Previous studies have shown the importance of DNA replication fork progression for the cytotoxicity of topoisomerase inhibitors. Nevertheless, while it was concluded that an interference of moving forks with drug-stabilized topo I-DNA complexes is critical for cell death, in the case of topo II only a partial contribution to cell killing was proposed. We have studied the influence of inhibition of DNA replication by aphidicolin on the production of chromosomal aberrations and SCE by topoisomerase inhibitors. Our results seem to indicate that fork progression is necessary for both cytogenetic endpoints. Pulsed field gel electrophoresis also confirmed this conclusion at the level of DNA breakage (double-strand breaks) efficiently induced by m-AMSA treatment alone, but not when aphidicolin was present. Differences found between topo I and topo II inhibitors (camptothecin and m-AMSA, respectively) could be explained as due to differences, in their persistence in trapping the 'cleavable complex'.
Mammalian DNA topoisomerases are the cel- lular targets for antitumor drugs with various structures such as the intercalating agent 4'-(9- acridinylamino) methanesulfon-m-anisidide (m- AMSA), which specifically inhibits topoisomerase II (topo II), or the plant alkaloid camptothecin (CPT), which does so with topoisomerase I (topo I) (Liu, 1989). A characteristic feature of these compounds is that they do not inhibit the cat- alytic activity of DNA topoisomerases but rather
* Corresponding author.
appear to interfere with their rejoining reaction by trapping an enzyme-DNA covalent intermedi- ate termed 'cleavable complex' (Liu, 1989; Smith, 1990). This unusual form of DNA damage, topo- isomerase-DNA cleavable complexes, seems to be responsible for the cytotoxicity of topoisomerase inhibitors as well as for their efficiency in the induction of sister-chromatid exchanges (SCE) and chromosomal aberrations (Pommier et al., 1988; Hsiang et al., 1989; Holm et al., 1989; Zhang et al., 1990), though the exact mechanisms involved are still largely unknown.
The drug-stabilized cleavable complex repre- sents reversible DNA damage. This feature offers
72
a unique opportunity to study the molecular events involved in the above mentioned end- points. On the other hand, such a reversible way of action of topoisomerase poisons raises intrigu- ing questions concerning how the cell killing sig- nal is triggered or how the covalent complexes can result in extensive chromosome breakage or elevated levels of SCE.
The necessity of DNA synthesis for the cyto- toxicity of topoisomerase inhibitors has been studied. Inhibition of DNA synthesis with aphidi- colin (APH) or hydroxyurea abolished the cyto- toxicity of CPT in mouse lymphoblastic leukemic cells (Hsiang et al., 1989) and Chinese hamster lung fibroblasts (Holm et al., 1989). It was con- cluded that an interference of moving DNA repli- cation forks with drug-stabilized topo I-DNA complexes is critical for cell death. Nevertheless, in the case of the topo II poisons VP-16 and m-AMSA, APH only partially reduced drug cyto- toxicity and it was concluded that arrest of repli- cation forks may only contribute partly to cell killing (Holm et al., 1989).
We have carried out experiments in Chinese hamster ovary (CHO) cells following protocols similar to those reported by Holm et al. (1989) in order to analyze the influence of inhibition of DNA replication by APH on both chromosomal aberrations and SCE induced by the topoisom- erase inhibitors CPT and m-AMSA. Our results seem to indicate that CPT has a completely re- versible effect, while the m-AMSA effect is more persistent and it is still able to induce damage after drug removal, though to a much lower ex- tent, presumably when the replication fork re- sumes its progression after APH treatment. Pulsed field gel electrophoresis (PFGE) seemed to confirm the protection exerted by APH on DNA from extensive damage (double-strand breaks) induced by m-AMSA. The implications of these observations for the possible mechanisms of cell killing, chromosome breakage and SCE by topoisomerase poisons are discussed.
