Mtltation Research, 288 (1993) 281-289 {3 1993 Elsevier Science Publishers B.V. All rights reserved 0027-5107/93/$06.00 281 MUT 05265 Cytogenetic effects of inhibition of topoisomerase i or H activities in the CHO mutant EM9 and its parental line AA8 Felipe Cort& a Joaqu~n Pifiero a and Fabrizio Palitti b a Department of Cell Biology, Faculty of Biology, E-41012 Seville, Spain and b Department of Agrobiology and Agrochemistry, University of Viterbo, Italy (Received 2 December 1992) (Revision received 17 February 1993) (Accepted 18 February 1993) Keywords: Topoisomerase inhibitors; CHO mutant; Sister-chromatid exchange; Aberrations Summary The possible relationship between topoisomerase activities and the occurrence of SCE and chromoso- mal aberrations was investigated in the CHO ethyl methanesulfonate-sensitive mutant EM9 and its parental line, AA8. The Topo II inhibitor m-AMSA induced fewer SCE in EM9 than in AAS, mainly when given during the first S period. When the Topo I inhibitor camptothecin was used, it showed a higher efficiency to induce both chromosomal aberrations and SCE in EM9 than in AA8. The impact of BrdU incorporated into parental DNA on topoisomerase activity was tested using nuclear extracts from both EM9 and AA8 assayed for their ability to decatenate kinetoplast DNA by Topo II and to relax supercoiled plasmid DNA by Topo I. Taken as a whole, the results seem to indicate that there are differences between the two cell lines, consistent with the hypothesis put forward by other investigators that topoisomerases are involved in the molecular mechanism of SCE. The CHO ethyl methanesulfonate (EMS)-sen- sitive mutant EM9 is also hypersensitive to killing by X~rays and UV-A radiation, and has an ex- tremely high baseline sister-chromatid exchange (SCE) frequency compared to its parental line, AA8 (Thompson et al., 1982; Dillehay et al., 1983; Churchill et al., 1991). It has been shown, Correspondence: Dr. F. Cort6s, Department of Cell Biology, Facul W of Biology, E-41012 Seville, Spain. at the molecular level, that EM9 is defective in the rate of rejoining DNA strand breaks after treatment with mutagens (Thompson et al., 1982; Churchill et al., 1991). Consistent with this, we have recently reported on the hypersensitivity of EM9 to EcoRI restriction endonuclease (Cort6s and Ortiz, 1991). However, both DNA ligase and apurinic/apyrimidinic endonuclease activities, as well as poly(ADP-ribose) metabolism appear nor- mal in EM9 (Chan et al., 1984; La Belle et al., 1984; Ikejima et al., 1984). Although the gene
Transcript
Mtltation Research, 288 (1993) 281-289 {3 1993 Elsevier Science Publishers B.V. All rights reserved 0027-5107/93/$06.00
281
MUT 05265
Cytogenetic effects of inhibition of topoisomerase i or H activities in the CHO mutant EM9 and its parental line AA8
Felipe Cort& a Joaqu~n Pifiero a and Fabrizio Palitti b a Department of Cell Biology, Faculty of Biology, E-41012 Seville, Spain and b Department of Agrobiology and Agrochemistry,
University of Viterbo, Italy
(Received 2 December 1992) (Revision received 17 February 1993)
(Accepted 18 February 1993)
Keywords: Topoisomerase inhibitors; CHO mutant; Sister-chromatid exchange; Aberrations
Summary
The possible relationship between topoisomerase activities and the occurrence of SCE and chromoso- mal aberrations was investigated in the CHO ethyl methanesulfonate-sensitive mutant EM9 and its parental line, AA8. The Topo II inhibitor m-AMSA induced fewer SCE in EM9 than in AAS, mainly when given during the first S period. When the Topo I inhibitor camptothecin was used, it showed a higher efficiency to induce both chromosomal aberrations and SCE in EM9 than in AA8. The impact of BrdU incorporated into parental DNA on topoisomerase activity was tested using nuclear extracts from both EM9 and AA8 assayed for their ability to decatenate kinetoplast DNA by Topo II and to relax supercoiled plasmid DNA by Topo I. Taken as a whole, the results seem to indicate that there are differences between the two cell lines, consistent with the hypothesis put forward by other investigators that topoisomerases are involved in the molecular mechanism of SCE.
