) doses of ionising radiation. Instead of radionuclide incorporation, DNA staining in the gel by SYBR-Gold was used, which lowered the background of DNA damage and could be applied to non-cycling cells. DSB level was defined as a product of a fraction of DNA released to the gel (FR) and a number of DNA fragments in the gel (DNA(fragm)) and expressed as a percentage above control value. The slope of the dose-response curve was two-fold higher compared to that with FR alone as DSB level indicator (31.4 versus 15.6% per Gy). Two alternative ways were proposed to determine the total amount of DNA, used for FR calculation: measurement of DNA content in a plug not subjected to electrophoresis, with the use of Pico-Green, or estimation of DNA released to the gel from a plug irradiated with 600 Gy of gamma-rays. The limit of DSB detection was 0.25 Gy for human G1-lymphocytes and 0.5-1 Gy for asynchronous cultures of human glioma M059 K and J or mouse lymphoma L5178Y-R and -S cells. Specificity of our PFGE assay to DSB was confirmed by the fact that no damage was detected after treatment of the cells with H(2)O(2), an inducer of single-strand DNA breaks (SSBs). On the contrary, the H(2)O(2) inflicted damage was detected by neutral comet assay, attaining 160% above control (equivalent to 2.5 Gy of X-radiation). DSB rejoining, measured in cells after X-irradiation with a dose of 10 Gy, generally proceeded faster than that measured previously after higher (30-50 Gy) doses of ionising radiation. Clearly seen were defects in DSB rejoining in radiosensitive M059 J and L5178Y-S cells compared to their radioresistant counterparts, M059 K and L5178Y-R. In some cell lines, a secondary post-irradiation increase in DSB levels was observed. The possibility is considered that these additional DSBs may accumulate during processing of non-DSB clustered DNA damage or/and represent early apoptotic events.     

Induction and rejoining of DNA double-strand breaks in human cervix carcinoma cell lines of differing radiosensitivity

Five recently established cell lines of human carcinoma of the cervix of varying radiosensitivity have been used to determine whether the induction or rejoining of DNA double-strand breaks (dsb) shows any correlation with radiosensitivity or radiation recovery capacity. Double-strand DNA breaks have been measured using neutral filter elution at pH 9.6. The number of breaks induced immediately after irradiation with doses of 10 to 40 Gy 60Co gamma rays appeared to show some correlation with radiosensitivity particularly after 10 Gy; the two more radiosensitive lines incurred more breaks than the more radioresistant lines. In addition, the shape of the induction curve with dose was linear for the two sensitive lines but curvilinear for the resistant lines. Despite the dose scales being different, this mirrored their respective cell survival curve shapes. After 30 or 50 Gy irradiation, rejoining of breaks appeared to be rapid and almost complete within 60 min at 37 degrees C for the three resistant lines. However, for the sensitive lines, one line (HX160c) in particular exhibited a reduced rate of dsb rejoining. In addition, a residual level of dsb was present in this line even after allowing rejoining for 3 h. While induction and rejoining of DNA dsb therefore appears to be a factor in determining radiosensitivity, at doses relevant to cellular survival (up to 10 Gy), the greater induction of DNA dsb in radiosensitive lines may play a significant role in determining the cellular response to ionizing radiation.     

The potential value of the neutral comet assay and the expression of genes associated with DNA damage in assessing the radiosensitivity of tumor cells

The assessment of tumor radiosensitivity would be particularly useful in optimizing the radiation dose during radiotherapy. Therefore, the degree of correlation between radiation-induced DNA damage, as measured by the alkaline and the neutral comet assays, and the clonogenic survival of different human tumor cells was studied. Further, tumor radiosensitivity was compared with the expression of genes associated with the cellular response to radiation damage. Five different human tumor cell lines were chosen and the radiosensitivity of these cells was established by clonogenic assay. Alkaline and neutral comet assays were performed in γ-irradiated cells (2-8Gy; either acute or fractionated). Quantitative PCR was performed to evaluate the expression of DNA damage response genes in control and irradiated cells. The relative radiosensitivity of the cell lines assessed by the extent of DNA damage (neutral comet assay) immediately after irradiation (4Gy or 6Gy) was in agreement with radiosensitivity pattern obtained by the clonogenic assay. The survival fraction of irradiated cells showed a better correlation with the magnitude of DNA damage measured by the neutral comet assay (r=-0.9; P<0.05; 6Gy) than evaluated by alkaline comet assay (r=-0.73; P<0.05; 6Gy). Further, a significant correlation between the clonogenic survival and DNA damage was observed in cells exposed to fractionated doses of radiation. Of 15 genes investigated in the gene expression study, HSP70, KU80 and RAD51 all showed significant positive correlations (r=0.9; P<0.05) with tumor radiosensitivity. Our study clearly demonstrated that the neutral comet assay was better than alkaline comet assay for assessment of radiosensitivities of tumor cells after acute or fractionated doses of irradiation.     

