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Induction of sister-chromatid exchanges by AluI, DNase I, benzon nuclease and bleomycin in Chinese hamster ovary (CHO) cells
0 .t/ Fundamental and Molecular Mechanisms of Mutagenesis
ELSEVIER
Mutation Research 307 (1994) 315-321
Induction of sister-chromatid exchanges by Alu I, DNase I, benzon nuclease and bleomycin in Chinese hamster ovary (CHO) cells G. Obe *, C. Schunck, C. Johannes University GH Essen, Department of Genetics, P.O. Box 45037, D-45117Essen, Germany (Received 28 October 1993; revision received 2 December 1993; accepted 2 December 1993)
Abstract
Various endonucleases (AluI, DNase I, benzon nuclease) and bleomycin induce sister-chromatid exchanges (SCE) in Chinese hamster ovary (CHO) cells. The frequencies of SCE are elevated in cells with chromosome-type aberrations, only slightly elevated in cells with chromatid exchanges, and in the control range in cells without chromosomal aberrations. These data indicate that SCE are produced when DNA breaks induced in G1 are either not repaired or misrepaired.
Key words: Sister-chromatid exchange; DNA break repair
1. Introduction
It has been shown that various restriction endonucleases (RE) producing DNA double-strand breaks (DSB) with blunt or overhanging ends induce sister-chromatid exchanges (SCE) in Chinese hamster ovary (CHO) cells in vitro (Natarajan et al., 1985; Stoilov et al., 1986; Darroudi and Natarajan, 1989; Folle et al., 1992; Balajee and Natarajan, 1993). The expression of RE-induced SCE needs a long recovery time of about 18 h, progressively shorter recovery times result in no or only slightly increased frequencies of SCE
* Corresponding author.
(Natarajan et aL, 1985; Morgan et al., 1989; Folle et al., 1992). Folle et al. (1992) proposed that apart from DSB, RE could induce single-strand breaks (SSB) in canonical structures formed by transiently occurring single-strand stretches in the chromosomal DNA or by cleavage of DNA-mRNA hybrids in G1 phase and that some of these SSB could give rise to SCE in the following S phase. In this communication we show that not only RE but also other endonucleases such as DNase I and benzon nuclease (Benzonase ®, Merck; BEN) as well as bleomycin (BLM) are able to induce high frequencies of SCE. Since these agents produce DNA strand breaks of different molecular types our results indicate that the types of breaks induced are not critical for the production of SCE.
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G. Obe et al. /Mutation Research 307 (1994) 315-321
The mechanisms of break production by RE, DNase I and BLM have been recently reviewed (Povirk and Austin, 1991; Obe et al., 1992). BEN is an extracellular endonuclease from Serratia marcescens which degrades single- and doublestranded D N A to di-, tri-, and tetranucleotides and a few mononucleotides (less than 2%) with 5'-phosphate ends (Eaves and Jeffries, 1963; Nestle and Roberts, 1969a,b; Ball et al., 1987; Biedermann et al., 1989; Biedermann and Nielsen, 1990). In C H O cells BEN induces chromosomal aberrations in a S phase-independent manner (Johannes and Obe, in preparation) similar to DNase I (Folle et al., 1991), R E (Obe et al., 1992) and BLM (Povirk and Austin, 1991). We report here that RE, DNase I, BEN and BLM induce SCE mainly in metaphases showing also chromosome-type aberrations, which indicates that directly induced D N A strand breaks of various types lead to SCE.
CHO-9 (from Dr. A.T. Natarajan, Leiden) and C H O - H P R T - (from Dr. J. Thomale, Essen) cells were grown in McCoy's 5A medium (Gibco) supplemented with 10% fetal calf serum (Gibco) and antibiotics (100 u n i t s / m l penicillin and 1 2 5 / x g / ml dihydrostreptomycin sulfate) in the presence of 5-bromodeoxyuridine (BrdUrd, final concentration 2 x 10 -5 M; Serva, Heidelberg) for 18 h. After incubation in BrdUrd, the cells were treated and recovered in the absence of BrdUrd, i.e., differentially stained metaphases represent first posttreatment ones.