The CHO ethyl methanesulfonate (EMS)-sen- sitive mutant EM9 is also hypersensitive to killing by X~rays and UV-A radiation, and has an ex- tremely high baseline sister-chromatid exchange (SCE) frequency compared to its parental line, AA8 (Thompson et al., 1982; Dillehay et al., 1983; Churchill et al., 1991). It has been shown,
Correspondence: Dr. F. Cort6s, Department of Cell Biology, Facul W of Biology, E-41012 Seville, Spain.
at the molecular level, that EM9 is defective in the rate of rejoining DNA strand breaks after treatment with mutagens (Thompson et al., 1982; Churchill et al., 1991). Consistent with this, we have recently reported on the hypersensitivity of EM9 to EcoRI restriction endonuclease (Cort6s and Ortiz, 1991). However, both DNA ligase and apurinic/apyrimidinic endonuclease activities, as well as poly(ADP-ribose) metabolism appear nor- mal in EM9 (Chan et al., 1984; La Belle et al., 1984; Ikejima et al., 1984). Although the gene
282
responsible for the EM9 repair defect has been cloned recently (Thompson et al., 1990), the func- tion of the gene product is not known.
The high rate of SCE observed in EM9 cells after 5-bromodeoxyuridine (BrdU) substitution of DNA is also characteristic of the autosomal re- cessive human disorder Bloom's syndrome (BS) (Chaganti et al., 1974) and so, the role of BrdU in both cell types deserves special attention for its possible implications in explaining the still un- known molecular process leading to SCE.
DNA topoisomerases are enzymes which cat- alyze the topological changes of DNA during many cellular processes such as replication and transcription by concerted breaking and rejoining of DNA strands. Topoisomerase I (Topo I) breaks and rejoins one DNA strand at a time, while topoisomerase 1I (Topo II) is able to do so with the two strands that make up duplex DNA (Wang, 1985). The unique unknotting activity of DNA Topo II is essential for segregating replicated daughter chromosomes.
SCE models involving topoisomerases have been proposed (Cleaver, 1981; Pommier et al., 1985; Dillehay et al., 1989), and topoisomerase inhibitors have been shown to induce SCEs and chromosomal aberrations efficiently (Liu, 1989). These inhibitors, some of which are potent anti- tumor drugs, stabilize a reaction intermediate, the so-called cleavable complex in such a way that the enzyme is covalently bound to DNA (Liu, 1989; Zhang et al., 1990; Smith, 1990).
The intercalating Topo 1I inhibitor amsacrine (m-AMSA) and the Topo I inhibitor camp- tothecin (CPT) were analyzed for their efficiency at inducing both SCEs and chromosomal aberra- tions in EM9 and the parental line AA8. To overcome the problem of the very high baseline SCE frequency after BrdU substitution in EM9, we have made use of an anti-BrdU monoclonal antibody combined with an immunoperoxidase method to detect very low levels of BrdU in chromosomes with a reduced yield of SCEs (Pinkel et al., 1985; Shiraishi and Ohtsuki, 1987). This methodology has allowed us to detect any increase in the frequency of SCEs induced by m-AMSA and CPT over baseline SCEs.
In order to test whether growing the cells in the presence of BrdU has any influence on Topo
II activity, as reported earlier for Bloom's syn- drome fibroblasts (Heartlein et al., 1987), nuclear extracts from both EM9 and AA8 were assayed for their abilit3~ to decatenate kinetoplast DNA (kDNA)o The efficiency in relaxation of SUper. coiled plasmid DNA by Topo I was analyzed as well.
dine (FdU), deoxythymidine (dT), deoxycytidine (dC) and camptothecin (CPT) were purchased from Sigma Chemical Co., St. Louis, MO (USA). 4'-(9-Acridinylamino)methanesulfon-m-anisidide (m-AMSA) (NSC-249992) was obtained from the Drug Synthesis & Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD (USA).
Cells The parental line AA8 and mutant EM9 were
obtained as a generous gift from Dr. L.H. Thompson (University of California, Lawrence Livermore National Laboratory, USA). The cells were maintained as monolayer cultures in Mc- Coy's 5A medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and the antibi- otics penicillin (50 units/ml) and streptomycin (50 /xg/ml). Cells were cultured at 37°C in 5% CO z in air.
