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Cytological characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6
Mutation Research, 177 (1987) 149-160 Elsevier
149
MTR 04314
Cytological characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6 II. Induction of sister-chromatid exchanges and chromosomal aberrations by X-rays and UV-irradiation and their modulation by inhibitors of poly(ADP-ribose) synthetase and a-polymerase F. Darroudi a and A.T. Natarajan a,b " Department of Radiation Genetics and Chemical Mutagenesls, State Unwerslty of Leiden, Wassenaarseweg 72, 2333 A L Letden (The Netherlands) and b J.A. Cohen Institute, Interunwerstty Institute of Radiation Protecnon, Letden (The Netherlands)
(Received 1 July 1986) (Revision received 21 October 1986 (Accepted 24 October 1986)
Key words. (Chinese hamster ovary cells); xrs 5, xrs 6; Chromosomal aberrations Sister-chromatid exchanges~ Poly(ADP-ribose) synthetase inhibitor; a-Polymerase inhibitors.
Summary The cell killing and induction of sister-chromatid exchanges (SCEs) by X-rays and short-wave ultraviolet (UV) irradiation in combination with inhibitors of DNA repair, 3-aminobenzamide (3AB), cytosine arabinoside (ara-C) or aphidicolin (APC) were studied in wild-type CHO-K1 and two X-ray-sensitive mutants, xrs 5 and xrs 6 cells. The spontaneous frequency of SCEs was similar in the mutants and the wild-type CHO-K1 cells (8.4-10.3 SCEs/cell). Though X-rays are known to be poor inducers of SCEs, a dose-dependent increase in the frequency of SCEs in xrs 6 cells (doubling at 150 rad) was found in comparison to a small increase in xrs 5 and no increase in wild-type CHO-K1 cells. 3AB, an inhibitor of poly(ADP-ribose) synthetase increased the spontaneous frequency of SCEs in all the cell types. 3AB did not potentiate the X-ray-induced frequency of SCEs in any of the cell lines. Ara-C, an inhibitor of DNA polymerase a, increased the frequency of SCEs in all the cell lines. In combined treatment with X-rays, ara-C had no synergistic effect in xrs 5 and xrs 6 cells, but the frequency of SCEs increased in X-irradiated wild-type CHO-K1 cells post-treated with ara-C. For the induced frequency of SCEs, CHO-K1 cells treated with X-rays plus ara-C behaved like xrs 6 cells treated with X-rays alone, suggesting a possible defect in DNA base damage repair in xrs 6 cells, in addition to the known defective repair of DNA double-strand breaks (DSBs). Survival experiments revealed higher sensitivity of xrs 5 and xrs 6 mutant cells to the cell killing effect of X-rays in S-phase when compared to wild-type CHO-K1 cells. The mutants responded with lesser sensitivity to cell killing effect of ara-C and APC than CHO-K1 cells, the relative sensitivity to ara-C or APC being CHO-K1 > xrs 5 > xrs 6 cells. When X-irradiation was coupled with ara-C, the results obtained for survival were similar to those of the SCE test, i.e., unlike Correspondence: Dr. F. Darroudi, Department of Radiation Genetics and Chemical Mutagenesis, State University of
Leiden, Wassenaarseweg 72, 2333 AL Leiden (The Netherlands).
0027-5107/87/$03.50 © 1987 Elsevier Science Publishers B.V (Biomedical Division)
150 wild-type CHO-K1, no synergistic effect was observed in xrs 5 or xrs 6 cells. After UV-irradiation, the frequency of SCEs increased similarly in wild-type CHO-K1 and xrs 6 cells, but xrs 5 cells responded with lower frequency of SCEs. When UV-irradiation was followed by post-treatment with ara-C, a synergistic effect was obtained in CHO-K1 cells, but not in xrs 5 or xrs 6 cells. UV alone in Gl-stage increased the frequencies of chromatid-type of aberrations in all cell lines, but the response was more pronounced in mutants xrs 5 and xrs 6 when compared to wild-type CHO-K1 cells. UV generally does not induce chromosome-type aberrations in (31. It induced chromosome-type aberrations when coupled with ara-C in CHO-K1 ( P < 0.01) and in xrs 5 ( P ~< 0.05), but no induction of chromosome-type aberrations was obtained in xrs 6 cells. On the basis of these findings, the mechanism of involvement of specific type of DNA lesion responsible for formation of SCE and chromosomal aberration in X- or UV-irradiated ceils is discussed.
Most of the clastogenic agents induce chromosomal aberrations and SCEs. Agents such as X-rays and bleomycin, which induce chromosomal aberrations independent of an intervening S-phase between treatment and mitosis are poor inducers of SCEs. On the other hand, agents such as UV and alkylating agents, which induce chromosomal aberrations dependent on the S-phase are potent inducers of SCEs. These observations suggest that D N A lesions and mechanisms which are responsible for formation of chromosomal aberrations and SCEs, may be at least partly different. Use of C H O cell lines deficient in specific pathways of D N A repair may lead to a better identification of D N A lesions responsible for different types of biological effect. Six X-ray-sensitive mutants have been isolated by Jeggo et al. (1982), which are deficient in repair of DSBs and proficient in repair of single-strand breaks (SSBs) (Kemp et al., 1984). We have cytologically characterized two of these mutants, xrs 5 and xrs 6 cells (Darroudi and Natarajan, 1987). On the basis of survival and induction of chromosomal aberrations in X-irradiated wild-type CHO-K1, mutant xrs 5 and xrs 6 cells, a good correlation between defect in repair of DSBs and induction of chromosomal aberrations was found. On the basis of the results obtained, i.e. induction of chromosome- and chromatid-type of aberrations following X-irradiation of (31 cells of xrs 5 and xrs 6, we suggested that these mutants are not only defective in repair of DSBs but may have also defects in repair of base damage (BD), or types of SSB which persist till the S-phase of the cell cycle and not detectable by the available biochemical techniques. A possible
relationship between X-ray-induced base damage and SCEs has been reported earlier (Uggla and Natarajan, 1983), based on the relative induction of SCEs and chromosomal aberrations in cells irradiated under aerobic and anaerobic conditions. In the present paper, we have examined (1) the spontaneous and induced frequency of SCEs m wild-type CHO-K1, mutant xrs 5 and xrs 6 cells after X-rays and UV-irradiation, (2) the effect of UV on the induction of chromosomal aberrations in G 1 cells, (3) the influence of 3AB and ara-C on the frequency of spontaneous and X-ray-induced SCEs, and the effect of ara-C on the frequency of UV-induced SCEs and chromosomal aberrations in G 1 cells and (4) the sensitivity of these cell strains to cell killing effect of X-rays, ara-C or APC and their combined treatments. Materials and methods Cells and culture condttlons
Wild-type CHO-K1 cells and radiosensitive mutants xrs 5 and xrs 6 (Jeggo et al., 1982) were kindly provided by Dr. P.A. Jeggo, National Institute for Medical Research, Mill Hill, London (Great Britain), Cells were cultured as monolayer in 9-cm petri dishes containing Ham's F10 medium (Boehringer, Mannheim) supplemented with 10% foetal calf serum (Gibco) and antibiotics, at 37 ° C with 5% CO 2. X-lrradiatton
X-Rays were generated by an E N R A F apparatus at 150 kV, 6 mA at a dose rate of 2.8 rad/sec,
151
L Analysis of sister-chromatid exchanges Spontaneous frequency of SCEs, and the effect of X-rays on the frequency of SCEs. Expt. A. Exponentially growing cells were X-irradiated with doses of 50, 100 and 150 rad. 5-Bromodeoxynridine (BrdUrd) (5 /~M) was added to the medium immediately after irradiation. Influence of 3AB on frequency of spontaneous and X-ray-induced SCEs. Expt. B. Exponentially growing cells were X-irradiated with 100 rad. BrdUrd (5 #M) and 3AB (3 mM) were added to the medium immediately after irradiation. In both Expts. A and B, cells were grown for two cell cycles in BrdUrd before fixation. Three fixations were made, namely at 24-28 h after treatment, at 2-h intervals. Expt. C. Exponentially growing cells were grown in medium containing BrdUrd (5 /~M) for 12 h, followed by X-irradiation (50 and 150 rad). Cells were recovered in fresh medium containing thymidine (TdR) (4 /xM) or thymidine + 3AB (3 mM) for additional 16 and 18 h.
Influence of ara-C post-treatment on spontaneous and X-ray-induced SCEs. Expt. D. Cells were synchronized by mitotic shake off and grown in medium containing BrdUrd (5/tM) for 12 h. They were then irradiated with doses of 50 and 150 rad, and recovered in fresh medium with T d R (4 ktM). Ara-C (10 -7 and 10 -6 M) was added to appropriate dishes, and fixations were carried out after 18 and 24 h. 11. Determination of cell survival Influence of X-rays, ara-C or APC alone or the effect of inhibitor post-treatment on the survival of X-irradiated cells. Cells were synchronized by mitotic shake off and 3 h after plating (S-phase) they were irradiated. Cells were subcultured, 200 cells were plated in each dish and 10 dishes were used for each dose. Cloning efficiency was calculated on the basis of non-irradiated ceils. (a) Non-irradiated cells were treated with ara-C or APC (10 -9, 10 -8, 10 -7, 10 -6 and 10 -5 M) for 24 h.
(b) The X-irradiated (30, 75 and 100 rad) cells were treated with ara-C (10 -8 and 10 -6 M) for 24 h. (c) The X-irradiated (30 and 100 rad) cells were treated with ara-C (10 -6, 10 -5 and 10 -4 M) for 4 h, and recovered in fresh medium containing deoxycytidine (10 -4 M) for 24 h. In all experiments, cells were grown in fresh medium for additional 7-8 days after treatment. All plates were rinsed with 0.9% sodium chloride and the colonies stained with 0.1% methylene blue and counted.
UV-irradiation UV-Irradiation was done with a germicidal lamp at a dose rate of 0.23 j / m 2 / s e c at 254 nm light calibrated by an erythemal radiometer. L Sister-chromatid exchanges CHO cells were synchronized by mitotic shake off, and plated in petri dishes. After 1.5 h (G1) they were rinsed twice in phosphate-buffered saline (PBS), drained and then irradiated with UV (2 and 4 J / m 2). They were recovered in fresh medium containing BrdUrd (5 #M). Ara-C (10 -7 and 10 -6 M) was added to appropriate dishes (treated or nontreated) for 6 h. The cells were fixed 30 h after irradiation. 11. Chromosomal aberrations CHO cells were synchronized by mitotic shake off. After 1.5 h (G~) they were rinsed twice in PBS, drained and then UV-irradiated (4 j / m 2 ) . Ara-C (10 -6 M) was added immediately after UV-irradiation for 4 h and then the cells were rinsed in PBS and recovered in fresh medium containing deoxycytidine (10 -4 M) for an additional 20 h. In all experiments, the cells blocked with colcemid (5.4 × 10 -6 M) for the last 2 h and routine air-dried preparations of hypotonically treated mitotic cells were made. For scoring SCEs, the slides were stained according to the F P G technique (Perry and Wolff, 1974). 25 second-division metaphases were counted in each treatment and all experiments were repeated twice. Student's ttest was used for statistical analysis of the data. For the analysis of chromosomal aberrations the slides were stained with 2% aqueous Giemsa solution and 100 metaphases were scored for each point.