(dC), the topo I inhibitor CPT, and the DNA synthesis inhibitor APH were purchased from Sigma Chemical Co., St. Louis, MO (USA). m- AMSA (NSC-249992) was obtained from the Drug Synthesis and Chemistry Branch, Division of Cancer Treatment, National Cancer Institute, Bethesda, MD (USA).
Cell culture Chinese hamster ovary (CHO6) cells were
grown as monolayers in McCoy's 5A medium with 10% fetal calf serum, 2 mM L-glutamine, and the antibiotics penicillin (50 U/ml ) and streptomycin (50 ~g/ml) . Cells were cultured at 37°C in 5% CO2 in air.
Treatment of cells Exponentially growing cells were grown in the
dark for two cell cycles (total 26 h, including the last 2 h in colcemid) in the presence of 5 /zM BrdU, 5/xM dT, 100/~M dC, and 1 /~M FdU in order to obtain differential staining of sister chro- matids in second mitosis. CPT or m-AMSA treat- ments were given for 30 min, 18 h after starting BrdU incorporation (second S). Treatment with the DNA synthesis inhibitor APH was started 5 min before CPT or m-AMSA treatments, contin- ued throughout the ensuing 30 min, and ended at the same time as the topoisomerase inhibitors (35 min total treatment time). At the end of drug treatments, cells were thoroughly washed three times with warm medium and given again the same mixture as previously to ensure BrdU sub- stitution until the addition of Colcemid (0.2 ~M final concentration) and harvesting. Cells were collected by mitotic shake off and then treated with hypotonic 0.075 M KCI (2-3 min) and finally fixed in two washes with methanol-acetic acid (3: 1). Standard cytological preparations were then made and either Giemsa-stained for the scoring of chromosomal aberrations or processed by a modified fluorescence plus Giemsa (FPG) technique as reported elsewhere (Cort6s et al., 1987).
Pulsed field gel electrophoresis (PFGE) of DNA Cells were treated with 1 ~M m-AMSA and /
or 10/~M APH as reported above, embedded in agarose inmediately after treatment, and DNA
73
TABLE 1
INFLUENCE OF THE DNA SYNTHESIS INHIBITOR APH ON CHROMOSOME DAMAGE AND SCE IN CHO CELLS TREATED WITH CPT (INHIBITOR OF TOPO I) AND m-AMSA (INHIBITOR OF TOPO II)
APH Treatment Abnormal M . A . Scorable Dicentrics SCE/metaphase (10 ~M) metaphases aberrations _+ SE
M.A., cells showing multiple aberrations. In the case of I ~M m-AMSA treatment, must have been underestimated.
100 metaphases were scored for chromosomal aberrations and SCE, except for a: 50 cells. scoring of SCE was only possible in the less damaged cells and so the figure (14.67_+0.75)
double - s t rand breaks analyzed by c lamped homo- geneous field ( C H E F ) gel electrophoresis .
The p rocedure was as follows: cells were mixed
at an equal vo lume with 1% low mel t ing agarose (Bio Rad, USA) solut ion p repared in buffer L (0.5 M E D T A , 0.01 M Tris, 0.02 M NaCI, pH 8.0) at 42°C, and agarose plugs con ta in ing about 4 × 10 6 cells finally ob ta ined by cooling down (4°C) in moulds. Subsequent ly , plugs were incuba ted for
lysis overnight at 50°C in buffer L solut ion con- ta in ing 0.5 m g / m l pro te inase K and 1% Sarkosyl,
with one buffer change. Af te r lysis, the agarose plugs were washed at least four t imes (2 h for each wash) with T E buffer (10 m M Tris, 1 m M
E D T A , p H 8.0). Elect rophores is was carr ied out in half-s t rength T B E buffer (45 m M Tris, 45 mM boric acid, 1.5 m M E D T A , p H 8.2). Agarose gels (0.5% Bio Rad Ul t ra Pure D N A grade) were p repared in T B E buffer, and Saccharomyces cere- visiae chromosomes were run in parallel as
molecular weight markers . The agarose plugs conta in ing the encapsula ted cel lular D N A were
Fig. 1. Cytogenetic effects of topoisomerase inhibitors. (a) Heavily damaged CHO cell after treatment with 1/~M m-AMSA. (b) 7.5 tzM CPT-treated cell showing a very high frequency of SCEs.
