Human Cdc45 is a proliferation-associated antigen
S. Pollok
1
, C. Bauerschmidt
2
,J.Sa
¨
nger
3
, H P. Nasheuer
4
and F. Grosse
1,5
1 Leibniz Institute for Age Research, Fritz Lipmann Institute, Jena, Germany
2 Radiation Oncology and Biology, University of Oxford, UK
3 Institute of Pathology, Bad Berka, Germany
4 National University of Ireland, Department of Biochemistry, Galway, Ireland
5 Center for Molecular Biomedicine, Friedrich Schiller University, Jena, Germany
In an adult human body only a small proportion of
cells actively progresses through the mitotic cell cycle
in self-renewing tissues [1]. The majority of cells have
ceased proliferation during growth and development
and have arrested temporarily or permanently in non-
proliferative states. Normal somatic cells require stimu-
lation by growth factors for continual proliferation.
After mitogen withdrawal, cells exit the cycle prior to
progression through the restriction point in G
1
and
enter into a quiescent state also called G
0

marker; senescence; terminal differentiation
Correspondence
F. Grosse, Leibniz Institute for Age
Research, Fritz Lipmann Institute e.V.,
Biochemistry, Jena, Germany
Fax: +49 3641-656288
Tel: +49 3641 656290
E-mail: fgrosse@fli-leibniz.de
(Received 9 March 2007, revised 21 May
2007, accepted 23 May 2007)
doi:10.1111/j.1742-4658.2007.05900.x
Cell division cycle protein 45 (Cdc45) plays a critical role in DNA replica-
tion to ensure that chromosomal DNA is replicated only once per cell
cycle. We analysed the expression of human Cdc45 in proliferating and
nonproliferating cells. Our findings show that Cdc45 protein is absent from
long-term quiescent, terminally differentiated and senescent human cells,
although it is present throughout the cell cycle of proliferating cells. More-
over, Cdc45 is much less abundant than the minichromosome maintenance
(Mcm) proteins in human cells, supporting the concept that origin binding
of Cdc45 is rate limiting for replication initiation. We also show that the
Cdc45 protein level is consistently higher in human cancer-derived cells
compared with primary human cells. Consequently, tumour tissue is pref-
erentially stained using Cdc45-specific antibodies. Thus, Cdc45 expression
is tightly associated with proliferating cell populations and Cdc45 seems to
be a promising candidate for a novel proliferation marker in cancer cell
biology.
Abbreviations
BrdU, 5-bromo-1-(2-deoxy-b-
D-ribofuranosyl) uracil; Cdc, cell division cycle; Cdt1, cdc10 target 1; CENP-F, centromer protein F; b-Gal,
senescence associated b-galactosidase; HEF, human embryonic fibroblasts; HRP, horseradish peroxidase; IP, immunoprecipitation; Mcm,

) [20]
demonstrates the relative low abundance of Cdc45 in
human cells and is further evidence that Cdc45 may be
a rate-limiting factor for the initiation of DNA replica-
tion [21,22].
Results
Level of Cdc45 protein is constant during the cell
cycle in proliferating cells
The central functions of Cdc45 in human DNA repli-
cation raised the question of whether Cdc45 is regula-
ted differently in proliferating and nonproliferating
cells. First, we analysed the expression and subcellular
distribution of human Cdc45 protein during the cell
cycle in proliferating HeLa S3 cells. To this end, cells
were arrested at the G
1
⁄ S border by a double thymi-
dine (TdR) block and released to continue the cell
cycle. Successful synchronization and cell-cycle distri-
bution was confirmed by flow cytometry (Fig. 1A). To
monitor cell-cycle progression of the synchronized
cells, levels of cyclin A and cyclin B1 were detected in
western blots and shown to fluctuate depending on
progression through the cycle (Fig. 1B). In parallel,
levels of origin recognition complex (Orc)2, Cdc45 and
b-actin (loading control) were determined to be rather
invariable in cycling HeLa S3 cells (Fig. 1B).
To verify the cell-cycle distribution of synchronized
cells, which were maintained under optimal growth
conditions, immunofluorescence studies were per-

