Tài liệu Báo cáo khoa học: Human Cdc45 is a proliferation-associated antigen pdf

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

[2]. The G
0
arrest is reversible and after addition of growth factors
cells re-enter the cell cycle [2]. In addition, cells can be
induced to enter a differentiation pathway [1]. More-
over, normal somatic cells have only a limited poten-
tial to divide. After a finite number of cell divisions
they irreversibly enter a senescent state [3]. In contrast
to quiescent cells, senescent cells fail to initiate DNA
replication in response to mitogens [4].
Previous reports have shown that licensing factors
are present in actively cycling cells but are downregu-
lated during quiescence, differentiation and senescence
[5–8]. The licensing reaction depends on the sequential
assembly of cell division cycle protein 6 (Cdc6), cdc10
target 1 (Cdt1) and minichromosome maintenance
(Mcm)2–7 at origins of replication in late mitosis and
early G
1
to form the so-called prereplicative complex
[9]. At the G
1
⁄ S transition prereplicative complexes are
converted into initiation complexes by the concerted
action of cyclin-dependent kinases and Cdc7 kinase
[10]. Following activation of these kinases, replication
factors such as Cdc45, GINS and Mcm10 are recruited
to the origins [11]. Cdc45 has a critical role in the
Keywords
half life; molecule number; proliferation

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,

minichromosome maintenance; Orc, origin recognition complex; PCNA, proliferating nuclear antigen; PMA, 4b-phorbol 12-myristate
13-acetate; RPA, replication protein A; TdR, thymidine.
FEBS Journal 274 (2007) 3669–3684 ª 2007 The Authors Journal compilation ª 2007 FEBS 3669
initiation and elongation steps of DNA replication
[12–14]. Chromatin association of Cdc45 requires
activity of both cyclin-dependent kinases and Cdc7
[15,16]. Studies in baker’s yeast and Drosophila
revealed that Cdc45 is part of a high molecular mass
complex, which was shown to possess helicase activity
[17,18] leading to the concept that Cdc45 is an auxili-
ary factor for the putative Mcm2–7 helicase [19].
In contrast to human replication licensing factors
such as the Mcm proteins, very little is known about
the expression of human Cdc45 during exit from
cell proliferation to nonproliferating states. Here, we
report on the analysis of human Cdc45 protein levels
during the mitotic cell cycle and in various nonprolifer-
ating states. Our data highlight that Cdc45 is a prolif-
eration-associated antigen that becomes undetectable
in long-term quiescent, terminal differentiated and sen-
escent human cells. Demonstration of good immunore-
activity of a Cdc45-specific antibody in formalin-fixed
paraffin-embedded tissues suggests that Cdc45 could
be used as a marker for cell proliferation. From our
analysis we estimated that the number of Cdc45 mole-
cules per human cell is 4.5 · 10
4
. Comparison with the
published molecule number for Mcm3 (1 · 10
6

) [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-

formed with the following phase-specific markers:
Ki-67 for G
1
phase (12 h after TdR block) and mitosis
(9 h after TdR block), 5-bromo-1-(2-deoxy-b-d-ribo-
furanosyl) uracil (BrdU) incorporation for the S phase
(3 h after TdR block) and centromer protein F
(CENP-F) for the G
2
phase (9 h after TdR block).
Human Cdc45 protein was exclusively present in the
nucleus from G
1
to G
2
, but was uniformly distributed
throughout the cell interior after breakdown of the
nuclear membrane in mitosis (Fig. 1C). Consistent
with our previous report [23], Cdc45 appeared in a
spot-like pattern in the S phase, which partially colo-
calized with BrdU signals. In metaphase, anaphase
and telophase, Cdc45 was found spread around the
condensed chromosomes (Fig. 1C). These prominent
changes in the subcellular localization of Cdc45,
together with an unchanged protein level during the
cell cycle, were also obtained with T98G cells (data
not shown). The data led us to conclude that human
Cdc45 protein is present at comparable amounts
throughout all phases of cycling cells.
Cdc45 protein is diminished in quiescent human