Treatment of cells For chromosome studies, cells were grown in
the dark for a first cell cycle (13 h for AA8 and 16 h for EM9) in the presence of 0.25 /,M BrdU, 4.75 /zM dT, 100 /xM dC, and 1 /xM FdU, to suppress endogenous dT synthesis (Pinkel et al., 1985) and, after thorough washing, grown for an additional round of replication in medium corn taining 100 /,M dT. Treatment with m-AMSA and CPT at different concentrations took place for 30 rain during either the first or the second S period. Colcemid (0.2 /.cM final concentration) was present for the last 3 h. Cells were collected by mitotic shake off and then treated with hypo o tonic 0.075 M KC1 (2-3 rain) and finally fixed in
two washes with methanol-acetic acid (3 : 1). Stan- dard cytological preparations were then made.
Immunological detection of SCEs (immunoperoxi- dase reaction)
After harvesting and fixing the cells as deo scribed above, a monoclonal antibody against BrdU-containing DNA was used. The procedure was as follows: slides were preincubated in saline sodium citrate (SSC, 5 × ) at 65°C for 90 rain before the denaturation step of 40 min incubation at room temperature in 2 N HC1 (Natarajan et al., !986)~ The slides were then washed with PBS for 2 min and with distilled water for 1 rain, followed by incubation with anti-BrdU mono- clonal antibody (BioCell, Switzerland, diluted 1 : 100) in PBS with 0.25% bovine serum albumin (BSA) and 2% Tween-20 for 60 min at room temperature in the dark. The slides were washed three times in PBS-BSA for 3 rain with continu- ous shaking and then incubated with rabbit anti- mouse HRP (Amersham Int. Plc., diluted 1:100) in PBS-BSA-Tween-20 for 30 min at room tem- perature in the dark. Subsequently, the slides were washed twice in PBS with shaking and once in 50 mM Tris-HC1 buffer (TB) pH 7.4. Develop- ing with 3,3'-diaminobenzidine • 4HC1 (DAB) re- action was carried out by immersing the slides in 0.02% DAB, containing 100 /xl of 5% H 2 0 2,
283
diluted with TB pH 7.4 for 10-20 min in com- plete darkness. Washing briefly in TB and tap water was the final step before air-drying at room temperature and mounting in DPX.
Assay for topoisomerases Cells were grown in the dark for one cell cycle
either in the absence or in the presence of BrdU and dT at different concentrations, making up a total of 5 # M in medium with t /aM FdU to ensure BrdU substitution levels in DNA of 5, 20, and 100%, and allowed to grow for another cell cycle in 100/xM dT.
The procedure followed to obtain nuclear ex- tracts to test topoisomerase activity was basically that described by Heartlein et al. (1987). Approx. 20 × 106 cells were suspended in 1 ml of 0.32 M sucrose, 0.01 M Tris-HC1 pH 7.5, 0.05 M MgC12, 1% Triton X-100 and thoroughly vortexed to lyse the cells. Nuclear pellets were obtained by cen- trifugation at 2000 rpm (Eppendorf centrifuge), for 5 min at 4°C. Nuclei were then washed in 1 ml of nucleus wash buffer (5 mM potassium phos- phate buffer, pH 7.0, 1 mM phenylmethylsulfonyl fluoride (PMSF), l mM /3-mercaptoethanol, and 0.5 mM dithiothreitol (DTT). The nuclei were pelleted as described above and resuspended in 50/xl of nucleus wash buffer and 5/xl of 40 mM EDTA was added.
d %
a , d b
Fig. 1. EM9 metaphases showing differentiation of sister chromatids and their characteristic high yield of SeEs. (a) Giemsa-stained cell grown in 5/xM BrdU. Note the presence of chromosomal aberrations. (b) EM9 metaphase grown in 0.25 p,M BrdU (anti-BrdU monoclonal antibody) showing a much reduced yield of SeE. This reduction in BrdU-induced SeEs is necessary to detect any
effect of topoisomerase inhibitors in this cell line.
284
Fol lowing incuba t ion at 0°C for 15 rain, the nuclei were lysed by adding 55 /xl of 2 M NaCI, 20 m M T r i s - H C 1 p H 7.5, 10 m M /3- m e r c a p t o e t h a n o l , 1 m M PMSF. Fo l lowing a 15- min incuba t ion at O°C, 50 /x l of 18% po lye thy lene glycol (PEG-6000) in 1 M NaC1, 50 m M Tris-HC1, p H 7.5, 10 m M ]3-mercaptoe thanol , and 1 m M P M S F was added . The suspens ion was i ncuba t ed for a fu r the r 40 rain at 0°C. The s u p e r n a t a n t f rom a 30-min cen t r i fuga t ion at 12,500 r p m at 4°C was then col lected. The total p ro te in concen t r a t i on in each extract (Bradford , 1976) was then de te r - m i n e d in a Beckman DU-64 s p e c t r o p h o t o m e t e r using bovine se rum a lbumin (BSA) as a s t andard .