74
inserted into the gel wells, which were sealed with agarose and subjected to electrophoresis.
Electrophoresis was carried out using a CHEF-DR II hexagonal apparatus (Bio Rad) and the temperature maintained at 14°C by circulat- ing the buffer through an external cooling unit. The conditions for electrophoresis were 200 V, 24 h with pulse times of 60 s (15 h) and 90 s (9 h). Finally, gel was stained with ethidium bromide (0.1 /xg/ml) for photography.
Results
Table 1 shows the efficiency of a 30-min treat- ment with different doses of the topo I inhibitor CPT (ranging from 2.5 to 7.5 ~M) or the topo II inhibitor m-AMSA (0.5 and 1 ~zM) in the induc- tion of chromosome damage and SCE as well as the influence of 10/~M APH given shortly before the addition of the topoisomerase inhibitors. As can be seen, m-AMSA was a much stronger in- ducer of chromosomal aberrations than CPT, causing extensive chromosome damage (cells showing multiple aberrations; Fig. la) at concen- trations well below those of CPT able to produce only a limited clastogenic effect. CPT, on the other hand, increased the frequency of SCE at these doses dramatically (Fig. lb), while the strong effect of m-AMSA on aberration yield at doses higher than 0.5 t~M made the scoring of SCE difficult.
DNA synthesis inhibition by a 35-min treat- ment with APH as the only treatment resulted in a moderate increase in the yield of aberrations and SCE (Table 1). Nevertheless, APH almost fully protected CHO cells from CPT effect on both cytogenetic endpoints for all concentrations tested. In contrast to the case of CPT, however, APH protected only partly against m-AMSA-in- duced damage, as shown by both aberration and SCE levels.
To examine whether the apparent protection provided by APH against chromosome damage induced by m-AMSA could be detected at the level of DNA, PFGE was carried out to detect DNA double-strand breaks in cells t reated with 1 /xM m-AMSA, previously shown to be able to induce extensive chromosome damage, or given APH to inhibit replication during treatment with
1 2 3 4 5 6 7 ~ ' ~ ~
J
Fig. 2. PFGE measurements of DNA double-strand breaks induced by m-AMSA and influence of DNA synthesis inhibi- tion by APH. Lanes 1 and 7, S. cerevisiae DNA marker. Lanes 2-6 contain DNA isolated from CHO cells irradiated with 50 Gy of X-rays (lane 2), treated with 10 tzM APH alone (lane 3), given 1 ~M m-AMSA and APH simultaneously (lane 4).
m-AMSA alone (lane 5) or untreated (lane 6).
the topo II inhibitor. As can be seen in Fig. 2, the amount of DNA migrating from the well, which gives a measure of DNA double-stand breakage, was clearly higher when cells were treated with m-AMSA than when APH was present simulta- neously.
Discussion
In recent years, it has been reported that many antitumor cytotoxic drugs with different chemical natures appear to interfere with the breakage-re- union reactions catalyzed by DNA topoisom- erases. These agents are able to stabilize the 'cleavable complexes' in which the enzyme is co- valently linked to the 3' (topo I) or 5' (topo II) termini of single- or double-strand DNA breaks (Liu, 1989; Smith, 1990). Possible mechanism(s) of cell killing by mammalian topoisomerase in- hibitors involving the collision between the mov- ing replication forks and the cleavable complexes have been a matter of discussion (revised by Liu,
75
1989). Consistent with the known S-phase speci- ficity of the topo I inhibitor CPT, DNA synthesis inhibition by APH resulted in a complete cancel- lation of CPT-induced cytotoxicity, while for the topo II inhibitors (VP-16 or m-AMSA) the pro- tection was only partial (Holm et al., 1989).
