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Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing

Abstract

The cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) is a cell-cycle checkpoint effector and inducer of senescence, regulated by p53. Yet, evidence suggests that p21 could also be oncogenic, through a mechanism that has so far remained obscure. We report that a subset of atypical cancerous cells strongly expressing p21 showed proliferation features. This occurred predominantly in p53-mutant human cancers, suggesting p53-independent upregulation of p21 selectively in more aggressive tumour cells. Multifaceted phenotypic and genomic analyses of p21-inducible, p53-null, cancerous and near-normal cellular models showed that after an initial senescence-like phase, a subpopulation of p21-expressing proliferating cells emerged, featuring increased genomic instability, aggressiveness and chemoresistance. Mechanistically, sustained p21 accumulation inhibited mainly the CRL4–CDT2 ubiquitin ligase, leading to deregulated origin licensing and replication stress. Collectively, our data reveal the tumour-promoting ability of p21 through deregulation of DNA replication licensing machinery—an unorthodox role to be considered in cancer treatment, since p21 responds to various stimuli including some chemotherapy drugs.

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Figure 1: p21 and Ki67 are co-expressed in a subset of atypical cells of high-grade/poorly differentiated, advanced human carcinomas and precancerous lesions.
Figure 2: Prolonged stimulation of p21 upregulates and stabilizes the RLFs CDC6 and CDT1 at the protein level.
Figure 3: The status of p53 defines the ability of p21 to regulate the levels of CDT1 and CDC6.
Figure 4: Sustained p21 expression triggers replication stress and DNA damage accumulation in a CDT1/CDC6-dependent manner in S phase.
Figure 5: Extended p21 overexpression mediates accumulation of replication intermediate lesions that are processed by MUS81–EME1 and repaired by a Rad52-dependent mechanism.
Figure 6: Deregulated upregulation of CDC6/CDT1 links p53-independent activation of p21 with senescence.
Figure 7: Prolonged p21 expression, in cells with p53 loss of function, overrides the senescence barrier.
Figure 8: p21-expressing cells that have overridden (escaped) the senescence barrier demonstrate genomic instability and aggressive behaviour.

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Acknowledgements

We would like to thank A. Kotsinas, K. Evangelou, T. Liloglou and A. Georgakilas for their valuable support to this work. We would like to thank A. Dutta for providing the vectors with the wt and PIP mutated domain of p21, G. Blandino for the H1299 p21–Ponasterone-ON cells and Z. Lygerou for the secondary antibodies employed in the IF analyses. We thank R. Allsopp, D. Coates, the Wessex Cancer Trust and Medical Research, UK, and the University of Southampton ‘Annual Adventures in Research’ fund for their support of the proteomics infrastructure and its use for this study. We are also indebted to the PRIDE team for the proteomics data processing–repository assistance. This work received funding from the European Union’s Seventh Framework Programmes ‘INsPiRE’ and ‘INTEGER’, the Greek GSRT programmes of Excellence I (Aristeia I - ‘STOCHAGEN’), the Greek GSRT programmes of Excellence II (Aristeia II - ‘TransProFeat CDC6’), the Greek GSRT Cooperation programme (‘NoisePlus’), the Hellenic Association for Molecular Cancer Research (HAMCR), and partial funding from the Research Institute for the Study of Genetic and Malignant Diseases in Childhood, ‘Aghia Sophia’ Children’s Hospital, Athens, Greece, the Danish National Research Foundation (Center of excellence project CARD), the Lundbeck Foundation and the Danish Council for Independent Research.

