Mutagenesis vol.16 no.2 pp.121–125, 2001
Detection of DNA primary damage by premature chromosome
condensation in human peripheral blood lymphocytes treated with
methyl methanesulfonate
C.Lorenti Garcia1, M.Carloni1, N.Palma de la Peña,
E.Fonti and F.Palitti1
Dipartimento di Agrobiologia, e Agrochimica, Università degli Studi della
Tuscia, Viterbo, Italy
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 dosedependent 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.
Introduction
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 N3methyladenine 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).
Materials and methods
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 (BioWhittaker). All steps were performed at room temperature. Isolated
lymphocytes were resuspended at final concentration of 1⫻106 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
whom correspondence should be addressed. Tel: ⫹39 0761 357206; Fax: ⫹39 0761 357242; Email: [email protected]
The first two authors contributed equally to this work
1To
© UK Environmental Mutagen Society/Oxford University Press 2001
121
C.Lorenti Garcia et al.
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.
Table I. Frequencies of damaged cells in MMS-treated G1 human
lymphocytes detected by the PCC technique and distribution of chromosome
fragments per cell
Treatment
(1 h)
Control
0.2 mM MMS
1.3 mM MMS
2.5 mM MMS
5 mM MMS
No. of
cells
scored
100
100
96
68
94
aHF, highly fragmented
bStatistically significant
Abnormal
cells (%)
0
0
39b
61b
95b
Distribution of fragments in
damaged cells (%)
47–57
58–68
HFa
0
0
25
38
6
0
0
10
5
17
0
0
4
9
77
(⬎20 fragments).
(P ⬍ 0.05, Fisher’s exact test) between control and
treated cells.
Analysis and quantification of cell death
For morphological analysis 5⫻105 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 1000⫻. 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 1⫻ 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 χ2 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,
6⫻105 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 0.5⫻ 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 λ ladder (50–1000 kb).
Results
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.
A significant dose-dependent increase in chromosome breaks
was found and at the highest dose (5 mM) the chromosomes
122
Fig. 1. Examples of PCC in untreated (A) and 2.5 mM MMS treated
HPBL (B).
were severely damaged (Figure 1); no chromosomal damage
was detected at the lowest dose (0.2 mM) (Table I).
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
Detection of DNA primary damage
Fig. 2. Analysis of cell death induction after 1 h of MMS exposure by nuclear morphology (A) and Trypan blue exclusion assay (B) in stimulated (⫹PHA)
and unstimulated (–PHA) HPBL at different recovery times. The results are reported as the means ⫾ SD of percentages of two donors.
nuclear morphology (Figure 2A) show a dose- and timedependent 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 χ2 test did not demonstrate a
significant difference (P ⬎ 0.01) in apoptotic cell death
induction between unstimulated and stimulated human
lymphocytes.
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
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C.Lorenti Garcia et al.
Fig. 3. HMW DNA fragments analysis by PFGE in stimulated (⫹PHA) and unstimulated (–PHA) HPBL after 1 h MMS treatment at different recovery times.
The photographs are an example of the results obtained from two donors. DNA size standards included the yeast chromosome (2200–225 kb) and a λ ladder
(50–1000 kb).
DNA fragmentation is qualitatively the same in both stimulated
and unstimulated cells.
Discussion
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
124
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
Detection of DNA primary damage
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.
Acknowledgements
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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Received on May 19, 2000; accepted on November 7, 2000
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