Induction and rejoining of gamma-ray-induced DNA single- and double-strand breaks in Chinese hamster AA8 cells and in two radiosensitive clones

The induction and rejoining of gamma-ray-induced DNA strand breaks were measured in a Chinese hamster ovary cell line, AA8, and in two radiosensitive clones (EM9 and NM2) derived from it. The kinetics of recovery from sublethal damage (SLD) and potentially lethal damage (PLD) has previously been characterized in each of these lines [vanAnkeren et al., Radiat. Res., 115, 223-237 (1988)]. No significant differences were observed among the cell lines in the yields of either DNA single-strand breaks (SSBs) or double-strand breaks (DSBs) as assayed by filter elution. Data for SSB rejoining in AA8 and NM2 cells irradiated with 7.5 Gy were fit by a biexponential process (t1/2 values of approximately 4 and 80 min). In comparison, SSB rejoining in EM9 cells was initially slower (t1/2 = 10 min) and a higher level of SSBs was unrejoined 6 h after irradiation. DSB rejoining in AA8 cells assayed at pH 9.6 was also biphasic (t1/2 values of 15 and 93 min), although when assayed at pH 7.0, most (approximately 80%) of the damage was rejoined at a constant rate (t1/2 = 45 min) during the first 2 h. EM9 cells exhibited a slower initial rate of DSB rejoining when assayed at pH 9.6 but showed no difference compared with AA8 cells in DSB rejoining when assayed at pH 7.0. These results indicate that radiosensitive EM9 cells, whose kinetics of recovery from SLD and PLD was the same as that of AA8 cells, have a defect in the fast phase of SSB rejoining but no measurable defect in DSB rejoining. Conversely, NM2 cells, which displayed a reduced shoulder width on their survival curve and decreased recovery from SLD, had no demonstrable defects in the rate or extent of rejoining of DSBs or SSBs. When compared with the SLD and PLD data reported previously, these results suggest that there is no direct correlation between either of these recovery processes and the rejoining of SSBs or DSBs as assayed here.     

Two-lesion kinetic model of double-strand break rejoining and cell killing

Radiobiological models, such as the lethal and potentially lethal (LPL) model and the repair-misrepair (RMR) model, have been reasonably successful at explaining the cell killing effects of radiation. However, the models have been less successful at relating cell killing to the formation, repair and misrepair of double-strand breaks (DSBs), which are widely accepted as the main type of DNA damage responsible for radiation-induced cell killing. A fully satisfactory model should be capable of predicting cell killing for a wide range of exposure conditions using a single set of model parameters. Moreover, these same parameters should give realistic estimates for the initial DSB yield, the DSB rejoining rate, and the residual number of unrepaired DSBs after all repair is complete. To better link biochemical processing of the DSB to cell killing, a two-lesion kinetic (TLK) model is proposed. In the TLK model, the family of all possible DSBs is subdivided into simple and complex DSBs, and each kind of DSB may have its own repair characteristics. A unique aspect of the TLK model is that break ends associated with both kinds of DSBs are allowed to interact in pairwise fashion to form irreversible lethal and nonlethal damages. To test the performance of the TLK model, nonlinear optimization methods are used to calibrate the model based on data for the survival of CHO cells for an extensive set of single-dose and split-dose exposure conditions. Then some of the postulated mechanisms of action are tested by comparing measured and predicted estimates of the initial DSB yield and the rate of DSB rejoining. The predictions of the TLK model for CHO cell survival and the initial DSB yield and rejoining rate are all shown to be in good agreement with the measured data. Studies suggest a yield of about 25 DSBs Gy(-1) cell(-1). About 20 DSBs Gy(-1) cell(-1) are rejoined quickly (15-min repair half-time), and 4 to 6 DSBs Gy(-1) cell(-1) are rejoined very slowly (10- to 15-h repair half-time). Both the slowly and fast-rejoining DSBs make substantial contributions to the killing of CHO cells by radiation. Although the TLK model provides a much more satisfactory formalism to relate biochemical processing of DSBs to cell killing than did the earlier kinetic models, some small differences among the measured and predicted CHO cell survival and DSB rejoining data suggest that additional factors and processes not considered in the present work may affect biochemical processing of DSBs and hence cell killing.     

