Molecular architecture of the DNA replication origin activation checkpoint
2010; Springer Nature; Volume: 29; Issue: 19 Linguagem: Inglês
10.1038/emboj.2010.201
ISSN1460-2075
AutoresSlavica Tudzarova, Matthew Trotter, Alex Wollenschlaeger, Claire M. Mulvey, Jasminka Godovac‐Zimmermann, Gareth Williams, Kai Stoeber,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle20 August 2010free access Molecular architecture of the DNA replication origin activation checkpoint Slavica Tudzarova Slavica Tudzarova Wolfson Institute for Biomedical Research, University College London, London, UK Search for more papers by this author Matthew W B Trotter Matthew W B Trotter Anne McLaren Laboratory for Regenerative Medicine and Department of Surgery, University of Cambridge, Cambridge, UK Search for more papers by this author Alex Wollenschlaeger Alex Wollenschlaeger Wolfson Institute for Biomedical Research, University College London, London, UK Search for more papers by this author Claire Mulvey Claire Mulvey Division of Medicine, Centre for Molecular Medicine, University College London, London, UK Search for more papers by this author Jasminka Godovac-Zimmermann Jasminka Godovac-Zimmermann Division of Medicine, Centre for Molecular Medicine, University College London, London, UK Search for more papers by this author Gareth H Williams Corresponding Author Gareth H Williams Wolfson Institute for Biomedical Research, University College London, London, UK Research Department of Pathology and UCL Cancer Institute, University College London, London, UK Search for more papers by this author Kai Stoeber Kai Stoeber Wolfson Institute for Biomedical Research, University College London, London, UK Research Department of Pathology and UCL Cancer Institute, University College London, London, UK Search for more papers by this author Slavica Tudzarova Slavica Tudzarova Wolfson Institute for Biomedical Research, University College London, London, UK Search for more papers by this author Matthew W B Trotter Matthew W B Trotter Anne McLaren Laboratory for Regenerative Medicine and Department of Surgery, University of Cambridge, Cambridge, UK Search for more papers by this author Alex Wollenschlaeger Alex Wollenschlaeger Wolfson Institute for Biomedical Research, University College London, London, UK Search for more papers by this author Claire Mulvey Claire Mulvey Division of Medicine, Centre for Molecular Medicine, University College London, London, UK Search for more papers by this author Jasminka Godovac-Zimmermann Jasminka Godovac-Zimmermann Division of Medicine, Centre for Molecular Medicine, University College London, London, UK Search for more papers by this author Gareth H Williams Corresponding Author Gareth H Williams Wolfson Institute for Biomedical Research, University College London, London, UK Research Department of Pathology and UCL Cancer Institute, University College London, London, UK Search for more papers by this author Kai Stoeber Kai Stoeber Wolfson Institute for Biomedical Research, University College London, London, UK Research Department of Pathology and UCL Cancer Institute, University College London, London, UK Search for more papers by this author Author Information Slavica Tudzarova1, Matthew W B Trotter2, Alex Wollenschlaeger1, Claire Mulvey3, Jasminka Godovac-Zimmermann3, Gareth H Williams 1,4 and Kai Stoeber1,4 1Wolfson Institute for Biomedical Research, University College London, London, UK 2Anne McLaren Laboratory for Regenerative Medicine and Department of Surgery, University of Cambridge, Cambridge, UK 3Division of Medicine, Centre for Molecular Medicine, University College London, London, UK 4Research Department of Pathology and UCL Cancer Institute, University College London, London, UK *Corresponding author. Wolfson Institute for Biomedical Research, University College London, The Cruciform Building, Gower Street, London WC1E 6BT, UK. Tel.: +44 20 7679 6304; Fax: +44 20 7388 4408; E-mail: [email protected] The EMBO Journal (2010)29:3381-3394https://doi.org/10.1038/emboj.2010.201 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Perturbation of DNA replication initiation arrests human cells in G1, pointing towards an origin activation checkpoint. We used RNAi against Cdc7 kinase to inhibit