Eur J Biochem 271, 3368–3378 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04271.x Cdt1 and geminin are down-regulated upon cell cycle exit and are over-expressed in cancer-derived cell lines Georgia Xouri1, Zoi Lygerou1, Hideo Nishitani2, Vassilis Pachnis3, Paul Nurse4,* and Stavros Taraviras5 Laboratory of General Biology, Medical School, University of Patras, Rio, Patras, Greece; 2Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan; 3Division of Molecular Neurobiology, National Institute for Medical Research, London, UK; 4Cell Cycle Laboratory, Cancer Research UK, London Laboratories, London, UK; 5Laboratory of Pharmacology, Medical School, University of Patras, Rio, Patras, Greece Licensing origins for replication upon completion of mitosis ensures genomic stability in cycling cells Cdt1 was recently discovered as an essential licensing factor, which is inhibited by geminin Over-expression of Cdt1 was shown to predispose cells for malignant transformation We show here that Cdt1 is down-regulated at both the protein and RNA level when primary human fibroblasts exit the cell cycle into G0, and its expression is induced as cells re-enter the cell cycle, prior to S phase onset Cdt1’s inhibitor, geminin, is similarly down-regulated upon cell cycle exit at both the protein and RNA level, and geminin protein accumulates with a 3–6 h delay over Cdt1, following serum re-addition Similarly, mouse NIH3T3 cells down-regulate Cdt1 and geminin mRNA and protein when serum starved Our data suggest a transcriptional control over Cdt1 and geminin at the transition from quiescence to proliferation In situ hybridization and immunohistochemistry localize Cdt1 as well as geminin to the proliferative compartment of the developing mouse gut epithelium Cdt1 and geminin levels were compared in primary cells vs cancer-derived human cell lines We show that Cdt1 is consistently over-expressed in cancer cell lines at both the protein and RNA level, and that the Cdt1 protein accumulates to higher levels in individual cancer cells Geminin is similarly over-expressed in the majority of cancer cell lines tested The relative ratios of Cdt1 and geminin differ significantly amongst cell lines Our data establish that Cdt1 and geminin are regulated at cell cycle exit, and suggest that the mechanisms controlling Cdt1 and geminin levels may be altered in cancer cells Genomic stability is maintained in proliferating cells through control mechanisms which ensure that the cell’s genetic content is duplicated entirely and only once in each cell cycle, and is correctly partitioned to the two daughter cells during mitosis [1] In eukaryotes, replication starts from multiple origins along each chromosome during S phase, and re-firing of the same origins is inhibited until mitosis is completed This is achieved through the replication licensing system [2], a regulatory system conserved in evolution from yeast to humans, which ÔlicensesÕ each origin for a single round of DNA replication This license is lost upon origin firing, and is reestablished only upon completion of mitosis, thereby preventing over-replication of the genome Recent studies, mostly using yeasts and a Xenopus laevis in vitro licensing system, have permitted an understanding of the licensing process at the molecular level (reviewed in [3–5]) A multisubunit complex is formed on origins of DNA replication upon completion of mitosis, by the stepwise association of licensing factors to origin sequences, which confers to each origin the license to replicate Origins are recognized by the six-subunit origin recognition complex (ORC), which, at least in lower eukaryotes, remains chromatin associated throughout the cell cycle Temporal regulation of origin licensing is achieved through the action of two loading factors, Cdc6/18 and Cdt1, which are required for the chromatin association of the six subunit mini chromosome maintenance (MCM) complex The MCM complex is believed to function as the replicative helicase [6], and its chromatin association confers to origins the license to replicate Cdt1 was recently identified as a factor essential for the chromatin loading of MCM proteins upon completion of mitosis in both lower and higher eukaryotes [7–11] The identification of the human homologue of Cdt1 permitted its analysis during the cell cycle in cultured human cells [12–14] Cdt1 is tightly regulated so that its protein accumulates only in G1, when licensing is legitimate This regulation is mediated mostly by targeted proteolysis of Cdt1 from S phase to mitosis, rather than by transcriptional controls [13] In addition, Cdt1 binds strongly to and is inhibited by geminin [12,15] Geminin, originally identified in Xenopus as an inhibitor of Correspondence to S Taraviras, Laboratory of Pharmacology, Medical School, University of Patras, 26500, Rio, Patras, Greece Fax: +30 2610 994720, Tel.: +30 2610 997638, E-mail: taraviras@med.upatras.gr and Z Lygerou, Laboratory of Biology, Medical School, University of Patras, 26500, Rio, Patras, Greece Fax: +30 2610 991769, Tel.: +30 2610 997621, E-mail: lygerou@med.upatras.gr Abbreviations: HFF, human foreskin fibroblasts; MCM, mini chromosome maintenance; ORC, origin recognition complex *Present address: The Rockefeller University, NY, USA (Received 19 April 2004, revised 17 June 2004, accepted 28 June 2004) Keywords: cancer; Cdt1; G0; geminin; licensing 1 Ó FEBS 2004 Cdt1 and geminin down-regulation in quiescence (Eur J Biochem 271) 3369 licensing specifically degraded at the end of mitosis [16], is believed to act through binding to Cdt1 [12,15] Cdt1 and geminin are, however, hardly coexpressed during the cell cycle in cultured human cells [13], raising the question of when and how geminin exerts its function In contrast to other cell cycle inhibitors, geminin was shown to be a marker of proliferating cells [17] Cells cease to proliferate and exit the cell cycle (G0 phase) in response to growth arrest and