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-

cent cells (Fig. 2A,C). Cdc6, Mcm2 and Mcm7 were
previously shown to be downregulated in G
0
and
Cdc45 expression in proliferation S. Pollok et al.
3670 FEBS Journal 274 (2007) 3669–3684 ª 2007 The Authors Journal compilation ª 2007 FEBS
served as controls for proteins that are absent in
nonproliferating cells (Fig. 2A,C) [6]. Logarithmically
growing T98G cells expressed Ki-67 and also Cdc45,
whereas quiescent cells, which were markedly smaller
in size, expressed neither Ki-67 nor Cdc45 (Fig. 2D).
Contrary to these findings, proliferating nuclear anti-
gen (PCNA), the DNA polymerase d subunits p125
and p50 and the replication protein A subunits p70
and p32 were present in G
0
cells even after 20 days
serum starvation, although the protein levels of some
were reduced slightly (Fig. 2B). These findings suggest
that the latter proteins might fulfil essential functions
in quiescent cells such as DNA repair. Furthermore,
Cdc45 protein was not detectable in an extract from
primary unstimulated lymphocytes isolated from fresh
blood of a healthy volunteer (see below), in accordance
with the reported resting G
0
state of primary periph-
eral blood T lymphocytes [28].
Regulation of Cdc45 in terminally differentiated
cells

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

24 h of PMA incubation HL60 cells showed several
monocyte ⁄ macrophage-like characteristics, such as an
intense clustered adherence of almost all cells on the
plastic surface, accompanied by the formation of prom-
inent pseudopodia (Fig. 3B). Terminal differentiation
to the monocyte ⁄ macrophage phenotype was also veri-
fied by a Nitro Blue tetrazolium reduction assay.
Monocytes ⁄ macrophages are able to generate reactive
oxygen species and this burst activity can be visualized
by the existence of blue–black diformazan granules
within the cell. Only 12 h after PMA incubation  35%
of HL60 cells were Nitro Blue tetrazolium-positive
(Fig. 3C,F) indicating the macrophage status [30]. Fur-
thermore, the arrest of PMA-treated HL60 cells was
monitored by the detection of p21
CIP1
and p27
KIP1
in
western blots (Fig. 3A). Exponentially growing HL60
cells expressed no detectable p21
CIP1
and only small
amounts of p27
KIP1
. Induction of p21
CIP1
and p27
KIP1
was detected at defined periods after PMA treatment

[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

same cell line, the Cdc45 protein became undetectable
60 h after incubation with 1a-25-dihydroxy-vitamin
D3, which induces differentiation of HL60 cells into
monocytes (supplementary Fig. S1B). In contrast to the
considerable downregulation of Cdc45 after induction
of differentiation, the licensing factors Cdc6, Mcm2,
Mcm4 and Mcm7 were still present in those differenti-
ated cells (Fig. 3A, supplementary Fig. S1).
Ninety-six hours after incubation with PMA, the
multipotential, haematopoietic malignant K562 cells
displayed morphological changes characteristic of mega-
karyocytic differentiation. Numerous cells were larger
and adhered on plastic surfaces compared with parental
suspension cells (Fig. 3E). In these cells, Cdc6 became
undetectable 24 h after PMA incubation (Fig. 3D), in
agreement with published results [29]. PMA-induced
differentiation along the megakaryocytic phenotype
was accompanied by downregulation of cyclin E [29]
and cyclin A, indicating cell-cycle arrest (Fig. 3D).
Forty-eight hours after PMA application Cdc45 pro-
tein was no longer detectable, whereas Mcm2 and
Mcm7 protein levels were still visible but significantly
AD
BE
CF
Fig. 3. Regulation of human Cdc45 protein during terminal differentiation. (A,D) Immunoblot analysis of whole-cell lysates of HL60 (A) and
K562 (D) cells treated with PMA for up to 96 h to induce terminal differentiation. b-Tubulin served as an internal control. (B,E) Changes of
cell morphology and attachment properties after PMA incubation of HL60 cells (B, ·100) and K562 cells (E, ·10). (C) Number of Nitro Blue
tetrazolium-positive HL60 cells in PMA time course. (F) Detection of Nitro Blue tetrazolium-positive HL60 cells after 12 h of PMA incubation.
(Left) magnification ·20, (right) magnification ·100.