152
Results
251 1
X-Irradiation
cn
2D
I. Sister-chromatid exchanges Expt. A. The results on the spontaneous frequency of SCEs and the effect of X-rays on the frequency of SCEs are presented in Fig. 1. The spontaneous frequency of SCEs in all the cell types was in the range of 8.4-9.6 SCEs/cell (Fig. 1). Exposure to X-rays had no effect on the frequency of SCEs in wild-type CHO-K1, the frequency of SCEs increased slightly in xrs 5 ( P ~< 0.05), whereas the frequency of SCEs increased in a dose-dependent manner in xrs 6 cells (Fig. 1).
IO
o
50
o
1 0
150
X-RAYS ( ~'ad )
Expt. B. The results of the effect of 3AB on spontaneous and X-ray-induced SCEs are presented in Tables 1 and 2. 3AB alone significantly increased the frequency of SCEs in all cell types. The increase was similar in the wild-type CHO-K1 and mutant xrs 6 cells, whereas the increase was slightly less in xrs 5 cells. The mean values of 3AB induced SCEs/cell from three fixations (24, 26 and 28 h after treatment) was 25.6, 26.7 and 22.6, for wild-type CHO-K1, xrs 6 and xrs 5, respectively (Table 1) (no significant differences have
Fig. 1 Frequency of S C E s + % by X-rays m CHO-K1 and xrs mutant cell hnes (pooled values of 3 fixations, 1 e., 24, 26 and 28 h). e , CHO-K1; III, xrs 5; v, xrs 6
been found between 3 fixations in any of 3 types of cell). X-Rays (100 rad) increased the frequency of SCEs only slightly in xrs 5 (P~<0.05) and significantly in xrs 6 cells ( P < 0.01). The combination of 3AB and X-rays did not show any synergism in xrs 5 and xrs 6 cells and no increase
TABLE 1 T H E F R E Q U E N C I E S OF SCEs IN CHO-K1, A N D M U T A N T S xrb 5 A N D xrs 6 CELLS W I T H O R W I T H O U T X - I R R A D I A T I O N A N D IN THE P R E S E N C E OR A B S E N C E OF 3AB Cell type
X-Rays
3AB
Fxxatlon
SCEs/cell
(rad)
(3 mM)
time (h)
~
So
CHO-K1 xrs 5 xrs 6
0 0 0
-
24, 26, 28 24, 26, 28 24, 26, 28
8.6 87 10.3
1.7 17 1.7
CHO-K1 xrs 5 xrs 6
0 0 0
+ + +
24, 26, 28 24, 26, 28 24, 26, 28
34.2 31.3 37.0
4.4 41 4.8
CHO-K1 xrs 5 xrs 6
100 100 100
-
28 28 28
10 0 12.0 17.2
18 17 26
CHO-K1 xrs 5 xrs 6
100 100 100
+ + +
28 28 28
34.0 35.8 40.3
4.6 4.3 5.0
Induced frequency
Induced frequency (X-rays + 3AB)
3AB
Observed
Expected
25 4 27 1 30 0
27.0 25 9 33 6
X-Rays
25 6 22.6 26.7 14 33 6.9
153 TABLE 2 I N F L U E N C E OF 3AB POST-TREATMENT ON THE FREQUENCIES OF X - R A Y - I N D U C E D SCEs IN CHO-K1 A N D MUTANTS xrs 5 A N D xrs 6 Cell type
X-Rays
3AB
Fixation
SCEs/cell
(rad)
(3 raM)
time (h)
,~
sd
Induced frequency
Induced frequency (X-rays + 3AB)
X-Rays
Observed
Expected
3AB
CHO-K1 xrs 5 xrs 6
0 0 0
-
16, 18 16, 18 16, 18
9.5 10.0 10.3
1.7 1.8 1.7
CHO-K1 xrs 5 xrs 6
50 50 50
-
16, 18 16, 18 16, 18
9.9 11.5 14.6
1.6 1.8 2.5
0.4 1.5 4.3
CHO-K1 xrs 5 xrs 6
150 150 150
-
16, 18 16, 18 16, 18
10.6 16.0 23.1
1.8 2.8 3.7
1.1 6.0 12.8
CHO-K1 xrs 5 xrs 6
0 0 0
+ + +
18 18 18
34.5 31.3 36.2
4.6 4.3 4.8
CHO-K1 xrs 5 xrs 6
50 50 50
+ + +
18 18 18
32.4 33.2 41.5
42 4.4 5.0
22.9 23.2 31.2
25.4 22.8 30.2
CHO-K1 xrs 5 xrs 6
150 150 150
+ + +
18 18 18
33.1 35.8 47.0
4.4 4.6 5.3
23.6 25.8 36.7
26.1 27.3 38.7
25.0 21.3 25.9
^ i0 ¢
10~ !
1O-lf 0
I..a I0
I_¢ 17 14 l-s 10 10" 10 10 CONCENTRATION OF ora-C ( H )
Fig. 2. Survival of CHO-K1 and mutant cell lines to cytosine arabinoside (24 h treatment). O, CHO-K1; II, xrs 5; v, xrs 6.
lo-1
I4 IO
I4 I0 CONCENTRATION
1.7
I
I
I0 10 I0 OF APHIDICOLINE ( M )
Fig. 3. Survival of CHO-K1 and mutant cell lines to aphidicolin (24 h treatment). O, CHO-K1; n, xrs 5; v, xrs 6.
154 TABLE 3 INFLUENCE OF ara-C POST-TREATMENT ON THE FREQUENCIES OF X-RAY-INDUCED SCEs IN WILD-TYPE CHO-K1 AND MUTANT xrs 5, xrs 6 CELLS Cell type
CHO-K1
xrs 5
xrs 6
X-Rays (rad)
ara-C (M)
Fixation time (h)
SCEs/cell ~ sd
Induced frequency ara-C X-Rays
0 0 0
0 10 7 10 6
24 18, 24 18, 24
86 12.5 14,1
15 20 22
39 55
50 50 50
0 10 v 10 6
18 18, 24 24
10,0 12.2 155
18 19 29
t4
150 150 150
0 10 7 10 6
18
10.1 19.7
17 29
15
24 18 a,24 "
0 0 0
0 10 7 10 6
24 18, 24 18, 24
87 12.3 14.5
16 20 27
50 50 50
0 10 7 l0 6
18 18, 24 24
ll 0 126 142
1.9 22 23
2.3
150 150 150
0 l0 7 10 6
18 18 24 18 ~,24
14.7 166 180
26 3.1 33
6.0
0 0 0
0 10 7 10 6
24 18, 24 18, 24
10.3 13.7 15.2
I7 2.3 27
50 50 50
0 l0 7 10-6
18 18, 24 24
14.5 15.4 17 2
23 26 3.1
42
150 150 150
0 10 7 10 6
18 18, 24 18",24
21.0 20 8 21.6
3.2 3.3 3,6
10 7
Induced frequency (X-rays + ara-C) Observed
Expected
3.6 6,9
53 69
ll 1
54
39 55
59 81
79 93
96 11.8
51 69
76 91
10 5 11 3
14 1 156
36 5.8
34 49
No differentiation was observed,
on the frequency of SCEs by X-rays in wild-type C H O - K 1 cells w a s o b s e r v e d ( T a b l e 1).
Expt. C. C e l l s p r e l a b e l e d w i t h B r d U r d f o r o n e cycle were X-irradiated. The results presented in T a b l e 2 s h o w t h a t X - i r r a d i a t i o n s i g n i f i c a n t l y ind u c e d S C E s i n x r s 6 cells ( d o s e o f 150 r a d d o u b l e d the frequency), while xrs 5 responded with slight i n d u c t i o n ( P < 0 . 0 5 ) i n S C E s a t 150 r a d , a n d n o i n c r e a s e w a s o b s e r v e d i n w i l d - t y p e C H O - K 1 cells. 3AB post-treatment had no effect on X-ray-ind u c e d S C E s i n all t h e s e cell s t r a i n s . The induction of chromosome- and chromatid-
types of aberrations following X-irradiation of G l - s t a g e o f m u t a n t cells (xrs 5 a n d xrs 6) ( D a r r o u d i a n d N a t a r a j a n , 1987), as well as i n d u c t i o n of SCEs by X-rays in these mutants, suggested a possible defect in the repair other than DSBs, such as b a s e d a m a g e a n d / o r S S B s i n t h e s e cell lines. S i n c e a r a - C is k n o w n to i n h i b i t r e p a i r o f D N A base damage and strand breaks, we studied the influence of ara-C on X-ray-induced chromosomal a l t e r a t i o n s i n t h e s e m u t a n t cell lines. T h e e f f e c t o f ara-C post-treatment on the frequencies of spontaneous and X-ray-induced SCEs are presented in T a b l e 3. C o n t i n u o u s t r e a t m e n t o f cells w i t h a r a - C
155 TABLE 4 I N F L U E N C E OF ara-C POST-TREATMENT (24 h) ON THE SURVIVAL (%) OF CHO-K1, A N D M U T A N T S xrs 5, xrs 6 CELLS I R R A D I A T E D W I T H X-RAYS IN S-PHASE X-Rays (rad)
ara-C (M) 0
0 30 75 100
10 -8
10 -6
CHO-K1
xrs 5
xrs 6
CHO-K1
xrs 5
xrs 6
CHO-K1
xrs 5
xrs 6
100 96 89 80
100 41 11.5 4.8
100 44 13.3 6.4
76 70 62 55
92 38.5 11 4.2
100 42 13 6
18 14 6.4 3
26 9.5 2 0.3
32.4 15 6 0.7
increased the frequency of SCEs in all the cell types, the induced frequency being similar. X-Rays alone had no effect on induction of SCEs in CHO-K1 cells, but a slight effect was observed in xrs 5 (at 150 tad) ( P < 0.05), and a profound effect was obtained in xrs 6 cells ( P < 0.01) similar to that obtained in Expt. B. Ara-C post-treatment significantly increased SCEs in X-irradiated wild-type CHO-K1 cells with a potentiation factor of 2 (150 rad plus 10 -7 M ara-C), an additive effect was observed in X-irradiated (50 rad) wildtype CHO-K1 cells post-treated with ara-C, which could be due to few aberrations induced by this dose of X-rays. No synergistic effect was observed in combined treatment of X-rays plus ara-C in mutant xrs 5 and xrs 6 cel!s. In this experiment the X-irradiated wild-type CHO-K1 cells posttreated with ara-C responded like xrs 6 cells treated with X-rays alone, suggesting that ara-C inhibits specific DNA-repair process in wild-type CHO-K1 cells leading to increase of SCEs and this response is absent in xrs 6 cells.
II. Cell-survival studies The response of CHO-K1, xrs 5 and xrs 6 cells to ara-C, APC, X-rays and their combination was studied with regard to cell killing. The cloning efficiency was 92, 82 and 81 for CHO-K1, xrs 5 and xrs 6, respectively. Expt. A. The relative survival response of the C H O cells treated for 24 h with ara-C (Fig. 2) and APC (Fig. 3) was xrs 6 > xrs 5 > CHO-K1. The survival (%) for ara-C (10 -8 M) was 75, 90 and 100 for CHO-K1, xrs 5 and xrs 6, respectively, and for APC (10 -8 M) was 89, 100 and 100 for CHO-K1, xrs 5 and xrs 6, respectively. The cell killing effect of APC was much lower at higher doses (10 -6 and 10 -5 M) than that of ara-C. In xrs 6 which proved to be the most insensitive cell line to the effects of these DNA-repair inhibitors, ara-C or APC (10 -6 M) induced 64 and 20 (%) cell killing, respectively. Expt. B.
