From the Department of Molecular Biology, Institute
of Cellular and Molecular Biology, the ¶ Third Department of
Anatomy, Okayama University Medical School, Okayama 700-8558, Japan and
the
Laboratory of Medical Mycology, Research Institute for
Disease Mechanism and Control, Nagoya University School of Medicine,
Nagoya 466-8550, Japan
Received for publication, September 18, 2000, and in revised form, November 29, 2000
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ABSTRACT |
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Two isoforms of DNA topoisomerase II (topo II)
have been identified in mammalian cells. While topo II DNA topoisomerase II (topo
II)1 is essential for cell
proliferation since some mitotic events such as condensation and
segregation of daughter chromosomes are entirely dependent on its
activity. It is also likely to be involved in other DNA transactions
like DNA replication, transcription, and recombination (1). The enzyme,
in a heart-shaped dimer form (2), catalyzes topological transformations
of DNA by transient cleavage of DNA double strands forming an
enzyme-operated gate, followed by passing a second double-stranded DNA
segment through the gate (3, 4). Only one form of topo II is present in
lower eukaryotes and invertebrates whereas in mammalian cells two
isoforms, the 170-kDa topo II In contrast to topo II The cerebellar cortex has a unique structure with laminar arrangement
of cells that is formed during the development in early postnatal
period. A number of mRNAs were identified to be expressed specifically at distinct stages of cerebellar development (22, 23). We
have shown previously using in situ hybridization that in
the first two postnatal weeks the signal levels for topo II Antibodies
Topoisomerase II Topoisomerase II Amphiphysin I--
IgG was purified from the serum of a breast
cancer patient with anti-amphiphysin I autoantibodies and
paraneoplastic sensory neuropathy (27).
Neurofilament--
A mouse monoclonal antibody to the rat
neurofilament proteins (160 K/210 K) was purchased from IBL (Gunma, Japan).
Western Blot Analysis
Nuclei were prepared from cerebellum, cerebral cortex, and
thymus of male Wistar rats as described previously (28). Nuclear samples were pretreated with 50 µg/ml DNase I at room temperature for
30 min prior to electrophoresis. Topo II-enriched fraction was
extracted from the purified nuclei with 20 mM Tris-HCl (pH 7.5), 0.3 M NaCl, and 140 mM 2-mercaptoethanol.
Cultured granule cells were directly lyzed in the loading buffer with a
brief sonication. Samples were heated at 95 °C for 1 min in a
loading buffer and then subjected to SDS-polyacrylamide gel
electrophoresis. Separated proteins in gels were transferred to
nitrocellulose membranes by electroblotting and processed for
immunodetection according to a standard procedure. Secondary antibodies
were peroxidase-conjugated goat antibodies (Bio-Rad) and the peroxidase
activity was visualized with 4-methoxy-1-naphthol and hydrogen peroxide.
Immunohistochemical Detection
Male Wistar rats under deep anesthesia were perfused
transcardially with 0.9% saline, followed by 4% paraformaldehyde and 0.2% picric acid in 100 mM sodium phosphate (pH 7.4). The
brain was dissected out, post-fixed with the same solution, and
cryoprotected with 20% sucrose in 100 mM sodium phosphate
(pH 7.4). After trimming, the brain blocks were frozen rapidly and
sectioned at a thickness of 10 µm on a cryostat.
Frozen sagittal sections were thaw-mounted on glass slides precoated
with 3-aminopropyltriethoxysilane, followed by washing with PBS (150 mM NaCl in 10 mM sodium phosphate, pH 7.4),
blocking with PBS containing 10% goat serum and 0.3% Triton X-100,
and incubation for 15 h with primary antibodies diluted in PBS
containing 0.3% Triton X-100 (TPBS). The incubation was performed
either at 37 °C (monoclonal antibodies against topo II isoforms) or
at room temperature (all the other antibodies). After washing with TPBS, the sections were incubated successively at room temperature with
biotinylated goat secondary antibodies (Vector Labs) and with the ABC
reagent (Vectastain ABC Kit, Vector Labs). The peroxidase reaction was
visualized with diaminobenzidine and hydrogen peroxide.
Cultured granule cells that were grown on coverslips precoated with
poly-L-lysine were fixed with 4% paraformaldehyde in PBS at room temperature for 15 min. The fixed cells were immunostained as
described above for tissue sections.
Immunofluorescence Microscopy and Confocal Microscopy
The brain tissue was perfused as described above except that the
solution did not contain picric acid. Frozen sagittal sections (14 µm) were mounted on glass slides as mentioned, washed with PBS,
permeabilized with 0.3% Triton X-100 in PBS, and blocked with 10%
goat serum and 10% horse serum in TPBS. After incubation with various
primary antibodies diluted in TPBS containing 3% horse serum for
15 h, sections were washed with PBS, followed by incubation with
fluorescent conjugates of goat secondary antibodies in PBS at room
temperature for 30 min. DNA was then stained with 0.25 µg/ml
4',6-diamidino-2-phenylindole in PBS. Stained sections were mounted in
an antifade solution (Prolong Antifade Kit, Molecular Probes, Eugene,
OR) and examined under an OLYMPUS epifluorescence microscope (BX60-FLB)
coupled to a cooled monochromatic CCD camera (Hamamatsu Photonics) or
OLYMPUS laser scanning microscope (FLUOVIEW-BX50). Recorded digital
images were processed later using Adobe Photoshop software.
