Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6 IV. Study of chromosomal aberrations and sister-chromatid exchanges by restriction endonucleases and inhibitors of DNA topoisomerase II

Mutation Research, 212 (1989) 137-148

137

Elsevier MTR 04764

Cytogenetical characterization of Chinese hamster ovary X-ray-sensitive mutant cells, xrs 5 and xrs 6 IV. Study of chromosomal aberrations and sister-chromatid exchanges by restriction endonucleases and inhibitors of D N A topoisomerase II F. D a r r o u d i a a n d A.T. N a t a r a j a n 1,2 1 Department of Radiation Genetics and Chemical Mutagenesis, State University of Leiden, 2333 AL Leiden (The Netherlands) and e J.A. Cohen Institute, lnterunioersity Research Institute for Radiopathology and Radiation Protection, Leiden (The Netherlands)

(Received 12 January 1989) (Revision received 10 February 1989) (Accepted 13 February 1989)

Keywords: xrs mutants; Restriction endonucleases; Inhibitors of topoisomerase II; Induction of DNA double-strand breaks,

chromosomal aberrations, sister-chromatid exchanges

Summary Induction of chromosomal aberrations and sister-chromatid exchanges (SCEs) was studied in wild-type Chinese hamster ovary (CHO-K1) cells and its 2 X-ray-sensitive mutants xrs 5 and xrs 6 (known to be deficient in repair of D N A double-strand breaks (DSBs)) by restriction endonucleases (REs) and inhibitors of D N A topoisomerase II known to induce D N A strand breaks, Five different types of REs, namely C f o I , E c o R I , H p a l I (which induce cohesive DSBs), H a e l I I and A l u I (which induce blunt DSBs) were employed. REs that induce blunt-end D N A DSBs were found to be more efficient in inducing chromosomal aberrations than those inducing cohesive breaks, xrs 5 and xrs 6 mutants responded with higher sensitivity (50-100% increase in the frequency of aberrations per aberrant cell) to these REs than wild-type C H O - K 1 cells. All these REs were also tested for their ability to induce SCEs. The frequency of SCEs increased in wild-type as well as mutant C H O ceils, the induced frequency being about 2-fold higher in xrs mutants than in the wild-type ceils. We also studied the effect of inhibitors of D N A topoisomerase II, namely 4'-(9-acridinylamino)methanesulfon-m-anisidide (m-AMSA) and etoposid (VP 16), at different stages of the cell cycle of these 3 types of cells. Both drugs increased the frequency of chromosomal aberrations in G 2 cells. The mutants showed increased sensitivity to m-AMSA and VP 16, xrs 6 cells being 10- and 2-fold more sensitive than wild-type C H O - K 1 ceils respectively, and xrs 5 responding with 2-fold higher sensitivity than xrs 6 cells. G 1 treatment of C H O cells with m-AMSA increased both chromosome- and chromatid-type aberrations, xrs mutants being about 3-fold more sensitive than C H O - K 1 cells.

Correspondence: Dr. F. Darroudi, Department of Radiation Genetics and Chemical Mutagenesis, State University of Lei-

den, Sylvius Laboratories, Wassenaarseweg72, 2333 AL Leiden (The Netherlands).

0027-5107/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)

138 The frequency of SCEs increased also after treatment of exponentially growing and S-phase CHO cells with m-AMSA and the higher sensitivity of xrs mutants (2-fold) was evident. The S-phase appeared to be a specific stage which is most prone for the induction of SCEs by m-AMSA. The results indicate that DNA DSBs induced by REs and inhibitors of D N A topoisomerase II correlate closely with induced chromosomal aberrations and SCEs in these cell lines, indicating that DSBs are responsible for the production of these 2 genetic endpoints.

There exists clear evidence for a direct relationship between induced chromosomal aberrations and cell death. Therefore it is important to establish the relation between different types of induced DNA lesions and chromosomal aberrations following treatment with physical and chemical agents. Bender et al. (1974) proposed a model for the formation of chromosomal aberrations, i.e., by the non-repair or misrepair of DNA double-strand breaks (DSBs) yielding deletions and exchanges respectively. Evidence for the involvement of DSB in the origin of chromosomal aberrations was later provided by Natarajan and Obe (1978), who showed that when additional DSBs were introduced into the DNA of X-irradiated mammalian cells by single-strand-specific endonuclease at the sites of the single-strand breaks (SSBs), a concurrent increase occurred in the yield of chromosomal aberrations. Introduction of restriction endonucleases (REs) into permeabilised mammalian cells that induce only 1 class of lesion (i.e., DSB) in the cellular D N A provided direct evidence for the involvement of DSB in the origin of chromosomal aberrations (Bryant, 1984; Natarajan and Obe, 1984). The efficiency of the induction of chromosomal aberrations by REs seems to be related to: (a) the ability of the enzyme to induce blunt rather than cohesive-type DSBs, (b) the number of recognition sites in the genome (Bryant, 1984, 1985; Natarajan and Obe, 1984; Obe and Natarajan, 1985). Furthermore, Natarajan et al. (1985) have shown that DNA DSBs induced during S-phase by REs led to an increase in the frequency of sister-chromatid exchanges (SCEs). Inhibitors of D N A topoisomerase II, a D N A intercalator, m-AMSA, and a non-intercalator, VP 16, have been shown to induce specific types of D N A strand breaks, and like X-rays they are S-independent (Deaven et al., 1978; Nelson et al.,