The mutant xrs 5 and xrs 6 cells were
TABLE 5 I N F L U E N C E OF ara-C POST-TREATMENT (4 h) ON THE SURVIVAL (%) OF CHO-K1, A N D MUTANTS xrs 5, xrs 6 CELLS I R R A D I A T E D WITH X-RAYS IN S-PHASE ( D E O X Y C Y T I D I N E 10 -4 M FOR 24 h) X-Rays (rad)
ara-C
(M)
0
0 30 100
10 - 6
10 - 5
10-4
CHO-K1
xrs 5
xrs 6
CHO-K1
xrs 5
xrs 6
CHO-K1
xrs 5
xrs 6
CHO-K1
xrs 5
xrs 6
100 98 84
100 41 5.4
100 42 7
65 58 32
100 39.7 5.1
100 43 6.7
50 40 20
90 37 4.8
98 41.8 6
35 17 8
65 17 3.8
85 31.8 4.8
156
very sensitive to cell-killing effect of X-rays when compared to wild-type CHO-K1 cells (Table 4). X-Rays (30 rad) induced 4, 59 and 56 (%) cell killing in CHO-K1, xrs 5 and xrs 6, respectively. The response of treatment of S-phase cells was slightly enhanced for cell killing than the treatment of G 1 cells. D o for the mutants was in the range of 33-35 rad in S-phase cells in comparison to 4 2 - 4 4 rad in G x cells reported earlier (Darroudi and Natarajan, 1987). Post-treatment of X-irradiated cells with ara-C did not produce synergistic effect in any of the 3 types of cell (Table 4). X-Irradiation followed by 4 h post-treatment with a high dose of ara-C led to a pronounced synergis-
tic effect ( P < 0.05) in CHO-K1 cells for cell killing. Such an effect was absent in xrs 5 and xrs 6 cells (Table 5).
U V-irradiation I. Stster-chromatid exchanges The results on induction of SCEs in UV-irradiated wild-type CHO-K1, mutants xrs 5 and xrs 6 cells are presented in Fig. 4. The frequency of SCEs increased in a dose-dependent manner in all 3 cell strains. The induced frequencies were similar in CHO-K1 and xrs 6 cells but higher than in xrs 5 cells. Ara-C alone (10-6 M) slightly in-
TABL E 6 I N F L U E N C E OF ara-C P O S T - T R E A T M E N T ON T H E F R E Q U E N C I E S OF U V - I N D U C E D SCEs IN CHO-K1 A N D M U T A N T S xrs 5, xrs 6 (G 1 T R E A T M E N T ) , CELLS W E R E F I X E D AT 30 h A F T E R I R R A D I A T I O N
Cell type
CHO-K1
xrs 5
xrs 6
UV dose
ara-C
SCEs/cell
Induced frequency
Induced frequency (UV + ara-C)
( J / m 2)
(M)
~
so
ara-C
Observed
Expected
0 0 0
0 I0 7 l0 6
90 91 11.6
15 16 19
01 2.6
2 2 2
0 l0 7 10 6
253 29 8 380
30 3.6 44
16.3 20 8 29.0
16.4 18 9
4 4 4
0 10 7 10 6
386 39.4 54.0
48 50 60
29 6 30 4 45.0
29 7 32.2
0 0 0
0 10 7 10 6
9.0 9.0 9.0
1.5 16 1.5
2 2 2
0 10 7 10 -6
18.8 18.5 21 1
2.7 28 28
98 95 121
98 98
4 4 4
0 10 7 10 6
27 5 271 27.8
3.9 37 37
185 I8 1 18 8
18 5 18 5
0 0 0
0 10 7 10 6
10.2 12.5 13.1
15 1.7 19
2 2 2
0 10 7 10 6
281 29.2 28 9
30 34 3.2
17.9 190 18 7
20 2 20.8
40.4
4 5
30.2
41.0 41.6
47 47
30.8 31.4
32.5 33.1
4 4 4
0 I0 7 10 -6
UV
0 0
23 2.9
157 50[
creased the s p o n t a n e o u s f r e q u e n c y of SCEs in C H O - K 1 an d xrs 6 cells, b u t no i n d u c t i o n was o b s e r v e d in xrs 5 cells (Table 6). These responses were different f r o m those o b s e r v e d in previous e x p e r i m e n t s ( T a b l e 3) w h i c h c o u ld be due to the shorter p e r i o d of t r e a t m e n t in this e x p e r i m e n t (6 h) in c o m p a r i s o n to 1 8 - 2 4 h a r a - C t r e a t m e n t in the p r e v i o u s one. In wild-type C H O - K 1 cells, a synergistic effect was o b t a i n e d with c o m b i n e d t r e a t m e n t o f U V a n d ara-C ( n a m e l y at 10 -6 M), b u t in xrs 5 an d xrs 6 cells the response was only additive (Table 6).
1[
11. Chromosomal aberrations U V is c o n s i d e r e d as an S - d e p e n d e n t agent and is k n o w n to i n d u c e p r e d o m i n a n t l y c h r o m a t i d - t y p e o f a b e r r a t i o n s w h e n cells are treated in G l - p h a s e o f the cell cycle. T h e results of the e x p e r i m e n t in w hi ch G 1 cells were U V - i r r a d i a t e d a n d posttreated with a r a - C (10 -6 M) for 4 h are p r e s e n t e d in T a b l e 7. A 4-h a r a - C t r e a t m e n t i n d u c e d very low frequencies of c h r o m o s o m a l aberrations in all the three C H O cell lines studied. A U V dose of 4 J//m 2 i n d u c e d significantly all types of c h r o m a t i d a b e r r a t i o n s in all the cell strains. T h e effect was m o r e p r o n o u n c e d in xrs 5 and xrs 6 than in the
UV floelnce ( J/M'2 )
Fig. 4. Induction of SCEs by UV-irradiation in CHO-K1 and mutant cell lines, e, CHO-K1; II, xrs 5; v, xrs 6.
wild-type C H O - K 1 cells. F o l l o w i n g a 4-h ar a- C p o s t - t r e a t m e n t of U V - i r r a d i a t e d C H O - K 1 cells (G1), the frequencies of b o t h c h r o m o s o m e - t y p e an d c h r o m a t i d - t y p e of ab er r at i o n s increased. A slight increase in the f r e q u e n c y of c h r o m o s o m e type of ab er r at i o n s were f o u n d in xrs 5 cells, and n o n e in xrs 6 cells.
TABLE 7 INFLUENCE OF ara-C (10 -6 M, 4 h) POST-TREATMENT ON THE FREQUENCIES OF UV-INDUCED CHROMOSOMAL ABERRATIONS IN CHO-K1, AND MUTANTS xrs 5 and xrs 6 (GFSTAGE TREATMENT), FIXED AT 24 h AFTER IRRADIATION Cell type
UV dose (4 J / m 2)
ara-C
CHO-K1 xrs 5 xrs 6
-
-
4 4 5
5 5 5
0 0 0
0 0 0
0 0 0
8 9 9
9 9 10
CHO-K1 xrs 5 xrs 6
-
+ + +
5 6 7
7 8 8
0 0 0
0 0 0
2 1 0
12 13 14
14 15 15
CHO-K1 xrs 5 xrs 6
+ + +
-
16 15 18
22 34 38
0 0 0
0 0 0
8 15 19
32 42 52
46 64 75
CHO-K1 xrs 5 xrs 6
+ + +
+ + +
28 16 18
50 37 42
10 3 0
2 1 0
24 17 20
62 46 54
114 74 80
(10 - 6 M)
Chromosomal aberrations/lO0 cells Gaps
Breaks
Dicentrics
Rings
Exchanges
Abnormal cell (%)
Total aberrations
158 Discussion The agents which induce chromosomal aberration in a n ' S ' dependent way (i.e. requiring an intervening S-phase for the formation of chromosomal aberrations) are potent inducers of SCEs, whereas S-independent agents, such as X-rays are poor inducers of SCEs. Thus, the lesions responsible for chromosomal aberrations and SCEs must at least in part be different in origin and in their repair. It has been suggested that base damage induced by ionizing radiation is responsible for the formation of SCEs (Andersson et al., 1981; Uggla and Natarajan, 1983), whereas DSBs appear to be the important lesion responsible for the origin of chromosomal aberrations induced by ionizing radiations (Natarajan and Obe, 1978, 1984; Bryant, 1984). In an attempt to correlate the role of different types of lesion induced by ionizing radiations to chromosomal aberrations and SCEs, we characterized two X-ray-sensitive mutants of CHO-K1 cells, xrs 5 and xrs 6 cells known to have a defect in repair of DSBs ( K e m p et al., 1984), and found a good correlation between impairment of DSB repair and chromosomal aberrations (Darroudi and Natarajan, 1987). In these mutants a dose-dependent increase in both chromosome and chromatid-types of aberrations was observed following G I irradiation, in contrast to the expected predominantly chromosome type of aberrations (Darroudi and Natarajan, 1987). The increase in the chromosome-type of aberrations could be due to the defective repair of DSBs but increase in the frequency of chromatid-type aberrations could come partly from defects in repair of base damage a n d / o r SSBs, though it has been reported that these mutants are proficient in repair of SSBs ( K e m p et al., 1984). Both mutants have similar frequencies of spontaneously occurring SCEs when compared to the wild-type CHO-K1 cells (Fig. 1). X-Irradiation had no effect on induction of SCEs in wild-type C H O - K 1 cells, slightly induced SCEs in xrs 5 cells ( P < 0.05), whereas a dose-dependent increase was obtained in X-irradiated xrs 6 cells. The induced frequency of SCEs was doubled at 150 rad. On the basis of data on X-ray-induced chromosomal aberrations in G I (Darroudi and Natarajan, 1987), and on SCE in this study, we suggest that xrs 5
and xrs 6 may also be defective in repair of SSBs, a n d / o r BD. The low induction of SCEs in xrs 5 cells, compared to xrs 6 might come from the direct involvement of DSBs in the SCE process and it is known that xrs 5 is most defective in DSB repair. Phenotypically SCE is a recombination event, probably involving exchange between D N A double strand breaks near the replication fork (Painter, 1980; Natarajan et al., 1985). Generally ionizing radiation is potent reducer of DNA-strand breaks but a poor inducer of SCEs, because radiation does not exclusively induce DSBs and those induced are random and low m number per genome of the cell. The xrs 5 cells are reported to be the most defective mutant in repair of DSBs ( K e m p et al., 1984), thus DSBs persisting till S-phase can lead to SCEs. Alternatively because X-irradiated G~ xrs 5 cells showed a considerable increase in chromatid-type aberrations, indicating persistence of lesions till S-phase, the reason for the SCE induction could also be due to defects in repair of persisting base damage. xrs 6 cells responded with dose-dependent increase m the frequency of SCEs with X-rays. We assume that induced base damage lead to SCEs in this cell type. This cell line responded to X-rays ( G : t r e a t m e n t ) , with very high frequency of chromatid-type aberrations, about 1.6-fold more than xrs 5 cells (Darroudi and Natarajan, 1987), which could be due to defect in repair of base damage a n d / o r SSBs. On the basis of these observations we suggest that xrs 6 cells are more defective in repair of lesions which need an S-dependent processing, such as BD a n d / o r SSBs than xrs 5 cells. The frequency of spontaneously occurring SCEs in all the 3 cell lines was potentiated by 3AB (Tables 1 and 2). No synergistic effect between X-rays and 3AB was observed. 3AB is known to potentiate the frequency of X-ray-induced chromosomal aberrations in these cell stratus (Darroudi and Natarajan, 1987). These observations taken together confirm that 3AB interfere with the repair of specific type of D N A lesions i.e. DSBs (Benjamin and Gill, 1980) which lead to chromosomal aberrations and it has no effect on the type of lesion such as base damage, which presumably leads to SCEs in X-irradiated cells (Zwelling et al., 1982). This is consistent with the
