DNA-break repair, radioresistance of DNA synthesis, and camptothecin sensitivity in the radiation-sensitive irs mutants: Comparisons to ataxia-telangiectasia cells

Mutation Research, 235 (1990) 49-58 DNA Repair Elsevier

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MUTDNA 06371

DNA-break repair, radioresistance of D N A synthesis, and camptothecin sensitivity in the radiation-sensitive irs mutants: Comparisons to ataxia-telangiectasia cells J o h n T h a c k e r a n d A n i l N. G a n e s h MRC Radiobiolog), Unit, Chilton, Didcot, Oxon 0)(11 ORD (Great Britain) (Received 9 March 1989) (Revision received 27 September 1989) (Accepted 6 October 1989)

Keywords: DNA-break repair; DNA synthesis, radioresistance; Camptothecin sensitivity; Irs mutants; Ataxia-telangiectasia cells

Summary Induction and rejoining of DNA single-strand breaks (ssb) and double-strand breaks (dsb) after y-irradiation were measured, respectively, by alkaline and neutral sucrose gradient sedimentation methods. The radiosensitive mutants irsl, irs2, and irs3 showed no significant difference from wild-type V79 hamster cells in ability to rejoin either ssb or dsb, while the previously-described xrs-1 mutant showed the expected defect in rejoining dsb. The resistance of DNA synthesis to y-irradiation was measured in the 3 irs mutants and, for comparative purposes, in transformed human cell lines from normal and ataxiatelangiectasia (A-T) individuals. The irs2 mutant was found to be very similar in response to the A-T lines, showing a marked decrease in inhibition of DNA synthesis, compared to V79 cells, in both time-course and dose-response experiments. However, irsl also had some decrease in inhibition at the higher doses used, while irs3 was similar to the wild-type V79 cells. Both irsl and irs2 were found to be considerably more sensitive to the DNA topoisomerase I-inhibitor camptothecin, while irs3 was only slightly more sensitive than the parent V79 line. These data place the irs mutants in a similar category of radiosensitive phenotype to A-T cells, but we view this as only the beginning of a useful classification of this type of mutant. The irs2 mutant has the strongest links to A-T cells, through its sensitivity profile to DNA-damaging agents and radioresistant DNA synthesis, but irsl in particular has other similarities to A-T.

The isolation and characterization of mutants with defined sensitivities to DNA-damaging agents will help understand the way in which those agents produce cell killing, mutation and other responses. While the characterization of mutants is vital to

Correspondence: Dr. J. Thacker, MRC Radiobiology Unit, Chilton, Didcot, Oxon O X l l 0RD (Great Britain).

gain such knowledge, it is often difficult to know the most appropriate tests to establish the nature of the mutated function, especially at the molecular level. We have recently described a series of new mutants, isolated from V79 hamster cells, which exhibit an approx. 3-fold sensitivity to X-rays (Jones et al., 1987). These mutants, called irsl, irs2 and irs3, were shown to have different spectra

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50 of sensitivities to other DNA-damaging agents and to fall into separate complementation groups (Jones et al., 1987, 1988). With one recent exception (see below) they are also genetically distinct from a number of other well-described radiationsensitive mutants of mammalian cells (Jones et al., 1988; J. Thacker and R.E. Wilkinson, unpublished). We have speculated previously (Jones et al., 1987) on the possible relationship between these mutants and cells derived from patients with the radiation-sensitive human disorder, ataxia telangiectasia (A-T). A-T cells are also about 3-fold more sensitive to X- and 3,-rays, and have similarities to at least one of the irs mutants in their response to other DNA-damaging agents. To characterize these mutants further, and to relate each to the A-T cellular phenotype, we have undertaken relevant DNA repair and synthesis assays after irradiation and exposed the mutants to the DNA topoisomerase 1-inhibitor camptothecin. DNA-strand breakage appears to be an important type of lesion induced by ionising radiations. In both bacteria and yeast, a number of radiation-sensitive mutants have defects in strand-break repair (Sargentini and Smith, 1986; Burtscher et al., 1988). Similarly, some radiosensitive mammalian cell mutants have been shown to have reduced levels of DNA single-strand break repair (Thompson et al., 1982; Fuller and Painter, 1988) or double-strand break repair (Kemp et al., 1984; Giaccia et al., 1985; Evans et al., 1987; Wlodek and Hittelman, 1987; Zdzienicka et al., 1988). However, A-T cells appear to have no detectable defect in the rejoining of DNA singleor double-strand breaks, when assayed with standard biochemical procedures such as sucrosegradient sedimentation or filter elution (review: McKinnon, 1987). On the other hand, the response of DNA synthesis to irradiation is abnormal in A-T cells; normal human cells show a rapid decrease in synthesis after irradiation while A-T cells show little decrease ("radioresistant DNA synthesis"; Painter, 1986). Therefore, we have investigated the rejoining of strand breaks (using alkaline and neutral sucrose gradient sedimentation) and the radioresistance of DNA synthesis in the 3 its mutants.

