Báo cáo khoa học: Cdk2 activity is dispensable for triggering replicon initiation after transient hypoxia in T24 cells docx

Cdk2 activity is dispensable for triggering replicon
initiation after transient hypoxia in T24 cells
Dirk Stabenow, Hans Probst and Maria van Betteraey-Nikoleit
Interfakulta
¨
res Institut fu
¨
r Biochemie der Universita
¨
tTu
¨
bingen, Germany
In eukaryotic cells, orderly cell cycle progression is
believed to be regulated by the action of Cdks and
their binding partners, i.e. the cyclins, their inhibitors,
and the E2F family of transcription factors [1]. The
initiation of the first replicons at the G
1
- to S-phase
transition, marks a key step of cell cycle progression.
In living cells, the stepwise assembly of proteins at the
replication origins of replicons prepares for initiation
[2–5]. First, the hexameric origin recognition complex
binds; this then recruits Cdc6 [6,7], Cdt1 [8,9] and the
minichromosome maintenance proteins [10]. This pre-
replication complex (pre-RC) is built up during the
G
1
-phase. The complex is suggested to be activated by
Cdk2 [11] and the Dbf4 ⁄ Cdc7 kinase [12] which is
required to load the initiation factor Cdc45 on the pre-

RC [13–15]. Commonly, Cdk2 in association with
cyclin E is thought to be essential for driving the cells
through this transition. Cdk2-mediated phosphoryla-
tions are reported to be important in numerous steps
preceding initiation of DNA replication; for example,
together with Cdk4, the phosphorylation of pRb and
the subsequent release of E2F results in the transcrip-
tion of essential factors of the replication initiation
complex such as MCM and Cdc6 [16,17]. Protein
phosphorylations by Cdk2 were suggested to be
important for initiation complex assembly and activa-
tion [17,18]. Cdk2 ⁄ cyclin A mediated phosphorylation
of Cdc6 is thought to induce its translocation from the
Keywords
hypoxia; reoxygenation; Cdk2; replication;
chromatin
Correspondence
M. van Betteraey-Nikoleit, Physiologisch-
Chemisches Institut der Universita
¨
t
Tu
¨
bingen, Hoppe-Seyler-Straße 4,
D-72076 Tu
¨
bingen, Germany
Fax: +49 7071293339
Tel: +49 70712973329
E-mail: maria.van-betteraey@

uni-tuebingen.de
(Received 6 July 2005, revised 16 August
2005, accepted 5 September 2005)
doi:10.1111/j.1742-4658.2005.04957.x
We examined whether the fast release of replicon initiation after sudden O
2
recovery of hypoxically incubated mammalian cells depends on kinase
activity of Cdk2. We used a system based on starved ⁄ refed T24 cells elab-
orated previously for such investigations [van Betteraey-Nikoleit M, Eisele
KH, Stabenow D & Probst H (2003) Eur J Biochem 270, 3880–3890]. Cells
subjected to hypoxia concurrently with refeeding accumulate the G
1
DNA
content within 5–6 h. In this state they are ready to perform, within 1–
2 min after O
2
recovery, a burst of replicon initiations that marks the start
of a synchronous S-phase. We found that Cdk2 binds to the chromatin
fraction within 4–6 h after refeeding with fresh medium, irrespective of
whether the cells were incubated normoxically or hypoxically. However,
inhibition of Cdk2 by olomoucine, roscovitine or the Cdk2 ⁄ cyclin inhibi-
tory peptide II had no influence on the synchronous burst of replicon initi-
ations. Cdc6 and pRb, possible targets of Cdk2 phosphorylation, behaved
differentially. Inhibition did not affect phosphorylation of Cdc6 after
reoxygenation, whilst chromatin bound pRb remained hypophosphorylated
beyond the initiation burst. Thus, neither Cdk2 activity, though present at
the end of the hypoxic period, nor pRb phosphorylation are necessary for
releasing the burst of replicon initiations upon oxygen recovery. Conse-
quentially, Cdk2 dependent phosphorylation(s) cannot be a critical trigger
of replicon initiation in response to reoxygenation after several hours of

hypoxia, at least in the T24 cells studied.
Abbreviations
FITC, fluorescein isothiocyanate; PCNA, proliferating cell nuclear antigen; Pre-RC, prereplication complex.
FEBS Journal 272 (2005) 5623–5634 ª 2005 FEBS 5623
nucleus to the cytoplasm, thus preventing reinitiation.
Altogether, these observations suggest that Cdk2 activ-
ity is necessary for progression of cells from G
1
to act-
ive DNA replication. The importance of Cdk2 has
been substantiated by a number of different approa-
ches. Microinjection of antibodies against Cdk2,
cyclin E or cyclin A and the antisense mRNA of Cdk2
block initiation of DNA synthesis in mammalian cells
[19,20]. In vitro initiation in G
1
nuclei is dependent on
Cdk2 ⁄ cyclin A and Cdk2 ⁄ cyclin E complexes [21]. Fur-
thermore, in general, elevated levels of the Cdk2 inhib-
itor p27 and decreased activity of Cdk2 in conjunction
with hypophosphorylated pRb have been shown to
result in growth arrest [22,23]. However, these results
have been challenged by the finding that Cdk2 knock-
out mice are viable [24]. In addition, Cdk4 can sub-
stitute for Cdk2 in pRb phosphorylation, and
proliferation of cancer cells that do not contain pRb,
may be completely independent of Cdk2 or Cdk4
activity [25].
In order to reveal whether Cdk2 activity is also
involved in the very fast (requiring a few min only)