159 data on the effect of 3AB on the repair of UV damage (Natarajan et al., 1983; Darroudi and Natarajan, 1985). Ara-C is known as an inhibitor of DNA synthesis. The inhibition of D N A replication by ara-C may be a consequence of a direct action on D N A polymerase a (Loeb et al., 1980). Ara-C can inhibit repair of base damage (Hiss and Preston, 1977) as well as DNA strand breaks (Iliakis and Bryant, 1983; Holmberg and Gumauskas, 1986; Natarajan et al., 1986). Ara-C is known to increase the frequency of SCEs (Ishii and Bender, 1980). In our study, ara-C alone increased the frequency of spontaneous SCEs in all cell types to a similar extent. In X-irradiated wild-type CHO cells, post-treatment with ara-C potentiated the frequency of SCEs by a factor of 2, but no such effect was found in xrs 5 and xrs 6 cells. We assume that ara-C could inhibit repair of specific types of lesion leading to SCEs in which the mutant cells are already defective (DSBs, BDs), thus leading to no increase of SCEs. If ara-C could block only the repair of base damage, it would be expected to give a synergistic effect in xrs 5 cells, but if it inhibited the repair of DSBs as well, no synergistic effect would be expected in these mutant cells. The mutants xrs 5 and xrs 6 responded with lesser sensitivity to ara-C and APC for cell killing when compared with wild-type CHO-K1 cells the relative sensitivity being CHOK1 > xrs 5 > xrs 6 (Figs. 2 and 3). xrs 5 and xrs 6 cells treated in S-phase, responded with higher sensitivity to the cell-killing effect of X-rays in comparison to G 1 irradiation (Darroudi and Natarajan, 1987). This could be due to the drastic inhibition of DNA synthesis, which has been observed in these mutant cells in comparison to the wild-type CHO-K1 cells (Jeggo, 1985). With X-rays plus ara-C treatment a synergistic effect was observed for cell killing in CHOK1 cells with higher doses of ara-C (4 h treatment) (Table 5). UV-irradiation UV-Irradiation increased the frequency of SCEs in all cell lines in a dose-dependent manner, the frequency of induced SCEs was slightly lower in xrs 5. Synergistic effect between UV and ara-C treatment was observed in wild-type CHO-K1
cells, but only additive effect was observed in xrs 5 and xrs 6 cells. When G 1 cells are exposed to UV, only chromatid-type of aberrations is encountered in the first mitosis. It has been shown by Ejima and Sasaki (1986) that UV irradiation of A - T or normal human fibroblast cells (G1) followed by incubation with ara-C resulted in the formation of chromosome-type of aberrations. When the wildtype CHO-K1, mutant xrs 5, and xrs 6 cells were irradiated with UV at Gl-stage, the frequency of chromatid-type aberrations increased significantly in all cell lines, the response being more pronounced in the mutant cells (Table 7). These data are in good agreement with survival data reported by Jeggo and Kemp (1983) which showed a higher sensitivity of xrs 5 and xrs 6 mutants compared to CHO-K1 cells to UV-irradiation. Post-treatment of UV-irradiated G a cells with ara-C induced both chromosome- and chromatidtype of aberrations in wild-type CHO-K1 cells. A slight potentiation effect obtained in xrs 5 and no effect was detected in xrs 6 cells (Table 7). Ara-C is known to inhibit the repair of UV-induced D N A damage through the blockage of the synthesis step of excision repair, thus accumulating SSBs (Hiss and Preston, 1977). These breaks could be converted into DSBs leading to formation of chromosome-type of aberrations in G 1 cells (Natarajan and Zwanenburg, 1982). The low induction of chromosome-type exchanges in xrs 5 in comparison to no induction in xrs 6 cells could be due to the difference in degree of endogenous defect of SSB repair in these mutant cells. In conclusion, it can be stated that X-rays are able to induce SCEs to a great extent in xrs 6 cells and to a lesser extent in xrs 5. This is probably due to the defects in the repair of persisting base damage a n d / o r SSBs, in these mutants. In comparison to wild-type cells, both xrs 5 and xrs 6 cells are less sensitive to cell killing by the DNA-repair inhibitors ara-C and APC. Both mutants are further characterized by the fact that post-treatments with ara-C have no influence on the frequencies of X-ray- or UV-induced SCEs. Both mutants are more sensitive to UV-irradiation when compared to wild-type CHO-K1 cells for induction of chromosomal aberrations, whereas less sensitive (xrs 5) and insensitive (xrs 6) to the
160
effect of ara-C on UV-induced chromosomal aberrations. Further biochemical characterization of repair of UV damage in the presence of inhibitors may help to understand the basic repair defect in these mutants. Acknowledgements This investigation was financially supported by the Euratom Contract B16-166-NL. We are grateful to Dr. L.H.F. Mullenders for critical reading of the manuscript and valuable discussions. References Andersson, H C., B.A Kahlman and F Pahttl (1981) Production of sister chromatld exchanges by X-rays under aerobic and anaerobic conditions, Heredltas, 94, 41-44 Benjamin, R.C., and D.M. Gill (1980) Poly(ADP-nbose) synthesis in vitro programmed by damaged DNA, A companson of DNA molecules containing different types of strand breaks, J. Biol. Chem., 255, 10502-10508. Bryant, P.E. (1984) Enzymatic restriction of mammahan cell DNA using Pvu II and Bam-Hl: Evidence for the double strand break origin of chromosomal aberrations, Int J Radiat. Biol., 46, 57-65. Darroudi, F., and A.T. Natarajan (1985) Cytological characterization of repair-deficient CHO cell line 43-3B, I. Induction of chromosomal aberrations and sister-chromatld exchanges by UV and its modulation with 3-aminobenzarnide, Mutation Res., 149, 239-247. Darroudl, F., and A.T Natarajan (1987) Cytological characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6, I. Induction of chromosomal aberrations by X-irradiation and its modulation with 3-aminobenzamide and caffeine, Mutation Res, 177, 133-148. Ejima, Y , and M.S. Sasaki (1986) Enhanced expressmn of X-ray- and UV-induced chromosome aberrations by cytosine arabinoside in ataxla telangiectasia cells, Mutation Res, 159, 117-123. Graham, F.L., and G.F. Whitmore (1970) Studies in mouse L-cells on the incorporation of 1-fl-D-arabinofuranosylcytosine with DNA and on inhibition of DNA polymerase by 1-fl-D-arabmofuranosylcytoslne-5'-triphosphate,Cancer Res., 30, 2636-2644. Hiss, E A , and R.J. Preston (1977) The effect of cytosine arablnoside on the frequency of single strand breaks m mammalian cells following irradiation or chemical treatment, Biochim. Biophys. Acta, 478, 1-8. Holmberg, M , and E. Gumauskas (1986) The role of short-hved DNA lesions in the production of chromosome-exchange aberrations, Mutation Res., 160, 221-229, Ihakis, G., and P.E Bryant (1983) Effect of nucleoside analogs
a-ara-A, fl-ara-A, fl-ara-C on cell growth and repair of potentially lethal damage and DNA double strand breaks in mammalian cell cultures, Cancer Res, 3, 143-150 Ishal, Y, and M.A Bender (1980) Effect of inhibitors of DNA synthesis on spontaneous and ultraviolet light-induced sister chromatid exchanges in Chinese hamster cells, Mutation Res, 79, 19-32. Jeggo, P.A. (1985) Genetic analysis of X-ray-sensitive mutants of CHO cell line, Mutation Res., 146, 265-270 Jeggo, P.A, and L.M. Kemp (1983) X-Ray-sensitive mutants of Chinese hamster ovary, cell line, Isolation and cross sensltwity to other DNA-damaging agents, Mutation Res, 112, 313-327. Jeggo, P.A, L.M. Kemp and R Holhday (1982) The application of the microbial "tooth-pick" technique to somatic cell genetics, and ItS use in the isolation of X-ray-sensitive mutants of Cl'nnese hamster ovary cells, Biochimie, 64, 713-715 Kemp, L M., S.G Sedwxck and P.A. Jeggo (1984) X-Ray-sensitive mutants of Chinese hamster ovary cells defective in double strand break rejoimng, Mutation Res, 132, 189-196 Loeb, L.A., S S. Agarwal, D K Duba, G S Gopinthan, C W Shearman, M.A. Sirover and E C Travaghrd (1980) Inhlbitots of mammahan DNA polymerases: possible chemotherapeutic approaches, in: P S Sann and R.C. Gallo (Eds.), Inhibitors of DNA and RNA polymerases, Pergamon, Oxford, pp 27-45. Natarajan, AT., and G Obe (1978) Molecular mechamsms involved in the production of chromosome aberrations, I Utihzat~on of Neurospora endonuclease for the study of aberration production In ()~ state of the cell cycle, Mutation Res, 52, I37-149 Natarajan, A.T., and T.S B. Zwanenburg (1982) Mechamsms for chromosomal aberrations in mammalian cells, Mutation Res., 95, 1-6 Natarajan, A T , A.A. van Zeeland and T S.B Zwanenburg (1983) Influence of inhibitors of poly(ADP-ribose) polymerase on DNA repair and chromosomal alterations, m M. Miwa, O. Hayashi, S Shall, S Snmlson and T. Sugimura (Eds.), ADP-ribosylation, DNA Repair, and Cancer, Japan Scientific Society Press, Tokyo, pp 227-242 Natarajan, A.T, L,H.F. Mullenders, M Meijers and U Mukherjee (1985) Induction of sister-chromatld exchanges by restriction endonucleases, Mutation Res, 144, 33-39 Natarajan, A.T, F. Darroudi, L.H.F. Mullenders and M Meijers (1986) The nature and repmr of DNA lesmns that lead to chromosomal aberrations induced by mnizing radiation, Mutation Res, 160, 231-236. Uggla, A.H., and A.T Natarajan (1983) X-Ray-induced SCEs and chromosome aberrations in CHO cells. Influence of nitrogen and mr dunng lrradiatmn in different stages of the cell cycle, Mutation Res., 122, 183-200. Zwelling, L A., D. Kerrigan and Y, Pommier (1982) Inhibmon of poly(adenosine diphosphoribose) synthesis slows the resealing rate of X-ray-induced DNA strand breaks, Biochem. Biophys Res. Comnmn, 104, 897-902
149
MTR 04314
Cytological characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6 II. Induction of sister-chromatid exchanges and chromosomal aberrations by X-rays and UV-irradiation and their modulation by inhibitors of poly(ADP-ribose) synthetase and a-polymerase F. Darroudi a and A.T. Natarajan a,b " Department of Radiation Genetics and Chemical Mutagenesls, State Unwerslty of Leiden, Wassenaarseweg 72, 2333 A L Letden (The Netherlands) and b J.A. Cohen Institute, Interunwerstty Institute of Radiation Protecnon, Letden (The Netherlands)
(Received 1 July 1986) (Revision received 21 October 1986 (Accepted 24 October 1986)
Key words. (Chinese hamster ovary cells); xrs 5, xrs 6; Chromosomal aberrations Sister-chromatid exchanges~ Poly(ADP-ribose) synthetase inhibitor; a-Polymerase inhibitors.
