Tài liệu Báo cáo khoa học: Analyzing changes of chromatin-bound replication proteins occurring in response to and after release from a hypoxic block of replicon initiation in T24 cells pptx

Analyzing changes of chromatin-bound replication proteins occurring
in response to and after release from a hypoxic block of replicon
initiation in T24 cells
Maria van Betteraey-Nikoleit, Karl-Heinz Eisele, Dirk Stabenow and Hans Probst
Physiologisch-Chemisches Institut der Universita
¨
tTu
¨
bingen, Germany
It was shown previously [Riedinger, H. J., van Betteraey-
Nikoleit, M & Probst, H. (2002) Eur. J. Biochem. 269,
2383–2393] that initiation of in vivo SV40 DNA replication
is reversibly suppressed by hypoxia in a state where viral
minichromosomes exhibit a nearly complete set of repli-
cation proteins. Reoxygenation triggers fast completion
and post-translational modifications. Trying to reveal such
fast changes of chromatin-bound replication proteins in the
much more complex replication of the cellular genome
itself, we developed a protocol to extend these studies using
the human bladder carcinoma cell line T24, which was
presynchronized in G
1
by starvation. Concomitantly with
stimulation of the cells by medium renewal, hypoxia was
established. This treatment induced T24 cells to contain a
large amount of replicons arrested in the ‘hypoxic preini-
tiation state’, ready to initiate replication as soon as normal
pO
2
was restored. Replicons in other stages of replicative
activity were not detectable. Consequently the arrested

replicons were rapidly released into synchronous initiation
and succeeding elongation. Extraction of T24 nuclei with a
Triton X-100 buffer yielded a fraction containing the
cellular chromatin, including DNA-bound replication
proteins, while unbound proteins were removed. The use-
fulness of this protocol was tested by the proliferation
marker PCNA. We demonstrate here that this protein
switches from the remainder cellular protein pool into the
Triton-extracted nuclear fraction upon reoxygenation.
Employing this protocol, analyses of chromatin-bound
MCM2, MCM3, Cdc6 and cdk2 suggests that the ‘classi-
cal’ prereplication complex is already formed during
hypoxia.
Keywords: chromatin; DNA replication; hypoxia; nuclei;
synchronization.
Apart from control by cell cycle signals, DNA replication in
mammalian cells is subject to a regulation which depends on
the O
2
tension in the cellular environment. Presumably, this
regulatory phenomenon, adapting the intensity of DNA
replication of growing cells to the supply of O
2
, is important
during embryonic growth and wound healing, and influen-
ces the propagation of malignant tumors. The O
2
-depend-
ent regulation concerns cells which are in S-phase or are
definitively committed to enter S-phase. When the concen-

tration of O
2
drops to about 0.2–0.02%, scheduled replicon
initiations are suppressed and already-active replication
forks are slowed down. Of the cell lines examined so far,
only Ehrlich ascites cells exhibit suppression of replicon
initiation without a significant slowing down of fork
progression [1–3]. During hypoxia cells accumulate repli-
cons in a state ready to initiate (almost instantaneously)
within a few minutes after oxygen recovery. Thus, sudden
reoxygenation after several hours of hypoxia triggers a
synchronous burst of initiations of the accumulated repli-
cons followed by normal replication. So far, this regulatory
phenomenon has been published for Ehrlich ascites, HeLa
and CCRF cells [2,4,5]. Further cell lines examined so far,
e.g.T24,A549,PC3,TC7,BHK,SW2,HL60andHUVEC
arealsosubjecttothefastO
2
-dependent control of
replication (G. Probst, H. Probst & M. van Betteraey-
Nikoleit, unpublished results). We therefore suggest that it
represents a general phenomenon in mammalian cells,
although the molecular mechanisms involved are still largely
obscure. The remarkably fast resumption of initiations after
reoxygenation suggests that the O
2
-dependent replication
control acts very directly on the replication apparatus itself.
As published earlier, replication of the SV40 genome in
virus infected cells also obeys the oxygen-dependent regu-

lation [6,7]. Reoxygenation of virus infected cells after
several hours of hypoxia triggers a burst of hypoxically
accumulated viral replicon initiations followed by a syn-
chronous round of completely regular replication of viral
genomes.
Studying several replication proteins bound to SV40
minichromosomes before and after reoxygenation, i.e.
before and after triggering initiation, we found that a large
number of polypeptides taking part in viral replication were
bound to the SV40 minichromosome already under hypo-
xia. However, the multiprotein complexes necessary for
unwinding, primer synthesis and elongation lacked essential
components and remained incomplete as long as hypoxia
lasted [8]. Reoxygenation triggered fast completion to a
Correspondence to M. van Betteraey-Nikoleit, Physiologisch-Chem-
isches Institut der Universita
¨
tTu
¨
bingen, Hoppe-Seyler-Straße 4,
D-72076 Tu
¨
bingen, Germany.
Fax: + 49 7071293339, Tel.: + 49 70712973329,
E-mail:
Abbreviations: PCNA, proliferating cell nuclear antigen; BrdU,
5¢-bromodeoxyuridine; FITC, fluorescein isothiocyanate.
(Received 30 April 2003, revised 24 July 2003,
accepted 28 July 2003)
Eur. J. Biochem. 270, 3880–3890 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03769.x