Determination of Topoisomerase Activities in Vivo
From male Wistar rats of P10, the cerebellum was dissected out
under deep anesthesia and immediately cut into 1-mm thick slices on ice
(about 10 sagittal slices), submerged in 1 ml of RPMI 1640 with or
without 100 µM etoposide, and incubated at 37 °C for
2 h in a CO2 incubator. The slices were homogenized
with 3 ml of 1% (v/v) Sarkosyl in 10 mM Tris-HCl (pH 7.5)
and 1 mM EDTA (TE buffer), and then fractionated by
centrifugation on a CsCl step gradient (2-ml layers of CsCl solutions
in TE buffer with densities of 1.82, 1.72, 1.50, and 1.37 g/cm3) according to Muller and Mehta (29). Density of the
gradient fractions (0.5 ml) was calculated from the refractive index of each fraction. DNA concentration was estimated by a spectrophotometric method based on the Warburg-Christian equation. Aliquots (47 µl for
topo II Primary Culture
Cerebellar granule cells were prepared from the cerebellum of P8
Wistar rats and processed for culture as described previously (30). The
tissue was treated briefly with trypsin and DNase I and the dispersed
cells were cultured at 37 °C in a humidified atmosphere of 5%
CO2 in Dulbecco's modified Eagle's medium containing 10%
fetal calf serum, 25 mM KCl, and 60 µg/ml kanamycin
sulfate. The cell suspension from one cerebellum was dispensed into 6 plastic culture dishes (60 mm in diameter) precoated with
poly-L-lysine. After 24 h in culture, proliferation of
non-neuronal cells was prevented by replacing the medium with that
containing 10 µM cytosine arabinoside. The medium was
refreshed at the end of day 3. Cells remained viable under these
conditions for at least 8 days in culture.
Northern Blot Analysis
Total cellular RNA was prepared from the cultured cells by using
a commercial kit (RNeasy, Quiagen). RNA samples (17 µg/lane) were
electrophoresed and blotted as described previously (11). cDNA
clones encoding the neuronal markers (amphiphysin I, amphiphysin II,
synaptophysin, and mRNA Expression Profiling
A cDNA macroarray system (Atlas Rat 1.2 Array, Clontec) was
used for determination of relative levels of mRNAs that are
expressed in granule neurons differentiating in vitro. Total
cellular RNA was isolated from untreated granule cells at day 1, at day
5, and ICRF-treated cells at day 5 as described above. The following procedures were performed according to the protocol supplied by the
manufacturer. Briefly, RNA preparations were first treated with DNase
I, followed by enrichment of poly(A)+ RNA on streptoavidin
magnetic beads. RNA templates bound to the beads were subjected to
cDNA synthesis with rat CDS primer mixture and
[ Expression of DNA Topoisomerase II Isoforms in Rat Cerebellar
Cortex at Postnatal Day 10 (P10)--
As we have shown previously,
mRNAs of both topo II Catalytic Activities of DNA Topoisomerase II Isoforms Detected in
Vivo during the Postnatal Development of the Cerebellum--
The
results presented above or simple immunohistochemical detection methods
with specific antibodies do not provide information on the abundance of
catalytically active topo II molecules. Although it is possible to
measure topo II activities in tissue extracts, this in vitro
activity on naked DNA does not reflect the topo II enzyme acting on DNA
in situ, since their accessibility is largely restricted
in vivo by local chromatin structures (31, 32). The way to
circumvent this problem is to take advantage of the unique property of
topo II enzymes. A class of topo II inhibitors, sometimes referred to
as "topo II poisons," such as etoposide (VP-16) binds specifically
to the enzyme and stabilizes the topo II-DNA covalent complex
(cleavable complex) by preventing the religation of transiently cleaved
DNA strands (33). Upon addition of strong detergents, the complex is
disrupted to liberate DNA fragments with denatured topo II protein
covalently attached to the 5' termini. For quantitative estimation of
functional topo II isoforms in the developing rat cerebellum,
cerebellar slices were incubated with etoposide as described under
"Experimental Procedures." The tissue was then homogenized in
Sarkosyl, and the functioning topo II molecules cross-linked to DNA
were separated from the free enzyme by CsCl step gradient
centrifugation. Gradient fractions were blotted onto membranes and
probed with the monoclonal antibodies to topo II
Fig. 2 shows a representative result
obtained with P10 rat cerebellum. When the tissue was incubated in the
absence of etoposide, both topo II
In rodents, the gross organization of cerebellar tissue initiates after
birth and is completed within 3 weeks. During this period, the
precursor for granule cells, the most abundant neurons in central
nervous system, proliferate in the outmost layer (external germinal
layer, EGL), then migrate inward through the molecular layer and the
Purkinje cell layer to form the granular layer, where they continue to
differentiate establishing neuronal connections (34). Using the
cross-linking technique, we next assessed the in vivo
activity of topo II isoforms and their total protein levels during the
postnatal development of rat cerebellum (Fig.
3). The difference in the densitometric
levels indicated on the ordinate roughly reflects the relative
abundance of topo II isoforms in the cerebellum. Topo II
To identify what cell populations contribute to the levels of total
enzyme and their in vivo activities, spacial distribution of
topo II isoforms in the developing cerebellar cortex was visualized by
immunostaining (Fig. 4). The topo
II Effects of Topo II-specific Inhibitors on the Granule Cells
Differentiating in Vitro--
The topographical and chronological
analyses on the levels of topo II
Granule cells prepared from P8 rat cerebellum by enzymatic dissociation
extend processes in vitro to form elaborate neurite networks
during the culture as shown by the staining with an antibody against
neurofilaments (Fig. 5). When cells were
stained with anti-topo II
Expression of representative genes underlying the differentiation
phenotypes of the granule neuron were then analyzed. Amphiphysin I is a
protein highly expressed in the central nervous system, including
cerebellum, and involved in synaptic membrane recycling at nerve
terminals (37). Levels of amphiphysin I was measured daily by
immunoblotting (Fig. 6). The expression
of amphiphysin I was markedly induced during the culture period, up to
more than 10 times at day 5 as compared with day 1. However, in the
continuous presence of ICRF-193, a topo II-specific inhibitor, the
induction was suppressed significantly. Under these conditions topo
II
In the experiment shown in Fig. 6, topo II inhibitors were first added
at day 1 and cells were kept exposed to the drugs thereafter by
supplementary additions. To see whether the inhibitory effect on
amphiphysin I expression depends on the initiation timing of the drug
treatment, the addition of ICRF-193 was delayed in order (Fig.