release of replicon initiations from the hypoxic arrest
described first by us [26], we studied the association of
Cdk2 with the chromatin fraction of T24 cells and its
enzymatic activity during the course of a starvation–
refeeding ⁄ hypoxia–reoxygenation experiment. As out-
lined in detail before and substantiated by cytofluoro-
metry, analysis of DNA replication at the level of
replicons and determination of the fraction of mitotic
cells [27] our especially elaborated starvation protocol
accumulates T24 cells in a G
1
arrest, from which they
can be released by medium renewal. Normally, the
cells then proceed to the S-phase within approximately
5 h, passing through a number of the above mentioned
regulatory steps until the start of orderly DNA synthe-
sis at the origins of replication (scheduled to be activa-
ted as the very first of the S-phase). We demonstrated
[27] that subjecting the cells to hypoxic conditions
directly after restimulation with fresh medium reversi-
bly interrupts this process at a state situated extremely
close to the actual occurrence of replicon initiation.
The cells accumulate in a state which we refer to as
the ‘hypoxic preinitiation’ state on the basis of prior
studies on DNA replication of a number of other cell
lines [27] and Simian virus 40 replication in vivo [28].
From this state, characterized by a still incomplete and
not yet functional set of replication proteins associated
with chromatin, they can be released within 1–2 min
by restoring atmospheric pO

2
, into a synchronous
wave of replicon initiations. During the transition trig-
gered by the reoxygenation event, chromatin bound
replication proteins become completed [28]. We have
demonstrated that, in the T24 system described before
[27], most cells pass through a normal synchronous
S-phase after reoxygenation.
In the present study we found that substantial chro-
matin associated Cdk2 emerged during the hypoxic
period. However, inhibitors of Cdk2 activity did not
affect replicon initiation after reoxygenation in vivo.
Cdc6 phosphorylation in the course of initiation of
DNA replication was also not affected, pRb remained
hypophoshorylated for at least 30 min beyond the ini-
tiation burst.
Thus, at least in the T24 cells studied, Cdk2 activity
as well as Rb phoshorylation is dispensable for the fast
release of hypoxically suppressed initiation of DNA
replication at the beginning of the S-phase.
Results
Hypoxia or reoxygenation do not interfere with
the emergence of chromatin bound Cdk2 activity
after restimulation by fresh medium
In [27] we found no Cdk2 protein in the chromatin
fraction of starved T24 cells by western blot analysis.
However, significant amounts exist 7 h after medium
renewal, irrespective of whether the cells were grown
normoxically or hypoxically. Immunofluorescence
staining of the cells using a Cdk2 specific antibody

(Fig. 1D, final column) confirmed this result. We then
examined the protein kinase activity of Cdk2 from dif-
ferent fractions of hypoxic and reoxygenated cells: The
medium of two cultures with starved T24 cells was
renewed and both were incubated hypoxically for a fur-
ther 7 h. One culture was subsequently reoxygenated
for 30 min. The immunoprecipitates of cytosolic,
NP40-extractable nucleosolic, and salt eluted chromatin
bound proteins were analysed for protein kinase activ-
ity as described in Eperimental procedures. The result-
ing autoradiograph (Fig. 1A) convincingly shows that
the highest concentration (relative to total protein pre-
sent) of kinase activity occurs in the chromatin bound
fraction and (slightly less) in the cytosolic fraction. The
nucleosol exhibits the lowest activity. There is obvi-
ously no difference between Cdk2 precipitated from
hypoxic and 30-min reoxygenated cells. Western blot
analysis of the same membrane (Fig. 1B), exhibited
analogous differences of general signal intensities, but
revealed two bands of different electrophoretic mobil-
ity, most probably reflecting two different phosphoryla-
tion states. We therefore suspect that chromatin bound
Cdk2 differs from the cytosolic form by its preferential
phosporylation state. However, both forms obviously
exhibit comparable protein kinase activity in vitro.
Cdk2 is dispensable for replicon initiation D. Stabenow et al.
5624 FEBS Journal 272 (2005) 5623–5634 ª 2005 FEBS
We next examined the inhibitory action of the Cdk2
inhibitors olomoucine, roscovitine and staurosporine,
all of which compete for the ATP-binding domain of

the kinase [29,30], and the Cdk2 ⁄ cyclin inhibitory pep-
tide II which is reported to inhibit specifically the
phosphorylation of substrates by Cdk2⁄ cyclin A and
Cdk2 ⁄ cyclin E complexes [31] in an in vitro kinase
assay. All four inhibitors were used at medium concen-
trations reported by others to reliably inhibit Cdk2
[31–34]. Starved T24 cells incubated normoxically after
medium stimulation were lysed. Subsequently, Cdk2
was immunoprecipitated. The kinase assay was per-
formed both in the absence and in the presence of the
inhibitors. Figure 1C shows that Cdk2 activity is signi-
ficantly decreased in the presence of the inhibitors.
Furthermore, to demonstrate that the Cdk2 ⁄ cyclin
inhibitory peptide II is cell permeable and localizes at
the sides of Cdk2, we used the Cdk2 ⁄ cyclin inhibitory
peptide II in a form carrying a fluorescence label at
the amino end (Fig. 1D, middle column). Starved T24
cells were stimulated by medium renewal (except for
the culture designated N–), and hypoxic or normoxic
gassing was started concurrently. The labelled peptide
was added (without interruption of the hypoxic incu-
bation) 4 h after the start of gassing. A further 3 h
later, one hypoxic culture was reoxygenated for
30 min. After this, cells were processed for Cdk2
immunostaining as described in Experimental proce-
dures. Finally, we found a strong fluorescence within
the cells colocalized with the Cdk2 immunostaining
(Fig. 1D, last column). Starved T24 cells (N–) expres-
sing barely detectable amounts of Cdk2 protein
A