The close relationship between gross chromo- somal damage and cell reproductive death has been demonstrated in mammalian cells (Grote et al., 1981; Joshi et al., 1982; Prosser et al., 1990). We have observed that APH completely abol- ished the effect of CPT on chromosomal aberra- tions, while only a partial protection against the topo II inhibitor m-AMSA was provided by the DNA synthesis inhibitor. It is worth mentioning that APH, at the concentration used in the pres- ent investigation (10 ~zM), has been reported to inhibit thymidine incorporation by more than 90% within the first 5 min of drug exposure (Holm et al., 1989).
Taking into account the above mentioned di- rect relationship between chromosomal aberra- tions and colony-forming ability, our results are in good agreement with those reported by Hsiang et al. (1989) and Holm et al. (1989). Furthermore, our observations seem to indicate the necessity for the progression of the replication fork for the production of chromosomal aberrations by topo- isomerase inhibitors. As regards the only partial reduction in the yield of aberrations observed for m-AMSA, the most likely explanation is that, contrasting with the clean reversible effect of CPT on topo I, a number of m-AMSA stabilized topo II-DNA cleavable complexes are still pres- ent when replication forks resume their move- ment after drug and APH removal from culture. This could explain, in our opinion, the reported differences between CPT and topo II inhibitors regarding the protection provided by APH and, on this basis, the collision between DNA replica- tion forks and topo II complexes could also be a determinant for cell killing.
Focusing on the still unclear mechanism of SCE production, on the other hand, the observa- tion that inhibition of DNA synthesis by APH results in a clear reduction in the yield of SCE induced by topoisomerase inhibitors, seems to support that: (a) they occur during replication, as first reported by Wolff et al. (1974), and (b) most
likely they take place at or near the replication fork, as proposed by other investigators (Latt and Loveday, 1978; Ishii and Bender, 1980).
Regarding the relative importance of different lesions in DNA for the production of chromoso- mal aberrations or SCE, it is noteworthy that, at the concentrations employed by us, CPT is ex- pected to induce DNA single-strand, but not dou- ble-strand breaks (Mattern et al., 1987), while m-AMSA is expected to produce both, but mainly double-strand breaks. As a result of DNA repli- cation on broken templates, however, double- strand breaks can arise after CPT treatment (Liu, 1989). This latter type of lesion is responsible for chromosomal aberrations according to different reports (Natarajan et al., 1980; Bryant, 1984; CortEs and Ortiz, 1991), while for SCE the pres- ence of modified bases in DNA during replica- tion seems to be a requisite (Wolff, 1982; CortEs et al., 1991). We have found a good correlation between reduction in extensive chromosome damage induced by 1 /~M m-AMSA when APH was present and protection provided by APH against the production of DNA double-strand breaks, as detected by PFGE.
SCE models involving topoisomerases have been proposed (reviewed by Dillehay et al., 1989). For some of these models (Ishii and Bender, 1980; Shafer, 1982), incisions in DNA by topoi- somerases at or near the replication forks would start the process leading to SCE. Interestingly, it has been reported that CPT induces DNA break- age at replication forks (Avemann et al., 1988).
The present investigation seems to support that the progression of replication forks is neces- sary for the occurrence of both chromosomal aberrations and SCE as a consequence of inhibi- tion of topoisomerases, and that SCE do occur in connection with this active replication by a mech- anism that might involve DNA topoisomerases.
The demonstration of the close relationship existing between the activity of replicons and the production of unrepaired double-strand breaks in DNA leading to chromosome damage by in- hibitors of DNA topoisomerases reported here, in our opinion opens the possibility of potentiat- ing the effectiveness of treatments with these antitumor drugs by means of a premature onset of replication such as that reported to occur as a
76
consequence of excess thymidine (Schvartzman and Krimer, 1985). Experiments are in progress in our laboratory to test this hypothesis.
Acknowledgements
We thank Dr. Antonio Carballo (Department of Genetics, University of Seville) for his continu- ous support and advice with PFGE. The excellent technical assistance of J.A. Rojano is also very much appreciated. 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). This work has been partly supported by grants from Junta de Andalucla (Spain).
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