Restriction endonuclease Bam H I induces chromosomal aberrations in Chinese hamster ovary (CHO) cells

Restriction endonuclease Bam H I induces chromosomal aberrations in Chinese hamster ovary (CHO) cells

Mutation Research, 175 (1986) 91-95 91 Elsevier MRLett 0899 Restriction endonuclease Barn H I induces chromosomal aberrations in Chinese hamster o...

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Mutation Research, 175 (1986) 91-95

91

Elsevier

MRLett 0899

Restriction endonuclease Barn H I induces chromosomal aberrations in Chinese hamster ovary (CHO) cells Bianca Gustavino, Christian Johannes and Giinter Obe Institut ff~r Genetik, Freie Universitiit Berlin, Arnimallee 5-7, D-IO00 Berlin 33 (F.R.G.) (Accepted 30 May 1986)

Summary Treatment of Chinese hamster ovary (CHO) cells with the restriction endonuclease Bam H I (recognition site: G/GATCC) leads to high frequencies of chromosomal aberrations. Experiments with bromodeoxyuridine-labelled chromosomes show that the aberrations occur nearly exclusively in first posttreatment metaphases. The results are interpreted to mean that only some of the cells take up the enzyme and that these cells are the ones showing the aberrations. Cells which do not take up the enzyme show up as differentially stained metaphases and have no aberrations. Why some cells take up the restriction enzyme and others not is not known, possibly this is dependent on the physiological condition of the cells.

The restriction endonuclease Bam H I produces DNA double-strand breaks (DSB) with sticky ends (recognition site: G/GATCC). In Chinese hamster V79 cells, permeabilized with inactivated Sendal virus, Bryant (1984, 1985) found that Barn H I is nearly inactive in inducing structural chromosomal aberrations; Pvu II (recognition site CAG/CTG), which produces blunt-ended DSB, induced high frequencies of chromosomal aberrations. Natarajan and Obe (1984), using virus-permeabilized Chinese hamster (CHO) cells, found aberrations with Barn H I, but the frequencies were much lower than the ones induced with Pvu II. These findings led to the conclusion that chromosomal aberrations are more effectively formed from DSB with blunt than from DSB with sticky ends. In further

experiments, Bryant (1985) showed that Pvu II, but not Barn H I, leads to cell killing in permeabilized V79 cells, and he argued that bluntended DSB, but not sticky-ended ones lead to a loss of cloning efficiency, and that cell killing produced by Pvu II is mediated by chromosomal aberrations. Using the DNA-unwinding method, Bryant (1984) found that Pvu II and Barn H I break the chromosomal DNA to the same extent; nevertheless, Barn H I induced only very few chromosomal aberrations. In this communication we show that Barn H I leads to high frequencies of chromosomal aberrations in CHO cells, indicating that in the experiments of Bryant (1984, 1985) and of Natarajan and Obe (1984) the conditions for the production of aberrations by this enzyme were not optimal.

All correspondence to G. Obe. 0165-7992/86/$ 03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

92

mixed by adding 6.3 #1 stock solution to 1.7 #1 NCS (lot No. 36; Expts. 2A, 7A, 8A), or 5 #1 stock solution to 3 #1 NCS (lot No. 40; Expts. 12A, 12B) in Eppendorf reaction vessels. The resulting 8/zl of fluid were added to the pellets. The cells were centrifuged for 5 min at 37°C (about 230 x g) and were incubated at 37°C for another 15 min. The cells were washed with prewarmed medium and 2 x 106 cells were incubated in 6-cm plastic petri dishes (Falcon) in medium with foetal calf serum (Seromed); 2 cultures were set up from each pellet. In Expts. 12A and 12B, one culture from each pellet was incubated with bromodeoxyuridine (BrdUrd; final concentration 2 x 10 -5 M; Serva, Heidelberg). The cells were fixed 22 h later, including a colcemid treatment of 2 h (final concentration 0.08 #g/ml). Controls were treated with 8 #l NCS or with a mixture of 6.3/A Barn H I ship-