1984; Pommier et al., 1984a, b). Both single- and double-strand breaks are induced by m-AMSA (Ross et al., 1979; Ross and Bradley, 1981). However, in contrast to X-rays and REs, the DNA strand breaks induced by m-AMSA are associated with protein and the ratio between SSBs and D N A - p r o t e i n cross-links is around 1 : 1 (Zwelling et al., 1981). VP 16 does not interact with DNA directly, but induces a similar spectrum of DNA lesions as m-AMSA, exerting its cytotoxic effect by inhibiting topoisomerase II leading to proteinassociated D N A strand breaks (Chen et al., 1984; Ross et al., 1984). It is therefore interesting to compare the effects of REs, which induce direct DNA DSB, and inhibitors of topoisomerase II, which induce protein-associated strand breaks, for their ability to induce chromosomal alterations. For this study we employed mutant cell lines of Chinese hamster ovary (CHO) cells deficient in DSB repair and their parental line CHO-K1 (Jeggo and Kemp, 1983). xrs 6 appeared to be moderately (40%) and xrs 5 most (70%) defective in the repair of D N A DSBs induced by X-irradiation in comparison with the wild-type CHO-K1 cells (Kemp et al., 1984). The response of these mutants to treatment with X-rays, UV, bleomycin, mono- and bi-functional alkylating agents (Jeggo and Kemp, 1983; Kemp and Jeggo, 1986; Darroudi and Natarajan, 1987a, b, 1989) has been studied earlier. All the data obtained suggested that the higher sensitivity of xrs 5 and xrs 6 cells as reflected in cell killing and induction of chromosomal aberrations resulted from the impairment of the repair of D N A DSBs. Recently, Bryant et al. (1987) reported that REs, specially those causing bluntended DSB (i.e., PvuII and EcoRV), were more effective in xrs 5 than in CHO-K1 cells for the induction of chromosomal aberrations. Though it is known that these mutants are sensitive to directly induced DSBs (by X-rays and REs), no

139 information is available on their response to protein-associated strand breaks. The results are presented and discussed in this paper with the aim of elucidating further the involvement of induced D N A DSBs in the formation of chromosomal aberrations and SCEs. Materials and methods

Cells and culture conditions The parental line C H O - K 1 and mutants xrs 5 ~nd xrs 6 were kindly provided by Dr. P.A. Jeggo (National Institute for Medical Research, Mill Hill, London). The cells were grown in H a m ' s F10 (deficient in hypoxanthine and thymidine), supplemented with 10% fetal calf serum (Gibco) and antibiotics, at 3 7 ° C in a 5% CO 2 atmosphere.

counted and divided into different tubes with 1.5-2 × 106 ceils in each. The cell pellets were treated directly in a suspension of 30 ~tl (buffer + enzyme) in a test tube at 4 ° C , they were incubated at 37 ° C for 30 rain. Following treatment, the cells were washed in prewarmed Hanks' balanced salt solution (HBSS) and were seeded into 60-mm petri dishes in complete growth medium supplemented with BrdUrd (final concentration 5 ~tM). The cells were fixed after 18 and 20 h. Control cultures were set up in the same way, but were treated with buffer alone.

(I) Restriction endonucleases The enzymes were purchased from Promega Biotec. (Leiden), and specifications of the REs are listed in Table 1. The appropriate buffer was used for each RE as specified by the manufacturer with the exclusion of mercaptoethanol and triton.

(II) Inhibitors of D N A topoisomerase H m - A M S A was a gift from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, N I H , Bethesda, M D (U.S.A.). VP 16 was purchased from Bristol Myers, Weesp (The Netherlands). The m-AMSA was dissolved in dimethyl sulfoxide (DMSO) (0.1 mM), and VP 16 was dissolved in PBS. The stock solutions of m - A M S A (0.1 mM) and VP 16 (1 m M ) were kept frozen at - 2 0 o C, and they were thawed immediately before treatment.

