Download Molecular mechanisms of the origin of micronuclei

Mutagenesis vol. 26 no. 1 pp. 119–123, 2011
doi:10.1093/mutage/geq053
REVIEW
Molecular mechanisms of the origin of micronuclei from extrachromosomal elements
Noriaki Shimizu*
Graduate School of Biosphere Science, Hiroshima University, 1-7-1
Kagamiyama, Higashi-Hiroshima 739-8521 Japan
*To whom correspondence should be addressed. Graduate School of Biosphere
Science, Hiroshima University, 1-7-1 Kagamiyama, Higashi-Hiroshima 7398521, Japan. Tel: þ81 824 24 6528; Fax: þ81 824 24 0759; Email: shimizu
@hiroshima-u.ac.jp
Hereafter, these micronuclei are called ‘chromosome-type
micronuclei’. On the other hand, I will focus on another type
of micronuclei; i.e. the ‘double minute (DM)-type micronuclei’, which are formed from the extrachromosomal DMs or,
possibly, from a variety of extrachromosomal elements in
general. The chromosome-type and the DM-type micronuclei
are distinct entities, and they may form independently in
a single cell by partially overlapping mechanisms.
Received on May 11, 2010; revised on July 15, 2010;
accepted on August 10, 2010
In addition to micronuclei that are formed from chromosomal material (the chromosome-type micronuclei), there
are also micronuclei formed from extrachromosomal
elements [the double minute (DM)-type micronuclei].
These two types of micronuclei are distinct entities, which
exist and arise independently in a cell. A DM is a large
extrachromosomal element that consists of amplified genes
that are commonly seen in cancer cells; the aggregates of
DMs can eventually be expressed as DM-type micronuclei.
The question of how the DM-type micronuclei arise was
answered by uncovering the quite unique intracellular
behaviour of DMs during the cell cycle progression. This
behaviour of DMs appeared to be common among the
broad spectrum of extrachromosomal elements of endogenous, exogenous or artificial origin. Therefore, studying
the biology of DM-type micronuclei will enable us to
understand how these extrachromosomal structures may
be retained within a cell or expelled from the nucleus and
eliminated from the cell. This knowledge could also be used
for the treatment of cancers and the development of a new
mammalian host–vector system.
The chromosome-type and the double minute-type
micronuclei
As reviewed in other parts of this special issue, micronuclei
may arise from an acentric chromosomal fragment or a centric
whole chromosome that was not bound by the spindle
microtuble or that was merotelically bound by the microtubles
from both spindle poles. Such chromatin lags behind the
separating anaphase chromosomes and generates a micronucleus in the cytoplasm after the cells enter the next interphase.
In addition to such chromatin laggards, a chromatin bridge
between the separating anaphase chromosomes can also
generate micronuclei if the bridge breaks during the anaphase
to the cytokinesis transition. Because these micronuclei are
generated during an abnormal mitosis and because their
presence reflect the damage of genomic materials, the micronuclei may be used as a biomarker for the presence of
genotoxic stress induced by drugs or environmental influences.
DMs and the DM-type micronuclei
DMs are numerous paired minute chromatin bodies that were
often detected among 4#,6-diamidino-2-phenylindole- or
Giemsa-stained metaphase chromosome spreads prepared from
human cancer cells (for recent review, see refs 1,2). The
DMs appear in various kinds of human cancer cells but not
in normal cells. DMs are autonomously replicating acentric
chromatin bodies composed of circular DNA that do not
require a telomeric end. If they were visible under light
microscopy, the DNA would be more than a megabase pair
long. However, a smaller submicroscopic episome, which may
differ from DMs only in its size, may also be present in cancer
cells. It was proposed that such episomes might be precursors
of DMs (3,4).
