Mutagenesis vol. 27 no. 3 pp. 323–327, 2012
Advance Access Publication 15 November 2011
doi:10.1093/mutage/ger082
DNA replication occurs in all lamina positive micronuclei, but never in lamina
negative micronuclei
Atsushi Okamoto, Koh-ichi Utani1 and Noriaki Shimizu*
Department of Biofunctional Science and Technology, Graduate School of
Biosphere Science, Hiroshima University,
Higashi-hiroshima, Hiroshima 739-8521, Japan,
1
Present address: Laboratory of Molecular Pharmacology, National Cancer
Institute, National Institute of Health, Bethesda, MD 20892, USA.
*
To whom correspondence should be addressed. Tel: þ81-824-24-6528;
Fax: þ81-824-24-0759; Email: [email protected]
Received on September 3, 2011; revised on October 7, 2011;
accepted on October 17, 2011
A micronucleus is a small nucleus-like structure found in the
cytoplasm of dividing cells that suffered from genotoxic stress.
It is generally hypothesised that micronuclei content is
eventually lost from cells, though the mechanism of
how this occurs is unknown. If DNA located within the
micronucleus is not replicated, it may explain the loss of
micronuclei content. Because there had been no compelling
evidence for this issue, we have addressed whether DNA
located within the micronucleus is replicated this issue. Pulse
labelling of bromodeoxyuridine revealed that DNA synthesis
takes place in a portion of micronuclei that contain nuclear
lamin B protein. By using iodine 3#-deoxyuridine/chlorodeoxyuridine double labelling, we found that all micronuclei
containing lamin B are replicated during one cell cycle,
whereas micronuclei lacking lamin B are never replicated.
This result suggests that the content of lamin B-negative
micronuclei is lost during cell division. Furthermore, we
simultaneously visualised sites of DNA synthesis, lamin B
and the extrachromosomal double minutes chromatin,
which contain amplified oncogenes. We found that
although the replication timing of double minutes was
generally preserved in micronuclei, at times it differed
greatly from the timing in the nucleus, which may perturb
the expression of the amplified oncogenes. Taken together,
these findings uncovered the DNA replication occurring
inside micronuclei.
broken chromatin bridge connecting the separating anaphase
chromosome. Furthermore, micronuclei can be generated from
aggregates of extrachromosomal elements such as double
minutes (DMs) chromatin bodies, which is a cytogenetic
manifestation of gene amplification (3). This type of micronuclei is referred to as DM-type, while the other micronuclei
are known as chromosome type (4). In addition to post-mitotic
generation, micronuclei are also formed during interphase by
nuclear budding (5). The nuclear buds may form through the
lamina break, which is coupled to cytoplasmic membrane
blebbing (Koh-ichi Utani, Atsushi Okamoto and Noriaki
Shimizu, submitted for publication). On the other hand, there
are micronuclei that lack the lamin B protein, a constituent of
nuclear lamina, as well as others that contain lamin B. It has
been suggested that micronuclei lacking lamin B are generated
by broken chromatin bridges (6) and/or by nuclear budding
through the lamina break (Koh-ichi Utani, Atsushi Okamoto and
Noriaki Shimizu, submitted for publication).
It is commonly hypothesised that the content of micronuclei is
ultimately eliminated from cells, though the mechanism of how
this occurs remains unknown. The micronuclei may be degraded
in situ in cytoplasm (7). Alternatively, they may be extruded out of
cells since extracellular micronuclei have been detected in culture
fluid (8). Another possibility is that no DNA replication occurs in
micronuclei, leading to the dilution of the unreplicated micronuclei
DNA during cell division. However, previous studies using shortterm bromodeoxyuridine (BrdU) pulse labelling revealed that at
least a portion of micronuclei underwent DNA synthesis, though
there were also micronuclei that did not incorporate BrdU (5,9,10).
Since DNA replication inside micronuclei has not been well
studied (reviewed in ref. 7), we used several modern in situ
techniques to examine this process more extensively.