(Johannes and Obe, 1991). In brief, cells were seeded into 94-mm Petri dishes (Greiner, Nfirtingen) containing 10 ml complete medium supplemented with BrdUrd. The cells were incubated for 18 h, trypsinized and washed once with medium. They were exposed to 100/xl of a mixture containing 10 units A l u I (Gibco-BRL, Eggenstein; 10 units//xl) or 20 units BEN (Merck, Darmstadt; 25 units//xl), 2.2 M glycerol (final concentration) and McCoy's 5A medium for 30 min at 37°C in a 5% CO 2 incubator. Controls were performed using the respective shipping buffer instead of enzyme in the treatment mixture or with 100 ~1 medium only. After treatment the cells were washed once with medium and recovered in 94-mm Petri dishes in 10 ml complete medium for 20 h including 2 h exposure to colcemid (Ciba; 0 . 0 8 / x g / m l ) . Electroporation. The treatments were principally done as described previously (Johannes and Obe, 1991). In brief, 94-mm Petri dishes in which the bottoms were fully covered with cells were trypsinized. Cells were washed once in sucrose buffer (phosphate-buffered sucrose: 272 mM sucrose, 7 mM KHzPO4, pH 7.4 and 1 mM MgC12) and suspended in 800 /xl sucrose buffer containing 2500 units DNase I (Boehringer, Mannheim), 20 units BEN (same as above) or 5 0 / x g / m l BLM (Mack, Illertissen). Controls were performed with 800/xl sucrose buffer or 100/~1 medium in 700/xl sucrose buffer. Cells were electroporated using the Gene Pulser from Bio-Rad, Munich with 400 kV and 25/zF. Following electroporation cuvettes containing the cells were kept at room temperature for 15 min. Cells were washed once in prewarmed medium and recovered in 94-mm Petri dishes in 10 ml complete medium for 20 h including 2 h exposure to colcemid (0.08 ~ g / m l ) .
2.2. Treatments
2.3. Preparations and scoring
Treatment using 2.2 M glycerol. Treatments were principally done as described previously
Preparations were made following a routine protocol and slides were stained differentially
2. Materials and methods 2.1. Cell culture
Fig. 1. Metaphases with SCE of CHO-9 (a, b, e, f) and HPRT- (c, d) cells followingtreatment with 2500 units DNase I (a, b, e), 10 units AluI (c), 20 units BEN (d) and 50 /~g/ml BLM (f). (a) Undamaged metaphase with a few SCE; (b) metaphase with chromatid-type exchanges; the frequency of SCE is slightly elevated; (c-f) metaphases with chromosome-typeaberrations and elevated SCE frequencies.
G. Obe et al. /Mutation Research 307 (1994) 315-3212e
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G. Obe et al. /Mutation Research 307 (1994) 315-321
318
(Hill and Wolff, 1982). Differentially stained metaphases were scored for SCE and for the presence of chromosome-type aberrations (polycentric chromosomes, rings, double minutes, fragments) or of chromatid-type exchanges (triradials, chromatid interchanges and intrachanges). Images of metaphases were taken using an IMACCCD camera (CompuLog, B6blingen) connected to a metaphase finder system (Meta Systems, Sandhausen). Brightness and contrast were adjusted using a photo finishing program (Corel Photopaint, Ottawa). The figures were printed with a Hewlett Packard (Frankfurt/Main) Deskjet 550C color printer.
3. Results and discussion
With all agents tested (AluI, DNase I, BEN, BLM) and with both treatment methods (glycerol, electroporation) the following results were obtained (Figs. 1 and 2, Table 1):
(1) The SCE frequencies of each cell line are similar in the different controls and therefore pooled data are given. CHO-HPRT- cells consistently have higher spontaneous frequencies of SCE than CHO-9 cells (Table 1). (2) In both cell types the frequencies of SCE following treatment with enzymes or BLM are considerably elevated when compared to controls. Treatment with DNase I seems to induce higher SCE frequencies in CHO-9 when compared to CHO-HPRT- cells (Table 1). In view of the fact that only one experiment was done with CHO-9 ceils we feel that the higher frequencies of SCE following treatment with DNase I rather reflect interexperimental variabilities and not a higher sensitivity of CHO-9 cells. (3) The elevated SCE frequencies were nearly exclusively found in cells with chromosome-type aberrations and in the few cells with chromosome- and chromatid-type aberrations. The intercellular distribution of SCE is characterized by a huge spread with some cells having more than 100 SCE (Figs. 1 and 2).
50
! 40
!
] !
• CHO-HPRT cells, Alul (GLY; 2 Expts.) • CHO-HPRT cells, BEN (GLY; 4 Expts.) CHO-HPRT
~30
tell Q.
cells, DNasel (EP; 2 Expts.)
[ ] CHO-9 cells, BLM (EP; 2 Expts.)
:E o
2o
O.
10
0-20
21-40
41-60
61410
81 -t O0
101-120
121-140
141 -t 60
161-180
Metaphases with different numbers of 8CE
Fig. 2. Intercellular distribution of SCE in cells with chromosome-type aberrations following treatment with 2500 units D N a s e I, 10 units AluI, 20 units B E N or 50 ~ g / m l BLM. Data from Table 1.
