Radiation induced DNA damage and damage repair in three human tumour cell lines

DNA Repair

ELSEVIER

Mutation Research 362

( 1996) 5 l-59

Radiation induced DNA damage and damage repair in three human tumour cell lines Elies C. Woudstra *, Jeanette F. Brunsting, Judith M. Roesink, Antonius W.T. Konings, Harm H. Kampinga Depurtment

of Radiobiologxs CJker.sit\‘ of Groningen, Bloemsingel I. 9712 BZ Groningm, Netherlands Received 3 1 March 1995; revised 30 May 1995: accepted 4 July 1995

Abstract Three human tumour cell lines (HX 142, RT 112 and MGH-U 1) with different radiosensitivities were tested for differences in the rate and/or extent of DNA unwinding in alkali as well as for differences in the induction of DNA double strand breaks by means of the pulsed field gel electrophoresis, after X-irradiation. Unlike that which has been found using the non-denaturing filter elution technique (NDE, McMillan et al., 1990), no differences in initial DNA damage (the extent of alkaline unwinding and the induction of double strand breaks) were found for the three cell lines. These data suggest that rather than a different number of DNA lesions per Da per Gy between these cell lines, structural differences in chromatin structure (related to radiosensitivity) might impair the detectability of lesions in some assays like the NDE. The nature of such structure differences remains unclear. However. the differences did not affect alkaline unwinding profiles, as all three cell lines showed identical rates of DNA unwinding after exposure to X-rays. Furthermore, the three cell lines did not show significant differences in the kinetics of DNA strand break rejoining nor in the amounts of damage remaining after 24 h repair. The results obtained in this study, together with other findings, suggest that the three cell lines may differ in their

‘presentation’ of DNA damage. 1Ye~~~~rrfs:Alkaline unwinding:

DNA strand breaks: CHEF: Chromatin

structure:

1. Introduction Variations in sensitivity to ionizing radiation amongst cell lines have often been related to the

Abbreviations: CHEF, clamped homogeneous electrical field electrophoresis; Da, dalton; ds DNA, double stranded DNA: PBS, phosphate buffered saline; PFGE, pulsed field gel electrophoresis; NDE. non-denaturing filter elution: SDS, sodium dodecyl sulfate: TBE, tris-base boric acid EDTA * Corresponding author. Tel.: *31 50-3632911: Fax: *31 503632913; e-mail: [email protected]. 0921.8777/96/$15.00 0 1996 Elsevier Science B.V. All rights reserved SSDi 092 1-8777(95)00032-I

Pulsed field gel electrophoresis;

Radiation

sensitivity

capacity of cells to repair radiation-induced DNA damage (George and Cramp, 1987; Kelland et al., 1988: Frankenberg-Schwager, 1989; McMillan et al., 1990; Ward, 1990: Dahm-Daphi et al.. 1993). The role of DNA repair in cellular radiation sensitivity has mostly been studied in radiosensitive mutants of cultured cell lines. Three of the nine complementation groups of mammalian X-ray sensitive mutant cells have been found to be severely defective in their repair of DNA double strand breaks, like the scid, V3, xrs-5, XR-V15B, and XR-V9B cell lines (Kemp et al., 1984; Biedermann et al., 1991; Smider

