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 [...]... 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 assessment of cellular proliferation in histological material is a valuable... formalin-fixed and paraffin-embedded tissues (Fig 9) Antibodies against PCNA and Cdc45 stained malignant cells in a comparable manner, e.g on invasive-lobular mamma carcinoma 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... through S phase Oncogene 24, 2827–2843 Hopwood B & Dalton S (1996) Cdc45p assembles into a complex with Cdc46p ⁄ Mcm5p, is required for minichromosome maintenance, and is essential for chromosomal DNA replication Proc Natl Acad Sci USA 93, 12309–12314 Mimura S, Masuda T, Matsui T & Takisawa H (2000) Central role for cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extracts Genes... washing three times with IP buffer Precipitated Cdc45 protein was detected after western blotting with the Cdc45specific 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 His6tagged protein using a recombinant... have been available Therefore, 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,... Gerdes J, Schwab U, Lemke H & Stein H (1983) Production of a mouse monoclonal antibody reactive with a human nuclear antigen associated with cell proliferation 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 proliferationassociated human nuclear antigen defined by the monoclonal antibody Ki-67 J Immunol 133, 1710–1715 28 Lea NC, Orr... 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 3684 Supplementary material The following supplementary material is available online: Fig S1 Regulation of human Cdc45. .. valuable component of conventional histopathological analysis and may be of major prognostic importance [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... JA, Labib K & Diffley JF (2000) DNA synthesis at individual replication forks requires the essential initiation factor Cdc45p Embo J 19, 2082–2093 Mimura S & Takisawa H (1998) Xenopus Cdc45dependent loading of DNA polymerase alpha onto chromatin under the control of S-phase Cdk Embo J 17, 5699–5707 Zou L & Stillman B (2000) Assembly of a complex containing Cdc45p, replication protein A, and Mcm2p at... Cy3, goat anti-(rat IgG) conjugated with horseradish peroxidase (HRP) (all Dianova, Hamburg, Germany) and goat anti-(mouse ⁄ rabbit 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, FEBS Journal 274 (2007) 3669–3684 ª 2007 The Authors Journal compilation . 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. normal human skin sections as well as on invasive-lobular mamma carcinoma and small cell bronchial carcinoma sections using standard immunohistochemical