Mutagenesis, Vol. 14, No. 1, 103-105,
January 1999
© 1999 UK Environmental Mutagen Society/Oxford University Press
The involvement of chromatin condensation in camptothecin-induced chromosome breaks in G0 human lymphocytes
Department of Agrobiology and Agrochemistry, University of Tuscia, Via San Camillo de Lellis s.n.c., 01100 Viterbo, Italy
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Abstract |
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In the present study we evaluated campthotecin (CPT)-induced chromosomal damage in human lymphocytes in the G0 phase of the cell cycle as revealed by the premature chromosome condensation technique. The results obtained here indicate that CPT was able to induce chromosome fragments in the G0 phase of the cell cycle of human lymphocytes as detected in prematurely condensed chromosomes. This result appears to be rather surprising, since the DNA lesions produced by CPT (e.g. `protein concealed' DNA single-strand breaks) should not produce any damage in G0. A possible explanation for this result could come from much evidence to suggest that chromatin condensation processes are significantly involved in the conversion of DNA lesions into chromosome breaks in prematurely condensed chromosomes. The unexpected clastogenic behaviour of CPT can be explained taking into account the chromosome condensation induced by mitosis promoting factors when human lymphocytes are fused in G0, thus converting the `protein concealed' DNA single-strand breaks induced by CPT into chromosome breaks. The same perspective should be taken into consideration for breaks induced by CPT under normal physiological conditions in the G2 phase of the cell cycle.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Camptothecin (CPT) and its derivative compounds are a new class of anticancer drugs that target DNA topoisomerase I. Like other anti-topoisomerase drugs, it can be classified as a clastogenic agent which has an indirect mechanism of action compared with one which damages DNA directly (Palitti et al., 1989
; Scott et al., 1991).
DNA breaks induced by anti-topoisomerase drugs are formed by an enzyme-mediated process and, being `protein concealed', they can be detected by the DNA filter elution methodology only if the cell lysate is digested with a proteinase before elution (Zwelling et al., 1981; Zwelling, 1985; Glisson and Ross, 1987).
Inhibitors of both DNA topoisomerases I and II act by stabilizing the DNA–topoisomerase complex known as the `cleavable complex': in the topoisomerase II reaction, both single-strand and double-strand DNA breaks (SSB and DSB) are formed, while in the topoisomerase I reaction, only SSB are generated.
Inhibitors of DNA topoisomerase II, which give rise to DSB, are able to induce chromosomal aberrations in all phases of the cell cycle, acting similarly to ionizing radiation and resembling an `S-independent' mechanism. They are also able to induce sister chromatid exchanges (SCE) when the treatment is performed in the S phase of the cell cycle (Andersson and Kihlman, 1989; Palitti et al., 1989). Only CPT and its derivatives are known to inhibit topoisomerase I (D'Arpa and Liu, 1989), giving rise to SSB only. While treatment with the topoisomerase I inhibitor CPT in human lymphocytes gives rise exclusively to chromatid-type aberrations when the drug is present in the S and G2 phases of the cell cycle, G1 treatments have no effect (Degrassi et al., 1989). Elevated frequencies of SCE are also observed, provided that CPT is present during the S phase (Dillehay et al., 1983).
Trapping of `the cleavable complex' by the topoisomerase I inhibitor CPT gives rise only to SSB, which per se are not expected to produce chromosomal aberrations. The chromatid-type aberrations obtained after S phase treatment could be the result of DSB derived from collision of the CPT-trapped `cleavable complex' with the replication fork (D'Arpa and Liu, 1989; Hsiang et al., 1989; Zhang et al., 1990; Ryan et al., 1991). The finding that CPT also induces chromatid aberrations during G2 phase, however, is unexpected, as no DNA synthesis should occur during this phase (Degrassi et al., 1989). This finding was also confirmed in Chinese hamster cells (Degrassi et al., 1989; Palitti, 1993) and in root tips of Vicia faba (Andersson and Kihlman, 1992).
Considering that the clastogenicity of CPT is dependent on DNA synthesis, it was suggested that in G2 phase residual DNA synthesis is still present but that it cannot be detected by conventional autoradiographic methods (Andersson and Kihlman, 1992; Palitti et al., 1994).
More recently, to test this hypothesis (Bassi et al., 1998), we investigated the localization of CPT-induced breakpoints in the G2 phase of a primary Chinese hamster cell culture among euchromatic and heterochromatic regions of chromosomes, taking into account the fact that if residual DNA synthesis was involved in the clastogenic effects induced by CPT in G2 phase, one would expect chromosome aberrations to occur preferentially in the heterochromatic regions, which are late replicating.