7A). The efficiency of
inhibition decreased successively as the timing of drug addition was
delayed. This phenomenon was more clearly illustrated by plotting the
starting time of drug treatment against a susceptibility index which
was calculated from the induction rate determined after 1.5 days (Fig.
7B). These data suggest that topo II
The induction of amphiphysin I protein was reflected by the mRNA
contents as estimated by Northern blotting (Fig.
8A). The amphiphysin I
transcript was also decreased by ICRF-193 to a similar extent as in the
protein level, suggesting that the effects of topo II inhibitors on
protein amounts are not due to a decreased rate of translation or an
accelerated protein degradation. Effects of ICRF-193 on transcript
levels were further analyzed with other gene probes and are also shown
in Fig. 8. All the transcripts were induced as granule cells
differentiate in culture. These proteins are directly involved in
neuronal functions such as exocytosis (synaptophysin) or endocytosis
(amphiphysins I and II) of synaptic vesicles, and a neurotransmitter
receptor protein (
If the mRNAs for amphiphysin I and synaptophysin are rapidly
turning over, the observed susceptibility to ICRF-193 might be caused
by a slight decrease in their transcription rates. To see whether or
not this is the case, mRNA turnover rates were measured by blocking
the RNA polymerase II-dependent transcription with 5,6-dichloro-1-
As only a limited number of gene transcripts were analyzed thus far, we
finally investigated inducible genes and their ICRF susceptibility
using a larger set of identified gene probes that are fixed on membrane
as a cDNA macroarray. Out of 300 detectable hybridization signals,
54 (18%) were increased, 36 (12%) were decreased, and 210 (70%) were
unchanged during the 5 days in culture. The identity of up-regulated
genes were summarized in decreasing order of inducibility (Fig.
10). As expected, multiple genes that are required for neuronal functions such as ion channels, receptors, and signal transduction molecules were noticeable in the list. Although
ICRF-193 showed little effect on majority (96%) of the constitutively
expressed genes, one-third of the inducible gene transcripts were
repressed by the drug (termed class I genes). In light of these
results, we propose that inducible genes can be classified into two
groups with respect to their susceptibility to ICRF-193 in
vitro. According to this criteria, therefore, amphiphysin I and
synaptophysin belong to the class I, and amphiphysin II and
Topo II Isoforms in Differentiating Neuronal Cells Are
Catalytically Active in Vivo--
Topo II is an enzyme that
transiently cleaves DNA double strands in one duplex segment to form an
enzyme-operated gate and rejoins the cleaved DNA ends after passing a
second double-stranded DNA segment through the gate. This enzymatic
activity is essential for the segregation of daughter chromosomes and
thus for cell proliferation. Growing evidence supports that the role is
exclusively played by topo II
Following the final division cycle and migration in embryonic days,
Purkinje cells start to develop dendrites at their final destination
beneath the EGL (34). It is these periods (P1-P5) when the expression
of topo II
Granule cells start to differentiate in the inner zone of EGL
(differentiating zone) soon after their final division in the mitotic
zone of EGL. The granule cell differentiation is most active around 10 days after birth (34). The expression of topo II Functional Topo II
Our experimental system, primary culture of cerebellar granule cells,
reflects an ongoing process of in vivo differentiation and
is clearly advantageous over cultured cell lines that are forced to
differentiate by addition of inducers. In the granule cell culture
majority of cells that can be maintained in vitro are
derived from the postmitotic neurons in EGL and possibly in ML but the
cells in GL do not survive at all (50). Because of this selection, the
culture starts with largely immature granule cells and their
differentiation proceeds semi-synchronously. The present study
demonstrates that the transcriptional induction of a number of genes is
markedly repressed by topo II inhibitors (termed class I genes). The
effect is caused neither by inhibition of topo II
Our study also corroborates a probable link between topo II Possible Mechanisms for the Topo II
In eukaryotes, particular genomic segments called matrix attachment
regions organize the genome into large looped domains that are
topologically independent. We have demonstrated recently that the
putative action sites of topo II is essential
for chromosome segregation in mitotic cells, in vivo
function of topo II
remains to be clarified. Here we demonstrate
that the nucleoplasmic topo II
, highly expressed in differentiating
cerebellar neurons, is the catalytically competent entity operating
directly on chromatin DNA in vivo. When the cells reached
terminal differentiation, this in vivo activity decreased
to a negligible level with concomitant loss of the nucleoplasmic
enzyme. Effects of topo II-specific inhibitors were analyzed in a
primary culture of cerebellar granule neurons that can mimic the
in vivo situation. Only the
isoform was expressed in
granule cells differentiating in vitro. ICRF-193, a
catalytic topo II inhibitor, suppressed the transcriptional induction
of amphiphysin I which is essential for mature neuronal activity. The
effect decreased significantly as the cells differentiate. Expression
profiling with a cDNA macroarray showed that 18% of detectable
transcripts were up-regulated during the differentiation and one-third
of them were susceptible to ICRF-193. The results suggest that topo
II
is involved in an early stage of granule cell differentiation by
potentiating inducible neuronal genes to become transcribable probably
through alterations in higher order chromatin structure.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and the 180-kDa topo II
, that are
encoded by separate genes have been identified (5-7). These isoforms
may share their roles in different cellular functions although they
show similar catalytic properties in vitro. From the
following observations, topo II
is considered to be the main isoform
involved in the mitotic processes. First, there is a positive
correlation between the cellular concentration of topo II
and the
rate of cell proliferation (8, 9). Second, the expression of mRNA
for topo II
is higher in tissues containing proliferating cells (10)
and is localized in brain regions enriched with mitotic cells (11, 12).
Third, the level of topo II
protein peaks at G2/M phase
during the cell cycle (9, 13) and finally, high affinity binding of
topo II
with chromatin at mitosis is essential for chromosome
condensation/segregation and topo II
cannot be substituted for topo
II
(14).