B
C
D
Fig. 1. Cdk2 activity does not decrease under hypoxia. (A) Phos-
phorylation of histone H1 by immunoprecipitated Cdk2 obtained
from the cytosolic, nucleosolic and salt eluted chromatin bound pro-
teins, after 7 h hypoxia or reoxygenation, respectively. Immuno-
precipitations and subsequent kinase assays were performed as
described. The kinase reaction was stopped by boiling in protein
buffer, and proteins were separated by SDS ⁄ PAGE (12% polyacryl-
amide). After blotting, the membrane was autoradiographed. (B)
Western blot analysis of immunoprecipitated Cdk2 from (A). H, 7 h
hypoxic incubation after medium renewal; 30¢, reoxygenated for
30 min after 7 h hypoxic incubation. (C) Phosphorylation of histone
H1 by immunoprecipitated Cdk2 from cell lysate prepared after 7 h
normoxic incubation following medium stimulation. Immunoprecipi-
tation was performed as described. Subsequently, the kinase assay
was performed in the absence or presence of olomoucine (20 l
M),
roscovitine (7 l
M), staurosporine (100 nM) or the Cdk2 ⁄ cyclin inhibi-
tory peptide II (50 l
M). The kinase reaction was stopped by boiling
in protein sample buffer, and the proteins were separated by
SDS ⁄ PAGE (12% polyacrylamide). After blotting, the membrane
was autoradiographed (upper row). Western blot analysis of the
membrane of immunoprecipitated Cdk2 was performed afterwards
(lower row). (D) Immunofluorescence staining of Cdk2 under norm-
oxic, hypoxic and reoxygenated incubation conditions in the pres-
ence of FITC labelled Cdk2 ⁄ cyclin inhibitor peptide II. T24 cells

were grown on coverslips for 44 h. The FITC labelled Cdk2 ⁄ cyclin
inhibitory peptide II was added 4 h before the end of the respective
incubation conditions. After fixation of the cells, Cdk2 was visual-
ized by using a Cdk2 antibody, followed by an anti-mouse Ig
labelled with Alexa FluorÒ 568. Total DNA was stained with bis-
benzimide. The respective incubation conditions are indicated
below the images. N–, 7 h normoxic incubation without medium
renewal; N+, 7 h normoxic incubation after medium renewal; H,
7 h hypoxic incubation after medium renewal; R 30¢, reoxygenated
for 30 min after 7 h hypoxic incubation.
D. Stabenow et al. Cdk2 is dispensable for replicon initiation
FEBS Journal 272 (2005) 5623–5634 ª 2005 FEBS 5625
showed significantly less fluorescence of the inhibitor
and as mentioned above Cdk2 immunofluorescence.
Both increased dramatically after feeding the cells.
Cdk2 inhibitors fail to prevent replicon initiation
after reoxygenation
As mentioned, our starvation–feeding ⁄ hypoxia proto-
col arrests T24 cells very close before the effective
entry into S-phase. During the hypoxic period, they
accumulate in a state separated by a few minutes only
from initiation of the replicons scheduled to be repli-
cated first [27]. Readmission of O
2
to a thus pretreated
T24 culture triggers a subsequent synchronous burst of
replicon initiations. Up to about 1 h thereafter, the
replicative activity in the reoxygenated culture is gov-
erned mainly by synchronous daughter strand growth
within the replicon cohort activated in response to the

reoxygenation event. Afterwards, initiations of repli-
cons scheduled to be activated later in the S-phase
succeed, followed by G
2
, mitosis and a further syn-
chronous cell cycle [27]. Alkaline sedimentation analy-
sis of the length distribution of pulse labelled daughter
strand DNA is suited to demonstrate the initial burst
as well as the succeeding synchronous daughter strand
elongation. On the basis of numerous prior studies
[26,35–40], 8-min [
3
H]dThd pulses applied 20 and
40–50 min after reoxygenation, proved to be most con-
venient for demonstrating initiation and succeeding
elongation.
We studied the influence of olomoucine, roscovitine
and the Cdk2 ⁄ cyclin inhibitory peptide II on the sedi-
mentation profiles of pulse labelled daughter strand
DNA 20 and 50 min after reoxygenation (Fig. 2).
Starved T24 cells were stimulated by medium renewal,
and hypoxic gassing was started concurrently. The
inhibitors were added (without interruption of the hyp-
oxic incubation) 4 h after the start of gassing. A fur-
ther 3 h later, all cultures were reoxygenated. After
continuing normoxic incubation for 20 or 50 min,
respectively, the cells were labeled by [
3
H]dThd pulses.
In controls, inhibitor addition was omitted.

The controls presented in the inset of Fig. 2 serve to
confirm the expected effect of hypoxia in the present
experiment. These alkaline sedimentation profiles
represent pulse labelled DNA of cells which were
Fig. 2. Inhibition of Cdk2 does not affect replicon initiation after reoxygenation. Alkaline sedimentation patterns of pulse-labelled T24 DNA
after lysis on top of the gradients. Eight minutes before the end of the respective incubation conditions, nascent daughter strand DNA
chains were pulse-labelled with 7 lCi [
3
H]dThdÆmL
)1
. Inset, Starved T24 cells were stimulated by medium renewal and incubated normox-
ically (n) or hypoxically (.)for7h;x,
14
C-labelled matured bulk DNA. Main figure, T24 cells were stimulated by medium renewal, incubated
hypoxically for 7 h and then reoxygenated for 20 min (open symbols) and 50 min (filled symbols), respectively. Olomoucine (20 l
M), roscovi-
tine (7 l
M) or the Cdk2 ⁄ cyclin inhibitory peptide II were added 3 h before reoxygenation. s, d, Reoxygenated for 20 min and 50 min,
respectively, after 7 h hypoxic incubation; h, n, reoxygenated for 20 min and 50 min, respectively, after 7 h hypoxic incubation in presence
of olomoucine; e, r, reoxygenated for 20 min and 50 min, respectively, after 7 h hypoxic incubation, in presence of roscovitine; n, m, reoxy-
genated for 20 min and 50 min, respectively, after 7 h hypoxic incubation, in presence of the Cdk2 ⁄ cyclin inhibitory peptide II.
Cdk2 is dispensable for replicon initiation D. Stabenow et al.
5626 FEBS Journal 272 (2005) 5623–5634 ª 2005 FEBS
incubated 7 h after medium renewal normoxically
(open triangles) and hypoxically (filled triangles). The
mature DNA of these cells contained a prelabel result-
ing from [
14
C]Thd added 44 h before. The
14