Contrary to expectation, the breakpoints were not localized in the late replicating regions. This suggests that CPT-induced chromatid-type aberrations arise in the G2 phase by a mechanism which does not involve DNA replication through collision of the CPT-trapped `cleavable complex' with the replication fork, but possibly that chromatin condensation could play a role.
In the present paper, in order to evaluate the role of chromatin condensation, we tested the effect of premature chromosome condensation (PCC) on the formation of chromosomal damage in human lymphocytes treated with CPT in the G0 phase of the cell cycle.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Chemicals
CPT, purchased from Sigma Chemical Co. (St Louis, MO) was dissolved in dimethyl sulphoxide (DMSO). Stock solutions were prepared at 2.5 mM and kept frozen at –20°C. For treatment of cultures 50 µl stock solution were added to 5 ml culture such that the final concentration of solvent did not exceed 1%. H2O2 (Carlo Erba, Milan, Italy) was diluted directly in culture medium at a final concentration of 100 µM. Polyethylene glycol (PEG), purchased from Sigma Chemical Co., was dissolved in distilled water at 1 g/ml in an oven. Spectroscopic grade DMSO was obtained from Fluka AG (Buchs, Switzerland). Colcemid (Gibco BRL) was used at a final concentration of 0.2 µg/ml.
Premature chromosome condensation technique
Cell preparation.
Mitotic Chinese hamster ovary (CHO) cells were used to induce PCC in human lymphocytes. CHO cells are routinely grown as monolayers in Ham's F10 culture medium containing 15% newborn calf serum, 2 mM L-glutamine, penicillin (50 µg/ml) and streptomycin (50 µg/ml) at 37°C in a humidified atmosphere of 5% CO2. These cells were grown for two cell cycles in complete culture medium supplemented with 5-bromodeoxyuridine (BrdU) at a final concentration of 5 µM, to allow differential staining of chromatids following FPG staining and to better differentiate lymphocyte PCC from mitotic CHO chromosomes. Colcemid, at a final concentration of 0.2 µg/ml, was added to exponentially growing CHO cells for 5 h. The accumulated mitotic cells were harvested by selective detachment (shake-off) and frozen at –80°C in culture medium supplemented with 10% DMSO until use. Human lymphocytes were obtained from one bag of buffy coat, supplied by a local hospital and generated from ~500 ml heparinized fresh venous whole blood drawn from a healthy male donor. The buffy coat blood was diluted in phosphate-buffered saline (1:1) and lymphocytes separated using Ficoll–Isopaque. Briefly, 15–20 ml diluted buffy coat were layered over 12.5 ml Ficoll–Isopaque in 50 ml tubes and centrifuged for 35 min at 450 g at room temperature. After centrifugation, mononuclear cells and platelets were in a fluffy white layer just under the plasma. The cell layers were collected with Pasteur pipettes and washed twice in fresh culture medium. After the last centrifugation the cell pellet was resuspended by gentle vortexing and a cell suspension at a final concentration of 5x106 cells/ml in Ham's F-10 supplemented with fetal calf serum (40%) and DMSO (10% v/v) was prepared. Aliquots of 1.5 ml were added to each ampoule and frozen under liquid nitrogen at –196°C. G0 human lymphocytes were thawed immediately before treatment directly in a water bath at 37°C. When slightly melted they were transferred to centrifuge tubes (15 ml) and 10 ml cold (4°C) Ham's F-10 supplemented with 40% fetal calf serum were added drop by drop over ~30 min to the suspension. The cells were centrifuged at 1000 r.p.m. for 10 min and the cell pellet resuspended in 5 ml Ham's F-10 supplemented with 5% fetal calf serum, ready for treatment.
Treatment. G0 human lymphocytes were incubated at 37°C with 25 µM CPT for 1 h or with 100 µM H2O2 for 0.5 h in complete culture medium. H2O2 served as a positive control. After treatment cells were washed with culture medium and utilized immediately for cell fusion.
Cell fusion.