Evidence to support the existence of efficient DNA double-strand break rejoining in a radiosensitive mutant of V79-4 following irradiation with 250 kVp X-rays or neutrons

The repair of ionising-radiation-induced DNA double-strand break type damage was measured by Kohn neutral elution in an X-ray-sensitive mutant of V79-4, irs1. This was done in order to investigate further the likelihood that irs1 carries a defect which leads to error-prone repair of DNA damage, and not simply a reduced ability to rejoin DNA double-strand breaks. The mutant displayed an equal increase in sensitivity to the lethal effects of neutrons, as compared to X-rays. Both irs1 and V79-4 showed an increased sensitivity to the killing effects of neutrons of around 2 at 10% survival. irs1 also showed an exponential survival after either X-rays or neutrons. The induction of DNA double-strand breaks was measured in both cell lines over a dose range of 10-40 Gy using Kohn neutral filter elution. Induction of breaks by X-rays in irs1 seemed to increase slightly with dose, relative to induction in V79-4, so that at 40 Gy 1.5 times more DNA double-strand breaks were measured in irs1 cells than in V79-4. Neutron irradiation resulted in a more similar level of induction in either strain after 10-40 Gy. This difference in induction of damage may be due to a different cell-cycle composition in either cell line. The rejoining of X-ray induced double-strand breaks showed a very similar pattern (on a percentage rejoined basis) in both cell lines, although from the induction data at 40 Gy, the dose at which rejoining was measured, fewer breaks were rejoined in V79-4 but also fewer breaks remained unsealed. Neutron-induced breaks, however, were rejoined more efficiently in irs1 again on a percentage basis, but also in absolute terms since similar induction was seen after 40 Gy. This data, together with the differences seen in the rejoining of X-ray compared to neutron induced breaks, may indirectly support the proposal that irs1 is a misrepair mutant.     

Misrejoining of DNA double-strand breaks in primary and transformed human and rodent cells: a comparison between the HPRT region and other genomic locations.

Many studies of radiation response and mutagenesis have been carried out with transformed human or rodent cell lines. To study whether the transfer of results between different cellular systems is justified with regard to the repair of radiation-induced DNA double-strand breaks (DSBs), two assays that measure the joining of correct DSB ends and total rejoining in specific regions of the genome were applied to primary and cancer-derived human cells and a Chinese hamster cell line. The experimental procedure involves Southern hybridization of pulsed-field gel electrophoresis blots and quantitative analysis of specific restriction fragments detected by a single-copy probe. The yield of X-ray-induced DSBs was comparable in all cell lines analyzed, amounting to about 1 x 10(-2) breaks/Mbp/Gy. For joining correct DSB ends following an 80 Gy X-ray exposure all cell lines showed similar kinetics and the same final level of correctly rejoined breaks of about 50%. Analysis of all rejoining events revealed a considerable fraction of unrejoined DSBs (15-20%) after 24 h repair incubation in the tumor cell line, 5-10% unrejoined breaks in CHO cells and complete DSB rejoining in primary human fibroblasts. To study intragenomic heterogeneity of DSB repair, we analyzed the joining of correct and incorrect break ends in regions of different gene density and activity in human cells. A comparison of the region Xq26 spanning the hypoxanthine guanine phosphoribosyl transferase locus with the region 21q21 revealed identical characteristics for the induction and repair of DSBs, suggesting that there are no large variations between Giemsa-light and Giemsa-dark chromosomal bands.     

