International Journal of Radiation Biology
ISSN: 0955-3002 (Print) 1362-3095 (Online) Journal homepage: http://www.tandfonline.com/loi/irab20
Effect of Heat on Induction and Repair of DNA Strand Breaks in X-irradiated CHO Cells E. Dikomey & J. Franzke To cite this article: E. Dikomey & J. Franzke (1992) Effect of Heat on Induction and Repair of DNA Strand Breaks in X-irradiated CHO Cells, International Journal of Radiation Biology, 61:2, 221-233 To link to this article: http://dx.doi.org/10.1080/09553009214550851
Published online: 03 Jul 2009.
Submit your article to this journal
Article views: 14
View related articles
Citing articles: 12 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=irab20 Download by: [University of Toronto Libraries]
Date: 01 December 2015, At: 02:18
INT . J . RADIAT . BIOL .,
1992, VOL . 61,
NO .
2, 221 -233
Effect of heat on induction and repair of DNA strand breaks in X-irradiated CHO cells E. DIKOMEY*t and J . FRANZKEt
Downloaded by [University of Toronto Libraries] at 02:18 01 December 2015
(Received 24 August 1990; second revision received 17 June 1991 ; accepted 28 June 1991)
Abstract. Chinese hamster ovary cells were exposed to various heat treatments followed by X-irradiation, and the induction and repair of DNA strand breaks was studied using the alkaline unwinding technique . Heat treatments alone were found to cause DNA strand breakage only for temperatures >43 ° C, whereas the number of radiation-induced strand breaks was unaffected by additional heating. Strand break repair was studied for irradiated cells preheated at temperatures ranging from 42 ° C to 45 °C . The total repair curve could be separated into three phases, a fast (t=0-15 min), an intermediate (t=15-120 min) and a slow (t,> 120 min) phase . All phases were altered when cells were heated either prior to or after irradiation. The fast and the intermediate phase could be well interpreted by the assumption that irradiation leads to both primary and secondary singlestrand breaks, the latter being generated by enzymatic incision at sites of damaged bases. For irradiation alone, the ratio of all secondary strand breaks to all primary breaks was .5 .°=1 s ±0 . 5 . This ratio was not altered by preceding heat treatments (mean f,.°=1 .7±0 .2) . The main effect of heating on the repair kinetics of single-strand breaks was an increase in the repair half-time of primary and secondary breaks (maximum increase by a factor of 3 . 4), whereas the generation of secondary breaks was only slightly retarded (factor 1 . 3) . The slow repair phase, which is assumed to represent the repair of DNA double-strand breaks, was best described by a single exponential component . The half-time of this component was found to increase from 4,j ., =170 ± 70 min for non-heated cells to i,Iow =345 ±80 min for cells heated at 45° C for 20 min, indicating that heat inhibited the repair of double-strand breaks . For irradiation alone, the initial fraction of the slow component was f,,OW =0 . 065±0 .004. This fraction was enhanced by additional heating, with a maximum increase by a factor of 2 . 7 for cells heated at 45 ° C for 20 min . This elevation cannot be the result of an enhanced induction of double-strand breaks, but must be associated with an additional formation of slowly repaired strand breaks during repair incubation . These additional strand breaks must arise from stand breaks which in nonheated cells are repaired during the fast or intermediate phase . These findings suggest that thermal radiosensitization results from an impaired repair of double-strand breaks and also from the formation of strand breaks which are slowly repaired with time after irradiation .
* Author for correspondence . t Institure of Biophysics and Radiobiology, University of Hamburg, Martinistrafie 52, D-2000 Hamburg 20, Germany .
1 . Introduction The sensitivity of mammalian cells to ionizing irradiation may be enhanced by additional heating . The mechanisms of this so-called thermal radiosensitization are not yet completely understood . It was assumed that thermal radiosensitization did not result from an enhanced induction of DNA lesions, since generally the number of single- and doublestrand breaks and base damage induced by irradiation was found to be unaffected by prior heat treatments (Corry et al. 1977, Warters and Roti Rod 1978, Clark et al . 1981, Lunec et al. 1981, Jorrritsma and Konings 1983, McGhie et al . 1983, Warters et al . 1987) . However, when DNA breakage was detected by the alkaline or neutral elution technique, either no effect (Iliakis et al. 1990) or an increase (Mills and Meyn 1981, Radford 1983, Warters et al. 1987) in the number of breaks was observed, when irradiation was combined with heat . It has often been suggested that the increase in radiosensitivity might be a consequence of impaired repair, since the repair of radiation-induced damage was generally reported to be reduced after heating (Ben Hur and Elkind 1974, Corry et al. 1977, Warters and Roti Roti 1978, Clark et al . 1981 Lunec et al . 1981 Dikomey 1982, Jorritsma and Konings 1983, McGhie et al. 1983, Warters et al . 1987, Iliakis et al. 1990) . However, it has to be taken into account that for some types of damage impaired repair does not necessarily result in an enhanced radiosensitivity, since not all types of lesions are involved in cell death . For instance, after ionizing irradiation cell death primarily results from double-strand breaks and probably also from DNA base damage but not from single-strand breaks (Ward 1985, 1986, Frankenberg-Schwager 1989) . While there are abundant data concerning the effect of heat on single-strand break repair (Ben Hur and Elkind 1974, Clark et al . 1981, Lunec et al. 1981, Dikomey 1982, Jorritsma and Konings 1983,
0020-7616/92 $3 .00 © 1992 Taylor & Francis Ltd
Downloaded by [University of Toronto Libraries] at 02:18 01 December 2015
222
E . Dikomey and J . Franzke
McGhie et al . 1983, Warters et al. 1987 Iliakis et al. 1990) there are only a few reports regarding the effect of heat on the repair of base damage or doublestrand breaks . Warters and Rod Roti (1978, 1979) reported that the excision of damaged bases was depressed after prior heating at 45°C for 20 min, whereas the enzymatic incision as tested by micrococcus endonuclease was only slightly retarded . The repair of double-strand breaks was reported to be depressed by pre-irradiation heat treatments at 43°C or 45°C (Correy et al. 1977, Radford 1983, Iliakis et al. 1990) . The present study was designed in order to obtain further information on DNA strand break repair and its modification by heat . We have shown previously that after ionizing irradiation the kinetics of strand break repair as measured by the alkaline unwinding technique was best described by a sum of three exponential components having different half-times (Dikomey and Franzke 1986) . The fast and intermediate components were interpreted as being due to the repair of two different classes of single-strand breaks, whereas the slow component was assigned to the repair of double-strand breaks . It was the aim of the present report to analyse these three components after heat treatments at different temperatures ranging from 42 to 45°C . For this purpose, cells were first heated and then irradiated on ice with X-rays followed by incubation at 37°C for up to 360 min . DNA strand breaks were measured by the alkaline unwinding technique . Preliminary results have been reported previously (Dikomey and Franzke 1988) .