Treatment with the RE. Induction of chromosomal aberrations and SCEs. Exponentially growing C H O cells were labelled with 5bromodeoxyuridine (BrdUrd, final concentration 5 ~M) for 13 h before treatment. Cells were trypsinised (0.05% trypsin/0.02% E D T A in phosphate-buffered saline (PBS) free of Mg 2+ and Ca 2+) for 2 min at 37°C, suspended in 10 ml complete growth medium. They were collected,

Treatment with m - A M S A and VP 16. (A) Induction of chromosomal aberrations by treatment of G2 ceils. Exponentially growing C H O were treated for 30 rain as monolayer with m - A M S A or VP 16, cells were washed twice with HBSS and allowed to recover in complete growth medium for an additional 2 h in the presence of Colcemid (final concentration 0 . 1 / t g / m l ) . Induction of chromosomal aberrations by treat-

TABLE 1 CHARACTERISTICSOF THE RESTRICTION ENDONUCLEASES EMPLOYED Enzyme

Recognition site

CfoI

5'... G CG/C. ,. 3' 3'... C/GC G...5' 5',.. G/AATT C...3' 3'... C TTAA/G... 5' 5'...C/CG G...3' 3'...G GC/C...5' 5'...AG/CT...3' 3'... TC/GA... 5' 5'... GG/CC... 3' 3'...CC/GG.., 5'

EcoRI HpaII AluI HaeIII

Number of base sequence 4

Termini

6

Cohesive (4-base overlap with 5' termini)

4

Cohesive (2-base overlap with 5' termini)

4

Blunt (4-base recognition sequence)

4

Blunt (4-base recognition sequence)

Cohesive (2-base overlap with 3' termini)

140 ment of G~ cells. C H O cells were synchronised by mitotic shake-off. They were treated in the G~ stage with m-AMSA for 1 h, washed twice with HBSS and allowed to recover in complete growth medium. Fixation was carried out after 18 and 20 h. To avoid the problem of persistence of the chemical (Kihlman and Palitti, personal communication), the medium was changed every 4 h until the fixation time and the medium collected was used to treat the C H O cells (independent experiment) which served as control (for the presence of the inhibitor) in addition to the untreated cultures. (B) Induction of SCEs by treatment of exponentially growing cells. Exponentially growing C H O cells were treated with m-AMSA for 1 h, washed twice with HBSS and allowed to recover in complete growth medium supplemented with BrdUrd (final concentration 5/~M) for 28 and 30 h. The medium was changed every 6 h until the fixation time. Induction of SCEs by S-phase treatment. C H O cells were labelled with BrdUrd for 12 h, they were synchronised by mitotic shake-off, and plated. Cells were grown in growth medium supplemented with thymidine (TdR, final concentration 4 /~M). They were treated as monolayer 6 h after plating with m-AMSA for 1 h, washed and fixed after 8 and 10 h recovery. The medium was changed subsequently as described for G1 cells.

Analysis of the chromosomal aberrations and SCEs In all of the experiments Colcemid (final concentration 0.1 g g / m l ) was added to the culture medium 2 h before fixation, followed by hypotonic treatment, and conventional air-dried chromosomal preparations were made. The slides were stained either with an aqueous Giemsa solution for the study of chromosomal aberrations or with Hoechst 33258 plus Giemsa (Perry and Wolff, 1974) for the study of SCEs. 100 metaphases were scored for the frequency of chromosomal aberrations, and 25-50 cells were scored for the evaluation of SCEs. Results

Treatment with the RE The results of the induction of chromosomal aberrations induced by different types of REs (see