Summary The cell killing and induction of sister-chromatid exchanges (SCEs) by X-rays and short-wave ultraviolet (UV) irradiation in combination with inhibitors of DNA repair, 3-aminobenzamide (3AB), cytosine arabinoside (ara-C) or aphidicolin (APC) were studied in wild-type CHO-K1 and two X-ray-sensitive mutants, xrs 5 and xrs 6 cells. The spontaneous frequency of SCEs was similar in the mutants and the wild-type CHO-K1 cells (8.4-10.3 SCEs/cell). Though X-rays are known to be poor inducers of SCEs, a dose-dependent increase in the frequency of SCEs in xrs 6 cells (doubling at 150 rad) was found in comparison to a small increase in xrs 5 and no increase in wild-type CHO-K1 cells. 3AB, an inhibitor of poly(ADP-ribose) synthetase increased the spontaneous frequency of SCEs in all the cell types. 3AB did not potentiate the X-ray-induced frequency of SCEs in any of the cell lines. Ara-C, an inhibitor of DNA polymerase a, increased the frequency of SCEs in all the cell lines. In combined treatment with X-rays, ara-C had no synergistic effect in xrs 5 and xrs 6 cells, but the frequency of SCEs increased in X-irradiated wild-type CHO-K1 cells post-treated with ara-C. For the induced frequency of SCEs, CHO-K1 cells treated with X-rays plus ara-C behaved like xrs 6 cells treated with X-rays alone, suggesting a possible defect in DNA base damage repair in xrs 6 cells, in addition to the known defective repair of DNA double-strand breaks (DSBs). Survival experiments revealed higher sensitivity of xrs 5 and xrs 6 mutant cells to the cell killing effect of X-rays in S-phase when compared to wild-type CHO-K1 cells. The mutants responded with lesser sensitivity to cell killing effect of ara-C and APC than CHO-K1 cells, the relative sensitivity to ara-C or APC being CHO-K1 > xrs 5 > xrs 6 cells. When X-irradiation was coupled with ara-C, the results obtained for survival were similar to those of the SCE test, i.e., unlike Correspondence: Dr. F. Darroudi, Department of Radiation Genetics and Chemical Mutagenesis, State University of
Leiden, Wassenaarseweg 72, 2333 AL Leiden (The Netherlands).
0027-5107/87/$03.50 © 1987 Elsevier Science Publishers B.V (Biomedical Division)
150 wild-type CHO-K1, no synergistic effect was observed in xrs 5 or xrs 6 cells. After UV-irradiation, the frequency of SCEs increased similarly in wild-type CHO-K1 and xrs 6 cells, but xrs 5 cells responded with lower frequency of SCEs. When UV-irradiation was followed by post-treatment with ara-C, a synergistic effect was obtained in CHO-K1 cells, but not in xrs 5 or xrs 6 cells. UV alone in Gl-stage increased the frequencies of chromatid-type of aberrations in all cell lines, but the response was more pronounced in mutants xrs 5 and xrs 6 when compared to wild-type CHO-K1 cells. UV generally does not induce chromosome-type aberrations in (31. It induced chromosome-type aberrations when coupled with ara-C in CHO-K1 ( P < 0.01) and in xrs 5 ( P ~< 0.05), but no induction of chromosome-type aberrations was obtained in xrs 6 cells. On the basis of these findings, the mechanism of involvement of specific type of DNA lesion responsible for formation of SCE and chromosomal aberration in X- or UV-irradiated ceils is discussed.
Most of the clastogenic agents induce chromosomal aberrations and SCEs. Agents such as X-rays and bleomycin, which induce chromosomal aberrations independent of an intervening S-phase between treatment and mitosis are poor inducers of SCEs. On the other hand, agents such as UV and alkylating agents, which induce chromosomal aberrations dependent on the S-phase are potent inducers of SCEs. These observations suggest that D N A lesions and mechanisms which are responsible for formation of chromosomal aberrations and SCEs, may be at least partly different. Use of C H O cell lines deficient in specific pathways of D N A repair may lead to a better identification of D N A lesions responsible for different types of biological effect. Six X-ray-sensitive mutants have been isolated by Jeggo et al. (1982), which are deficient in repair of DSBs and proficient in repair of single-strand breaks (SSBs) (Kemp et al., 1984). We have cytologically characterized two of these mutants, xrs 5 and xrs 6 cells (Darroudi and Natarajan, 1987). On the basis of survival and induction of chromosomal aberrations in X-irradiated wild-type CHO-K1, mutant xrs 5 and xrs 6 cells, a good correlation between defect in repair of DSBs and induction of chromosomal aberrations was found. On the basis of the results obtained, i.e. induction of chromosome- and chromatid-type of aberrations following X-irradiation of (31 cells of xrs 5 and xrs 6, we suggested that these mutants are not only defective in repair of DSBs but may have also defects in repair of base damage (BD), or types of SSB which persist till the S-phase of the cell cycle and not detectable by the available biochemical techniques. A possible
relationship between X-ray-induced base damage and SCEs has been reported earlier (Uggla and Natarajan, 1983), based on the relative induction of SCEs and chromosomal aberrations in cells irradiated under aerobic and anaerobic conditions. In the present paper, we have examined (1) the spontaneous and induced frequency of SCEs m wild-type CHO-K1, mutant xrs 5 and xrs 6 cells after X-rays and UV-irradiation, (2) the effect of UV on the induction of chromosomal aberrations in G 1 cells, (3) the influence of 3AB and ara-C on the frequency of spontaneous and X-ray-induced SCEs, and the effect of ara-C on the frequency of UV-induced SCEs and chromosomal aberrations in G 1 cells and (4) the sensitivity of these cell strains to cell killing effect of X-rays, ara-C or APC and their combined treatments. Materials and methods Cells and culture condttlons
Wild-type CHO-K1 cells and radiosensitive mutants xrs 5 and xrs 6 (Jeggo et al., 1982) were kindly provided by Dr. P.A. Jeggo, National Institute for Medical Research, Mill Hill, London (Great Britain), Cells were cultured as monolayer in 9-cm petri dishes containing Ham's F10 medium (Boehringer, Mannheim) supplemented with 10% foetal calf serum (Gibco) and antibiotics, at 37 ° C with 5% CO 2. X-lrradiatton
X-Rays were generated by an E N R A F apparatus at 150 kV, 6 mA at a dose rate of 2.8 rad/sec,
151
L Analysis of sister-chromatid exchanges Spontaneous frequency of SCEs, and the effect of X-rays on the frequency of SCEs. Expt. A. Exponentially growing cells were X-irradiated with doses of 50, 100 and 150 rad. 5-Bromodeoxynridine (BrdUrd) (5 /~M) was added to the medium immediately after irradiation. Influence of 3AB on frequency of spontaneous and X-ray-induced SCEs. Expt. B. Exponentially growing cells were X-irradiated with 100 rad. BrdUrd (5 #M) and 3AB (3 mM) were added to the medium immediately after irradiation. In both Expts. A and B, cells were grown for two cell cycles in BrdUrd before fixation. Three fixations were made, namely at 24-28 h after treatment, at 2-h intervals. Expt. C. Exponentially growing cells were grown in medium containing BrdUrd (5 /~M) for 12 h, followed by X-irradiation (50 and 150 rad). Cells were recovered in fresh medium containing thymidine (TdR) (4 /xM) or thymidine + 3AB (3 mM) for additional 16 and 18 h.
Influence of ara-C post-treatment on spontaneous and X-ray-induced SCEs. Expt. D. Cells were synchronized by mitotic shake off and grown in medium containing BrdUrd (5/tM) for 12 h. They were then irradiated with doses of 50 and 150 rad, and recovered in fresh medium with T d R (4 ktM). Ara-C (10 -7 and 10 -6 M) was added to appropriate dishes, and fixations were carried out after 18 and 24 h. 11. Determination of cell survival Influence of X-rays, ara-C or APC alone or the effect of inhibitor post-treatment on the survival of X-irradiated cells. Cells were synchronized by mitotic shake off and 3 h after plating (S-phase) they were irradiated. Cells were subcultured, 200 cells were plated in each dish and 10 dishes were used for each dose. Cloning efficiency was calculated on the basis of non-irradiated ceils. (a) Non-irradiated cells were treated with ara-C or APC (10 -9, 10 -8, 10 -7, 10 -6 and 10 -5 M) for 24 h.
(b) The X-irradiated (30, 75 and 100 rad) cells were treated with ara-C (10 -8 and 10 -6 M) for 24 h. (c) The X-irradiated (30 and 100 rad) cells were treated with ara-C (10 -6, 10 -5 and 10 -4 M) for 4 h, and recovered in fresh medium containing deoxycytidine (10 -4 M) for 24 h. In all experiments, cells were grown in fresh medium for additional 7-8 days after treatment. All plates were rinsed with 0.9% sodium chloride and the colonies stained with 0.1% methylene blue and counted.
UV-irradiation UV-Irradiation was done with a germicidal lamp at a dose rate of 0.23 j / m 2 / s e c at 254 nm light calibrated by an erythemal radiometer. L Sister-chromatid exchanges CHO cells were synchronized by mitotic shake off, and plated in petri dishes. After 1.5 h (G1) they were rinsed twice in phosphate-buffered saline (PBS), drained and then irradiated with UV (2 and 4 J / m 2). They were recovered in fresh medium containing BrdUrd (5 #M). Ara-C (10 -7 and 10 -6 M) was added to appropriate dishes (treated or nontreated) for 6 h. The cells were fixed 30 h after irradiation. 11. Chromosomal aberrations CHO cells were synchronized by mitotic shake off. After 1.5 h (G~) they were rinsed twice in PBS, drained and then UV-irradiated (4 j / m 2 ) . Ara-C (10 -6 M) was added immediately after UV-irradiation for 4 h and then the cells were rinsed in PBS and recovered in fresh medium containing deoxycytidine (10 -4 M) for an additional 20 h. In all experiments, the cells blocked with colcemid (5.4 × 10 -6 M) for the last 2 h and routine air-dried preparations of hypotonically treated mitotic cells were made. For scoring SCEs, the slides were stained according to the F P G technique (Perry and Wolff, 1974). 25 second-division metaphases were counted in each treatment and all experiments were repeated twice. Student's ttest was used for statistical analysis of the data. For the analysis of chromosomal aberrations the slides were stained with 2% aqueous Giemsa solution and 100 metaphases were scored for each point.