cells
Stoeber et al. [6] demonstrated that human Mcm2,
Mcm3 and Mcm5 proteins were completely down-
regulated when WI-38 cells stopped proliferation and
entered into quiescence. Furthermore, Cdc6, Mcm2,
Mcm3, Mcm5 and Mcm7 proteins were not detectable
in quiescent mouse NIH 3T3 cells [6]. Also, another
initiation factor, human Cdt1 protein, was not
expressed in quiescent human foreskin fibroblasts [24].
Arata et al. reported that Cdc45 protein was below
detectable levels in quiescent mouse NIH cells [25].
These findings led us to investigate whether the human
replication factor Cdc45 is also downregulated when
human cells exited from the cell cycle and entered into
the G
0
phase. Therefore, T98G glioblastoma cells and
human embryonic fibroblasts (HEF) were growth-
arrested by serum starvation in combination with
contact inhibition for up to 20 days and cells were
collected at the indicated times for later analyses.
After 7 days of serum starvation the majority of
T98G and HEF cells reached quiescence, as monitored
by the absence of cyclin A and upregulation of the
p27
KIP1
protein (Fig. 2A,C). Expression of the Ki-67
protein is associated with proliferating cells and is
undetectable in quiescent cells [26,27]. We found that
Cdc45 protein became undetectable in long-term quies-

To explore the regulation of human Cdc45 in nonpro-
liferating cells in more detail, the differentiation of
human cells was used as a second system. Human
Cdc6 protein was completely downregulated during
in vitro differentiation of K562 cells to cells with a
megakaryocytic phenotype [29]. Furthermore, the
amount of human Mcm3 protein was significantly
reduced after induction of HL60 differentiation into
monocytes ⁄ macrophages [7]. Mcm protein expression
was absent in adult neurons and cardiac myocytes [6].
A
C
B
Fig. 1. Expression of human Cdc45 protein in proliferating cells. (A) Flow cytometry analysis of HeLa S3 cells after release from a double TdR
block. Asynchronously growing cells (log) served as a control for the classification of cell-cycle phases. (B) Immunoblot analysis was performed
from whole-cell lysates of asynchronously proliferating cells (log) and cells in a time course after release from a double TdR block. Cdc45,
Orc2, cyclin A and cyclin B1 were detected with specific primary antibodies, HRP-coupled secondary antibodies, followed by the standard
enhanced chemoluminescence technique. b-Actin served as a control for equal loading. (C) Immunofluorescence analysis of the subcellular dis-
tribution of Cdc45 throughout the cell-cycle phases. The yellow bar in the phase contrast ⁄ DAPI stain indicates 10 lm(·100). The upper panel
shows phase contrast and DAPI staining, the middle panel displays Cdc45 in green and the lower panel shows in red either Ki-67 (G
1
phase:
12 h after TdR block, and mitosis: 9 h after TdR block), BrdU (S phase: 3 h after TdR block) or CENP-F (G
2
phase: 9 h after TdR block).
S. Pollok et al. Cdc45 expression in proliferation
FEBS Journal 274 (2007) 3669–3684 ª 2007 The Authors Journal compilation ª 2007 FEBS 3671
To test whether Cdc45 is regulated during terminal
differentiation both HL60 and K562 cells were treated
with 4b-phorbol 12-myristate 13-acetate (PMA). After

[31] indicating that the cells stopped cycling after
induction of differentiation (Fig. 3A). Changes in
morphology of cycle-arrested cells were accompanied
by a rapid decrease in immunological detectable
Cdc45 within 36 h after PMA application; 12 h later
the level of Cdc45 protein was almost completely abol-
ished (Fig. 3A). Similarly, immunofluorescence studies
with HL60 cells revealed that Cdc45 protein became
AC
B
D
Fig. 2. Regulation of human Cdc45 protein following exit into the G
0
phase. (A,C) Immunoblot analysis of Mcm2, -4, -7, Cdc6, Cdc45, cyclin
A and p27
KIP1
in whole-cell lysates of asynchronously proliferating (log) and serum-starved T98G cells (A) and human embryonic fibroblasts
(HEF) (C). (B) Immunoblot analysis of DNA polymerase d p125 and p50 subunits, PCNA and replication protein A p70 and p32 subunits in
whole-cell lysates of serum-starved T98G cells. (D) Immunofluorescence analysis of Cdc45 in logarithmic (log) or 10 days serum-starved
T98G cells (G
0
). The upper panel shows phase contrast and Ki-67 in red, the lower panel shows Cdc45 in green (·20).
Cdc45 expression in proliferation S. Pollok et al.
3672 FEBS Journal 274 (2007) 3669–3684 ª 2007 The Authors Journal compilation ª 2007 FEBS
undetectable after 48 h of PMA incubation (data not
shown). In addition, a significant downregulation of
human Cdc45 protein was also detected after incuba-
tion of HL60 cells with all trans retinoic acid, which
causes terminal differentiation along the granulocyte
phenotype (supplementary Fig. S1A). Moreover, in the