Braunschweig, Germany) . Sealed plastic flasks containing the cells were completely immersed in a precision-controlled water bath (±0 .05°C) . The temperature of the medium approximated the desired temperature (difference 15 min the number of strand breaks increased again, reached a maximum by t = 25 min, and declined afterwards . This curve demonstrated the existence of two different kinetics : (1) an initial exponential decline, and (2) a complex kinetics rising fom `zero' breaks up to a maximum and descending thereafter . Curve (1) represents the repair kinetics of strand breaks induced directly by irradiation, termed primary breaks . Curve (2) represents the kinetics of those breaks, which are generated and repaired after irradiation, termed secondary strand breaks . Thus, the repair curve shown in Figure 8d demonstrates clearly the occurrence of primary and secondary strand breaks . Assuming that these two types of breaks are also present in non-heated cells and in cells heated prior to irradiation the repair kinetics of the fast repaired primary and secondary strand breaks may be described by an equation proposed by Nelson (1982) :
228
E. Dikomey and J. Franzke
Downloaded by [University of Toronto Libraries] at 02:18 01 December 2015
Nprim + Nsec = NO,prim x exp (-ln2t ) + No,.. Tfast,r Tfast,r t x x [exp (-1 n2 ) Tfast,in - Tfast,r Tfast,in t -exp(- 1n2Tfast,r)] (4) where No,prim is the total number of fast repaired primary strand breaks and No,, the total number of those lesions which are converted into secondary strand breaks ; Tfast, in is the half-time of this conversion and Tfast,r the half-time at which primary and secondary strand breaks are repaired . Equation (4) was fitted to the data shown in Figure 8 by least square fit . The results obtained for the repair kinetics for non-heated cells (Figure 8a) and for cells heated at 43°C for 40 min (Figure 8b) showed an excellent agreement between the experimental data and the calculated curves (Figures 8a and b, solid lines) . The identical result was obtained for the repair curve measured after prior heating at 42 or 43°C for 20 min (data not shown) . It becomes obvious from this analysis that the initial decline (termed fast phase of repair) mainly reflects the repair of primary strand breaks with the half-time Tfast,r . The intermediate phase mainly represents the formation and repair of secondary breaks, and since the formation is the rate-limiting step the half-time, Tfast,in is equal to the half-time by which the number of strand breaks declines during the intermediate phase . In order to fit the experimental data obtained for cells heated at 45°C for 20 min prior to the irradiation (Figure 8c) it was necessary to assume that repair of primary and secondary strand breaks was delayed for the time interval td = 4 .4 + 0 . 3 min, whereas the formation of secondary breaks started immediately after irradiation without any delay . Due to this assumption, the number of primary strand breaks remained constant for the time td, while the number of secondary breaks went up, thus leading to the observed result that the total number of strand breaks increased at the beginning of repair incubation followed by a decline later on (Figure 8c) . Similar assumptions had to be made for cells preheated at 43°C for 60 min and 44°C for 20 min (data not shown), the corresponding delay times were td =1 .3 ± 0 . 4 min and td = 2 . 9 + 0 . 3 min, respectively. In addition, from curve fitting the values for the total number of secondary strand breaks, No,sec, (see Equation (4)) could be obtained . From these numbers the relative frequency fsec = No,seclNtot was calculated, where Ntot represents the total number of
all primary strand breaks including fast and slowly repaired primary strand breaks . The calculated ratios for heated cells are in the range of 1 .0 and 2 .5 (Table 1) . There might be a slight tendency that ec increases with increasing temperature, but due to the large scatter of the data this increase was statistically not significant . The mean value calculated from the fsec ratios for heated cells was eC =1 . 7 ± 0 .2, which is similar to fsec = 1 .5 + 0 .2 for nonheated cells . The values of Tfast,ln and Tfast,r obtained from curve fitting are plotted in Figure 7 (diamonds and squares) . Both parameters were found to increase with duration of heat treatment and with temperature, but the effect for Tfast,in was smaller as compared to Tfast,r . For example, after a prior heating at 45°C for 20 min Tfast,in was increased by a factor of 1 . 3 (Tfast,in = 22 . 3 + 1 . 1 min versus Tfast,in = 17 + 5 min for non-heated cells), whereas a 3 .4-fold increase was observed for the repair half-time Tfast,r (Tfast,r = 7 .8 + 0 . 1 min versus rfast,,, = 2 .3 + 0 .2 min for non-heated cells) . This result indicates that the formation of secondary strand breaks was only slightly affected by heat, whereas the repair was inhibited more strongly. Consequently, in heated cells more secondary breaks accumulated with time after irradiation than in non-heated cells . This becomes obvious from Figure 8 (a-c) showing that the maximum number of secondary strand breaks (broken lines) increased with the duration and the temperature of the heat treatment applied which, in addition, leads to the gradual disappearance of the inflection on the decline of the repair curve . The parameters Tfast,r, Tfast,in and fsec were obtained after subtracting a hypothetical curve constructed for the slow phase, which was based on a fast initial rise within 2 min (Figure 6) . Adopting a slow rise within 80 min, the calculated values would increase by less than 10-15% (data not shown) . Thus, the values obtained should be regarded as lower limits . The repair curve obtained for cells heated at 45°C immediately after irradiation (Figure 8d) could not be analysed by curve fitting, since Equation (4) implies that the cells were incubated at 37°C after irradiation but not at elevated temperatures .
f
f
4. Discussion The present study showed that heat did not affect the induction of DNA strand breaks by Xirradiation but substantially altered the repair kinetics
Downloaded by [University of Toronto Libraries] at 02:18 01 December 2015
Strand break repair and heat
DNA strand breaks were measured using the alkaline unwinding technique (Ahnstrom and Erixon 1981, Rydberg 1975) . It was found that the kinetics of DNA unwinding in alkaline solution was the same for heated and non-heated cells (Figure 3) . This result showed that the enhanced amount of proteins associated with the DNA in heated cells (Roti Rod et al. 1979) did not alter the rate of unwinding . As a consequence, the relationship between the fraction of DNA remaining doublestranded after alkaline denaturation for 30 min and the number of X-ray-induced strand breaks as determined for non-heated cells (Equation (3)) could also be applied for heated cells . The occurrence of strand breaks was observed after heat treatment alone, but temperatures of 43-45°C were required to induce a measurable number of strand breaks during a 1 h period (Figures I and 2) . This is in agreement with the results of a previous work (Dikomey 1982) and of other investigators (Jorritsma and Konings 1984, Mitchel and Birnboim 1985, Warters et al. 1985, Warters and Brizgys 1987) . Our result that the total number of strand breaks induced by X-irradiation was unaffected by preceding heat treatments at 40-46 °C (Figures 1 and 2) is in accordance with previous reports, where strand breaks were detected either by the alkaline unwinding or the alkaline sedimentation technique (Corry et al . 1977, Clark et al. 1981, Lunec et al . 1981, Jorritsma and Konings 1983, McGhie et al . 1983, Warters et al . 1987) . However, when strand breaks were measured by the alkaline or neutral elution technique, additional hyperthermia either had no effect (Iliakis et al . 1990) or caused an increase in the number of strand breaks induced by irradiation (Mills and Meyn 1981, Radford 1983, Warters et al . 1987) . The latter results might be due to the fact that the elution techniques strongly depend on the amount of proteins associated with the DNA (Okayasu and Iliakis 1989), and this amount is known to be enhanced by heat (Roti Roti and Winward 1980, Mills and Meyn 1981) . Strand break repair was studied after an X-ray dose of 9 Gy combined with various heat treatments (Figures 4-6) . Independently of the heat treatment applied the total repair curve could be described by three phases : a fast (t=0-15 min), an intermediate (t=15-120 min) and a slow phase (t>, 120 min) . It was previously demonstrated for irradiated cells that the slow phase represents the repair kinetics of DNA double-strand breaks, since the slow repair component measured by the alkaline unwinding technique for several different conditions agreed with the
229
repair curve of double-strand breaks measured by the neutral sedimentation technique (Bryant and Blocher 1980, 1982, Blocher et al. 1983 Dikomey and Franzke 1986) . For double-strand breaks detected by the method of premature chromosome condensation, the repair curve could also be described by a single exponential component (Johnson et al. 1982, Cornforth and Bedford 1983, Iliakis et al. 1988, Wlodek and Hittelman 1988), which is in accordance with the results obtained by the alkaline unwinding or neutral sedimentation technique . In contrast, when doublestrand breaks were measured by the neutral elution technique, the repair kinetics consisted of a sum of two exponential components ; a fast component with an initial fraction of 70-90% and a half-time of 2-5 min and a slow component with an initial fraction of 10-30% and a half-time of 90-170 min (see, for instance, Weibezahn and Coquerelle 1981, Rowley and Kort 1988) . This strong dependence of the results on the methods applied is a well-documented fact (Peak et al. 1988, Hutchinson 1989), but is not understood . It could be shown that the substrates used by the neutral sedimentation and the alkaline unwinding technique must be different from the substrates used by the neutral elution technique (Costa and Bryant 1990) . Moreover, it can be excluded that the slow repair phase found here (Figures 4-6) represents the component of the slowly repaired double-strand breaks detected by the neutral elution technique, since, in this case, the fraction (Table 1) had to be multiplied by a factor of three to nine in order to obtain the initial fraction of all double-strand breaks . This would lead to values in the range of 18-50%, which is much too high, since the initial fraction of DNA double-strand breaks is generally about 5% for sparsely ionizing irradiation . The most prominent effect of heat on the slow repair phase was an elevation of its backextrapolation value, ( Figures 5 and 6, table 1) . The elevation could not be the result of an enhanced induction of DNA double-strand breaks, since this elevation was also observed if heat was applied after irradiation (Figure 6) . Therefore, the number of double-strand breaks induced in heated and nonheated cells must be the same . However, in heated cells the number of slowly repaired strand breaks must increase during the incubation period after irradiation . This increase shows that a certain number of lesions normally repaired during the fast or intermediate phase, are repaired in heated cells at a rate similar to that of double-strand breaks or are converted into real double-strand breaks . Either