Repair of radiation-induced heat-labile sites is independent of DNA-PKcs, XRCC1 and PARP

Ionizing radiation induces a variety of different DNA lesions; in addition to the most critical DNA damage, the DSB, numerous base alterations, SSBs and other modifications of the DNA double-helix are formed. When several non-DSB lesions are clustered within a short distance along DNA, or close to a DSB, they may interfere with the repair of DSBs and affect the measurement of DSB induction and repair. We have shown previously that a substantial fraction of DSBs measured by pulsed-field gel electrophoresis (PFGE) are in fact due to heat-labile sites within clustered lesions, thus reflecting an artifact of preparation of genomic DNA at elevated temperature. To further characterize the influence of heat-labile sites on DSB induction and repair, cells of four human cell lines (GM5758, GM7166, M059K, U-1810) with apparently normal DSB rejoining were tested for biphasic rejoining after gamma irradiation. When heat-released DSBs were excluded from the measurements, the fraction of fast rejoining decreased to less than 50% of the total. However, the half-times of the fast (t(1/2) = 7-8 min) and slow (t(1/2) = 2.5 h) DSB rejoining were not changed significantly. At t = 0, the heat-released DSBs accounted for almost 40% of the DSBs, corresponding to 10 extra DSBs per cell per Gy in the initial DSB yield. These heat-released DSBs were repaired within 60-90 min in all cells tested, including M059K cells treated with wortmannin and DNA-PKcs-defective M059J cells. Furthermore, cells lacking XRCC1 or poly(ADP-ribose) polymerase 1 (PARP1) rejoined both total DSBs and heat-released DSBs similarly to normal cells. In summary, the presence of heat-labile sites has a substantial impact on DSB induction and DSB rejoining rates measured by pulsed-field gel electrophoresis, and heat-labile sites repair is independent of DNA-PKcs, XRCC1 and PARP.     

Rejoining of DNA strand breaks by T4 DNA ligase in mammalian cells

We have tested the ability of T4 DNA ligase to rejoin radiation-induced DNA strand breaks in living hamster cells (CHO-K1, EM9, xrs-5). T4 DNA ligase was introduced into cells by electroporation prior to x-irradiation. Single- and double-strand breaks were measured by the alkaline comet assay technique, and double-strand breaks (DSBs) were evaluated by the pulsed-field gel electrophoresis method. In the comet assay, the three cell lines showed reduced tail moments following pretreatment with T4 DNA ligase, both directly after irradiation and after repair incubation for 4 h. Similarly, the results obtained from pulsed-field gel electrophoresis showed reduced DSB frequencies after pretreatment with T4 DNA ligase. We conclude that exogeneous T4 ligase contributes to rejoining of radiation-induced strand breaks.     

Incomplete rejoining of DNA double-strand breaks in unstimulated normal human lymphocytes

We investigated the repair kinetics of DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) in unstimulated normal human peripheral blood lymphocytes (HPBL). SSBs and DSBs induced by gamma-irradiation (at 0 degree C) were assayed without radiolabel by alkaline and neutral filter elution, respectively. Incubation of irradiated cells at 37 degrees C for various lengths of time demonstrated that the percent DNA rejoined increased until it reached a plateau at approximately 60 min; this repair plateau underwent no substantial change when incubation continued for 20-24 h. The level of the plateau indicated how closely the elution profile of DNA from cells irradiated and incubated (experimental) resembled the elution profile of DNA from unirradiated cells (control). After 6 Gy and 60 min incubation, the alkaline elution profile of DNA from experimental cells from 5 donors was indistinguishable from that seen in DNA from control cells, suggesting that rejoining of SSBs was complete. In contrast after 100 Gy and 60 min incubation the neutral elution profile of DNA from cells from the same donors demonstrated that, compared to DNA from control cells, rejoining of DSBs was approximately two-thirds complete. In the range of 2-8 Gy, 85-104% of SSBs were rejoined after 60 min incubation; in the range of 30-120 Gy, 46-80% of DSBs were rejoined after 60 min incubation. These unexpected results stand in contrast to our previous studies with confluent normal human diploid fibroblasts (HDF), in which rejoining of both SSBs and DSBs was greater than 90% complete by 60 min repair incubation and 100% complete after 18-24 h.     

Radioprotection of cultured mammalian cells by the aminothiols WR-1065 and WR-255591: correlation between protection against DNA double-strand breaks and cell killing after gamma radiation

We compared the effects of the radioprotective aminothiols WR-1065 and WR-255591 on the induction of DNA double-strand breaks (DSBs) and on the survival of aerated Chinese hamster ovary cells exposed to 60Co gamma radiation. DSBs were measured using the pH 9.6 neutral elution method. In agreement with earlier studies, protection factors for both drugs measured using the end point of clonogenic cell survival were significantly greater than the protection factors for DSB induction when DSBs were measured after gamma-ray doses ranging from 20 to 90 Gy. However, when DSBs and cell survival measurements were made on the same cell populations after low radiation doses (between 3 and 30 Gy) using the replicate plating method, there appeared to be a close correlation between the modification of DSB induction and the modification of cell survival produced by both drugs. The major influence accounting for the differences between these and previously obtained results appears to be the range of radiation doses used, suggesting that protection against DSB induction is radiation-dose dependent.     