The antitumour drug camptothecin is thought to exert its cytotoxic effect through an interaction with DNA topoisomerase I (Hsiang and Liu, 1988), and camptothecin-resistant mammalian cells have alterations in the level and kinetic properties of topoisomerase I (Andoh et al., 1987; Gupta et al., 1988; Kjeldsen et al., 1988a). The specificity of the drug is shown by recent experiments with yeast: inactivation of the TOP1 gene to give no topoisomerase I activity effectively makes these cells resistant to camptothecin (Nitiss and Wang, 1988). Topoisomerase I catalyses the transient breakageand-rejoining of single strands in duplex DNA, and it is thought that camptothecin interferes with its ability to rejoin these breaks by forming a d r u g - e n z y m e - D N A complex (Hsiang and Liu, 1988; Kjeldsen et al., 1988b). Very recently, it has been shown that A-T cells are sensitive to camptothecin, relative to cells of normal ionising radiosensitivity, although A-T cells do not show altered levels of topoisomerase I (Smith et al., 1989). Thus, these authors considered that the A-T sensitivity may relate to an inability to resolve this type of DNA break. Since this is a potentially important new phenotype of A-T cells, we have also measured camptothecin sensitivity in the irs mutants. Materials and methods Cell cultures

V79-4 Chinese hamster cells (Thacker, 1981) and the mutant derivatives irsl, irs2 and irs3 (Jones et al., 1987) were grown in Eagle's minimal essential medium (MEM) supplemented with 10% foetal calf serum, 2 mM glutamine and antibiotics. Cells were grown as monolayers in 5% CO2:95% air at 37°C and harvested with 0.1% trypsin and 0.04% EDTA. The transformed human cell cultures MRC5V1 (Huschtscha and Holliday, 1983), A T 5 B I V A (Day et al., 1980), and A T 4 B I / N E 1 (kindly supplied by the MRC Cell Mutation Unit, Brighton) were grown similarly. The xrs-1 mutant (kindly supplied by Dr. P.A. Jeggo, NIMR, London) was grown in a-MEM as described previously (Thacker and Stretch, 1985). Cells were taken from a freezer stock and grown for 2-3 days before each experiment.