fg,ow
fjoq,
E. Dikomey and J . Franzke
Downloaded by [University of Toronto Libraries] at 02:18 01 December 2015
230
process should result in an elevated backextrapolation value of the slow repair phase as was observed here (Table 1) . In accordance with the results presented here, Corry et al. (1977) and Iliakis et al . (1990) also observed that the number of double-strand breaks induced during irradiation was not altered by heat, whereas Radford (1983) reported on an increase after a prior exposure to 43°C for 1 h . The half-time of the slow phase, ;low, was found to increase with increasing duration and temperature of the heat treatment (Figure 7), which indicates that the repair of double-strand breaks was slowed down in irradiated cells after a prior heat treatment . This result is in accordance to previous reports by Corry et al . (1977), Radford (1983) and Iliakis et al . (1990) . In addition, it was shown here that doublestrand break repair was also slowed down in cells heated immediately after irradiation (Figure 6, triangles) . Adopting the interpretation that the slow repair phase represents the repair of DNA double-strand breaks, the kinetics obtained after subtracting this phase from the total kinetics should represent the repair kinetics of DNA single-strand breaks only (Figure 8) . These kinetics came out differently for the various heat treatments applied . Nevertheless, these different curves could be interpreted by assuming that X-irradiation leads to both primary and secondary strand breaks . As a consequence, an alternative idea discussed previously (Dikomey and Franzke 1986), that the kinetics of single-strand break repair might be a sum of two exponential components describing the repair of two different classes of primary single-strand breaks can be discarded . It was also suggested by other investigators (Bryant et al. 1984) that the kinetics of strand break repair measured after X-irradiation reflects repair of both primary as well as secondary strand breaks . On this basis, and in accordance with the results presented here, we suggest that the initial phase of the repair curve represents mainly the repair of primary breaks, while the intermediate phase is dominated by the formation and repair of secondary strand breaks. The relative frequency of all lesions converted into secondary strand breaks was Sec = 1-5+0-5 for nonheated cells (Table 1) . Secondary strand breaks occur during the excision repair, when the DNA is incised at the sites of base damage by a specific endonuclease . When 'endonuclease-sensitive sites' were measured by the y-endonuclease technique, the relative frequency was 1 .7 ± 0 . 6 (Fohe and Dikomey 1991) . This directly measured value agrees quite
f
well with that determined by curve fitting, which might be taken as a further indication that an analysis of the data by separating primary from secondary single-strand breaks leads to an adequate description of the data . The relative frequency, ec, was not significantly altered by heat (Table 1) . That means that the amount of radiation-induced base damage, which is converted into secondary strand breaks, is not altered by prior heat treatment . Similar results were also obtained by Radford (1986) for mouse L cells preheated at 43°C for 60 min, when base damage was measured by the y-endonuclease technique . The rate at which secondary strand breaks were formed from base damage was found to be only slightly reduced in preheated cells, while the repair of secondary breaks was depressed to a greater extent (Figure 7) . The effect of heat on base damage repair was previously studied by Warters and Roti Roti (1978, 1979) in CHO cells using the thymine damage of the t'-type as a model . Prior heat treatment at 45 ° C for 15 min strongly inhibited the removal of this thymine damage, while the enzymatic incision as tested by micrococcus endonuclease was not, or only slightly, reduced . Since the removal of a damaged base requires an incision as well as further repair steps, the data of Warters and Roti Roti (1978, 1979) indicate that heat affects base damage repair primarily by inhibiting the repair steps, whereas the incision step is not, or only slightly, retarded . This conclusion is in full accordance with our results. The half-time of strand break repair, Tfast,r, was found to increase with increasing duration and temperature of the heat treatment applied (Figure 7), which indicates that the repair of primary singlestrand breaks was slowed down by heat . Similar observations were previously made by other investigators using different cell lines and different techniques (Corry et al . 1977, Clark et al. 1981, Lunec et al . 1981, Jorritsma and Konings 1983, McGhie et al . 1983, Warters et al. 1987, Iliakis et al . 1990) . In cells heated at 45°C immediately after the irradiation strand break repair was found to be as fast as in nonheated cells (Figure 8d) . This is in accordance with previous reports, which showed that incubation at elevated temperatures after irradiation did not affect the rate of repair but even caused an acceleration of strand break repair (Ben Hur and Elkind 1974, Weniger et al . 1979, Dikomey 1982) . After prior heat treatments at 44°C and 45 ° C, the number of strand breaks was found to increase after irradiation followed by a decline after 2-4 min (Figures 5 and 6) . A similar initial increase was previously observed by Jorritsma and Konings
f
Downloaded by [University of Toronto Libraries] at 02:18 01 December 2015
Strand break repair and heat
(1983, 1984) and by Iliakis et al . (1990) which was attributed to an interaction phenomenon between heat and radiation (Iliakis et al. 1990) . It is now shown here, that this increase can also be explained by a delay in the repair of primary and secondary strand breaks . This delay might indicate that the relative rate of repair was rather low at the beginning, but increased when the number of strand breaks decreased and finally approached a constant value leading to exponential kinetics. This increase in the relative rate of repair with decreasing number of strand breaks suggested that in heated cells strand break repair was saturated even after a dose as low as 9 Gy . This saturation in strand break repair might be a consequence of a heat-induced reduction in the activity of DNA repair enzymes such as DNA polymerase a and fl (Spiro et al . 1982, Jorritsma et al . 1986, Dikomey et al . 1987) . In conclusion we show here that the changes in the total kinetics of strand break repair induced by heating either prior to or after irradiation (Figures 4-6) can be interpreted by a depressed repair of primary and secondary single-strand breaks, while the formation of secondary breaks was only slightly affected . Furthermore, heat was found to reduce the rate of double-strand break repair and, in particular, to increase the number of slowly repaired strand breaks during repair incubation . DNA double-strand breaks are regarded to be the most relevant type of damage leading to radiationinduced cell death (Frankenberg-Schwager 1989) . Thus, the alterations found here for the slow repair phase might help to interpret the increase in radiosensitivity which is generally observed for radiation combined with heat (Dikomey and Jung 1991) . The present results indicate that heat causes both a depression in double-strand break repair and also a formation of slowly repaired strand breaks with time after irradiation . As a consequence, in heated cells more double-strand breaks are present and persist for a longer time as compared to non-heated cells, and this might lead to an increase in cell killing and thus to an increase in radiosensitivity, when radiation is combined with heat . Acknowledgements The authors are grateful to Mrs Jutta Schafer for skilfully performing part of the DNA analysis and to Gabriel Bond for writing the computer programs used for data analysis. Appreciation is expressed to Jochen Dahm-Daphi, Christian Fohe and Professor Horst Jung for valuable suggestions and discussions. This investigation was supported by Bundesminis-
231
terium fur Forschung and Technologie, Bonn, by grant number 01 VF8516 .
References AHNSTROM, G . and ERIXON, K ., 1981, Measurement of strand breaks by alkaline denaturation and hydroxyapatite chromatography . In DNA Repair, edited by E. C . Fried-
berg and P. C. Hanawalt (New York and Basel : Marcel Dekker), pp. 403-418 . BEN HUR, E . and ELKIND, M . M ., 1974, Thermally enhanced radioresponse of cultured Chinese hamster cells : damage and repair of single-stranded DNA and a DNA complex . Radiation Research, 59, 484-495 . BLOCHER, D. NvsSE, M . and BRYANT, P . E ., 1983, Kinetics of double-strand break repair in the DNA of X-irradiated synchronized mammalian cells . International Journal of Radiation Biology, 43, 579-584. BRYANT, P. E . and BLOCHER, D., 1980, Measurement of the kinetics of DNA double-strand break repair in Ehrlich ascites tumour cells using the unwinding method . International Journal of Radiation Biology, 38, 335-347 .
BRYANT, P. E . and BLOCHER, D ., 1982, The effect of 9-fl-Darabinofuranosyladenine on the repair of DNA strand breaks in X-irradiated Ehrlich ascites tumour cells. International Journal of Radiation Biology, 42, 385-394 .
BRYANT, P . E ., WARRING, R. and AHNSTROM, G ., 1984, DNA repair kinetics after low doses o(X-rays: a comparison of results obtained by the unwinding and nucleosid sedimentation methods . Mutation Research, 131, 19-26 . CLARK, E . P ., DEWEY, W . C . and LETT, T., 1981, Recovery of CHO cells from hyperthermic potentiation to X-rays : repair of DNA and chromatin . Radiation Research, 85, 302-313 . CORNFORTH, M . N . and BEDFORD, J . S ., 1983, X-ray-induced breakage and rejoining of human interphase chromosomes . Science, 222, 1141-1143 . CORRY, P. M ., ROBINSON, S . and GETZ, S ., 1977, Hyperthermic effects on DNA repair mechanisms . Radiology, 123, 475-482 . COSTA, N . D. and BRYANT, P . E ., 1990, Neutral elution detects only limited inhibition of double-strand break repair by 9-fl-D-arabinofuranosyladenine . Mutation Research, 235, 217-223 . DIKOMEY, E . 1982, Effect of hyperthermia at 42 and 45 ° C on repair of radiation-induced DNA strand breaks in CHO cells International Journal of Radiation Biology, 41, 603-614. DIKOMEY, E . and FRANZKE, J ., 1986, Three classes of DNA strand breaks induced by X-irradiation and internal Prays International Journal of Radiation Biology, 50, 893-908 . DIKOMEY, E . and FRANZKE, J ., 1988, Effect of hyperthermia (42-45 ° C) on repair of DNA strand breaks induced by ionizing irradiation . Hyperthermic Oncology 1988, Vol 1, edited by T. Sugahara and M . Saito (London and Philadelphia : Taylor & Francis), pp 154-155 . DIKOMEY, E . and JUNG, H ., 1991, Thermal radiosensitization in CHO cells by prior heating at 41-46°C . International Journal of Radiation Biology, 59, 815-825 .