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

[32]. In agreement
with the literature [33], the protein level of p21
CIP1
accumulated when the cells were growth arrested and
then decreased when the cells achieved senescence
(Fig. 4A,B), whereas the p16
INK4A
protein level peaked
when the fibroblasts had reached senescence (Fig. 4A).
To verify the presence of senescent cells, the activity
of senescence-associated b-galactosidase (b-Gal) was
determined (Fig. 4C) [34]. Approximately 65% of
MRC-5 and WI-38 cells in passage 26 were b-Gal-pos-
itive (Fig. 4A,B; percentages above the passage num-
ber). In WI-38 cells of passage 28 this was increased to
83% (Fig. 4B). Immunoblot analysis of total extracts
revealed that Cdc45 protein was no longer detectable
in late passage and in senescent fibroblasts, where it
followed a similar expression course as Mcm7, which
was determined in parallel (Fig. 4A,B).
In cells induced to proliferate Cdc45 protein is
expressed just prior to the S phase
The apparent absence of Cdc45 from nonproliferating
cells raised the question of the time point of de novo
Cdc45 protein expression in a reversible system, such
as in cells released from G
0
to start proliferation. To
this end, T98G cells were made quiescent by a com-
bination of serum starvation and contact inhibition.

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

B
C
Fig. 4. Regulation of human Cdc45 protein during exit into senes-
cence. (A,B) Immunoblot analysis of whole-cell lysates of MRC-5
(A) and WI-38 (B) fibroblasts with the indicated passage numbers.
b-Actin served as a control for equal loading. The percentage of
senescence-associated b-Gal-positive cells (b-Gal) is depicted above
the passage number. (C) Detection of senescence-associated b-Gal
activity in WI-38 cells of passage 18 and 28 cells (·20).
Cdc45 expression in proliferation S. Pollok et al.
3674 FEBS Journal 274 (2007) 3669–3684 ª 2007 The Authors Journal compilation ª 2007 FEBS
3 h after serum stimulation in early G
1
, cyclin A
after 18 h of serum stimulation at the G
1
⁄ S trans-
ition, and cyclin B1  9 h later during the S phase
(Fig. 5C). Flow cytometry analysis and BrdU incor-
poration, together with cyclin A accumulation, indica-
ted that cells started replication at 18–21 h after
serum stimulation (Fig. 5). As described, Cdc45 pro-
tein was not expressed in G
0
T98G cells (Figs 2,5C
time point 0 h), but became detectable at  15 h after
serum re-addition in late G
1
phase, which was  3h
after Cdc6 expression but 3 h before the p180 sub-

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

In human cells, the half-life of Cdc6 is very short,
whereas that of Mcm3 is significantly higher [7,37].
Here, we determined the half-life of human Cdc45
protein in logarithmically growing HeLa S3 cells by
performing [
35
S]-pulse-chase labelling of proteins.
Approximately equal amounts of protein were taken
from cell extracts after the indicated chase periods with
unlabelled cysteine. These samples were immunoprecip-
itated and analysed by SDS gel electrophoresis, west-
ern blotting and autoradiography (Fig. 6). Western
blotting demonstrated that the precipitates contained
approximately equal amounts of the human Cdc45
protein. Autoradiography of these samples showed a
significant reduction in the radiolabelled Cdc45 protein
to  40% after a chase period of 12 h (Fig. 6A).
Therefore, endogenous Cdc45 can be described as a
stable protein with a half-life of  10 h in proliferating
HeLa S3 cells (Fig. 6B).
The number of molecules of replication proteins in
HeLa cells varies from 1 · 10
6
for Mcm3 [20] to
3 · 10
4
for both Cdt1 and geminin [24]. Here we deter-
mined the number of Cdc45 molecules in HeLa S3 and
T98G cells by loading known amounts of recombinant
His