functional complex, indicating a specific influence of
O
2
-dependent cellular changes on critical steps of the
assembly of a functional viral replication machinery.
Consequently, we wanted to extend these studies from
the SV40 model to the far more complex replication of
the cellular genome of mammalian (human) systems. For
this purpose we had to meet two demands. Firstly to
find a cell line that can be induced to bear a maximal
number of replicons arrested in the ‘hypoxic preinitiation
state’ and as few as possible in other states of replication,
and secondly the elaboration of a protocol for preparing
a cell fraction that contains the cellular chromatin and
specifically retains the functionally bound (replication)
proteins.
In this communication we demonstrate that by a simple
starvation procedure followed by stimulation with fresh
medium and concomitant establishment of hypoxia, the
human bladder carcinoma cell line T24 can be induced to
accumulate replicons scheduled to initiate in the early
S-phase in most cells, while other stages of replicon
activity are virtually absent. Reoxygenation triggers these
replicons to initiate replication at a high degree of
synchrony, followed by subsequent normal elongation.
The immediate answer to sudden reoxygenation resembles
in principle that of SV40 replicons in virus infected cells.
However, replicons started in the noninfected T24 cells are
much longer and not identical. Extraction of T24 nuclei
with a Triton X-100 buffer yields a fast sedimenting

nuclear fraction containing the cellular DNA and proteins
associated with replicating chromatin. We take this
material as a functional equivalent to SV40 minichromo-
somes elutable from the nuclei of virus infected cells and
operationally define replication proteins remaining after
the Triton X-100 extraction (in accordance with [9]) as
functionally chromatin-bound. To justify this view we
examined whether reoxygenation triggers the association
of PCNA, the processivity clamp of polymerase delta in
mammalian replication, with the fast sedimenting Triton-
extracted fraction.
In a first experiment we employed this protocol thereby
demonstrating that important components of the ‘classical’
prereplication complex [10] become bound to chromatin
already during the hypoxic incubation. This suggests that
the ‘hypoxic preinitiation state’ is similar to the known
‘classical’ prereplication complex, and that hypoxia directly
influences mechanisms activating this complex.
Materials and methods
Cell culture, transient hypoxia, reoxygenation and
radioactive labeling
T24 cells (gift from Altana Pharma, Konstanz, Germany)
were grown in plastic flasks in DMEM supplemented with
10% fetal bovine serum and penicillin/streptomycin (100 U/
100 lgÆmL
)1
). The cells were subcultured when they
reached confluence. Under these conditions the cells exhib-
ited a partially tetraploid caryotype.
For synchronization, the desired number of glass Petri

dishes was seeded from an almost confluent large culture
with 150 000 cellsÆmL
)1
(35 mm, 1.5 mL; 145 mm,
25 mL) 44 h before the start of an experiment. Thereby,
most cells became arrested in G
1
due to starvation. When
a
14
C prelabel was desired, the seeding medium was
supplemented with 2.5 nCiÆmL
)1
[
14
C]Thd. Experiments
started with stimulation of the cells by a complete
exchange of the culture medium with prewarmed fresh
medium supplemented with 10% (v/v) fetal bovine serum.
Subsequent gassing of the cell cultures was performed
with a continuous flow of humidified artificial air
containing 5% (v/v) CO
2
for normoxic incubations, and
with 0.02% O
2
,5%CO
2
, and Ar to 100% for hypoxic
gassing. For gassing, the equipment and the procedures

described by [7] were used. For reoxygenation 0.25
volumes of medium equilibrated with 95% O
2
/5% CO
2
(v/v) were added to hypoxic cell cultures, and gassing was
continued with artificial air.
[Methyl-
3
H]deoxythymidine was added either directly to
the cells, or under hypoxic culture conditions by plunging a
spatula carrying the appropriate quantity in dried form into
the culture medium. To stop incubations medium was
removed by aspiration and the cells were washed once with
ice-cold phosphate-buffered saline (NaCl/P
i
: 150 m
M
NaCl,
10 m
M
NaHPO
4
, pH 7) and either processed for determin-
ation of acid insoluble radioactivity as described [11] or
otherwise for analyses as described below.
Alkaline sedimentation analyses of cellular DNA
For analyzing the length distribution of growing daughter
strands of T24 DNA, cultures on 35 mm glass Petri dishes
were pulse-labeled for 8 min with 7 lCi [methyl-

3
H]deoxy-
thymidineÆmL
)1
. Labeling was stopped by washing the cells
with ice cold phosphate-buffered saline (NaCl/P
i
: 150 m
M
NaCl, 10 m
M
NaHPO
4
, pH 7). The cells were trypsinized
for 5 min at 4 °C and layered onto the top of 10–30%
alkaline sucrose gradients [12]. After denaturation of
the DNA for 6 h, centrifugation was performed at
20 000 r.p.m., 23 °Cfor10hinaBeckmanSW28rotor.
1.2 mL fractions were collected from the top of the gradient
and processed to analyze acid insoluble radioactivity.
DNA cytofluorometry
For cytofluorometry of cellular DNA cells were trypsinized,
washed with NaCl/P
i
and fixed with 90% methanol.
Histograms of DNA contents were recorded with a
FACSCalibur (Becton-Dickinson) after staining the cells
with propidium iodide (0.05 mgÆmL
)1
in 0.1% sodium

citrate) and simultaneous RNase digestion (1 mgÆmL
)1
)for
30 min at 37 °C.
Cell fractionation
Cells were washed once with NaCl/P
i
and twice with
hypotonic buffer (20 m
M
Hepes, pH 7.5, 20 m
M
NaCl,
5m
M
MgCl
2
) and suspended in 10 mL of hypotonic buffer.
After 10 min on ice, cells were disrupted to free nuclei by
25 strokes with the tight fitting pestle of a dounce
homogenisator and were then centrifuged for 5 min and
1500 g at 4 °C to separate the cytosolic supernatant from
the nuclear pellet. Nuclei were resuspended in extraction
buffer (50 m
M
Hepes, pH 7.5, 100 m
M
KCl, 0.25% Triton
X-100, 2.5 m
M