differentiation signals or when deprived of growth factors The vast majority of cells in multicellular organisms exist in Ôout of cycleÕ states, either temporarily resting in G0, from which they can respond to stimuli for cell cycle re-entry or differentiation, or in terminally differentiated or arrested (senescent) states Defects in the mechanisms that ensure the timely proliferation of human cells are key events in the development of neoplasia Cells exit the cell cycle from the G1 phase and previous work has shown that licensing is lost when G1 cells exit to G0 Nuclei isolated from early G0 cells fail to replicate in a Xenopus in vitro system, similar to G2 nuclei [18–21] ORC2-5 proteins persist on G0 chromatin, but MCM proteins and Cdc6/18 rapidly dissociate from chromatin and are gradually lost from G0 cells [20–22] When cells re-enter the cell cycle, expression of Cdc6/18 and MCM proteins is induced [22,23] Cdc6/18, Orc1 and several of the MCM proteins have been shown to be transciptionally regulated by E2F at the transition from quiescence to proliferation [24–28] MCM proteins have been proposed as sensitive proliferation markers for the detection of premalignant and malignant states [29–31] In this study we examine whether Cdt1, a factor essential for licensing across evolution and tightly controlled during the cell cycle, is negatively regulated in quiescent cells We studied Cdt1 and its inhibitor geminin at the transition from quiescence to proliferation in cultured primary human cells and NIH3T3 cells and we compared their expression patterns in tissue sections and their expression levels in primary and normal diploid vs cancer cell lines Our data show a correlation of Cdt1 and geminin expression levels with cell proliferation Materials and methods Cell culture Human foreskin fibroblast (HFF), HeLa, MDAMB231, MCF7, HT1080, U2OS, MRC5 and LNCAP cells were grown in DMEM/high glucose medium with 10% (v/v) fetal bovine serum NIH3T3 cells were grown in DMEM/ high glucose medium with 10% (v/v) calf serum Most cell lines used were provided by the Cancer Research UK cell line facility For serum starvation, HFF or NIH3T3 cells were incubated in the presence of 0.1% (v/v) serum for 48 h Cells were then induced to re-enter the cell cycle by addition of 20% (v/v) serum For contact inhibition, NIH3T3 cells were cultured in the presence of 10% (v/v) calf serum until confluent (day 0) and then for the indicated number of days following confluency To induce cell cycle re-entry following contact inhibition, days following confluency cells were split : 10 Plasmids Mouse Cdt1 and geminin full-length cDNAs were compiled by combining EST entries in the nucleotide databases Based on the deduced sequences, specific oligonucleotides were designed for PCR cloning the fulllength open reading frames of mouse Cdt1 and mouse geminin into BamHI/HindIII and EcoRI/BamHI sites of pBluescript KS, respectively These were used to generate specific probes for Northern hybridization on total RNA extracted from NIH3T3 cells and mouse in situ hybridization Antibodies, Western blotting, immunofluorescence Antibodies against hCdt1 were described previously [13] Affinity purified anti-hCdt1 Ig was used for all experiments Anti(h-geminin) serum raised in rabbits against the C-terminal 94 amino acids of human geminin (expressed as a 6· His fusion protein in Escherichia coli and purified on an Ni-column) was affinity purified using the same recombinant fragment These affinity purified antibodies raised against geminin will be referred to hereafter as anti-Gem2 A major band with the expected apparent molecular mass for h-geminin (around 30 kDa) was detected by Western blotting using anti-Gem2 on HeLa total cell extracts RNAi directed against human geminin resulted in complete disappearance of this band (data not shown), verifying that it indeed corresponds to human geminin For Western blotting, total cell lysates were prepared by lysing cell pellets directly in SDS/PAGE loading buffer and boiling Antibodies were used at the following dilutions: anti-hCdt1, : 500; anti(h-geminin) (Santa Cruz), : 500; anti-Gem2, : 2000, anti-hCdc6/18 (Upstate Biotechnology), : 1000; anti-cyclin A (Upstate Biotechnology), : 2000, and anti(a-tubulin) (Sigma), : 10 000 Immunofluorescence on HFF and HeLa cells, using affinity purified anti-Cdt1 Ig (1 : 200 dilution), or antiGem2 Ig (1 : 1000) was carried out as previously described [13] Unrelated rabbit IgG or pre-immune serum was used as a negative control For BrdU staining, cells were incubated for 30 in the presence of 20 lM BrdU (Sigma) added directly to the culture medium prior to collection Cells were then washed twice with ice-cold NaCl/Pi, fixed in 3.8% (v/v) formaldehyde for 10 min, washed twice with NaCl/Pi, and permeabilized with 0.3% (v/v) Triton X-100 in NaCl/Pi After washing cells three times with NaCl/Pi and once with double distilled H2O, DNA was denatured by incubation in M HCl for h at room temperature Cells were then washed for in 0.1 M Tris/HCl, pH 8.8, to neutralize the pH, and three times with NaCl/ Pi containing 0.1% (v/v) Tween Cells were treated with blocking buffer [3% (w/v) bovine serum albumin/10% (v/v) goat serum in NaCl/Pi] for 30 and incubated with anti-BrdU (Sigma B2531, : 150) in blocking buffer, overnight in a wet chamber Cells were washed in NaCl/Pi containing 0.1% (v/v) Tween three times and incubated with an Alexa 488 conjugated goat anti-mouse secondary antibody (Molecular Probes) After washing, DNA was stained briefly with Hoechst 33258 3370 G Xouri et al (Eur J Biochem 271) Quantitation of protein levels in cancer cell lines and primary cells To calculate the number of hCdt1 and h-geminin molecules present per HeLa cell, full-length hCdt1 (HisT7Cdt1) and full-length h-geminin (Hisgeminin) were expressed as His-tagged proteins in E coli (using vectors pET28a-Cdt1 and pQE-geminin), purified on Ni-agarose, and protein amounts of each