C profile
obtained (crosses) typically exhibits a peak in the last
third of the gradient. The
3
H-profile of the normox-
ically incubated culture exhibits a sedimentation profile
attributable to asynchronously acting replicons and
reflects the normal steady-state of asynchronous initi-
ation, elongation, and termination [26,38,41]. The gra-
dient of hypoxic cells contains practically no
3
H-label,
indicating almost total absence of replicative activity.
Twenty minutes after reoxygenation (Fig. 2, main,
open symbols), four almost coincident prominent
radioactivity peaks at fraction 7–8 (35–41 S
20w
) indi-
cate the presence of growing daughter strands of about
35–50 kb length in both inhibitor treated and control
cells. Assuming a bidirectional strand elongation rate
of 1.5 kbÆmin
)1
at either end of strands, this size is
compatible with daughter strands of replicons initiated
10–20 min before. Minor shoulders around fractions
11–12 (51–67 S
20w
) indicate the additional presence of
small portions of longer growing strands. This shoul-

der was more or less prominent in different independ-
ent experiments of this type (not shown). As in prior
studies, we attribute them to replicons in few cells
which could no more escape the late S-phase during
starvation and are hit by hypoxia in a still active state.
Reoxygenation probably reactivates these late S-phase
replicons. However, the shoulder is no more present in
the profiles obtained by analysing the cells 50 min after
reoxygenation (Fig. 2, filled symbols). All four profiles
exhibit nearly identical narrow single peaks around
fraction 13 (about 62 S
20w
or 140–150 kb). Relative to
the main peaks obtained at 20 min, the observed shift
is again compatible with an elongation rate of about
1.5 kbÆmin
)1
. Thus, the inhibitors had no detectable
influence, neither on the replicon initiation burst trig-
gered by reoxygenation, nor on the succeeding growth
of daughter strands.
Changes of chromatin bound pRb and Cdc6 after
reoxygenation
Starved T24 cells were stimulated by medium renewal
(except for the culture designated N–) and incubated
normoxically or hypoxically. Further cultures were
reoxygenated after hypoxic incubation. After fraction-
ation of the cells, chromatin bound proteins Cdc6 and
pRb were examined by western blot analysis.
Western blot analysis of pRb using a pRb antibody

which recognizes an epitope between amino acids 332–
344 (Fig. 3A, upper row) and an antibody specific for
underphosphorylated pRb (Figs 3A, row 2 from top)
concurrently suggested that pRb is hypophosphorylated
in starved cells and becomes phosphorylated within
7 h after medium stimulation and further normoxic
incubation (Fig. 3B shows the effect of phosphatase
A
B
C
Fig. 3. Phosphorylation of Cdc6 and pRb after reoxygenation. (A)
Western blot analysis of chromatin bound pRb, Cdc6, and PCNA
from 7 h normoxic, hypoxic, and 30 min reoxygenated T24 cells.
(B) Lambda protein phosphatase digestion of immunoprecipitated
Cdc6 and pRb. (–), Immunoprecipitated Cdc6 and pRb prior phos-
phatase digestion; (+), immunoprecipitated Cdc6 and pRb after
phosphatase digestion according to the manufacturer’s instructions
(New England Biolabs: Lambda protein phosphatase). (C) Western
blot and subsequent immunological detection of chromatin bound
PCNA and Cdc6. Staurosporine (10 l
M) was added 30 min before
reoxygenation where indicated. N–, 7 h normoxic incubation with-
out medium renewal; N+, 7 h normoxic incubation after medium
renewal; H, 7 h hypoxic incubation after medium renewal; 5¢, reoxy-
genated for 5 min after 7 h hypoxic incubation; 30¢, reoxygenated
for 30 min after 7 h hypoxic incubation.
D. Stabenow et al. Cdk2 is dispensable for replicon initiation
FEBS Journal 272 (2005) 5623–5634 ª 2005 FEBS 5627
digestion on pRb immunoprecipitated from N+ T24
cells). In comparison with normoxic cells, a larger

portion seems to remain hypophosphorylated in the
hypoxic cells. Reoxygenation apparently causes no
detectable change of pRb phosphorylation, at least
during the following 30 min.
Cdc6 protein was not detectable in starved cells and
appeared after medium stimulation, whereby more
Cdc6 accumulated during hypoxic than under normox-
ic incubation conditions (Fig. 3A, row 3 from top).
After reoxygenation, the amount of chromatin bound
Cdc6 decreased within 30 min below the level of the
normoxic control. Chromatin bound Cdc6 obtained
from hypoxic cells seems to migrate slightly faster
compared with that occurring in reoxygenated cells.
Confirmed by the effect of phosphatase digestion
(Fig. 3B), this points to a phosphorylation after re-
oxygenation. The suspected phosphorylation could
(nonspecifically) be prevented by adding 10 lm
staurosporine before reoxygenation (Fig. 3C). The lat-
ter experiment again clearly shows that the amount of
chromatin bound Cdc6 is high at the end of the hyp-
oxic period and distinctly decreases within 30 min after
reoxygenation. The remainder is apparently converted
into the slower migrating form. This significant
decrease and conversion to the slower migrating form
is inhibited by 10 lm staurosporine.
The presence of substantial amounts of chromatin-
bound proliferating cell nuclear antigen (PCNA)
[27,28] can, in conformity with the function of PCNA
as processivity factor of polymerase delta, serves as an
indicator of active replicative DNA strand elongation