152
Results
251 1
X-Irradiation
cn
2D
I. Sister-chromatid exchanges Expt. A. The results on the spontaneous frequency of SCEs and the effect of X-rays on the frequency of SCEs are presented in Fig. 1. The spontaneous frequency of SCEs in all the cell types was in the range of 8.4-9.6 SCEs/cell (Fig. 1). Exposure to X-rays had no effect on the frequency of SCEs in wild-type CHO-K1, the frequency of SCEs increased slightly in xrs 5 ( P ~< 0.05), whereas the frequency of SCEs increased in a dose-dependent manner in xrs 6 cells (Fig. 1).
IO
o
50
o
1 0
150
X-RAYS ( ~'ad )
Expt. B. The results of the effect of 3AB on spontaneous and X-ray-induced SCEs are presented in Tables 1 and 2. 3AB alone significantly increased the frequency of SCEs in all cell types. The increase was similar in the wild-type CHO-K1 and mutant xrs 6 cells, whereas the increase was slightly less in xrs 5 cells. The mean values of 3AB induced SCEs/cell from three fixations (24, 26 and 28 h after treatment) was 25.6, 26.7 and 22.6, for wild-type CHO-K1, xrs 6 and xrs 5, respectively (Table 1) (no significant differences have
Fig. 1 Frequency of S C E s + % by X-rays m CHO-K1 and xrs mutant cell hnes (pooled values of 3 fixations, 1 e., 24, 26 and 28 h). e , CHO-K1; III, xrs 5; v, xrs 6
been found between 3 fixations in any of 3 types of cell). X-Rays (100 rad) increased the frequency of SCEs only slightly in xrs 5 (P~<0.05) and significantly in xrs 6 cells ( P < 0.01). The combination of 3AB and X-rays did not show any synergism in xrs 5 and xrs 6 cells and no increase
TABLE 1 T H E F R E Q U E N C I E S OF SCEs IN CHO-K1, A N D M U T A N T S xrb 5 A N D xrs 6 CELLS W I T H O R W I T H O U T X - I R R A D I A T I O N A N D IN THE P R E S E N C E OR A B S E N C E OF 3AB Cell type
X-Rays
3AB
Fxxatlon
SCEs/cell
(rad)
(3 mM)
time (h)
~
So
CHO-K1 xrs 5 xrs 6
0 0 0
-
24, 26, 28 24, 26, 28 24, 26, 28
8.6 87 10.3
1.7 17 1.7
CHO-K1 xrs 5 xrs 6
0 0 0
+ + +
24, 26, 28 24, 26, 28 24, 26, 28
34.2 31.3 37.0
4.4 41 4.8
CHO-K1 xrs 5 xrs 6
100 100 100
-
28 28 28
10 0 12.0 17.2
18 17 26
CHO-K1 xrs 5 xrs 6
100 100 100
+ + +
28 28 28
34.0 35.8 40.3
4.6 4.3 5.0
Induced frequency
Induced frequency (X-rays + 3AB)
3AB
Observed
Expected
25 4 27 1 30 0
27.0 25 9 33 6
X-Rays
25 6 22.6 26.7 14 33 6.9
153 TABLE 2 I N F L U E N C E OF 3AB POST-TREATMENT ON THE FREQUENCIES OF X - R A Y - I N D U C E D SCEs IN CHO-K1 A N D MUTANTS xrs 5 A N D xrs 6 Cell type
X-Rays
3AB
Fixation
SCEs/cell
(rad)
(3 raM)
time (h)
,~
sd
Induced frequency
Induced frequency (X-rays + 3AB)
X-Rays
Observed
Expected
3AB
CHO-K1 xrs 5 xrs 6
0 0 0
-
16, 18 16, 18 16, 18
9.5 10.0 10.3
1.7 1.8 1.7
CHO-K1 xrs 5 xrs 6
50 50 50
-
16, 18 16, 18 16, 18
9.9 11.5 14.6
1.6 1.8 2.5
0.4 1.5 4.3
CHO-K1 xrs 5 xrs 6
150 150 150
-
16, 18 16, 18 16, 18
10.6 16.0 23.1
1.8 2.8 3.7
1.1 6.0 12.8
CHO-K1 xrs 5 xrs 6
0 0 0
+ + +
18 18 18
34.5 31.3 36.2
4.6 4.3 4.8
CHO-K1 xrs 5 xrs 6
50 50 50
+ + +
18 18 18
32.4 33.2 41.5
42 4.4 5.0
22.9 23.2 31.2
25.4 22.8 30.2
CHO-K1 xrs 5 xrs 6
150 150 150
+ + +
18 18 18
33.1 35.8 47.0
4.4 4.6 5.3
23.6 25.8 36.7
26.1 27.3 38.7
25.0 21.3 25.9
^ i0 ¢
10~ !
1O-lf 0
I..a I0
I_¢ 17 14 l-s 10 10" 10 10 CONCENTRATION OF ora-C ( H )
Fig. 2. Survival of CHO-K1 and mutant cell lines to cytosine arabinoside (24 h treatment). O, CHO-K1; II, xrs 5; v, xrs 6.
lo-1
I4 IO
I4 I0 CONCENTRATION
1.7
I
I
I0 10 I0 OF APHIDICOLINE ( M )
Fig. 3. Survival of CHO-K1 and mutant cell lines to aphidicolin (24 h treatment). O, CHO-K1; n, xrs 5; v, xrs 6.
154 TABLE 3 INFLUENCE OF ara-C POST-TREATMENT ON THE FREQUENCIES OF X-RAY-INDUCED SCEs IN WILD-TYPE CHO-K1 AND MUTANT xrs 5, xrs 6 CELLS Cell type
CHO-K1
xrs 5
xrs 6
X-Rays (rad)
ara-C (M)
Fixation time (h)
SCEs/cell ~ sd
Induced frequency ara-C X-Rays
0 0 0
0 10 7 10 6
24 18, 24 18, 24
86 12.5 14,1
15 20 22
39 55
50 50 50
0 10 v 10 6
18 18, 24 24
10,0 12.2 155
18 19 29
t4
150 150 150
0 10 7 10 6
18
10.1 19.7
17 29
15
24 18 a,24 "
0 0 0
0 10 7 10 6
24 18, 24 18, 24
87 12.3 14.5
16 20 27
50 50 50
0 10 7 l0 6
18 18, 24 24
ll 0 126 142
1.9 22 23
2.3
150 150 150
0 l0 7 10 6
18 18 24 18 ~,24
14.7 166 180
26 3.1 33
6.0
0 0 0
0 10 7 10 6
24 18, 24 18, 24
10.3 13.7 15.2
I7 2.3 27
50 50 50
0 l0 7 10-6
18 18, 24 24
14.5 15.4 17 2
23 26 3.1
42
150 150 150
0 10 7 10 6
18 18, 24 18",24
21.0 20 8 21.6
3.2 3.3 3,6
10 7
Induced frequency (X-rays + ara-C) Observed
Expected
3.6 6,9
53 69
ll 1
54
39 55
59 81
79 93
96 11.8
51 69
76 91
10 5 11 3
14 1 156
36 5.8
34 49
No differentiation was observed,
on the frequency of SCEs by X-rays in wild-type C H O - K 1 cells w a s o b s e r v e d ( T a b l e 1).
Expt. C. C e l l s p r e l a b e l e d w i t h B r d U r d f o r o n e cycle were X-irradiated. The results presented in T a b l e 2 s h o w t h a t X - i r r a d i a t i o n s i g n i f i c a n t l y ind u c e d S C E s i n x r s 6 cells ( d o s e o f 150 r a d d o u b l e d the frequency), while xrs 5 responded with slight i n d u c t i o n ( P < 0 . 0 5 ) i n S C E s a t 150 r a d , a n d n o i n c r e a s e w a s o b s e r v e d i n w i l d - t y p e C H O - K 1 cells. 3AB post-treatment had no effect on X-ray-ind u c e d S C E s i n all t h e s e cell s t r a i n s . The induction of chromosome- and chromatid-
types of aberrations following X-irradiation of G l - s t a g e o f m u t a n t cells (xrs 5 a n d xrs 6) ( D a r r o u d i a n d N a t a r a j a n , 1987), as well as i n d u c t i o n of SCEs by X-rays in these mutants, suggested a possible defect in the repair other than DSBs, such as b a s e d a m a g e a n d / o r S S B s i n t h e s e cell lines. S i n c e a r a - C is k n o w n to i n h i b i t r e p a i r o f D N A base damage and strand breaks, we studied the influence of ara-C on X-ray-induced chromosomal a l t e r a t i o n s i n t h e s e m u t a n t cell lines. T h e e f f e c t o f ara-C post-treatment on the frequencies of spontaneous and X-ray-induced SCEs are presented in T a b l e 3. C o n t i n u o u s t r e a t m e n t o f cells w i t h a r a - C
155 TABLE 4 I N F L U E N C E OF ara-C POST-TREATMENT (24 h) ON THE SURVIVAL (%) OF CHO-K1, A N D M U T A N T S xrs 5, xrs 6 CELLS I R R A D I A T E D W I T H X-RAYS IN S-PHASE X-Rays (rad)
ara-C (M) 0
0 30 75 100
10 -8
10 -6
CHO-K1
xrs 5
xrs 6
CHO-K1
xrs 5
xrs 6
CHO-K1
xrs 5
xrs 6
100 96 89 80
100 41 11.5 4.8
100 44 13.3 6.4
76 70 62 55
92 38.5 11 4.2
100 42 13 6
18 14 6.4 3
26 9.5 2 0.3
32.4 15 6 0.7
increased the frequency of SCEs in all the cell types, the induced frequency being similar. X-Rays alone had no effect on induction of SCEs in CHO-K1 cells, but a slight effect was observed in xrs 5 (at 150 tad) ( P < 0.05), and a profound effect was obtained in xrs 6 cells ( P < 0.01) similar to that obtained in Expt. B. Ara-C post-treatment significantly increased SCEs in X-irradiated wild-type CHO-K1 cells with a potentiation factor of 2 (150 rad plus 10 -7 M ara-C), an additive effect was observed in X-irradiated (50 rad) wildtype CHO-K1 cells post-treated with ara-C, which could be due to few aberrations induced by this dose of X-rays. No synergistic effect was observed in combined treatment of X-rays plus ara-C in mutant xrs 5 and xrs 6 cel!s. In this experiment the X-irradiated wild-type CHO-K1 cells posttreated with ara-C responded like xrs 6 cells treated with X-rays alone, suggesting that ara-C inhibits specific DNA-repair process in wild-type CHO-K1 cells leading to increase of SCEs and this response is absent in xrs 6 cells.
II. Cell-survival studies The response of CHO-K1, xrs 5 and xrs 6 cells to ara-C, APC, X-rays and their combination was studied with regard to cell killing. The cloning efficiency was 92, 82 and 81 for CHO-K1, xrs 5 and xrs 6, respectively. Expt. A. The relative survival response of the C H O cells treated for 24 h with ara-C (Fig. 2) and APC (Fig. 3) was xrs 6 > xrs 5 > CHO-K1. The survival (%) for ara-C (10 -8 M) was 75, 90 and 100 for CHO-K1, xrs 5 and xrs 6, respectively, and for APC (10 -8 M) was 89, 100 and 100 for CHO-K1, xrs 5 and xrs 6, respectively. The cell killing effect of APC was much lower at higher doses (10 -6 and 10 -5 M) than that of ara-C. In xrs 6 which proved to be the most insensitive cell line to the effects of these DNA-repair inhibitors, ara-C or APC (10 -6 M) induced 64 and 20 (%) cell killing, respectively. Expt. B.