, functional aspect of topo II
remains
obscure (1, 15). Our previous studies demonstrated a significant level
of topo II activity in isolated nuclei from post-mitotic neuronal cells
(16, 17), that was shown later to be attributable to the
isoform
(11). Expression of topo II
was also shown in other nonproliferative
or fully differentiated tissues at both the transcript (10) and the
protein levels (18, 19). Cell cycle analysis in cultured cells showed
that the level of topo II
was not altered significantly and, in a
sharp contrast to topo II
, it rather increased slightly as cells
reached a plateau phase (9, 13, 20). Expression of topo II
in these
immortalized cell lines, however, is probably deregulated. Topo II
is even dispensable for cell proliferation and survival in
vitro since in some cell lines the enzyme is not expressed at all
(21). These observations suggest that topo II
is not required for
maintenance of general cellular activities but rather involved in more
specific processes in vivo.
mRNA
increased on the cerebellar granule cells actively differentiating in
the granular layer, suggesting a link between the topo II
expression
and the cerebellar development (12). The cerebellar system also appears
to be ideal in elucidating the physiological function of topo II
since the control of its expression in cultured cell lines may well be
aberrant. Parenthetically, there have been no detailed studies on
temporo-spatial expression of topo II isoforms in living tissues. In
the present study, we discriminated the catalytically competent entity
of topo II isoforms that is acting on chromatin DNA in the developing
cerebellar tissue. Postnatal expression levels and intranuclear
localization of topo II
were also traced in the cerebellar neuronal
lineages, Purkinje cells and granule cells. We finally assessed the
requirement of topo II
activity in the expression of neuronal genes
in a primary culture of differentiating granule cells by use of topo
II-specific inhibitors. Our results are consistent with the defects in
embryonic neural development identified recently in topo II
knockout mice (24).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
--
Full-length cDNA for human topo
II
was expressed in Escherichia coli, and mice were
immunized with purified protein to prepare monoclonal antibodies as
described previously (25). One of the isolated clones, 4E12, recognized
a NH2-terminal fragment (residues numbers 1-40), and was
cross-reactive to rodent topo II
(26).
--
Full-length cDNA for human topo
II
was expressed and mouse monoclonal antibodies were prepared as
above. One of the isolated clones, 3B6, was also reactive to rodent
topo II
(26), recognizing a COOH-terminal fragment (residues numbers
1178-1273).
detection and 12 µl for topo II
detection) of each fraction were diluted 1:3 with TE buffer and blotted onto
nitrocellulose membranes using a slot blotting apparatus (Bio-Dot SF,
Bio-Rad). The membranes were then processed for immunostaining with
topo II isoform-specific monoclonal antibodies according to the
procedure described above. Stains on the membrane were scanned on a
scanner and topo II isoforms in each fraction were quantified using a densitometric software, Intelligent Quantifier (Bio Image).
-aminobutyric acid receptor
6) were first obtained by polymerase chain reaction amplification of a rat brain cDNA library (Uni-Zap XR, Stratagene) with unique primer sets designed to amplify 500-1500-base pair regions of the cDNAs.
Products were cloned into the pGEM-T vector (Promega) and verified by
nucleotide sequencing. Hybridization probes were prepared by polymerase
chain reaction amplification of the cloned cDNA fragments with the
same primer sets. The purified products were labeled by a random-primed synthesis with [
-32P]dCTP. Hybridization was carried
out under standard conditions at 42 °C overnight in a solution
containing 50% (v/v) formamide.
-32P]dATP. Purified cDNA probes (>6.5 × 106 cpm) were hybridized to the arrayed rat cDNA
membranes under recommended conditions. Washed membranes were exposed
against the imaging plate for 12 h at room temperature and
hybridization signals were quantified on the BAS 2000 analyzer using an
array-analysis software ArrayGauge (Fuji Film).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
and topo II
were detected by in
situ hybridization in the P10 rat cerebellum (11). To confirm the
expression of these enzymes at the protein level, nuclei isolated from
rat tissues were first subjected to Western blot analysis. A monoclonal
antibody, 3B6, raised against human topo II
specifically recognized
a 180-kDa polypeptide in rat cerebellum (Fig.
1, lane 5). A band with the
same mobility was also detected in cerebral cortex (lane 6)
but not in P14 rat thymus (lane 4) unless the color
development was prolonged. In contrast to topo II
, Western blot
signals were quite low when total nuclear proteins were probed with a
monoclonal antibody, 4E12, raised against human topo II
. However,
when nuclear salt extracts enriched with topo II were used for the
analysis, the antibody detected an immunoreactive band of 170 kDa, the
molecular mass expected for canonical topo II
, in both thymus and
cerebellum (Fig. 1, lanes 1 and 2), but still
failed to detect any reactive bands in cerebral cortex (lane
3). These results are consistent with the levels of transcripts
estimated previously by Northern blot analysis and further support the
idea that the expression of topo II
is tightly associated with cell
proliferation whereas that of topo II
is not (11). These results
also confirmed that both isoforms of topo II are expressed in P10 rat
cerebellum and that the monoclonal antibodies, 4E12 and 3B6, can be
used as a specific probe for topo II
and topo II
,
respectively.
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Fig. 1.
Specific detection of topo II isoforms in rat
nuclei by immunoblotting. Nuclear proteins extracted with 0.3 M NaCl and 140 mM 2-mercaptoethanol
(lanes 1-3, 30 µg of protein/lane) or whole nuclei
treated with DNase I (lanes 4-6, 5 µg of protein/lane),
were subjected to 6% SDS-polyacrylamide gel electrophoresis and
blotted onto a nitrocellulose membrane. Blotted membranes were probed
with mouse monoclonal antibodies raised against human topo II , 4E12
(lanes 1-3, 2 µg of IgG/ml) or human topo II
, 3B6
(lanes 4-6, 1 µg of IgG/ml). Lanes 1 and
4, P14 rat thymus nuclei; lanes 2 and
5, P10 rat cerebellar nuclei; lanes 3 and
6, P10 rat cerebral cortex nuclei; lane M,
molecular weight markers.
or topo II
, thus
enabling the isoform-specific detection.
and topo II
were completely
recovered in fractions with a density of typical proteins (Fig. 2,
A and C). In the presence of etoposide, however,
55% of topo II
and 22% of topo II
were recovered in the DNA
peak fractions at around 1.69 g/cm3 (Fig. 2, B
and D). The immunoreactive material in the DNA peak fractions represents the topo II-DNA complexes accumulated during the
incubation with etoposide, and thus reflects an in vivo
activity of enzymes that are accessible to chromatin DNA. The density
of these complexes is comparable to that of bulk DNA because of the small topo II versus DNA mass ratio. When the lysate DNA was
fragmented by sonication before loading the gradient, the peak of topo
II-DNA complexes shifted toward the right depending on the extent of sonication and became separated from the DNA peak. By estimating the
average length of DNA fragments, it was calculated that under these
conditions one or two topo II monomers are cross-linked to a few
kilobase pairs of DNA.