51

DNA-strand break rejoining Freshly-grown cells were respread at 106 per flask in 25-cm 2 flasks containing 629 kBq/ml [Me-3H]thymidine (925 GBq/mmole; Amersham International). The next day the radioactive medium was removed, the cell monolayer washed with MEM, and the cells reincubated for 1 h with 5 ml fresh medium containing 8 / t g / m l thymidine. The cells were then harvested by trypsinization and irradiated suspended in complete medium on ice. A 60Co v-ray source was used for irradiations at approx. 18 Gy/min. After irradiation the cell suspension was split into (i) control: held on ice for a time equal to the longest incubation time, and (ii) incubated samples: cells replaced into 25-cm2 flasks and incubated at 37°C for various time periods. The cells were then harvested and resuspended in 200 #1 buffer (10 mM Tris, 1 mM EDTA, 150 mM NaC1, pH 7.5) for counting. A volume equivalent to 2 × 104 cells (about 30 #1) was added to 120 #1 lysis buffer per gradient. Different types of strand break were estimated as follows: (a) Single-strand breaks (based on the methods of Millar et al., 1981). Lysis buffer (0.5 M NaOH, 10 mM EDTA, 1% Sarkosyl) and cells were loaded onto 5-20% sucrose gradients containing 1 M NaC1, 1 mM EDTA, 100 mM NaOH and 0.1% Sarkosyl. Loaded gradients were held for 4 h in the rotor (Beckman SW 55) to allow for cell dissociation and denaturation of DNA prior to centrifugation at 10000 rpm for 14 h at 20°C. T4 phage was used as a molecular weight standard to calibrate the gradients. (b) Double-strand breaks (based on the methods of Bloecher, 1982). Lysis buffer (2 mg/ml proteinase K, 50 mM Tris, 10 mM EDTA pH 8.7, 10% Sarkosyl, 10% SDS, 5% sodium deoxycholate) and cells were placed in 1-ml syringes (the tips of which had been cut off, to avoid sheafing forces, and then sealed with plastic film). The syringes were held on ice for 30 min, then at 70°C for 30 min, and lastly at 50°C for about 18 h. The lysed cells were transferred to 5-20% sucrose gradients containing 100 mM NaC1, 0.1% Sarkosyl, 10 mM Tris, 1 mM EDTA (pH 7.5). Loaded gradients were held for 4 h in the rotor (as above) and centrifuged at 3000 rpm for 65 h at 20°C. The linearity of the sucrose gradients was checked prior to use by refractive index measure-

ments of fractions. After centrifugation of the DNA samples, fractions were collected onto Whatman 3MM filters; these were soaked twice in 10% TCA, washed in ethanol and dried for scintillation counting. Gradient profiles were analysed on the assumption that the DNA is randomly broken, to give the weight-averaged molecular weight using a curve-fitting procedure described by Fox (1976).

Post-irradiation DNA synthesis Freshly-grown cells were respread at 105/flask in 25-cm 2 flasks with 5 ml complete medium and incubated at 37°C for 24 h. At this time 925 Bq [Me-14Clthymidine (1.9 GBq/mmole; Amersham International) were added to each flask, followed by reincubation for a further 24 h. The medium was then replaced and the cells incubated for 30 min at 37°C, before irradiation. The medium was replaced with fresh ice-cold medium, the flasks irradiated (as described above), and the medium was again replaced with fresh ice-cold medium (to keep the cells under relatively constant conditions). The cells were held in their flasks on ice until all irradiations were completed and then placed at 37°C; 370 kBq [Me-3H]thymidine (3.3 T B q / mmole; Amersham International) were added to each flask at an appropriate time for DNA synthesis measurement (see text). After incubation, the flasks were placed on ice; using ice-cold solutions, the cells were washed twice with saline, scraped into 1 ml saline and added to 1 ml 10% TCA. After holding on ice for 15 min, the solution was filtered onto Whatman G F / C filters, rinsed and dried for scintillation counting. Cells exposed to [14C]thymidine alone or [3H]thymidine alone were always run as controls, from which the dualcounting windows were established for both unirradiated and irradiated cells. After appropriate correction the results were expressed as the ratio of [3Hlthymidine incorporation (post-irradiation synthesis) to [14C]thymidine incorporation (overall synthetic activity as a measure of culture growth). In time-course experiments, unirradiated samples were measured at the same time intervals as irradiated cultures and the results are expressed as irradiated relative to unirradiated incorporation levels. In dose-response experiments, incorporation was measured in duplicate irradiated flasks

52

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and similarly expressed relative to unirradiated flasks; on average, within-experiment duplicates varied by less than 10%.

Carnptothecin sensitivity Camptothecin (Sigma) was dissolved immediately before use in fresh dimethyl sulphoxide (ACS grade; Sigma) at 1 m g / m l , and then diluted in MEM to give stocks at 100 times final concentrations. Camptothecin was added to cells in complete medium after one-day's growth from 106/75-cm 2 flask (1 flask per treatment concentration). After 24 h treatment at 37°C, the cells were washed once with MEM, then harvested, centrifuged and counted. After appropriate dilution, cells were respread in 9-cm dishes with complete medium and incubated for colony formation as described previously (Jones et al., 1987).