DIKOMEY, E ., BECKER, W. and WIELCKENS, K ., 1987, Reduction of DNA polymerase fi activity of CHO cells by single and combined heat treatments . International Journal of Radiation Biology, 52, 775-785. F6HE, C, and DIKOMEY, E ., 1991, The induction of gamma-
Downloaded by [University of Toronto Libraries] at 02:18 01 December 2015
232
E. Dikomey and J. Franzke
endonuclease sensitive sites in X-irradiated CHO cells (Abstract) . International Journal of Radiation Biology, 59, 572 . FRANKENBERG-SCHWAGER, M ., 1989, Review article : review of repair kinetics for DNA damage induced in eukaryotic cells in vitro by ionizing radiation. Radiotherapy and Oncology, 14, 307-320 . GRAUBMANN, S . and DIKOMEY, E ., 1983, Induction and repair of DNA strand breaks in CHO cells irradiated in various phases of the cycle. International Journal of Radiation Biology, 43, 475-483 . HAGEN U ., 1973, Strahlenwirkung auf Struktur and Funktion der Desoxyribonukleinsaure . Biophysik, 9, 279-289 . HUTCHINSON, F., 1989, On the measurement of DNA doublestrand breaks by neutral elution Radiation Research, 120, 182-186 . ILIAKIS, G ., PANTELIAS, G . E . and SEANER, R., 1988, Effect of arabinofuranosyladenine on radiation-induced chromosome damage in plateau-phase CHO cells measured by premature chromosome condensation : implications for repair and fixation of a-PLD . Radiation Research, 114, 361-378 . ILIAKIS, G., SEANER R . and OKAYASU, R., 1990, Effects of hyperthermia on the repair of radiation-induced DNA single- and double-strand breaks in DNA double-strand break repair-deficient and repair-proficient cell lines . International Journal of Hyperthermia, 6, 813-833 . JOHNSON, R . T., COLLINS, A . R . S . and WALDREN, C . A ., 1982, Prematurely condensed chromosomes and the analysis of DNA and chromosome lesions . Premature Chromosome Condensation, edited by P . N . Rao, R . T . Johnson and K . Sperling (New York, London : Academic Press), pp 253-308 . JORRITSMA, J . B . M . and KoNINGS, A. W . T ., 1983, Inhibition of repair of radiation induced strand breaks by hyperthermia, and its relationship to cell survival after hyperthermia alone . International Journal of Radiation Biology, 43, 505-516 . JORRITSMA, J . B . M . and KoNINGS, A . W. T ., 1984 The occurrence of DNA strand breaks after hyperthermic treatments of mammalian cells with and without radiation . Radiation Research, 98, 198-208 . JORRITSMA, J . B. M . BURGMAN, P., KAMPINGA, H . H . and KONINGS, A. W. T ., 1986 DNA polymerase activity in heat killing and hyperthermic radiosensitization of mammalian cells as observed after fractionated heat treatments . Radiation Research, 105, 307-319 . LUNEC. L ., HESSLEWOOD, I . P., PARKER, R. and LEAPER, S ., 1981, Hyperthermic enhancement of radiation cell killing in HeLa S3 cells and its effect on the production and repair of DNA strand breaks . Radiation Research, 85, 116-125 . MCGHIE, J . B., WOLD, E ., PETTERSEN, E . O . and MOAN, J ., 1983, Combined electron radiation and hyperthermia . Repair of DNA strand breaks in NHIK 3025 cells irradiated and incubated at 37, 42 . 5, or 45 ° C . Radiation Research, 96, 31-40 . MILLS, M . D . and MEYN, R . E., 1981, Effects of hyperthermia on repair of radiation-induced DNA strand breaks . Radiation Research, 87, 314-328 . MITCHEL, R . E . J . and BIRNBOIM, H . C., 1985, Triggering of DNA strand breaks by 45 ° C hyperthermia and its influence on the repair of y-radiation damage in human white blood cells . Radiation Research, 45, 2040-2045 . NELSON, S . J ., 1982, Models for DNA damage formation and
repair in mammalian cells exposed to ionizing radiation, Radiation Research, 92, 120-145 . OKAYASU, R . and ILIAKIS, G., 1989, Linear DNA elution dose response curves obtained in CHO cells with nonunwinding filter elution after appropriate selection of the lysis conditions . International Journal of Radiation Biology, 55, 569-581 . PEAK, M . J., PEAK, J. G. and BLAZEK, E . R ., 1988, Symposium report . Radiation-induced DNA damage and repair : Argonne National Laboratory Symposium, Argonne Illinois 60439, 15 April 1988 . International Journal of Radiation Biology, 54, 513-520 . RADFORD. I . R ., 1983, Effects of hyperthermia on the repair of X-ray induced DNA double-strand breaks in mouse L cells. International Journal of Radiation Biology, 43, 551-557 . RADFORD . I . R ., 1986, Effect of radiomodifying agents on the ratios of X-ray-induced lesions in cellular DNA : use in lethal lesion determination . International Journal of Radiation Biology, 49, 621-637 . RoTI ROTI, J . L . and WINWARD . R . T ., 1980, Factors affecting the heat-induced increase in protein content of chromatin . Radiation Research, 81, 138-144. Ron Ron, J . L ., HENLE, K . J . and WINWARD, R . T., 1979, The kinetics of increase in chromatin protein content in heated cells : a possible role in cell killing. Radiation Research, 78, 522-531 . ROWLEY, R . and KORT, L ., 1988, The effect of modulators of radiation-induced G2 arrest on the repair of radiationinduced DNA damage detectable by neutral filter elution. International Journal of Radiation Biology, 54, 749-759 . RYDBERG, B ., 1975, The rate of strand separation in alkali of DNA of irradiated mammalian cells . Radiation Research, 61, 274-287 . Sxov, K . A., 1984, The contribution of hydroxyl radical to radiosensitization : a study of DNA damage . Radiation Research, 99, 502-510 . SPIRO, I . J ., DENMAN, D. L . and DEWEY, W . C ., 1982, Effect of hyperthermia on CHO DNA polymerase a and P. Radiation Research, 89, 134-149 . WARD, J . F ., 1985, Biochemistry of DNA lesions . Radiation Research, 102, S-103-S-111 . WARD, J . F., 1986, Mechanisms of DNA repair and their potential modification for radiotherapy . International Journal
of Radiation
Oncology,
Biology, Physics,
12,
1027-1032. WARTERS, R . L . and ROTI ROTI J . L ., 1978, Production and excision of 5', 6'-dihydroxydihydrothymine type products in the DNA of preheated cells . International Journal of Radiation Biology, 34, 381-384 . WARTERS, R . L. and RoTI Ron J . L., 1979, Excision of X-ray induced thymine damage in chromatin from heated cells . Radiation Research, 79, 113-121 . WARTERS, R . L . and BRIZGYS, L . M ., 1987, Apurinic site induction in DNA of cells heated at hyperthermic temperatures . Journal of Cellular Physiology, 133, 144-150 . WARTERS, R . L., BRIZGYS, L . M . and AXTELL-BARTLETT, J ., 1985, DNA damage production in CHO cells at elevated temperatures . Journal of Cellular Physiology, 124, 481-486 . WARTERS, R . L., LYONS, B . W. and AXTELL-BARTLETT, J ., 1987, Inhibition of repair of radiation-induced DNA damage by thermal shock in Chinese hamster ovary cells . International Journal of Radiation Biology, 51, 505-517 .
Strand break repair and heat
233
Downloaded by [University of Toronto Libraries] at 02:18 01 December 2015
WLODEK, D. and HrrTELMAN, W . N ., 1988, The relationship of WEIBEZAHN, K . F. and COQUERELLE, T ., 1981, Radiation induced DNA double-strand breaks are rejoined by DNA and chromosome damage to survival of synchroligation and recombination processes. Nucleic Acids nized X-irradiated L5178Y cells . II . repair. Radiation Research, 9, 3139-3150 . Research, 115, 566-575. WENIGER, P., WAWRA, E . and DOLEJIS, 1 ., 1979, Die Wirkung von Hyperthermie auf DNA-Reparaturvorgange . Radiation and Environmental Biophysics, 16, 135-141 .