T o p o I I and T o p o I activit ies in nuc lea r exo t rac ts were assayed using T o p o G e n (Columbus , O H , U S A ) assay kits based upon d e c a t e n a t i o n of k ine top las t D N A ( k D N A ) (Mul le r et al., 1988) and re laxa t ion of superco i led p la smid D N A , re- spectively. R e a c t i o n p roduc t s were resolved using agarose gel e l ec t rophores i s of D N A . A f t e r diges- t ion (40 min at 37°C for Topo II; 30 min for T o p o I) the samples were l oaded onto 1% agarose gels and sub jec ted to e lec t rophores i s for e i the r 2.5 h
at 100 V (Topo II assay) or 3.5 h at 50 V Cl'Opo D. Final ly, gels were s t a ined with 0.5 g g / m l e th id ium bromide , des t a ined (30 min) in distilled wate r and p h o t o g r a p h e d using a s t anda rd photo, dyne set up.
Results
SCEs and chromosomal aberrations induced by m-AMSA and camptothecin in EM9 and AA8 cells
In the absence of d rug t r e a tmen t , EM9 cells showed the charac te r i s t i c high level of SCEs (Fig. 1 and Table 1) as c o m p a r e d to its pa ren ta l line AA8. In a g r e e m e n t with tha t r e p o r t e d by Pinkel et al. (1985), the yield of SCEs in EM9 was dras t ica l ly r e d u c e d when the cells were grown in a concen t ra t ion of B r d U as low as 0.25 ~ M , only cytological ly de t ec t ab l e by the immunological m e t h o d (Fig. 1), bu t still the f requency of SCEs was about 5 - 6 - f o l d h igher than tha t observed in A A 8 (Table 1, cont ro l values) ,
Tab le 1 shows the ef fec t iveness of a single 30-min pulse with d i f fe ren t concen t r a t ions of the t opo i somera se inhib i tors m - A M S A and C P T given
TABLE 1
EFFECT OF A SINGLE 30-min PULSE WITH DIFFERENT CONCENTRATIONS OF m-AMSA AND CPT GIVEN DURING EITHER THE FIRST OR THE SECOND S PERIOD ON THE YIELD OF SCE AND CHROMOSOMAL ABERRATIONS IN EM9 AND AA8
EM9 AA8
S1 $2 $1 $2
SCE/Met % Damaged SCE/Met % Damaged SCE/Met % Damaged SCE/Met % Damaged +_ SE metaphses +_ SE metaphases +_ SE metaphases ± SE metaphases
50 or 100 metaphases were scored for SCE scored; b 24 metaphases scored; (MA), total control (p < 0.001 or p < 0.05 respectively),
and chromosomal aberrations, respectively, unless stated otherwise; a 34 metaphases number of scored cells showing multiple aberrations. *'** Statistically different from Student's t test. Controls were always sham-treated cultures.
during either the first or the second S period to induce SCEs and chromosomal aberrations in EM9 and AA8. As can be seen, both inhibitors were able to induce SCEs and chromosome dam= age in the mutant and the parental cell line but, apparently, the Topo II inhibitor m-AMSA was inefficient at inducing SCEs above control values in EM9 when given during the first replication period (S1) in spite of its strong clastogenic ef- fect. This absence of SCE-inducing effect of m- AMSA during $1 in EM9 contrasted with the observed efficiency of the Topo I inhibitor CPT (Table 1). On the other hand, in agreement with our previous results on the effectiveness of CPT in G2 to induce chromosome damage (Palitti et al., 1993), S-treated EM9 cells appeared more sensitive than AA8.
In order to test the hypothesis that the lack of an effect upon SCEs in EM9 during the first S could be due to a failure to treat the cells at their sensitive period (Dillehay et al., 1987), cells were given 30-rain pulses with 2 /xM m-AMSA throughout either their first round of replication (in the presence of BrdU) or their second S period (in dT). As can be seen in Table 2, EM9 cells exposed to m-AMSA at 2, 4, 6 or 8 h after the addition of dT (second cell cycle) showed a drug-induced increase in the yield of SCEs over their high baseline level though always lower than that observed in AA8 treated with the Topo II inhibitor (Table 1). However, in agreement with our previous results, no significant increase over control SCE frequency was observed when EM9 cells were pulse-treated with 2 /xM m-AMSA at
£,gN,~
B.DNA
a b c d e f g h
285
AA8
L. DNA
a b e d e f g h
.D,A E M 9
O,ONA L.DNA
Fig. 2. kDNA decatenat ing activities (Topo II activity) of nuclear extracts from AA8 and EM9. Cells were grown in the absence or presence of BrdU and nuclear extracts were pre- pared as described under Materials and methods. The assays were carried out for 40 rain at 37°C. Lane a, 0.4 /xg of catenated kDNA (C.DNA); lanes b - e , 100 ng of nuclear protein plus 0.2 /xg of C .kDNA (b, no BrdU; c, 0.25 /xM BrdU; d, 1 p,M BrdU; e, 5 /zM BrdU); lanes f -h , D N A controls (f, decatenated kDNA (D.DNA); g, linear kDNA
(L.DNA); h, HindlI2 cut phage lambda DNA).