Cell fusion between mitotic CHO cells and G0 human lymphocytes was mediated by PEG (Pantelias and Maillie, 1983). Briefly, mitotic cells and human lymphocytes were mixed in a ratio of 1:3 in a round-bottomed culture tube. After centrifugation, the cells were suspended in 2 ml Ham's F-10 medium without serum and centrifuged again. The supernatant was discarded without disturbing the pellet and 0.25 ml PEG was added (all at once) and left for 1 min. For the next 3 min, 2 ml phosphate-buffered saline were added dropwise; the tube was gently shaken after each drop. The cell suspensions were centrifuged and resuspended in 0.5 ml culture medium containing colcemid (0.2 µg/ml) and incubated at 37°C for 1 h (Pantelias and Maillie, 1984; Darroudi and Natarajan, 1989). Finally, cells were treated with hypotonic KCl solution (75 mM) for 10 min and fixed in methanol:acetic acid (3:1). The cell suspension was dropped onto pre-cleaned wet slides and stained by the FPG method to allow discrimination between mitotic CHO chromosomes and PCC lymphocytes. Prior to scoring, to avoid bias, microscope slides were coded and randomized. PCC lymphocytes were analysed by light microscopy. The number of prematurely condensed chromosomes was scored in each cell (2n = 46) and an excess >46 represented chromosomal damage. Cells containing from one to four fragments were grouped as `cells containing 47–50 elements'; cells containing from five to 10 fragments were grouped as `cells with >50 elements'; cells containing >10 fragments were classified as `highly fragmented' (HF). For each sample, the chromosomal damage was analysed in the PCC of at least 100 lymphocytes.
For statistical evaluation of the data the numbers of cells bearing aberrations in the control and treated cultures were compared using Fisher's exact test.
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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The results obtained are collated in Table I
and indicate that both CPT, which produces exclusively `protein concealed' DNA SSB, and the positive control H2O2, which effectively produces DNA SSB (Ward et al., 1987), induced significant increases in chromosome breaks in the G0 phase of the cell cycle.
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This result appears to be rather surprising, since the DNA lesions produced by CPT (e.g. `protein concealed' DNA SSB) and by H2O2 (mainly DNA SSB) should not produce any damage in G0. It has been reported that DSB increase as a function of H2O2 concentration up to ~200 µM and reach a maximum at ~1 mM. Nevertheless, up to dose levels of 100 µM (which is the dose employed in our experiments) the quantity of `eluted DNA', which is a measure of DSB, was negligible (Iliakis et al., 1992
). Furthermore, induction of chromosome breaks by H2O2 analysed by means of PCC in CHO cells was reported by the same authors to be similar to that observed in human lymphocytes in our experiments.
A possible explanation for this result could come from evidence which suggests that chromatin condensation–decondensation processes are significantly involved in the conversion of DNA lesions into chromosome breaks in PCC. Using PCC, it was realized that changes in chromatin conformation soon after irradiation strongly affect the conversion of DNA lesions into chromosome breaks in PCC (Pantelias, 1989). Additional evidence for the involvement of changes in chromatin conformation comes from the observation that higher concentrations of mitosis promoting factors (MPF) in the mitotic PCC inducer cells, which are mainly responsible for the changes in chromatin condensation associated with PCC, induce higher levels of fragments (Cheng et al., 1993).
More recently, it has been shown that the increased radiosensitivity of the G2 phase of the cell cycle compared with the G0 phase does not reflect different intrinsic features of the chromatin structure which determine a more efficient conversion of DNA lesions into chromosome damage, but may be related to chromatin condensation during the G2/M transition and subsequent efficient conversion of DNA damage into chromosomal breaks as the cells progress to mitosis (Terzoudi and Pantelias, 1997).
All this evidence suggests a significant involvement of chromatin condensation–decondensation in the conversion of DNA lesions into chromosomal damage.
The unexpected clastogenic behaviour of CPT can be explained taking into account the chromosome condensation induced by MPF when human lymphocytes are fused in G0, thus converting the `protein concealed' SSB induced by CPT into chromosome breaks by mechanical stress. The same perspective should be taken into consideration for breaks induced in the G2 phase of cell cycle.
This view provides further support for the idea that DNA SSB could be directly converted into chromosomal damage following chromatin condensation either prematurely induced with MPF or under normal physiological conditions in the G2 phase of the cell cycle.
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Acknowledgments |
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This paper is dedicated to Prof. Günter Obe on the occasion of his 60th birthday. We would like to thank Mr Angelo Schinoppi for expert technical assistance. MURST (40%) and EU contract no. F14P-CT95-0001 supported this work.
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Notes |
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1 To whom correspondence should be addressed. Tel: +39 761 357257; Fax: +39 761 357242; Email: palitti{at}unitus.it
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References |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Received on May 13, 1998; accepted on July 27, 1998.
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