7.67_+0.59 (39/1) 14.55_+0.57 (65/2)
. .
63.28_+4.15(102) 39.64_+2.66(110)
60.36+3.13 (213) n.d.
53.23 5:6.03 (48) n.d.
73.875:6.60 (53) 50.405:3.11 (159)
31.655:4.50(37) 29.885:2.36 (123)
. .
Damaged cells
63.825:4.91 (73) 42.475:3.26 (85)
60.66_+3.41(190) n.d.
55.28 5:6.56 (43) n.d.
73.875:6.60 (53) 60.485:3.98 (103)
34.005:5.60(23) 34.895:3.40 (80)
. .
Cells with chromosome type aberrations . .
12.005:5.00 (2) 20.675:3.92 (6)
8.50-+ 2.50 (2) n.d.
9.00 (1) n.d.
15.36_+ 1.52 (25)
11.33:1: 0.80(6) 14.10+ 1.31 (20)
Cells with chromatid type aberrations
65.63± 8.05 (27) 32.955:3.55 (19)
62.57_+ 7.18 (21) n.d.
42.25 +_11.86 (4) n.d.
45.19+_ 6.00 (31)
40.135:11.93(8) 26.17-1- 2.32 (23)
Cells with both types of aberrations
a Solved in 800/~1 sucrose buffer. b The following controls are included: GLY (100/.d medium without additives, AluI shipping buffer in 2.2 M glycerol or BEN shipping buffer in 2.2 M glycerol); EP (800/~1 sucrose buffer or 100 #1 medium+700/zl sucrose buffer). c The following controls are included: GLY (100/zl medium without additives or BEN shipping buffer in 2.2 M glycerol); EP (800/.d sucrose buffer).
EP
DNaseI a
8.39 + 0.21 (228/2) n.d.
EP 7.58-+0.31 (90/2) n.d.
7.695:0.16 (333/2) 11.625:0.20 (441/4)
GLY
BEN
EP
8.235:0.53(30/1) 11.975:0.25 (211/2)
GLY
AluI
BLM a
7.21 5:0.14 b (394/8) 11.285:0.19 c (452/6)
-
Controls
Undamaged cells
Type of treatment
Treatment
Table 1 SCE induced in CHO cells following exposure to 10 units AluI, 2500 units DNaseI, 20 units benzon nuclease (BEN) or 50/~g/ml bleomycin (BLM) in the presence of 2.2 M glycerol (GLY) or by electroporation (EP). The cells were cultured in the presence of BrdUrd for 18 h, treated and recovered in the absence of BrdUrd for 20 h. SCE per metaphase_+ standard errors are given. In brackets the numbers of cells analyzed and the numbers of independent experiments are shown. First rows, CHO-9; second rows CHO-HPRT-. n.d. = not done
I
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320
G. Obe et al. /Mutation Research 307 (1994) 315-321
(4) Cells having exclusively chromatid-type aberrations have only slightly elevated SCE frequencies (Fig. 1, Table 1). (5) The SCE frequencies in undamaged cells are in the control range (Fig. 1, Table 1). Our data indicate that direct induction of breaks in the chromosomal DNA leads to SCE. The occurrence of SCE in metaphases containing chromosome-type aberrations indicates that the breaks giving rise to SCE were induced in the G1 phase of the cell cycle (Folle et al., 1992). Cells exposed in the S phase of the cell cycle which contain chromatid-type aberrations show only a slight elevation of SCE frequencies (Table 1; Figs. 1 and 2). The exposed but undamaged cells could represent cells in which induced breaks are fully repaired, or cells which did not take up the enzymes or BLM. AluI, DNase I, and BEN are proteins and cannot pass the cell membrane freely. Their uptake is mediated in this work by hypertonic concentrations of glycerol, or by electroporation (Johannes et al., 1992; Obe et al., 1992). BLM enters cells only to a limited extent, its uptake can be potentiated by electroporation (Weissenborn et al., 1994). Our data show that the molecular structures of strand breaks, namely, DSB with 5'-phosphate and 3'-OH blunt or overhanging ends (AluI, BEN, DNase I), SSB with 5'-phosphate and 3'-OH ends (BEN, DNase I), and DSB or SSB with modified ends (BLM), are not critical for the induction of SCE. Sparsely ionizing radiation such as X-rays belong to the S phase-independent chromosome-breaking agents but nevertheless they are poor inducers of SCE (Perry and Evans, 1975; Littlefield et al., 1979; Nagasawa and Little, 1979; Morgan and Crossen, 1980; Obe et al., 1982; Nagasawa et al., 1990). Probably the number of breaks induced by reasonable doses of X-rays is not high enough to effectively induce SCE. Our data do not support the assumption that X-ray-induced breaks as such cannot induce SCE. Treatment of cells with BLM without permeabilization only leads to a small elevation of SCE (Perry and Evans, 1975; Gebhart and Kappauf, 1978; Obe et al., 1982). As shown in this paper BLM applied to cells via electroporation is a very effective inducer of SCE.