which, for its part, depends on successful replicon initi-
ation. Therefore, its increase following reoxygenation
as shown in Fig. 3A,C indicates successful replicon ini-
tiation. Treatment with 10 lm staurosporine (Fig. 3C)
abolishes this increase. This suggests that, in accord-
ance with earlier findings of our group [42], replicon
initiation was suppressed by the elevated concentration
of staurosporine.
Effects of Cdk2 inhibition on pRb, Cdc6 and
PCNA in vivo
In Fig. 4 we analysed the effects of the Cdk2 inhibitors
olomoucine, roscovitine and staurosporine (at 100 nm)
on Cdk2 activity and its substrates pRb and Cdc6
in vivo. Starved T24 cells were stimulated by medium
renewal (except for the culture designated N–). Subse-
quently, the cells were incubated normoxically or
hypoxically, or were reoxygenated with or without
prior addition of the inhibitors. A concentration of
100 nm staurosporine suffices to inhibit Cdk2 accord-
ing to [43], but does not suppress replicon initiation
in cultured cells [42]. Accordingly, chromatin bound
PCNA increased after reoxygenation under 100 nm
staurosporine as well as under olomoucine and rosco-
vitine.
As already found in the experiment shown in
Fig. 3A, the hypophosphorylated form of pRb (migra-
ting faster) is prominent in starved cells and mostly
changes to a higher phosphorylation state after feeding
with fresh medium (Fig. 4A, top row). As before
(Fig. 3) this change is also suppressed by hypoxic gas-

sing started concurrently with feeding. Equally, the
changes in appearance and mobility of Cdc6 were
reproduced. The decrease of Cdc6 in response to reoxy-
genation is strengthened until 30 min, whereas chro-
matin bound PCNA increases, reflecting increasing
DNA strand elongation (Fig. 4A, bottom row).
A
B
Fig. 4. Western blot analysis of pRb, Cdc6 and PCNA after inhibi-
tion of Cdk2 activity by olomoucine, roscovitine and staurosporine
in vivo. (A) Western blot analysis of chromatin bound pRb, Cdc6,
and PCNA from 7 h normoxic, hypoxic, 5 min, and 30 min reoxy-
genated T24 cells. Cdk2 inhibitors were added 3 h before reoxygen-
ation where indicated. (B) Western blot analysis of total cellular
pRb, Cdc6, and PCNA from 7 h normoxic, hypoxic, 5 min, and
30 min reoxygenated T24 cells. Cdk2 inhibitors were added 3 h
before reoxygenation where indicated. N–, Normoxic incubation
without medium renewal; N+, 7 h normoxic incubation after med-
ium renewal; H, 7 h hypoxic incubation after medium renewal; 5¢,
reoxygenated for 5 min after 7 h hypoxic incubation; 30¢, reoxygen-
ated for 30 min after 7 h hypoxic incubation.
Cdk2 is dispensable for replicon initiation D. Stabenow et al.
5628 FEBS Journal 272 (2005) 5623–5634 ª 2005 FEBS
However, neither addition of olomoucine, roscovitine
or staurosporine (at 100 nm) before reoxygenation pro-
duced, until 30 min thereafter, any change of chroma-
tin bound pRb, Cdc6 or PCNA in comparison to the
untreated reoxygenated sample. Application of the
Cdk2 ⁄ cyclin inhibitory peptide II in a separate experi-
ment according to the same schedule (data not shown)

yielded identical results. This suggests that initiation
and further replication proceeded normally despite the
presence of the Cdk2 inhibitors.
Figure 4B, on the other hand, shows that the differ-
ent treatments of the cells in this experiment caused
no visible quantitative differences of total cellular pRb
and PCNA. Both proteins, apparently, are relatively
abundant among total cellular proteins. The change of
the electrophoretic mobility of pRb in response to
feeding with fresh medium (attributed by us to phos-
phorylation) obviously concerns the total cellular pro-
tein including the chromatin associated proteins. On
the other hand the relative intensity of the Cdc6 signal
in Fig. 4B is very low. Because all wells of the gel for
Fig. 4 were loaded with strictly equal amounts of pro-
tein and the fraction of chromatin associated proteins
represents only 8.2% of total proteins, the distinctly
higher Cdc6 signal intensities in Fig. 4A indicate that,
in contrast to PCNA and pRb, a substantial portion
of Cdc6 was concentrated in the chromatin fraction.
The high content of PCNA in the starved cells (N– in
Fig. 4B) indicates that these cells rather reside in a
G
1
-arrest than in a G
0
-phase [44].
Discussion
This study continues prior work on molecular mecha-
nisms governing the initiation of replication units in

mammalian cells and its regulation in vivo. The dis-
covery of the fast acting O
2
-dependent control of
mammalian replicon initiation [26,39] and the suc-
ceeding detailed evaluation of the relevant conditions
(ranges of O
2
partial pressures, etc. [38–40,45]) provi-
ded a useful tool for this purpose: exposing cultures
of growing mammalian cells for several hours to hyp-
oxic conditions accumulates replicons (scheduled to
be initiated within the hypoxic period), in a state
from which they can easily be released within a few
minutes into a synchronous wave of initiations, by
abruptly restoring atmospheric pO
2
. The state of repl-
icon origins accumulated under hypoxia was opera-
tionally called by us ‘hypoxic preinitiation state’.
Having characterized the changes occurring at the
level of the viral DNA replication in response to
reoxygenation [46], we developed a protocol for
analogous studies on cellular mammalian DNA
replication and its regulation, based on human T24
bladder carcinoma cells [27]. Because we followed this
protocol exactly in this study, we explicitly refer to
the latter communication with respect to data charac-
terizing cell cycle, DNA synthesis and cell fraction-
ation into cytosolic, nucleosolic and chromatin