replication initiation and dissect this checkpoint in fibroblasts. We show that the checkpoint response is dependent on three axes coordinated through the transcription factor FoxO3a. In arrested cells, FoxO3a activates the ARF-∣Hdm2-∣p53 → p21 pathway and mediates p15INK4B upregulation; p53 in turn activates expression of the Wnt/β-catenin signalling antagonist Dkk3, leading to Myc and cyclin D1 downregulation. The resulting loss of CDK activity inactivates the Rb-E2F pathway and overrides the G1-S transcriptional programme. Fibroblasts concomitantly depleted of Cdc7/FoxO3a, Cdc7/p15, Cdc7/p53 or Cdc7/Dkk3 can bypass the arrest and proceed into an abortive S phase followed by apoptosis. The lack of redundancy between the checkpoint axes and reliance on several tumour suppressor proteins commonly inactivated in human tumours provides a mechanistic basis for the cancer-cell-specific killing observed with emerging Cdc7 inhibitors. Introduction An estimated 30 000 replication origins are spread along human chromosomes and it is understood that chromatin structure, adjacent sites of transcription and epigenetic parameters all affect origin selection (Mechali, 2001; Biamonti et al, 2003). Initiation of DNA replication is a two-step process. In early G1, the origin recognition complex (ORC) cooperates with Cdc6 and Cdt1 in loading the Mcm2–7 helicase to form a 'licensed' pre-replicative complex pre-RC). In late G1, the origin is 'fired' by CDKs and Cdc7/Dbf4 kinase. Cdc7 phosphorylates the Mcm2, 4 and 6 subunits, thereby inducing a conformational change that stimulates MCM helicase activity and exposes a domain of Mcm5 required for Cdk2-dependent loading of Cdc45 and the replisome containing RPA, PCNA and DNA polymerase α-primase (Sclafani and Holzen, 2007). In addition to its highly conserved function in origin firing, other, less well understood, functions have been reported for Cdc7 kinase. These include activation of the ATR-Chk1 pathway in response to DNA damage and DNA replication stress (Takeda et al, 1999; Costanzo et al, 2003; Dierov et al, 2004; Tenca et al, 2007; Kim et al, 2008), cohesin loading onto chromatin required for chromosomal segregation in mitosis (Takahashi et al, 2008), regulation of exit from mitosis (Miller et al, 2009) and double-strand break formation during meiotic recombination (Matos et al, 2008). As the two-step-replication model excludes the formation of replication-competent origins once S phase has started, it has been argued on the basis of experimental evidence that a putative cell cycle checkpoint could delay progression from G1 into S phase if replication initiation is perturbed (Blow and Gillespie, 2008). In breast epithelial cells, for example, RNAi against ORC2 impairs DNA replication, causing G1 arrest with low cyclin E–Cdk2 activity (Machida et al, 2005). Inhibition of pre-RC assembly by overexpressing a stable form of geminin causes G1 arrest associated with low CDK activity in fibroblasts (Shreeram et al, 2002). Blocking activation of the MCM helicase through RNAi against CDC7 also causes G1 arrest in fibroblasts and leads to elevated p53 levels, p21 induction and hypo-phosphorylated Rb (Montagnoli et al, 2004). These findings, therefore, suggest that somatic cells can respond directly to impairment of the DNA replication initiation machinery by blocking S phase entry (Blow and Gillespie, 2008). In contrast, inhibition of origin licensing or firing has been shown to cause apoptotic cell death in a range of different cancer cell lines. This is thought to arise as a result of transformed cells entering S phase with inadequate numbers of competent origins to complete chromosomal replication, arguing for loss of the putative origin activation checkpoint in cancer. As only a limited number of replication forks can be established when replication initiation is perturbed, it is plausible that apoptosis is triggered as a result of fork stalling/collapse in cancer cells with active intra S phase checkpoint mechanisms or mitotic catastrophe arising from partially replicated chromosomes in more transformed cells (Blow and Gillespie, 2008). The cancer-cell-specific killing reported for emerging pharmacological Cdc7 inhibitors, while normal cells undergo a non-genotoxic