Materials and methods
Introduction
Cell and cell culture
Human colorectal tumour COLO 320DM (CCL 220) cells (11) and human
cervical tumour HeLa cells (6) were obtained and maintained as described
previously. For the experiments shown in Figure 2, HeLa cells at the mitotic
phase were collected by the mitotic shake-off. The cells were cultured for 14 h
in normal conditions and then chlorodeoxyuridine (CldU) and cytochalasin B
(4 lg/ml; Sigma, St Louis, MO, USA) were added to the culture.
The micronucleus is a small nucleus-like structure that forms
within cytoplasm, separately from the main nucleus (1,2). It
can be detected in almost all growing mammalian cells in
response to a wide variety of stimuli that cause genome
damage. Therefore, the presence of micronuclei is widely used
as a hallmark of genotoxic stress. Micronuclei are generated
by several different mechanisms. They can be generated postmitotically from the lagging chromatid, and such ‘chromatin
laggards’ can be derived from (i) an acentric chromosome
fragment generated by drugs or radiation that induce DNA
breaks, (ii) a whole chromosome that was not bound to
or merotelically bound to the spindle microtubule or (iii) a
Cytochemical procedure
BrdU (Sigma), iodine 3#-deoxyuridine (IdU) (Sigma) or CldU (ICN pharmaceuticals, Inc., Irvine, CA, USA) is a halogenated thymidine analogue, and it was used
to label the sites of DNA synthesis. Indirect immunofluorescence detection of
BrdU or the simultaneous detection of IdU and CldU was performed using
antibodies and the protocols described in our previous paper (12). The
incorporated CldU was detected with a monoclonal rat anti-BrdU antibody (6
lg/ml; OBT0030; Oxford Biotechnology), and IdU was detected with a monoclonal mouse anti-BrdU antibody (1.5 lg/ml, cat. no. 347580; Becton Dickinson).
The former antibody binds to both BrdU and CldU but not to IdU, while the latter
antibody strongly binds to both BrdU and IdU and weakly to CldU. Therefore,
samples were first incubated with the rat anti-BrdU antibody for 1 h at 37°C to
detect CldU and then incubated with the mouse anti-BrdU antibody for 1 h at 37°C
to detect IdU. These primary antibodies were detected by Alexa Fluor
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647-conjugated donkey anti-mouse IgG (Invitrogen, Co.) and Alexa Fluor 488conjugated donkey anti-rat IgG (Invitrogen, Co.).
In the experiment shown in Figures 3 and 4, DNA synthesis was detected by
EdU (5-ethynyl-2#-deoxyuridine) labelling. Because fluorescent detection of
EdU employs only a chemical reaction, it enabled the simultaneous detection of
lamin B by immunofluorescence and of DMs by fluorescence in situ
hybridization, both of which require multiple antibody reactions. Labelling
and detection of EdU was performed as described previously (13). In short,
EdU (Invitrogen) was added to the culture to a final concentration of 10 lM and
incubated for the indicated times. After labelling EdU, the cells were harvested,
washed with phosphate-buffered saline (PBS) and fixed with 3% paraformaldehyde (PFA) for 10 min at room temperature. Then, the cells were
cytocentrifuged onto poly L-lysine-coated slides. The slides were reacted with 1
mM CuSO4, 100 mM ascorbic acid and 10 lM Alexa Fluor 647 azide (Invitrogen)
for 30 min at room temperature and were washed with PBS. Denaturation of
DNA, hybridisation with DIG-labelled probe prepared from c-myc cosmid DNA,
and simultaneous detection of the hybridised probe and the lamin B protein was
performed using the same procedure as described previously (10).
Immunofluorescent detection of lamin B protein used 5 lg/ml Goat anti
Lamin B (M-20; Santa Crutz Biotechnology, Inc.) and 10 lg/ml Texas redconjugated rabbit anti-goat IgG (EY Laboratories).