M a t e r i a l s and m e t h o d s

CHO cells were treated in pellets of 4 x 106 cells as described (Obe and Winkel, 1985). Cells were grown in McCoy's 5A medium supplemented with 10070 (v/v) newborn calf serum (NCS) (both Seromed, Berlin), 100 units penicillin (Hoechst, Frankfurt) and 0.128 mg/ml dihydrostreptomycin sulphate (Heyl, Berlin) in 10-cm petri dishes (Falcon). The cells were trypsinized for 2-6 min with 0.05070 trypsin/0.02070 EDTA, Ca 2+- and Mg2+-free (Seromed, Berlin), resuspended in McCoy's medium without serum and counted. 4 × 106 cells from 1-, 2- or 3-day-old cultures were pelleted, resuspended in 1 ml NCS and pelleted again. As much of the supernatant as possible was discarded and 100 units Barn H I (Biolab, 2 different charges) were added. The 100 units were

TABLE 1 CHROMOSOMAL ABERRATIONS INDUCED BY THE RESTRICTION ENDONUCLEASE Barn H I IN CHO CELLS The cells were grown for 1, 2 or 3 days before they were treated with the enzyme. Fixation 22 h after the treatment. Dose of enzyme tested: 100 units/4 x 106 cells. In the controls pellets were treated with 8/A NCS. Experiments (time the cells were grown before treatment in in days)

Number of metaphases analysed

Controls: 2A; 7A; 8A (1, 2, 3)

700

Bam H I: 7A; 8A; (l)

200

Aberrant metaphases including AL (%)

6.4

33

Aberrations/100 metaphases AL

B'

3.4

1.1

4.5

2

B"

RB'

Triradials

CI

Polycentric chromosomes (number of centromeres)

Rings and minutes

1.1

0.1

0

0

0.6(2)

0.1

140.5

0.5

i

0

29.5 (2)

48.5

3.5 (3) 3

(4)

0.5 (5) 0.5 (6) Barn H I: 2A; 7A; 8A (2)

400

44.5

4

3.5

143.8

6.5

1.3

0.8

45 (2) 5.8 (3) 1.8 (4)

60

Bam H I: 7A; 8A (3)

200

56.5

3

4

439

0.5

9.5

0.5

92.5 (2) 26.5 (3) 5 (4)

117

1 1

(5) (6)

93 TABLE 2 POLYCENTRIC CHROMOSOMES INDUCED BY THE RESTRICTION ENZYME Barn H 1 IN CHO CELLS After the treatments the cells were incubated in BrdUrd, and first (MI) and second (M2) post-treatment metaphases were analysed for aberrations in differentially stained preparations. Fixation 22 h after the treatments. Dose of enzyme: 100 units (5/tl stock solution plus 3 ~1 NCS). The buffer controls were treated with a mixture of 6.3/~1 buffer and 1.7 t~l NCS. The results of 2 Expts. (12A, 12B) are given. Treatment Days First post-treatcells were ment metagrown phases a (%) before treatment

Number of metaphases analysed for aberrations (% aberrant metaphases, including AL)

Polycentric chromosomes/100 metaphases (number of centromeres)

M1

Ml

12A

M2

12B

12A

12B

12A

12B

1

11

10

Bam H 1 1

12

4

Buffer

2

37

32

100(18)

50 (18)

50 (12)

100(5)

Barn H 1 2

49

21

100 (45)

100 (47)

50 (10)

100 (9)

Buffer

3

40

17

65 (30.7) 50 (22)

50 (12)

70 (10)

Bam H 1 3

47

20

110 (78.2) 50 (46)

50 (10)

76 (9.2)

Serum

l0

19

Buffer

3

50 (26)

50 (18)

73 (54.8) 72 (61)

50 (12)

50 (14)

50 (8)

100 (lO)

146(9.6)

126 (8.7)

50 (2)

100(4)

M2

12A

12B

12A

12B

0

5 (2)

2.7 (2)

2.4 (2)

0

2 (2)

4 (2)

5 (2)

0

1.4 (2)