S. Pollok et al. Cdc45 expression in proliferation
FEBS Journal 274 (2007) 3669–3684 ª 2007 The Authors Journal compilation ª 2007 FEBS 3673
downregulated, and Mcm4 levels did not change at all
in the differentiated megakaryocytic-like cells compared
with undifferentiated K562 cells (Fig. 3D). Collectively,
in the tested differentiation systems there seem to exist a
(slightly) unequal regulation of the various DNA repli-
cation factors, whereas the time dependence of Cdc45
expression was remarkably similar.
Level of Cdc45 protein is abolished in human
cells entering senescence in vitro
After a finite number of cell divisions normal somatic
cells irreversibly arrest in G
1
with a senescent pheno-
type. Stoeber et al. [6] showed that human Mcm2,
Mcm3, Mcm5 and Cdc6 proteins were downregulated
in senescent WI-38 fibroblasts, whereas Orc2 protein
levels remained largely unaffected [6]. However, to
date, nothing has been reported about the regulation
of Cdc45 protein in cells entering replicative senes-
cence.
Senescent MRC-5 and WI-38 fibroblasts were
obtained by continuously culturing them up to passage
26 or 28. Intracellularly, senescence was exerted and
maintained through the function of the cyclin-kinase
inhibitors p21
CIP1
and p16
INK4A

Cell-cycle re-entry was induced by the addition of
10% fetal bovine serum and the subsequent splitting
of culture cells to enhance proliferation. The trans-
ition from G
0
to proliferation was monitored by flow
cytometry (Fig. 5A; percentage of cell population in
G
0,
G
1
,S,G
2
⁄ M) and BrdU incorporation into cells
(Fig. 5B). In addition, the expression of cyclin D1,
cyclin A, cyclin B1 and p27
KIP1
was determined by
western blotting (Fig. 5C). p27
KIP1
was reported to
be elevated in quiescent cells [35] and to become
degraded by the ubiquitin–proteasome pathway after
stimulation of cells with growth factors [36]. A signi-
ficant decrease in the p27
KIP1
protein level was seen
6 h after serum addition (Fig. 5C). Cyclin expression
started in a defined order beginning with cyclin D1
A

unit of DNA polymerase a showed up (Fig. 5C).
These results indicate that human Cdc45 protein is
synthesized de novo after G
0
release prior to the
S-phase entry in consistence with its requirement for
the initiation of DNA replication. Remarkably, the
observed time course of expression of Cdc6, Cdc45
and DNA polymerase a seems to mirror the time
course of loading of these replication factors to the
origins of replication.
A
B
C
Fig. 5. Expression of human Cdc45 protein after serum stimulation. T98G cells were arrested by serum starvation in the G
0
phase and sti-
mulated with 10% fetal bovine serum to re-enter the cell cycle. Samples were taken at the indicated time points after serum stimulation
and from asynchronously proliferating cells (log). (A) In order to assess cell-cycle progression, flow-cytometry analysis was performed. (B) To
determine the percentage of replicating cells, the cells were pulse labelled with BrdU. (C) SDS ⁄ PAGE and western blotting were performed
with whole-cell lysates from 2 · 10
5
cells for each time point after serum stimulation. p27
KIP1
, cyclin D1, cyclin A and cyclin B1 were ana-
lysed to determine entry into and passage through cell cycle.
S. Pollok et al. Cdc45 expression in proliferation
FEBS Journal 274 (2007) 3669–3684 ª 2007 The Authors Journal compilation ª 2007 FEBS 3675
Half-life of Cdc45 protein and number of Cdc45
molecules in proliferating human cells