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DNA strand-break rejoining D N A single-strand breaks were measured in cells exposed to 100 Gy -/-rays immediately after irradiation and after 30 min post-irradiation incubation at 37°C. The immediate reduction in molecular weight of single-stranded D N A in all

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cell lines was to about 5 × 10 7 d, corresponding to about 20 breaks/1011 d / G y (similar to published values; review, Lehmann, 1978). A sizeable fraction of such damage (85-90%) was rejoined in 30 rain and no difference in the amount of rejoining was found for the V79 wild type cells and for the mutants irsl, irs2, its3 and xrs-1 (data not shown). The data for xrs-1 were consistent with those already published by Kemp et al. (1984). The measurement of D N A double-strand breaks gave more variable results, and time courses of post-irradiation incubation were carried out for up to 8 h. Combined data for all cell lines following a dose of 200 Gy are shown in Fig. 1; analysis of variance on the data for V79, irsl, irs2 and irs3 provided no significant evidence of a difference between these lines. Similarly, curves fitted to the

53

data for each line except xrs-1 did not differ significantly. As anticipated from published data (Kemp et al., 1984; Weibezahn et al., 1985), the xrs-1 mutant showed a much reduced ability to rejoin double-strand breaks.

20 Gy or 40 Gy under constant temperature (4 o C) conditions (see Materials and Methods). These experiments (Figs. 2 and 3) showed, for hamster cells: (a) inhibition curves are multiphasic and are affected little by the temperature at irradiation (see Fig. 3 where the X symbols are test irradiations at room temperature, showing a slightly faster course of inhibition but no overall change in the curve shape); (b) increasing the y-ray dose affects the initial slope of inhibition (i.e., up to 2-3 h after irradiation) but in most cases particularly affects the relative level to which synthesis declines, especially distinguishing irs2 cells from V79 and the other irs mutants, Other differences are also apparent, however: the degree of dose-dependence for synthesis inhibition is least for irsl cells (compare Figs. 2 and 3), and irs3 shows greater recovery potential than V79 especially after 40 Gy (Fig. 3). Dose-response data, obtained by labelling with [3H]thymidine for periods covering different parts of the time courses, were broadly consistent with time-course data. Thus, A-T and normal human cells could be distinguished when labelled for 2 h periods, starting either 45 min after irradiation to measure mostly the initial slope (Fig. 4a) or 2.5 h after irradiation to measure the recovery phase

Post-irradiation DNA synthesis

Enhanced resistance of DNA synthesis to radiation has been found, with few exceptions (see Discussion), to be a characteristic of cells from the radiosensitive disorder ataxia-telangiectasia (A-T). In this study, post-irradiation DNA synthesis was measured in transformed lines of A°T and normal human cells for comparison to V79 and the irs mutants. However, measuring synthesis for 20 min at 37°C immediately after irradiation (5-40 Gy at room temperature) gave very poor data repeatability and did not allow good distinctions to be made between any of the cell lines, including A-T and normal cells. This result was not improved by giving a 30-min post-irradiation incubation before labelling with [3H]thymidine to allow for replicons which had initiated synthesis before irradiation to complete chain elongation (Painter, 1981). In an attempt to define appropriate conditions for measurement, time courses of DNA synthesis at 37°C were carried out following irradiations at

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(not shown). Similarly, irs2 cells were clearly less inhibited than V79 cells when labelled for 2 h starting at 45 min after irradiation (Fig. 4b) but less well separated especially at the higher doses by measurements including the recovery phase (Fig. 5 and data not shown). Post-irradiation synthesis in irsl cells showed consistently a dosedependence which is similar to that of V79 at low doses but tending towards that of irs2 at the higher doses used, while irs3 synthesis is closest to V79 over the whole dose range (Fig. 5 and data not shown). Camptothecin sensitivity Responses to a 24-h exposure to camptothecin, followed by respreading into medium without drug, are shown in Fig. 6. It is evident that all the mutants show some carnptothecin sensitivity, compared to V79 cells, but that irsl and irs2 are especially sensitive to this drug (approx. 5-6-fold increase in sensitivity at 37% survival). Interestingly, on a semi-log plot, irsl shows an approximately linear response while that for irs2 is curvi-