Low-dose hyper-radiosensitivity is not caused by a failure to recognize DNA double-strand breaks

One of the earliest cellular responses to radiation-induced DNA damage is the phosphorylation of the histone variant H2AX (gamma-H2AX). gamma-H2AX facilitates the local concentration and focus formation of numerous repair-related proteins within the vicinity of DNA DSBs. Previously, we have shown that low-dose hyper-radiosensitivity (HRS), the excessive sensitivity of mammalian cells to very low doses of ionizing radiation, is a response specific to G(2)-phase cells and is attributed to evasion of an ATM-dependent G(2)-phase cell cycle checkpoint. To further define the mechanism of low-dose hyper-radiosensitivity, we investigated the relationship between the recognition of radiation-induced DNA double-strand breaks as defined by gamma-H2AX staining and the incidence of HRS in three pairs of isogenic cell lines with known differences in radiosensitivity and DNA repair functionality (disparate RAS, ATM or DNA-PKcs status). Marked differences between the six cell lines in cell survival were observed after high-dose exposures (>1 Gy) reflective of the DNA repair capabilities of the individual six cell lines. In contrast, the absence of functional ATM or DNA-PK activity did not affect cell survival outcome below 0.2 Gy, supporting the concept that HRS is a measure of radiation sensitivity in the absence of fully functional repair. No relationship was evident between the initial numbers of DNA DSBs scored immediately after either low- or high-dose radiation exposure with cell survival for any of the cell lines, indicating that the prevalence of HRS is not related to recognition of DNA DSBs. However, residual DNA DSB damage as indicated by the persistence of gamma-H2AX foci 4 h after exposure was significantly correlated with cell survival after exposure to 2 Gy. This observation suggests that the persistence of gamma-H2AX foci could be adopted as a surrogate assay of cellular radiosensitivity to predict clinical radiation responsiveness.     

Mechanism of radiosensitization by halogenated pyrimidines: effect of BrdU on repair of DNA breaks, interphase chromatin breaks, and potentially lethal damage in plateau-phase CHO cells.

There is evidence suggesting that radiosensitization induced in mammalian cells by substitution in the DNA of thymidine with BrdU has a component that relies on inhibition of repair and/or fixation of radiation damage. Here, experiments designed to study the mechanism of this phenomenon are described. The effect of BrdU incorporation into DNA was studied on cellular repair capability, rejoining of interphase chromosome breaks, as well as induction and rejoining of DNA double- and single-stranded breaks (DSBs and SSBs) in plateau-phase CHO cells exposed to X rays. Repair of potentially lethal damage (PLD), as measured by delayed plating of plateau-phase cells, was used to assay cellular repair capacity. Rejoining of interphase chromosome breaks was assayed by means of premature chromosome condensation (PCC); induction and rejoining of DNA DSBs were assayed by pulsed-field gel electrophoresis and induction and rejoining of DNA SSBs by DNA unwinding. A decrease was observed in the rate of repair of PLD in cells grown in the presence of BrdU, the magnitude of which depended upon the degree of thymidine replacement. The relative increase in survival caused by PLD repair was larger in cells substituted with BrdU and led to a partial loss of the radiosensitizing effect compared to cells tested immediately after irradiation. A decrease was also observed in the rate of rejoining of interphase chromosome breaks as well as in the rate of rejoining of the slow component of DNA DSBs in cells substituted with BrdU. The time constants measured for the rejoining of the slow component of DNA DSBs and of interphase chromosome breaks were similar both in the presence and in the absence of BrdU, suggesting a correlation between this subset of DNA lesions and interphase chromosome breaks. It is proposed that a larger proportion of radiation-induced potentially lethal lesions becomes lethal in cells grown in the presence of BrdU. Potentially lethal lesions are fixed via interaction with processes associated with cell cycle progression in cells plated immediately after irradiation, but can be partly repaired in cells kept in the plateau-phase. It is hypothesized that fixation of PLD is caused by alterations in chromatin conformation that occur during normal progression of cells throughout the cell cycle.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ionizing radiation damage to DNA: molecular aspects