cultures with BrdU for 2 h to label the DNA replication sites,
fixed the cells and visualised the BrdU-labelled DNA and the
nuclear lamin B protein simultaneously. Representative images
of HeLa cells are shown in Figure 1A–F. We observed both
lamin B-positive and negative micronuclei. Presence of both
type was reported for chromosome-type (6,14–16) or DMtype micronuclei (10,16). While we were able to detect DNA
replication in some lamin B-positive micronuclei (Figure 1A and
B), we did not detect it in another portion of lamin B-positive
micronuclei (Figure 1C and D) nor in any lamin B-negative
micronuclei (Figure 1E and F), despite the fact that DNA
replication occurred within the main nucleus. To address
whether this was due to cells being in different cell cycle stages,
we classified the cells into early, middle and late S phase as
well as G1/G2 phase according to the nuclear distribution
Microscopy
The images appearing in Figures 1 and 2 were obtained with an Olympus
FV10-ASW confocal system on FV1000D-IX81 with a 60 objective
(UPLSAPO NA 1.35, oil). The images appearing in Figure 3 were obtained
with a Nikon inverted microscope (TE2000-E; Nikon, Tokyo) with a 60
objective (Nikon, Tokyo, Plan Apo VC 1.40, oil). All images were processed
and assembled using Adobe Photoshop CS version 8.0.1 (Adobe Systems Inc.).
Results and discussion
BrdU pulse labelling revealed that DNA replication occurs in
lamin B-positive micronuclei
About 5% of logarithmically growing cultures of cells (HeLa
or COLO 320DM) contained micronuclei. We treated these
Fig. 1. BrdU pulse labelling revealed that DNA replication occurs in
micronuclei. HeLa cells (A–G) or COLO 320DM cells (H) were pulse treated
with 100 lM BrdU for 2 h and then harvested and fixed with PFA. The
location of BrdU incorporation and the lamin B protein was visualised
simultaneously, and confocal images were obtained (A–F). Micronuclei were
classified according to lamin B status and BrdU incorporation. After
examination of 149 (HeLa) or 115 (COLO 320DM) micronuclei, each type of
micronuclei were quantified and plotted in G and H.
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Fig. 2. DNA replication occurs in all lamin B-positive micronuclei but not in
lamin B-negative micronuclei. (A) Outline of the experimental procedure.
Logarithmically growing COLO 320DM or cell cycle-synchronised HeLa cells
were cultured in the presence of 30 lM CldU and 4 lg/ml cytochalasin B for
12 h. Then, 100 lM IdU was added during the last 20 min. The cells were
harvested and fixed with PFA. Incorporated CldU and IdU as well as lamin B
protein were simultaneously detected, and DNA was counterstained by
4#,6-diamidino-2-phenylindole (DAPI). The representative images are shown
in B–G. Mononucleated cells that were CldU-labelled, but not IdU-labelled
(D–F), were designated as G2 phase cells. The COLO 320DM and the HeLa
culture contained 5.5 and 32.5% G2 phase cells, respectively. The number of
micronuclei in G2 phase cells, as classified by the presence or absence of CldU
signal and lamin B within micronuclei, were plotted (H and I).
DNA replication in micronuclei
shown). However, labelling the cells with BrdU for an entire
cell cycle (24 h) resulted in the delay of cell cycle
progression, perhaps due to the cytotoxic effect of BrdU.
Furthermore, continuous BrdU labelling induced DNA damage
in the labelled chromatin, which generated BrdU-positive
micronuclei after mitosis that hindered analysis. To overcome
this technical obstacle, we used a double labelling technique in
the following section.
Fig. 3. Simultaneous imaging of DNA replication sites, lamin B and DMs
revealed the timing of replication in DM-type and chromosome-type
micronuclei. Logarithmically growing COLO 320DM cells were treated with
EdU for 2 h and fixed with PFA. The incorporated EdU, lamin B and DMs
were simultaneously detected by different fluorescent colours, and DNA was
counterstained with 4#,6-diamidino-2-phenylindole (DAPI). The representative
images are shown in A–E. COLO 320DM (F) or HeLa (G) cells were cultured
in the presence of 4 lg/ml of cytochalasin B for 24 h. The cells were treated
with 100 lM BrdU for 30 min and then fixed with PFA. BrdU was detected as
in above and counterstained with DAPI. The binucleated cells showing
differential replication timing are shown.