full-length protein were quantified by comparison with increasing amounts of bovine serum albumin on an SDS/PAGE stained with Coomassie brilliant blue Increasing amounts of each recombinant protein were then loaded on an SDS/PAGE alongside a total cell extract corresponding to 1.5 · 105 asynchronously growing HeLa cells and immunoblotted using anti-Cdt1 and anti-geminin specific antibodies Comparison of the Western blot signals showed that approximately 0.4 ng of Cdt1 protein and 0.2 ng of geminin protein are present in 1.5 · 105 HeLa cells Because the molecular mass of Cdt1 is nearly twice that of geminin, it was calculated that about 30 000 molecules of each protein are present on average in each HeLa cell It should be noted, however, that Cdt1 is present in cells that are in G1 while geminin is present in cells in S to M phases In order to quantify the amount of Cdt1 detected by immunofluorescence in individual HFF and HeLa cells, indirect immunofluorescence was carried out as described above and signal intensity was quantified using IPLAB software (Scanalytics Inc., Fairfax, VA, USA) The mean fluorescence intensity for 25 high-power fields (40· magnification) for HFF cells and 25 high-power fields for HeLa cells, from three independent experiments, was quantified by defining the respective area as a region of interest and after applying background correction Western blots were quan6 tified using the QUANTIFY ONE program (BioRad) Northern blot analysis and semiquantitative RT-PCR analysis Total cell RNA was prepared by the TrizolTM method (Invitrogen) and 10 lg of total RNA per sample was used for Northern blot analysis Northern blot analysis was as described [13,32] Probes specific for the mouse Cdt1 and geminin cDNAs, generated by random priming, were used for NIH3T3 cells (see above) while probes specific for the human geminin gene were generated by random priming using the complete open reading frame of the human geminin cDNA A probe directed against the actin mRNA served as a loading control A blot containing total mRNA from human tumor and normal samples was purchased from ResGen (Invitrogen Corporation) Northern blots were quantified using the QUANTIFY ONE program (BioRad) Semiquantitative RT-PCR analysis was performed to examine hCdt1 and h-geminin mRNA levels in HFF cells Total RNA was isolated from cycling, serum deprived and re-stimulated HFF cells using the Trizol method (Invitrogen) Reverse transcription was performed using 1–5 lg total RNA and random primers according to the manufacturer’s protocol (Superscript; Invitrogen) cDNA was amplified by PCR using specific sets of primers for hCdt1, h-geminin and h-actin Primers used were: 5¢-AAGGATC CCGCCTACCAGCGCTTCC-3¢ and 5¢-CCAAGCTTGA AGGTGGGGACACTG-3¢ for hCdt1 (288 nucleotide Ĩ FEBS 2004 product); 5¢-CTTCTGTCTTCACCATCTACA-3¢ and 5¢-AGTGGAGGTAAACTTCGGCAG-3¢ for h-geminin (710 nucleotide product) and 5¢-CACCTTCTACAATG AGCTGC-3¢ and 5¢-AGGCAGCTCGTAGCTCTTCT-3¢ for h-actin (437 nucleotide product) PCR was performed under the following conditions: denaturation at 94 °C for 45 s, annealing for 30 s at 65 °C for hCdt1 and 62 °C for h-geminin and h-actin, extension at 72 °C for min; 26 cycles were used for the amplification of hCdt1 and h-geminin cDNAs and 20 cycles for the amplification of h-actin cDNA The number of cycles was adjusted to ensure that the reaction was in the linear range PCR products were analyzed by agarose gel electrophoresis Two PCRs with twofold dilution of cDNA were performed for each sample, to show linearity in detection In situ hybridization and immunohistochemistry Non-radioactive in situ hybridization was performed on fresh-frozen sections of E17 mouse embryos Embryos were obtained from timed pregnancies of outbred (Parkes) mice All animal work was performed according to the Home Office (UK) and local (NIMR-MRC) Ethical Commitee guidelines Frozen sections were postfixed for 10 at room temperature with 4% (v/v) paraformaldehyde Subsequently the slides were pretreated with 0.25% (w/v) acetic anydride for 10 and hybridization was carried out in a 5· NaCl/Cit humidified chamber overnight at 65 °C The slides were then washed at high stringency (0.2· NaCl/Cit at 65 °C) for h and transferred to 0.2· NaCl/Cit at room temperature for Slides were blocked for h at room temparature, with 10% (v/v) sheep serum in 0.1 M Tris/HCl pH 7.5/0.15 M NaCl and incubated overnight at °C with anti-DIG Ig (1 : 5000 dilution, Roche) in 0.1 M Tris/HCl pH 7.5/0.15 M NaCl Slides were rinsed in 0.1 M Tris/HCl pH 7.5/0.15 M NaCl, equilibrated in 0.1 M Tris/ HCl pH 9.5/0.1 M NaCl/50 mM MgCl2 and incubated with 262.5 lgỈmL)1 Nitro Blue tetrazolium (Roche) and 175 lgỈmL)1 5-bromo-4-chloroindol-2-yl phosphate 10 (Roche) in 0.1 M Tris/HCl pH 9.5/0.1 M NaCl/50 mM MgCl2 for 3–6 h [33] Antisense riboprobes were generated using the full-length open reading frames of mouse Cdt1 and geminin as templates and the T3 and T7 polymerase, respectively, while sense probes served as negative controls and were generated using T7 and T3 polymerase E17 dpc mouse embryos used for immunohistochemistry experiments were fixed overnight with 4% (v/v) paraformaldehyde, transferred to a 30% (v/v) sucrose solution in NaCl/Pi for 24 h and embedded in OCTTM 11 compound (BDH) Immunohistochemistry was performed on consecutive fresh-frozen sections that were postfixed in 4% (v/v) paraformaldehyde in NaCl/Pi, washed with NaCl/Pi and permeabilized with 0.3% (v/v) Triton X-100 in NaCl/Pi Horseradish peroxidase activity was quenched by a 10 incubation in 10% methanol/10% H2O2 (v/v) Sections were then blocked in 3% (w/v) bovine serum albumin, 10% (v/v) goat serum in NaCl/Pi for h and incubated overnight at °C with anti-hCdt1 or antigeminin Ig (Santa Cruz) at : 100 dilution in blocking buffer Secondary antibodies, anti-rabbit or anti-goat horseradish peroxidase conjugated (Roche), were used Incubation with the pre-immune serum or secondary Ó FEBS 2004 Cdt1 