Radioresistant tumor cells are found in tumor specimens from patients in whom radiotherapy has failed or whose tumors have recurred after therapy. This suggests that inherent cellular radioresistance may in part underlie the failure of radiotherapy, and therefore determination of the presence of resistant cells within a tumor might be a useful predictor of response to radiation therapy. Most standard clonogenic assays of radiation response are time-consuming, and alternative assays of radiation response are being sought. In an earlier publication (J. L. Schwartz et al., Int. J. Radiat. Oncol. Biol. Phys. 15, 907-912, 1988), we reported that radioresistant human tumor cells rejoin DNA double-strand breaks, as measured by DNA neutral filter elution (pH 9.6), faster than more sensitive cell lines. To determine whether DNA elution might have potential as a rapid predictive assay, we examined the relationship between the rate of DNA double-strand break rejoining and radiosensitivity in nine first-passage-after-explant squamous cell carcinomas under conditions that minimized the influence of nontumor and nonclonogenic cells. The frequency of DNA double-strand breaks measured 1 h after irradiation with 100 Gy 60Co gamma rays was used as an estimate of relative rejoining rate. This number is a reflection of both the initial DNA double-strand break frequency and the amount of repair that occurs in 1 h. The relative break frequency was compared to radiosensitivity as measured by standard clonogenic survival assays in later passages (p3-p14) of these same cells. A significant relationship (r = 0.61, P less than 0.01) was found between break frequency measured in first-passage cells and radiosensitivity measured in later passages, suggesting that the neutral elution assay as described here has some promise as a relatively rapid assay of the radiosensitivity of human tumor cells.     

CHK1 Affecting Cell Radiosensitivity is Independent of Non-Homologous End Joining

CHK1 is one of the most important checkpoint proteins in mammalian cells for responding toDNA damage. Cells defective in CHK1 are sensitive to ionizing radiation (IR). The mechanismby which CHK1 protects cells from IR-induced killing remains unclear. DNA double strandbreaks (DSBs) induced by IR are critical lesions for cell survival. Two major complementaryDNA DSBs repair pathways exist in mammalian cells, homologous recombination repair (HRR)and non-homologous end joining (NHEJ). By using CHK1 kinase dead human cell linesestablished in our laboratory, we show here that although these human cell lines have differentCHK1 activities with different sensitivities to IR-induced killing and G2 accumulation, all thesecell lines show similar inductions and rejoining rates of DNA DSBs. These results indicate thatthe different radiosensitivities and G2 checkpoint responses in these cell lines are independent ofNHEJ, suggesting that CHK1-regulated checkpoint facilitates HRR and therefore protects cellsfrom IR-induced killing.     

No change in DNA damage or repair of single- and double-strand breaks as human diploid fibroblasts age in vitro

Using the in vitro human diploid fibroblast model, we tested theories of aging which hypothesize that either accumulation of DNA damage or decreased DNA repair capacity is causally related to cellular senescence. Between population doubling level (PDL) 32 and 71, fetal lung-derived normal diploid human fibroblasts (IMR 90) were assayed for both DNA single-strand breaks (SSBs, spontaneous and induced by 6 Gy) and DNA double-strand breaks (DSBs, spontaneous and induced by 100 Gy). After gamma-irradiation cells were kept on ice unless undergoing repair incubation at 37 degrees C for 7.5-120 min or 18-24 h. To assay DNA strand breaks we used the filter elution technique in conjunction with a fluorometric determination of DNA which is not biased in favor of proliferating aging cells as are radioactive labelling methods. We found no change with in vitro age in the accumulation of spontaneous SSBs or DSBs, nor in the kinetics or completeness of DNA strand rejoining after gamma-irradiation. Cells at varying PDLs rejoined approx. 90% of SSBs and DSBs after 60 min repair incubation and 100% after 18-24 h repair incubation. We conclude that aging and senescence as measured by proliferative lifespan in IMR 90 cells are neither accompanied nor caused by accumulation of DNA strand breaks or by diminished capacity to rejoin gamma-radiation-induced SSBs or DSBs in DNA.     