MgCl
2
,1m
M
dithiothreitol) containing
Ó FEBS 2003 Chromatin-bound replication proteins (Eur. J. Biochem. 270) 3881
aprotinine (1 l
M
), leupeptine (50 l
M
), 4-(2-aminoethyl)-
bezenesulfonylfluoride/HCl (1 m
M
)andNaF(10m
M
)and
centrifuged for 3 min and 600 g at 4 °C. Nuclei were
resuspended in extraction buffer three more times to fully
lyse the nuclear envelope and complete extraction. Super-
natants were combined and yielded nucleosolic proteins.
The remaining pellet contains all DNA and structure bound
proteins and is further referred to as chromatin-fraction.
Electrophoresis of proteins and Western blotting
Cytosolic and nucleosolic proteins were precipitated from
the respective supernatants by adding five volumes of ice-
cold acetone. Proteins remaining in Triton-extracted nuclei
were recovered after nuclease digestion with DNase and
RNase. Nuclei prepared as above were suspended in
extraction buffer containing DNase (0.1 mgÆmL
)1

), RNase
(0.025 mgÆmL
)1
)andMgCl
2
(5 m
M
). Digestion was for 1 h
on ice. Proteins for Western blot analyses were then either
isolated by phenol extraction and subsequent acetone
precipitation as described [13] or directly denatured and
solubilized with SDS electrophoresis sample buffer, as it
turned out that remaining DNA fragments did not
interfere with the running properties of the proteins in
SDS-gels.
Proteins were separated on an 8% SDS/polyacrylamide
gel [14], blotted to Nylon-P membrane (Amersham) and
subsequently immunodetected using the ECL Western
blotting procedure (Amersham) according to the manufac-
turer’s instructions. Dilution of antibodies used were as
follows: PCNA (mouse monoclonal antibody, Santa Cruz)
1 : 3000, Cdc6 (mouse monoclonal antibody, Santa Cruz
Biotechnologies) 1 : 500, MCM2 (rabbit polyclonal anti-
body, Transduction Laboratories, Heidelberg, Germany)
1 : 10,000, MCM3 (rabbit polyclonal antibody, Transduc-
tion Laboratories, Heidelberg, Germany) 1: 3000, Cdk2
(rabbit polyclonal antibody, Santa Cruz Biotechnologies)
1 : 500.
Immunofluorescence staining of total PCNA
and chromatin-bound PCNA

Cells grown on coverslips were washed once with ice-cold
NaCl/P
i
. For subsequent staining of total PCNA, cells were
directly fixed with ice-cold acetone/methanol (1 : 1, v/v) for
10 min at 4 °C. When only chromatin-bound PCNA had to
be stained, soluble proteins were extracted by washing the
cells three times withextraction buffer (see Cell fractionation)
and afterwards fixed with acetone/methanol (1 : 1, v/v)
for 10 min at 4 °C. Subsequently all coverslips were
processed for detection of PCNA after air drying. Cells
were blocked with 1% (w/v) BSA in NaCl/P
i
for 20 min and
incubated with anti-PCNA Ig (Boehringer Mannheim,
dilution 1 : 100) in NaCl/P
i
/BSA for 1 h at room tempera-
ture. After washing three times with NaCl/P
i
for 5 min they
were further incubated for 30 min with anti-mouse IgG
labeled with Alexa FluorÒ 586 (Molecular Probes, dilution
1 : 200) in NaCl/P
i
/BSA. Cells were again washed three
times for 5 min with NaCl/P
i
. During the last wash total
DNA was stained with bisbenzimide (2 lgÆmL

)1
in NaCl/
P
i
). Finally PCNA (Alexa FluorÒ 568 stain) and total
DNA (bisbenzimide stain) were visualized with a Zeiss
fluorescence microscope (Axioskop) using the appropriate
filter combinations.
Immunofluorescence staining of chromatin-bound PCNA
and of replicating DNA
Cells grown on coverslips were labeled by adding 15 l
M
5¢-bromodeoxyuridine (BrdU) 15 min before the end of
the respective incubation conditions, in case of hypoxic
labeling by plunging a spatula carrying the appropriate
quantity in dried form into the cell culture medium. To
stop incubations cells were washed once with NaCl/P
i
.
For extraction of soluble proteins cells were washed three
times with extraction buffer (see Cell fractionation) and
subsequently fixed with methanol for 10 min at 4 °C.
Cells were then sequentially stained and fixed as reported
previously in [15]. Briefly, cells were blocked with 1%
(w/v) BSA in NaCl/P
i
for 20 min, incubated with anti-
PCNA Ig (Boehringer Mannheim, dilution 1 : 100) in
NaCl/P
i

/BSA for 1 h at room temperature and for 30 min
with Alexa FluorÒ 568 antibody (red fluorescence, dilu-
tion 1 : 200) in NaCl/P
i
/BSA. The primary and secondary
antibodies were fixed in place with 4% (v/v) formaldehyde
for 20 min at room temperature. Subsequently cells were
washed twice with NaCl/P
i
. For DNA denaturation cells
were treated with 2
M
HCl at 37 °Cfor1h.After
neutralization with NaCl/P
i
they were finally incubated
for one h with a fluorescein isothiocyanate (FITC)-labeled
anti-BrdU Ig (green fluorescence, Boehringer Mannheim,
dilution 1 : 50). Between the antibody incubation steps
cells were washed three times for 5 min with NaCl/P
i
.
During the last wash total DNA was stained with
bisbenzimide (2 lgÆmL
)1
in NaCl/P
i
). Finally PCNA
(Alexa FluorÒ 568 stain), replicating DNA (FITC stain)
and total DNA (bisbenzimide stain) were visualized with a