and geminin down-regulation in quiescence (Eur J Biochem 271) 3371 antibody only was used to determine the specificity of the primary antibodies used Results A Anti-Cdt1 DAPI Anti-geminin DAPI Pr Cdt1 protein levels are low in quiescent cells Our previous work showed that Cdt1, a DNA licensing factor, is tightly controlled by proteolysis during the cell cycle in human cells, accumulating only during the G1 phase, when licensing is legitimate [13] When cells exit the cell cycle, licensing in lost, and is established again as cells prepare to reenter the cell cycle While a previous study did not detect a significant down-regulation of Cdt1 in serum deprived cells [14], a different study showed that cells which express higher levels of Cdt1 in G0 exhibit a quicker entry into S phase and are predisposed for malignant transformation [34] We therefore investigated whether Cdt1 is down-regulated upon cell cycle exit To this effect, HFF cells were deprived of serum for 48 h to induce cell cycle exit Serum was then re-added and samples taken as cells progressed into the cell cycle Total Cdt1 levels were measured by Western blotting (Fig 1A) Cyclin A and Cdc6 served as controls of proteins previously shown to be down-regulated during G0, while tubulin served as a loading control Cdt1 is markedly down-regulated upon cell cycle exit and then quickly re-accumulates as cells re-enter the cell cycle Antibodies against hCdt1 were used to assess by immunofluorescence the percentage of cells expressing Cdt1 in asynchronously growing HFF cells, and at different time points during the transition from quiescence to proliferation (Fig 2A, left panels, immunofluorescence images; Fig 2B, quantitation) The percent of BrdU positive cells at each 0h 6h 9h 12 h 15 h 18 h 21 h 24 h B Fig Cdt1 protein expression in human fibroblasts (HFF) at the transition from quiescence (G0) to proliferation Total cell extracts from HFF cells were analyzed by Western blotting using Cdt1, cyclin A, Cdc6 and tubulin specific antibodies Lane 1, proliferating HFF cells; lane 2, HFF cells deprived of serum for 48 h; lanes 3–8, serum deprived HFF cells induced to re-enter the cell cycle by addition of serum and collected at 6, 12, 15, 18, 21 and 24 h, respectively The band corresponding to Cdt1 has been marked by an arrow, while a cross-reacting band running above Cdt1 is indicated by an asterisk The position of migration of the 66-kDa molecular mass marker band is indicated at the left of the Cdt1 blot Fig Cdt1 and geminin protein levels and localization at the transition from quiescence to proliferation Proliferating HFF cells (Pr), HFF cells deprived of serum for 48 h (0 h), or serum deprived cells induced to reenter the cell cycle by serum re-addition for 6, 9, 12, 15, 18, 21 and 24 h were processed for indirect immunofluorescence using anti-Cdt1 and anti-geminin (anti-Gem2) Ig (A) Microscopy images (recorded with identical exposure settings for all time points) (B) Percentage of cells 14 showing staining for Cdt1 (white bars), BrdU (black bars) or geminin (hatched bars) in each time point Over 200 cells were measured for each time point In order to assess cell cycle progression, cells were incubated with BrdU 30 prior to fixation, and processed for BrdU staining as described in Materials and methods Percent of BrdU positive cells in each time point was scored 3372 G Xouri et al (Eur J Biochem 271) time-point is also shown in Fig 2B for comparison Cdt1 is detected in the nucleus of approximately one-third of cells from an asynchronous population but its levels are markedly decreased in cells cultured in the absence of serum Cells staining for Cdt1 appear around 12 h following serum readdition, several hours before the peak of cells in S phase (21–24 h) We wished to compare the behavior of Cdt1 to that of its inhibitory molecule, geminin, during cell cycle exit and re-entry To that effect, we assessed the levels of the geminin protein by immunofluorescence, in proliferating HFF cells, upon serum withdrawal and upon serum readdition, in parallel to Cdt1 immunofluorescence detection described above (Fig 2A, right panels, immunofluorescence images; Fig 2B quantitation) Geminin was detected in the nucleus of around one-quarter of asynchronously growing HFF cells Upon serum withdrawal geminin levels were markedly decreased, similar to Cdt1 Geminin staining first re-appeared in a small number of cells around 15 h following serum readdition, and peaked at 21–24 h together with the peak of cells in S phase, as judged by the percentage of BrdU positive cells Geminin appeared in the cell nucleus significantly later upon serum re-addition than Cdt1 (a 3–6 h delay) and its accumulation paralleled the accumulation of BrdU positive cells Our data suggest that both Cdt1 and geminin are downregulated in quiescent HFF cells When cells re-enter the cell cycle, Cdt1 is expressed first, as cells prepare for a new round of S phase, while geminin accumulates as cells enter S phase Geminin, an inhibitor of Cdt1 is severely down-regulated upon cell cycle exit Cdt1 is negatively regulated by geminin [12,15], and, during the cell cycle, geminin accumulates in S phase and G2, when Cdt1 levels are low, while Cdt1 accumulates in G1, when Ó FEBS 2004 geminin is undetectable [12,13,16] Our immunofluorescence findings, showing that upon serum starvation of human fibroblasts geminin is down-regulated similar to Cdt1, were somewhat surprising, as geminin might have been expected to be up-regulated upon cell cycle exit We therefore wished to examine this point more carefully The low levels of geminin protein and mRNA present in HFF cells however (see below) hampered a detailed analysis in these cells We therefore turned to mouse NIH3T3 cells, which express geminin to levels similar to HeLa cells (Fig 3, left: compare lanes and 2), but can be induced to exit the cell cycle by serum withdrawal or contact inhibition Figure shows the levels of Cdt1 