(2) 64 (2) (3) 4 (3) (4) 2 (4) (5)

4 (2)

5.3 (2)

4 (2)

2 (2)

3 (2)

4 (2)

6 (2)

67.1 (2) 88.9 (2) 9.6 (3) 5.6 (3) 1.4 (4) 5 (2) 65 10 2 2

(2) (3) (4) (5)

7.7 (2) 115.5 22.7 4.5 0.9

2 (2)

2 (3) 79 (2) 15 (3) l (4) 1 (5) 0

a 100 metaphases analysed per entry.

ping buffer (50 mM KC1; l0 mM Tris-HCl (pH 7.4); 0.1 mM EDTA; 1 mM dithiothreitol; 200 /zg/ml bovine serum albumin; 50070 (v/v) glycerol) and 1.7 #l NCS. Metaphases were prepared using a routine protocol and were stained with Giemsa stain, or differentially following the method of Hill and Wolff (1982). In most of the cases equal numbers of metaphases were scored from the parallel cultures for chromosomal aberrations as follows: achromatic lesions (AL); chromatid breaks (B'); isochromatid/chromosome breaks (B"); chromatid interchanges (RB'); chromatid intrachanges (CI); triradials; polycentric chromosomes; ring chromsomes and minutes. Fragments which may belong to polycentrics or

centric rings were included in the B " . In the differentially stained preparations, aberrations were scored in first (M 1, uniformly stained) and second (M2, differentially stained) post-treatment rectaphases, and in the latter the frequencies of sisterchromatid exchanges (SCEs) were determined.

Results and discussion The results are compiled in Tables 1-4. Table 1 includes the data from Expts. 2A, 7A and 8A; the results of these experiments were similar and the data were pooled. Treatment with Barn H I leads to high frequencies of aberrations mainly of the

94 chromosome type. This result indicates that Bam H I, like Alu I (Obe and Winkel, 1985) produces chromosomal aberrations independent from the S phase o f the cell cycle. There are differences in the aberration frequencies when cells grown for 1, 2, or 3 days were treated. The longer the cells were cultured before treatment with Barn H I the more aberrations that were found. Table 2 shows the results obtained when the aberrations were analysed in M1 and M2 cells. In Table 2 we present the frequencies of aberrant metaphases and the polycentric chromosomes found in 100 metaphases; the aberration spectrum was principally the same as that shown in Table 1. The abberations occur nearly exclusively in M1 cells. With only one exception the aberration frequencies are similar irrespective o f the time the cells were cultured before the treatment with the enzyme. The frequencies of aberrant cells in the buffer-treated cells are between 18 and 30.7070 (Table 2). Most of the damaged cells have only few aberrations and heavily damaged cells were found only rarely. This contrasts with the enzyme-treated cells in which the aberration frequencies are higher, and generally there are m a n y more aberrations per damaged cell than in the buffer-treated cells. Polycentric chromosomes are relatively rare in buffer-treated cells, but are frequent in the Bam H I-treated cells. Table 3 shows the frequencies of polycentric chromosomes found in Expts. 12A and 12B in uniformly stained metaphases (without BrdUrd). The frequencies are lower when compared to the ones found in M1 from the same experiments (see Table 2). Using the data from Table 2, we calculated how m a n y polycentrics are expected in uniformly stained preparations, at least in some cases the agreement of the respective values is very good (Table 3). It can he speculated that only a certain amount o f cells take up the enzyme and that these cells are in M1 after a recovery time o f 22 h. Why only some cells seem to take up the enzyme is not known. It could be that the number of cells (4 x 106) is too high for the a m o u n t o f enzyme solution added (8 #1). Another possibility is that it is dependent on the physiological status of the cells