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Fig. 2.
Fractionation of topo II-DNA covalent
complexes induced by etoposide. P10 rat cerebellar slices were
incubated with (B and D) or without (A
and C) 100 µM etoposide, lysed, and
fractionated by CsCl density gradient centrifugation as described under
"Experimental Procedures." Aliquots from the fractions were blotted
onto nitrocellulose membranes and the topo II isoforms were detected
separately with the isoform-specific monoclonal antibodies (3 µg/ml).
After scanning the stained membranes, topo II isoforms were quantified
by densitometry. A and B, topo II ;
C and D, topo II
. Open circles,
amounts of topo II in densitometric units; closed circles,
DNA concentrations. Fraction densities were determined by refractometry
and shown here by broken lines.
linked to
DNA increased rapidly during the first 5 days after birth, reaching a
peak at P9, and sharply decreased thereafter in the second week to a
negligible level at P20 (Fig. 3A). More than 50% of topo
II
was cross-linked to DNA between P5 and P10 when its expression
level was also high. At P27, topo II
was no longer detectable in
both the DNA peak fractions and free protein fractions. On the other
hand, topo II
showed a triphasic time course during the postnatal
development (Fig. 3B). In both its total amount and the
DNA-linked forms, the enzyme gave a high signal already at P1 and after
a transient decrease at P5, turned to increase again (second phase).
The topo II
-DNA complex stayed at a high level during the first 2 weeks, decreased gradually in the third week, and became almost
undetectable at P27. Interestingly, the peak of granule cell
differentiation in the second week coincides with the period when topo
II
is most active on chromatin. The total amount of topo II
was
also maintained at an elevated level during the first 2 weeks, then decreased to about half the maximum level at P20, and in contrast to
topo II
, this level was maintained at P27 (third phase) and even in
adulthood. The time course of topo II
expression was also confirmed
by a conventional Western blotting of tissue lysates (Fig. 3B,
open triangles).
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Fig. 3.
Time course of the topo II isoform expression
and their in vivo activity during the postnatal
cerebellar development. Cerebellar slices from P1 through P27 rats
were incubated with or without 100 µM etoposide and the
DNA-linked topo II was separated from free enzyme by CsCl density
gradient centrifugation. Topo II isoforms in the gradient fractions
were quantified as in Fig. 2. A, topo II ; B,
topo II
. Densitometric values plotted here had been normalized by
total DNA amounts loaded on the gradient. Note the difference in the
ordinate scale magnitudes. Topo II proteins cross-linked to
DNA are shown by closed circles. Open circles indicate total
topo II protein in the gradient. Open triangles in
B designate relative amounts of topo II
in tissue
homogenates as determined directly by Western blotting.
-positive cells in EGL were already present at P1, increased in
number to peak at P10, and decreased to disappear at P25 (Fig.
4A). This changing pattern closely parallels the monophasic
time course of its in vivo activity (Fig. 3A),
which can thus be ascribed to the granule cell progenitors proliferating in the mitotic zone of EGL. Topo II
signal was not
detected in Purkinje cells throughout the postnatal period, being
consistent with the fact that their final cell division occurs in an
embryonic stage. With respect to the in vivo activity of
topo II
(Fig. 3B), the initial phase is likely due to the multilayered Purkinje cells that constitute the major population of
topo II
-positive cells at P1 (Fig. 4A). The fluorescence
and confocal microscopy at a higher magnification revealed that both nucleoplasm and nucleolus are brightly stained in P1 Purkinje cells
(Fig. 4B). As they mature, the topo II
signal decreased significantly in nucleoplasm but persisted in nucleoli. Granule cells
should contribute little to the topo II
activity at P1 since its
population size is still small and granular layer is not generated yet.
However, the second phase of the topo II
activity that peaks around
P9 is clearly accounted for by the growing population of
differentiating granule cells. Granule cell nuclei in the granular layer were highly labeled with anti-topo II
antibody at P10 but the
signal was attenuated significantly at P25 when granule cells reach
maturity (Fig. 4B). After the completion of cerebellar
architecture, there is an intriguing discrepancy between the amount of
topo II
expressed in nuclei and that actually acting on DNA. At this stage, a large proportion of the enzyme appears to be localized in
nucleoli (Fig. 4B).
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Fig. 4.
Immunolocalization of topo II isoforms during
the postnatal cerebellar development. A,
diaminobenzidine staining with the isoform-specific antibodies.
Arrows indicate Purkinje cells. B, fluorescence
micrographs of 4',6-diamidino-2-phenylindole (DAPI) staining
and topo II staining (paired panels in the left) and
confocal images for topo II
staining (right panels). PL,
Purkinje cell layer; ML, molecular layer; GL, granular layer;
Pc, Purkinje cells; gc, granule cells.
expression and its in
vivo activity suggested that the isoform
may play a critical
role in the differentiation process of cerebellar neurons. To further
investigate this possibility, we used a primary culture system for
cerebellar granule cells that has been well established and used
successfully in many studies. Expression of various genes required for
neuronal maturation was shown to be tightly regulated in the course of
culture, mimicking the in vivo situation (35, 36).
antibody, only a small proportion of cell
nuclei (<4%) was positive for topo II
after 1 day in culture
(indicated by arrow). The positive cells decreased further in the
following days. This is consistent with the notion that cells surviving in the culture are mostly postmitotic granule neurons. However, the
level of topo II
expression was already significant on day 1 in a
large population of cell nuclei and appeared to increase as the cells
differentiate in vitro, resembling the changing profile observed in vivo. The enhanced staining might be caused in
part by an increased accessibility of the antibody since only a
moderate increase in the levels of topo II
protein and its mRNA
was observed (data not shown).