pattern of BrdU labelling (5). DNA replication takes place at
the inner euchromatin during early S phase (Figure 1B, C, D
and F), at the peri-nuclear and the peri-nucleolar heterochromatin during middle S phase (Figure 1A and E) and at the
internal large heterochromatin during late S phase (not shown
in Figure 1 and see Figure 2B). We counted micronuclei with/
without lamin B and with/without BrdU labelling at each cell
cycle stage (Figure 1G and H) and found that no lamin Bnegative micronuclei displayed a BrdU signal at any point
within the cell cycle. On the other hand, we were able to detect
DNA replication in lamin B-positive micronuclei, most frequently during early S phase. However, even during early S, the
BrdU signal was detected in only a portion of the lamin Bpositive micronuclei. There are two possible explanations for
this observation (i) DNA replication is permitted in only a
portion of lamin B-positive micronuclei or (ii) DNA replication
is permitted in all lamin B-positive micronuclei, but the limited
BrdU labelling time (2 h) may prevent all lamin B-positive
micronuclei from being labelled since S phase typically spans
10 h. We found that the number of BrdU-positive micronuclei
increased when the BrdU labelling time increased (data not
DNA replication occurs in all lamin B-positive micronuclei but
not in lamin B-negative micronuclei
To determine whether every micronucleus is replicated during
S phase (10 h), we performed a double pulse-chase experiment
using IdU and CldU, instead of a single BrdU pulse labelling.
Namely, we cultured logarithmically growing COLO 320DM
cells in the presence of CldU and cytochalasin B for 12 h,
added IdU during the last 20 min of culture (Figure 2A) and
then harvested the cells. We fixed the cells with PFA and
detected the incorporated CldU, IdU and lamin B protein
simultaneously by using specific antibodies. CldU or IdU, as
BrdU, was a halogenated thymidine analogue that label the
site of DNA replication. Its labelling pattern among the nucleus
correlates to the position in the S phase, as shown above for
BrdU-labelling. When cells in late S phase were harvested, the
IdU label showed a late S phase pattern and the CldU labelling
represented DNA in the main nucleus that was replicated
during the 12 h prior to harvest (Figure 2B). If the cells passed
through mitosis, they appeared as binucleated cells since
cytochalasin B inhibited cytokinesis (Figure 2C). Therefore,
G2 phase cells were identified as cells containing a CldU signal
in the absence of an IdU signal. The representative image
shown in Figure 2D shows that a CldU signal was detected
in the lamin B-positive micronucleus. Out of the 85 lamin Bpositive micronuclei (Figure 2H), 84 were labelled with CldU,
while only one was not (Figure 2F). It is possible that S phase
progression was partly delayed in that the cell lacking a CldU
signal and thus required .12 h of CldU labelling. Apart from
this exception, we concluded that all lamin B-positive micronuclei are replicated during S phase. By contrast, lamin Bnegative micronuclei in G2 phase were not labelled with CldU
(Figure 2E and H), suggesting that DNA replication does not
occur in lamin B-negative micronuclei. Similar results were
obtained for cell cycle-synchronised HeLa cells (Figure 2I).
DNA replication timing of DM-type and chromosome-type
micronuclei
During the above experiment, we noticed that some micronuclei actively incorporated IdU despite the fact that its
neighbouring nucleus had already completed DNA synthesis
(Figure 2G). Therefore, we examined the replication timing
of not only the usual chromosome-type micronuclei but also
DM-type micronuclei in COLO 320DM cells. DMs are extrachromosomal, transcriptionally active euchromatin (16,17) that
are replicated during early S phase within the nucleus (18). To
detect DMs, lamin B and the DNA replication sites simultaneously, we labelled DNA replication sites with EdU. We
used EdU instead of halogenated thymidine analogue, because
detection of EdU required only chemical reaction, and it enabled
the simultaneous detection of lamin B and DMs. The representative images are shown in Figure 3A–E, and all the images
contain lamin B-positive DM-type micronuclei. In Figure 3A
and B, DNA replication was detected in the micronucleus when
the nucleus was at early S phase (A) or middle S phase (B). As
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A. Okamoto et al.