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55 linear to give a greater sensitivity at the higher concentrations used. Discussion

The stimulus behind these experiments was to characterize the newly-isolated irs mutants with methods related to their ionising radiation sensitive phenotype, and in particular to compare these mutants to A-T cells. It was found that none of the its mutants have defective repair of single- or double-strand breaks within the detection limits of the techniques used. However, the measurement of post-irradiation DNA synthesis did distinguish some of the irs mutants from normal V79 cells. While the lack of detection of a DNA strandbreak repair defect does not distinguish the its mutants from each other, it does liken these mutants to cells from the human A-T disorder. In common with the it's mutants, A-T cells show an approx. 2-3-fold increase in cell lethality on exposure to X-rays when compared with normal human cells (Taylor et al., 1975; Cox and Masson, 1980; compared to Jones et al., 1987). Thus, as a group, A-T and the irs mutants may be contrasted at present to radiosensitive mutants which show measurable DNA strand-break repair defects (see Introduction). The contrast is particularly strong for hamster cell mutants with impaired doublestrand break repair since these also have more extreme radiosensitivity (6-fold or more; Jeggo and Kemp, 1983; Stamato et al., 1983; Zdzienicka et al., 1988), but it may be necessary to develop more sensitive methods to verify that the its mutants are completely 'normal' in DNA strandbreak processing. Indeed, the irsl mutant does show a correlation with one line of A-T cells (AT5BIVA) in a plasmid-based assay: both cell lines show a reduced fidelity in rejoining enzyme-induced DNA dsb when compared to normal cells (review: Thacker, 1989). However, irsl also shows a greatly enhanced sensitivity to mitomycin C compared to the parent V79 cells (Jones et al., 1987), while no consistent hypersensitivity to this agent has been found for A-T cells (review: Lehmann, 1982). It is tempting to speculate that the camptothecin sensitivity shown by irsl and its2 may comment further on the response of these mutants to

agents causing DNA-strand breakage: as noted in the Introduction, camptothecin appears to affect cells primarily through the 'fixation' of topoisomerase I-associated breaks. However, as with most other inhibitors, it is not yet clear whether other mechanisms may operate in mammalian cells (cf. Downes and Johnson, 1988). Interestingly, the order of sensitivity to camptothecin for the irs mutants is the same as that to ionising radiations (irs2 > irsl > irs3; Jones et al., 1987), although the relative sensitivities differ. Thus, irsl and irs2 are relatively more sensitive to camptothecin than to X-rays (about 5-6-fold compared to 3-fold, respectively, at 37% survival), while irs3 is relatively more sensitive to X-rays than to camptothecin. Additionally, the shape of survival curves, on a semi-log plot, for irsl is linear for both X-rays and for camptothecin, in contrast to the curvilinear shapes of survival curves for irs2 and irs3 (Fig. 6 and Jones et al., 1987). These factors may relate to a common mechanistic basis for their sensitivity to X-rays and camptothecin. A-T cells also show a somewhat higher relative sensitivity to camptothecin than to X-rays (Smith et al., 1989), again suggesting their similarity to irsl and its2. Additionally, it has been found that A-T cells show some increase in relative sensitivity to topoisomerase II inhibitors, such as etoposide (Henner and Blaska, 1986; Smith et al., 1986), or to less specific inhibitors such as novobiocin (Debenham et al., 1987; Thacker and Debenham, 1988). This sensitivity does not correlate to topoisomerase II levels, since different A-T cell lines may show either higher- or lower-than-normal enzyme activity or amounts (Singh et al., 1988; Smith and Makinson, 1989), again leading to the possibility that the repair of inhibitor-associated DNA breakage may be defective in A-T cells. In contrast to A-T cells it has been found that its2 shows no enhanced sensitivity to novobiocin (unpublished data) or to etoposide (P.A. Jeggo, personal communication) when compared to V79 cells, while irsl shows an approx. 2-fold enhanced sensitivity to either inhibitor. Measurement of the radioresistance of DNA synthesis prompts a comparison of the irs2 mutant, in particular, with A-T cells. The dose-response curves of Figs. 4 and 5 show that, when compared to their respective parents, irs2 and A-T cells