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Fig. 5.
Expression of neurofilaments and topo II
isoforms in the cerebellar granule cells differentiating in
vitro. Cerebellar tissue from P8 rats was processed for
primary culture according to the procedure described under
"Experimental Procedures." Granule cells grown on coverslips were
fixed at the days indicated with 4% paraformaldehyde and immunostained
with antibodies to neurofilament proteins, topo II , and topo
II
.
activity was inhibited to <5% of
control.2 Similar effect on
the amphiphysin I induction was exerted with etoposide at a
noncytotoxic concentration. Although these inhibitors do not
discriminate topo II isoforms, one can assume that topo II
is the
only target of the drug under these conditions in the absence of topo
II
expression. Immunocytochemical staining revealed that the
induction of amphiphysin I in the cell body and neurite extensions was
susceptible to ICRF-193 (not shown). It should be pointed out that
those topo II inhibitors with different action mechanisms bring about
similar results. ICRF-193 and other bisdioxopiperazines inhibit topo II
activity without interfering with the DNA rejoining step but by binding
to the closed-clamp form of the enzyme-ATP complex to prevent its
conversion to the open-clamp form (38). Presence of cell lines that are
selectively resistant to this class of drugs and express topo II
having a point mutation in its ATP-binding site (39) strongly suggests
that topo II is the single molecular target of ICRF-193. Also deserve
mentioning here is that the cells stayed healthy and viable until the
final day in culture in the presence of ICRF-193. Generally, it is much less cytotoxic compared with topo II poisons like etoposide which induce DNA strand breaks. Cytochemical staining of apoptotic cells detected only a low level of apoptosis in both the ICRF-treated and
control cultures.
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Fig. 6.
Effects of topo II inhibitors on the
induction of amphiphysin I in the granule cell culture. Cells were
harvested at the times indicated and analyzed for protein expression by
immunoblotting (control). In separate series of cultures, 1 µM etoposide (VP-16) or 10 µM ICRF-193 was
added at the first, third, and fifth days to ensure continuous
treatment. This treatment schedule was adopted to minimize the
cytotoxicity of etoposide and maximize the effect of ICRF-193 which was
shown to inhibit topo II activity in vitro completely at 10 µM (54) and inhibit the growth of U-937 cells for at
least 3 days in culture with a single addition at 0.6 µM
(55). Protein blots were probed with a human autoantibody against
amphiphysin I. Immunoreactive bands on the membrane (shown
below the graph) were scanned and quantified by
densitometry. As upper bands of the doublet were identified as a
phosphorylated form of amphiphysin I, they were also included into the
calculation. Data points were normalized by the total protein amounts
loaded onto the gel and expressed as induction folds relative to day
1.
activity is involved
in an early stage of granule cell differentiation in vitro
that potentiates the induction process of amphiphysin I. Topo II
activity appears to be essential for this stage to initiate since even
in cells cultured for 4.5 days in the presence of ICRF-193, amphiphysin I was induced at a normal rate upon removal of the drug (Fig. 7A, f).
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Fig. 7.
Effects of delayed addition or removal of
ICRF-193 on the amphiphysin I induction. A, amphiphysin
levels were analyzed as described in the legend to Fig. 6. No drug
control (a); 10 µM ICRF-193 was added daily
starting from the culture days 0.5 (b), 2.5 (c),
4.5 (d), and 6.5 (e). In f, cells were
treated with the drug as in b until day 4.5 when the medium
was replaced with drug-free medium. B, ICRF susceptibility
of the amphiphysin induction was calculated by {1 (increment
during 1.5 days in culture with drug)/(increment during 1.5 days in
culture without drug)} × 100, and plotted against the starting
time of the drug treatment. One-hundred % susceptibility indicates
complete repression of the amphiphysin induction during the first 1.5 days in the presence of ICRF-193.
-aminobutyric acid receptor
6 subunit). The
inducible genes were either susceptible to the drug (Fig. 8,
A and B) or not susceptible at all (Fig. 8,
C and D). Genes that are already expressed
in vivo at P8 and some housekeeping genes tested so far were
not suppressed by ICRF-193 (data not shown). Amphiphysin II is an
isoform of amphiphysin I encoded by a distinct gene (37). They have
been suggested to cooperate by forming a heterodimer (40).
Unexpectedly, they behave differently in the presence of the topo II
inhibitor (Fig. 8, A and C). These results from
the transcript analysis were confirmed at the protein level by Western
blotting.
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Fig. 8.
Differential effects of ICRF-193 on the
transcript levels of neuronal genes that are induced in granule
cells. Total cellular RNA extracted from control cells or the
cells treated with 10 µM ICRF-193 as described in the
legend to Fig. 6 was subjected to Northern hybridization with
32P-labeled cDNA probes: A, amphiphysin I;
B, synaptophysin; C, amphiphysin II; and
D, -aminobutyric acid receptor
6. Radioactive mRNA
bands visualized on the autoradiogram are shown in the inset
(
, control; +, ICRF-treated). Bands were scanned, quantified by
densitometry, and the relative densitometric units, day 1 as unity,
were plotted (open circles, control; closed
circles, ICRF-treated).
-D-ribofuranosylbenzimidazole at an early
stage of chain elongation (Fig. 9).