The mechanism of how DNA breaks are transformed to SCE is not known. Our data indicate that breaks which are either not repaired or misrepaired in G1 could be vital for SCE formation. In this context it is interesting that densely ionizing radiations such as d(42MeV)-Be neutrons and plutonium-238 a-particles but not Xrays have been shown to induce SCE when unstimulated GO human lymphocytes are exposed (Aghamohammadi et al., 1988; Savage and Holloway, 1988). Though the induced frequencies of SCE are much lower than following treatment of CHO cells with RE, DNase I, BEN, or BLM, a similar mechanism of action may be operating in these cases. The studies of Nagasawa and coworkers (Nagasawa et al., 1990; Nagasawa and Little, 1992) show that very low doses of a-particles can induce SCE in G1 CHO cells. The doses are so low that most of these SCE cannot be the result of a particle transfer through the nucleus, rather they seem to be induced by indirect effects provoked by the irradiation (Nagasawa and Little, 1992). In conclusion, our data indicate that the induction of DNA strand breaks with different molecular structures in the G1 phase of the cell cycle may lead to SCE in the ensuing metaphase.
4. Acknowledgements This work was financially supported by the European Community (CI1-0435-D). We thank Dr. Frank M6rsberger, Merck, Darmstadt for kindly giving us Benzonase ~ and Mack, Illertissen for kindly giving us bleomycin.
5. References Aghamohammadi, S.Z., D.T. Goodhead and J.R.K. Savage (1988) Induction of sister chromatid exchanges (SCE) in GO lymphocytes by plutonium-238 a-particles, Int. J. Radiat. Biol., 53, 909-915. Balajee, A.S., and A.T. Natarajan (1993) Restriction endonucleases do induce sister-chromatid exchanges in Chinese hamster ovary cells, Mutation Res., 302, 25-31. Ball, T.K., P.N. Saurugger and M.J. Benedik (1987) The
G. Obe et al. / M u t a t i o n Research 307 (1994) 315-321
extracellular nuclease gene of Serratia marcescens and its secretion from Escherichia coli, Gene, 57, 183-192. Biedermann, K., and B.R. Nielsen (1990) Homogeneity analysis of a nuclease secreted by E. coli, BioPharm, June, 34-39. Biedermann, K., P.K. Jepsen, E. Riise and I. Svendsen (1989) Purification and characterization of a Serratia marcescens nuclease produced by Escherichia coli, Carlsberg Res. Commun., 54, 17-27. Darroudi, F., and A.T. Natarajan (1989) 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 Res., 212, 137-148. Eaves, G.N., and C.D. Jeffries (1963) Isolation and properties of an exocellular nuclease of Serratia marcescens, J. Bacteriol., 85, 273-278. Folle, G.A., C. Johannes and G. Obe (1991) Induction of chromosomal aberrations by DNase I, Int. J. Radiat. Biol., 59, 1371-1378. Folle, G.A., C. Johannes, B.H. Mechoso, I.I. Medina and G. Obe (1992) The restriction endonuclease AluI induces sister chromatid exchanges in Chinese hamster ovary cells, Mutagenesis, 7, 291-294. Gebhart, E., and H. Kappauf (1978) Bleomycin and sisterchromatid exchange in human lymphocyte chromosomes, Mutation Res., 58, 121-124. 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. Johannes, C., and G. Obe (1991) Induction of chromosomal aberrations with the restriction endonuclease AluI in Chinese hamster ovary cells: comparison of different treatment methods, Int. J. Radiat. Biol., 59, 1379-1393. Johannes, C., R. Boes and G. Obe (1992) Uptake of the restriction endonuclease A l u I by Chinese hamster ovary cells measured by frequencies of induced chromosomal aberrations: effect of hypertonic concentrations of glycerol and sorbitol, Mutagenesis, 7, 225-232. Littlefield, L.G., S.P. Colyer, E.E. Joiner and R.J. DuFrain (1979) Sister chromatid exchanges in human lymphocytes exposed to ionizing radiation during GO, Radiat. Res., 78, 514-521. Morgan, W.F., and P.E. Crossen (1980) X irradiation and sister chromatid exchange in cultured human lymphocytes, Environ. Mutagen., 2, 149-155. Morgan, W.F., H.W. Chung, J.W. Phillips and R.A. Winegar (1989) Restriction endonucleases do not induce sister-