Importantly, in addition to the chromosomal homogeneously
staining region, the extrachromosomal DMs and the episome
are the sites where the amplified genes reside. The amplification of several kinds of oncogenes or therapeutic drug-resistant
genes plays a pivotal role in the malignant transformation of
human cancer cells through the overproduction of specific
protein products. Therefore, the presence of DMs has important
implications for the cancer cell phenotype. Because of the
importance of gene amplification in malignant transformation
of cells, the decrease in the number of the amplified genes or in
the number of DMs in cancer cells might result in the loss of
the cancer cell phenotype, the proliferation arrest, the
expression of the differentiation markers or an increase in
apoptotic cell death (5–11). Therefore, elimination of DMs
from cancer cells may provide the possibility to cure many
cancers (12).
Such loss of DMs, or loss of amplified genes on DMs, might
be induced by the treatment of the cells with low concentration
of hydroxyurea (HU; ref. 13). Notably, such treatment also
induced the cytoplasmic micronuclei that were enriched with
the DMs, i.e. the ‘DM-type micronuclei’ (5), which were
detected by fluorescence in situ hybridisation (FISH) that
detects the amplified sequence on the DMs. Because micronuclei usually tend to be eliminated from the cells, the
generation of DM-type micronuclei may mediate the elimination of DMs. The DM-type micronuclei can be induced by
a broad range of DNA replication inhibitors at lower doses that
do not completely stop the cell cycle progression (14) or
radiation therapy (10).
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119
N. Shimizu
Intracellular behaviour of DMs and the generation of
DM-type micronuclei during the mitosis
The DM-type micronuclei are distinct entities from the
chromosome-type micronuclei, because they arise independently in a single cell, and an intermediate mixed type was
rarely detected (15). Furthermore, purified DM-type micronuclei contained essentially pure DM’s DNA (16). The reason
for the appearance of such DM-type micronuclei was obtained
from the novel intracellular behaviour of DMs during the cell
cycle progression (Figure 1).
The DMs are acentric chromatin, but they may segregate
stably to the daughter cells by sticking to the chromosome arms
during mitosis (Figure 1a; refs 17–20). Such a manner of
segregation is called ‘hitchhike’ or ‘hitchhiking’ (21,22). The
mechanism how the DMs stick to the chromosome arm is
virtually unknown. One publication suggests that the nucleolus-derived material that is located at the perichromosomal
sheath during the mitosis might mediate the binding of the
DMs to the chromosome (17).
Importantly, many kinds of viral nuclear episomes or
artificial episomes that bear the viral genes for replication/
segregation also use a similar hitchhike for their segregation
during cell division (21,22). Therefore, the hitchhike appears to
be a widespread mechanism that supports the transmission of
extrachromosomal elements to the nucleus of the daughter
cells. In the case of several viruses, the viral-encoded protein
and the chromosome targets that were bound by the viral
protein was identified. For example, (i) the Epstein-Barr virusencoded Epstein-Bar nuclear antigen-1 protein (18,23,24)
binds both the viral latent origin of replication (ori-P) and the
chromosomal EBP2 protein (25), (ii) Kaposi’s sarcomaassociated herpesvirus-encoded latency-associated nuclear
antigen protein binds to the nucleosomal histone H2A-H2B
(26) and (iii) the E2 protein of human papilloma virus (ref. 27)
e’
g
e
f
4
a
3
1
2
b
M
G2
G1
S
b
d
h
c
+HU
j
i
Fig. 1. Intracellular behaviour of DMs and the generation of DM-type
micronuclei. A novel intracellular behaviour of DMs during the cell cycle
progression, which explains how the DM-type micronuclei are generated, is
depicted. Green dots, DMs; blue, chromatin; red, nuclear lamina; solid arrow,
almost proven process by published data; dashed arrow, unproven hypothetical
process. This scheme may be applied to explain the behaviour of a broad
spectrum of extrachromosomal elements in general (For a–j, see the text).
120
or the bovine papilloma virus (BPV; refs 28–30) binds to the
C-terminal domain (CTD) of the chromosomal Brd4 protein
(30). These studies showed that the viral hitchhike target
proteins did not share their molecular function other than their
localisation at the chromosomal surface. Therefore, it is
probable that the docking of DMs onto chromosomes will be
different from that reported in the study of virus hitchhiking.