and geminin in NIH3T3 cells, which are induced to exit the cell cycle either by serum deprivation or contact inhibition Cdc6/18 and cyclin A protein levels serve as controls for proteins previously shown to be down-regulated upon cell cycle exit, while tubulin serves as a loading control As shown above for HFF cells, Cdt1 protein levels are significantly reduced in serum starved NIH3T3 cells and re-accumulate upon addition of serum Cdt1 protein levels are much less affected by contact inhibition (still present days following confluency) Geminin is severely down-regulated by both serum deprivation and contact inhibition, similar to cyclin A and more dramatically than Cdt1 For example, geminin protein levels are undetectable upon serum starvation and are already reduced from the first day following confluency, when Cdt1 levels are still unaffected We conclude that geminin is dramatically down-regulated in NIH3T3 cells in G0, consistent with our findings with HFF human cells Cdt1 and geminin mRNAs are down-regulated in G0 During the cell cycle, Cdt1 and geminin mRNA levels are mostly stable and protein levels are primary controlled by Fig Expression of Cdt1 and geminin proteins in quiescent and proliferating NIH3T3 cells NIH3T3 cells were induced to exit the cell cycle by serum starvation (–S) or contact inhibition (Ci) Total cell extracts at the conditions indicated below were prepared and Western blot analysis was performed using antibodies that recognize specifically Cdt1, geminin (Santa Cruz), cyclin A, Cdc6 and tubulin proteins Lane 1, proliferating HeLa cells; lane 2, proliferating NIH3T3 cells; lane 3, serum deprived NIH3T3 cells (cultured for 48 h in low serum); lane 4, NIH3T3 cells induced to re-enter the cell cycle upon serum addition for h; lanes 5–8, contact inhibited NIH3T3 cells, 2, and days following confluency; lane 9, NIH3T3 cells induced to re-enter cell cycle by splitting : 10, days after confluency The arrow on the cyclin A blot indicates the band corresponding to cyclin A while the asterisk marks a cross-reacting band Mouse Cdt1 migrates slower than human Cdt1 1 Ó FEBS 2004 Cdt1 and geminin down-regulation in quiescence (Eur J Biochem 271) 3373 Fig Transcriptional control of Cdt1 and geminin in quiescent cells (A) Total cell RNA prepared from proliferating NIH3T3 cells (lane 1), from cells deprived of serum for 48 h (lane 4) or from cells first serum deprived for 48 h and then cultured for or 12 h in the presence of 20% (v/v) serum (lanes and 2, respectively) was subjected to Northern blot analysis using a probe specific for human Cdt1 (upper), human geminin (middle) or actin as a loading control (lower) (B) Northern blotting analysis was performed using total cell RNA prepared from NIH3T3 cells that were grown in the presence of serum (lane 1), in the absence of serum for 12, 24, 32, 40 and 48 h (lanes 2–6) or for 18 h after re-addition of serum to cells which were previously serum deprived for 48 h (lane 7) and hybridized using specific probes for Cdt1, geminin and actin (C) Total RNA extracted from HFF cells was subjected to reverse transcription and PCR amplification with oligonucleotides specific for the hCdt1 and h-geminin cDNAs PCR with oligonucleotides specific for actin served as a loading control Twofold dilutions of starting cDNA were used to show linearity (data not shown) Lane 1, proliferating HFF cells; lane 2, HFF cells deprived of serum for 48 h; lanes 3–7, serum deprived HFF cells upon serum readdition for 6–24 h proteolysis [13] Given the down-regulation of both proteins upon serum deprivation, we wished to examine their respective mRNA levels In Fig 4A, the mRNA levels of Cdt1, geminin and actin (as loading control) are shown in serum deprived NIH3T3 cells, and and 12 h following serum re-addition, and compared with mRNA levels in proliferating cells Both Cdt1 and geminin mRNAs are down-regulated upon serum deprivation and re-accumulate as cells prepare for S phase Densitometry scanning and data normalization against the actin control shows that geminin mRNA is at background levels in serum deprived NIH3T3 cells and at h following serum re-addition, while at 12 h it has returned to the level detected in proliferating cells Cdt1 mRNA levels are reduced twofold and similarly return to the levels detected in proliferating cells by 12 h following serum readdition We then examined how quickly upon serum deprivation Cdt1 and geminin mRNA levels are reduced (Fig 4B) Densitometry analysis showed that geminin mRNA levels appear significantly reduced already at early time points (12 h minus serum, twofold reduction) and are further reduced when cells are cultured longer in the absence of serum (reaching a sevenfold reduction at 40 h minus serum) Cdt1 mRNA levels show a twofold reduction 24 h following serum deprivation and remain at approximately the same level to the end of the time course In order to reproduce our findings also in human HFF cells, and given that Cdt1 and geminin mRNAs were hardly detectable in these cells by Northern blotting (see below) we employed reverse-transcription followed by semiquantitative PCR amplification (RT-PCR) of human Cdt1 and geminin mRNAs in cycling, serum deprived and re-stimulated HFF cells (Fig 4C) Consistent with our finding with NIH3T3 cells, geminin mRNA levels were dramatically down-regulated in serum starved HFF cells and geminin mRNA accumulated again around 18 h following serum re-addition Cdt1 mRNA levels were also decreased in serum starved HFF cells and re-accumulated from 12 h following serum re-addition Similar to our findings with NIH3T3 cells, mRNA fluctuations upon serum withdrawal in HFF cells appeared less dramatic for Cdt1 than geminin We conclude that Cdt1 and geminin mRNA levels are reduced in quiescent cells, suggesting that in contrast to their regulation during the cell cycle [13], upon exit and entry to the cell cycle both genes are controlled, at least in part, transcriptionally