G1 arrest, has generated widespread interest in small molecule inhibitors of the DNA replication initiation machinery (Jackson, 2008; Montagnoli et al, 2008; Swords et al, 2010). However, very little is known about the molecular architecture and circuitry of the proposed origin activation checkpoint on which tumour specificity is dependent. Here, we have used RNAi against CDC7 to inhibit replication initiation and elucidate the molecular architecture of the checkpoint in human fibroblasts. Results Cdc7 depletion in IMR90 fibroblasts causes cell cycle arrest in G1 We set out to determine whether Cdc7 depletion can activate a checkpoint response to impaired DNA replication initiation by transfecting IMR90 cells with three different siRNAs with sequences corresponding to the CDC7 cDNA. Notably, two of the CDC7 siRNAs have been characterized in a previous study (Montagnoli et al, 2004), whereas the third has been validated by the manufacturer (Ambion, Warrington, UK) (Supplementary Table 1 and Supplementary Figure 1A–D). All three oligos efficiently reduced CDC7 mRNA levels (Supplementary Figure 1B). On the basis of the highest knock-down score and consistency in replicate experiments (Supplementary Figure 1B–D), oligo CDC7-A (referred to here as 'CDC7-siRNA') was used for all experiments shown, except those in which siRNA specificity was shown with an alternative siRNA (oligo CDC7-B). Relative to control-siRNA (CO), transfection of IMR90 cells with CDC7-siRNA reduced CDC7 mRNA levels by 65% 48 h post-transfection, by 85% at 72 h and by >95% at 96 h (Figure 1A). Correspondingly, in whole cell extracts (WCE), Cdc7 protein levels started to fall by 24 h and were undetectable from 48 h until 120 h post-transfection (Figure 1B). Consistent with efficient Cdc7 depletion, we noted a decrease in total Mcm2 protein levels and a shift from hyper-phosphorylated to slower migrating, hypo-phosphorylated Mcm2 isoforms (Montagnoli et al, 2004) (Figure 1B). Downregulation of Cdc7 expression caused a cessation of cell proliferation with cell numbers reaching a plateau 48 h post-transfection (Figure 1C). The majority of CDC7-siRNA-transfected cells accumulated with G1 DNA content. Although a small fraction of cells showed a G2/M DNA content, cells with less than 2C DNA content were not detected (Figure 1D), indicating that Cdc7-depleted cells remained viable. In cells that were synchronized by release from double thymidine block and directly transfected with CDC7-siRNA, the majority of cells again showed a G1 DNA content (90%), whereas the small G2/M peak noted for asynchronous cells (9%) was lost with remaining cells equally distributed between the S and G2/M fractions (5% each) (Figure 1E). To provide further evidence that Cdc7 depletion is causing a G1 arrest, cells were transfected with CDC7-siRNA and control-siRNA, pulsed with BrdU and immunostained with anti-BrdU antibodies. In keeping with the cell cycle profile, the percentage of BrdU-incorporating cells dropped from 22% in cells transfected with control-siRNA (23% in untreated cells) to 2% in Cdc7-depleted cells (Figure 1F). Interestingly, 6 days after transfection with CDC7-siRNA, IMR90 cells re-expressed Cdc7 and resumed cell proliferation, showing a degree of confluency seen in control cells (Supplementary Figure 1E–G). Figure 1.Cdc7 depletion in IMR90 fibroblasts causes cell cycle arrest in G1. (A) Time course of CDC7 mRNA knock-down (KD) in IMR90 cells relative to cells transfected with control-siRNA (CO). (B) Whole cell extracts (WCE) prepared from untreated (UT), CO and Cdc7KD cells were analysed by immunoblotting with the indicated antibodies (β-actin—loading control). (C) At the indicated time points, cell number was measured in UT, CO and Cdc7KD cell populations. (D) DNA content of UT, CO and Cdc7KD cells at 96 and 120 h post-transfection. (E) DNA content of double thymidine-arrested cells (DTB), CO and Cdc7KD cells 48 h after release from double thymidine block and transfection, and asynchronous Cdc7KD cells 48 h post-transfection. (F) At 96 h post-transfection, cells were pulsed for 2 h with BrdU, fixed and detected with an FITC-conjugated anti-BrdU antibody. DNA was stained with propidium iodide (PI). Numbers show the percentage of cells