Zeiss fluorescence microscope (Axioskop, Zeiss, Go
¨
ttin-
gen, Germany) using the appropriate filter combinations.
Results
Inducing the ‘hypoxic preinitiation state’ in cellular
replicons
About 35 h after infection with SV40 virus, CV1 cells
replicate almost exclusively SV40 minichromosomes at high
intensity. These represent a highly homogenous population
of conveniently small subcellular entities. During a hypoxic
period of 6–7 h, a large amount of them is arrested in the
‘hypoxic preinitiation state’ [6,7] and can be released within
2–3 min into effective initiation and a succeeding synchron-
ous replication round by reoxygenation. Thereby, among
total viral genomes exhibiting replicative activity (‘viral
replicons’), the fraction of hypoxically synchronized repli-
cons reaches > 90%. Consequently, the equipment of the
minichromosomes with replication proteins reflects with
sufficient reliability the state of the replication machinery
before and after oxygen recovery, respectively.
Cellular replicons on the other hand, are highly hetero-
geneous in their sizes and replication states within an
asynchronous cell cycle. Therefore in addition to ‘hypoxic
pre-initiation states’, 7 h of hypoxia accumulated significant
amounts of replicons hit by hypoxia in other states of
3882 M. van Betteraey-Nikoleit et al.(Eur. J. Biochem. 270) Ó FEBS 2003
activity [4]. Thus, accumulation of cellular ‘hypoxic prein-
itiation states’ cannot be achieved as easily as that of SV40.
Therefore we tried to subject cell populations enriched with

G
1
cells to hypoxia, as successfully performed previously
with Ehrlich ascites cells, by selecting G
1
cells by zonal
zentrifugation [3,16]. In the course of investigating several
cell lines (see Discussion), we came across the human
bladder carcinoma cell line T24, which is easily arrested in
G
1
by starvation [17]. Using this cell line, we developed an
appropriate protocol. Briefly, cells were grown for 44 h after
seeding which caused shortage of nutrients and growth
factors in the medium. Starved cells were stimulated by
exchanging the medium with prewarmed fresh medium,
followed by hypoxic or normoxic gassing of the cells. The
experiments described below demonstrate that replicative
activity released immediately after O
2
admission to pre-
treated hypoxic T24 cells represents almost exclusively
synchronous replicon initiation followed by normal elon-
gation.
DNA synthesis rate
The course of the [methyl-
3
H]deoxythymidine incorpor-
ation rate into DNA of starved T24 cells was monitored
after stimulation by medium renewal under normoxic,

hypoxic and reoxygenated incubation conditions. Figure 1
shows that, in normoxically incubated cells, the incorpor-
ation rate remained relatively low up to 4 h after medium
exchange and then gradually increased up to 10 h, when
maximal incorporation was attained. This was followed by
a decrease. Under hypoxia, in contrast, incorporation
decreased to a background level during the first 2 h and
remained at this level until reoxygenation. Immediately after
reoxygenation, incorporation of radioactivity increased
strongly within a very short interval and decreased 6–8 h
later. The profile of [
3
H]Thd incorporation after reoxygen-
ation appears double-peaked. The first peak is possibly
caused by cells that proceded to the end of G
1
phase during
the 7 h hypoxic gassing, accumulating replicons ready to
initiate immediately after reoxygenation. Cells causing the
second peak possibly had not yet reached this border during
the 7 h hypoxic period.
Alkaline sedimentation analyses of growing daughter
strands
A fast increase of the DNA synthesis rate either reflects
release of replicon initiations or stimulation of elongation,
or both. To determine the cause of the increase in Fig. 1, we
analyzed the chain length distribution of pulse-labeled
nascent daughter strands by means of alkaline sedimenta-
tion. Synchronous replicon initiations first produce homo-
geneously sized small daughter strands, which subsequently

grow homogeneously to longer sizes, thus causing a
synchronous shift of growing DNA chains to higher
S-values.
Figure 2A shows a survey of alkaline sedimentation
profiles of acid-insoluble radioactivity from pulse labels
applied to normoxic, hypoxic and reoxygenated T24
cells.
The cells were prelabeled with [
14
C]Thd when seeded,
44 h before the start of the experiment. The resulting
[
14
C]Thd profile (Fig. 2B, crosses) typically exhibits a peak
in the last third of the gradient representing matured bulk
DNA. The [
14
C]Thd gradients were omitted from Fig. 2A
for clarity. After medium exchange the normoxically
incubated cultures exhibited a sedimentation profile
(Fig. 2A, first profile) attributable to asynchronously acting
replicons, because of a typical label distribution across the
gradient, resulting from the normal steady-state of asyn-
chronous initiation, elongation and termination. The
gradient of hypoxically treated T24 cells contains almost
no [
3
H]Thd, as expected according to the incorporation
curve (Fig. 1). As soon as 15 min after reoxygenation, a
strong incorporation of [

3
H]Thd into growing daughter
strands occurs, preferentially sedimenting in the first third
of the gradient and attributable to short chains originating
from newly initiated replicons. In the course of further
25 min of reoxygenated growth, the incorporation of
[
3
H]Thd still increased, while the peak shifted to higher
S-values. To visualize the chain growth between 15 and
40 min better, the last two profiles of Fig. 2A are depicted
as the percentage of total c.p.m. in Fig. 2B. From 15 to
40 min after reoxygenation the peak distinctly shifted to
higher S-values. The extent of the shift reflects the chain
elongation during 25 min and can be calculated as about
0.5 lmÆmin
)1
at either end of growing daughter strands.
This is a very common elongation rate for mammalian
cells. Note that up to 15 min after reoxygenation there is
hardly any incorporation into fast sedimenting ‘old’
daughter strands. Thus, almost no active replicons occur
that have been initiated before reoxygenation. The shapes
of the two gradient profiles are narrow and very similar,
suggesting that the cellular replicons grow synchronously
at relatively homogenous elongation rates. Thus, alkaline
Fig. 1. Rate of [
3
H]Thd incorporation into DNA of starved T24 cells
under normoxic (s) and hypoxic/reoxygenated (d) incubation condi-