associated material. Clearly, T24 is an abnormal can-
cer cell line having a permanently active H-ras [47]
which conceivably could bypass some regulatory
checkpoints of normal cells. We choose these cells
because they, best of all cell lines examined, permitted
a selective accumulation of a defined cohort of repli-
cons (i.e. that is scheduled to be activated as first of
the S-phase) in the ‘hypoxic preinitiation state’, in
preferable absence of other activated replicons [27]. A
main intention of this work was to study the mechan-
ism of the transition of the latter state to actual initi-
ation. We emphasize that some of the results
obtained, possibly, could only be valid for T24 cells
and not for normal mammalian cells. On the other
hand, the hypoxic suppression of replicon initiation
(situated very close before actual initiation), obvi-
ously, is a widespread property of proliferating mam-
malian cells, possibly serving basal functions, such as
protection against metabolic catastrophes during
embryonic development or wound healing and, unfor-
tunately, also in tumour growth [38]. So far, we
found it in all types of cells examined in this respect
during the past 20 years, ranging from cells replica-
ting a virus (SV40) over a diversity of tumour cell
lines to normal human primary explanted from umbi-
lical cord vein (HUVEC) and nasal epithelium
(HNEpC) (G. Probst, H. Probst & V. Gekeler, unpub-
lished observations). We suggest, therefore, that the
data presented here have more general significance.
The ‘hypoxic preinitiation complex’ as defined by us

[27] appears very similar to the preinitiation complex
described by Dutta and Bell [2,48]. We suspected that
the mechanisms activating the ‘hypoxic preinitiation
complex’ and the ‘classical prereplication complex’ are
similar, and that hypoxia interferes with the switch
from preinitiation to initiation. The molecular mode of
action of the suspected switch is, so far, completely
obscure. However, as mentioned, fast recruitment of
regulatory proteins such as protein kinases to the ori-
gins of replicons and ⁄ or modifications of other pro-
teins associated with them, by the still inactive
initiation complex are involved. A likely candidate was
thus Cdk2. Numerous reports suggested that Cdk2
activity is essential for growth, and that the absence of
Cdk2 activity leads to growth arrest. In addition, hyp-
oxia has been reported to suppress Cdk2 activity
through elevated expression of the Cdk inhibitor p27,
D. Stabenow et al. Cdk2 is dispensable for replicon initiation
FEBS Journal 272 (2005) 5623–5634 ª 2005 FEBS 5629
thus preventing pRb phosphorylation and activation
[49].
We found that Cdk2 associates with cellular chro-
matin within 4–6 h, when cells arrested in G
1
are re-
stimulated by feeding under normoxic as well as under
hypoxic conditions. Cdk2 protein kinase activity was
immunoprecipitable from the chromatin and cytosolic
fraction of hypoxic as well as of reoxygenated cells.
Total cellular Cdk2 protein kinase activity was found

to be susceptible to inhibition by olomoucine, roscovi-
tine, staurosporine or the Cdk2 ⁄ cyclin inhibitory
peptide II in vitro. However, administration of the
inhibitors in vivo before reoxygenation to the hypoxic
cells had virtually no influence on the DNA replication
occurring after reoxygenation, neither on the synchron-
ous burst of replicon initiations, nor on the succeeding
daughter strand elongation (Fig. 2). The inhibitors had
no detectable influence on the behaviour of total cellu-
lar and chromatin associated pRb, Cdc6 and PCNA
after the reoxygenation either.
Hypoxia, started concurrently with feeding with
fresh medium, suppressed pRb phosphorylation at
least until 30 min after reoxygenation (Figs 3 and 4),
despite the initiation burst occurring within these
30 min. However, because the pRb phoshorylation
appearing after cell feeding without imposed hypoxia
emerges in the course of 6–7 h, it is possible through-
out that a pRb phoshorylation, susceptible to the
inhibitors, occurs some hours later after reoxygenation,
but then clearly cannot be causal for triggering the ini-
tiation burst already passed by. Obviously, increased
Cdc6 accumulation occurred in the chromatin associ-
ated protein fraction when, after feeding, hypoxia was
imposed on the cells. Subsequent reoxygenation
caused, within 30 min, a significant decrease of chro-
matin associated Cdc6 accompanied by a phosphoryla-
tion of a part of it. The decrease of chromatin
associated Cdc6 as well as the phosphorylation, was
not prevented by 100 nm staurosporine (sufficient to

inhibit Cdk2), or by olomoucine, roscovitine or the
Cdk2 ⁄ cyclin inhibitory peptide II. Staurosporine at
10 lm, however, distinctly inhibited this decrease and
the presumed phosphorylation. At the same time, the
increase of chromatin associated PCNA, indicating
emerging processive DNA strand elongation after
reoxygenation, failed to appear (Fig. 3C). The high
staurosporine concentration also suppressed the burst
of replicon initiations released by reoxygenation in the
T24 system observable by alkaline sedimentation (data
not shown). In this context it is tempting to speculate
that Cdc6 may be an in vivo substrate of a protein kin-
ase other than Cdk2, and its phosphorylation may be
involved in the eventual release of replicon initiation.
This phosphorylation possibly abolishes binding of
Cdc6 to the still incomplete initiation complex, thus,
perhaps, allowing recruitment of still missing proteins.
However, despite the recent data that Cdk2 may be
dispensable for proliferation in a mouse model [24],
our data may seem contradictory to published studies
on the prerequisites of initiation of DNA replication
starting from different states present in G
1
or G
0
nuclei.
It has been reported that the initiation of DNA
replication is dependent on the coordinate activity
of Cdk2 ⁄ cyclin E and Cdk2 ⁄ cyclin A complexes
[18,21,50]. The mentioned results are obtained with

