Mutagenesis, Vol. 16, No. 2, 121-125,
March 2001
© 2001 UK Environmental Mutagen Society/Oxford University Press
Detection of DNA primary damage by premature chromosome condensation in human peripheral blood lymphocytes treated with methyl methanesulfonate
Dipartimento di Agrobiologia, e Agrochimica, Università degli Studi della Tuscia, Viterbo, Italy
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Abstract |
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Methyl methanesulfonate (MMS) is a direct acting methylating agent which produces apurinic sites that are transformed into DNA single-strand breaks by base excision repair. MMS-induced DNA lesions have to be transformed by DNA synthesis in order to give rise to chromosomal damage. In this study the premature chromosome condensation (PCC) technique was used in G1 human lymphocytes treated with MMS to investigate whether, with this technique, chromosomal damage could be detected without the cell needing to undergo DNA synthesis. A dose-dependent increase in chromosomal fragmentation was indeed observed in G1 lymphocytes. MMS treatment at 1.3, 2.5 and 5 mM was characterized by the appearance of highly fragmented chromosomes. This observation induced us to further investigate whether this effect was more connected with triggering of apoptotic cell death than a consequence of the PCC technique. Data obtained by nuclear morphology analysis, by Trypan blue exclusion assay and pulsed field gel electrophoresis seem to suggest that the observed chromosome fragmentation could be due to the onset of apoptosis. Consequently, one should bear in mind that the PCC technique can overestimate chromosomal damage when apoptosis is also induced.
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Introduction |
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The premature chromosome condensation technique (PCC) can be used to address the possible relationship between DNA primary lesions, DNA repair and formation of chromosomal aberrations. There is evidence for a possible involvement of chromatin conformation changes in the conversion of DNA lesions into chromosome aberrations. The efficiency of chromatin condensation and decondensation soon after irradiation greatly affect transformation of DNA lesions into chromosome breaks (Pantelias, 1989
). An unexpected clastogenic effect of camptothecin (CPT), an inhibitor of DNA topoisomerase I, was found when PCC was performed in CPT-treated G0 human lymphocytes, since protein-concealed DNA single-strand breaks (SSBs) induced by CPT were converted into chromosome breaks (Mosesso et al., 1999). Usually CPT-induced chromatid-type aberrations arise as a consequence of collision of the replication fork with a CPT-trapped cleavable complex (D'Arpa and Liu, 1989).
In order to further investigate the possible conversion of DNA SSBs into DNA double-strand breaks (DSBs), PCC was performed in G1 human peripheral blood lymphocytes (HPBL) treated with methyl methanesulfonate (MMS). MMS is a direct acting methylating agent yielding as the major DNA adduct N7-methylguanine and several minor adducts, such as N3-methyladenine and O6-methylguanine (Hemminki, 1983). Direct alkylation of the phosphodiester chain is a very infrequent event (Lawley and Brookes, 1963). Depuration of methylated nucleotides yields DNA with apurinic sites (AP), which are further transformed into SSBs by spontaneous or AP endonuclease-catalyzed hydrolysis (Kohn and Spears, 1967; Singer and Brent, 1981). MMS-induced DNA lesions, which give rise indirectly to SSBs, are able to induce chromosome damage by an `S-dependent' mechanism (Evans, 1977), i.e. only chromatid-type aberrations are induced. It has been shown that G2 treatment with MMS does not induce chromosomal damage, but if the cells are post-treated with Neurospora endonuclease the SSBs are transformed in DSBs, giving rise to chromatid aberrations (Natarajan and Obe, 1978).
Our results show that PCC in MMS-treated G1 HPBL produces chromosome fragmentation. In order to determine whether this effect was an indication of ongoing apoptosis, nuclear morphological analysis (Kerr et al., 1972; Wyllie et al., 1980), Trypan blue exclusion assay and pulsed field gel electrophoresis (PFGE) were performed in the same cultures to detect the pattern of high molecular weight (HMW) DNA fragments, typical of the apoptotic process (Filipski et al., 1990; Oberhammer et al., 1993; Cohen et al., 1994; Walker et al., 1994).