incorporating BrdU. Download figure Download PowerPoint To control for siRNA specificity, we studied an alternative CDC7-siRNA (oligo CDC7-B; Supplementary Table 1 and Supplementary Figure 1B and C) targeting a different region of the transcript. Oligos CDC7-B and CDC7-A showed comparable gene silencing efficacy (mRNA and protein reduction) and induced similar phenotypic effects (accumulation of cells with G1 DNA content) (Figure 1; Supplementary Figure 2A and B). Moreover, rescue experiments were performed in which the RNAi effect was reversed through expression of a CDC7 gene variant refractory to silencing by oligo CDC7-A. Under these conditions, IMR90 cells were able to recover from the cell cycle arrest caused by Cdc7 depletion, as shown by flow cytometry and BrdU-incorporation data strongly resembling those of control cells (Supplementary Figure 3A–C). We, therefore, reasoned that CDC7 oligos A and B are equivalent and that the cell cycle arrest phenotype directly correlates with Cdc7 depletion and is unlikely to be due to concomitant non-specific downregulation of an unknown gene (off-target effects). Taken together, these data show that after Cdc7 depletion diploid human fibroblasts arrest cell cycle progression in G1, remaining in a non-proliferative state from which they can re-enter the cell cycle after Cdc7 levels have been restored. CDK activity and replication initiation factors are downregulated in response to Cdc7 depletion The Mcm2–7-replicative helicase is an important target for S phase-promoting kinases during origin activation, and the Mcm2 subunit, in particular, has been shown to be a substrate for both CDK and Cdc7. Phosphosites have been mapped in the N-terminal tail of Mcm2 for Cdc7 (Ser-40, Ser-53 and Ser-108) and Cdk2 (Ser-13, Ser-27 and Ser-41) in vitro and in vivo (Montagnoli et al, 2006). To test whether the Cdc7-depletion-induced checkpoint response involves downregulation of S phase-promoting CDK activity, we transfected IMR90 cells with CDC7-siRNA and studied Mcm2 phosphorylation at the mapped CDK and Cdc7 phosphosites. In keeping with a previous study (Montagnoli et al, 2004), Cdc7 depletion caused a decrease in Mcm2 levels and reduced the electrophoretic mobility of the protein in polyacrylamide gels, showing the presence of hypo-phosphorylated Mcm2 isoforms (Figure 2A). Although Mcm2 total protein levels consistently dropped in Cdc7-depleted cells, the extent of Mcm2 reduction varied between experiments. The average reduction in the intensity of Mcm2 protein bands (relative to control-siRNA-transfected cells) was 45% at 48 h, 62% at 96 h and 76% at 120 h post-transfection (Image J densitometry analysis). Mcm2 Ser-40/41 and Ser-53 phosphorylation was abolished when Cdc7 kinase was downregulated (48–120 h post-transfection), whereas phosphorylation at the CDK phosphosites Ser-27 and Ser-41 was significantly reduced after 96 h (Figure 2A). To confirm the loss of CDK activity in Cdc7-depleted cells, we carried out in vitro kinase assays with immunoprecipitated Cdk2 (Figure 2B). As expected, in vitro phosphorylated, recombinant truncated Rb protein was detected with an anti-Thr-821-phosphorylated Rb antibody in IMR90 and MDA-MB231 breast cancer cells (controls), but not in IMR90 cells transfected with CDC7-siRNA or treated with the CDK inhibitor roscovitine. Figure 2.CDK activity and replication initiation factors are downregulated in response to Cdc7 depletion. (A) WCE prepared from IMR90 cells transfected with control-siRNA (CO) and CDC7-siRNA (Cdc7KD) were analysed by immunoblotting with the indicated antibodies (β-actin—loading control). (B) WCE prepared from CO and Cdc7KD cell populations and from CO cells treated with 200 μM roscovitine (ROS) for 24 h (negative control) and MDA-MB231 breast cancer cells (positive control) were immunoprecipitated with anti-cyclin A and anti-cyclin E antibodies. Cdk2 immunoprecipitates (Cdk2 IP) were subjected to an in vitro kinase assay using recombinant truncated Rb protein (p56) as substrate. In vitro phosphorylation was detected with a specific anti-Rb-phospho-Thr-821 antibody. Note that lanes 1–8 were run on the same polyacrylamide gel and proteins transferred to the same PVDF membrane by semi-dry electroblotting. The membrane