Table 1) are compiled in Tables 2-4. The results of REs with cohesive termini are presented in Tables 2 and 3. C H O cells were treated with CfoI, EcoRI or HpaII for 30 min and fixed after 18 and 20 h. The data are pooled from both fixation times. The cultures treated with appropriate buffer that served as control showed a similar frequency of chromosomal aberrations compared to untreated cultures in all C H O cell lines. Due to the fact that not every cell was permeable and could be affected by the REs, we have determined the frequency of aberrations per aberrant cell which is used to compare the data. In C H O - K 1 cells treated with 50 units EcoRI, the frequency of chromosomal aberrations did not increase, but a slight induction was observed at a dose of 100 U (Table 2). The mutants responded to EcoRI (50 U) with a slight increase in the frequency of both chromosome- and chromatid-type aberrations (Table 2). These data are in good agreement with results previously obtained with EcoRI in CHO-K1 and xrs 5 cells by Bryant et al. (1987). Following treatment of C H O cells with HpalI (50 U) (Table 2) and CfoI (25, 50 and 100 U) (Table 3), the frequency of both chromosome- and chromatidtype aberrations increased. The chromatid-type aberrations were more frequent than chromosometype in the mutants and 30-40% of chromatid exchanges were triradials. Mutants xrs 5 and xrs 6 responded with around 50-100% higher sensitivity than C H O - K 1 cells, and the increased sensitivity was obvious at both fixation times. The results of the chromosomal aberration studies with REs which induce blunt DSBs are presented in Table 4. Following treatment of C H O cells with AluI and HaelII, the frequency of both chromosome- and chromatid-type aberrations increased, of which the latter were predominant in xrs mutant cells. The mutants responded with a 75-100% higher frequency of chromosomal aberrations when compared to CHO-K1 cells. The sensitivity of C H O cells to REs that induce blunt breaks was around 50-100% higher than to REs that induce cohesive breaks at the comparable doses.

Induction of SCEs by REs The results are presented in Tables 5-7. The data are pooled values of 2 experiments and 2

141 TABLE 2 F R E Q U E N C Y OF C H R O M O S O M A L A B E R R A T I O N S IN W I L D - T Y P E A N D xrs M U T A N T C H O CELLS T R E A T E D W I T H T H E R E S T R I C T I O N E N Z Y M E S EcoRI A N D HpalI Cell type

CHO-K1

Enzyme

-

EcoRI EcoRl EcoRI Hpa II Hpall xrs 5

-

EcoRi EcoRI Hpall HpalI xrs 6

-

Eco RI EcoRI Hpa II Hpa II

Dose

Abnormal

Chromosomal aberrations/100 cells

(units)

cells/ 100 cells

Chromatid and isochromatid breaks

Chromosome exchanges

Chromatid exchanges

Total aberrations

-

4

4

0

0

4

buffer 50 100 buffer 25

5 7 13 4 10

5 8 30 4 17

0 2 3 0 5

0 2 17 0 8

5 12 50 4 30

-

5

5

0

0

5

buffer 50 buffer 25

5 18 6 25

5 48 6 63

0 5 0 12

0 9 0 35

5 62 6 110

-

5

5

0

O

5

buffer 50 buffer 25

6 17 5 20

6 38 6 47

0 10 0 17

O 12 0 27

6 60 6 91

Aberration(s)/ aberrant cell 1.0 1.0 1.7 2.8 1.0 3.0 1.0 1.0 3.5 1.0 4.4 1.0 1.0 3.5 1.2 4.6

TABLE 3 F R E Q U E N C Y O F C H R O M O S O M A L A B E R R A T I O N S IN W I L D - T Y P E A N D xrs M U T A N T C H O CELLS T R E A T E D W I T H T H E R E S T R I C T I O N E N Z Y M E CfoI Cell type

Enzyme

Dose

Abnormal

Chromosomal aberrations/100 cells

CfoI

(units)

cells/ 100 cells

Chromatid and isochromatid breaks

Chromosome exchanges

25 50 100

5 5 10 22 29

5 5 17 65 119

0 0 3 14 18

25 50 100

4 4 0 6 6 0 18 44 11 33 140 21 not enough metaphases to score

CHO-K1 buffer + + + xrs 5 buffer + + + xrs 6

4

buffer + + +

25 50 100

5

0

5 6 0 16 31 9 31 125 22 not enough metaphases to score

Chromatid exchanges

Total aberrations

Aberration(s)/ aberrant cell

0 0 10 50 65

5 5 30 129 202

1.0 1.0 3.0 5.9 7.0

0 0 26 110

4 6 81 271

1.0 1.0 4.5 8.2

0 0 33 115

5 6 73 262

1.3 1.2 4.6 8.4

142 TABLE 4 F R E Q U E N C Y OF CHROMOSOMAL ABERRATIONS IN WILD-TYPE A N D xrs M U T A N T CHO CELLS TREATED WITH THE RESTRICTION ENZYMES AluI A N D HaeIII Cell type

CHO-KI

Enzyme

-

-

Chromosomal aberrations/100 cells

cells/ 100 cells

Chromatid and isochromatid breaks

buffer 25 buffer 25 -

Alul AluI HaelII HaellI xrs 6

Abnormal

-

/lluI AluI Haelll HaelII xrs 5

Dose (units)