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) [

35
S]-pulse-chase labelling
and IP with Cdc45-specific antibody was performed as described in
Experimental procedures. Briefly, HeLa S3 cells were labelled with
[
35
S]-methionine and -cysteine and were collected after the indica-
ted chase periods. Then whole-cell extracts were prepared and
1 mg extract for each time point was subjected to IP. The precipi-
tates were separated on a 10% SDS polyacrylamide gel and trans-
ferred onto a poly(vinylidene difluoride) membrane. Cdc45 signals
were determined by immunoblotting and by autoradiography as
indicated. (B) The autoradiographic Cdc45 bands were quantified
with the program
PHORETIX 1D ADVANCED, and depicted in a graph.
A
B
Fig. 7. Calculation of the number of Cdc45 molecules per HeLa S3
and T98G cell. (A) Human Cdc45 was expressed as His
6
-tagged
protein in High five
TM
insect cells, purified on Co-Talon
TM
. MS ana-
lysis revealed that the band marked with an asterisk was recombin-
ant Cdc45, the band above Cdc45 was heat shock protein 70 and
the bands below were cytokeratin 1 and 9. Four microlitres of the
eluted fraction was loaded onto a SDS gel together with declining

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

nature of the Cdc45 double band is still in progress.
Although Cdc45 was found in actively cycling cells
it became undetectable in nonproliferating cells
(Figs 1–4 and Fig. 8B, lane 1). These observations led
us to investigate whether the mAb C45-3G10 raised
against human Cdc45 [23,38] can be used for histologi-
cal sections. The antibody was tested on formalin-fixed
paraffin-embedded normal human skin sections as well
as on invasive-lobular mamma carcinoma and small
cell bronchial carcinoma sections using standard
immunohistochemical procedures (Fig. 9). Expression
of Ki-67 and PCNA, both approved markers for pro-
liferating cells [39], were stained on parallel sections of
the same preparation (Fig. 9A,B).
The Cdc45-specific antibody C45-3G10 worked well
for immunohistochemical staining on formalin-fixed
paraffin-embedded tissues (Fig. 9). In normal human
skin sections there were fewer Cdc45-positive than
PCNA- or Ki-67-positive cells. Cdc45 immunoreactivi-
ty was mostly nuclear although a weak cytoplasmic
staining was also seen (Fig. 9A). In a series of inva-
sive-lobular mamma carcinoma sections the antibodies
against Cdc45 and PCNA intensely stained in a very
similar manner tumour-associated cells (Fig. 9B). Some
proliferating fibroblasts or activated lymphocytes adja-
cent to the malignant cells also showed Cdc45 staining.
The Ki-67 signal was associated only with a small frac-
tion of the malignant cell population (Fig. 9B). The
lower percentage of Ki-67-stained cells in comparison
with Cdc45 or PCNA might have been caused by 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

to G
2
, but was distributed throughout the whole cell
following breakdown of the nuclear membrane in
mitosis. In S-phase cells, the Cdc45 signal changed
from a dispersed distribution to local accumulations,
which colocalized with BrdU signals (Fig. 1C), as
reported for HeLa S3 cells [23].
When the cells exited the proliferative cycle and
entered a nonproliferative state the licensing factors
Cdt1, Cdc6 and Mcms were downregulated [6,8,24,
44,45]. This contrasts to a persistence of the Orc2
protein in nonproliferating cells [6,7,46], which points to
other functions of Orc proteins in addition to DNA
replication, for example, transcriptional silencing. Here,
we show that Cdc45 was downregulated completely
when human cells ceased proliferation and entered into
A
B
Fig. 8. Cdc45 is highly expressed in human cancer-derived cell
lines. Immunoblot analysis of the Cdc45 protein level in whole-cell
lysates of various human cell lines. The level of b-tubulin served to
monitor equal loading. (A) Lanes 1–8: MRC-5 (human embryonic
lung fibroblasts), WI-38 (human embryonic lung fibroblasts),
HeLa S3 (cervix carcinoma), HEp2 (cervix carcinoma), MCF-7
(breast carcinoma), BT-20 (breast carcinoma), Saos-2 (osteosarco-
ma) and T98G (glioblastoma), respectively. (B) Lanes 1–8: PBL (pri-
mary unstimulated blood lymphocytes isolated from fresh blood
of a healthy volunteer), HEF (human embryonic lung fibroblasts),
WI-38 (human embryonic lung fibroblasts), CEM (acute lymphoblas-