different times during the first round of replica- tion, in the presence of BrdU. Contrasting with the apparent lack of effectiveness to induce SCEs,
TABLE 2
EFFECT OF A SINGLE 30-min PULSE W I T H 2 /xM m - A M S A GIVEN AT D I F F E R E N T TIMES D U R I N G E I T H E R THE FIRST OR T H E SECOND S P E R I O D ON T HE YIELD OF SCE AND C H R O M O S O M A L A B E R R A T I O N S IN EM9
$1 $2
S C E / M e t + SE % DM M A CA S C E / M e t +_ SE % D M MA CA
50 or 100 metaphases were scored for showing multiple aberrations; CA, total p < 0.05 respectively), Student 's t test.
SCE and chromosomal aberrations, respectively. DM, damaged metaphases; MA, cells scorable chromosomal aberrations. * ' ** Statistically different from control (p < 0.002 or
286
chromosome damage was readily induced under these latter experimental conditions (Table 2).
Topoisornerase H and I actiuities in A A 8 and EM9 Measurements of Topo II activity were made
using nuclear extracts from AA8 and EM9 after growing the cells in the absence of BrdU, or for one cell cycle in the presence of 0.25, 1, or 5/xM BrdU (5, 20 and 100% BrdU substitution in DNA, respectively) followed by another cell cycle in 100 /xM dT. The assay carried out is based upon decatenation of kinetoplast DNA (kDNA) (Muller et al., 1988), and it is specific for type II activity (not type i). Reaction of Topo II with the cate- hated networks of kDNA results in the release of double-stranded DNA minicircles.
As shown in Fig. 2, nuclear extracts from both cell lines showed a clear decatenating activity except when cells had been grown in 0.25 /xM BrdU (5% BrdU substitution in DNA), which showed a very reduced activity.
Topo I activity was measured in the same nuclear extracts tested for Topo II. Topo I, in the absence of ATP, is able to catalyze the relaxation of supercoiled plasmid DNA. As can be seen in Fig. 3, EM9 extracts showed an apparently higher efficiency than AA8 extracts for the relaxation reaction. Growing AA8 cells in the presence of BrdU, however, resulted in an increased relax- ation efficiency as compared to cells grown in the absence of BrdU.
Discuss ion
An argument against the importance of topo- isomerases for the EM9 mutant phenotype is the assignment of the human repair gene which cor- rects the high SCE frequency in EM9 to chromo- some 19 (Siciliano et al., 1986), while human DNA topoisomerases I and II are encoded by single-copy genes located on chromosome 20 and 17, respectively (Liu, 1989). Nevertheless, the site of the primary defect in EM9 could well not be directly responsible for all the phenotypic alter- ations observed in this CHO mutant.
The major aim of the present study was to investigate the possible involvement of DNA topoisomerases in the extraordinarily high fre- quency of SCE and increased yield of chromoso-
f s
a b c d e f
AA8
a b c d e f
EM9
Fig. 3. Relaxation of supercoiled pRYG DNA by nuclear extracts from AA8 and EM9 (Topo I assays). The assays were carried out for 30 rain at 37°C, as described in Materials and methods. AAS: lane a, supercoiled pRYG DNA, 0.5/xg; lanes b-e, 100 ng of nuclear protein plus 0.25 /xg of supercoiled pRYG DNA (b, no BrdU; c, 0.25/xM BrdU; d, 1 /xM BrdU; e, 5 /xM BrdU); lane f, HindIII cut phage lambda DNA. EM9: lanes a-d, incubations with nuclear extracts (a, no BrdU; b, 0.25/zM BrdU; c, 1/xM BrdU; d, 5/xM BrdU); lane e, supercoiled pRYG DNA, 0.25 /xg; lane f, HindIII cut
phage lambda DNA.