The mutant xrs 5 and xrs 6 cells were
TABLE 5 I N F L U E N C E OF ara-C POST-TREATMENT (4 h) ON THE SURVIVAL (%) OF CHO-K1, A N D MUTANTS xrs 5, xrs 6 CELLS I R R A D I A T E D WITH X-RAYS IN S-PHASE ( D E O X Y C Y T I D I N E 10 -4 M FOR 24 h) X-Rays (rad)
ara-C
(M)
0
0 30 100
10 - 6
10 - 5
10-4
CHO-K1
xrs 5
xrs 6
CHO-K1
xrs 5
xrs 6
CHO-K1
xrs 5
xrs 6
CHO-K1
xrs 5
xrs 6
100 98 84
100 41 5.4
100 42 7
65 58 32
100 39.7 5.1
100 43 6.7
50 40 20
90 37 4.8
98 41.8 6
35 17 8
65 17 3.8
85 31.8 4.8
156
very sensitive to cell-killing effect of X-rays when compared to wild-type CHO-K1 cells (Table 4). X-Rays (30 rad) induced 4, 59 and 56 (%) cell killing in CHO-K1, xrs 5 and xrs 6, respectively. The response of treatment of S-phase cells was slightly enhanced for cell killing than the treatment of G 1 cells. D o for the mutants was in the range of 33-35 rad in S-phase cells in comparison to 4 2 - 4 4 rad in G x cells reported earlier (Darroudi and Natarajan, 1987). Post-treatment of X-irradiated cells with ara-C did not produce synergistic effect in any of the 3 types of cell (Table 4). X-Irradiation followed by 4 h post-treatment with a high dose of ara-C led to a pronounced synergis-
tic effect ( P < 0.05) in CHO-K1 cells for cell killing. Such an effect was absent in xrs 5 and xrs 6 cells (Table 5).
U V-irradiation I. Stster-chromatid exchanges The results on induction of SCEs in UV-irradiated wild-type CHO-K1, mutants xrs 5 and xrs 6 cells are presented in Fig. 4. The frequency of SCEs increased in a dose-dependent manner in all 3 cell strains. The induced frequencies were similar in CHO-K1 and xrs 6 cells but higher than in xrs 5 cells. Ara-C alone (10-6 M) slightly in-
TABL E 6 I N F L U E N C E OF ara-C P O S T - T R E A T M E N T ON T H E F R E Q U E N C I E S OF U V - I N D U C E D SCEs IN CHO-K1 A N D M U T A N T S xrs 5, xrs 6 (G 1 T R E A T M E N T ) , CELLS W E R E F I X E D AT 30 h A F T E R I R R A D I A T I O N
Cell type
CHO-K1
xrs 5
xrs 6
UV dose
ara-C
SCEs/cell
Induced frequency
Induced frequency (UV + ara-C)
( J / m 2)
(M)
~
so
ara-C
Observed
Expected
0 0 0
0 I0 7 l0 6
90 91 11.6
15 16 19
01 2.6
2 2 2
0 l0 7 10 6
253 29 8 380
30 3.6 44
16.3 20 8 29.0
16.4 18 9
4 4 4
0 10 7 10 6
386 39.4 54.0
48 50 60
29 6 30 4 45.0
29 7 32.2
0 0 0
0 10 7 10 6
9.0 9.0 9.0
1.5 16 1.5
2 2 2
0 10 7 10 -6
18.8 18.5 21 1
2.7 28 28
98 95 121
98 98
4 4 4
0 10 7 10 6
27 5 271 27.8
3.9 37 37
185 I8 1 18 8
18 5 18 5
0 0 0
0 10 7 10 6
10.2 12.5 13.1
15 1.7 19
2 2 2
0 10 7 10 6
281 29.2 28 9
30 34 3.2
17.9 190 18 7
20 2 20.8
40.4
4 5
30.2
41.0 41.6
47 47
30.8 31.4
32.5 33.1
4 4 4
0 I0 7 10 -6
UV
0 0
23 2.9
157 50[
creased the s p o n t a n e o u s f r e q u e n c y of SCEs in C H O - K 1 an d xrs 6 cells, b u t no i n d u c t i o n was o b s e r v e d in xrs 5 cells (Table 6). These responses were different f r o m those o b s e r v e d in previous e x p e r i m e n t s ( T a b l e 3) w h i c h c o u ld be due to the shorter p e r i o d of t r e a t m e n t in this e x p e r i m e n t (6 h) in c o m p a r i s o n to 1 8 - 2 4 h a r a - C t r e a t m e n t in the p r e v i o u s one. In wild-type C H O - K 1 cells, a synergistic effect was o b t a i n e d with c o m b i n e d t r e a t m e n t o f U V a n d ara-C ( n a m e l y at 10 -6 M), b u t in xrs 5 an d xrs 6 cells the response was only additive (Table 6).
1[
11. Chromosomal aberrations U V is c o n s i d e r e d as an S - d e p e n d e n t agent and is k n o w n to i n d u c e p r e d o m i n a n t l y c h r o m a t i d - t y p e o f a b e r r a t i o n s w h e n cells are treated in G l - p h a s e o f the cell cycle. T h e results of the e x p e r i m e n t in w hi ch G 1 cells were U V - i r r a d i a t e d a n d posttreated with a r a - C (10 -6 M) for 4 h are p r e s e n t e d in T a b l e 7. A 4-h a r a - C t r e a t m e n t i n d u c e d very low frequencies of c h r o m o s o m a l aberrations in all the three C H O cell lines studied. A U V dose of 4 J//m 2 i n d u c e d significantly all types of c h r o m a t i d a b e r r a t i o n s in all the cell strains. T h e effect was m o r e p r o n o u n c e d in xrs 5 and xrs 6 than in the
UV floelnce ( J/M'2 )
Fig. 4. Induction of SCEs by UV-irradiation in CHO-K1 and mutant cell lines, e, CHO-K1; II, xrs 5; v, xrs 6.
wild-type C H O - K 1 cells. F o l l o w i n g a 4-h ar a- C p o s t - t r e a t m e n t of U V - i r r a d i a t e d C H O - K 1 cells (G1), the frequencies of b o t h c h r o m o s o m e - t y p e an d c h r o m a t i d - t y p e of ab er r at i o n s increased. A slight increase in the f r e q u e n c y of c h r o m o s o m e type of ab er r at i o n s were f o u n d in xrs 5 cells, and n o n e in xrs 6 cells.
TABLE 7 INFLUENCE OF ara-C (10 -6 M, 4 h) POST-TREATMENT ON THE FREQUENCIES OF UV-INDUCED CHROMOSOMAL ABERRATIONS IN CHO-K1, AND MUTANTS xrs 5 and xrs 6 (GFSTAGE TREATMENT), FIXED AT 24 h AFTER IRRADIATION Cell type
UV dose (4 J / m 2)
ara-C
CHO-K1 xrs 5 xrs 6
-
-
4 4 5
5 5 5
0 0 0
0 0 0
0 0 0
8 9 9
9 9 10
CHO-K1 xrs 5 xrs 6
-
+ + +
5 6 7
7 8 8
0 0 0
0 0 0
2 1 0
12 13 14
14 15 15
CHO-K1 xrs 5 xrs 6
+ + +
-
16 15 18
22 34 38
0 0 0
0 0 0
8 15 19
32 42 52
46 64 75
CHO-K1 xrs 5 xrs 6
+ + +
+ + +
28 16 18
50 37 42
10 3 0
2 1 0
24 17 20
62 46 54
114 74 80
(10 - 6 M)
Chromosomal aberrations/lO0 cells Gaps
Breaks
Dicentrics
Rings
Exchanges
Abnormal cell (%)
Total aberrations
158 Discussion The agents which induce chromosomal aberration in a n ' S ' dependent way (i.e. requiring an intervening S-phase for the formation of chromosomal aberrations) are potent inducers of SCEs, whereas S-independent agents, such as X-rays are poor inducers of SCEs. Thus, the lesions responsible for chromosomal aberrations and SCEs must at least in part be different in origin and in their repair. It has been suggested that base damage induced by ionizing radiation is responsible for the formation of SCEs (Andersson et al., 1981; Uggla and Natarajan, 1983), whereas DSBs appear to be the important lesion responsible for the origin of chromosomal aberrations induced by ionizing radiations (Natarajan and Obe, 1978, 1984; Bryant, 1984). In an attempt to correlate the role of different types of lesion induced by ionizing radiations to chromosomal aberrations and SCEs, we characterized two X-ray-sensitive mutants of CHO-K1 cells, xrs 5 and xrs 6 cells known to have a defect in repair of DSBs ( K e m p et al., 1984), and found a good correlation between impairment of DSB repair and chromosomal aberrations (Darroudi and Natarajan, 1987). In these mutants a dose-dependent increase in both chromosome and chromatid-types of aberrations was observed following G I irradiation, in contrast to the expected predominantly chromosome type of aberrations (Darroudi and Natarajan, 1987). The increase in the chromosome-type of aberrations could be due to the defective repair of DSBs but increase in the frequency of chromatid-type aberrations could come partly from defects in repair of base damage a n d / o r SSBs, though it has been reported that these mutants are proficient in repair of SSBs ( K e m p et al., 1984). Both mutants have similar frequencies of spontaneously occurring SCEs when compared to the wild-type CHO-K1 cells (Fig. 1). X-Irradiation had no effect on induction of SCEs in wild-type C H O - K 1 cells, slightly induced SCEs in xrs 5 cells ( P < 0.05), whereas a dose-dependent increase was obtained in X-irradiated xrs 6 cells. The induced frequency of SCEs was doubled at 150 rad. On the basis of data on X-ray-induced chromosomal aberrations in G I (Darroudi and Natarajan, 1987), and on SCE in this study, we suggest that xrs 5
and xrs 6 may also be defective in repair of SSBs, a n d / o r BD. The low induction of SCEs in xrs 5 cells, compared to xrs 6 might come from the direct involvement of DSBs in the SCE process and it is known that xrs 5 is most defective in DSB repair. Phenotypically SCE is a recombination event, probably involving exchange between D N A double strand breaks near the replication fork (Painter, 1980; Natarajan et al., 1985). Generally ionizing radiation is potent reducer of DNA-strand breaks but a poor inducer of SCEs, because radiation does not exclusively induce DSBs and those induced are random and low m number per genome of the cell. The xrs 5 cells are reported to be the most defective mutant in repair of DSBs ( K e m p et al., 1984), thus DSBs persisting till S-phase can lead to SCEs. Alternatively because X-irradiated G~ xrs 5 cells showed a considerable increase in chromatid-type aberrations, indicating persistence of lesions till S-phase, the reason for the SCE induction could also be due to defects in repair of persisting base damage. xrs 6 cells responded with dose-dependent increase m the frequency of SCEs with X-rays. We assume that induced base damage lead to SCEs in this cell type. This cell line responded to X-rays ( G : t r e a t m e n t ) , with very high frequency of chromatid-type aberrations, about 1.6-fold more than xrs 5 cells (Darroudi and Natarajan, 1987), which could be due to defect in repair of base damage a n d / o r SSBs. On the basis of these observations we suggest that xrs 6 cells are more defective in repair of lesions which need an S-dependent processing, such as BD a n d / o r SSBs than xrs 5 cells. The frequency of spontaneously occurring SCEs in all the 3 cell lines was potentiated by 3AB (Tables 1 and 2). No synergistic effect between X-rays and 3AB was observed. 3AB is known to potentiate the frequency of X-ray-induced chromosomal aberrations in these cell stratus (Darroudi and Natarajan, 1987). These observations taken together confirm that 3AB interfere with the repair of specific type of D N A lesions i.e. DSBs (Benjamin and Gill, 1980) which lead to chromosomal aberrations and it has no effect on the type of lesion such as base damage, which presumably leads to SCEs in X-irradiated cells (Zwelling et al., 1982). This is consistent with the