et al., 1994; Taccioli et al.. 1994; Troelstra and Jaspers, 1994). Mutants in complementation group 1 (EM-9, Thompson et al., 19901 have been found to have a defect in single strand break repair. The reason for the sensitivity to X-rays for the other groups is either not known or may relate to perturbed cell cycle arrest patterns after irradiation (Ataxia Telangiectasia, Lavin and Schroeder. 1988). Some of the genes responsible for the hypersensitivity have been cloned (for review see Troelstra and Jasper& 1994) from which a picture on repair of DNA damage after ionizing radiation in eukaryotes is beginning to emerge. How to divide different human tumour cell lines with varying radiosensitivity (Deacon et al., 1984; Peacock et al., 1989) into these different complementation groups is as yet unclear. In some cases differences in sensitivity of tumour cells to ionizing radiation have been related to differences in DNA double strand break repair (Olive et al., 19941, while in other cases no differences in DNA damage repair were observed in various tumour cell types, despite (large) differences in radiosensitivity (McMillan et al., 1990; Smeets et al., 1993; Olive et al.. 1994). Besides differences in the ability to repair DNA damage, variations in the susceptibility to the induction of DNA damage by ionizing radiation have been proposed to relate to differences in cellular radiosensitivity. Using the neutral filter elution technique (NDE assay; pH 9.6) various investigators showed correlations between initial damage and radiosensitivity (Radford, 1986; Kelland et al., 1988; MC Millan et al., 1990). Differences in intracellular scavenger concentration between cell lines can in theory be a reason for damage induction differences; this would, however, require marked local concentration differences in scavengers, which is unlikely (Ward. 1990). Variations in damage induction may also be related to distinct variations in chromatin structures between cell lines (Olive et al., 1986; Ward, 1990; Ljungman, 1991; Schwartz et al., 1992). E.g., stepwise removal of proteins from the chromatin dramatically increases the amount of radiation-induced strand breaks in human fibroblasts (Ljungman, 1991). The alkaline unwinding assay measures several kinds of DNA damage (e.g. single and double strand breaks) and allows the measurement of not only the extent of unwinding (proportional to the number of

lesions) but also the rate of unwinding of the DNA from the sites of damage. It has been indicated that chromatin structure may influence the rate of alkaline unwinding (Jorgensen et al., 1990; Olive et al., 1986; Ljungman. 1991). Therefore, the assay might reveal differences between cell lines in (features of1 DNA organization. In the current study, three human tumour cell lines (HX142. RTl12 and MGH-Ul) were used that showed differences in the rate of elution in NDE (presumably proportional to DNA double strand breaks) relating to their differences in radiosensitivity (McMillan et al., 1990). Therefore. it seemed that HX142 cells might carry a greater burden of double strand breaks after a given dose of radiation compared to the other two cell lines. Assuming that double strand breaks and single strand breaks are produced in a constant ratio by ionizing radiation, it would be predicted that HX142 cells would also show elevated levels of single strand breaks (extent of unwinding in alkali). Yet, our current data show that this was found not to be the case. Also, no differences in the rate of unwinding could be found. No indications were obtained to suggest an altered ratio of double strand break to single strand break induction in HX 142 versus RTl12 or MGH-Ul cells as no differences in induction were found with pulsed field gel electrophoresis either (measuring double strand breaks only). These results provide support for the view that rather than differences in the number of lesions induced, differences in chromatin structure between HX 142, RTl12 and MGH-U 1 lead to a different ‘presentation’ of lesions, in certain assays (e.g. NDE). This difference may affect the cells ability to repair the damage properly. Since no differences in repair kinetics were observed in the current study the different ‘presentation’ of the damage may affect the fidelity of repair.

2. Materials

and methods

2.1. Cell culturing und cell labelling procedures Three human tumour cell lines were used in this study. HX142. a neuroblastoma cell line is more sensitive to ionizing radiation than RTI 12, or MGHUl. which both are bladder carcinoma cell lines. Cells were grown at 37°C. as monolayer, in Costar

E.C. Woudstra et al. /Mutation

plastic flasks containing HAMS F12 medium (Life Technologies) supplemented with 10% foetal calf serum (Life Technologies). Cells were incubated in an atmosphere of 3% O?, 5% CO,. and 92% N2. The experiments were performed with exponentially and with confluently grown cells. Uniform DNA labelling (3 to 4 days) was obtained by adding ‘H- or “C-thymidine (DuPont. NEN) to the medium at a final concentration of 3 PCi ‘H-TdR/ml or 1 PCi “C-TdR/ml. Cold thymidine (8 pg/mlI was added at the same time. All our standard laboratory chemicals were purchased from Sigma (St Louis, USA) or Merck (Darmstadt, West Germany).