HaellI

HaelII

Chromatid exchanges

Total aberrations

5

5

0

0

5

6 14 6 13

6 40 6 37

0 3 0 3

0 16 0 14

6 59 6 54

4

5

0

0

5

buffer 25 buffer 25

6 24 5 22

6 118 6 110

0 10 0 9

0 58 0 63

6 186 6 182

5

6

0

0

6

buffer 25 buffer 25

6 27 6 25

7 127 6 120

0 12 0 7

0 68 0 70

7 207 6 197

-

Alul AluI

Chromosome exchanges

Aberration(s)/ aberrant cell 1.0 1.0 4.2 1.0 4.2 1.3 1.0 7.8 1.2 8.3 1.2 1.2 7.7 1.0 7.9

TABLE 5 FREQUENCY OF SCEs IN WILD-TYPE A N D xrs M U T A N T CHO CELLS T R E A T E D W I T H T H E RESTRICTION ENZYME

Cfo I Cell type

CHO-K1

xrs 5

xrs 6

Cfo I (units)

SCEs/ cell a

-

8.8

Buffer 25 50 100

9.1 13.1 16.5 28.1

-

9.1

Buffer 25 50 100 b

9.1 16.8 23.2 9.2

-

8.7

Buffer 25 50 100 c

8.8 17.5 24.3 20.6

Induced frequency -

0.3 4.0 7.4 19.0 -

0 7.7 14.1 0.1 -

0.1 8.7 15.5 11.8

Distribution of SCEs/cell 0-7

8-13

14-19

20-25

26-31

32-37

38-43

6

44

0

0

0

0

0

7 2 4 0

43 25 14 0

0 20 24 4

0 2 4 12

0 1 2 24

0 0 1 5

0 0 1 5

6

44

0

0

0

0

0

4 2 0 4

46 17 0 6

0 20 28 0

0 10 8 0

0 1 7 0

0 1 5 0

0 1 2 0

9

41

0

0

0

0

0

10 2 0 2

40 13 0 5

0 28 10 1

0 3 18 1

0 2 14 8

0 2 4 3

0 0 4 0

a The data are pooled from scoring of 50 differentially stained metaphases. b The data are pooled from scoring of 10 differentially stained metaphases. c The data are pooled from scoring of 20 differentially stained metaphases.

143 TABLE 6 FREQUENCY OF SCEs IN WILD-TYPE AND xrs MUTANT CHO CELLS TREATED WITH THE RESTRICTION ENZYMES EcoRl AND HpalI Cell type

CHO-K1

Enzyme

-

-

Induced

Distribution of SCEs/ceU

frequency

0-7

8-13

-

5

45

0

0

0

0

0

0.3 4.1 0 4.7

5 0 6 0

44 26 44 32

1 22 0 14

0 2 0 4

0 0 0 0

0 0 0 0

0 0 0 0

-

6

44

0

0

0

0

0

0 8.5 0.1 8.2

8 0 6 0

42 6 44 10

0 32 0 32

0 8 0 4

0 3 0 2

0 1 0 1

0 0 0 1

-

8

42

0

0

0

0

0

8 0 7 0

42 4 43 10

0 12 0 24

0 20 0 10

0 6 0 4

0 6 0 2

0 2 0 0

(buffer) 50 (buffer) 25 -

EcoRI EcoRI HpalI HpalI xrs 6

SCEs/ cell

-

EcoRI EcoRl HpalI HpaII xrs 5

Dose (units)

-

(buffer) 50 (buffer) 25 -

EcoRl EcoRi HpalI Hpall

(buffer) 50 (buffer) 25

8.9

9.2 13.3 8.9 13.6 9.1

9.1 17.6 9.2 17.4 8.5

8.5 19.7 8.7 18.0

0 11.2 0.2 9.3

fixation times. All 5 enzymes studied increased the

14-19

20-25

26-31

32-37

38-43

frequency of SCEs a n d xrs m u t a n t s were a b o u t

i n g S C E s i n all C H O cells. T h e r e a s o n c o u l d b e the number of nucleotides needed for recognition

1.5-2-fold

cells.

( s e e T a b l e 1). I t a p p e a r s t h a t m o r e b r e a k s a r e

A l u I and H a e l I I w e r e m o r e e f f i c i e n t (at c o m p a r a b l e d o s e s ) t h a n E c o R I i n i n d u c -

i n d u c e d w h e n t h e l e n g t h o f t h e r e c o g n i t i o n seq u e n c e is 4 i n s t e a d o f 6 n u c l e o t i d e s , w h i c h c o u l d

