Cdk2⁄ cyclin A mediated phosphorylation of Cdc6 is thought to induce its translocation from the Keywords hypoxia; reoxygenation; Cdk2; replication; chromatin Correspondence M.. In order
Trang 1initiation after transient hypoxia in T24 cells
Dirk Stabenow, Hans Probst and Maria van Betteraey-Nikoleit
Interfakulta¨res Institut fu¨r Biochemie der Universita¨t Tu¨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 G1- 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
G1-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 replicatranscrip-tion initiatranscrip-tion 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 O2 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 G1 DNA content within 5–6 h In this state they are ready to perform, within 1–
2 min after O2recovery, 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.
Trang 2nucleus to the cytoplasm, thus preventing reinitiation.
Altogether, these observations suggest that Cdk2
activ-ity is necessary for progression of cells from G1to
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 G1 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 G1 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 demonstraactiva-ted
[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 pO2, 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) convincresult-ingly 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
Trang 3We 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 n M ) 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 Fluor 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.
Trang 4showed 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 O2to 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 G2, 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 [3H]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 [3H]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 [3H]dThdÆmL)1 Inset, Starved T24 cells were stimulated by medium renewal and incubated normox-ically (n) or hypoxnormox-ically (.) for 7 h; 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.
Trang 5incubated 7 h after medium renewal normoxically
(open triangles) and hypoxically (filled triangles) The
mature DNA of these cells contained a prelabel
result-ing from [14C]Thd added 44 h before The 14C profile
obtained (crosses) typically exhibits a peak in the last
third of the gradient The 3H-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, elonginiti-ation, and termination [26,38,41] The
gra-dient of hypoxic cells contains practically no3H-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 S20w)
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 S20w) indicate the additional presence of
small portions of longer growing strands This
shoul-der was more or less prominent in different
independ-ent experimindepend-ents 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 S20w 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¢, reoxyreoxy-genated for 30 min after 7 h hypoxic incubation.
Trang 6digestion 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 medmed-ium renewal; 5¢, reoxygenated for 5 min after 7 h hypoxic incubation; 30¢, reoxygen-ated for 30 min after 7 h hypoxic incubation.
Trang 7However, 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 treatmdiffer-ents of the cells in this experimdiffer-ent 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 propro-teins 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 propro-teins
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
G1-arrest than in a G0-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 O2-dependent control of
mammalian replicon initiation [26,39] and the
suc-ceeding detailed evaluation of the relevant conditions
(ranges of O2 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 pO2 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,
Trang 8thus preventing pRb phosphorylation and activation
[49]
We found that Cdk2 associates with cellular
chro-matin within 4–6 h, when cells arrested in G1 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 G1 or G0 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 menfrac-tioned 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 repliciniti-ation
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 menincuba-tionable 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 O2 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
Trang 9well 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 G1
due to starvation For prelabelling of DNA, the seeding
medium was supplemented with 5 nCiÆmL)1[14C]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) CO2in case of normoxic,
and with 0.0075% (v⁄ v) O2, 5% (v⁄ v) CO2, Ar ad 100% in
case of hypoxic incubations This hypoxic gassing protocol
diminished the pO2in 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% O2⁄ 5% CO2(v⁄ v) were added to hypoxic cell cultures,
and gassing was continued with artificial air⁄ 5% (v ⁄ v) CO2
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⁄ Pi
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⁄ Pi 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 23C 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 Na3Vo4(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 MgCl2; 1 mm dithiothreitol;
20 mm b-glycerolphosphate; containing aprotinin (1 lm); leupeptin (50 lm); AEBSF (1 mm); NaF (10 mm); and
Na3Vo4 (1 mm)] Excess solution was removed completely and beads were incubated for 30 min at 30C 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 lL 2· 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
Trang 10solubilized chromatin bound proteins, containing 300 lg of
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 Phosphatprote-ase 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, propro-teins
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⁄ Pi 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 ⁄ Pifor 20 min and incubated with the
Cdk2-antibody (Pharmingen, Clone 55) in NaCl⁄ Pi⁄ BSA
for 1 h at room temperature After washing three times
with NaCl⁄ Pi for 5 min they were further incubated for
30 min with a mouse-antibody labelled with Alexa Fluor
586 (Molecular Probes, dilution 1 : 200; Eugene, OR, USA)
in NaCl⁄ Pi⁄ BSA Cells were again washed three times for
5 min with NaCl⁄ Pi During the last wash total DNA was
stained with bisbenzimide (2 lgÆmL)1 in NaCl⁄ Pi) 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
References
1 Gitig DM & Koff A (2001) Cdk pathway: Cyclin-depen-dent kinases and cyclin-depenCyclin-depen-dent kinase inhibitors Mol Biotechnol 19, 179–188
2 Dutta A & Bell SP (1997) Initiation of DNA replication
in eukaryotic cells Annu Rev Cell Dev Biol 13, 293–332
3 Findeisen M, El Denary M, Kapitza T, Graf R & Straus-feld U (1999) Cyclin A-dependent kinase activity affects chromatin binding of ORC, Cdc6, and MCM in egg extracts of Xenopus laevis Eur J Biochem 264, 415–426
4 Lei M & Tye BK (2001) Initiating DNA synthesis: from recruiting to activating the MCM complex J Cell Sci
114, 1447–1454
5 Takisawa H, Mimura S & Kubota Y (2000) Eukaryotic DNA replication: from pre-replication complex to initia-tion complex Curr Opin Cell Biol 12, 690–696
6 Cocker JH, Piatti S, Santocanale C, Nasmyth K & Diff-ley JF (1996) An essential role for the Cdc6 protein in forming the pre-replicative complexes of budding yeast Nature 379, 180–182
7 Liang C, Weinreich M & Stillman B (1995) ORC and Cdc6p interact and determine the frequency of initiation
of DNA replication in the genome Cell 81, 667–676
8 Nishitani H, Lygerou Z, Nishimoto T & Nurse P (2000) The Cdt1 protein is required to license DNA for replica-tion in fission yeast Nature 404, 625–628
9 Nishitani H, Taraviras S, Lygerou Z & Nishimoto T (2001) The human licensing factor for DNA replication Cdt1 accumulates in G(1) and is destabilized after initia-tion of S-phase J Biol Chem 276, 44905–44911
10 Maine GT, Sinha P & Tye BK (1984) Mutants of
S cerevisiae defective in the maintenance of mini-chromosomes Genetics 106, 365–385
11 Strausfeld UP, Howell M, Rempel R, Maller JL, Hunt
T & Blow JJ (1994) Cip1 blocks the initiation of DNA