ISSN: 0955-3002 (Print) 1362-3095 (Online) Journal homepage: http://www.tandfonline.com/loi/irab20
Effect of Heat on Induction and Repair of DNA Strand Breaks in X-irradiated CHO Cells E. Dikomey & J. Franzke To cite this article: E. Dikomey & J. Franzke (1992) Effect of Heat on Induction and Repair of DNA Strand Breaks in X-irradiated CHO Cells, International Journal of Radiation Biology, 61:2, 221-233 To link to this article: http://dx.doi.org/10.1080/09553009214550851
Published online: 03 Jul 2009.
Submit your article to this journal
Article views: 14
View related articles
Citing articles: 12 View citing articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=irab20 Download by: [University of Toronto Libraries]
Date: 01 December 2015, At: 02:18
INT . J . RADIAT . BIOL .,
1992, VOL . 61,
NO .
2, 221 -233
Effect of heat on induction and repair of DNA strand breaks in X-irradiated CHO cells E. DIKOMEY*t and J . FRANZKEt
Downloaded by [University of Toronto Libraries] at 02:18 01 December 2015
(Received 24 August 1990; second revision received 17 June 1991 ; accepted 28 June 1991)
Abstract. Chinese hamster ovary cells were exposed to various heat treatments followed by X-irradiation, and the induction and repair of DNA strand breaks was studied using the alkaline unwinding technique . Heat treatments alone were found to cause DNA strand breakage only for temperatures >43 ° C, whereas the number of radiation-induced strand breaks was unaffected by additional heating. Strand break repair was studied for irradiated cells preheated at temperatures ranging from 42 ° C to 45 °C . The total repair curve could be separated into three phases, a fast (t=0-15 min), an intermediate (t=15-120 min) and a slow (t,> 120 min) phase . All phases were altered when cells were heated either prior to or after irradiation. The fast and the intermediate phase could be well interpreted by the assumption that irradiation leads to both primary and secondary singlestrand breaks, the latter being generated by enzymatic incision at sites of damaged bases. For irradiation alone, the ratio of all secondary strand breaks to all primary breaks was .5 .°=1 s ±0 . 5 . This ratio was not altered by preceding heat treatments (mean f,.°=1 .7±0 .2) . The main effect of heating on the repair kinetics of single-strand breaks was an increase in the repair half-time of primary and secondary breaks (maximum increase by a factor of 3 . 4), whereas the generation of secondary breaks was only slightly retarded (factor 1 . 3) . The slow repair phase, which is assumed to represent the repair of DNA double-strand breaks, was best described by a single exponential component . The half-time of this component was found to increase from 4,j ., =170 ± 70 min for non-heated cells to i,Iow =345 ±80 min for cells heated at 45° C for 20 min, indicating that heat inhibited the repair of double-strand breaks . For irradiation alone, the initial fraction of the slow component was f,,OW =0 . 065±0 .004. This fraction was enhanced by additional heating, with a maximum increase by a factor of 2 . 7 for cells heated at 45 ° C for 20 min . This elevation cannot be the result of an enhanced induction of double-strand breaks, but must be associated with an additional formation of slowly repaired strand breaks during repair incubation . These additional strand breaks must arise from stand breaks which in nonheated cells are repaired during the fast or intermediate phase . These findings suggest that thermal radiosensitization results from an impaired repair of double-strand breaks and also from the formation of strand breaks which are slowly repaired with time after irradiation .
* Author for correspondence . t Institure of Biophysics and Radiobiology, University of Hamburg, Martinistrafie 52, D-2000 Hamburg 20, Germany .
1 . Introduction The sensitivity of mammalian cells to ionizing irradiation may be enhanced by additional heating . The mechanisms of this so-called thermal radiosensitization are not yet completely understood . It was assumed that thermal radiosensitization did not result from an enhanced induction of DNA lesions, since generally the number of single- and doublestrand breaks and base damage induced by irradiation was found to be unaffected by prior heat treatments (Corry et al. 1977, Warters and Roti Rod 1978, Clark et al . 1981, Lunec et al. 1981, Jorrritsma and Konings 1983, McGhie et al . 1983, Warters et al . 1987) . However, when DNA breakage was detected by the alkaline or neutral elution technique, either no effect (Iliakis et al. 1990) or an increase (Mills and Meyn 1981, Radford 1983, Warters et al. 1987) in the number of breaks was observed, when irradiation was combined with heat . It has often been suggested that the increase in radiosensitivity might be a consequence of impaired repair, since the repair of radiation-induced damage was generally reported to be reduced after heating (Ben Hur and Elkind 1974, Corry et al. 1977, Warters and Roti Roti 1978, Clark et al . 1981 Lunec et al . 1981 Dikomey 1982, Jorritsma and Konings 1983, McGhie et al. 1983, Warters et al . 1987, Iliakis et al. 1990) . However, it has to be taken into account that for some types of damage impaired repair does not necessarily result in an enhanced radiosensitivity, since not all types of lesions are involved in cell death . For instance, after ionizing irradiation cell death primarily results from double-strand breaks and probably also from DNA base damage but not from single-strand breaks (Ward 1985, 1986, Frankenberg-Schwager 1989) . While there are abundant data concerning the effect of heat on single-strand break repair (Ben Hur and Elkind 1974, Clark et al . 1981, Lunec et al. 1981, Dikomey 1982, Jorritsma and Konings 1983,
0020-7616/92 $3 .00 © 1992 Taylor & Francis Ltd
Downloaded by [University of Toronto Libraries] at 02:18 01 December 2015
222
E . Dikomey and J . Franzke
McGhie et al . 1983, Warters et al. 1987 Iliakis et al. 1990) there are only a few reports regarding the effect of heat on the repair of base damage or doublestrand breaks . Warters and Rod Roti (1978, 1979) reported that the excision of damaged bases was depressed after prior heating at 45°C for 20 min, whereas the enzymatic incision as tested by micrococcus endonuclease was only slightly retarded . The repair of double-strand breaks was reported to be depressed by pre-irradiation heat treatments at 43°C or 45°C (Correy et al. 1977, Radford 1983, Iliakis et al. 1990) . The present study was designed in order to obtain further information on DNA strand break repair and its modification by heat . We have shown previously that after ionizing irradiation the kinetics of strand break repair as measured by the alkaline unwinding technique was best described by a sum of three exponential components having different half-times (Dikomey and Franzke 1986) . The fast and intermediate components were interpreted as being due to the repair of two different classes of single-strand breaks, whereas the slow component was assigned to the repair of double-strand breaks . It was the aim of the present report to analyse these three components after heat treatments at different temperatures ranging from 42 to 45°C . For this purpose, cells were first heated and then irradiated on ice with X-rays followed by incubation at 37°C for up to 360 min . DNA strand breaks were measured by the alkaline unwinding technique . Preliminary results have been reported previously (Dikomey and Franzke 1988) .