Comet assay evaluation of DNA single- and double-strand breaks induction and repair in C3H10T1/2 cells

Ionizing radiation is a potent inducer of DNA damage because it causes single- and double-strand breaks, alkali-labile sites, base damage, and crosslinks. The interest in ionizing radiation is due to its environmental and clinical implications. Single-strand breaks, which are the initial damage induced by a genotoxic agent, can be used as a biomarker of exposure, whereas the more biologically relevant double-strand breaks can be analyzed to quantify the extent of damage. In the present study the effects of ¹³⁷Cs γ-radiation at doses of 1, 5, and 10 Gray on DNA and subsequent repair by C3H10T1/2 cells (mouse embryo fibroblasts) were investigated. Two versions of the comet assay, a sensitive method for evaluating DNA damage, were implemented: the alkaline one to detect single-strand breaks, and the neutral one to identify double-strand breaks. The results show a good linear relation between DNA damage and radiation dose, for both single-strand and double-strand breaks. A statistically significant difference with respect to controls was found at the lowest dose of 1 Gy. Heterogeneity in DNA damage within the cell population was observed as a function of radiation dose. Repair kinetics showed that most of the damage was repaired within 2 h after irradiation, and that the highest rejoining rate occurred with the highest dose (10 Gy). Single-strand breaks were completely repaired 24 h after irradiation, whereas residual double-strand breaks were still present. This finding needs further investigation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Radioprotective action of WR-1065 on radiation-induced DNA strand breaks in cultured Chinese hamster ovary cells

We have examined the radioprotective effect of WR-1065 on cultured Chinese hamster ovary cells. The effects of the drug on the induction and rejoining of gamma-ray-induced DNA single-strand breaks (SSBs) and double-strand breaks (DSBs) were measured using alkaline (pH 12.1) and neutral (pH 7.0) elution, respectively. Molecular protection factors (PFs) calculated from these data allowed us to determine whether the degree of modification of strand breakage accurately predicted the PFs measured using the biological end point of cell survival. The drug did protect against the induction of both SSBs and DSBs, although to an extent that did not appear to fully account for the degree of radioprotection in terms of cell killing measured under identical conditions. It is therefore unlikely that radioprotection by WR-1065 occurs simply as a consequence of a general lowering of all types of gamma-ray-induced DNA lesions, and it is possible that the drug could differentially protect against the induction of subsets of these DNA lesions. The rate of SSB rejoining was retarded following preirradiation treatment of cells with WR-1065, but there was no effect on DSB rejoining. Postirradiation treatment with WR-1065 also appeared to retard SSB rejoining but without an accompanying effect on either DSB rejoining or cell survival; however, this effect was largely reversed by the addition of catalase and was therefore probably a result of H2O2 generated by autoxidation of the drug. Based on these observations, it would appear that the molecular actions of aminothiol radioprotective compounds that lead to reduced cell killing are much more complex than previously thought.     

Potentiation of cell killing by low-dose-rate radiation by camptothecin is related to an increase in the level of DNA double-strand breaks

We investigated the ability of camptothecin to potentiate cell killing by low-dose-rate irradiation and whether this potentiation was associated with an increase in the level of residual DNA double-strand breaks (DSBs). Human melanoma (Sk-Mel-3) cells, grown to the confluent phase, were treated with low-dose-rate radiation (0.88 cGy/min) alone, camptothecin alone, or concurrent camptothecin and low-dose-rate radiation. Cell survival was determined using a clonogenic assay. The interactions between camptothecin and low-dose-rate radiation were analyzed further using isobolograms. DNA DSBs were determined using the neutral comet assay. We found that 10 and 25 microM camptothecin, but not 1 microM, camptothecin potentiated cell killing significantly relative to that seen with low-dose-rate radiation alone. Unexpectedly, the potentiation of the effects of low-dose-rate radiation by camptothecin was accompanied by large increases in the alpha parameter of the linear-quadratic fit rather than in the beta parameter. This suggests a modification of intrinsic radiosensitivity rather than of repair of sublethal damage. From isobologram analysis, low-dose-rate radiation interacted either additively or supra-additively with 25 or 10 microM camptothecin. Conversely, the interaction of low-dose-rate radiation with 1 microM camptothecin was subadditive. Finally, there were strong correlations (correlation coefficients >0.9) between surviving fraction and either comet tail length or comet tail moment after concurrent treatment with 25 microM camptothecin and low-dose-rate radiation. This suggests that the level of residual DNA DSBs was a good indicator of cell killing after treatment with low-dose-rate radiation plus 25 microM camptothecin.