6
–Cdc45 onto an SDS gel alongside total cell
lysates from 2 · 10
5
asynchronously growing cells.
Quantification of the western blot signals revealed that
 1 ng of Cdc45 protein was present in 2 · 10
5
HeLa S3 as well as in T98G cells (Fig. 7B). Because
the molecular mass of Cdc45 is 65.5 kDa it can be cal-
culated that  4.5 · 10
4
molecules were present in each
cell of these two human cell lines. It should be kept in
mind that the Cdc45 protein was detectable in all sta-
ges of the cell cycle of proliferating cells (Fig. 1B,C).
Cdc45 is overexpressed in cancer-derived cell
lines and can serve as a biomarker for tumour
cells using immunohistology
After showing a positive correlation between Cdc45
expression and cell proliferation, we examined the
expression levels of the protein in different cancer-
derived cell lines in comparison with primary cells. Cell
extracts were prepared from the primary cells WI-38,
MRC-5 and HEF in low passage numbers, as well as
A
B
Fig. 6. Estimation of Cdc45 protein half-life. Metabolic labelling of
logarithmically growing HeLa S3 cells was performed to measure
the half-life of human Cdc45 protein. (A) [

amounts of BSA. After staining with PageBlue
TM
protein-staining
solution (Fermentas) the bands were quantified with the program
PHORETIX 1D ADVANCED. (B) Whole cell extracts of 2 · 10
5
asynchro-
nously proliferating HeLa S3 and T98G cells were run alongside
with declining amounts of recombinant human His
6
-Cdc45. The gel
was immunoblotted and probed with the anti-Cdc45 serum and an
HRP-coupled secondary antibody using the enhanced chemolumi-
nescence technique. The positions of endogenous and recombinant
His
6
-tagged Cdc45 are marked. The protein bands were quantified
with the program
PHORETIX 1D ADVANCED.
Cdc45 expression in proliferation S. Pollok et al.
3676 FEBS Journal 274 (2007) 3669–3684 ª 2007 The Authors Journal compilation ª 2007 FEBS
from human cell lines that represented carcinoma-,
sarcoma-, leukaemia- and lymphoma-derived cells (for
details see description in Fig. 8). Western blot analysis
revealed that cancer-derived cell lines had consistently
higher Cdc45 levels than the tested primary lines
(Fig. 8A,B). Interestingly, Cdc45 was exclusively detec-
ted as a double band in HL60 cells (Figs 3A,8B),
whereas Cdc45 appeared as a single band in other
leukaemia-derived cell lines. The investigation of the

fact that the specimen consisted of a relatively slow
growing cell population with a high number of cells in
the G
1
phase. Ki-67 is a short-lived protein [40] pre-
dominantly expressed during the S, G
2
and M phases
[27], whereas Cdc45 is present in comparable amounts
throughout the cell cycle (Fig. 1).
Discussion
Previous reports have shown that the amounts of
human Mcm2, -3, -5, -7 and Orc2 remain constant
during the cell cycle [6,41], whereas levels of Cdc6 and
Cdt1 fluctuate [37,42]. Here, we showed that in
HeLa S3 (Fig. 1B) and T98G cells (data not shown)
levels of human Cdc45 protein remained constant dur-
ing the cell cycle. This confirms a previous report, in
which, according to western blot analysis of HeLa
cells, the protein level of Cdc45 remained unchanged
during the cell cycle, whereas the amount of Cdc45
mRNA peaked at G
1
⁄ S [43]. Although Cdc45 protein
levels remained constant in proliferating cells (Fig. 1B),
we detected significant changes in subcellular localiza-
tion over the cell cycle (Fig. 1C). In HeLa S3 (Fig. 1C)
and T98G glioblastoma cells (data not shown), Cdc45
protein was found exclusively in the nucleus during G
1

tic leukaemia), Jurkat (acute T-cell leukaemia), HL60 (acute pro-
myelocytic leukaemia), K562 (chronic myelogenous leukaemia), and
U-937 (histiocytic leukaemia), respectively.
S. Pollok et al. Cdc45 expression in proliferation
FEBS Journal 274 (2007) 3669–3684 ª 2007 The Authors Journal compilation ª 2007 FEBS 3677
quiescence, terminal differentiation or senescence
(Figs 2–4, supplementary Fig. S1). Furthermore, both
in mouse cells [25] and human cells leaving the G
0
phase,
Cdc45 reappeared shortly before a new S phase started
(Fig. 5). Downregulation of the essential replication fac-
tor Cdc45 seems to reflect an additional control mechan-
ism over the breakdown of licensing factors to ensure
the inactivity of replication origins in cells that had left
the mitotic cycle. Previous reports clearly exhibited that
E2F-regulated promoters were transcriptional silenced
in quiescent as well as in senescent cells [47,48].
E2F-binding sites were identified in the promoter
regions of mammalian MCM genes [41], the CDC6 gene
[49] and the gene for DNA polymerase a [50]. Because
an ‘E2F-binding site’-like element was found on human
Cdc45 cDNA [25] and Cdc45 protein was completely
absent from nonproliferating cells (Figs 2–4), it is
reasonable to assume that expression is regulated via the
pRb-E2F pathway. However, the observed consecutive
expression of Cdc6, Cdc45 and polymerase a (Fig. 5C)
A
B
Fig. 9. Immunohistochemical detection of human Cdc45, PCNA and Ki-67. The proteins Cdc45, Ki-67 and PCNA were detected in formalin-