After DNA labelling the cells were washed with complete medium, trypsinized (2 min for HX142 and MGH-Ul cells or 5 min for RTl12 cells at 37°C. centrifuged for 5 min at 150 g) and resuspended in fresh complete medium at a density of lo6 cells/ml. Irradiations were performed on ice using a PhilipsMuller MG 300 X-ray machine, operating at 200 kV and 15 mA at a dose rate of 5 Gy/min. The dosimetry was performed with an ionization chamber (Philips 37489/10) calibrated with a “Sr source (Philips XL 201 I /OO). 2.3. Determination

of cell surr~il~al

Cell survival (colony forming ability) was determined by plating 1 ml of an appropriately diluted sample to Costar triplicate plastic flasks, containing 4 ml of complete growth medium. 1 X 10’ Feeder cells (lethally (> 150 Gy) irradiated) were added at the time of plating. Culture flasks were gassed with 3% O,, 5% CO,. and 92% N, and after 14 days of incubation in a humidified 37°C incubator, colonies were fixed with 70% ethanol and stained with 0.5% crystal violet. Colonies containing more than 50 cells were counted. Plating efficiencies were about 40% for HX142 and RTI 12, and about 90% for MGH-UI cells. 2.4. Alkaline

unwinding

DNA strand breakage by X-rays was determined using the slightly modified. alkaline unwinding

Research 362 (19%) S/-S9

53

method of Ahnstrijm and Edvardsson (1974). Cells from one cell line labelled with “H-thymidine and cells from another cell line with “C-thymidine were pooled followed by irradiation on ice with different doses of X-rays. The mixing was performed to be able to detect even small differences amongst the cell lines. The choice of the label (‘H or IIC) per cell line was random and did not affect the outcome of the results (data not shown). For repair studies, cells were incubated at 37°C for varying periods of time (O-24 h). For each treatment. triplicate samples containing 1 X 10’ cells were rapidly lysed in a freshly made ice-cold solution (pH > 12) of 0.03 M NaOH. 0.9 M KC1 and 0.008 mM sodium phosphate buffer (pH 6.8). Alkaline unwinding was performed in the dark at 30°C. The time of unwinding was 30 min in most experiments, unless indicated otherwise. DNA single strand breaks and double strand breaks, as well as alkali-labile sites. will serve as starting points for DNA unwinding during strand separation in alkaline solution. After rapid neutralization with 3 ml of 0.015 M HCl. 1.8 ml SDS (2.5% weight/volume) was added and the samples were stored at -20°C. After thawing the samples. they were sonicated with a Branson sonifier for 20 s at 75 W. Two ml of each sample was heated to 60°C and loaded onto hydroxylapatite columns (150 mg DNA-grade Bio-Gel HTP hydroxylapatite, Biorad). which were maintained at 60°C in an aluminium block. The columns were pre-washed with 2 ml 0.0125 M sodium phosphate buffer (pH 6.8). Single stranded DNA was subsequently eluted with 4 ml 0.15 M sodium phosphate buffer (pH 6.8). followed by double stranded DNA (ds DNA) elution with 0.4 M sodium phosphate buffer (pH 6.8). All buffers were pre-heated at 60°C before applying to the columns. The eluates were mixed with 8 ml of Ultima Gold (Hewlet Packard) and the radioactivity was measured in a liquid scintillation counter. For DNA repair, the percentage ds DNA was converted to Gy-equivalents (on the basis of induction curves run in parallel in the same experiment). 2.5. Pulsed ,field gel electroplwresis (PFGE) For PFGE, we used the CHEF gel electrophoresis described by Blocher et al. (1989) using the detection method of Rosemann et al. ( 1993). In short, cells at a concentration of 2 X 10’ cells/ml were

54

EC.