Braunschweig, Germany) . Sealed plastic flasks containing the cells were completely immersed in a precision-controlled water bath (±0 .05°C) . The temperature of the medium approximated the desired temperature (difference 15 min the number of strand breaks increased again, reached a maximum by t = 25 min, and declined afterwards . This curve demonstrated the existence of two different kinetics : (1) an initial exponential decline, and (2) a complex kinetics rising fom `zero' breaks up to a maximum and descending thereafter . Curve (1) represents the repair kinetics of strand breaks induced directly by irradiation, termed primary breaks . Curve (2) represents the kinetics of those breaks, which are generated and repaired after irradiation, termed secondary strand breaks . Thus, the repair curve shown in Figure 8d demonstrates clearly the occurrence of primary and secondary strand breaks . Assuming that these two types of breaks are also present in non-heated cells and in cells heated prior to irradiation the repair kinetics of the fast repaired primary and secondary strand breaks may be described by an equation proposed by Nelson (1982) :
228
E. Dikomey and J. Franzke
Downloaded by [University of Toronto Libraries] at 02:18 01 December 2015
Nprim + Nsec = NO,prim x exp (-ln2t ) + No,.. Tfast,r Tfast,r t x x [exp (-1 n2 ) Tfast,in - Tfast,r Tfast,in t -exp(- 1n2Tfast,r)] (4) where No,prim is the total number of fast repaired primary strand breaks and No,, the total number of those lesions which are converted into secondary strand breaks ; Tfast, in is the half-time of this conversion and Tfast,r the half-time at which primary and secondary strand breaks are repaired . Equation (4) was fitted to the data shown in Figure 8 by least square fit . The results obtained for the repair kinetics for non-heated cells (Figure 8a) and for cells heated at 43°C for 40 min (Figure 8b) showed an excellent agreement between the experimental data and the calculated curves (Figures 8a and b, solid lines) . The identical result was obtained for the repair curve measured after prior heating at 42 or 43°C for 20 min (data not shown) . It becomes obvious from this analysis that the initial decline (termed fast phase of repair) mainly reflects the repair of primary strand breaks with the half-time Tfast,r . The intermediate phase mainly represents the formation and repair of secondary breaks, and since the formation is the rate-limiting step the half-time, Tfast,in is equal to the half-time by which the number of strand breaks declines during the intermediate phase . In order to fit the experimental data obtained for cells heated at 45°C for 20 min prior to the irradiation (Figure 8c) it was necessary to assume that repair of primary and secondary strand breaks was delayed for the time interval td = 4 .4 + 0 . 3 min, whereas the formation of secondary breaks started immediately after irradiation without any delay . Due to this assumption, the number of primary strand breaks remained constant for the time td, while the number of secondary breaks went up, thus leading to the observed result that the total number of strand breaks increased at the beginning of repair incubation followed by a decline later on (Figure 8c) . Similar assumptions had to be made for cells preheated at 43°C for 60 min and 44°C for 20 min (data not shown), the corresponding delay times were td =1 .3 ± 0 . 4 min and td = 2 . 9 + 0 . 3 min, respectively. In addition, from curve fitting the values for the total number of secondary strand breaks, No,sec, (see Equation (4)) could be obtained . From these numbers the relative frequency fsec = No,seclNtot was calculated, where Ntot represents the total number of
all primary strand breaks including fast and slowly repaired primary strand breaks . The calculated ratios for heated cells are in the range of 1 .0 and 2 .5 (Table 1) . There might be a slight tendency that ec increases with increasing temperature, but due to the large scatter of the data this increase was statistically not significant . The mean value calculated from the fsec ratios for heated cells was eC =1 . 7 ± 0 .2, which is similar to fsec = 1 .5 + 0 .2 for nonheated cells . The values of Tfast,ln and Tfast,r obtained from curve fitting are plotted in Figure 7 (diamonds and squares) . Both parameters were found to increase with duration of heat treatment and with temperature, but the effect for Tfast,in was smaller as compared to Tfast,r . For example, after a prior heating at 45°C for 20 min Tfast,in was increased by a factor of 1 . 3 (Tfast,in = 22 . 3 + 1 . 1 min versus Tfast,in = 17 + 5 min for non-heated cells), whereas a 3 .4-fold increase was observed for the repair half-time Tfast,r (Tfast,r = 7 .8 + 0 . 1 min versus rfast,,, = 2 .3 + 0 .2 min for non-heated cells) . This result indicates that the formation of secondary strand breaks was only slightly affected by heat, whereas the repair was inhibited more strongly. Consequently, in heated cells more secondary breaks accumulated with time after irradiation than in non-heated cells . This becomes obvious from Figure 8 (a-c) showing that the maximum number of secondary strand breaks (broken lines) increased with the duration and the temperature of the heat treatment applied which, in addition, leads to the gradual disappearance of the inflection on the decline of the repair curve . The parameters Tfast,r, Tfast,in and fsec were obtained after subtracting a hypothetical curve constructed for the slow phase, which was based on a fast initial rise within 2 min (Figure 6) . Adopting a slow rise within 80 min, the calculated values would increase by less than 10-15% (data not shown) . Thus, the values obtained should be regarded as lower limits . The repair curve obtained for cells heated at 45°C immediately after irradiation (Figure 8d) could not be analysed by curve fitting, since Equation (4) implies that the cells were incubated at 37°C after irradiation but not at elevated temperatures .
f
f
4. Discussion The present study showed that heat did not affect the induction of DNA strand breaks by Xirradiation but substantially altered the repair kinetics
Downloaded by [University of Toronto Libraries] at 02:18 01 December 2015
Strand break repair and heat
DNA strand breaks were measured using the alkaline unwinding technique (Ahnstrom and Erixon 1981, Rydberg 1975) . It was found that the kinetics of DNA unwinding in alkaline solution was the same for heated and non-heated cells (Figure 3) . This result showed that the enhanced amount of proteins associated with the DNA in heated cells (Roti Rod et al. 1979) did not alter the rate of unwinding . As a consequence, the relationship between the fraction of DNA remaining doublestranded after alkaline denaturation for 30 min and the number of X-ray-induced strand breaks as determined for non-heated cells (Equation (3)) could also be applied for heated cells . The occurrence of strand breaks was observed after heat treatment alone, but temperatures of 43-45°C were required to induce a measurable number of strand breaks during a 1 h period (Figures I and 2) . This is in agreement with the results of a previous work (Dikomey 1982) and of other investigators (Jorritsma and Konings 1984, Mitchel and Birnboim 1985, Warters et al. 1985, Warters and Brizgys 1987) . Our result that the total number of strand breaks induced by X-irradiation was unaffected by preceding heat treatments at 40-46 °C (Figures 1 and 2) is in accordance with previous reports, where strand breaks were detected either by the alkaline unwinding or the alkaline sedimentation technique (Corry et al . 1977, Clark et al. 1981, Lunec et al . 1981, Jorritsma and Konings 1983, McGhie et al . 1983, Warters et al . 1987) . However, when strand breaks were measured by the alkaline or neutral elution technique, additional hyperthermia either had no effect (Iliakis et al . 1990) or caused an increase in the number of strand breaks induced by irradiation (Mills and Meyn 1981, Radford 1983, Warters et al . 1987) . The latter results might be due to the fact that the elution techniques strongly depend on the amount of proteins associated with the DNA (Okayasu and Iliakis 1989), and this amount is known to be enhanced by heat (Roti Roti and Winward 1980, Mills and Meyn 1981) . Strand break repair was studied after an X-ray dose of 9 Gy combined with various heat treatments (Figures 4-6) . Independently of the heat treatment applied the total repair curve could be described by three phases : a fast (t=0-15 min), an intermediate (t=15-120 min) and a slow phase (t>, 120 min) . It was previously demonstrated for irradiated cells that the slow phase represents the repair kinetics of DNA double-strand breaks, since the slow repair component measured by the alkaline unwinding technique for several different conditions agreed with the
229
repair curve of double-strand breaks measured by the neutral sedimentation technique (Bryant and Blocher 1980, 1982, Blocher et al. 1983 Dikomey and Franzke 1986) . For double-strand breaks detected by the method of premature chromosome condensation, the repair curve could also be described by a single exponential component (Johnson et al. 1982, Cornforth and Bedford 1983, Iliakis et al. 1988, Wlodek and Hittelman 1988), which is in accordance with the results obtained by the alkaline unwinding or neutral sedimentation technique . In contrast, when doublestrand breaks were measured by the neutral elution technique, the repair kinetics consisted of a sum of two exponential components ; a fast component with an initial fraction of 70-90% and a half-time of 2-5 min and a slow component with an initial fraction of 10-30% and a half-time of 90-170 min (see, for instance, Weibezahn and Coquerelle 1981, Rowley and Kort 1988) . This strong dependence of the results on the methods applied is a well-documented fact (Peak et al. 1988, Hutchinson 1989), but is not understood . It could be shown that the substrates used by the neutral sedimentation and the alkaline unwinding technique must be different from the substrates used by the neutral elution technique (Costa and Bryant 1990) . Moreover, it can be excluded that the slow repair phase found here (Figures 4-6) represents the component of the slowly repaired double-strand breaks detected by the neutral elution technique, since, in this case, the fraction (Table 1) had to be multiplied by a factor of three to nine in order to obtain the initial fraction of all double-strand breaks . This would lead to values in the range of 18-50%, which is much too high, since the initial fraction of DNA double-strand breaks is generally about 5% for sparsely ionizing irradiation . The most prominent effect of heat on the slow repair phase was an elevation of its backextrapolation value, ( Figures 5 and 6, table 1) . The elevation could not be the result of an enhanced induction of DNA double-strand breaks, since this elevation was also observed if heat was applied after irradiation (Figure 6) . Therefore, the number of double-strand breaks induced in heated and nonheated cells must be the same . However, in heated cells the number of slowly repaired strand breaks must increase during the incubation period after irradiation . This increase shows that a certain number of lesions normally repaired during the fast or intermediate phase, are repaired in heated cells at a rate similar to that of double-strand breaks or are converted into real double-strand breaks . Either