4
Cdc45 molecules exist per
HeLa S3 or T98G cell (Fig. 7). Previous reports showed
that  1 · 10
6
Mcm3 [20] and  3 · 10
4
Cdt1 ⁄ geminin
molecules were present per HeLa cell [24]. Thus, the
ratio of extractable Mcm3 to Cdc45 was 22 : 1, suggest-
ing that Mcm proteins are in great excess in human
cells. Because there are  2.5 · 10
4
initiation events per
somatic cell [54], there are about two molecules Cdc45
per origin. This low abundance of human Cdc45 further
supports the idea that Cdc45 may be a rate-limiting
factor for replication initiation [21,22].
Also, we observed overexpression of endogenous
Cdc45 in human cancer cell lines from various sources
(Fig. 8). A characteristic hallmark of cancer cells is a
deregulation of cellular proliferation [55]. The assess-
ment of cellular proliferation in histological material is
a valuable component of conventional histopathologi-
cal analysis and may be of major prognostic import-
ance [56]. Proliferation in immunohistochemical
sections can be measured in different ways [39,56]: on
the one hand, by detecting cells in mitosis (mitotic
index) or S phase (S-phase fraction) and, on the other
hand, by detecting proliferation-associated proteins

The indicated proteins were analysed using the following
sera: anti-(Cdc45, C45-3G10) [38]; anti-(b-tubulin Clone
SAP.4G5), anti-(b-actin Clone AC-15) (both Sigma,
St Louis, MO), anti-(Cdc6 sc13136), anti-(PCNA PC-10),
anti-(Mcm4 H-300), anti-Mcm7, anti-(Cdc45 sc20685), anti-
(cyclin A sc751), anti-(cyclin B1 sc245), anti-(cyclin D1
sc8396), anti-(cyclin E sc481), anti-(p27
KIP1
sc1641) (all
Santa Cruz Biotechnology, Santa Cruz, CA); anti-(Mcm2
BM28), anti-(p16
INK4
clone G175-1239), anti-(BrdU clone
3D4) (all BD Bioscience, Erembodegem, Belgium); anti-
(Orc2 M055-3S) (MoBiTec, Gottingen, Germany); anti-
(p21
CIP1
OP68) (Calbiochem, San Diego, CA); anti-(pol d
p125 PDG-5G1), anti-(pol d p50 PDK-7B4), anti-(pol a
p180 2CT25) [61]; anti-(Ki-67 MIB-1), anti-(PCNA PC-10)
(Dako cytomation, Carpinteria, CA); anti-(CENP-F
NB500-101) (Novus, Littleton, CO); biotinylated rabbit
anti-(ratIgG) ⁄ biotinylated horse anti-(mouse IgG) (LIN-
ARIS, Wertheim, Germany); donkey ⁄ goat anti-(mouse ⁄ rab-
bit ⁄ rat IgG) conjugated with Cy2 ⁄ Cy3, goat anti-(rat IgG)
conjugated with horseradish peroxidase (HRP) (all
Dianova, Hamburg, Germany) and goat anti-(mouse ⁄ rab-
bit IgG) conjugated HRP ⁄ alkaline phosphatase (Promega,
Madison, WI).
Cell culture and synchronization