tions after medium renewal. T24 cells were prelabeled with [
14
C]Thd
and grown for 44 h. Subsequently the medium was renewed and cells
were either incubated normoxically for 7 h, or hypoxically for 7 h and
then reoxygenated. At the times indicated cells were pulse-labeled for
8min with 7lCiÆmL
)1
[
3
H]Thd while maintaining the respective
incubation conditions during labeling and processed for measuring the
ratio between acid-insoluble
3
Hand
14
C radioactivity.
Ó FEBS 2003 Chromatin-bound replication proteins (Eur. J. Biochem. 270) 3883
gradient centrifugation confirmed that replicon initiation is
inhibited under hypoxia. Upon reoxygenation, suppressed
initiations are released very fast in a highly synchronous
fashion.
DNA cytofluorometry
A large portion of the partially tetraploid T24 cells exhibited
G
1
DNA content at 44 h growth after seeding (Fig. 3A).
Subjecting such cells after medium renewal to a 7-h hypoxic
period markedly increased the cell fraction with G
1

DNA
content (Fig. 3B). Three hours after reoxygenation, the
majority had entered the S-phase while a minor part still
exhibited G
1
DNA content (Fig. 3C). This result also
supports the assumption that after medium renewal most of
the cells proceed during hypoxia through G
1
up to the point
at which the first replicons of a (scheduled) S-phase would
normally be activated. However, for a minor part of the cells
a 7-h hypoxic incubation following medium exchange does
not seem to be sufficient to accumulate replicons ready to
initiate immediately after O
2
recovery. Perhaps these cells
already were in G
0
at the time of medium exchange. Yan
et al. [18] presented flow cytometric analyses of T24 cells
4 days after seeding in high density and following release
from contact inhibition. In the ATCC catalogue T24 cells
are described as hypertriploid with 8% polyploidy. In
contrast to the diploid T24 cells used by Yan et al. [18], the
T24 cells we used were tetraploid for unknown reasons.
Nevertheless their flow cytometric analyses also show that
the cells are arrested with a G
1
DNA content. As they enter

S-phase about 20 h after replating, the cells must have been
in a G
0
state before this. We intended to arrest the cells in
G
1
, from where they can proceed to DNA synthesis within
about 6 h. As shown in Fig. 3C, the majority of the cells
exhibiting S-phase DNA content 3 h after reoxygenation
probably only experienced a G
1
arrest. These cells are
obviously identical to those initiating immediately upon
reoxygenation, and may be the cause of the first peak in
Fig. 1 and the sedimentation profiles shown in Fig. 2B.
Mitotic index
To demonstrate that after release of the hypoxic block T24
cells further proceed through the cell cycle normally and at
high synchrony, we determined the percentage of mitotic
cells. Figure 4 shows that after medium exchange and
further normoxic gassing first mitotic cells appear after
about 13 h, their number increases within the next 5 h and
decreases again at longer incubation. A similar increase of
DNA synthesis occurs in the same cells 8–10 h before
(Fig. 1), compatible with an elapse of a S- and G2-phase.
Cells exposed to hypoxia directly after medium renewal and
reoxygenated 7 h later exhibited sharp rise of mitotic cells
10 h after reoxygenation, which resembles the sharp rise in
the DNA synthesis rate directly after reoxygenation. The
Fig. 3. Histograms of cellular DNA content recorded by flow cyto-

fluorometry. T24 cells were grown for 44 h. Subsequently the medium
was renewed and the cells were incubated hypoxically for 7 h or
reoxygenated for 3 h thereafter. After stopping the respective incu-
bation conditions, cells were trypsinized, fixed and stained as described
in the Materials and methods. (A) Cells after medium renewal; (B) cells
after medium renewal and 7 h of hypoxia (200 p.p.m.); (C) the same
cells after 7 h of hypoxia (200 p.p.m) and 3 h aerated incubation.
Fig. 2. Alkaline sedimentation patterns of pulse-labeled T24 DNA after
lysis on top of the gradients. T24 cells were grown for 44 h, after which
the medium was renewed and cells were either incubated normoxically
or hypoxically for 7 h, or reoxygenated after 7 h of hypoxia. Nascent
daughter DNA chains were pulse-labeled with 10 lCi [
3
H]ThdÆmL
)1
8 min before the end of the respective incubation conditions. (A)
Comparison of the gradient profiles of normoxic, hypoxic, 15 min and
40 min reoxygenated T24 cells. Profiles are depicted consecutively in
total c.p.m. Normoxia, 97215 c.p.m.; hypoxia, 365 c.p.m.; reoxygen-
ated 15 min, 57735 c.p.m.; reoxygenated 40 min, 176754 c.p.m. Each
profile consists of 31 fractions. (B) Comparison of the profiles of
15 min (m)or40min(d) reoxygenated cells (same as in Fig. 2A) in
percentage of total c.p.m. ·,Matured
14
C-labeled bulk DNA of T24
cells. Sedimentation was from left to right.
3884 M. van Betteraey-Nikoleit et al.(Eur. J. Biochem. 270) Ó FEBS 2003
mitotic index also exhibits a double-peaked profile similar to
the profile of the [
3