more

sensitive than

CHO-K1

CfoI, H p a l I ,

TABLE 7 FREQUENCY OF SCEs IN WILD-TYPE AND xrs MUTANT CHO CELLS TREATED WITH THE RESTRICTION ENZYMES

AluI AND HaelII Cell type

CHO-K1

Enzyme

-

AluI Alul HaelII HaelII xrs 5

-

AluI Alul HaelII HaelII xrs 6

-

AluI AluI HaellI HaelII

Dose (units) -

(buffer) 25 (buffer) 25 -

(buffer) 25 (buffer) 25 -

(buffer) 25 (buffer) 25

SCEs/

Induced

Distribution of SCEs/ceU

cell

frequency

0-7

8-13

-

7

43

0

0

0

0

0

0 5.4 0.2 6.6

8 3 7 4

42 19 43 18

0 22 0 20

0 6 0 5

0 0 0 2

0 0 0 1

0 0 0 0

-

5

45

0

0

0

0

0

6 0 6 0

44 9 44 6

0 30 0 18

0 6 0 20

0 3 0 3

0 2 0 2

0 0 0 1

9

41

0

0

0

0

0

8 0 6 0

42 9 44 5

0 31 0 20

0 7 0 17

0 2 0 2

0 0 0 4

0 1 0 2

8.8

8.8 14.2 9.0 15.6 9.0

8.8 17.5 8.6 20.5 8.3

8.6 17.1 8.8 21.0

8.7 11.9 -

0.3 8.5 0.5 12.2

14-19

20-25

26-31

32-37

38-43

144

in turn lead to the higher frequencies of chromosomal aberrations (Tables 2 and 3) and SCEs. A linear relationship was found in C H O - K 1 cells between dose of CfoI and the frequency of SCEs (Table 5). In the xrs 6 mutant the frequency of SCEs saturated at the highest dose of enzyme (100 U), in xrs 5 ceils it remained at the level of controls. In these cells we determined the frequencies of mitotic index (MI) and cells in the first, second and third stages of the cell cycle. In CHOK1 ceils frequencies of MI and M I I cells were higher than in xrs mutants, and the order was CHO-K1 > xrs 6 > xrs 5 cells. This indicates that treatment with CfoI (100 U) induces a high number of DSBs which remain unrepaired in xrs mutant cells, and only unaffected (xrs 5) or slightly affected cells (xrs 6) could reach mitosis at the time of fixation. Unlike in the chromosomal aberration studies, no obvious differences were observed in the frequency of SCEs between REs inducing cohesive (Tables 5 and 6) or blunt-end (Table 7) DSBs.

Inhibitors of topoisomerase H

(A) Chromosomal aberrations after Ge treatment The results are presented in Table 8. Treatment (30 rain) of C H O cells with m-AMSA or VP 16 increased the frequency of chromosomal aberrations in a dose-dependent manner. A positive correlation between defects in D N A DSB repair and the induced frequency of chromosomal aberrations was found, xrs 5 responded to m-AMSA treatment with a 2-fold increase in sensitivity in comparison to xrs 6 which was 10-fold more sensitive than wild-type C H O - K 1 cells, xrs 5 and xrs 6 cells were also more sensitive to VP 16, 4 and 2 times respectively when compared to CHO-K1 cells.

Chromosomal aberrations after Gj treatment. Following treatment of G1 cells with m-AMSA (1 h), the frequencies of both chromosome- and chro-

TABLE 8 I N D U C T I O N OF CHROMOSOMAL ABERRATIONS IN CHO-K1, xrs 5 A N D xrs 6 CELLS TREATED W I T H I N H I B I T O R OF TOPOISOMERASE II, m-AMSA A N D VP 16 (G 2 STAGE) Cell type

CHO-K1

Inhibitor

DMSO m-AMSA

VP 16

xrs 5

DMSO m-AMSA

VP 16

xrs 6

DMSO m-AMSA

VP 16

Dose

Abnormal

Chromosomal aberrations/100 ceils

( # M)

ceils/ 100 cells

Breaks

1% 0.2 1.0 2.0 15.0 30.0

4 8 18 40 20 36

1% 0.2 1.0 2.0

4 57 82 100

15.0 30.0

60 78

1% 0.2 1.0 2.0

5 33 58 95

15.0 30.0

40 62

4 10 20 51 20 37

Chromatid exchanges 0 0 4 16 4 14

Total aberrations 4 10 24 67 24 51

4 0 4 82 21 103 196 80 276 75% of the cells had multi-aberrations (more than 10 aberrations/cell) 65 26 91 142 62 204 5 0 5 46 12 58 114 32 146 40% of,the cells had multi-aberrations (more than 10 aberrations/cell) 42 15 57 90 40 130

145 TABLE 9

INDUCTION OF C H R O M O S O M A L A B E R R A T I O N S (G 1 STAGE) BY A N I N H I B I T O R O F T O P O I S O M E R A S E II (m-AMSA) IN CHO-K1, xrs 5 A N D xrs 6 CELLS Cell type