56 respond with remarkable similarity. This result is supported by studies on another set of radiationresistant mutants isolated by Zdzienicka et al. (1987): we have recently shown that their mutants V-C4, V-E5 and V-G8 are in the same genetic group as its2 (J. Thacker and R.E. Wilkinson, unpublished) and these mutants have also been found to show radioresistant DNA synthesis (M.Z. Zdzienicka, personal communication). In terms of their sensitivity to other DNA-damaging agents, the irs2 mutants also correlate well with the A-T cellular phenotype; both cell types show little enhancement of sensitivity to ultraviolet light, alkylating agents or mitomycin C (Lehmann, 1982; Jones et al., 1987). However, the measurement and interpretation of radioresistant D N A synthesis is worthy of further comment. We experienced difficulty in measuring post-irradiation D N A synthesis reproducibly especially with relatively short labelling periods (see Results). Similarly, Young and Painter (1989) have recently reported between-experiment variability (with little within-experiment variability) in these measurements but found that they could always distinguish A-T from normal human cells within a given experiment. Further, it may be noted that discrepancies occur in data for other human syndromes on the extent a n d / o r presence of radioresistant D N A synthesis (e.g., for Down's syndrome: Barenfeld et al., 1986; Ganges et al., 1988; Young and Painter, 1989). These examples suggest that the apparent simplicity of the D N A synthesis assay may be misleading; unrecognized variables are present which may influence the feasibility of showing a difference between two cell hnes. Some of these variables may reside in the experimental protocol: in published data, for example, the length of the D N A labelling period has been varied between 10 rain and 4 h, so that different parts of the inhibitory curve will be sampled. In the present experiments we were able to show that, relative to their respective normal counterparts, irs2 and A-T cells had a similar reduction in DNA synthesis inhibition under a variety of measurement protocols (Figs. 2-5 and data not shown). Is there, however, any significance to the differences found for the other irs mutants? It seems unlikely that irs3 can be distinguished from V79, but irsl shows consistently a

trend of normal inhibition at low doses and A - T / irs2-1ike inhibition at the higher doses used (e.g.,

Fig. 5). Thus the final slope of the dose-response curve is less steep than normal in irsl cells. In other cell systems, this final slope has been interpreted as a reflection of inhibition of DNA-chain elongation, while the initial slope results from inhibition of replicon initiation (review: Painter, 1986). If this was true for irsl cells, then chain elongation is more resistant than normal. However, our confidence in such interpretation is weakened by the fact that chain elongation has been found to be especially resistant in A-T cells (Painter, 1986), yet they do not show the same dose-response kinetics as irsl. In summary, we are beginning to see a more complex functional categorization of ionising radiation-sensitive mutants. We noted above that the irs mutants could be grouped with A-T cells in a category of "moderately-sensitive" mutants, in contrast to more sensitive mutants showing overt D N A double-strand break repair defects. We are now beginning to see further sub-division of these mutant groups, with irs2 having the greatest similarity to A-T cells, but with the other irs mutants also having some common features. Note. While this paper was being revised, similar DNA strand-break repair and radioresistant D N A synthesis data for the independently-isolated mutants V-C4, V-E5 and V-G8 were reported to those found here for irs2 (Zdzienicka et al., 1989).

Acknowledgements This work was supported in part by CEC Contract BI6-E-144-UK. We thank Terry Jenner and Peter O'Neill for advice on DNA-strand-break measurements, David Papworth for statistical analysis, and all those who supplied us with cell lines.

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