H]Thd incorporation. The double peak
of the
3
H incorporation curve is therefore possibly caused
by cells entering S-phase in succession.
Separating a cell fraction containing DNA
bound proteins
Entire replicative SV40 minichromosomes bearing func-
tionally bound replication proteins can be eluted from
nuclei of virus infected cells by hypotonic buffer [19]. The
DNA of mammalian chromatin, however, is organized into
loops of about 5–150 kb firmly attached to the nuclear
matrix [20]. Thus, intact cellular chromatin cannot be eluted
from isolated nuclei. Interrupting the continuity of the
DNA (e.g. by suitable endonucleases) yields elutable
chromatin fragments preferably originating from regions
far from matrix attachment points. As DNA replication foci
are probably located near the nuclear matrix, preferably
nonreplicative chromatin fragments might be eluted while
replicative chromatin regions remain attached. Therefore,
preserving the natural chromatin/matrix relations and
extracting unbound replication proteins from the nuclei
seemed to be more appropriate for studying the influence of
oxygen recovery after a hypoxic period on DNA-bound
proteins. For this purpose, we adopted a protocol described
in [9] with some modifications. The modified protocol yields
three fractions which are denoted according to the proteins
they contain. Fraction 1 includes all ‘non-nuclear proteins’,
i.e. cytosolic proteins separated during hypotonic prepar-
ation of nuclei, fraction 2 contains ‘soluble nuclear proteins’

which are extractable from nuclei by Triton X-100 contain-
ing buffer, and in fraction 3, the chromatin fraction, all
proteins remain that resist Triton extraction. We supposed
that the latter fraction included, besides the common
chromatin proteins, functionally DNA-bound replication
proteins. Because the PCNA protein is loaded by a well-
defined actively controlled process onto replicative DNA
structures [21], it can be taken as an example of replication
proteins recruited to DNA according to the demands of
replication.
Western blot analyses
T24 cells synchronized by starvation/hypoxia were fract-
ionated as described. Equal amounts of protein from each
fraction were separated by SDS gel electrophoresis, blotted
onto a Nylon-P membrane and PCNA was immunodetec-
ted. Figure 5 shows the results obtained from cells incuba-
ted hypoxically for 7 h and then stopped or reoxygenated
for 5 min, 30 min or 1 h.
In cytosolic and soluble nuclear proteins, the amounts of
PCNA did not vary under any incubation conditions. By
contrast, in hypoxic chromatin only very little PCNA was
detected. However PCNA increased strongly as soon as
5 min after reoxygenation and continued to increase after
30 min and 1 h. The pattern of chromatin-bound PCNA
suggests that the protein is recruited to DNA as soon as its
function in replication is required, after replicon initiation
had taken place.
Immunofluorescence staining of total cellular and
chromatin-bound PCNA
T24 were grown and incubated on coverslips. Two sets of

hypoxic cells and of cells reoxygenated for 10 min and
30 min were prepared. One set of cells was directly fixed
after the incubation. From the second set, soluble proteins
were extracted by washing with buffer containing Triton
X-100 prior to fixation. As shown in Fig. 6A, directly fixed
cells show a very similar PCNA content after any
incubation condition. No visible differences exist between
hypoxically incubated and reoxygenated cells. The mainly
nuclear localization of PCNA is due to the fixation
procedure. Acetone fixation leads to cell shrinkage and
loss of membranes. Therefore cytosolic PCNA is not as
prominent as in the Western blot (Fig. 5). In contrast,
when the cells were extracted prior to fixation by Triton
Fig. 4. Mitotic index of starved T24 cells under normoxic (s)and
hypoxic/reoxygenated (d) incubation conditions after medium renewal,
respectively. T24 cells were grown on coverslips for 44 h. The medium
was then renewed and cells were either incubated normoxically for 7 h,
or hypoxically for 7 h and then reoxygenated. At the times indicated
incubations were stopped, cells were fixed with acetone/methanol and
total DNA was stained with bisbenzimide. Subsequently cells were
photographed and counted. The percentage of mitotic cells was
calculated as indicated.
Fig. 5. Western blot analyses of cytosolic, soluble nucleosolic and
chromatin-bound PCNA from hypoxic and reoxygenated T24 cells.
Cytosolic, soluble nucleosolic and chromatin-bound proteins were
prepared after the indicated incubation conditions (for details see
Materials and methods) and equal amounts were separated on an 8%
SDS/polyacrylamide gel. After blotting onto Hybond-P membrane
(Amersham) PCNA was visualized with an anti-PCNA Ig (Santa Cruz
Biotechnologies) using the ECL detection procedure. H, hypoxic; 5¢,

5minreoxygenated;30¢, 30 min reoxygenated; 1 h, 1 h reoxygenated.
Ó FEBS 2003 Chromatin-bound replication proteins (Eur. J. Biochem. 270) 3885
Fig. 6. Immunofluorescence staining of total cellular PCNA and chromatin-bound PCNA. Cells were grown on coverslips for 44 h, after which the
medium was renewed and cells were incubated hypoxically for 7 h or subsequently reoxygenated for the indicated periods. Cells were then either
fixed directly or washed three times with extraction buffer to remove soluble proteins prior to fixation. PCNA was visualized using anti-PCNA Ig
(Boehringer Mannheim, dilution 1 : 100) as the primary and Alexa FluorÒ 568 (dilution 1 : 200) as the secondary antibody. Total DNA was
stained with bisbenzimide. (A) PCNA immunfluorescence staining of directly fixed T24 cells. (B) PCNA immunfluorescence staining of T24 cells
extracted prior to fixation. The respective incubation conditions of cell cultures are indicated below the images. B
0
corresponds to B
1
and is shown in
this special case to visualize all cell nuclei present. The other bisbenzimide images are not shown as the red PCNA fluorescence is almost identical to
the blue DNA fluorescence.
3886 M. van Betteraey-Nikoleit et al.(Eur. J. Biochem. 270) Ó FEBS 2003
buffer, PCNA was barely detectable in nuclei from hypoxic
cells but became visible in nuclei as soon as 10 min after
reoxygenation. The proportion of unextractable PCNA
increased significantly from hypoxic to reoxygenated
incubations. These results again confirm that PCNA
becomes chromatin-bound only when required for DNA
synthesis.
Simultaneous staining of replicating DNA
and DNA-bound PCNA
To demonstrate the connection between active DNA
replication and the appearance of bound PCNA in nuclei,
simultaneous immunodetection of replicating DNA after
BrdU incorporation and PCNA was performed. T24 cells
grown on coverslips were incubated hypoxically and then
stopped or reoxygenated for 30 min. Labeling with 15 l