was subsequently cut as indicated for optimized immunodetection. (C) WCE and chromatin-bound protein fractions (CBF) prepared from untreated (UT), CO and Cdc7KD cells (96 h post-transfection) were analysed by immunoblotting with the indicated antibodies (β-actin and histone H1—loading controls). (D) WCE from UT, CO and Cdc7KD cells (48 and 96 h post-transfection) were analysed by western blotting with the indicated antibodies (β-actin—loading control). (E) WCE from UT, CO and Cdc7KD cells (48 and 96 h post-transfection) and from cells treated for 24 h with 17 μM cisplatin (Pt) were analysed by western blotting with the indicated antibodies (β-actin—loading control). Download figure Download PowerPoint The decline in Mcm2 levels in Cdc7-depleted cells (Figure 2A) raises the possibility that perturbation of origin activation may result in downregulation of replication initiation factors and/or affect the stability on chromatin of pre-RCs already formed. To address this question, IMR90 cells transfected with CDC7- and control-siRNAs were biochemically fractionated into WCE and chromatin-bound fractions (CBF) and immunoblotted with antibodies against replication initiation factors (Figure 2C). Orc4 levels in untreated and siRNA-transfected cells did not vary over the course of the experiment. On the contrary, in Cdc7 depleted, but not control cells, protein levels of Cdc6, Cdt1, Mcm2, Dbf4, Mcm10 and Cdc45 were significantly downregulated, whereas levels of these replication initiation factors associated with chromatin were also reduced. Notably, the DNA polymerase processivity factor PCNA was undetectable in CBF from Cdc7-depleted cells (Figure 2C). Next, we sought to establish whether the Cdc7-depletion-induced checkpoint response involves any known cell cycle regulators of the G1-S transition. Compared with control cells, at 96 h post-transfection Cdc7-depleted cells showed a marked increase in cyclin E levels, whereas cyclin A levels were reduced below the detection limit (Figure 2D). The loss of cyclin A (Figure 2D) and lack of chromatin-bound PCNA (Figure 2C) further support the notion of a late G1 arrest in Cdc7-depleted cells. The Cdc7-depleted cells also showed early loss of Rb phosphorylation at Ser-807/811, thought to be either Cdk4 or Cdk2 phosphorylation sites (Connell-Crowley et al, 1997; Chi et al, 2008), slightly raised p16 levels, p53 stabilization and increased levels of p21 (Figure 2D). Phosphorylation of p53 at Ser-15 (Figure 2E) and Chk1 at Ser-345 (Supplementary Figure 4) was not detected in Cdc7-depleted or control cells, indicating that the ATM/ATR checkpoint pathways were not activated. These results show that Cdc7 depletion results in downregulation of replication initiation factors and low CDK activity, consistent with the observed increase in p21 levels and the appearance of hypo-phosphorylated Rb. Cdc7 depletion affects expression of genes required for cell cycle progression and proliferation To investigate signalling pathways affected by CDC7 knock-down, we performed gene expression microarray (GEM) analysis on samples prepared from IMR90 cells transfected with CDC7-siRNA and control-siRNA (84 h post-transfection; see Supplementary Materials and Methods). Differentially regulated genes were analysed according to their membership of Kyoto Encyclopedia of Genes and Genomes human signalling pathways (Supplementary Table 2). The overall expression profile of genes in the cell cycle cluster was significantly altered between control cells and Cdc7-depleted cells (P<0.0001). Genes encoding Cdc6 and Mcm2–7, a number of mitosis regulatory factors, A-, B- and D-type cyclins, and Cdk1 and Cdk6 were all significantly downregulated in Cdc7-depleted cells, whereas CDKN2B (p15INK4B) was strongly upregulated (Supplementary Figure 5A). Genes in the p53 network cluster were also found to be differentially regulated (P<0.0001; Supplementary Figure 5B). The DNA damage checkpoint kinases ATM and ATR were strongly downregulated in Cdc7-depleted cells (Supplementary Figure 5B), consistent with the absence of p53 phosphorylation at Ser-15 (Figure 2E) and Chk1 phosphorylation at Ser-345 (Supplementary Figure 4). Although the pro-apoptotic genes BAX and FAS were upregulated, caspase 9 (CASP9) and