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 prolifera
Trang 1S Pollok1, C Bauerschmidt2, J Sa¨nger3, H.-P Nasheuer4and F Grosse1,5
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 G1 and
enter into a quiescent state also called G0 [2] The G0
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 G1 to form the so-called prereplicative complex [9] At the G1⁄ 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.
Trang 2initiation 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· 104 Comparison with the
published molecule number for Mcm3 (1· 106) [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 G1⁄ 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 G1phase (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 G2 phase (9 h after TdR block) Human Cdc45 protein was exclusively present in the nucleus from G1 to G2, 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 G0 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 p27KIP1 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 G0 and
Trang 3served 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 G0 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 G0 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).
Trang 4To 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 p21CIP1 and p27KIP1 in western blots (Fig 3A) Exponentially growing HL60 cells expressed no detectable p21CIP1 and only small amounts of p27KIP1 Induction of p21CIP1 and p27KIP1 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
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).
Trang 5undetectable 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
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.
Trang 6downregulated, 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 G1 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 p21CIP1 and p16INK4A [32] In agreement
with the literature [33], the protein level of p21CIP1
accumulated when the cells were growth arrested and
then decreased when the cells achieved senescence
(Fig 4A,B), whereas the p16INK4Aprotein 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 G0 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 G0 to proliferation was monitored by flow cytometry (Fig 5A; percentage of cell population in
G0, G1, S, G2⁄ M) and BrdU incorporation into cells (Fig 5B) In addition, the expression of cyclin D1, cyclin A, cyclin B1 and p27KIP1 was determined by western blotting (Fig 5C) p27KIP1 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 p27KIP1 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).
Trang 73 h after serum stimulation in early G1, cyclin A
after 18 h of serum stimulation at the G1⁄ 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 starindica-ted replication at 18–21 h after
serum stimulation (Fig 5) As described, Cdc45
pro-tein was not expressed in G0 T98G cells (Figs 2,5C
time point 0 h), but became detectable at 15 h after
serum re-addition in late G1 phase, which was 3 h
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 G0 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.
Trang 8Half-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 [35S]-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) Westwest-ern
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· 106 for Mcm3 [20] to
3· 104for 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 His6–Cdc45 onto an SDS gel alongside total cell lysates from 2· 105 asynchronously growing cells Quantification of the western blot signals revealed that
1 ng of Cdc45 protein was present in 2 · 105 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 · 104 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 1 D 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 1 D 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 His6-tagged Cdc45 are marked The protein bands were quantified with the program PHORETIX 1 D ADVANCED
Trang 9from 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 populafrac-tion (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 G1 phase Ki-67 is a short-lived protein [40] pre-dominantly expressed during the S, G2 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 G1⁄ 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 G1
to G2, 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 (glioblasto(osteosarco-ma), 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.
Trang 10quiescence, terminal differentiation or senescence
(Figs 2–4, supplementary Fig S1) Furthermore, both
in mouse cells [25] and human cells leaving the G0phase,
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)
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).