Dose

Abnormal

Chromosomal aberrations/lO0 cells

(/~M)

cells/ 100 cells

Breaks

CHO-K1

0 0.25 0.50 1.0 2.0

2 11 17 38 58

2 9 14 21 52

0 0 1 10 22

0 2 5 20 42

2 11 20 51 116

xrs 5

0 0,25 0.50 1,0 2,0

2 21 39 63 92

2 15 34 64 152

0 5 10 18 34

0 7 24 73 162

2 27 68 155 348

x.rs 6

0 0.25 0.50 1.0 2.0

3 20 43 56 85

2 16 38 58 127

0 5 11 23 62

1 6 26 60 94

3 27 75 141 283

Dicentric + Rings

matid-type aberrations increased in wild-type as well as in mutant CHO cells. The xrs mutants responded with a 3-fold higher sensitivity to mAMSA in comparison to CHO-K1 cells (Table 9).

Chromatid exchanges

Total aberrations

wide distribution in the number of SCEs was found for each tested dose of m-AMSA, which indicates that cells at different stages of the cell cycle responded differently to the m-AMSA treatment. The data presented in Fig. 1B indicate that in CHO ceils treated at S-phase with m-AMSA for 1 h, the frequency of SCEs increased very efficiently, and the higher sensitivity of xrs mutants was obvious (2-fold). The distribution of SCEs between these cell lines treated in S-phase was more uniform in comparison to treatment of ex-

(B) Induction of SCEs Following 1 h treatment of exponentially growing CHO cells with m-AMSA, the frequency of SCEs increased in the wild-type and xrs mutants, the mutants being 2-fold more sensitive than CHO-K1 cells (Fig. 1A). It should be noted that a

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Fig. 1. Induced frequency of SCEs ( ± SD) by m - A M S A in (A) exponentially growing cells and (B) S-phase cells of C H O - K 1 and xrs mutants. C H O - K 1 (A); xrs 5 (o); xrs 6 (e).

146 ponentially growing cells, in which case the distribution was overdispersed. The frequency of chromosomal aberrations and SCEs in cultures incubated with medium collected before washing remained at the level of the untreated control, indicating that no inhibitors of topoisomerase II or their active metabolites were present in that medium. Discussion

The results presented in this paper support the idea that induced D N A DSBs are the ultimate lesions which lead to chromosomal aberrations and SCEs induced by REs and inhibitors of topoisomerase II. xrs mutants were more sensitive (1.5-2-fold) to REs for induction of chromosomal aberrations and SCEs. The pattern of chromosomal aberrations (see Tables 2-4) induced by REs is very similar to that induced by X-rays and bleomycin (Kemp and Jeggo, 1986; Darroudi and Natarajan, 1987a, 1989). The treatment of exponentially growing cells led to the induction of both chromosome- and chromatid-type aberrations in the same cell even within one aberration. This indicates that the G1-S transition phase may be very sensitive to treatment with REs (Natarajan and Obe, 1984). It is known from X-ray experiments that at the G1-S stage both chromosome- and chromatid-type aberrations are induced (Evans and Savage, 1963; Scott and Evans, 1967; Luchnik et al., 1976). REs causing blunt-ended DSBs were more efficient in inducing chromosomal aberrations than REs with cohesive-ended DSBs, which is in good agreement with previous results (Bryant, 1984, 1985; Bryant et al., 1987; Natarajan and Obe, 1984). The increase in the frequency of breaks in the xrs mutants is likely to be due to a defect in the rejoining of induced DSBs in these mutants, and a higher number of exchanges would result if a higher number of DSBs remained unrepaired for longer periods of time, thus giving DSBs more opportunity to interact with each other to form exchanges. Treatment of C H O cells with REs increased the frequency of SCEs (Tables 5-7). Xrs mutants defective in DSB rejoining responded with a 1.5-2-fold increase in the frequency of SCEs corn-