321
chromatid exchanges in Chinese hamster ovary cells, Mutation Res., 226, 203-209. Nagasawa, H., and J.B. Little (1979) Effect of tumor promoters, protease inhibitors, and repair processes in X-ray induced sister chromatid exchanges in mouse cells, Proc. Natl. Acad. Sci. USA, 76, 1943,-1947. Nagasawa, H., and J.B. Little (1992) Induction of sister chromatid exchanges by extremely low doses of a-particles, Cancer Res., 52, 6394-6396. Nagasawa, H., J. Robertson and J.B. Little (1990) Induction of chromosomal aberrations and sister chromatid exchanges by alpha particles in density-inhibited cultures of mouse 10T½ and 3T3 cells, Int. J. Radiat. Biol., 57, 35-44. Natarajan, A.T., L.H.F. Mullenders, M. Meijers and M. Mukherjee (1985) Induction of sister-chromatid exchanges by restriction endonucleases, Mutation Res., 144, 33-39. Nestle, M., and W.K. Roberts (1969a) An extracellular nuclease from Serratia marcescens. I. Purification and some properties of the enzyme, J. Biol. Chem., 244, 5213-5218. Nestle, M., and W.K. Roberts (1969b) An extracellular nuclease from Serratia marcescens. II. Specificity of the enzyme, J. Biol. Chem., 244, 5219-5225. Obe, G., S. Kalweit, C. Nowak and F. Ali-Osman (1982) Liquid holding experiments with human peripheral lymphocytes. I. Effects of liquid holding on sister chromatid exchanges induced by trenimon, diepoxybutane, bleomycin and X-rays, Biol. Zent. BI., 101, 97-113. Obe, G., C. Johannes and D. Schulte-Frohlinde (1992) DNA double-strand breaks induced by sparsely ionizing radiation and endonucleases as critical lesions for cell death, chromosomal aberrations, mutations and oncogenic tranformation, Mutagenesis, 7, 3-12. Perry, P., and H.J. Evans (1975) Cytological detection of mutagen-carcinogen exposure by sister-chromatid exchange, Nature, 258, 121-125. Povirk, L.F., and M.J.F. Austin (1991) Genotoxicity of bleomycin, Mutation Res., 257, 127-243. Savage, J.R.K., and M. Holloway (1988) Induction of sisterchromatid exchanges by d(42 MeV)-Be neutrons in unstimulated human-blood lymphocytes, Br. J. Radiol., 61, 231-234. Stoilov, L., L.H.F. Mullenders and A.T. Natarajan (1986) Influence of bromodeoxyuridine substitution on sisterchromatid exchanges and chromosomal aberrations induced by restriction endonucleases, Mutation Res., 174, 295-301. Weissenborn, U., C. Johannes, C. Raesfeld and G. Obe (1994) Altered cytogenetic effects of bleomycin in mammalian cells by modified uptake conditions, Biol. Zent.bl., in press.
ELSEVIER
Mutation Research 307 (1994) 315-321
Induction of sister-chromatid exchanges by Alu I, DNase I, benzon nuclease and bleomycin in Chinese hamster ovary (CHO) cells G. Obe *, C. Schunck, C. Johannes University GH Essen, Department of Genetics, P.O. Box 45037, D-45117Essen, Germany (Received 28 October 1993; revision received 2 December 1993; accepted 2 December 1993)
Abstract
Various endonucleases (AluI, DNase I, benzon nuclease) and bleomycin induce sister-chromatid exchanges (SCE) in Chinese hamster ovary (CHO) cells. The frequencies of SCE are elevated in cells with chromosome-type aberrations, only slightly elevated in cells with chromatid exchanges, and in the control range in cells without chromosomal aberrations. These data indicate that SCE are produced when DNA breaks induced in G1 are either not repaired or misrepaired.
Key words: Sister-chromatid exchange; DNA break repair
1. Introduction
It has been shown that various restriction endonucleases (RE) producing DNA double-strand breaks (DSB) with blunt or overhanging ends induce sister-chromatid exchanges (SCE) in Chinese hamster ovary (CHO) cells in vitro (Natarajan et al., 1985; Stoilov et al., 1986; Darroudi and Natarajan, 1989; Folle et al., 1992; Balajee and Natarajan, 1993). The expression of RE-induced SCE needs a long recovery time of about 18 h, progressively shorter recovery times result in no or only slightly increased frequencies of SCE
* Corresponding author.