shown in Figure 3C, DNA replication was not detected in the
micronucleus, when the nucleus was in late S phase. Strikingly,
there were other micronuclei that replicated while the nucleus
was in late S phase (Figure 3D) and in G1/G2 (no nuclear EdUlabelling; Figure 3E). These events were quantified, and the
frequencies were plotted in Figure 4A. The micronuclei containing EdU signals were most frequently associated with nuclei
in early or middle S phase. Because DMs are early replicating
euchromatin in the nucleus (18), the results suggest that the
timing of replication in the nucleus was similar to the one in
the micronuclei. However, a portion of DM-type micronuclei
replicated while the nucleus was in late S to G1/G2 phase
(Figures 3D and E and 4A), consistent with Figure 2G,
suggesting that the replication timing of some DMs is not
synchronised with nuclear replication. On the other hand, the
replication timing of the chromosome-type micronuclei appeared to be distributed more randomly because approximately
half of the micronuclei replicated at each time point examined
(Figure 4B).
Differentiation of replication timing between the micronucleus
and the nucleus
As described above, we demonstrated that the timing of
micronuclei replication differed from that of nuclear replication, despite being within the same cytoplasm. Interestingly,
we also found that the timing of DNA replication also differed
between two daughter nuclei within the same cytoplasm, i.e.
binucleated cells, generated by cytochalasin B treatment
(Figure 3F and G). When 180 binucleated cells that contained
at least one nucleus in S phase were examined, we found that
the replication timing was identical between sister nuclei in 162
cells but was different in 18. A similar phenomenon has been
reported previously (19,20). Thus, it appears that the difference
in replication timing between the micronucleus and the nucleus
parallels the difference in replication timing between the two
nuclei of binucleated cells. Importantly, the time when DMs
are replicated may differ from the time when the same DMs are
replicated in the nucleus, i.e. early S phase. This difference in
replication timing may affect transcriptional control and may
ultimately influence tumour cell phenotype.
Implications of this study
Here, we showed for the first time that lamin B-positive
micronuclei replicate during the cell cycle and likely prevents
the dilution of micronuclear content during cell division. DNA
replication within a micronucleus does not necessarily mean
Fig. 4. DNA replication timing was conserved in micronuclei, but at times
differed significantly from the timing in the nucleus. Slides prepared similarly
to slides from Figure 3A–E were examined. The cell cycle stage of the nucleus
was estimated by the EdU labelling pattern. Of the cells in the culture, 5.5%
contained micronuclei. A total of 109 micronuclei with DMs (A; DM type) and
114 micronuclei without DMs (B; chromosome type) were classified according
to the presence or absence of lamin and EdU signal within micronuclei.
326
that the resulting chromosome fragments will segregate
properly to the daughter cells after the cell division. Thus,
the next task will be to determine whether the sister chromatids
generated in micronuclei are segregated and distributed equally
to the daughter cells during the next mitosis. However, this
may be difficult to determine since we previously showed that
cells containing micronuclei frequently produced cells with
additional micronuclei after undergoing mitosis (6).
By contrast, DNA replication was not detected in laminnegative micronuclei, indicating that their chromatin may be
lost during cell division. These lamin-negative micronuclei
were likely generated from broken chromatin bridges during
anaphase (6) or from interphase nuclear budding through the
lamina break (Koh-ichi Utani, Atsushi Okamoto and Noriaki
Shimizu, submitted for publication). Notably, transcription was
not detected inside these micronuclei (16). We reported that the
nuclear localisation signal-bearing protein did not enter the
lamin-negative micronuclei (16). Therefore, the protein required for replication and transcription may be missing in these
micronuclei. Understanding the nature of DNA replication and
segregation within micronuclei will be important for determining how the entrapment of genetic material within inert
micronuclei may contribute to the malignant nature of human
cancer cells.
Funding
This work was supported in part by a Grant-in-Aid for
Scientific Research (B) (17370002) and a Grant-in-Aid for
Challenging Exploratory Research (21657051) both from the
Japan Society for the Promotion of Science to N.S. and
a Grant-in-Aid for Scientific Research on Priority Areas—
Nuclear dynamics (19038016) from the Ministry of Education,
Science, Sports and Culture of Japan to N.S.
Acknowledgements
Conflict of interest statement: None declared.
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