M
BrdU was started 15 min before the end of either incuba-
tion. The cells were extracted prior to fixation and
processed for BrdU and PCNA immunodetection. As
shown in Fig. 7, hypoxic cells exhibit neither visible BrdU
incorporation nor bound PCNA. However, 30 min after
reoxygenation, BrdU incorporation into replicating DNA
was detectable and the amount of PCNA not extractable by
Triton buffer was high in the same cells. These results clearly
show that the PCNA staining is colocated with the BrdU
staining and this again signifies that PCNA is only bound to
chromatin portions where actively replicating DNA is
present.
Recruitment of proteins involved in replication
to chromatin during the hypoxic period
In contrast to starved T24 cells, which begin to initiate
replication after about 4 h following medium stimulation,
T24 cells that were exposed to hypoxia after medium
exchange start replicon initiation immediately upon reoxy-
genation. This suggests that the ‘classical’ prereplication
complex was already formed under hypoxia. We applied
the elaborated protocol to investigate the binding of
MCM2, MCM3 and Cdc6, which are known to be
important components of the prereplication complex as
well as Cdk2, which is considered to be (one of) the
activating kinase(s) of the complex, after medium renewal
before and at the end of hypoxic gassing as well as under
normoxic conditions.
As shown in Fig. 8 MCM3 and Cdc6 are not, and
MCM2 and Cdk2 are barely, detectable on chromatin of

starved T24 cells (lane 1). This may be caused partly by
different sensitivities of the antibodies used. However,
Fig. 7. Immunofluorescence staining of replicative T24 DNA and chromatin-bound PCNA under hypoxic and reoxygenated incubation conditions. T24
cells were grown on coverslips for 44 h. The medium was renewed prior to hypoxic gassing. Replicative DNA was labeled by incubating the cells for
15minwith15 l
M
BrdU at the end of the respective incubation. Cytosolic and soluble nuclear proteins were extracted prior to fixation by washing
the cells three times with extraction buffer (see Materials and methods). BrdU incorporated in replicating DNA was visualized after denaturation
with anti-BrdU–FITC conjugated Ig . PCNA was visualized by using anti-PCNA Ig, followed by anti-mouse IgG labeled with Alexa FluorÒ 568.
Total DNA was stained with bisbenzimide. The respective incubation conditions are indicated below the images.
Ó FEBS 2003 Chromatin-bound replication proteins (Eur. J. Biochem. 270) 3887
after medium renewal these proteins become obviously
bound to chromatin under hypoxic and normoxic condi-
tions. The signal intensities are slightly stronger under
hypoxic than under normoxic conditions. This seems
reasonable, as hypoxic suppression of replicon initiation
accumulates prereplication complexes which disappear
after initiation is completed. The latter gradually occur
in cultures not subjected to hypoxia after medium
renewal. The lack of Cdc6 may explain the 4 h lag phase
in replication after medium exchange under normoxic
conditions, since prior to replicon initiation prereplication
complexes have to be formed. This requires certain
proteins that have to be translated before (especially
proteins with a short half life, such as Cdc6) or whose
mRNA has to be transcribed first.
Nevertheless, the known ‘classical’ prereplication com-
plex seems to be formed under hypoxia, rendering the cells
ready to activate scheduled replicon initiations immedi-
ately upon reoxygenation. In this context it is noteworthy

that the prereplication complex activating kinase Cdk2
becomes bound to chromatin during hypoxia. The band-
ing pattern shows differences compared to Cdk2 of
normoxic cells. Under hypoxia the form of the protein
that migrates faster seems to predominate. Under norm-
oxic conditions both forms seem to be present at roughly
equal proportions.
Discussion
Although common interest focuses on the replication of
cells’ own genome, replication of SV40 DNA frequently
serves as a convenient model of mammalian (human) DNA
replication. However, when the cellular replication equip-
ment is abused for viral multiplication, cellular mechanisms
are often falsified or put out of function, in particular the
regulatory mechanisms involved. Decisive experiments
concerning regulatory phenomena have to be performed
in a cellular system in the long term.
The aim of the present study was to establish means for
extending a recent study [8] on changes of replication
proteins bound to SV40 minichromosomes, occurring in
the context of the fast O
2
-dependent regulation of replica-
tion [6–8], from the viral system to a (preferably human)
cellular system. Thus we were confronted with two main
problems. Firstly, inducing in as many as possible cellular
replicons the ‘hypoxic preinitiation state’ and excluding as
completely as possible active replicons in other states.
Secondly, preparing a cell fraction containing only those
replication proteins which are functionally associated with