(Natarajan et aL, 1985; Morgan et al., 1989; Folle et al., 1992). Folle et al. (1992) proposed that apart from DSB, RE could induce single-strand breaks (SSB) in canonical structures formed by transiently occurring single-strand stretches in the chromosomal DNA or by cleavage of DNA-mRNA hybrids in G1 phase and that some of these SSB could give rise to SCE in the following S phase. In this communication we show that not only RE but also other endonucleases such as DNase I and benzon nuclease (Benzonase ®, Merck; BEN) as well as bleomycin (BLM) are able to induce high frequencies of SCE. Since these agents produce DNA strand breaks of different molecular types our results indicate that the types of breaks induced are not critical for the production of SCE.
0027-5107/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 2 7 - 5 1 0 7 ( 9 3 ) E 0 2 2 6 - G
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G. Obe et al. /Mutation Research 307 (1994) 315-321
The mechanisms of break production by RE, DNase I and BLM have been recently reviewed (Povirk and Austin, 1991; Obe et al., 1992). BEN is an extracellular endonuclease from Serratia marcescens which degrades single- and doublestranded D N A to di-, tri-, and tetranucleotides and a few mononucleotides (less than 2%) with 5'-phosphate ends (Eaves and Jeffries, 1963; Nestle and Roberts, 1969a,b; Ball et al., 1987; Biedermann et al., 1989; Biedermann and Nielsen, 1990). In C H O cells BEN induces chromosomal aberrations in a S phase-independent manner (Johannes and Obe, in preparation) similar to DNase I (Folle et al., 1991), R E (Obe et al., 1992) and BLM (Povirk and Austin, 1991). We report here that RE, DNase I, BEN and BLM induce SCE mainly in metaphases showing also chromosome-type aberrations, which indicates that directly induced D N A strand breaks of various types lead to SCE.
CHO-9 (from Dr. A.T. Natarajan, Leiden) and C H O - H P R T - (from Dr. J. Thomale, Essen) cells were grown in McCoy's 5A medium (Gibco) supplemented with 10% fetal calf serum (Gibco) and antibiotics (100 u n i t s / m l penicillin and 1 2 5 / x g / ml dihydrostreptomycin sulfate) in the presence of 5-bromodeoxyuridine (BrdUrd, final concentration 2 x 10 -5 M; Serva, Heidelberg) for 18 h. After incubation in BrdUrd, the cells were treated and recovered in the absence of BrdUrd, i.e., differentially stained metaphases represent first posttreatment ones.
(Johannes and Obe, 1991). In brief, cells were seeded into 94-mm Petri dishes (Greiner, Nfirtingen) containing 10 ml complete medium supplemented with BrdUrd. The cells were incubated for 18 h, trypsinized and washed once with medium. They were exposed to 100/xl of a mixture containing 10 units A l u I (Gibco-BRL, Eggenstein; 10 units//xl) or 20 units BEN (Merck, Darmstadt; 25 units//xl), 2.2 M glycerol (final concentration) and McCoy's 5A medium for 30 min at 37°C in a 5% CO 2 incubator. Controls were performed using the respective shipping buffer instead of enzyme in the treatment mixture or with 100 ~1 medium only. After treatment the cells were washed once with medium and recovered in 94-mm Petri dishes in 10 ml complete medium for 20 h including 2 h exposure to colcemid (Ciba; 0 . 0 8 / x g / m l ) . Electroporation. The treatments were principally done as described previously (Johannes and Obe, 1991). In brief, 94-mm Petri dishes in which the bottoms were fully covered with cells were trypsinized. Cells were washed once in sucrose buffer (phosphate-buffered sucrose: 272 mM sucrose, 7 mM KHzPO4, pH 7.4 and 1 mM MgC12) and suspended in 800 /xl sucrose buffer containing 2500 units DNase I (Boehringer, Mannheim), 20 units BEN (same as above) or 5 0 / x g / m l BLM (Mack, Illertissen). Controls were performed with 800/xl sucrose buffer or 100/~1 medium in 700/xl sucrose buffer. Cells were electroporated using the Gene Pulser from Bio-Rad, Munich with 400 kV and 25/zF. Following electroporation cuvettes containing the cells were kept at room temperature for 15 min. Cells were washed once in prewarmed medium and recovered in 94-mm Petri dishes in 10 ml complete medium for 20 h including 2 h exposure to colcemid (0.08 ~ g / m l ) .
2.2. Treatments
2.3. Preparations and scoring
Treatment using 2.2 M glycerol. Treatments were principally done as described previously
Preparations were made following a routine protocol and slides were stained differentially
2. Materials and methods 2.1. Cell culture
Fig. 1. Metaphases with SCE of CHO-9 (a, b, e, f) and HPRT- (c, d) cells followingtreatment with 2500 units DNase I (a, b, e), 10 units AluI (c), 20 units BEN (d) and 50 /~g/ml BLM (f). (a) Undamaged metaphase with a few SCE; (b) metaphase with chromatid-type exchanges; the frequency of SCE is slightly elevated; (c-f) metaphases with chromosome-typeaberrations and elevated SCE frequencies.