Half-lives were estimated to be 10 and 14.3 h for amphiphysin I
and synaptophysin mRNAs, respectively, indicating that both
messages are quite stable. Also, ICRF-193 did not facilitate their
degradation since the turnover rates remained basically unchanged in
its presence. Furthermore, the drug exerted little effect on the
transcription initiation of both genes in late stage granule cells, as
revealed by a nuclear run-on assay in that the cells at culture day 6 were treated with ICRF-193 for 24 h (not shown). This is also
consistent with the results at the protein level (Fig. 7). These data
suggest that the differential effect of ICRF-193 on mRNA levels is
not due to differences in mRNA turnover rates. Therefore, it is
most conceivable that the inhibition of topo II
activity interferes
with some upstream events, resulting in the selective suppression on
the transcriptional induction of a subset of inducible genes.
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Fig. 9.
Determination of mRNA turnover rates in
the presence and absence of ICRF-193. Granule cells at day 5 in
culture were treated with 20 µg/ml
5,6-dichloro-1- -D-ribofuranosylbenzimidazole
(DRB) to arrest general mRNA synthesis. To one-half of
the culture plates, 10 µM ICRF-193 was also added. Total
cellular RNA was prepared after 0, 3, 6, 12, and 24 h of
incubation, and then subjected to Northern analysis as in Fig. 8 with
labeled probes for amphiphysin I (A) or synaptophysin
(B). From the regression lines to semi-logarithmic plots
(without ICRF treatment), half-lives of mRNA for amphiphysin I and
synaptophysin were calculated to be 10.0 and 14.3 h,
respectively.
-aminobutyric acid receptor
6 belong to the class II genes (Fig.
8). The underlying mechanism for the difference is unclear at present
(see "Discussion"). In conclusion, topo II
may thus be involved
in an early stage of differentiation and regulate the transcriptional
induction of a subset of neuronal genes that are to be up-regulated
during the course of differentiation.
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Fig. 10.
Identification and ICRF susceptibility of
the genes that are up-regulated during the granule cell differentiation
in vitro. Three RNA preparations were isolated
from untreated granule cells at day 1, at day 5, and ICRF-treated cells
at day 5, respectively. Relative levels of specific transcripts in
these preparations were determined by hybridization to a cDNA
macroarray. Gene transcripts that displayed 2-fold or greater changes
were scored as significant changes. Only 36 induced genes are shown
here with GenBankTM accession number, protein identity,
induction fold, and the ICRF-inhibition index defined as {(signal
increment during 5 days in culture with drug)/(signal increment during
5 days in culture without drug)} × 100. The inducible genes
were sorted into two groups with different susceptibilities to ICRF-193
by applying preset thresholds for the ICRF-inhibition index: less than
50% (class I genes), and between 70 and 130% (class II genes).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
in mammalian cells (1). Our
immunohistochemical data showing that topo II
in the developing
cerebellum localizes predominantly in cells situated in the mitotic
zone are consistent with this view. A large proportion of topo II
was found in covalent complexes with DNA after the treatment with
etoposide, suggesting that topo II
expressed in dividing EGL cells
is catalytically involved in vivo in proliferative processes
such as chromosomal condensation and segregation (41, 42).
in nucleoplasm of Purkinje cells reaches the highest
level. Immunofluorescence and confocal microscopy revealed fine
granulo-fibrous or reticulo-punctate localization of topo II
throughout the nucleoplasm. Similar nucleoplasmic distribution of topo
II
was reported in cultured cells such as A431 human epidermoid
cells (43) and Chinese hamster ovary fibroblasts (44). The transient
decrease in the total amount of topo II
that occurred at around P5
(Fig. 3) may be caused by the declining topo II
content in Purkinje
cells. At P5, topo II
expressed in granule cells seems to contribute
only partially to the total enzyme amount.
in the nucleoplasm
follows a similar time course and so does the immunoreactive material
recovered from the CsCl gradient (Fig. 3). Intranuclear localization of
topo II
showed a fine granulo-fibrous pattern as seen in the
differentiating Purkinje cells. In both Purkinje cells and granule
cells, therefore, intensity of topo II
staining in the nucleoplasm
increased as cellular differentiation proceeds, reaching a maximal
level when the cells are most actively involved in differentiation. At
the peak of granule cell differentiation, about a quarter of total topo
II
was recovered as topo II-DNA covalent complexes after treating
the cerebellar tissue with etoposide. This is a clear indication of
topo II
being actively engaged in its catalytic reaction in the
differentiating neuronal cells. Since the gene expression in developing
cerebellum is strictly controlled in a temporo-spatial manner (45), one
can speculate that topo II
in differentiating neurons might be a
part of machinery that controls gene expression according to the
developmental program, probably through altering the superstructure of
genomic DNA. As the cerebellar architecture came close to its fully
developed adult form toward the end of the third week, the
catalytically competent topo II
decreased gradually to reach a
detection limit and the nucleoplasmic topo II
in granule cells also
decreased while the staining of nucleolus-like structures remained
unchanged. Their identity as nucleoli has been demonstrated by double
immunostaining of antigens that are known to localize in
nucleolus.3 Expressed level
of topo II
protein also decreased in the third postnatal week, but
the decrease leveled off at about half the maximum level. These results
imply that the topo II
in nucleoplasm is the catalytically competent
entity that is actively involved in the neuronal differentiation and
disappears once neurons are terminally differentiated. The residual
topo II
in the terminally differentiated neurons that localizes
mainly in nucleoli is likely to be inaccessible to chromatin DNA
although it is still active on naked DNA in vitro (16).