Cdt1 and geminin are highly expressed in proliferating cells in vivo The experiments with cultured cells have suggested that Cdt1 and geminin mRNA and protein are down-regulated upon cell cycle exit and are progressively up-regulated upon 3374 G Xouri et al (Eur J Biochem 271) re-entry to the cell cycle A recent report showed that geminin protein levels are positively correlated with cell proliferation [17] We investigated the in vivo expression of Cdt1 and compared it to that of geminin in the developing mouse gut epithelium, a tissue in which a proliferating and a differentiating zone can be distinguished histologically Gut epithelium differentiation is initiated after E14dpc in mouse embryos and continues postnatally We determined Cdt1 and geminin mRNA and protein expression using in situ hybridization and immunohistochemistry, respectively, on sections from the gastrointestinal tract of an E17dpc mouse embryo At this stage of development, the gut epithelium is organized into villi, which are separated at their bases by a proliferating compartment known as the intervillus epithelium Cdt1 mRNA is mainly localized at the bases of the developing intestinal villi (the intervillus epithelium), where the proliferating cells of the intestinal epithelium are localized (Fig 5A) Geminin mRNA has a distribution similar to Cdt1 in the small and large intestine Immunohistochemistry using antibodies specific for Cdt1 and geminin (Fig 5B) showed that both proteins are detected in the proliferating cell layer of the developing gut epithelium in a similar expression pattern Fig Cdt1 and geminin are expressed in proliferating cells of the 15 gastrointestinal tract In situ hybridization (·5, ·10) (A) and immunohistochemical (·5) (B) analysis of Cdt1 (left) and geminin (right) expression on frozen section of an E17 dpc mouse embryo Cdt1 and geminin mRNA and protein expression show a similar expression profile Consecutive sections of the gastroinestinal tract are shown Ó FEBS 2004 Therefore, Cdt1 and geminin mRNA and protein reveal a similar distribution along the gastrointestinal tract, localizing mainly in the proliferating zone of the gut epithelium Cdt1 and geminin are over-expressed in cancer cells Given the correlation we observed between Cdt1 and geminin mRNA and protein levels and proliferation, we wished to examine the expression levels of these two genes in different tumor cell lines and compare them to primary cells Cellular lysates were prepared from human foreskin fibroblasts, a primary cell line, and the tumorigenic cell lines Saos, MDAMB231, MCF7, HeLa and LNcap Western blot analysis using anti-Cdt1 Ig showed that Cdt1 protein is detected at much lower levels in the primary HFF cells compared with all the tumorigenic cell lines tested (Fig 6A) Western blotting with commercial antibodies against geminin showed that geminin protein levels are also increased in cancer cell lines, while different cancer cell lines appear to over-express geminin to varying degrees In order to more carefully compare the relative levels of Cdt1 and geminin in different primary and cancer cell lines, we utilized a more sensitive antibodies against geminin (anti-Gem2) and included primary endothelial cells (Huvec), the normal diploid human cell line MRC5 and two more cancer cell lines (HT1080 and U2OS), in addition to HFF, HeLa and MCF7 analyzed in Fig 6A As shown in Fig 6B, cancer cell lines appear to consistently over-express Cdt1 in comparison with primary and normal diploid cell lines (for lanes 5–8, a higher exposure is shown for the Cdt1 blot, as evident by the intensity in lanes and 8, which both correspond to HeLa cell, to permit detection of Cdt1 in the normal diploid MRC5 cells) Quantitation of the blots in Fig 6A,B showed that the majority of cancer cell lines express Cdt1 over 10-fold more than primary cell lines Geminin is hardly detectable in the primary cell lines, and accumulates to higher levels in the majority of cancer cell lines tested It is noteworthy, however, that geminin levels vary significantly amongst the cancer cell lines tested (compare, for example, HeLa, lane 2, to MCF7, lane 3), suggesting that the relative amount of Cdt1 and its inhibitor geminin may differ in different cell lines In order to further investigate this point, we estimated how many molecules of Cdt1 and geminin are present on average per cell in an asynchronous population of HeLa cells To this end, known amounts of recombinant full-length Cdt1 (HisT7-Cdt1) and recombinant full-length geminin (His-geminin) were loaded on an SDS/PAGE gel alongside total cell extract from 1.5 · 105 asynchronously growing HeLa cells, and Western blotted with anti-Cdt1 and antigeminin Ig (Fig 6C) We estimate that approximately 30 000 molecules of Cdt1 and an equal number of geminin molecules are present on average per HeLa cell (Materials and methods), suggesting that rather similar levels of Cdt1 and its inhibitor are produced in this cell line, though at different cell cycle stages [13] In contrast, we calculate a ratio of Cdt1 to geminin of approximately 10 : for MCF7 cells The difference in Cdt1 protein levels which is consistently observed between primary and cancer cell lines tested could have been due to the larger Ôin cycleÕ pool of the cancer cell lines In order to address this, we used immunofluorescence to assess whether Cdt1 is over-expressed in individual cells Ó FEBS 2004 Cdt1 and geminin down-regulation in quiescence (Eur J Biochem 271) 3375 Fig Cdt1 and geminin are highly expressed in cancer cells (A,B) Western blotting analysis was used to determine the expression of Cdt1, geminin and tubulin as a loading control in cellular extracts from different human cell lines (A) Lanes 1–6, HFF, Saos, MDAMB231, MCF7, HeLa and LNcap, respectively (B) Lanes 1–8, HFF, HeLa, MCF7, Huvec, HT1080, U20S, MRC5 and HeLa, respectively A commercial anti-geminin Ig (Santa Cruz) was used for (A), while anti-Gem2, which