G. Obe et al. /Mutation Research 307 (1994) 315-3212e
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G. Obe et al. /Mutation Research 307 (1994) 315-321
318
(Hill and Wolff, 1982). Differentially stained metaphases were scored for SCE and for the presence of chromosome-type aberrations (polycentric chromosomes, rings, double minutes, fragments) or of chromatid-type exchanges (triradials, chromatid interchanges and intrachanges). Images of metaphases were taken using an IMACCCD camera (CompuLog, B6blingen) connected to a metaphase finder system (Meta Systems, Sandhausen). Brightness and contrast were adjusted using a photo finishing program (Corel Photopaint, Ottawa). The figures were printed with a Hewlett Packard (Frankfurt/Main) Deskjet 550C color printer.
3. Results and discussion
With all agents tested (AluI, DNase I, BEN, BLM) and with both treatment methods (glycerol, electroporation) the following results were obtained (Figs. 1 and 2, Table 1):
(1) The SCE frequencies of each cell line are similar in the different controls and therefore pooled data are given. CHO-HPRT- cells consistently have higher spontaneous frequencies of SCE than CHO-9 cells (Table 1). (2) In both cell types the frequencies of SCE following treatment with enzymes or BLM are considerably elevated when compared to controls. Treatment with DNase I seems to induce higher SCE frequencies in CHO-9 when compared to CHO-HPRT- cells (Table 1). In view of the fact that only one experiment was done with CHO-9 ceils we feel that the higher frequencies of SCE following treatment with DNase I rather reflect interexperimental variabilities and not a higher sensitivity of CHO-9 cells. (3) The elevated SCE frequencies were nearly exclusively found in cells with chromosome-type aberrations and in the few cells with chromosome- and chromatid-type aberrations. The intercellular distribution of SCE is characterized by a huge spread with some cells having more than 100 SCE (Figs. 1 and 2).
50
! 40
!
] !
• CHO-HPRT cells, Alul (GLY; 2 Expts.) • CHO-HPRT cells, BEN (GLY; 4 Expts.) CHO-HPRT
~30
tell Q.
cells, DNasel (EP; 2 Expts.)
[ ] CHO-9 cells, BLM (EP; 2 Expts.)
:E o
2o
O.
10
0-20
21-40
41-60
61410
81 -t O0
101-120
121-140
141 -t 60
161-180
Metaphases with different numbers of 8CE
Fig. 2. Intercellular distribution of SCE in cells with chromosome-type aberrations following treatment with 2500 units D N a s e I, 10 units AluI, 20 units B E N or 50 ~ g / m l BLM. Data from Table 1.
7.67_+0.59 (39/1) 14.55_+0.57 (65/2)
. .
63.28_+4.15(102) 39.64_+2.66(110)
60.36+3.13 (213) n.d.
53.23 5:6.03 (48) n.d.
73.875:6.60 (53) 50.405:3.11 (159)
31.655:4.50(37) 29.885:2.36 (123)
. .
Damaged cells
63.825:4.91 (73) 42.475:3.26 (85)
60.66_+3.41(190) n.d.
55.28 5:6.56 (43) n.d.
73.875:6.60 (53) 60.485:3.98 (103)
34.005:5.60(23) 34.895:3.40 (80)
. .
Cells with chromosome type aberrations . .
12.005:5.00 (2) 20.675:3.92 (6)
8.50-+ 2.50 (2) n.d.
9.00 (1) n.d.
15.36_+ 1.52 (25)
11.33:1: 0.80(6) 14.10+ 1.31 (20)
Cells with chromatid type aberrations
65.63± 8.05 (27) 32.955:3.55 (19)
62.57_+ 7.18 (21) n.d.
42.25 +_11.86 (4) n.d.
45.19+_ 6.00 (31)
40.135:11.93(8) 26.17-1- 2.32 (23)
Cells with both types of aberrations
a Solved in 800/~1 sucrose buffer. b The following controls are included: GLY (100/.d medium without additives, AluI shipping buffer in 2.2 M glycerol or BEN shipping buffer in 2.2 M glycerol); EP (800/~1 sucrose buffer or 100 #1 medium+700/zl sucrose buffer). c The following controls are included: GLY (100/zl medium without additives or BEN shipping buffer in 2.2 M glycerol); EP (800/.d sucrose buffer).