May be Required for the Induction of a Subset
of Neuronal Genes--
Topo II-specific inhibitors have been used in
various cell systems to investigate the involvement of topo II in cell
differentiation. However, the results from these studies, especially
those done in early times without knowing the presence of isoforms, are
difficult to interpret. Additional ambiguity originates from the fact
that usually both topo II isoforms are expressed in cultured cells and
they are both susceptible to such inhibitors. This makes it unable to
assess which enzyme is responsible for modulated gene expression
although levels of topo II
and topo II
can be estimated separately using isoform-specific antibodies. For instance, etoposide was shown to induce the differentiation of human promonocytic leukemia
U-937 cells as revealed by an increase in the production of reactive
oxygen species and surface expression of differentiation-specific antigens (46). It was also shown that the cells treated with etoposide
were arrested at the G2 phase and ceased to proliferate. It
appears, therefore, that the observed alteration in gene expression is
attributable to the growth arrest caused by inhibition of topo II
and similar effects may be expected when etoposide is replaced with
mitotic inhibitors such as mitomycin C (47). Topo II
may have some
action points in differentiation pathways downstream the growth arrest
but it is difficult to prove this because of the inherent experimental
drawback mentioned above. The same situation applies to other studies
using topo II inhibitors in differentiating culture cells (48, 49).
which is not
expressed in these cells nor by inhibition of general transcription as
supported by the predominance of class II genes and many other genes
that are constitutively expressed and not susceptible to ICRF-193. The
incomplete inhibition of class I genes may be explained by a subtle
difference in the timing between the onset of induction and the
addition of topo II inhibitors. As substantial topo II
expression is
already present in many cells at day 1 (Fig. 5), the putative process
requiring topo II
activity might have been partially completed when
inhibitors were added, being consistent with the result that ICRF-193
exerted less effect on the amphiphysin I induction when the addition
was further delayed (Fig. 7). The result also implies that the action point of the drug is not in the transcription per se which
is still active in the later stages of differentiation and should be
equally susceptible to the drug. These observations are consistent with
the involvement of topo II
in early processes of granule cell
differentiation when class I genes are likely to be potentiated for
transcription. The potentiation step for class II genes is either
completed already in vivo or not required at all for their transcriptional activation. To our knowledge these results are the
first unequivocal implication that catalytically competent topo II
is required for a transcriptional regulation of differentiation-related genes. Attempts to clarify the molecular mechanisms underlying these
processes are in progress.
and
embryonic neural development in light of the recent report on a gene
disruption mutant for topo II
(24). The
top2
/
mouse embryos show no apparent
defect in neurogenesis and overall growth until E15.5. At E18.5,
however, normal innervation pattern of the diaphragm and limb muscles
is absent in that motor neuron axons fail to branch and contact muscle
fibers. Development of sensory axons in the spinal cord is also
defective in the top2
/
embryos. It is
interesting to note that the mutation affects only late stages of
embryogenesis and not all the neural developments are involved since
the growth of motor axons is not impaired in the intercostal muscles.
Therefore, topo II
is not a general but rather a specialized factor
that is essential for neuronal differentiation. This view is consistent
with our results that induction of only a subset of genes is interfered
by topo II inhibitors in the granule cell culture. The mutant mice die
shortly after birth and it is not feasible to analyze the postnatal
events including cerebellar development. Unfortunately, abnormalities
in the central nervous system of the
top2
/
embryos have not been assessed in
detail. The absence of topo II
may as well affect the early
development of the Purkinje cell lineage occurring around E15, as we
have observed a concomitant induction of topo II
and calbindin, a
marker protein for Purkinje cells, in the postmitotic progenitor
cells.3
-mediated Gene
Regulation--
We have shown in the present study that the
nucleoplasmic topo II
is actively involved in DNA transactions
underlying the granule cell differentiation that accompany activations
of gene expression. Induction of developmentally regulated genes like the
-globin gene cluster is believed to proceed in two steps (51). Prior to the transcriptional activation enabled by specific interactions between regulatory cis elements and
trans-acting protein factors, chromatin conformation of the
gene-containing region must be modified to a less condensed state to
ensure such interactions (gene potentiation). Although there have been
no direct evidence for chromatin decondensation catalyzed by topo II
, the decondensation of sperm head chromatin in Xenopus
egg interphase extract was shown to be inhibited by ICRF-193 (52). It
is quite possible that topo II
assists the decondensation of
chromatin which is essential for some genes to be expressed according
to the developmental program.
in the P10 rat cerebellum are
predominantly AT-rich noncoding sequences frequently associated with
matrix attachment regions.4
Topological effects exerted by topo II
acting on matrix attachment regions can be propagated throughout the whole loop. We also found that
the average nuclear volume of granule cells increases by 30% after 7 days in culture (results not shown). Nuclear size of nondividing cells
is generally correlated with the state of chromatin condensation and
the differentiating granule cells indeed showed more dispersed
chromatin when DNA was stained with Hoechst 33342. Decondensation of
chromatin have also been noticed in the maturation process of granule
cells observed in tissue sections (34). As expected, the nuclear
enlargement in vitro was suppressed by ICRF-193. Therefore,
topo II
appears to modulate the superstructure of chromatin in a
detectable scale. This may involve the nucleosomal structure as well,
because a recent study showed that Drosophila topo II is a
component of a chromatin-remodeling factor designated CHRAC (53).
Connection between the chromatin decondensation and the transcriptional
induction of specified genes will be demonstrated by further analysis
including the detection of altered chromatin conformation and detailed
mapping of topo II
target sites within the class I gene. These
experiments will reveal the local environment of individual genes and
also clarify the reason why the induction of class II genes is
independent of topo II
activity.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank N. Nozaki (Kanagawa Dental College) for providing the topo II isoform-specific monoclonal antibodies and M. Miyaike (Mitsubishi Kasei Institute of Life Sciences) for determining the epitope regions of these antibodies. We are grateful to R. Takeuchi and M. Ishimaru for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (project number 11239206).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence should be addressed: Dept. of Molecular Biology, Institute of Cellular and Molecular Biology, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. Tel.: 81-86-235-7386; Fax: 81-86-235-7392; E-mail: tsukken@cc.okayama-u.ac.jp.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M008517200
2 K. Sano, K. Tsutsui, and K. Tsutsui, unpublished observation.
3 K. Tsutsui, K. Tsutsui, O. Hosoya, K. Sano, and A. Tokunaga, J. Comp. Neurol. (2001), in press.
4 K. Tsutsui, K. Sano, K. Tsutsui, A. Kikuchi, and A. Tokunaga, submitted for publication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: topo II, DNA topoisomerase II; EGL, external germinal layer; PBS, phosphate-buffered saline.
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