Woudstra et al./Mutation

mixed with an equal volume of 1% low melting agarose (Bio-Rad) in phosphate buffered saline, pH = 7.4 (PBS) at 37°C. Plugs were formed by pipetting 35 ~1 of this mixture into a Bio-Rad plug mold. After 15 min gelation at 4°C the plugs were placed in Eppendorf vials containing fresh medium and put on ice for irradiation. After irradiation (plus repair), cells in the plugs were lysed in a solution of 2% Sarkosyl, 0.5 mg/ml Proteinase K (Sigma, cat nr P-0390) and 500 mM EDTA (pH 7.6) in PBS (2 h 0°C + 16 h 37°C). Next, the plugs were washed twice with PBS and treated for 1 h with RNAse A (0.2 mg/ml in PBS, Sigma, cat nr R-5000) at 37°C. The plugs were inserted into the wells of a 0.5% agarose gel (Bio-Rad Chromosomal Grade) and CHEF electrophoresis was carried out at 14°C using a Bio-Rad DR-II system in TBE buffer (45 mM Tris-base; 45 mM boric acid: 2 mM EDTA, pH = 8.2) at 40 V for 25 h using a switching interval of 75 min. After staining with ethidium bromide, the gel and the plugs were analyzed for fluorescence intensity of the DNA. The percentage of DNA migrating from the plug into the lane (% DNA,,,,) was used as a measure of radiation-induced double strand breaks (for details see Rosemann et al., 1993).

Research 362 (19%)

51-59

1.ooq

J

4

0

8

10

12

X-raydose (Gy) Fig. 1. Radiosensitivity of HX142 (0). RTl12 (A ). and MGH-UI ( T ) cells. Cells were trypsinized prior to the exposure of graded doses of irradiation (0- 10 Gy). Colony forming ability was measured as described in the Materials and methods section. The data are the mean f standard error of the mean kern) of 3 independent experiments.

hydroxylapatite column chromatography and plotted against the X-ray dose. The percentage of ds DNA decreases exponentially with increasing dose. No differences in initial damage for the three cell lines were found, as determined by the extent of DNA

3. Results 3. I. Cell surr~iual studies The cell survival curves of exponentially grown cells are shown in Fig. 1. Our data confirm the higher sensitivity to ionizing radiation of HX142 compared to the other two human tumour cell lines. The surviving fractions after 2 Gy of X-rays for HX 142, RTl12 and MGH-Ul are 0.09. 0.70, and 0.61, respectively. Cells grown to confluency revealed a similar picture (data not shown). 3.2. Alkaline unwinding

studies

0

4

0 X-ray

A full dose-response curve (O-20 Gy) for initial DNA strand breaks. as measured in exponentially grown HX142, RTl12, and MGH-UI cells, is shown in Fig. 2. After irradiation the cells were subjected to lysis and alkaline unwinding for 30 min. Thereafter the percentages of ds DNA were measured following

12 dose (Gy)

l6

20

! 24

Fig. 2. Alkaline unwinding: dose response curves for HX142 (0 ). RTl 12 (A ). and MGH-UI ( v ) cells. Cells were exposed to graded doses of irradiation (O-20 Cy). followed by 30 min DNA unwinding in alkaline solution at 3-0°C. The fraction ds DNA was determined by hydroxylapatite column chromatography and plotted versus the X-ray dose, The data are the mean + sem of 3-5 independent experiments.

E.C. Woudstra et (11./Mutation

Rrsenrch

362

55

f 19%)51-59

9 GY

12 Gy

1 0

s

v

1

30

16

Time

64

46

of unwlndlng

0

76

(min)

unwinding in alkali. The slopes of the damage induction curves are indicated in Table 1. To test whether the cell lines differ in the kinetics of alkaline DNA unwinding, exponentially grown cells were X-irradiated with 12 Gy (and compared with unirradiated controls). Subsequently they were subjected to lysis and alkaline unwinding for between 0 and 60 min at 20°C and the percentage of ds DNA was plotted against the lysis time (Fig. 3). The

SF,

HXl42 RT112 MGH-U 1

8.6 f 2.7 69.6 + 13.5 61.2 + 17.6

of HX142. RTl12.