0
for up to 20 days. T98G cells starved for
10 days were induced to re-enter the cell cycle by a 1:3 or
1:4 split into new medium supplemented with 10% (v ⁄ v)
serum. Cell-cycle status was analysed by an EPICS XL
MCL flow cytometer (Beckman-Coulter, Krefeld, Ger-
many) after propidium iodide (Sigma) staining of DNA.
Induction of in vitro differentiation and
assessment of HL60 differentiation by Nitro Blue
tetrazolium reduction assay
A stock solution of PMA (Sigma) at 160 lm was prepared
in dimethylsulfoxide, and stored at )20 °C in the dark.
To stimulate HL60 cells to differentiate into a mono-
cyte ⁄ macrophage-like phenotype [63] and K562 into a
megakaryocyte phenotype [64] PMA was added for up to
96 h to a final concentration of 16 nm. Control cultures
received an equivalent final concentration of dimethylsulf-
oxide (0.01%). Cells that became attached to the flask
during differentiation were removed mechanically using a
cell scraper and pooled with the cells floating in the
culture media.
Differentiation of PMA-treated HL60 cells was con-
firmed by the appearance of an oxidative burst as well as
adhesive properties. Generation of superoxide was meas-
ured by converting a colourless chemical, Nitro Blue tetra-
zolium, to a deep blue colour. A stock solution was
prepared by suspending Nitro Blue tetrazolium (Roth,
Karlsruhe, Germany) in culture medium at a concentra-
tion of 6 mgÆmL
)1

2
. The number of blue-stained senescent cells was
counted in five microscope fields with a ·20 magnification.
Cell-extract preparation and western blotting
Whole protein extracts were prepared in lysis buffer contain-
ing 1% NP-40, 500 mm NaCl, NaCl ⁄ Tris pH 7 and phenyl-
methanesulfonyl fluoride, aprotinin and leupeptin. Protein
extracts were resolved by SDS ⁄ PAGE, transferred to
poly(vinylidene difluoride) membranes (Immobilon-P, Milli-
pore Corp., Bedford, MA), and detected by using HRP- or
alkaline phosphatase-conjugated secondary antibodies.
Immunofluorescence
Cells were grown in quadriPERM dishes (Sartorius,
Go
¨
ttingen, Germany) on coverslips (Roth). For BrdU
(Sigma) pulse-labelling cells were incubated for 15 min in
the presence of 32 lm BrdU added directly to the culture
medium prior to collection. Cells were washed twice in
fresh medium followed by a NaCl ⁄ P
i
washing step. Cells
were fixed in 4% (w ⁄ v) para-formaldehyde (Sigma) for
10 min, washed twice with NaCl ⁄ P
i
and then permeabi-
lized with 0.25% (v ⁄ v) Triton X-100 in NaCl ⁄ P
i
followed
by washing twice in NaCl ⁄ P

incubated for another 4 h. Cells were washed twice with
NaCl ⁄ P
i
and incubated in Dulbeccco’s modified Eagle’s
medium supplemented with 10% fetal bovine serum, 5 mm
l-methionine and 5 mml-cysteine (Sigma) for the periods
indicated in the figure. After the chase, the cells were lysed
and Cdc45 was immunoprecipitated in immunoprecipitation
(IP) buffer (50 mm Hepes pH 7.5, 150 mm NaCl, 0.5%
NP-40). The antibody C45-3G10 was mixed with pro-
tein G–Sepharose
TM
4 Fast Flow (GE Healthcare) for
30 min at 4 °C, followed by blocking with Perfect Block
(MoBiTec) overnight. After a preclearing step 1 mg total
cell lysate was loaded onto the blocked Sepharose and incu-
bated overnight at 4 °C. Unbound proteins were removed
by washing three times with IP buffer. Precipitated Cdc45
protein was detected after western blotting with the Cdc45-
specific antibody to monitor equal loading. Radiolabelled
Cdc45 was visualized by autoradiography.
Number of Cdc45 molecules per HeLa S3 and
T98G cell
To calculate the number of Cdc45 molecules present per
HeLa S3 or T98G cell, human Cdc45 was expressed as His
6
-
tagged protein using a recombinant baculovirus (vector
pFastBac
TM

Immunohistochemistry of normal and tumour tissues was
performed according to the avidin–-biotin complex peroxi-
dize method [66] with some modifications. Briefly, 4 mm
paraffin sections were transferred to glass slides and dried
overnight. After deparaffination and rehydration, endo-
genous peroxidase activity was quenched by incubation in
0.5% hydrogen peroxide for 30 min. Nonspecific binding
was reduced by incubation with normal horse serum for
20 min. To facilitate antigen retrieval a preheating step and
proteinase K incubation were performed. Sections were
immersed in 0.01 m citrate buffer, pH 6.0, and irradiated in
a microwave oven at 640 W for 16 min. A proteinase K
incubation (1 mgÆmL
)1
in 0.05 m Tris ⁄ HCl pH 7.5) was per-
formed for 3 min. After washing with NaCl ⁄ P
i
the primary
antibodies C45-3G10 [anti-(human Cdc45) 1 : 10 dilution],
MIB-1 [anti-(Ki-67) 1: 50 dilution, Dako cytomation]
and PC10 (anti-PCNA 1 : 100 dilution, Dako cytomation),
diluted in 1% (v ⁄ v) BSA in 0.05 m NaCl ⁄ P
i
, pH 7.5, were
applied for 12 h at 4 °C. Thereafter, sections were rinsed
carefully with NaCl ⁄ P
i
and incubated with a secondary
biotinylated antibody in NaCl ⁄ P
i