real aberrations observed in the CHO mutant strain EM9 after BrdU substitution of DNA (Thompson et al., 1982). DNA topoisomerases are good candidates to play a role in the still unknown molecular mechanism of SCE for sev- eral reasons. (a) Stimulation of SCE has been observed with all of the antitumor topoisomerase inhibitors tested so far (Pommier et al., 1988; Degrassi et al., 1989). (b) E. coli DNA gyrase (Ikeda et al., 1982) and bacteriophage T4 DNA topoisomerase (Ikeda, 1986), both type II en- zymes, can promote illegitimate recombination in vitro, and a possible role for the eukaryotic Topo I has also been proposed (Champoux and Bul-
lock, 1988); (c) Incision(s) in the DNA backbone, such as those produced by topoisomerases or endonucleases, must be a prerequisite for SCE (Dillehay et al., 1989). It is worth mentioning, however, that apurinic/apyrimidinic endonucle- ase activities appear normal in EM9 (La Belle et al., 1984). And (d) Exchange of duplex DNA could be carried out directly through exchange of Topo II subunits covalently bound to DNA, as proposed by Pommier et al. (1985).
Apart from Pommier's model, the normal functioning or misfunctioning of DNA topoiso- merases has been proposed in several models of SCE formation (for review, see Dillehay et al., 1989). Painter (1980) postulated that SCE were formed at the junction of a replicated replicon cluster and an adjacent one which had not yet completed replication, and that Topo II was in- volved. In their replication detour model, Ishii and Bender (1980) discussed the possible involve- ment of a Topo II activity in mediating the SCE at the replication forks, while Cleaver (1981) pro- posed that SCE occur within replicons between the diverging replication forks and result from errors in the unraveling of the intertwined daugh- ter duplexes by Topo II.
Heartlein et al. (1987) reported a reduction in Topo II extractable activity associated with the BrdUodependent increase in SCE in Bloom's syn- drome fibroblasts, and concluded that SCE in these cells may be mediated by an effect of BrdU substitution of template DNA on Topo II activity. Nevertheless, contrasting with this hypothesis, Pommier et al. (1991) have proposed that BrdU incorporation into DNA induces alkali-labile le- sions that are independent of topoisomerase II activity in Bloom and normal cells.
Our cytogenetic results seem to indicate that differences do exist between the mutant EM9 and the parental line AA8 concerning their re- sponse to topoisomerase inhibitors. This conclu- sion is based, on the one hand, upon the low effectiveness of the Topo I1 inhibitor m-AMSA to induce SCE in EM9 compared to AA8, mainly when given during the first S. The Topo I in- hibitor CPT, on the other hand, appears to be more efficient at inducing both chromosomal aberrations and SCE in EM9 than in AA8 (Palitti et al., 1993, and the present results). Since EM9
287
has been reported to be defective in the repair of single-strand breaks (Thompson et al., !982), such as those resulting from stabilization of Topo 1 cleavable complex by CPT, this latter result is not unexpected~
Our results on extractable topoisomerase activ- ities in cells grown in the absence of BrdU, or for a first cell cycle in BrdU followed by another one in dT (when, according to many reports, most of the SCE take place on BrdU-substituted template DNA), show an apparently higher Topo I activity in EM9, which could also provide an explanation of the cytogenetic results reported above. As re- gards Topo II activity, on the other hand, no clear differences between the cell lines were ob- served, contrasting with our results on the effeco tiveness of m-AMSA to induce SCE and aberra- tions. Growing the cells in BrdU, on the other hand, resulted in alterations in the extractable activity of Topo I (increase, as seen in AA8), and Topo II (reduction for 5% BrdU substitution, both cell lines).
In our opinion, taking into account the argu- ments discussed earlier and the present results, the hypothesis of a participation of topoiso- merases in SCE formation and a possible alter- ation of normal functioning of topoisomerases as responsible for the high SCE frequency and chro- mosome instability in EM9 seems attractive. It is noteworthy that, even in the absence of BrdU or mutagens, the level of chromosome breakage in EM9 is elevated, Another interesting feature of EM9 is its high spontaneous frequency of en- doreduplication, which could be explained by a failure of Topo II to unravel daughter duplexes during either S or G2, with subsequent triggering of a new replication. It has been proposed that EM9 could be lacking some 'factor' involved in altering the conformation of chromatin that is essential for completion of the enzymatic repair steps that directly involve the damaged DNA molecule (Chan et al~, 1984). Another interesting observation is that EM9 ceils show a defect in recombination (Hoy et al., 1987) and, as men- tioned earlier, the involvement of topoiso- merases, mainly Topo I1, in recombination is well documented (Ikeda et al., 1982; Ikeda, 1986; Champoux and Bullock, 1988). The above notwithstanding, determination of whether SCE
288
actually occur by any topoisomerase-mediated mechanism needs a better understanding of the rote that these enzymes play in DNA replication and repair.