20

40

Repair

time

60

(mln)

three cell lines show comparable background percentages of single strandedness (0 Gy curve). After irradiation (I 2 Gy) the percentage of ds DNA decreases with lysis time and reaches a plateau after about 15 min. No differences in the rates of DNA

and MGH-Ul

Damage induction

Repair characteristics

initial damage

(9 Gy: min)

-0.058 + 0.018 - 0.06 1 f 0.009 - 0.065 + 0.0 13

MGH-Ul

Fig. 4. Kinetics of DNA strand break rejoining of HX142 (O), RT 112 ( A ). and MGH-U 1 ( v ) cells exposed to an X-ray dose of 9 Gy. After irradiation, repair was allowed by incubating the cells at 37°C for 0 to 90 min. followed by 30 min DNA unwinding in alkaline solution at 20°C. The fraction ds DNA was determined as indicated in Fig. 2 and converted to Gy equivalents on the basis of an induction curve. which was run in parallel in the same experiment. The percentage of DNA damage remaining was calculated relative to the Gy equivalents at the zero-repair time point ( = 100%) and plotted versus repair time in minutes. The data are the mean i sem of 3-4 independent experiments.

Fig. 3. Kinetics of DNA unwinding in alkali for unirradiated (dashed lines) and 12 Gy irradiated (solid lines) HX142 (0). RTI 12 (A 1, and MGH-Ul (V ) cells. Cells were subjected to lysis and alkaline unwinding for O-60 min at 20°C. The fraction of ds DNA was determined as indicated in Fig. 2 and plotted versus unwinding/lysis time. The data are the mean + sem of 3-4 independent experiments.

Table 1 DNA damage and repair characteristics Fig. IFig. 2Fig. 3Fig. 4Fig. 5Fig. 6

HX142

A RT112

cells. in relation to their radiosensitivity

(90 Gy: mitt)

I/’ trZJ\;

+/z ,I,tCr

l,‘,df

5.2 (0.7) 6.1 (0.74) 5.7 (0.76)

81.4 (0.3) 88.7 (0.26) 147.42 (0.26)

203.4 f 117.6 232.2 + 116.4 227.4 + 131.4

(SF, ): data derived from

res. dam. after 120 Gy (Gy eq)

I .42 2.22 1.68

SF,: surviving fraction (%‘c)at 2 Gy, calculated after an LQ-fit of the survival data (Fig. 1). Initial damage: given are the slopes of the dose-response curves (Fig. 2) obtained by regression analysis. Repair characteribtics: the repair data after 9 or 90 Gy were fitted by bi- or mono-exponential functions respectively according to: Y = ueeL” + bemL” were Y is the fraction of damage remaining. kl and k2 are the rates of rejoining. and CIand h indicate the percentage of damage rejoined according to the fdSt (kl) and intermediate (k2) repair kinetics. respectively. Half times of strand break rejoining were subsequently calculated by ln2/k as well as the fraction of damage repaired with these kinetics fin brackets). The residual damage after 120 Gy of X-rays and 24 h of repair was calculated by linear quadratic curve fitting.

E.C. Woudstrn et al. / Mutath

56

”

0 HX142

.z 5

,D

0 P

8

73 P ‘E ‘i E ’ e

A RT112 V MGH-Ul

L E $

2

Resrarch 362 ( 19%) 51-59

the first 2 h of repair was 6 Gy (RTI 12). 6.7 Gy (HXl42). and 9 Gy (MGH-Ul) equivalents. From 2-8 h after irradiation. the amount of damage remaining declined exponentially, with about the same repair kinetics in the three cell lines. Half times of rejoining were in the range of 3.39-3.87 h (Table I). Finally. the residual damage was measured 24 h at 37°C after exposure of the cells to 15- 120 Gy. The residual damage increased with dose (Fig. 6) and was not significantly different in the three cell lines. A trend could be inferred towards less residual damage in the more radiosensitive cell line. HX142. 3.3. Pulsed,field