Interestingly, expression of Brd4-CTD released the viral DNA
from mitotic chromosomes in BPV-1-transformed cells, which
resulted in the complete elimination of the viral DNA and the
morphological reversion of BPV-1-transformed cells (31).
This strikingly resembled the case of DMs, where the
elimination of DMs resulted in the reversion of the tumour
cell phenotype (5–7).
The DMs that were delivered to the daughter cells by
hitchhiking frequently localised at the periphery of the nucleus
(Figure 1b; ref. 32). Curiously, the nuclear periphery is usually
occupied by heterochromatin, whereas DMs are euchromatin
because their genes are actively transcribed (15), and most of
the DMs are replicated at the early S phase (33). From the
apparently mismatched place, DMs relocate to the nuclear
interior during their replication in the early S phase (Figure 1c;
ref. 32), when the DMs themselves are replicated. Remarkably,
if the cells were treated with a low concentration of HU during
the early S phase, DMs tended to aggregate and detached from
the chromosome at the subsequent M phase (Figure 1d and e;
ref. 20,34).
HU is an inhibitor of the ribonucleotide reductase and
inhibits DNA replication. Various DNA replication inhibitors
induced the DM-type micronuclei, if used at a concentration
lower than that completely inhibited the DNA replication (14).
The treatment with a lower concentration of HU increased the
fraction of cells at S phase indicating transient cell cycle arrest
(14). Later study revealed that the lower concentration of HU
induced many gamma H2AX foci in the nucleus at the early S
phase (34). This suggested that HU induced DNA damage as
a result of the replication fork collapse. If the DMs were
simultaneously detected with the gamma H2AX, the latter
signal rarely coincided with the DMs because numerous
gamma H2AX foci appeared randomly throughout the nucleus.
If those cells were placed in fresh medium without HU, the
numerous gamma H2AX foci gradually disappeared, which
suggested the repair of DNA damage. Notably in these cells,
the remaining gamma H2AX signals associated with the
aggregated DMs (34). It suggests that the DNA repair in the
extrachromosomal DMs may be different from the one in
the chromosome arm and that the DNA damage in the
extrachromosomal DMs may induce their aggregation without
repair. The aggregated DMs are left behind the separating
anaphase chromosomes and they generate micronuclei at the
subsequent interphase (34).
We established cell lines that bear green fluorescent protein
(GFP)-tagged DMs (35), which were constructed by amplifying the lactose-operator sequence on DMs and visualising them
by the expression of the lactose repressor-GFP fusion protein.
Time-lapse observation of such cells revealed that the
aggregated DMs that lag behind the anaphase chromosome
actually formed micronuclei (Figure 1e). Furthermore, aggregates of DMs sometimes formed a bridge between anaphase
chromosomes (Figure 1e’; K. Utani and N. Shimizu, unpublished results). This was probably caused by the mutual
attachment of DMs to themselves and the attachment of the
DMs to the segregating chromosome. Severing of such a bridge
The DM-type micronucleus
at two points may also generate the DM-type micronuclei. The
former (Figure 1e) and the latter (Figure 1e’) mechanism
resemble the lagging chromatid and the chromatin bridge that
generate the chromosome-type micronuclei, respectively.
However, the aggregates of DMs or the bridges of DMs are
formed independently from the chromatin laggard or the
chromatin bridge, thus the DM-type and the chromosome-type
micronuclei are distinct entities.
Generation of DM-type micronuclei during the interphase
Most of the chromosome-type micronuclei are generated after
an abnormal mitosis, and the extensive live cell observation of
the micronuclei generation confirmed this argument (36–38).
On the other hand, because nuclear bud-shaped structure could
be detected among cytogenetically fixed preparations, the
generation of micronuclei during the interphase also appeared
to be possible. However, careful time-lapse studies using the
HeLa cell line that had GFP-tagged chromatin (HeLa H2BGFP) could not prove the possibility that the buds may produce
micronuclei (37,38).