fg,ow
fjoq,
E. Dikomey and J . Franzke
Downloaded by [University of Toronto Libraries] at 02:18 01 December 2015
230
process should result in an elevated backextrapolation value of the slow repair phase as was observed here (Table 1) . In accordance with the results presented here, Corry et al. (1977) and Iliakis et al . (1990) also observed that the number of double-strand breaks induced during irradiation was not altered by heat, whereas Radford (1983) reported on an increase after a prior exposure to 43°C for 1 h . The half-time of the slow phase, ;low, was found to increase with increasing duration and temperature of the heat treatment (Figure 7), which indicates that the repair of double-strand breaks was slowed down in irradiated cells after a prior heat treatment . This result is in accordance to previous reports by Corry et al . (1977), Radford (1983) and Iliakis et al . (1990) . In addition, it was shown here that doublestrand break repair was also slowed down in cells heated immediately after irradiation (Figure 6, triangles) . Adopting the interpretation that the slow repair phase represents the repair of DNA double-strand breaks, the kinetics obtained after subtracting this phase from the total kinetics should represent the repair kinetics of DNA single-strand breaks only (Figure 8) . These kinetics came out differently for the various heat treatments applied . Nevertheless, these different curves could be interpreted by assuming that X-irradiation leads to both primary and secondary strand breaks . As a consequence, an alternative idea discussed previously (Dikomey and Franzke 1986), that the kinetics of single-strand break repair might be a sum of two exponential components describing the repair of two different classes of primary single-strand breaks can be discarded . It was also suggested by other investigators (Bryant et al. 1984) that the kinetics of strand break repair measured after X-irradiation reflects repair of both primary as well as secondary strand breaks . On this basis, and in accordance with the results presented here, we suggest that the initial phase of the repair curve represents mainly the repair of primary breaks, while the intermediate phase is dominated by the formation and repair of secondary strand breaks. The relative frequency of all lesions converted into secondary strand breaks was Sec = 1-5+0-5 for nonheated cells (Table 1) . Secondary strand breaks occur during the excision repair, when the DNA is incised at the sites of base damage by a specific endonuclease . When 'endonuclease-sensitive sites' were measured by the y-endonuclease technique, the relative frequency was 1 .7 ± 0 . 6 (Fohe and Dikomey 1991) . This directly measured value agrees quite
f
well with that determined by curve fitting, which might be taken as a further indication that an analysis of the data by separating primary from secondary single-strand breaks leads to an adequate description of the data . The relative frequency, ec, was not significantly altered by heat (Table 1) . That means that the amount of radiation-induced base damage, which is converted into secondary strand breaks, is not altered by prior heat treatment . Similar results were also obtained by Radford (1986) for mouse L cells preheated at 43°C for 60 min, when base damage was measured by the y-endonuclease technique . The rate at which secondary strand breaks were formed from base damage was found to be only slightly reduced in preheated cells, while the repair of secondary breaks was depressed to a greater extent (Figure 7) . The effect of heat on base damage repair was previously studied by Warters and Roti Roti (1978, 1979) in CHO cells using the thymine damage of the t'-type as a model . Prior heat treatment at 45 ° C for 15 min strongly inhibited the removal of this thymine damage, while the enzymatic incision as tested by micrococcus endonuclease was not, or only slightly, reduced . Since the removal of a damaged base requires an incision as well as further repair steps, the data of Warters and Roti Roti (1978, 1979) indicate that heat affects base damage repair primarily by inhibiting the repair steps, whereas the incision step is not, or only slightly, retarded . This conclusion is in full accordance with our results. The half-time of strand break repair, Tfast,r, was found to increase with increasing duration and temperature of the heat treatment applied (Figure 7), which indicates that the repair of primary singlestrand breaks was slowed down by heat . Similar observations were previously made by other investigators using different cell lines and different techniques (Corry et al . 1977, Clark et al. 1981, Lunec et al . 1981, Jorritsma and Konings 1983, McGhie et al . 1983, Warters et al. 1987, Iliakis et al . 1990) . In cells heated at 45°C immediately after the irradiation strand break repair was found to be as fast as in nonheated cells (Figure 8d) . This is in accordance with previous reports, which showed that incubation at elevated temperatures after irradiation did not affect the rate of repair but even caused an acceleration of strand break repair (Ben Hur and Elkind 1974, Weniger et al . 1979, Dikomey 1982) . After prior heat treatments at 44°C and 45 ° C, the number of strand breaks was found to increase after irradiation followed by a decline after 2-4 min (Figures 5 and 6) . A similar initial increase was previously observed by Jorritsma and Konings
f
Downloaded by [University of Toronto Libraries] at 02:18 01 December 2015
Strand break repair and heat
(1983, 1984) and by Iliakis et al . (1990) which was attributed to an interaction phenomenon between heat and radiation (Iliakis et al. 1990) . It is now shown here, that this increase can also be explained by a delay in the repair of primary and secondary strand breaks . This delay might indicate that the relative rate of repair was rather low at the beginning, but increased when the number of strand breaks decreased and finally approached a constant value leading to exponential kinetics. This increase in the relative rate of repair with decreasing number of strand breaks suggested that in heated cells strand break repair was saturated even after a dose as low as 9 Gy . This saturation in strand break repair might be a consequence of a heat-induced reduction in the activity of DNA repair enzymes such as DNA polymerase a and fl (Spiro et al . 1982, Jorritsma et al . 1986, Dikomey et al . 1987) . In conclusion we show here that the changes in the total kinetics of strand break repair induced by heating either prior to or after irradiation (Figures 4-6) can be interpreted by a depressed repair of primary and secondary single-strand breaks, while the formation of secondary breaks was only slightly affected . Furthermore, heat was found to reduce the rate of double-strand break repair and, in particular, to increase the number of slowly repaired strand breaks during repair incubation . DNA double-strand breaks are regarded to be the most relevant type of damage leading to radiationinduced cell death (Frankenberg-Schwager 1989) . Thus, the alterations found here for the slow repair phase might help to interpret the increase in radiosensitivity which is generally observed for radiation combined with heat (Dikomey and Jung 1991) . The present results indicate that heat causes both a depression in double-strand break repair and also a formation of slowly repaired strand breaks with time after irradiation . As a consequence, in heated cells more double-strand breaks are present and persist for a longer time as compared to non-heated cells, and this might lead to an increase in cell killing and thus to an increase in radiosensitivity, when radiation is combined with heat . Acknowledgements The authors are grateful to Mrs Jutta Schafer for skilfully performing part of the DNA analysis and to Gabriel Bond for writing the computer programs used for data analysis. Appreciation is expressed to Jochen Dahm-Daphi, Christian Fohe and Professor Horst Jung for valuable suggestions and discussions. This investigation was supported by Bundesminis-
231
terium fur Forschung and Technologie, Bonn, by grant number 01 VF8516 .
References AHNSTROM, G . and ERIXON, K ., 1981, Measurement of strand breaks by alkaline denaturation and hydroxyapatite chromatography . In DNA Repair, edited by E. C . Fried-
berg and P. C. Hanawalt (New York and Basel : Marcel Dekker), pp. 403-418 . BEN HUR, E . and ELKIND, M . M ., 1974, Thermally enhanced radioresponse of cultured Chinese hamster cells : damage and repair of single-stranded DNA and a DNA complex . Radiation Research, 59, 484-495 . BLOCHER, D. NvsSE, M . and BRYANT, P . E ., 1983, Kinetics of double-strand break repair in the DNA of X-irradiated synchronized mammalian cells . International Journal of Radiation Biology, 43, 579-584. BRYANT, P. E . and BLOCHER, D., 1980, Measurement of the kinetics of DNA double-strand break repair in Ehrlich ascites tumour cells using the unwinding method . International Journal of Radiation Biology, 38, 335-347 .
BRYANT, P. E . and BLOCHER, D ., 1982, The effect of 9-fl-Darabinofuranosyladenine on the repair of DNA strand breaks in X-irradiated Ehrlich ascites tumour cells. International Journal of Radiation Biology, 42, 385-394 .
BRYANT, P . E ., WARRING, R. and AHNSTROM, G ., 1984, DNA repair kinetics after low doses o(X-rays: a comparison of results obtained by the unwinding and nucleosid sedimentation methods . Mutation Research, 131, 19-26 . CLARK, E . P ., DEWEY, W . C . and LETT, T., 1981, Recovery of CHO cells from hyperthermic potentiation to X-rays : repair of DNA and chromatin . Radiation Research, 85, 302-313 . CORNFORTH, M . N . and BEDFORD, J . S ., 1983, X-ray-induced breakage and rejoining of human interphase chromosomes . Science, 222, 1141-1143 . CORRY, P. M ., ROBINSON, S . and GETZ, S ., 1977, Hyperthermic effects on DNA repair mechanisms . Radiology, 123, 475-482 . COSTA, N . D. and BRYANT, P . E ., 1990, Neutral elution detects only limited inhibition of double-strand break repair by 9-fl-D-arabinofuranosyladenine . Mutation Research, 235, 217-223 . DIKOMEY, E . 1982, Effect of hyperthermia at 42 and 45 ° C on repair of radiation-induced DNA strand breaks in CHO cells International Journal of Radiation Biology, 41, 603-614. DIKOMEY, E . and FRANZKE, J ., 1986, Three classes of DNA strand breaks induced by X-irradiation and internal Prays International Journal of Radiation Biology, 50, 893-908 . DIKOMEY, E . and FRANZKE, J ., 1988, Effect of hyperthermia (42-45 ° C) on repair of DNA strand breaks induced by ionizing irradiation . Hyperthermic Oncology 1988, Vol 1, edited by T. Sugahara and M . Saito (London and Philadelphia : Taylor & Francis), pp 154-155 . DIKOMEY, E . and JUNG, H ., 1991, Thermal radiosensitization in CHO cells by prior heating at 41-46°C . International Journal of Radiation Biology, 59, 815-825 .