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-

fixed paraffin-embedded serial sections and visualized by the avidin–biotin complex technique (for details see Experimental procedures). (A)
Cdc45, PCNA and Ki-67 were detected as indicated in serial sections of normal human skin. The scale bar represents 50 lm(·40 objective
and ·2.5 projective). (B) Cdc45, PCNA and Ki-67 were detected in serial sections of invasive-lobular mamma carcinoma. The scale bar repre-
sents 200 lm(·10 objective and ·2.5 projective).
Cdc45 expression in proliferation S. Pollok et al.
3678 FEBS Journal 274 (2007) 3669–3684 ª 2007 The Authors Journal compilation ª 2007 FEBS
asks for a not yet understood temporal regulation of the
pRb-E2F-mediated transcription events.
Investigations on the stability of licensing factors
demonstrated that human Cdc6 protein is a short-lived
protein with a half-life of  2 h [37], whereas human
Mcm3 seems to be more stable with a half-life of 24 h
[7]. To date, no data for the turn-over of the essential
replication factor Cdc45 have been available. There-
fore, we evaluated the half-life of endogenous Cdc45
by radioactive metabolic labelling of HeLa S3 cells.
The measured half-life of  10 h (Fig. 6) indicates that
Cdc45 is relatively stable in proliferating human cells.
Indeed, the stabilizing residue methionine is found
at the N-terminus of the human Cdc45 protein
(NCBI NP 003495). This is in accordance with the so
called N-end-rule, where the presence of the N-ter-
minal residue affects the half-life of the protein [51].
For Xenopus oocytes, it was reported that one mole-
cule of Orc2, two molecules of Mcm10, one molecule of
Cdc45 and 40 molecules of Mcm3 bound per origin
[42,52,53]. The scarcity Cdc45 protein found in lower
eukaryotes led to the hypothesis that Cdc45 may be a
rate-limiting factor for replication [21]. Our analysis
revealed that  4.5 · 10

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

using immunohistochemistry. Proliferation markers
are, in most cases, Ki-67 and PCNA [39]. Proliferation
markers should be antigens that are expressed in all
cell types, are present throughout all cell-cycle stages
and are absent from nonproliferative states [57]. In
recent years, advanced understanding of the regulation
of replication factors has provided new sources for
possible proliferation markers. In this regard, anti-
bodies specific for various Mcm proteins, Cdc6 and
geminin were tested in immunohistochemistry [58–60].
Because of the precise correlation between cell prolifer-
ation and Cdc45 expression (Figs 1–4) immunohisto-
chemical detection of Cdc45 may extend the repertoire
of proliferation markers in routine pathology. Initial
experiments demonstrated the good immunoreactivity
of a Cdc45-specific antibody on formalin-fixed and
paraffin-embedded tissues (Fig. 9). Antibodies against
PCNA and Cdc45 stained malignant cells in a compar-
able manner, e.g. on invasive-lobular mamma carci-
noma sections (Fig. 9B). Currently, we are testing the
feasibility of the Cdc45-specific antibody on normal
tissue specimens and tumour entities and compare the
Cdc45 signals with the classical proliferation markers
PCNA and Ki-67, but also with the novel markers
Mcm proteins and geminin. Hopefully, the combined
use of different biomarkers helps assess tumour pro-
liferation and the potential prognosis of tumorigenic
diseases.
Experimental procedures
Antibodies

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

Cell lines were purchased from ATCC with the following
exceptions: BT-20 (J. Clement, University Hospital Jena,
S. Pollok et al. Cdc45 expression in proliferation
FEBS Journal 274 (2007) 3669–3684 ª 2007 The Authors Journal compilation ª 2007 FEBS 3679
Germany; HEp2 (P. Hemmerich, FLI Jena, Germany);
Jurkat (L. Wollweber, FLI Jena, Germany) and HEF
(U. Schmidt, HKI Jena, Germany). Cells were either grown
in Dulbecco’s modified Eagle’s medium or in RPMI med-
ium supplemented using 10% fetal bovine serum and 20%
fetal bovine serum for HL60 cells. High five
TM
insect cells
were obtained from Invitrogen (Carlsbad, CA) and main-
tained in TC-100 medium (Cambrex Bio Science, Verviers,
Belgium) supplemented with 10% fetal bovine serum.
Human primary blood lymphocytes, obtained from a
healthy volunteer, were isolated using Ficoll
Ò
(Biochrom,
Berlin, Germany) density gradient centrifugation.
HeLa S3 cells were synchronized at the G
1
⁄ S transition
by a double TdR block, as follows: 16 h block with 5 mm
TdR (Sigma), 10 h release followed by the second block for
16 h [62].
T98G cells and HEF became quiescent by a combination
of serum starvation (0.5% v⁄ v serum supplemented med-
ium) and density-dependent growth arrest, leaving them to
accumulate in G