Fig, 5. Kinetics of DNA strand break rejoining of HXl42 (0 ). RTI 12 (A ), and MGH-Ui ( 7) cells exposed to an X-ray dose of 90 Gy. Repair was allowed by incubating the cells at 37°C for 2-8 h, followed by 30 min DNA unwinding in alkaline solution at 20°C. The fraction ds DNA was determined and converted to Gy equivalents as before. The damage remainmg (in Gy equivalents) was subsequently plotted versus repair time in hours. The data are the mean + sem of 3 independent experiments

unwinding between the three cell lines could be detected. Also, the percentage of ds DNA at the plateau was invariable for the three cell lines confirming the data in Fig. 2. Cells grown to confluency showed similar results (data not shown). In all further experiments DNA unwinding was limited to 30 min. In order to see whether the differences in radiosensitivity can still be explained at the DNA level, three sets of experiments were performed to test for possible differences in repair. First, cells were irradiated with 9 Cy followed by between 0 and 60 min repair at 37°C (Fig. 4). Strand break rejoining was found to follow biphasic kinetics. Both the rates of these two phases as well as their relative contribution in the repair were the same for the three cell lines. If anything, a trend towards a slower second phase of repair was seen for MGH-Ul cells (Table I). The slow component of strand break rejoining was studied by irradiating the cells with 90 Gy (Fig. 5). The component of fast rejoining was completed in the first 2 h; repair at 37°C was subsequently monitored for 2-24 h. The damage remaining after

gel electsophoresis

(PFGE)

As our data on induction profiles were inconsistent with the NDE data, we tested whether any differences in induction of DNA damage were detectable with the PFGE technique. which is - like the NDE assay - thought to specifically measure damage proportional to double strand breaks only. Plotting the percentage of DNA extracted from the lane as a function of radiation dose, however. revealed no significant differences between the three

3

24 hrs repair

3 3 2,5 P E 2 P ‘E ‘Z E I,5 0L

A v

RT112 MGH-Ul

& s

’

: g 0,5 0 0

IZO

60

150

X-raydose(Gy) Fig. 6. DNA damage remaining after 24 h repair of HX142 co), RTI 12 (A ). and MGH-Ul ( v ) cells exposed to graded doses of irradiation (O-120 Gy). Repair was allowed by incubating the cell5 at 37°C for 24 h, followed by 30 min DNA unwinding in alkaline soiution at 20°C. The fraction ds DNA was determined and converled to Gy equivalents as before. The damage remaining (in Gy eqmvalents) was subsequently plotted versus the X-ray dose. The data are the mean f sem of 3 independent experiments.

E.C.

._

0 HX142

Woudstm

et al. / Mutcltion

I

X-ray dose (Gy)

Fig. 7. PFGE: dose response curves for HX142 (0 ). RTI 12 (A ). and MGH-UI (T ) cells. Cells were exposed to graded doses of irradiation (O-25 Gy). lysed, washed twice with PBS. treated for 1 h with RNAse A. and washed twice with TBE. After CHEF electrophoresis and gel staining. the percentage of DNA migrating into the lane (% DNA,,,,) was used as a measure of radiation-induced DSBs and plotted verhus the X-ray dose. Insert shows O-150 Gy dose response. The data are the mean + sem of 3 independent experiments.

cell lines (Fig. 7). Also, no differences in the rates of double strand break rejoining (O-2 h) were found (data not shown).