On the other hand, if the DMs were detected by the FISH
among methanol/acetic acid-fixed cells (14) or paraformaldehyde-fixed cells (20), the DMs sometimes appeared as nuclear
buds (14). This suggested that, in addition to the mitotic
mechanism, an interphase mechanism might generate the DMtype micronuclei. The simultaneous detection of the nuclear
lamina and the DMs showed that the DMs might localise at the
cytoplasm as a nuclear bud-shaped structure without lamin
association (Figure 1g; ref. 20). Our recent time-lapse
observation of cells bearing GFP-tagged DMs suggested that
the DMs might move from the nucleus to the cytoplasm under
a DNA damage inducing condition, i.e. the presence of low
concentration of HU (Figure 1, arrow 4; K. Utani and
N. Shimizu, unpublished results).
In order to know the general rule that governs the
intranuclear behaviour of an extrachromosomal element,
DNA with several physical structures was microinjected and
its fate in the nucleus of several cell lines with different genetic
background was studied (39). The DNA injected at the nuclear
environment initially diffused depending on its length. The
long DNA (2800 to 15 000 bp) could not move from the
injected site, whereas the shorter DNA (400 bp) moved by
diffusion. In any case, the injected DNA was rapidly
aggregated, which suggested the presence of a mechanism that
aggregates negatively charged foreign DNA. Such aggregates
might remain in the nucleus for a long time and formed the
micronucleus-like structure at the cytoplasm after the cells
passed the mitosis. On the other hand, within few minutes after
the injection, a portion of the aggregate in the nucleus moved to
the cytoplasm (39). This image was very close to the abovementioned image of DMs (Figure 1g). On the other hand, one
study showed that the damaged fragmented DNA that was
bound by Rad 51 repair protein might move from the nucleus
to the cytoplasm (40). Therefore, these observations suggested
the presence of a mechanism that might actively eliminate the
extrachromosomal or the foreign damaged DNA from the
nucleus to the cytoplasm.
The heterogeneity among the micronuclei
Both the chromosome-type and the DM-type micronuclei are
heterogeneous in respect to their size, their chromatin
condensation level and their possession of a nuclear lamina.
This may relate to the origin of the individual micronucleus
(i.e. the lagging chromatid, the chromatin bridge or the
interphase buds). For example, the lagging chromatid most
likely generates the micronuclei with lamina and with relaxed
chromatin, whereas the chromatin bridge tends to generate the
micronuclei without lamina and with condensed chromatin
(38). Because gene expression is restricted to the micronuclei
with lamina (15), it was consistent with a report that the
anaphase bridge breakage produced genetically inert micronuclei (36). Another reason that determines the heterogeneity
among micronuclei may be obtained from the position in the
anaphase cells where the chromatin that generates micronuclei
locates. It was reported that histone modification was critically
different between the spindle mid-zone and the region around
the anaphase chromosome (41) and such histone modification
might determine the heterogeneity of micronuclei. Furthermore, the heterogeneity may also relate to the cell cycle
position. Namely, the fraction of the DM-type micronuclei
without lamina was low in the G1 phase, and the micronuclei
with lamina increased after the S phase begun, when the
synthesis and large-scale rearrangement of lamin B protein
progresses (arrow 3 in Figure 1; ref. 20).
Presence of lamina around the micronuclei had important
implications because transcription (15) or DNA replication
(A. Okamoto and N. Shimizu, unpublished results) was
detected only in the micronuclei with lamin B protein. Because
the DMs had amplified genes that determine the malignant
phenotype of the cancer cells, its abnormal transcription in the
lamina-positive micronuclei should have important impact on
cancer cells; alternatively, its transcriptional silencing in the
lamina-negative micronuclei may also have impact on the cells
by changing the gene expression balance.