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

. PMA stimulated HL60 cells were
allowed to adhere on coverslips (Roth). Every 12 h slips
were removed and incubated with 1 mgÆmL
)1
Nitro Blue
tetrazolium solution for 30 min at 37 °C. Cells containing
blue black diformazan deposits were counted by light
microscopy and the percentage of Nitro Blue tetrazolium-
positive cells was calculated.
Induction of replicative senescence and b-Gal
staining
WI-38 and MRC-5 fibroblasts were brought to replicative
senescence by continuous culturing until passage 16–28.
Cells were passaged by detaching them from the plastic sur-
face and splitting them into new cell culture flasks.
Cells were grown on coverslips and washed with
NaCl ⁄ P
i
, fixed for 10 min at room temperature in 2%
formaldehyde, washed again and incubated overnight at
37 °C (without CO
2
) with a fresh staining solution to deter-
mine senescence-associated b-Gal activity: 1 mgÆmL
)1
of
5-bromo-4-chloro-3-indolyl-b-d-galactosidase, 40 mm citric
acid, sodium phosphate pH 6.0, 5 mm potassium ferrocya-
nide, 5 m m potassium ferricyanide, 150 mm NaCl, 2 mm
MgCl

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

i
. For BrdU detection, DNA
was denatured by incubation with 1 m HCl for 30 min at
room temperature.
Cells were treated with blocking buffer (5% BSA in
NaCl ⁄ P
i
) overnight in a wet chamber. Cells were incubated
for 1 h with primary antibodies followed by incubation
with the secondary antibodies for another 1 h. After wash-
ing, DNA was briefly stained with DAPI. Immunofluores-
cence was observed at a ·20 or ·100 magnification with a
Zeiss Axiovert 135 microscope in connection with a Sony
charge-coupled device colour video camera.
[
35
S]-Pulse-chase labelling
HeLa S3 cells were incubated for 1 h in methionine- and
cysteine-free Dulbeccco’s modified Eagle’s medium (Invitro-
Cdc45 expression in proliferation S. Pollok et al.
3680 FEBS Journal 274 (2007) 3669–3684 ª 2007 The Authors Journal compilation ª 2007 FEBS
gen) supplemented with 10% fetal bovine serum. l-[
35
S]-
Pro-Mix
TM
(GE Healthcare, Freiburg, Germany) was
added to this medium (0.1 mCiÆmL
)1
), and the cells were

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

HTb with human Cdc45 cDNA was a kind gift
of I. Kukimoto) [65]. Recombinant His
6
–Cdc45 protein was
expressed in High five
TM
insect cells (Invitrogen) and purified
on Co
TM
-Talon (Clontech, Heidelberg, Germany). The
amount of His
6
–Cdc45 was quantified by comparison with
decreasing amounts of BSA on an SDS ⁄ PAGE stained with
PageBlue
TM
protein-staining solution (Fermentas, St Leon-
Rot, Germany). Decreasing amounts of the recombinant
His
6
–Cdc45 were loaded onto an SDS gel together with total
cell extract corresponding to 2 · 10
5
asynchronously grow-
ing HeLa S3 or T98G cells and immunoblotted using the
anti-Cdc45 specific serum. Signals were quantified with
the program phoretix 1d advance (Nonlinear Dynamics
Limited, Newcastle, UK).
Immunohistochemical staining of paraffin-
embedded tissues