caspase 3 (CASP3) were downregulated (Supplementary Figure 5B), suggesting that Cdc7 depletion may sensitize fibroblasts to pro-apoptotic signals, but does not activate the cell death effector machinery. Among p53-target genes, SIAH1, known to ubiquitinate β-catenin (Matsuzawa and Reed, 2001), and Dickkopf homolog 3 (DKK3), which blocks β-catenin accumulation in the nucleus (Wei et al, 2006; Lee et al, 2009), were strongly upregulated (Supplementary Figure 5B and C), pointing towards possible coupling between the p53 network and Wnt/β-catenin signalling pathway after CDC7 knock-down. This supposition was further supported by the notion that the overall expression profile of genes in the Wnt-signalling cluster was significantly altered between control and Cdc7-depleted cells (P=0.014; Supplementary Table 2; Supplementary Figure 5C). The GEM data indicate that the Cdc7-depletion-induced checkpoint overrides the transcriptional programme in a way that tilts the balance in favour of cell cycle arrest and reduced competency for cell proliferation. The GEM data used here may be found in the Array Express data repository (http://www.ebi.ac.uk/arrayexpress) under accession number E-MEXP-2115. The Cdc7-depletion-induced checkpoint is p53 dependent As the ATM/ATR checkpoint pathway does not appear to be active in Cdc7-depleted cells, we reasoned that the balance between Hdm2 and p14ARF may constitute the main mechanism for controlling p53 levels. Immunoblot analysis of nuclear extracts (NEs) prepared from Cdc7-depleted cells confirmed an increase in ARF levels, which correlated with loss of Hdm2 and p53 stabilization (Supplementary Figure 6A). Hdm2 protein was also not detectable in nucleolar subfractions prepared from Cdc7-depleted cells (data not shown). In keeping with the biochemical data, Cdc7-depleted cells showed strong ARF immunostaining compared with control cells (Supplementary Figure 6B). Hdm2 transcript levels increased two-fold in Cdc7-depleted cells relative to control-transfected cells 72 h post-transfection and were comparable at later time points, arguing against transcriptional downregulation of Hdm2 because of siRNA off-target effects (Supplementary Figure 7). A previous study in dermal fibroblasts showed that the Cdc7-depletion-induced checkpoint is dependent on p53 (Montagnoli et al, 2004). To confirm an active function for p53 in our experimental system, we used RNAi to downregulate p53 in IMR90 cells previously arrested by Cdc7 depletion (Supplementary Figure 8A). Notably, whereas Mcm2 phosphorylation at Ser-27 was abolished in Cdc7-depleted cells, phosphorylation at this mapped Cdk2 phosphosite was detectable in doubly depleted Cdc7/p53 cells. On the contrary, Mcm2 phosphorylation at the mapped Cdc7 phosphosite was strongly reduced in both Cdc7- and Cdc7/p53-depleted cells (Supplementary Figure 9). These data show that S phase-promoting CDK activity was restored in doubly depleted Cdc7/p53 cells. In keeping with the findings reported by Montagnoli et al, doubly depleted Cdc7/p53 cells failed to arrest cell cycle progression and instead progressed through S/G2 (Supplementary Figure 6C and D). Failure to elicit the Cdc7-depletion-induced checkpoint under CDC7 and P53 double knock-down conditions, however, eventually resulted in apoptotic cell death (Supplementary Figure 6E and F). Interestingly, cytoplasmatic protein fractions prepared from doubly depleted Cdc7/p53 cells revealed reduced levels of the β-catenin antagonist Dkk3 (Supplementary Figure 6G), supporting the notion of possible coupling between the p53 network and Wnt/β-catenin signalling pathway after Cdc7 depletion (Supplementary Table 2 and Supplementary Figure 5B and C). Note that in keeping with a previous report (Hsieh et al, 2004), peptide blocking and N-glycanase treatment identified the different bands detected with Dkk3 antibody as N-glycosylated Dkk3 isoforms (Supplementary Figure 10). The Cdc7-depletion-induced checkpoint is dependent on p53 activity upstream of Wnt/β-catenin signalling antagonist Dkk3 to downregulate Myc and cyclin D1 expression Dkk3 is known to block nuclear accumulation of β-catenin (Lee et al, 2009), resulting in downregulation of its downstream targets including CCND1 (cyclin D1) and MYC (Clevers, 2006). As DKK3 upregulation in Cdc7-depleted cells is dependent on p53 (Supplementary Figures 5C and 6G), we reasoned that p53 may affect cell cycle progression by indirectly blocking Wnt/β-catenin signalling. Indeed, immunoblot analysis of cytoplasmatic fraction (CF) and NE prepared from Cdc7-depleted cells showed reduced nuclear β-catenin and low Myc and cyclin D1 levels in conjunction with an increase in the inducible, faster migrating isoform of Dkk3 (Figure 3A). Consistent with the biochemical data, Cdc7-depleted cells showed only weak Myc and cyclin D1 immunostaining compared with control cells (Figure 3B). Notably, inducible Dkk3 expression and reduced nuclear β-catenin and cyclin D1 protein levels were not detected in IMR90 cells arrested through specific activation of the p53 pathway by low dose actinomycin D (Choong et al, 2009) (Supplementary Figure 11), further supporting a close relationship between p53-dependent Dkk3 upregulation and DNA replication control. Figure 3.p53-dependent upregulation of Wnt/β-catenin signalling antagonist Dkk3 is required for Cdc7-depletion-induced cell cycle arrest. (A) Cytoplasmatic protein fractions (CF) and crude nuclear extracts (NE) from untreated (UT), control-siRNA (CO) and CDC7-siRNA (Cdc7KD)-transfected IMR90 cells (72 h post-transfection) were analysed by immunoblotting with the indicated antibodies (β-actin and Orc4—loading controls). (B) At the same time point, CO and Cdc7KD cells were fixed and β-catenin, Myc and cyclin D1 detected by indirect immunofluorescence using a fluorescein-labelled secondary antibody. (C, D) CF and NE samples prepared from CO, Cdc7KD and doubly depleted Cdc7/Dkk3 (Cdc7KD/Dkk3KD) cells 48 and 72 h post-transfection were analysed by immunoblotting with the indicated antibodies. (E) 72 h post-transfection CO, Cdc7KD and Cdc7KD/Dkk3KD cells were pulsed for 2 h with BrdU, fixed and detected with an FITC-conjugated anti-BrdU antibody. Chromatin-bound PCNA and γH2A.X (inset: higher magnification) were detected by indirect immunofluorescence with anti-PCNA and anti-γH2A.X antibodies and a fluorescein-labelled secondary antibody. DNA was stained with propidium iodide (BrdU) or DAPI (PCNA and γH2A.X). Apoptotic cell death was detected in doubly depleted Cdc7KD/Dkk3KD cells by (F) phase contrast microscopy and by (G) immunoblot analysis of CF and NE with the indicated antibodies (β-actin and Orc4—loading controls). Download figure Download PowerPoint To directly test whether the p53 → Dkk3-∣β-catenin axis is essential for the checkpoint response, we downregulated Dkk3 through RNAi in IMR90 cells previously arrested by Cdc7 depletion (Supplementary Figure 8B). Immunoblot analysis of CF and NE prepared at 48 and 72 h post-transfection shows loss of the inducible form of Dkk3 in the Cdc7-depleted background and confirms maintenance of elevated p53 and p21 levels (Figure 3C). Note that downregulation of the slower migrating, heavily glycosylated Dkk3 isoforms (upper bands) only occurs at later time points (Supplementary Figure 10B and C). Cdc7 depletion alone diminished the pool of nuclear β-catenin and reduced Myc and cyclin D1 levels (Figure 3D). As expected, this resulted in G1 arrest as shown by cells failing to incorporate BrdU (Figure 3E), loss of chromatin-bound PCNA (Figure 3E) and cyclin A and histone H3 Ser-10 phosphorylation becoming undetectable (Figure 3D). Importantly, in doubly depleted Cdc7/Dkk3 cells, nuclear levels of β-catenin, Myc and cyclin D1 were restored 72 h post-transfection (Figure 3D). In contrast to Cdc7-depleted cells, doubly depleted Cdc7/Dkk3 cells did not arrest in G1 and instead progressed into S phase, as shown by high levels of chromatin-bound PCNA (Figure 3E), BrdU incorporation (Figure 3E), cyclin A detection (Figure 3D) and flow cytometry (Supplementary Figure 12C). Notably, doubly depleted Cdc7/Dkk3 cells exhibited γH2A.X immunostaining indicative of double-strand breaks (Figure 3E) and did not appear to progress to G2/M as shown by the lack of histone H3 Ser-10 phosphorylation (Figure 3D). Induction of apoptosis in doubly depleted Cdc7/Dkk3 cells was confirmed morphologically (Figure 3F), through detection of caspase 3 activation and PARP-1 cleavage (Figure 3G), and b
Referência(s)