and maintenance of cellular diversity. Development 106,
619–633.
2 Pardee AB (1989) G1 events and regulation of cell pro-
liferation. Science 246 , 603–608.
3 Hayflick L & Moorhead PS (1961) The serial cultiva-
tion of human diploid cell strains. Exp Cell Res 25,
585–621.
4 Campisi J (1996) Replicative senescence: an old lives’
tale? Cell 84, 497–500.
5 Williams GH, Romanowski P, Morris L, Madine M,
Mills AD, Stoeber K, Marr J, Laskey RA & Coleman
N (1998) Improved cervical smear assessment using
S. Pollok et al. Cdc45 expression in proliferation
FEBS Journal 274 (2007) 3669–3684 ª 2007 The Authors Journal compilation ª 2007 FEBS 3681
antibodies against proteins that regulate DNA replica-
tion. Proc Natl Acad Sci USA 95, 14932–14937.
6 Stoeber K, Tlsty TD, Happerfield L, Thomas GA,
Romanov S, Bobrow L, Williams ED & Williams GH
(2001) DNA replication licensing and human cell prolif-
eration. J Cell Sci 114, 2027–2041.
7 Musahl C, Holthoff HP, Lesch R & Knippers R (1998)
Stability of the replicative Mcm3 protein in proliferating
and differentiating human cells. Exp Cell Res 241, 260–
264.
8 Kingsbury SR, Loddo M, Fanshawe T, Obermann EC,
Prevost AT, Stoeber K & Williams GH (2005) Repres-
sion of DNA replication licensing in quiescence is inde-
pendent of geminin and may define the cell cycle state
of progenitor cells. Exp Cell Res 309, 56–67.
9 Bell SP & Dutta A (2002) DNA replication in eukaryot-

replisome progression complexes at eukaryotic DNA
replication forks. Nat Cell Biol 8, 358–366.
18 Moyer SE, Lewis PW & Botchan MR (2006) Isolation
of the Cdc45 ⁄ Mcm2–7 ⁄ GINS (CMG) complex, a candi-
date for the eukaryotic DNA replication fork helicase.
Proc Natl Acad Sci USA 103, 10236–10241.
19 Aparicio T, Ibarra A & Mendez J (2006) Cdc45–MCM–
GINS, a new power player for DNA replication. Cell
Division 1, 18.
20 Burkhart R, Schulte D, Hu D, Musahl C, Go
¨
hring F &
Knippers R (1995) Interactions of human nuclear
proteins P1Mcm3 and P1Cdc46. Eur J Biochem 228
,
431–438.
21 Dolan WP, Sherman DA & Forsburg SL (2004) Schizo-
saccharomyces pombe replication protein Cdc45 ⁄ Sna41
requires Hsk1 ⁄ Cdc7 and Rad4 ⁄ Cut5 for chromatin
binding. Chromosoma 113, 145–156.
22 Forsburg SL (2004) Eukaryotic MCM proteins: beyond
replication initiation. Microbiol Mol Biol Rev 68,
109–131.
23 Bauerschmidt C, Pollok S, Kremmer E, Nasheuer HP &
Grosse F (2007) Interactions of human Cdc45 with the
Mcm2–7 complex, the GINS complex and DNA poly-
merase d and e. Genes Cells in press.
24 Xouri G, Lygerou Z, Nishitani H, Pachnis V, Nurse P
& Taraviras S (2004) Cdt1 and geminin are down-regu-
lated upon cell cycle exit and are over-expressed in can-