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

, supplemented with 0.1%
(v ⁄ v) BSA, at a dilution of 1 : 200 for 30 min at room
temperature. After three washes with NaCl ⁄ P
i
, sections were
incubated for 30 min with preformed macromolecular
complex consisting of avidin and biotinylated peroxidase
(Vector Laboratories, Burlingame, CA), which retains
biotin-binding sites. The staining reaction was performed by
incubation with a solution of 1.5 lm 3,3¢-diaminobenzidine,
10 mm hydrogen peroxide and 0.05 m Tris-buffered saline
pH 7.6 at 25 °C for 5 min. Slides were then lightly counter-
stained with 2% Harris hematoxylin for 5 min, dehydrated
in ethanol, and cleared in xylene. Slides were then mounted
with Canada balsam. Microscopic images were acquired with
an Olympus BH-2 microscope in connection with a digital
camera (Olympus Camedia C3030) and the images were
analysed with the olympus dp-soft imaging software v. 3.0.
Acknowledgements
We thank A. Schneider and J. Fuchs for excellent tech-
nical assistance. We are indebted to N. Baum and
B. Schlott for performing the MA analysis. This work
was supported by the Deutsche Forschungsgemein-
schaft (SFB 604). The Leibniz Institute for Age
Research is funded by the State of Thuringia and the
Bundesministerium fu
¨
r Forschung und Technologie.
References
1 Hall PA & Watt FM (1989) Stem cells: the generation

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-

ic cells. Annu Rev Biochem 71, 333–374.
10 Pasero P & Schwob E (2000) Think global, act local –
how to regulate S phase from individual replication ori-
gins. Curr Opin Genet Dev 10, 178–186.
11 Takeda DY & Dutta A (2005) DNA replication and
progression through S phase. Oncogene 24, 2827–2843.
12 Hopwood B & Dalton S (1996) Cdc45p assembles into
a complex with Cdc46p ⁄ Mcm5p, is required for mini-
chromosome maintenance, and is essential for chromo-
somal DNA replication. Proc Natl Acad Sci USA 93,
12309–12314.
13 Mimura S, Masuda T, Matsui T & Takisawa H (2000)
Central role for cdc45 in establishing an initiation com-
plex of DNA replication in Xenopus egg extracts. Genes
Cells 5, 439–452.
14 Tercero JA, Labib K & Diffley JF (2000) DNA synthe-
sis at individual replication forks requires the essential
initiation factor Cdc45p. Embo J 19, 2082–2093.
15 Mimura S & Takisawa H (1998) Xenopus Cdc45-
dependent loading of DNA polymerase alpha onto
chromatin under the control of S-phase Cdk. Embo J
17, 5699–5707.
16 Zou L & Stillman B (2000) Assembly of a complex con-
taining Cdc45p, replication protein A, and Mcm2p at
replication origins controlled by S-phase cyclin-depend-
ent kinases and Cdc7p–Dbf4p kinase. Mol Cell Biol 20,
3086–3096.
17 Gambus A, Jones RC, Sanchez-Diaz A, Kanemaki M,
van Deursen F, Edmondson RD & Labib K (2006)
GINS maintains association of Cdc45 with MCM in

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-

cer-derived cell lines. Eur J Biochem 271, 3368–3378.
25 Arata Y, Fujita M, Ohtani K, Kijima S & Kato JY
(2000) Cdk2-dependent and -independent pathways in
E2F-mediated S phase induction. J Biol Chem 275,
6337–6345.
26 Gerdes J, Schwab U, Lemke H & Stein H (1983) Pro-
duction of a mouse monoclonal antibody reactive with
a human nuclear antigen associated with cell prolifer-
ation. Int J Cancer 31, 13–20.
27 Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U &
Stein H (1984) Cell cycle analysis of a cell proliferation-
associated human nuclear antigen defined by the mono-
clonal antibody Ki-67. J Immunol 133, 1710–1715.
28 Lea NC, Orr SJ, Stoeber K, Williams GH, Lam EW,
Ibrahim MA, Mufti GJ & Thomas NS (2003) Commit-
ment point during G0 fi G1 that controls entry into
the cell cycle. Mol Cell Biol 23, 2351–2361.
29 Bermejo R, Vilaboa N & Cales C (2002) Regulation of
CDC6, geminin, and CDT1 in human cells that undergo
polyploidization. Mol Biol Cell 13, 3989–4000.
30 Podlich D & Kemme M (2000) Expression of human
tissue kallikrein in differentiated HL-60 cells. Biol Chem
381, 629–632.
31 Das D, Pintucci G & Stern A (2000) MAPK-dependent
expression of p21 (WAF) and p27 (kip1) in PMA-induced
differentiation of HL60 cells. FEBS Lett 472, 50–52.
32 Lundberg AS, Hahn WC, Gupta P & Weinberg RA
(2000) Genes involved in senescence and immortaliza-
tion. Curr Opin Cell Biol 12, 705–709.
33 Stein GH, Drullinger LF, Soulard A & Dulic V (1999)

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.