shows a higher sensitivity, was used for (B) For Cdt1, a higher exposure is shown for lanes 5–8 in respect to lanes 1–4, to allow visualization of Cdt1 in MRC5 cells For lanes 1–4, a higher exposure of the geminin blot (Gem long) is also shown at the bottom of the panel, to permit visualization of geminin in the primary HFF cells (C) Estimation of the number of Cdt1 and geminin molecules present in HeLa cells Western blotting of total cell extract from 1.5 · 105 asynchronously growing HeLa cells (marked HeLa) was run alongside known amounts of recombinant full-length Cdt1 and geminin (HisT7-geminin and His-geminin, amount of recombinant protein run in each lane in ng is indicated) and immunoblotted with anti-Cdt1 and anti-geminin Ig See Materials and methods for calculations of a tumor cell line population in comparison with primary cells (Fig 7) The number of HeLa cells staining positive for Cdt1 was higher (approximately 50% of HeLa cells in comparison with 35% of HFF cells), consistent with a higher percentage of HeLa cells actively cycling In addition to that however, the staining observed in individual HeLa cells was higher than the staining observed in individual HFF cells (Fig 7A) Quantitation of 25 high-power fields each for HFF and HeLa immunostainings shows that a range of expression levels are observed in both HFF and HeLa cells, as expected for a protein whose levels fluctuate during the cell cycle, individual HeLa cells, however, express on average over twofold higher levels of Cdt1 than HFF cells (Fig 7B) These data show that Cdt1 is expressed to higher levels in individual cycling cancer cells in comparison with cycling primary cells To address whether Cdt1 and geminin up-regulation in cancer cell lines would also occur at the mRNA level, we used Northern blot analysis with total cell RNA extracted from the primary HFF cells and different tumor cell lines Similar to protein levels, both Cdt1 and geminin mRNA was markedly increased in all the tumor cell lines tested compared with the primary HFF cells (Fig 8A) We employed densitometry in order to compare the levels of hCdt1 and h-geminin mRNA amongst the different cancer cell lines tested When compared with HeLa cells, 3376 G Xouri et al (Eur J Biochem 271) Ó FEBS 2004 Fig Quantitative immunofluorescence to compare Cdt1 protein levels in individual HFF and HeLa cells Triplicates of HFF and HeLa cells grown on coverslips were subjected to immunofluorescence with antiCdt1 Ig (A) Microscopy images recorded under identical conditions are shown (B) A scatter plot of the expression values of Cdt1 in 25 different high-power fields each for HFF and HeLa cells is shown Relative expression values were measured with IPLAB software in arbitrary units MDAMB231 cells showed somewhat decreased levels for both hCdt1 and h-geminin (approximately threefold reducFig Cdt1 and geminin mRNA levels in cancer cell lines and tumours tion) while MCF7 cells showed increased levels of hCdt1 16 (A) Northern blotting analysis was performed to determine mRNA mRNA (twofold) and decreased levels of h-geminin mRNA levels of hCdt1, h-geminin and actin in different human cell lines (threefold reduction), supportive of our findings concerning Lanes 1–5, HeLa, MDAMB231, LNcap, MCF7 and HFF cells, hCdt1 and h-geminin protein levels in this cell line respectively (B) Northern blot bearing total mRNA from human Therefore, while all cancer cell lines tested express much kidney, liver and lung tumors and corresponding normal specimens higher levels of hCdt1 and h-geminin mRNAs when was hybridized with a geminin specific probe, and an actin specific compared with primary cells, cancer cell lines may differ probe as loading control in the levels of overexpression of these mRNAs In Fig 8B, a Northern blot containing total RNA extracted from tumorigenic and matched normal specimens thereby licensing DNA for a further round of DNA (kidney cancer, liver cancer and lung cancer and respective replication When G1 cells exit the cell cycle and enter normal specimens) was hybridized with an h-geminin quiescence, licensing is gradually lost We show here that specific probe and actin as a loading control Densitometry Cdt1 is down-regulated when cells are induced to transit analysis shows that h-geminin mRNA is increased in the from G1 into G0 by serum deprivation Cdt1 protein levels tumor specimens tested (approximately twofold) in comare low in serum deprived human primary fibroblast HFF cells and NIH3T3 cells Cdt1 appears again as cells are parison with the matched normal specimens, consistent with induced to re-enter the cell cycle upon serum re-addition, our findings with cancer cell lines before a new round of S phase is initiated, consistent with a requirement for Cdt1 in re-licensing G0 chromatin for a new Discussion round of DNA replication Cdt1 protein levels are not appreciably affected early upon contact inhibition, suggestIn cycling cells, Cdt1 is specifically expressed during the G1 ing that the degree of down-regulation of Cdt1 may vary phase of the cell cycle and is believed to act together with depending on how cells have entered the quiescent state Cdc6 to load the MCM protein complex onto chromatin, Ó FEBS 2004 Cdt1 and geminin down-regulation in quiescence (Eur J Biochem 271) 3377 This would explain why a significant down-regulation of Cdt1 in G0 was not previously detected [14] Cdc6, Cdt1’s partner for DNA licensing, has been shown to be downregulated in G0 cells and induced upon cell cycle re-entry [23] and to be under the transcriptional control of E2F [24– 26] We show here that, similar to Cdc6, Cdt1 mRNA levels are reduced in G0 and re-accumulate at the G0 to cell cycle transition, suggesting that, in this transition, Cdt1 may be controlled transcriptionally The presence of predicted E2F binding sites on the putative Cdt1 promoter (D Kougiou and S Taraviras, unpublished observation) attests to a possible E2F mediated regulation of