cellular chromatin and not those located in cytosolic or
nucleosolic fractions.
With respect to the first problem, we initially tried to use
Ehrlich ascites cells. With these cells we first demonstrated
the existence of the fast O
2
-dependent regulation of
replication [12,22,23]. We had already developed means to
select vital G
1
cells from cell cultures by a zonal centrifu-
gation procedure [16] and succeeded to bring them homo-
geneously to hypoxic arrest in which they bore exclusively
early S-phase replicons in the desired ‘hypoxic preinitiation
state’ [3]. Although resuming the old experiments principally
confirmed the suitability of the Ehrlich ascites cell system for
the present purpose, we searched for alternatives because
the selection procedure is complicated, time consuming and
works only with a mouse cell line (i.e. Ehrlich ascites), while
most available antibodies are directed against human
replication proteins.
Consequently we next examined a set of human cell lines,
e.g. CCRF, HeLa [4], PC3, A549, BHK, TC7, SW2, HL60
and HUVEC with respect to their response to hypoxia and
reoxygenation. The alkaline sedimentation profiles of HeLa
and CCRF cells after hypoxia and reoxygenation already
revealed [4] that hypoxic incubation caused significant
accumulation of initiation competent replicons, which could
be released into a more or less synchronous round of
replication upon reoxygenation. However the extent of

replicon synchrony attained by the hypoxic incubation
alone, i.e. absence of active replicons in the state of
elongation, turned out to be insufficient for examining the
transition reaction between the hypoxic and the reoxygen-
ated state in a satisfying specific manner. The same problem
occurred with the other cell lines examined. Inhibitors such
as thymidine or aphidicolin were not used, as they inhibit
elongation and not replicon initiation. Furthermore, we had
shown previously that initiation is not blocked in SV40-
infected CV1 cells treated with aphidicolin prior to reoxy-
genation [6].
Fortunately, we observed that in the human bladder
cancer cell line T24 the effect of hypoxia/reoxygenation
could be intensified five- to 10-fold when the medium was
renewed prior to hypoxia. We suspected that these cells had
been (at least partly) arrested in G
1
simply by preceding
starvation as formerly described by Prescott [17]. Our
experiments confirmed this suspicion. After the optimal
starvation conditions were found, starved T24 cells were
incubated hypoxically directly after stimulation by medium
renewal. This treatment accumulated cellular replicons
Fig. 8. Western blot analyses of chromatin-bound MCM2, MCM3,
Cdc6 and Cdk2 from normoxic and hypoxic T24 cells. Chromatin-
bound proteins were prepared after the indicated incubation condi-
tions (for details see Materials and methods) and equal amounts were
separated on an 8% SDS/polyacrylamide gel. After blotting onto
Hybond-P membrane (Amersham) the respective proteins were
immunodetected using the ECL detection procedure. Lane 1, norm-

oxia without medium renewal (i.e. beginning of the experiment); lane 2,
normoxia 7 h after medium renewal; lane 3, 7 h hypoxia after medium
renewal.
3888 M. van Betteraey-Nikoleit et al.(Eur. J. Biochem. 270) Ó FEBS 2003
almost exclusively in the ‘hypoxic preinitiation state’. It
should be mentioned that T24 cells proceed normally
through the cell cycle after hypoxia/reoxygenation for
several days. No signs of apototic cell death could be
detected by the CaspaTag
TM
Caspase (VAD) Activity Kit
(Intergen, Oxford, UK) during and after the hypoxic
treatment (data not shown).
With respect to the second problem, we demonstrated
by means of the PCNA example that T24 nuclei extracted
by Triton X-100 buffer contain functionally chromatin-
bound replication proteins, switching from another cellular
compartment into the chromatin fraction (Figs 5–7) or
undergoing changes of modifications (e.g. phosphoryla-
tion) in response to O
2
recovery of hypoxic cells. PCNA
seemed most suitable because elongation is not affected [2]
or just slowed down under hypoxia [4] and ongoing
elongation is dependent on functional PCNA. We suggest
that the absence of chromatin-bound PCNA under
hypoxia is rather a direct consequence of missing initi-
ation, i.e. lost activation of the ‘hypoxic preinitiation
complex’, than an impairment of ‘clamp loading’ by
replication factor C.

Since T24 cells start to replicate immediately upon
reoxygenation, transcriptional or translational processes
can be excluded as cause of the hypoxic arrest. It was
already shown for Ehrlich ascites cells that the expression of
growth related mRNA is not influenced during transient
hypoxia [1].
DNA replication in eukaryotes is initiated by the stepwise
assembly of proteins to the replication origin [10,24,25].
First the hexameric origin recognition complex binds [26],
which then recruits Cdc6 [27,28], cdt1 [29,30] and the
minichromosome maintenance proteins [31]. This prerepli-
cation complex is built up during G
1
of the cell cycle. The
complex is presumably activated by cyclin-dependent kinase
Cdk2 [32,33] and the Dbf4/cdc7 [34] kinase, which is
required to load the initiation factor Cdc45 on the
prereplication complex [35–37]. To investigate whether this
prereplication complex is built under hypoxia we performed
a first experiment using the above described protocol. We
show that MCM2/MCM3 and Cdc6, as well as the
activating kinase Cdk2, present in two modifications with
different electrophoretic mobilities, become bound to chro-
matin already under hypoxia, thus enabeling hypoxic cells
to initiate as soon as the hypoxic suppression of replicon
initiation is released. The relative intensities of the two Cdk2
bands differ under hypoxia and normoxia. Possibly, this
represents a modification of the kinase influencing its
activity/inactivity. Post-translational processes such as
modifications (e.g. phosphorylations or dephosphoryla-

tions) of proteins have already been found to be important
regulators in SV40 replication [38,39].
Our ongoing work now focuses on changes arising in the
pattern of further chromatin-bound proteins of hypoxic and
reoxygenated cell T24 cells. We use classical Western blot
analyses with immunodetection of replication proteins or
regulators as well as high resolution 2D-gels.
With the aid of the T24 system presented here, we hope to
characterize the special state of the protein equipment of the
replication of human cells under the hypoxic block and to
elucidate the fast events occurring as an effect of oxygen
recovery.
Acknowledgements
We thank G. Probst for critical reading of the manuscript.
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