40 Heidebrecht HJ, Buck F, Haas K, Wacker HH &
Parwaresch R (1996) Monoclonal antibodies Ki-S3 and
Ki-S5 yield new data on the ‘Ki-67’ proteins. Cell Prolif
29, 413–425.
41 Tsuruga H, Yabuta N, Hashizume K, Ikeda M, Endo
Y & Nojima H (1997) Expression, nuclear localization
and interactions of human MCM ⁄ P1 proteins. Biochem
Biophys Res Commun 236, 118–125.
42 Wohlschlegel JA, Dwyer BT, Dhar SK, Cvetic C,
Walter JC & Dutta A (2000) Inhibition of eukaryotic
DNA replication by geminin binding to Cdt1. Science
290, 2309–2312.
43 Saha P, Thome KC, Yamaguchi R, Hou Z, Were-
mowicz S & Dutta A (1998) The human homolog of
Saccharomyces cerevisiae CDC45. J Biol Chem 273,
18205–18209.
44 Williams RS, Shohet RV & Stillman B (1997) A human
protein related to yeast Cdc6p. Proc Natl Acad Sci USA
94, 142–147.
45 Madine MA, Swietlik M, Pelizon C, Romanowski P,
Mills AD & Laskey RA (2000) The roles of the MCM,
ORC, and Cdc6 proteins in determining the replication
competence of chromatin in quiescent cells. J Struct Biol
129, 198–210.
46 Stoeber K, Mills AD, Kubota Y, Krude T, Romanow-
ski P, Marheineke K, Laskey RA & Williams GH
(1998) Cdc6 protein causes premature entry into S phase
in a mammalian cell-free system. Embo J 17, 7219–7229.
47 Ogawa H, Ishiguro K, Gaubatz S, Livingston DM &
Nakatani Y (2002) A complex with chromatin modifiers

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

and apoptosis in cancer. Nature 411, 342–348.
56 Hall PA & Levison DA (1990) Review: assessment of
cell proliferation in histological material. J Clin Pathol
43, 184–192.
57 Scholzen T & Gerdes J (2000) The Ki-67 protein: from
the known and the unknown. J Cell Physiol 182,
311–322.
58 Semple JW & Duncker BP (2004) ORC-associated repli-
cation factors as biomarkers for cancer. Biotechnol Adv
22, 621–631.
59 Gonzalez MA, Tachibana KE, Laskey RA & Coleman
N (2005) Control of DNA replication and its potential
clinical exploitation. Nat Rev Cancer 5, 135–141.
60 Laskey R (2005) The Croonian Lecture 2001: Hunting
the antisocial cancer cell: MCM proteins and their
exploitation. Phil Trans R Soc Lond B Biol Sci 360,
1119–1132.
61 Zlotkin T, Kaufmann G, Jiang Y, Lee MY, Uitto L,
Syva
¨
oja J, Dornreiter I, Fanning E & Nethanel T
(1996) DNA polymerase epsilon may be dispensable for
SV40 – but not cellular – DNA replication. Embo J 15,
2298–2305.
62 Bootsma D, Budke L & Vos O (1964) Studies on syn-
chronous division of tissue culture cells initiated by
excess thymidine. Exp Cell Res 33, 301–309.
63 Huberman E & Callaham MF (1979) Induction of ter-
minal differentiation in human promyelocytic leukemia
cells by tumor-promoting agents. Proc Natl Acad Sci

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.
This material is available as part of the online article
from
Please note: Blackwell Publishing is not responsible
for the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corres-
ponding author for the article.
Cdc45 expression in proliferation S. Pollok et al.
3684 FEBS Journal 274 (2007) 3669–3684 ª 2007 The Authors Journal compilation ª 2007 FEBS