in vitro replication systems from HeLa and ⁄ or mouse
3T3 cells by mixing isolated nuclei and cytosolic
extracts from different cell cycle phases. Our in vivo
situation is a quite different one: the relevant intervals
of time between mixing cytosolic extracts with nuclei
and emergence of additional replicating nuclei are in
the order of hours. In contrast, our protocol produces,
in living cells, a defined state of replicons situated
before origin unwinding (as shown in the SV40 system
[46]) and characterized with respect to the absence of
the proliferation marker PCNA in the chromatin frac-
tion [27]. Moreover, in the mentioned studies, syn-
chronous cells for the preparation of nuclei and
cytosolic extracts were obtained by different protocols.
It can be asked whether the kind of synchronization is
critical for the need of Cdk2 ⁄ cyclin complexes in initi-
ation of replication.
It was also demonstrated that Cdk2 activity is
diminished in hypoxic cells [49,51,52]. These studies
examined Cdk2 preparations immunoprecipitated from
whole cells. The observed decrease in Cdk2 activity
was probably mainly due to elevated levels of the
Cdk2 inhibitory protein p27 obviously expressed under
hypoxia in the studied cells and coimmunoprecipitated
with Cdk2. With T24 cells we found did neither eleva-
ted levels of p27 after several hours of hypoxic incuba-
tion nor mentionable amounts of p27 coprecipitated
with Cdk2 under any incubation condition (data not
shown). This suggests that in T24 cells inhibition of
the pRb pathway is not the direct cause of the hypoxic

suppression of initiation in replicons. Moreover, in
cells replicating SV40 DNA pRb is inactivated by
binding to the large T-antigen; nevertheless, their repli-
cation of viral genomes obeys the fast O
2
dependent
control of replicon initiation [53].
The elucidation of the in vivo molecular processes
leading from the hypoxic preinitiation state, within a
few minutes, to actual initiation was the sole objective
of this work. At the time, the T24 cell based protocol,
to our knowledge, accomplishes that comparatively
Cdk2 is dispensable for replicon initiation D. Stabenow et al.
5630 FEBS Journal 272 (2005) 5623–5634 ª 2005 FEBS
well. However, we may have observed phenomena that
are characteristic only of this cell line and possibly for
some further (abnormal) cells carrying comparable
genetic defects. The general validity of our results
depends on the demonstration of the described effects
in a broader range of cell types, including noncancer
cells.
Experimental procedures
Cell culture, transient hypoxia, reoxygenation,
and radioactive labelling
T24 cells (ATCC No. HTB-4) were grown in plastic flasks
in DMEM supplemented with 10% (v ⁄ v) fetal bovine
serum and 100 UÆmL
)1
penicillin ⁄ 100 lgÆmL
)1

streptomy-
cin. The cells were subcultured when they reached conflu-
ence. For synchronization, the desired number of glass
Petri dishes was seeded from an almost confluent large cul-
ture with 150 000 cellsÆmL
)1
(35 mm, 1.5 mL; 145 mm,
25 mL) 44 h before the start of an experiment, as reported
previously [27]. As a result, most cells were arrested in G
1
due to starvation. For prelabelling of DNA, the seeding
medium was supplemented with 5 nCiÆ mL
)1
[
14
C]thymidine.
Experiments were started by stimulation of the cells by
complete medium change, using prewarmed fresh medium,
supplemented with 10% (v ⁄ v) fetal bovine serum. Then, the
cultures were gassed by a continuous flow of humidified
artificial air containing 5% (v ⁄ v) CO
2
in case of normoxic,
and with 0.0075% (v ⁄ v) O
2
,5%(v⁄ v) CO
2
, Ar ad 100% in
case of hypoxic incubations. This hypoxic gassing protocol
diminished the pO

2
in the cultures within 1.5–2 h to about
0.1% and within 6–7 h to about 0.02%. For gassing, the
equipment and the procedures described by [53] were used.
For reoxygenation, 0.25 vols 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 ⁄ 5% (v ⁄ v) CO
2
.
Hypoxic addition of inhibitors or radioactive precursors
was performed by plunging a spatula carrying the appropri-
ate quantity into the culture medium without opening the
gassing vessel. To stop incubations, medium was removed
by aspiration, and the cells were washed once with ice-cold
NaCl ⁄ P
i
.
Alkaline sedimentation analysis of cellular DNA
To analyse the length distribution of growing daughter
strands of T24 DNA, cultures on 35-mm glass Petri dishes
were pulse labelled for 8 min with 7 lCi [methyl-
3
H]deoxy-
thymidineÆmL
)1
. Labelling was stopped by washing the cells

with ice cold NaCl ⁄ P
i
. The cells were trypsinized and
layered onto the top of 15–30% alkaline sucrose gradients.
After denaturation of the DNA for 6 h, centrifugation was
performed at 20 000 r.p.m., at 23 °C for 10 h in a Beckman
SW28 rotor (Krefeld, Germany). Fractions of 1.2 mL were
collected from the top of the gradient and processed to ana-
lyse acid insoluble radioactivity.
Cell fractionation
Fractions containing cytosolic, nucleosolic, and chromatin
bound proteins of the cells were prepared as described pre-
viously [27]. Modified buffers were used for immunoprecipi-
tation and kinase assays. The cytosolic proteins were
obtained after incubation of the cells in buffer A [50 mm
Hepes pH 7.5; 20 mm b-glycerolphosphate; 1 mm dithio-
threitol; containing aprotinin (1 lm); leupeptin (50 lm);
4-(2-aminoethyl)-benzenesulfonylfluoride HCl (AEBSF)
(1 mm); NaF (10 mm); and Na
3
Vo
4
(1 mm)] and subsequent
disruption of the cellular envelope with 20 strokes of a tight
fitting pestle. Nucleosolic proteins were obtained after incu-
bation in buffer B (buffer A containing 0.1% NP40). The
remaining chromatin bound proteins were solubilized by
incubating the chromatin in IP buffer (buffer B containing
450 mm NaCl) for 1 h on ice and subsequent centrifuga-
tion. The fraction remaining after extraction of nuclei con-