pared to the parental CHO-K1 cells. The results presented demonstrate and confirm the involvement of DSBs induced by REs in the origin of SCEs (Natarajan et al., 1985). In this respect REs behaved differently from X-rays. Generally ionising radiation is a poor inducer of SCEs, radiation-induced DSBs are randomly distributed and low in number per genome. In contrast, DSBs induced by REs are specific, much higher in number and non-randomly distributed. This is the basis for the increased efficiency of RE-induced DSBs to induce SCEs (Natarajan et al., 1985). Following treatment of CHO cells with CfoI (Table 5), a dose-dependent increase in SCEs was observed in CHO-K1 cells. In xrs mutants the frequency of SCEs increased at lower doses more efficiently than in CHO-K1 ceils, but at the highest dose (100 U), it decreased to the control level in xrs 5 and remained at the level of 50 U in xrs 6 cells. This was accompanied by a very low mitotic index and frequency of differentially stained metaphases. For SCEs to be visualised the cells have to pass through 2 cell cycles. RE treatment at high concentrations led to a high frequency of aberrations in xrs cells with a low mitotic index. These data could explain the results of Gustavino et al. (1986), who reported a high frequency of chromosomal aberrations following treatment with BamHI inducing cohesive-ended DSBs at the very high dose of 12,500 U / m l , with no increase in the frequency of SCEs in CHO cells. Inhibitors of topoisomerase II, m-AMSA and VP 16, induce chromosomal alterations effectively in xrs mutants and the parental CHO-K1 cells. D N A topoisomerases are enzymes that can modify and may regulate the topological state of D N A through concerted breaking and rejoinittg of D N A single and double strands (for review, see Drlica and Franco, 1988; Downes and Johnson, 1988). m-AMSA binds to D N A and, like X-rays, acts in an S-independent manner (Pommier et al., 1984a, b). However, VP 16 does not bind to DNA and exerts its effect by impairment of the strandrejoining activity of topoisomerase II (Ross et al., 1984). In view of the D N A strand-breaking activity of VP 16, Gupta (1983) concluded that the cellular effects of VP 16 are similar to those of X-rays. Therefore it was of interest to see the relation between the strand-breaking activity of

147 these inhibitors and the formation of chromosomal aberrations and SCEs in xrs mutant cells. Our data show that in m-AMSA- or VP 16-treated C H O cells aberrations of the chromatid type were found in cells exposed during the G 2 phase (Table 8), and aberrations of chromosome and chromatid type when cells were exposed during the G 1 phase (Table 9). In the G2 experiment, a direct relation between the degree of impairment of repair of D N A DSBs and the induced frequency of chromosomal aberrations was found, xrs 5 and xrs 6 cells showed about 20- and 10-fold higher sensitivity to m-AMSA and about 4- and 2-fold higher sensitivity to VP 16 respectively, in comparison to wild-type CHO-K1 cells. The difference in the extent of response between m - A M S A and VP 16 is due to their mode of action, m - A M S A binds to D N A (Pommier et al., 1984a, b) and consequently induces a higher number of D N A DSBs, whereas VP 16 acts indirectly in the regulation of the strand breaks (Ross et al., 1984). The G~ cells of xrs mutants responded with a higher sensitivity (3-fold) to m-AMSA than C H O K1 cells (Table 9). In G 1 cells m-AMSA-induced D N A lesions gave rise to chromosome and chromatid aberrations. It has been reported that in h u m a n lymphocytes treated at the G1 stage with m-AMSA, if washed repeatedly following treatment, only chromosome-type aberrations are induced (Kihlman and Palitti, personal communication). In our study, the cells were thoroughly washed, which rules out the influence of persisting residual metabolites acting later in the S phase leading to chromatid aberrations. The frequency of SCEs was also increased by m - A M S A in wild-type and xrs mutant C H O cells, and the induced sensitivity of xrs mutants (2-fold) was obvious (Fig. 1). D N A topoisomerase II has been hypothesised to be involved in the formation of SCEs (Ishii and Bender, 1980). Pommier et al. (1985) have postulated that the SCE results from exchange of topoisomerase II subunits while they are covalently bound to adjacent double strands. rn-AMSA inhibits topoisomerase II by trapping the enzyme in the complex (Nelson et al., 1984), and results in an SCE when topoisomerase dissociates into subunits and each subunit then reassociates with a subunit from the other double strand (Pommier et al., 1985). Deficiency in legitimate

restitution of the induced D N A DSBs would lead to the higher frequency of SCEs. We have observed a wide distribution in the number of SCEs following treatment of exponentially growing C H O cells compared to the cells treated at early S phase with m-AMSA, which indicates and confirms Sphase sensitivity for induction of SCEs by mAMSA (Dillehay et al., 1987). In conclusion, it can be stated that repair-deficient xrs 5 and xrs 6 cells are more sensitive to REs and inhibitors of D N A topoisomerase II (mA M S A and VP 16) than wild-type C H O - K 1 ceils as judged by the induced frequencies of chromosomal aberrations and SCEs. We conclude that the DSBs induced by REs and inhibitors of topoisomerase II, although widely different in their origin and nature, lead to the formation of chromosomal aberrations and SCEs.

Acknowledgement This investigation was supported by the Euratom contract with the State University of Leiden (B 16-166-NL).

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