4. Discussion 4.1. Initial number qf DNA lesiom Using the neutral elution filter assay McMillan et al. ( 1990) found that a higher percentage of DNA eluted for the radiosensitive cell line HX142 compared to the other two cell lines, suggesting HX142 cells were more sensitive to damage induction by X-rays. However, our analysis of the extent of alkaline unwinding (Fig. 21, the percentage DNA fragmented in the pulsed field gel electrophoresis technique (Fig. 7), as well as the analysis of the tail moment in the alkaline comet assay (data not shown) revealed no significant differences in induced DNA single and double strand breakage between HX142. MGH-Ul and RTl12 cells. Thus it seems that the number of lesions induced per Gy per Da DNA is indifferent for the three cell lines. Therefore, the

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362 (I 9961 51-59

57

possibility of a varying concentration of scavengers leading to damage induction differences is unlikely. During the course of preparation of this manuscript, a report on PFGE data appeared (Whitaker et al., 199.5) showing evidence for (small) differences in DSB induction between HX142 and RT112. Although there are differences in the PFGE protocol and data analysis between these investigators and our work. we cannot yet explain the conflicting data. One possible explanation for the different results found in the various assays is that variations in chromatin structure between the three tumour cell lines (and the extent by which they are destroyed in the various protocols) might influence the detection of (apparent) DNA damage in some, but not all, DNA damage detecting assays. Indeed, in particular the NDE has been reported to be strongly influenced by features of DNA organization (Olive et al., 1994). It was expected that putative chromatin structure differences would have affected the me of DNA unwinding in alkali. This expectation was based on findings by e.g. Olive et al., (1986) and Ljungman (1991) indicating that the folding of the chromatin into higher-order structure and the presence of DNA-bound proteins of nuclear and nucleoid monolayers influences the rate of alkaline unwinding. However. the current data obtained with the alkaline unwinding assay demonstrate that the radiosensitive HX142 cells were identical in their response to radiation compared to the more radioresistant RTl 12 and MGH-U 1 cells. With increasing time of unwinding as well as with increasing dose, the percentage of ds DNA in all cell lines decreased exponentially with the same rate. So, if any differences in chromatin structure exist between the cell lines tested. they must be different from those reported by Ljungman ( 199 1J. Also, they must be distinct from those features that have led to different alkaline unwinding profiles after irradiating HeLa and Ewing Sarcoma cells (Jorgensen et al., 1990) or CHO-Kl and xrs-5 cells (Schwartz et al.. 1992). In conclusion, the presented data suggest that neither altered radiation-induced strand breakage per Gy per Da DNA nor differences in chromatin structure, which would have led to altered radiation-induced alkaline unwinding profiles. can explain the differences in radiosensitivity between the cell lines HX 142. RTl 12 and MGH-U 1. It is still feasible that

S8

E.C. Wotrdstra et al. /Mzrtution

variations in chromatin structure of HX142, RT112. and MGH-Ul exist. It can be speculated that such alterations in the higher order chromatin structure may lead to a different ‘presentation’ of the damage in some (NDE) but not other assays. 4.2. Repair

of DNA dumage

Our data with the alkaline unwinding (Figs. 4-6), the PFGE (data not shown). and with the alkaline comet assay (data not shown) also revealed no significant differences in repair between HX 142, RTI 12. and MGH-Ul cells. Thus, the variation in radiation sensitivity between the three cell lines cannot be explained by a difference in the rate of rejoining of single and double strand breaks. In other words, the putative differences in the chromatin of the three cell lines apparently seem to have no impact on the rate of repair of DNA lesions. It is possible that repair quality (fidelity) differs for the three cell lines. Plasmid reconstitution indeed occurs with low fidelity in HX142 cells (Powell and McMillan. 1994). Differential presentation of the same number of lesions due to chromatin structure differences may thus not only explain the differences in the detection of initial damage. They also may explain how differences in chromatin structure may lead to different consequences for the cells in situ, namely the higher sensitivity to radiation-induced cell killing of HXl42 cells e.g. because of lowered repair fidelity.

Acknowledgements This work was supported by a grant from the Interuniversitair instituut voor Radiopathologie en Stralenbescherming (IRS 7.24), by a grant from the European Community (SC 1*-CT9 l-0663 (NLA) and by a fellowship from the Dutch Cancer Society (NKB/KWF). The authors are indebted to John H. Peacock and Trevor J. McMillan for providing the cell lines and for valuable discussions.