DIKOMEY, E ., BECKER, W. and WIELCKENS, K ., 1987, Reduction of DNA polymerase fi activity of CHO cells by single and combined heat treatments . International Journal of Radiation Biology, 52, 775-785. F6HE, C, and DIKOMEY, E ., 1991, The induction of gamma-
Downloaded by [University of Toronto Libraries] at 02:18 01 December 2015
232
E. Dikomey and J. Franzke
endonuclease sensitive sites in X-irradiated CHO cells (Abstract) . International Journal of Radiation Biology, 59, 572 . FRANKENBERG-SCHWAGER, M ., 1989, Review article : review of repair kinetics for DNA damage induced in eukaryotic cells in vitro by ionizing radiation. Radiotherapy and Oncology, 14, 307-320 . GRAUBMANN, S . and DIKOMEY, E ., 1983, Induction and repair of DNA strand breaks in CHO cells irradiated in various phases of the cycle. International Journal of Radiation Biology, 43, 475-483 . HAGEN U ., 1973, Strahlenwirkung auf Struktur and Funktion der Desoxyribonukleinsaure . Biophysik, 9, 279-289 . HUTCHINSON, F., 1989, On the measurement of DNA doublestrand breaks by neutral elution Radiation Research, 120, 182-186 . ILIAKIS, G ., PANTELIAS, G . E . and SEANER, R., 1988, Effect of arabinofuranosyladenine on radiation-induced chromosome damage in plateau-phase CHO cells measured by premature chromosome condensation : implications for repair and fixation of a-PLD . Radiation Research, 114, 361-378 . ILIAKIS, G., SEANER R . and OKAYASU, R., 1990, Effects of hyperthermia on the repair of radiation-induced DNA single- and double-strand breaks in DNA double-strand break repair-deficient and repair-proficient cell lines . International Journal of Hyperthermia, 6, 813-833 . JOHNSON, R . T., COLLINS, A . R . S . and WALDREN, C . A ., 1982, Prematurely condensed chromosomes and the analysis of DNA and chromosome lesions . Premature Chromosome Condensation, edited by P . N . Rao, R . T . Johnson and K . Sperling (New York, London : Academic Press), pp 253-308 . JORRITSMA, J . B . M . and KoNINGS, A. W . T ., 1983, Inhibition of repair of radiation induced strand breaks by hyperthermia, and its relationship to cell survival after hyperthermia alone . International Journal of Radiation Biology, 43, 505-516 . JORRITSMA, J . B . M . and KoNINGS, A . W. T ., 1984 The occurrence of DNA strand breaks after hyperthermic treatments of mammalian cells with and without radiation . Radiation Research, 98, 198-208 . JORRITSMA, J . B. M . BURGMAN, P., KAMPINGA, H . H . and KONINGS, A. W. T ., 1986 DNA polymerase activity in heat killing and hyperthermic radiosensitization of mammalian cells as observed after fractionated heat treatments . Radiation Research, 105, 307-319 . LUNEC. L ., HESSLEWOOD, I . P., PARKER, R. and LEAPER, S ., 1981, Hyperthermic enhancement of radiation cell killing in HeLa S3 cells and its effect on the production and repair of DNA strand breaks . Radiation Research, 85, 116-125 . MCGHIE, J . B., WOLD, E ., PETTERSEN, E . O . and MOAN, J ., 1983, Combined electron radiation and hyperthermia . Repair of DNA strand breaks in NHIK 3025 cells irradiated and incubated at 37, 42 . 5, or 45 ° C . Radiation Research, 96, 31-40 . MILLS, M . D . and MEYN, R . E., 1981, Effects of hyperthermia on repair of radiation-induced DNA strand breaks . Radiation Research, 87, 314-328 . MITCHEL, R . E . J . and BIRNBOIM, H . C., 1985, Triggering of DNA strand breaks by 45 ° C hyperthermia and its influence on the repair of y-radiation damage in human white blood cells . Radiation Research, 45, 2040-2045 . NELSON, S . J ., 1982, Models for DNA damage formation and
repair in mammalian cells exposed to ionizing radiation, Radiation Research, 92, 120-145 . OKAYASU, R . and ILIAKIS, G., 1989, Linear DNA elution dose response curves obtained in CHO cells with nonunwinding filter elution after appropriate selection of the lysis conditions . International Journal of Radiation Biology, 55, 569-581 . PEAK, M . J., PEAK, J. G. and BLAZEK, E . R ., 1988, Symposium report . Radiation-induced DNA damage and repair : Argonne National Laboratory Symposium, Argonne Illinois 60439, 15 April 1988 . International Journal of Radiation Biology, 54, 513-520 . RADFORD. I . R ., 1983, Effects of hyperthermia on the repair of X-ray induced DNA double-strand breaks in mouse L cells. International Journal of Radiation Biology, 43, 551-557 . RADFORD . I . R ., 1986, Effect of radiomodifying agents on the ratios of X-ray-induced lesions in cellular DNA : use in lethal lesion determination . International Journal of Radiation Biology, 49, 621-637 . RoTI ROTI, J . L . and WINWARD . R . T ., 1980, Factors affecting the heat-induced increase in protein content of chromatin . Radiation Research, 81, 138-144. Ron Ron, J . L ., HENLE, K . J . and WINWARD, R . T., 1979, The kinetics of increase in chromatin protein content in heated cells : a possible role in cell killing. Radiation Research, 78, 522-531 . ROWLEY, R . and KORT, L ., 1988, The effect of modulators of radiation-induced G2 arrest on the repair of radiationinduced DNA damage detectable by neutral filter elution. International Journal of Radiation Biology, 54, 749-759 . RYDBERG, B ., 1975, The rate of strand separation in alkali of DNA of irradiated mammalian cells . Radiation Research, 61, 274-287 . Sxov, K . A., 1984, The contribution of hydroxyl radical to radiosensitization : a study of DNA damage . Radiation Research, 99, 502-510 . SPIRO, I . J ., DENMAN, D. L . and DEWEY, W . C ., 1982, Effect of hyperthermia on CHO DNA polymerase a and P. Radiation Research, 89, 134-149 . WARD, J . F ., 1985, Biochemistry of DNA lesions . Radiation Research, 102, S-103-S-111 . WARD, J . F., 1986, Mechanisms of DNA repair and their potential modification for radiotherapy . International Journal
of Radiation
Oncology,
Biology, Physics,
12,
1027-1032. WARTERS, R . L . and ROTI ROTI J . L ., 1978, Production and excision of 5', 6'-dihydroxydihydrothymine type products in the DNA of preheated cells . International Journal of Radiation Biology, 34, 381-384 . WARTERS, R . L. and RoTI Ron J . L., 1979, Excision of X-ray induced thymine damage in chromatin from heated cells . Radiation Research, 79, 113-121 . WARTERS, R . L . and BRIZGYS, L . M ., 1987, Apurinic site induction in DNA of cells heated at hyperthermic temperatures . Journal of Cellular Physiology, 133, 144-150 . WARTERS, R . L., BRIZGYS, L . M . and AXTELL-BARTLETT, J ., 1985, DNA damage production in CHO cells at elevated temperatures . Journal of Cellular Physiology, 124, 481-486 . WARTERS, R . L., LYONS, B . W. and AXTELL-BARTLETT, J ., 1987, Inhibition of repair of radiation-induced DNA damage by thermal shock in Chinese hamster ovary cells . International Journal of Radiation Biology, 51, 505-517 .
Strand break repair and heat
233
Downloaded by [University of Toronto Libraries] at 02:18 01 December 2015
WLODEK, D. and HrrTELMAN, W . N ., 1988, The relationship of WEIBEZAHN, K . F. and COQUERELLE, T ., 1981, Radiation induced DNA double-strand breaks are rejoined by DNA and chromosome damage to survival of synchroligation and recombination processes. Nucleic Acids nized X-irradiated L5178Y cells . II . repair. Radiation Research, 9, 3139-3150 . Research, 115, 566-575. WENIGER, P., WAWRA, E . and DOLEJIS, 1 ., 1979, Die Wirkung von Hyperthermie auf DNA-Reparaturvorgange . Radiation and Environmental Biophysics, 16, 135-141 .