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Materials and methods |
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Cell isolation and cultures
Human peripheral blood was collected from two healthy male individuals (age range 25–45 years). Lymphocytes were separated using Ficoll-Histopaque 1077 (Sigma) gradients and washed twice in Ham's F10 medium (Bio-Whittaker). All steps were performed at room temperature. Isolated lymphocytes were resuspended at final concentration of 1x106 cells/ml in complete medium [Ham's F10 medium plus heat-inactivated 20% foetal calf serum (Bio-Whittaker) supplemented with 2 mM L-glutamine, 50 IU/ml penicillin, and 50 µg/ml streptomycin] and grown at 37°C in a 5% CO2 atmosphere at 80% humidity. Phytohaemagglutinin (PHA) (Murex) was added to complete medium at a concentration of 2% to stimulate cell growth.
Drug treatments
MMS (Sigma Chemical Co.) was dissolved in phosphate-buffered saline immediately before use at a concentration of 1 M and was than diluted directly in complete culture medium to final concentrations of 0.2, 0.4, 0.6, 1.3, 2.5 and 5 mM.
Experiments to study chromosomal damage with PCC
One hour after PHA stimulation lymphocytes were treated for 1 h with the different doses of MMS and after washing analysed by PCC.
PCC technique
The technique used for PCC was that of Pantelias and Maillie (1984) and Darroudi and Natarajan (1989). Briefly, Chinese hamster ovary (CHO-K1) mitotic cells, obtained by the shaking off technique, and lymphocytes were washed separately with Ham's F10 medium without serum and mixed in the ratio 1:5 in a 10 ml round bottomed culture tube. After centrifugation at 200 g for 5 min the supernatant was discarded without disturbing the pellet and 0.15 ml of 40% (w/v) polyethylene glycol (mol. wt 1450; Sigma Chemical Co.) prepared in Ham's F10 medium without serum, was added immediately and held for 1 min. Subsequently, 2.5 ml of Ham's F10 was added drop by drop and the cell suspension was mixed very gently by shaking the tube and centrifuged at 200 g for 5 min. The supernatant was discarded and the pellet resuspended in 0.7 ml of culture medium containing colcemid (at a final concentration of 3 mg/ml).
Cell fusion was complete after 60 min at 37°C and chromosome preparations were obtained by standard procedures. The cell suspension was dropped onto pre-cleaned wet slides and stained with an aqueous 3% Giemsa solution to allow discrimination between mitotic CHO chromosomes and PCC lymphocytes. Damage was analysed in 30–50 cells for each dose and each experiment for each individual.
In scoring, cells containing 46 chromosomes were considered normal and >46 represented chromosomal damage. Only dislocated fragments were considered as damage. Cells containing >20 fragments were classified as `highly fragmented' (HF). The data were statistically analysed by comparing the number of cells bearing aberrations in the control and treated cultures using Fisher's exact test.
Experiments to study apoptosis
Unstimulated and 1 h PHA-stimulated lymphocytes were exposed to MMS for 1 h in complete medium. At the end of treatment the cells were washed once with Ham's F10 and resuspended in complete medium. Immediately after treatment or 12 or 24 h later the cells were analysed for induction of apoptosis by PFGE and nuclear morphology analysis and cell viability was determined by Trypan blue exclusion assay. All experiments were repeated several times, showing the same trend and good reproducibility.
Analysis and quantification of cell death
For morphological analysis 5x105 cells were resuspended and fixed in 4% (v/v) paraformaldehyde, loaded on a gelatinized slide, stained with 0.1% Mayers haematoxylin (Sigma) and analysed by direct optical microscopy at a magnification of 1000x. Apoptosis was quantified by scoring cells with condensed and fragmented nuclei. At least 500 cells in random selected fields were scored.
The cells were assessed by staining with Trypan blue, by mixing (1:1) with 4% Trypan blue (diluted in 1x phosphate-buffered saline) and counting the number cells which excluded Trypan blue in a Burker chamber within 5 min after staining. Cells excluding Trypan blue were considered viable and in the early stages of apoptosis in which the cytoplasmic membrane is intact.