Elimination of the micronuclear content
It is generally thought that the DNA content of micronuclei
tends to be diminished or the micronuclei eliminated from the
cells. However, the mechanism is not clarified. There are few
possibilities. The first of which is that the micronuclei is
degraded in situ. It may be plausible, but others and we could
not detect such event during the time-lapse observation of the
cells with GFP-tagged chromatin (37,38), and at least the
observed micronuclei were maintained in the cells quite stably
during one cell cycle. The second possible mechanism is that
the content of micronucleus is diluted during the cell division if
it is not replicated. Replication in the micronuclei was
suggested because there were micronuclei that had multiple
copies of the X chromosome (ranging from 4 to 10) in cultured
human lymphocytes (42). More directly, incorporation of
bromodeoxyuridine (BrdU) was detected in situ in the
micronuclei (20). Furthermore, a carefully conducted BrdUincorporation experiment suggested that the lamina-positive
micronuclei are replicated during one cell cycle, whereas the
lamina-negative micronuclei are not replicated (A. Okamoto
and N. Shimizu, unpublished results). Therefore, the DNA
content of lamina-negative micronuclei will probably be lost
during cell division.
Another mechanism for the elimination of micronuclear
content is the direct extrusion of the micronuclei from the
cells to the outside of the cell. We showed already that
extruded DM-type micronuclei were detected in the culture
medium (Figure 1j; ref. 43). Such micronuclei had both
121
N. Shimizu
a nuclear lamina and a cytoplasmic membrane. They were
enriched with DMs and the DNA inside them was not
degraded. Recent time-lapse observation of the cells bearing
GFP-tagged DMs suggested that the micronuclei extrusion
was mediated by the cytoplasmic membrane blebbing (Figure
1i; K. Utani and N. Shimizu, unpublished results). Because
the extruded DM-type micronuclei had aggregates of DMs
and possessed a cytoplasmic membrane (43), it is possible
that a membrane fusion event will enable the transfer of the
micronuclear content between cells. If it actually occurs, it
will explain the genotype diversification observed in tumour
tissue or it will enable horizontal gene transfer of extrachromosomal elements like viruses and will provide an alternative
approach for gene transfer.
Recommendations for future research
The studies reviewed in here have wide implications because
the proposed mechanisms and the newly uncovered questions
might be relevant not only to the maintenance and the
elimination of DMs but also to the maintenance and the
elimination of a variety of extrachromosomal elements. These
elements may include endogenous elements excised from the
chromosome arm, exogenous elements derived from viruses or
an artificially introduced vector used in experimental, industrial
or therapeutical purposes. The artificial extrachromosomal
plasmid actually generated the DM-type micronuclei (44,45).
Such universality may derived from that the hitchhiking way is
the only known way by which acentric extrachromosomal
elements might be delivered to the daughter nuclei and that
several kinds of extrachromosomal elements co-localise in the
nucleus (46,47 and N. Shimizu et al., unpublished results)
perhaps at the common inter-chromosomal domains in the
nucleus (44).
In order to develop such a field of research, many future
tasks remain. For example, we need to understand the
molecular details how the DMs attach to the mitotic
chromosome and how DMs aggregate. Additionally, the
mechanism of how DMs move from the nucleus to the
cytoplasm during the interphase is an interesting theme.
Furthermore, we need to answer the questions of what does
the structural heterogeneity of micronuclei mean. Understanding how micronucleus elimination proceeds is also the future
most important task. Interestingly, our experiments have
suggested that the stability of extrachromosomal elements
may be quite different between the cell lines (unpublished
results), and the generation of extrachromosomal elements is
limited to the cells with a specific genetic background.
Determining the genes involved in the stable maintenance
of an extrachromosomal element would enable us to understand and to develop a stable episomal vector in mammalian
cells.
Funding
This work was supported in part by a Grant-in-Aid for
Scientific Research (B) (17370002) from the Japan Society for
the Promotion of Science to N.S.; 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.; a Grant-in-Aid for challenging Exploratory
Research (21657051) from the Japan Society for the Promotion
of Science to N.S.
122
Acknowledgements
I acknowledge Ms Rita Kapoor from Osnabruck University (Germany) for her
reading the manuscript and kind suggestions on it.
Conflict of interest statement: None declared.
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