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

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Nikoleit, unpublished results).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-

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

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

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

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

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

/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

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,

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 [

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

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-

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’

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¢,

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

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

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-

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-

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