The 
2 test was used to compare the number of apoptotic cells in unstimulated versus stimulated lymphocytes.
DNA fragmentation studied by PFGE
The cells were processed as described by Zhivotousky et al. (1994). Briefly, 6x105 lymphocytes were resuspended in 50 µl of a solution containing 0.15 M NaCl, 2 mM KH2PO4/KOH (pH 6.8), 1 mM EGTA and 5 mM MgCl2. An equal volume of liquefied 1% low melting point agarose was added to the cell suspension and the mixture was aliquoted into gel plug casting forms. The resulting agarose blocks were placed in lysis buffer (10 mM NaCl, 10 mM Tris–HCl, pH 9.5, 25 mM EDTA, 1% lauryl sarcosine) supplemented with proteinase K (1 mg/ml) and incubated at 50°C for 24 h. The agarose blocks were then introduced into a 1% agarose gel and contour clamped homogeneous electric field electrophoresis was carried out using a Gel Navigator horizontal chamber and a Pulsator 2015 controller (Pharmacia LKB). Total run time was 19 h at 180 V at 9°C in 0.5x TBE (45 mM Tris, 1.25 mM EDTA, 45 mM borate, pH 8.0). Pulses were applied as follows: 180 s pulses for 4 h; 90 s pluses for 3 h; 45 s pulses for 6 h; 20 s pluses for 6 h. After the run the gel was stained with ethidium bromide (1 µg/ml) (Sigma), visualized under a 312 nm light source and photographed using polaroid 667 positive film. DNA size standards included the yeast chromosome (2200–225 kb) and a 
ladder (50–1000 kb).
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Results |
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Experiments to study chromosomal damage with PCC
Data from two donors, showing the same trend, were grouped and are reported in Table I
, which shows the frequency of damaged cells and the distribution of chromosome fragments. Cells containing from one to 10 fragments were grouped as cells with 47–57 elements and in this range no cells with less than five fragments were found; cells containing from 10 to 20 fragments were grouped as cells with 57–68 elements.
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A significant dose-dependent increase in chromosome breaks was found and at the highest dose (5 mM) the chromosomes were severely damaged (Figure 1
); no chromosomal damage was detected at the lowest dose (0.2 mM) (Table I
).
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Experiments to study apoptosis
Analysis and quantification of cell death
Figure 2A and B shows the results from the same experiment performed for PCC analysis. Data obtained by analysis of nuclear morphology (Figure 2A) show a dose- and time-dependent increase in fragmented and pycnotic nuclei after treatment with MMS for 1 h. Moreover, at later recovery times an increase in the frequency of Trypan blue-positive cells was found (Figure 2B), indicating that the apoptotic process was in the final stages, in which membrane and cytoplasmic damage determine accessibility of the dye, resembling features typical of necrosis (secondary necrosis), already at 24 h after treatment. Statistical analysis by the 2 test did not demonstrate a significant difference (P > 0.01) in apoptotic cell death induction between unstimulated and stimulated human lymphocytes.
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DNA fragmentation study by PFGE
Figure 3
shows dose- and time-dependent HMW DNA fragmentation. For the lower doses (0.4 or 0.6 mM) the DNA fragments had different molecular sizes, mostly corresponding to 2200–600 kb, immediately after a 1 h MMS treatment. At later sampling times (12 and 24 h) a decrease in the sizes of the fragments to 300–50 kb, typical hallmarks of apoptosis, was detected. For the highest doses (1.3 and 2.5 mM) the apoptotic process induced by MMS seems to be much faster, since fragment sizes of 300–50 kb were found soon after the 1 h treatment. These DNA fragments are then further degraded (<50 kb) at later recovery times. The pattern of DNA fragmentation is qualitatively the same in both stimulated and unstimulated cells.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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As it has previously been shown that DNA lesions produced by CPT (e.g. `protein-concealed' DNA SSBs) induce chromosome fragments in prematurely condensed G0 HPBL (Mosesso et al., 1999
), in this work we wanted to study the role of PCC chromatin condensation in the conversion of MMS-induced SSBs to DSBs in G1 human lymphocytes. Exposure of cells to alkylating agents results in formation of DNA modifications that can be detected as SSBs after denaturation of the DNA in alkaline medium with the elution technique (Kohn et al., 1976). MMS belongs to the class of compounds requiring an intervening S phase for the production of chromatid-type aberrations. Therefore, we would not expect any induction of chromosomal aberration in G1. As in the case of CPT G1 treatment, chromosome fragments were also observed in G1 MMS-treated HPBL by the PCC technique performed in G1 cells.