Cdt1 Indeed E2F was recently reported to regulate the transcription of Cdt1 [34] In contrast, in cycling cells, Cdt1 appears to be controlled mostly post-transcriptionally [13] The difference in Cdt1 regulation we observe between serum deprived and contact inhibited NIH3T3 cells may indicate a regulation of Cdt1 by growth factors present in the serum In that respect it is interesting to note that consensus sites for myc and TCF-1 A are also present on the Cdt1 promoter In addition, we detected slower migrating forms of Cdt1 in serum deprived HFF cells upon longer exposure (data not shown), suggesting that an additional control of Cdt1 at the posttranslational level may also operate in G0 Geminin is believed to act as a cell cycle inhibitor, with a role to prevent untimely licensing by specifically binding to Cdt1 and inhibiting its MCM loading function [12,15,16] During the cell cycle, geminin is expressed in S and G2 phases, when licensing should be inhibited and when the Cdt1 protein is undetectable [13,16] Based on these findings, one might have expected geminin to be induced when G1 cells exit the cell cycle into G0 In contrast, however, we show here that geminin levels are extremely low in quiescent cells, with a decrease even more pronounced than that observed for Cdt1 For example, geminin is already undetectable in NIH3T3 cells in early confluency, when Cdt1 levels are not affected Geminin levels closely mirror the levels of cyclin A in these experiments Geminin mRNA levels also appear to be reduced early upon serum withdrawal and more dramatically than Cdt1 mRNA levels These findings are consistent with a recent publication linking geminin expression to the proliferating cell [17] An E2F binding site is also present on the predicted geminin promoter (D Kougiou and S Taraviras, unpublished observations) suggesting that Cdt1 and its inhibitor might be under similar transcriptional regulatory mechanisms Indeed, while this manuscript was under review, regulation of geminin by E2F and RB was reported [34,35] Geminin protein accumulates in HFF cells re-entering the cell cycle from G0 3–6 h later than Cdt1, and as cells enter into S phase (Fig 2) The accumulation of geminin may inhibit further licensing and define the Ôwindow of opportunityÕ for licensing at the transition from quiescence to proliferation The distribution of Cdt1 and geminin in the gut epithelium mirrors our findings with cultured cells We show by in situ hybridization and immunohistochemistry that both Cdt1 and geminin are expressed in the proliferative compartment of the developing mouse gut epithelium, consistent with a down-regulation of these factors upon cell cycle exit This is the first report of the distribution of Cdt1 in a mammalian tissue, while our findings are consistent with a previous report on geminin’s localization [17] Cancer cells have defects in the control mechanisms regulating cell cycle exit and therefore divide uncontrollably In addition, cancer cells often exhibit genomic instability and are aneuploid A recent report showed that over-expression of Cdt1 can predispose cells to a malignant transformation [36], thereby identifying Cdt1 as a putative oncogene In addition, over-expression of Cdt1 together with Cdc6 has been shown to result in re-replication and genomic instability in both yeast and human cells [7,37] We wished to examine whether Cdt1 is over-expressed in cancer cells in culture, in comparison with normal cycling cells We show that both Cdt1 protein and mRNA accumulate to much higher levels in cancer cells Immunofluorescence experiments showed that Cdt1 is present in higher levels in individual cancer cells, vs cycling primary fibroblasts Over-expression of Cdt1 in cancer cell lines can therefore not be solely accounted for by the larger Ôin cycleÕ fraction of cancer cells It would be interesting to investigate the mechanism that leads to over-accumulation of Cdt1 in cancer cells and the functional significance of this over-expression for malignant transformation The presence of increased mRNA levels suggests that overexpression may be at least partly due to increased transcription or increased gene copy number Over-expression of a stable form of geminin was recently shown to induce cell cycle arrest or apoptosis in human cell lines [17,38] However, endogenous geminin is over-expressed in the majority of cancer cells tested, in comparison with normal cells This is observed both in cancer cell lines and human tumors and is consistent with a recent report [17] The degree of over-expression of the geminin protein differs between cell lines, resulting in gross differences in relative amounts of Cdt1 and its inhibitor in different cell lines It would be interesting to investigate whether geminin and/or Cdt1 levels or the ratio of Cdt1 to its inhibitor geminin may show a correlation with the type, aggressiveness or molecular pathology of a given tumor Acknowledgements We would like to thank A Pyriohou, D Kalatzis, M Iliou and 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Wagle, N., Hwang, D.S & Dutta, A (2003) A p53-dependent checkpoint pathway prevents rereplication Mol Cell 11, 997– 1008 38 Shreeram, S., Sparks, A., Lane, D.P & Blow, J.J (2002) Cell typespecific responses of human cells to inhibition of replication licensing Oncogene 21, 6624–6632  ... accumulates as cells enter S phase Geminin, an inhibitor of Cdt1 is severely down-regulated upon cell cycle exit Cdt1 is negatively regulated by geminin [12,15], and, during the cell cycle, geminin accumulates... down-regulated in G0 During the cell cycle, Cdt1 and geminin mRNA levels are mostly stable and protein levels are primary controlled by Fig Expression of Cdt1 and geminin proteins in quiescent and proliferating... blotting with commercial antibodies against geminin showed that geminin protein levels are also increased in cancer cell lines, while different cancer cell lines appear to over-express geminin