References Ahnstriim, G. and Edvardsson. K-A. (I 974) Radiation-induced single-strand breaks in DNA determined by rate of alkaline

Rrsrcrrch 362 (lYYt5) 51-59 strand separation and hydroxylapatite chromatography: an alternative tn velocity sedimentation. Int. J. Radiat. Biol.. 26, 393-397. Biedermann. K.. Sun. J.. Giaccia. A.. Tosto, L. and Brown J.M. ( 199 I ) The scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double strand break repair. Proc. Natl. Acad. Sci. USA. 88. 1394-1397. Bliicher. D., Einspenner. M.. Zajackowski, J. (1989) CHEF electrophoresis, a sensitive technique for the determination of DNA double strand breaks. Int. J. Radiat. Biol., 56, 337-448. Dahm-Daphi. J.. Dikomey, E., Pyttlik. C. and Jeggo. P.A. (1993) Reparable and non-reparable DNA strand breaks induced by X-irradiation in CHO Kl cells and the radiosensitive mutants .rrrl and vrs5. Int. J. Radiat. Biol.. 64. 19-26. Deacon. J.M.. Prckham, M.J. and Steel. G.G. (1984) The radioresponsivene\c of human tumours and the initial slope of the cell survival curve. Radiother. Oncol.. 2, 3 17-323. Frankenberg-Schwager. M. ( 1989) Review of repair kinetics for DNA damage induced in eukaryotic cells in vitro by ionizing radiation. Radiother. Oncol.. 14. 307-320. George, A.M. and Cramp. W.A. (1987) The effects of ionizing radiation on structure and function of DNA. Prog. Biophys. Mol. Biol.. 50. 121-169. Jorgensen, T.J.. Praaad. S.C., Brennan, T.P. and Dritschilo. A. (1990) Constraint!, to DNA unwinding near radiation-induced strand breaks in Ewing’s sarcoma cells. Radiation Res.. 123, 320-324. Krlland. L.R.. Edwards, S.M. and Steel. G.G. (1988) Induction and rejoining of DNA double-strand breaks in human cervix carcinoma cell lines of differing radiosensitivity. Radiation Reh., Ilk. S76-538. Kemp. L.M.. Sedgwick. S.G. and Jeggo. P.A. ( 1984) X-ray sensitive mutant of Chinese hamster ovary cells defective in double-3trand break rejoining. Mutation Res.. 132. l89- 196. Lavin. M.F. and Schroeder. A.L. ( 1988) Damage-resistant DNA synthesis m eukaryotes. Mutation Res., 193, 193-206. Ljungman. M. (I 99 I) The influence of chromatin structure on the frequency of radiation-induced DNA strand breaks: a study using nuclear and nucleoid monolayers. Radiation Rea., 126. 58-64. McMillan, T.J.. Cassoni, A.M.. Edwards. S.. Holmes, A. and Peacock. J.H. (1990) The relationship of DNA double-strand break induction to radiosensitivity in human tumour cell lines. Int. J. Radiat. Biol.. 58. 427-438. Olive, P.L., Hilton. J. and Durand. R.E. (1986) DNA conformation of Chinese Hamster V79 cells and sensitivity to ionizing radiation, Radiation Res.. 107. 115-124. Olive. P.L.. Banath. J.P. and MacPhail, H.S. (1994) Lack of a correlation between radiosensitivity and DNA double-strand break induction or rejoining in six human tumor cell lines. Cancer Res., 54. 3939-3946. Peacock, J.H.. Eady. J.J., Edwards, S.. Holmes, A.. McMillan. T.J. and Steel, G.G. (1989) Initial damage or repair as the major determinant of cellular radiosensitivity. Int. J. Radiat. Biol.. 56. 543-547. Powell, S.N. and McMillan, T.J. (1994) The repair fidelity of restriction enzyme-induced double strand breaks in plasmid

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DNA

correlates

with radioresistance

in human

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