AcknowJedgements
We are grateful for gifts of m-AMSA from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD (USA). Cell lines were kindly pro- vided by Dr. L.H. Thompson (Lawrence Liver- more National Laboratory, Livermore, CA (USA). The excellent technical assistance of J.A. Rojano as well as the photographic work of FJ . Fuentes are very much appreciated. This work was sup- ported by grants from Junta de Andalucfa and Subdireccidn General de Cooperacidn Internao cional (Spanish-Italian Joint Action), Spanish Ministry of Education and Science.
References
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem., 72, 248-254.
Chaganti, R.S.K., S. Schonberg and J. Gerraan (1974) A manyfold increase in sister cbromatid exchanges in Bloom's syndrome lymphocytes, Proc. Natl. Acad. Sci. USA, 71, 4508-4512.
Champoux, J.J., and P.A. Bullock (1988) Possible role for the eucaryotic type I topoisomerase in illegitimate recombina- tion, in: R. Kucherlapati and G,R. Smith (Eds.), Genetic Recombination, pp. 655-666.
Chan, J.Y.H., L.H. Thompson and F.F. Becker (1984) DNA- ligase activities appear normal in the CHO mutant EM9, Mutation Res., 131, 209-214.
Churchill, M.E., J.G. Peak, and M.J. Peak (1991) Repair of near-visible- and blue-light-induced DNA single-strand breaks by the CHO cell lines AA8 and EMg, Photochem. Photobiol., 54, 639-644.
Cleaver, J.E. (1981) Correlations between sister chromatid exchange frequencies and replicon sizes, a model for the mechanism of SCE production, Exp. Cell Res., 136, 27-30.
Cort6s, F. and T. Ortiz (1991) Induction of chromosomal aberrations in the CHO mutant EM9 and its parental line AA8 by EcoRI restriction endonuclease: electroporation experiments, Mutation Res., 246, 221-226
Degrassi, F., R. De Salvia, C. Tanzarella and F. Palitti (1989) Induction of chromosomal aberrations and SCE by camp- tothecin, an inhibitor of mammalian topoisomerase I, Mu- tation Res., 211, 125-130.
Dillehay, L.E., L.H. Thompson, J.L. Minkler and A.V. Car-
rano (1983) The relationship between sister-chromatid ex- change and perturbations in DNA replication in nmtant EM9 and normal CHO cells, Mutation Res, 109, 283-296.
Dillehay, L.E., S.C. Denstman and J.R. Williams (1987) Cell cycle dependence of sister chromatid exchange induction by DNA topoisomerase II inhibitors in Chinese hamster V79 cells, Cancer Res., 47, 206 209.
Dillehay, L.E., D. Jacobson-Kram and J.R. Williams (i989) DNA topoisomerases and models of sister-chromatid ex- change, Mutation Res., 215, 15-23.
Heartlein, M.W., H. Tsuji and S.A. Latt (1987) 5-Bromoode. oxyuridine-dependent increase in sister chromatid ex- change formation in Bloom's syndrome is associated with reduction in topoisomerase I1 activity, Exp. Cell Res., 169, 245-254.
Hoy, C.A., J.C. Fuscoe and L.H. Thompson (t987) Recombi- nation and ligation of transfected DNA in CHO mutant EMg, which has high levels of sister chromatid exchange, Mol. Cell. Biol., 7, 2007-2011.
Ikeda, H. (1986) Bacteriophage T4 DNA topoisomerase medi- ates illegitimate recombination in vitro, Proc. Natl. Acad. Sci. USA, 83, 922-926.
Ikeda, H., K. Aoki and A. Naito (1982) Illegitimate recombi. nation mediated in vitro by DNA gyrase of Escherichia coli: structure of recombinant DNA molecules, Proc. Natl. Acad. Sci. USA, 79, 3724-3728.
Ikejima, M., D. Bobannon, D.M. Gill and L.H. Thompson (1984) Poly(ADP-ribose) metabolism appears normal in EMg, a mutagen-sensitive mutant of CHO cells, Mutation Res., 128, 213 220.