159 data on the effect of 3AB on the repair of UV damage (Natarajan et al., 1983; Darroudi and Natarajan, 1985). Ara-C is known as an inhibitor of DNA synthesis. The inhibition of D N A replication by ara-C may be a consequence of a direct action on D N A polymerase a (Loeb et al., 1980). Ara-C can inhibit repair of base damage (Hiss and Preston, 1977) as well as DNA strand breaks (Iliakis and Bryant, 1983; Holmberg and Gumauskas, 1986; Natarajan et al., 1986). Ara-C is known to increase the frequency of SCEs (Ishii and Bender, 1980). In our study, ara-C alone increased the frequency of spontaneous SCEs in all cell types to a similar extent. In X-irradiated wild-type CHO cells, post-treatment with ara-C potentiated the frequency of SCEs by a factor of 2, but no such effect was found in xrs 5 and xrs 6 cells. We assume that ara-C could inhibit repair of specific types of lesion leading to SCEs in which the mutant cells are already defective (DSBs, BDs), thus leading to no increase of SCEs. If ara-C could block only the repair of base damage, it would be expected to give a synergistic effect in xrs 5 cells, but if it inhibited the repair of DSBs as well, no synergistic effect would be expected in these mutant cells. The mutants xrs 5 and xrs 6 responded with lesser sensitivity to ara-C and APC for cell killing when compared with wild-type CHO-K1 cells the relative sensitivity being CHOK1 > xrs 5 > xrs 6 (Figs. 2 and 3). xrs 5 and xrs 6 cells treated in S-phase, responded with higher sensitivity to the cell-killing effect of X-rays in comparison to G 1 irradiation (Darroudi and Natarajan, 1987). This could be due to the drastic inhibition of DNA synthesis, which has been observed in these mutant cells in comparison to the wild-type CHO-K1 cells (Jeggo, 1985). With X-rays plus ara-C treatment a synergistic effect was observed for cell killing in CHOK1 cells with higher doses of ara-C (4 h treatment) (Table 5). UV-irradiation UV-Irradiation increased the frequency of SCEs in all cell lines in a dose-dependent manner, the frequency of induced SCEs was slightly lower in xrs 5. Synergistic effect between UV and ara-C treatment was observed in wild-type CHO-K1
cells, but only additive effect was observed in xrs 5 and xrs 6 cells. When G 1 cells are exposed to UV, only chromatid-type of aberrations is encountered in the first mitosis. It has been shown by Ejima and Sasaki (1986) that UV irradiation of A - T or normal human fibroblast cells (G1) followed by incubation with ara-C resulted in the formation of chromosome-type of aberrations. When the wildtype CHO-K1, mutant xrs 5, and xrs 6 cells were irradiated with UV at Gl-stage, the frequency of chromatid-type aberrations increased significantly in all cell lines, the response being more pronounced in the mutant cells (Table 7). These data are in good agreement with survival data reported by Jeggo and Kemp (1983) which showed a higher sensitivity of xrs 5 and xrs 6 mutants compared to CHO-K1 cells to UV-irradiation. Post-treatment of UV-irradiated G a cells with ara-C induced both chromosome- and chromatidtype of aberrations in wild-type CHO-K1 cells. A slight potentiation effect obtained in xrs 5 and no effect was detected in xrs 6 cells (Table 7). Ara-C is known to inhibit the repair of UV-induced D N A damage through the blockage of the synthesis step of excision repair, thus accumulating SSBs (Hiss and Preston, 1977). These breaks could be converted into DSBs leading to formation of chromosome-type of aberrations in G 1 cells (Natarajan and Zwanenburg, 1982). The low induction of chromosome-type exchanges in xrs 5 in comparison to no induction in xrs 6 cells could be due to the difference in degree of endogenous defect of SSB repair in these mutant cells. In conclusion, it can be stated that X-rays are able to induce SCEs to a great extent in xrs 6 cells and to a lesser extent in xrs 5. This is probably due to the defects in the repair of persisting base damage a n d / o r SSBs, in these mutants. In comparison to wild-type cells, both xrs 5 and xrs 6 cells are less sensitive to cell killing by the DNA-repair inhibitors ara-C and APC. Both mutants are further characterized by the fact that post-treatments with ara-C have no influence on the frequencies of X-ray- or UV-induced SCEs. Both mutants are more sensitive to UV-irradiation when compared to wild-type CHO-K1 cells for induction of chromosomal aberrations, whereas less sensitive (xrs 5) and insensitive (xrs 6) to the
160
effect of ara-C on UV-induced chromosomal aberrations. Further biochemical characterization of repair of UV damage in the presence of inhibitors may help to understand the basic repair defect in these mutants. Acknowledgements This investigation was financially supported by the Euratom Contract B16-166-NL. We are grateful to Dr. L.H.F. Mullenders for critical reading of the manuscript and valuable discussions. References Andersson, H C., B.A Kahlman and F Pahttl (1981) Production of sister chromatld exchanges by X-rays under aerobic and anaerobic conditions, Heredltas, 94, 41-44 Benjamin, R.C., and D.M. Gill (1980) Poly(ADP-nbose) synthesis in vitro programmed by damaged DNA, A companson of DNA molecules containing different types of strand breaks, J. Biol. Chem., 255, 10502-10508. Bryant, P.E. (1984) Enzymatic restriction of mammahan cell DNA using Pvu II and Bam-Hl: Evidence for the double strand break origin of chromosomal aberrations, Int J Radiat. Biol., 46, 57-65. Darroudi, F., and A.T. Natarajan (1985) Cytological characterization of repair-deficient CHO cell line 43-3B, I. Induction of chromosomal aberrations and sister-chromatld exchanges by UV and its modulation with 3-aminobenzarnide, Mutation Res., 149, 239-247. Darroudl, F., and A.T Natarajan (1987) Cytological characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6, I. Induction of chromosomal aberrations by X-irradiation and its modulation with 3-aminobenzamide and caffeine, Mutation Res, 177, 133-148. Ejima, Y , and M.S. Sasaki (1986) Enhanced expressmn of X-ray- and UV-induced chromosome aberrations by cytosine arabinoside in ataxla telangiectasia cells, Mutation Res, 159, 117-123. Graham, F.L., and G.F. Whitmore (1970) Studies in mouse L-cells on the incorporation of 1-fl-D-arabinofuranosylcytosine with DNA and on inhibition of DNA polymerase by 1-fl-D-arabmofuranosylcytoslne-5'-triphosphate,Cancer Res., 30, 2636-2644. Hiss, E A , and R.J. Preston (1977) The effect of cytosine arablnoside on the frequency of single strand breaks m mammalian cells following irradiation or chemical treatment, Biochim. Biophys. Acta, 478, 1-8. Holmberg, M , and E. Gumauskas (1986) The role of short-hved DNA lesions in the production of chromosome-exchange aberrations, Mutation Res., 160, 221-229, Ihakis, G., and P.E Bryant (1983) Effect of nucleoside analogs
a-ara-A, fl-ara-A, fl-ara-C on cell growth and repair of potentially lethal damage and DNA double strand breaks in mammalian cell cultures, Cancer Res, 3, 143-150 Ishal, Y, and M.A Bender (1980) Effect of inhibitors of DNA synthesis on spontaneous and ultraviolet light-induced sister chromatid exchanges in Chinese hamster cells, Mutation Res, 79, 19-32. Jeggo, P.A. (1985) Genetic analysis of X-ray-sensitive mutants of CHO cell line, Mutation Res., 146, 265-270 Jeggo, P.A, and L.M. Kemp (1983) X-Ray-sensitive mutants of Chinese hamster ovary, cell line, Isolation and cross sensltwity to other DNA-damaging agents, Mutation Res, 112, 313-327. Jeggo, P.A, L.M. Kemp and R Holhday (1982) The application of the microbial "tooth-pick" technique to somatic cell genetics, and ItS use in the isolation of X-ray-sensitive mutants of Cl'nnese hamster ovary cells, Biochimie, 64, 713-715 Kemp, L M., S.G Sedwxck and P.A. Jeggo (1984) X-Ray-sensitive mutants of Chinese hamster ovary cells defective in double strand break rejoimng, Mutation Res, 132, 189-196 Loeb, L.A., S S. Agarwal, D K Duba, G S Gopinthan, C W Shearman, M.A. Sirover and E C Travaghrd (1980) Inhlbitots of mammahan DNA polymerases: possible chemotherapeutic approaches, in: P S Sann and R.C. Gallo (Eds.), Inhibitors of DNA and RNA polymerases, Pergamon, Oxford, pp 27-45. Natarajan, AT., and G Obe (1978) Molecular mechamsms involved in the production of chromosome aberrations, I Utihzat~on of Neurospora endonuclease for the study of aberration production In ()~ state of the cell cycle, Mutation Res, 52, I37-149 Natarajan, A.T., and T.S B. Zwanenburg (1982) Mechamsms for chromosomal aberrations in mammalian cells, Mutation Res., 95, 1-6 Natarajan, A T , A.A. van Zeeland and T S.B Zwanenburg (1983) Influence of inhibitors of poly(ADP-ribose) polymerase on DNA repair and chromosomal alterations, m M. Miwa, O. Hayashi, S Shall, S Snmlson and T. Sugimura (Eds.), ADP-ribosylation, DNA Repair, and Cancer, Japan Scientific Society Press, Tokyo, pp 227-242 Natarajan, A.T, L,H.F. Mullenders, M Meijers and U Mukherjee (1985) Induction of sister-chromatld exchanges by restriction endonucleases, Mutation Res, 144, 33-39 Natarajan, A.T, F. Darroudi, L.H.F. Mullenders and M Meijers (1986) The nature and repmr of DNA lesmns that lead to chromosomal aberrations induced by mnizing radiation, Mutation Res, 160, 231-236. Uggla, A.H., and A.T Natarajan (1983) X-Ray-induced SCEs and chromosome aberrations in CHO cells. Influence of nitrogen and mr dunng lrradiatmn in different stages of the cell cycle, Mutation Res., 122, 183-200. Zwelling, L A., D. Kerrigan and Y, Pommier (1982) Inhibmon of poly(adenosine diphosphoribose) synthesis slows the resealing rate of X-ray-induced DNA strand breaks, Biochem. Biophys Res. Comnmn, 104, 897-902