TABLE 3 POLYCENTR1C CHROMOSOMES INDUCED BY THE RESTRICTION ENDONUCLEASE Bam H I IN CHO CELLS The data are from Expts. 12A and 12B (seeTable 2) but the cells were incubated in the absence of BrdUrd. The frequencies of polycentric chromosomes found in uniformly stained preparations are compared with the frequencies expected from the frequencies of M 1 and M2 and from the polycentricchromosomes found in MI and M2 as given in Table 2. Only the data for the Barn H I-treated cells are given. Dose of enzyme: 100 units. Experi- Days cells Polycentric chromosomes/100 ment weregrown metaphases (number of centromeres) before treatment From uniformly Calculated from the stained metapha- frequencies of MI ses and the frequencies of polycentric chromosomes in M1 and M2 as given in Table 2 4 (2)

12A

10.4 (2)

1.2 (3) 12B

10 (2)

5.9 (2) 0.2 (3) 0.I (4)

12A

35 (2) 3 (3)

33.9 (2) 4.1 (3) 0.8 (4) 0.8 (5)

12B

21 (2) 2 (3)

20.5 (2) 3.2 (3) 0.2 (4) 0.2 (5)

49 (2) 13 (3) 2 (4)

48.6 (2) 9.1 (3) 1.8 (4) 0.4 (5)

12A

3

12B

3

5.5 (2)

17.0 (2) 0.8 (3) 0.4 (4)

whether the enzyme can enter the cell or not. In Barn H I-treated cells we always found a few metaphases with very complex chromatid interchanges, which are not included in Table 1. In differentiaily stained preparations the chromosomes in such metaphases showed multiple-handing pat-

95 TABLE 4 SISTER-CHROMATED EXCHANGES (SCEs) IN CHO CELLS TREATED WITH Barn H I, BUFFER, OR SERUM (Expts. 12A, 12B, see Table 2) The results from different experiments were nearly identical and the data were pooled. S.E.M., standard error of the mean from 6 cultures each (3 cultures each from Expts. 12A and 12B, set up with cells cultured for 1, 2 or 3 days before treatment. Treatment

Number of M2 cells analysed

SCEs/metaphases ( ± S.E.M.)

Bam H I

548

8.85 (0.13)

Buffer

420

8.88 (0.44)

Serum

400

7.40 (0.31)

rations in a mixture of M1 and M2 metaphases from cells cultured in the absence of BrdUrd leads to an underestimation of the chromosomebreaking activity of Barn H I.

Acknowledgements A fellowship by the Anna Villa Rusconi Foundation to one of us (B.G.) is gratefully acknowledged. This work was financially supported by the Deutsche Forschungsgemeinschaft (Ob 49/2-1).

References terns, probably these cells are in M1, exposed to Barn H I in the early S phase. The frequencies of SCEs found in M2 of Expts. 12A and 12B were in no case elevated over the control levels obtained when pellets were treated with 8 #1 NCS (Table 4). Our data clearly show that Bam H I is able to produce chromosome-type aberrations in CHO cells treated in the G~ phase of the cell cycle. Data obtained from differentially stained metaphases indicate that only a few cells are able to take up the enzyme, these cells are in MI after a recovery time of 22 h and exhibit chromosomal aberrations. Cells which do not accept the enzyme are already in M2 when the cells which accepted the enzyme are still in M 1. The analysis of chromosomal aber-

Bryant, P.E. (1984) Enzymatic restriction of mammalian cell DNA using Pvu II and Barn H I: Evidence for the doublestrand break origin of chromosomal aberrations, Int. J. Radiat. Biol., 46, 57-65. Bryant, P.E. (1985) Enzymatic restriction of mammalian cell DNA: Evidence for double-strand breaks as potentially lethal lesions, Int. J. Radiat. Biol., 48, 55-60. Hill, A., and S. Wolff (1982) Increased induction of sister chromatid exchanges by diethylstilbestrol in lymphocytes from pregnant and premenopausal women, Cancer Res., 42, 893-896. Natarajan, A.T., and G. Obe (1984) Molecular mechanisms involved in the production of chromosomal aberrations, lIl. Restriction endonucleases, Chromosoma, 90, 120-127. Obe, G., and E.-U. Winkel (1985) The chromosome-breaking activity of the restriction endonuclease Alu I in CHO cells is independent of the S phase of the cell cycle, Mutation Res., 152, 25-29. Communicated by F.H. Sobels

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