Ishii, Y., and M.A Bender (1980) Effects of inhibitors of DNA synthesis on spontaneous and ultraviolet-induced sister- chromatid exchanges in Chinese hamster cells, Mutation Res., 79, 19-32.
La Belle, M., S. Linn and L.H. Thompson (1984) Apuri- nic/apyrimidinic endonuclease activities appear normal in the CHO-cell ethyl methanesulfonate-sensitive mutant, EM9, Mutation Res., 141, 41-44.
Liu, L.F. (1989) DNA topoisomerase poisons as antitumor drugs, Annu. Rev. Biochem., 58, 351-375.
Muller, M.T., J.R. Spitzner, J.H. DiDonato, V.B. Mehta, K. Tsutsui and K. Tsutsui (1988) Single-strand DNA cleav- ages by eucaryotic topoisomerase II, Biochemistry, 27, 8369-8379.
Natarajan, A.T., A.H.M. Rotteveel, J. van Pieterson and M.G. Schliermann (1986) Influence of incorporated 5-bromo-de- oxyuridine on the frequencies of spontaneous and induced sister-chromatid exchange, detected by immunological methods, Mutation Res., 163, 51-55.
Painter, R.B. (1980) A replication model for sister-chromatid exchange, Mutation Res., 70, 337-341.
Palitti, F., F. Cort6s, L. Bassi, D. Di Chiara, M. Fiore, and J. Pif~ero (1993) Higher G2 sensitivity in the CHO mutant EM9 than in its parental line AA8 to the induction of chromosomal damage by camptothecin, an inhibitor of DNA topoisomerase I, Mutation Res., 285, 281-285.
Pinkel, D., L.H. Thompson, J.W., Gray and M. Vanderlaan (1985) Measurement of sister chromatid exchanges at very
low bromodeoxyuridine substitution levels using a mono- clonal antibody in Chinese hamster ovary" cells, Cancer Res., 45, 5795-5798.
Pommier, Y., L.A. Zwelling, C.-S. Kao-Shan, J. Whang-Peng and M.O. Bradley (1985) Correlations between intercala- to>induced DNA strand breaks and sister chromatid ex- changes, mutations, and cytotoxicity in Chinese hamster cells, Cancer Res., 45, 3143-3149.
Pommier, Y., D. Kerrigan, J.M. Covey, C.-S. Kao-Shan and J. Whang-Peng (1988) Sister chromatid exchanges, chromo- somal aberrations, and cytotoxicity produced by antitumor topoisomerase II inhibitors in sensitive (DC3F) and resis- tant (DC3F/9-OHE) Chinese hamster cells, Cancer Res., 48, 512-516.
Pommier, Y., T.M. Riinger, D. Kerrigan and K.H. Kraemer (1991) Relationship of DNA strand breakage produced by bromodeoxyuridine to topoisomerase II activity in Bloom- syndrome fibroblast, Mutation Res., 254, 185-190.
Shiraishi, Y., and Y. Ohtsuki (1987) SCE levels in Bloom-syn- drome cells at very low bromo-deoxyuridine (BrdU) con- centrations: monoclonal anti-BrdU antibody, Mutation Res., 176, 157-164.
289
Siciliano, M.J., A.V. Carrano and L.H. Thompson (1986) Assignment of a human DNA-repair gene associated with sister-chromatid exchange to chromosome 19, Mutation Res., 174, 303-308.
Smith, P.J. (1990) DNA topoisomerase dysfunction: a new goal for antitumor chemotherapy, BioEssays, 12, 167-172.
Thompson, L.H., K.W. Brookman, L.E. Dillehay, A.V. Car- rano, J.A. Mazrimas, C.L. Mooney and J.L. Minkler (1982) A CHO strain having hypersensitivity to mutagens, a de- fect in DNA strand-break repair, and an extraordinary baseline frequency of sister-chromatid exchange, Mutation Res., 95, 427-440.
Thompson, L.H., K.W. Brookman, N.J. Jones, S.A. Allen and A,V. Carrano (1990) Molecular cloning of the human XRCC1 gene, which corrects defective DNA strand break repair and sister chromatid exchange, Mol. Cell. Biol., 10, 6160- 6171.
Wang, J.C. (1985) DNA topoisomerases, Annu. Rev. Biochem., 54, 665-697.
Zhang, H., P. D'Arpa and L.F. Liu (1990) A model for tumor cell killing by topoisomerase poisons, Cancer Cells, 2, 23-27.