tains about 8.2% of total protein recovered and 96.6% of
total DNA.
Immunoprecipitation, kinase assay and
phosphatase digestion
For immunoprecipitation of Cdk2 and the subsequent kin-
ase assay, solutions of cytosolic, nucleosolic, solubilized
chromatin bound or total cellular proteins were adjusted to
equal protein content (300 lg) and incubated with 2 lg
Cdk2 antibody (Santa Cruz, Clone M2; Santa Cruz, CA,
USA) for 40 min on ice. Then, 20 lL of a slurry of Protein
G-agarose beads (Roche, Mannheim, Germany) equili-
brated with IP buffer were added, and incubation was con-
tinued overnight under continuous rotation.
Subsequently, the beads were washed three times with
500 lL IP buffer and three times with 500 lL kinase buffer
[50 mm Tris pH 7.5; 10 mm MgCl
2
;1mm dithiothreitol;
20 mm b-glycerolphosphate; containing aprotinin (1 lm);
leupeptin (50 lm); AEBSF (1 mm); NaF (10 mm); and
Na
3
Vo
4
(1 mm)]. Excess solution was removed completely
and beads were incubated for 30 min at 30 °C in a mix of
20 lL kinase buffer containing 2.5 lg H1; 0.2 mm ATP;
and 10 lCi [
32 (or33)
P]ATP[cP] in the absence or presence of

Cdk2 inhibitors.
Incubation was stopped by adding 20 lL2· protein sam-
ple buffer. After denaturation, the reaction products were
separated by an SDS ⁄ PAGE on a 12% polyacrylamide gel,
blotted onto a Nylon-P membrane (Amersham, Bucks, UK)
and subsequently exposed to Hyperfilm MP (Amersham).
For immunoprecipitation and subsequent phosphatase
digestion of Cdc6 or pRb, respectively, solutions of
D. Stabenow et al. Cdk2 is dispensable for replicon initiation
FEBS Journal 272 (2005) 5623–5634 ª 2005 FEBS 5631
solubilized chromatin bound proteins, containing 300 lgof
protein, were incubated with 2 lg Cdc6 antibody (MoBi-
Tec, Clone DCS-180), or pRb antibody (Pharmingen,
Clone G3-245; BD Biosciences Pharmingen, Canada) for
40 min on ice. Then, 20 lL of a slurry of Protein A-agarose
beads (Bio-Rad, Hercules, CA, USA) equilibrated with IP
buffer were added, and incubation was continued for 3 h
(Cdc6) or overnight (pRb) under continuous rotation. Sub-
sequently, the beads were divided in two equal parts and
washed three times with IP buffer. Protein sample buffer
(20 lL) was added to one part afterwards. The other part
was washed three times with phosphatase buffer (New Eng-
land Biolabs, Beverly, MA, USA) supplemented with prote-
ase inhibitors. Phosphatase digestion was performed for
30 min at 30° with 200 U Lambda phosphatase (New Eng-
land Biolabs). After a final wash with IP buffer 20 lL pro-
tein sample buffer were added. After denaturation, proteins
were separated on an 8% (49 : 1, Cdc6, PCNA and total
pRb) or 10% (149 : 1, chromatin bound-pRb) SDS ⁄ poly-
acrylamide gel, blotted onto Hybond-P (Amersham). Pro-

teins were visualized as described below.
Immunofluorescence staining of Cdk2
Cells grown on coverslips and incubated with the fluoresc-
ein isothiocyanate (FITC) labelled Cdk2 ⁄ cyclin inhibitory
peptide II during the respective incubations, washed once
with ice-cold NaCl ⁄ P
i
. For subsequent immunostaining of
Cdk2, cells were directly fixed with ice cold acetone ⁄ meth-
anol (1 : 1, v ⁄ v) for 10 min at 4 °C and processed for detec-
tion of Cdk2 after air drying. Cells were blocked with 1%
(w ⁄ v) BSA in NaCl ⁄ P
i
for 20 min and incubated with the
Cdk2-antibody (Pharmingen, Clone 55) in NaCl ⁄ P
i
⁄ BSA
for 1 h at room temperature. After washing three times
with NaCl ⁄ P
i
for 5 min they were further incubated for
30 min with a mouse-antibody labelled with Alexa FluorÒ
586 (Molecular Probes, dilution 1 : 200; Eugene, OR, USA)
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
Cdk2, the Cdk2 ⁄ cyclin inhibitory peptide II and total DNA
(bisbenzimide stain) were visualized with a Zeiss fluores-
cence microscope (Axioskop; Jena, Germany) using the
appropriate filter combinations.
Electrophoresis of proteins and western blotting
Cytosolic and nucleosolic proteins were precipitated from
the respective supernatants using the procedure described
by Wessel-Flu
¨
gge [54]. Chromatin bound proteins for west-
ern blot analysis were either separated from the DNA by
treatment with 450 mm NaCl in buffer B and subsequent
Wessel-Flu
¨
gge precipitation as described above, or chroma-
tin was directly denatured and solubilized with SDS electro-
phoresis sample buffer.
After determination of protein concentration (BioRad
DC protein assay) equal amounts were separated on appro-
priate SDS⁄ polyacrylamide gels, blotted onto a Nylon-P
membrane and subsequently immunodetected using the
ECL western blotting procedure (both Amersham), accord-
ing to the manufacturer’s instructions. Dilution of antibod-
ies used was as follows: Cdk2 (Pharmingen, Clone 55)
1 : 2,500, Cdc6 (Santa Cruz, 180.2) 1 : 500, pRb (Pharmin-
gen, Clone G3-245) 1 : 1,000, hypophosphorylated pRb

(Pharmingen, Clone G99-549), PCNA (Santa Cruz, clone
PC10) 1 : 10 000.
Acknowledgements
We thank Hubert Kalbacher for generous gift of the
Cdk2 ⁄ cyclin inhibitory peptide II. This work was sup-
ported by the Wilhelm-Schuler Stiftung.
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