Differential roles for cyclin-dependent kinase inhibitors
p21 and p16 in the mechanisms of senescence and
differentiation in human fibroblasts. Mol Cell Biol 19,
2109–2117.
34 Dimri GP, Lee X, Basile G, Acosta M, Scott G,
Roskelley C, Medrano EE, Linskens M, Rubelj I,
Pereira-Smith O et al. (1995) A biomarker that identifies
Cdc45 expression in proliferation S. Pollok et al.
3682 FEBS Journal 274 (2007) 3669–3684 ª 2007 The Authors Journal compilation ª 2007 FEBS
senescent human cells in culture and in aging skin
in vivo. Proc Natl Acad Sci USA 92, 9363–9367.
35 Kato JY, Matsuoka M, Polyak K, Massague J &
Sherr CJ (1994) Cyclic AMP-induced G1 phase arrest
mediated by an inhibitor (p27Kip1) of cyclin-dependent
kinase 4 activation. Cell 79 , 487–496.
36 Sherr CJ & Roberts JM (2004) Living with or without
cyclins and cyclin-dependent kinases. Genes Dev 18,
2699–2711.
37 Petersen BO, Wagener C, Marinoni F, Kramer ER,
Melixetian M, Lazzerini Denchi E, Gieffers C, Matt-
eucci C, Peters JM & Helin K (2000) Cell cycle- and cell
growth-regulated proteolysis of mammalian CDC6 is
dependent on APC–CDH1. Genes Dev 14, 2330–2343.
38 Pollok S, Stoepel J, Bauerschmidt C, Kremmer E &
Nasheuer HP (2003) Regulation of eukaryotic DNA
replication at the initiation step. Biochem Soc Trans 31,
266–269.
39 van Diest PJ, Brugal G & Baak JP (1998) Proliferation
markers in tumours: interpretation and clinical value.
J Clin Pathol 51, 716–724.

that occupies E2F- and Myc-responsive genes in G0
cells. Science 296, 1132–1136.
48 Narita M, Nunez S, Heard E, Lin AW, Hearn SA,
Spector DL, Hannon GJ & Lowe SW (2003)
Rb-mediated heterochromatin formation and silencing
of E2F target genes during cellular senescence. Cell 113,
703–716.
49 Ohtani K, Tsujimoto A, Ikeda M & Nakamura M
(1998) Regulation of cell growth-dependent expression
of mammalian CDC6 gene by the cell cycle transcrip-
tion factor E2F. Oncogene 17, 1777–1785.
50 Pearson BE, Nasheuer HP & Wang TS (1991) Human
DNA polymerase alpha gene: sequences controlling
expression in cycling and serum-stimulated cells. Mol
Cell Biol 11, 2081–2095.
51 Varshavsky A (1996) The N-end rule: functions, myster-
ies, uses. Proc Natl Acad Sci USA 93, 12142–12149.
52 Edwards MC, Tutter AV, Cvetic C, Gilbert CH, Prok-
horova TA & Walter JC (2002) MCM2–7 complexes
bind chromatin in a distributed pattern surrounding
the origin recognition complex in Xenopus egg extracts.
J Biol Chem 277, 33049–33057.
53 Woodward AM, Gohler T, Luciani MG, Oehlmann M,
Ge X, Gartner A, Jackson DA & Blow JJ (2006) Excess
Mcm2–7 license dormant origins of replication that can
be used under conditions of replicative stress. J Cell Biol
173, 673–683.
54 Hand R (1978) Eucaryotic DNA: organization of the
genome for replication. Cell 15, 317–325.
55 Evan GI & Vousden KH (2001) Proliferation, cell cycle

USA 76, 1293–1297.
S. Pollok et al. Cdc45 expression in proliferation
FEBS Journal 274 (2007) 3669–3684 ª 2007 The Authors Journal compilation ª 2007 FEBS 3683
64 Whalen AM, Galasinski SC, Shapiro PS, Nahreini TS
& Ahn NG (1997) Megakaryocytic differentiation
induced by constitutive activation of mitogen-activated
protein kinase kinase. Mol Cell Biol 17 , 1947–1958.
65 Kukimoto I, Igaki H & Kanda T (1999) Human
CDC45 protein binds to minichromosome maintenance
7 protein and the p70 subunit of DNA polymerase
alpha. Eur J Biochem 265, 936–943.
66 Hsu SM, Raine L & Fanger H (1981) Use of avidin–
biotin–peroxidase complex (ABC) in immunoperoxidase
techniques: a comparison between ABC and unlabeled
antibody (PAP) procedures. J Histochem Cytochem 29,
577–580.
Supplementary material
The following supplementary material is available
online:
Fig. S1. Regulation of human Cdc45 protein during
terminal differentiation.
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from
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Cdc45 expression in proliferation S. Pollok et al.
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