EP
DNaseI a
8.39 + 0.21 (228/2) n.d.
EP 7.58-+0.31 (90/2) n.d.
7.695:0.16 (333/2) 11.625:0.20 (441/4)
GLY
BEN
EP
8.235:0.53(30/1) 11.975:0.25 (211/2)
GLY
AluI
BLM a
7.21 5:0.14 b (394/8) 11.285:0.19 c (452/6)
-
Controls
Undamaged cells
Type of treatment
Treatment
Table 1 SCE induced in CHO cells following exposure to 10 units AluI, 2500 units DNaseI, 20 units benzon nuclease (BEN) or 50/~g/ml bleomycin (BLM) in the presence of 2.2 M glycerol (GLY) or by electroporation (EP). The cells were cultured in the presence of BrdUrd for 18 h, treated and recovered in the absence of BrdUrd for 20 h. SCE per metaphase_+ standard errors are given. In brackets the numbers of cells analyzed and the numbers of independent experiments are shown. First rows, CHO-9; second rows CHO-HPRT-. n.d. = not done
I
",.!
320
G. Obe et al. /Mutation Research 307 (1994) 315-321
(4) Cells having exclusively chromatid-type aberrations have only slightly elevated SCE frequencies (Fig. 1, Table 1). (5) The SCE frequencies in undamaged cells are in the control range (Fig. 1, Table 1). Our data indicate that direct induction of breaks in the chromosomal DNA leads to SCE. The occurrence of SCE in metaphases containing chromosome-type aberrations indicates that the breaks giving rise to SCE were induced in the G1 phase of the cell cycle (Folle et al., 1992). Cells exposed in the S phase of the cell cycle which contain chromatid-type aberrations show only a slight elevation of SCE frequencies (Table 1; Figs. 1 and 2). The exposed but undamaged cells could represent cells in which induced breaks are fully repaired, or cells which did not take up the enzymes or BLM. AluI, DNase I, and BEN are proteins and cannot pass the cell membrane freely. Their uptake is mediated in this work by hypertonic concentrations of glycerol, or by electroporation (Johannes et al., 1992; Obe et al., 1992). BLM enters cells only to a limited extent, its uptake can be potentiated by electroporation (Weissenborn et al., 1994). Our data show that the molecular structures of strand breaks, namely, DSB with 5'-phosphate and 3'-OH blunt or overhanging ends (AluI, BEN, DNase I), SSB with 5'-phosphate and 3'-OH ends (BEN, DNase I), and DSB or SSB with modified ends (BLM), are not critical for the induction of SCE. Sparsely ionizing radiation such as X-rays belong to the S phase-independent chromosome-breaking agents but nevertheless they are poor inducers of SCE (Perry and Evans, 1975; Littlefield et al., 1979; Nagasawa and Little, 1979; Morgan and Crossen, 1980; Obe et al., 1982; Nagasawa et al., 1990). Probably the number of breaks induced by reasonable doses of X-rays is not high enough to effectively induce SCE. Our data do not support the assumption that X-ray-induced breaks as such cannot induce SCE. Treatment of cells with BLM without permeabilization only leads to a small elevation of SCE (Perry and Evans, 1975; Gebhart and Kappauf, 1978; Obe et al., 1982). As shown in this paper BLM applied to cells via electroporation is a very effective inducer of SCE.
The mechanism of how DNA breaks are transformed to SCE is not known. Our data indicate that breaks which are either not repaired or misrepaired in G1 could be vital for SCE formation. In this context it is interesting that densely ionizing radiations such as d(42MeV)-Be neutrons and plutonium-238 a-particles but not Xrays have been shown to induce SCE when unstimulated GO human lymphocytes are exposed (Aghamohammadi et al., 1988; Savage and Holloway, 1988). Though the induced frequencies of SCE are much lower than following treatment of CHO cells with RE, DNase I, BEN, or BLM, a similar mechanism of action may be operating in these cases. The studies of Nagasawa and coworkers (Nagasawa et al., 1990; Nagasawa and Little, 1992) show that very low doses of a-particles can induce SCE in G1 CHO cells. The doses are so low that most of these SCE cannot be the result of a particle transfer through the nucleus, rather they seem to be induced by indirect effects provoked by the irradiation (Nagasawa and Little, 1992). In conclusion, our data indicate that the induction of DNA strand breaks with different molecular structures in the G1 phase of the cell cycle may lead to SCE in the ensuing metaphase.
4. Acknowledgements This work was financially supported by the European Community (CI1-0435-D). We thank Dr. Frank M6rsberger, Merck, Darmstadt for kindly giving us Benzonase ~ and Mack, Illertissen for kindly giving us bleomycin.
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