Chromosome fragmentation was dose dependent (Table I) and characterized by heavily damaged cells at the highest dose (Figure 1). This observation induced us to investigate further whether this effect was more connected with the triggering of apoptotic cell death than a consequence of the PCC technique.
In order to prove this hypothesis the Trypan blue exclusion assay, nuclear morphology analysis and PFGE were carried out. The results obtained by nuclear morphology analysis clearly indicate that MMS triggers induction of apoptosis: at the lower doses the apoptotic cell frequency is already high. At the latest recovery time after drug exposure (24 h) the apoptotic programme was completely executed. In fact, at this time the Trypan blue assay revealed a considerable percentage of cells that had lost integrity of the cytoplasmic membrane (TB+), a terminal step of apoptosis (Wyllie et al. 1980; Wyllie, 1981). These events were the same in both unstimulated and stimulated lymphocytes, indicating that the induction of apoptosis by MMS is not modified during the cell proliferation induced by PHA.
The PFGE analysis shows that MMS induces widespread DNA degradation (Figure 3). At lower doses (0.4 and 0.6 mM) DNA is cleaved into fragments with molecular sizes in the 2200–600 kb range immediately after treatment. Subsequently, at later sampling times, these DNA fragments seem to be temporally and hierarchically degraded to molecular sizes that are characteristic of apoptotic cell death (300–50 kb) (Walker et al., 1994; Beer et al., 1995). At the higher doses of MMS the execution of apoptosis was so rapid that the generation of 50–300 kb fragments occurred during the MMS treatment. There was no difference between unstimulated and stimulated lymphocytes, in agreement with the data from the nuclear morphology analysis (Figure 2).
These results suggest that the chromosome fragmentation observed in PCC G1 HPBL is connected more with the onset of apoptotic cell death induced by MMS treatment than with conversion of SSBs to DSBs caused by the PCC technique. Other possibilities cannot be excluded, such as disruption of chromosomes at sites where SSB are near to each other on both DNA strands or disruption of repair intermediates.
Walker et al. (1997) demonstrated that DNA fragmentation in apoptosis could be initiated and propagated by SSBs that are most likely attached to the nuclear matrix, therefore it is plausible that MMS-induced SSBs have the same effect. Another possible pathway by which MMS could induce apoptosis is activation of the membrane Fas/APO-1 (CD95) receptor (Faris et al., 1998; Kasibhatia et al., 1998), leading to the death-signalling cascade by expression of the JNK/SAPKs and p38 protein kinases (van Dam et al., 1995; Liu et al., 1996; Wilhelm et al., 1997; Kolbus et al., 2000).
In conclusion, these findings assert that the PCC technique can detect chromosome fragmentation which reflects, in some circumstances, pre-apoptotic stages and therefore using this technique one should consider that the observed chromosome fragmentation could be overestimated.
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Acknowledgments |
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We gratefully thank Ms B.Fazzini for the photographic reproductions. N.P. de la P. was visiting the University of Tuscia as a PhD student with a scholarship for the Erasmus project. This work was supported by MURST and the University of Tuscia (60%).
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Notes |
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1 To whom correspondence should be addressed. Tel: +39 0761 357206; Fax: +39 0761 357242; Email: palitti{at}unitus.it
The first two authors contributed equally to this work
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Received on May 19, 2000; accepted on November 7, 2000.
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