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Authors

Contributions

P.G., K.V. and D.W.: cell culture and manipulations, siRNA/plasmid/viral transfections/transductions/infections, immunoblots, cell growth, RT–PCR, ChIP, comet, IHC and IF assays. D.W. and C.S.S.: PFGE analysis. A.M.-M. and J.B.: DNA fibre spreading assay. A.K.A., R.Z., S.H. and M.L.: electron microscopy and cell culture for electron microscopy. E.J.H. and J.J.B.: FACS analyses. B.C., A.I. and A.N.: time-lapse video analyses. D.K.: MTT, soft agar, invasion and kinase assays. F.-M.R. and S.G.: molecular cytogenetic analyses. A.P., A.K. and D.T.: whole-genome and RNA sequencing. M.T. and E.K.: aCGH analyses. K.V., S.D.G. and P.T.: proteomic analysis. I.B.R.: data analysis and cell line production. K.V. and A.P.: transcriptomic and bioinformatic analyses. J.J.B., C.S.S., A.N. and J.B.: data analysis and interpretation, and assistance in manuscript preparation. V.G.G.: experimental design, guidance, manuscript preparation and writing.

Corresponding authors

Correspondence to Jiri Bartek or Vassilis G. Gorgoulis.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1

(a) Representative factors affected by p21WAF1/Cip1 induction at transcriptional and translational level. Representative real-time RT-PCR analyses to validate the high-throughput expression results (see also Fig. 2) (p < 0.01, t-test, error bars indicate mean ± SDs, n = 3 experiments). i. Mitotic factors: PLK1, AURKB, BUB1, BUB1B, KIF23 and the pro-apoptotic factor GLIPR1 along with the suppressor of the p21WAF1/Cip1 mediated effects ID1 are transcriptionally downregulated at the indicated time points in Saos2 p21WAF1/Cip1 Tet-ON induced cells. Growth factor IGFBP5, the ion channel encoding gene TRPM8 and the poly-A binding protein PABPC1L are upregulated. PBGD: Porphobilinogen deaminase (house-keeping gene) ii. Representative immunoblots that validate the proteome. Actin serves as a loading control. (PLK1: Polo-like kinase-1; AURKB: Aurora kinase B; BUB1: budding uninhibited by benzimidazoles 1 homolog; KIF23: kinesin family member 23; GLIPR1: Glioma pathogenesis related 1; ID1: inhibitor of DNA binding 1; IGFBP5: insulin-like growth factor binding protein 5; TRPM8: transient receptor potential cation channel subfamily M member 8; PABPC1L: poly(A) binding protein, cytoplasmic 1-like; TOP2A: topoisomerase 2A). (b) Timeline of senescence appearance in Saos2 p21WAF1/Cip1 Tet-ON and Li–Fraumeni p21WAF1/Cip1 Tet-ON induced cells. Activation of the senescence barrier occurs at approximately day 3 of induction in both cellular systems and increases gradually, reaching its highest value at around day 10, while no signs of senescence are evident in untreated cells grown for the same time period (as corresponding graphs depict). p21WAF1/Cip1 was confirmed by western blot (upper right panel). (c) i. E2F1 is upregulated while Chk1 is activated upon prolonged p21WAF1/Cip1 induction. Lysates from Saos2 p21WAF1/Cip1 Tet-ON cells, after treatment with 1 μg ml−1 Doxycycline for the depicted time points, were separated by SDS-PAGE and immunoblotted to detect the indicated proteins. ii. Silencing of Cdh-1/FZR-1 leads to increase in E2F1 expression in the p53 null H1299 cells. iii. A decline of Cdk2 activity is observed following p21WAF1/Cip1 induction. Histogram depicting decreased Cdk2 activity at days 4 after p21WAF1/Cip1 induction. (d) MCM2-7 chromatin loading is increased following p21WAF1/Cip1 induction in Saos2 and Li–Fraumeni cells. i. Diagram describing cell fractionation experimental algorithm. iiiii. All fractions were separated by SDS-PAGE and were analyzed by IB in Saos2 cells (ii) and Li–Fraumeni cells (iii). Lamin-B serves as fractionation control, while β-tubulin as loading control (n = 3 experiments). iv. FACS analysis of MCM2 chromatin loading in induced Saos2 p21WAF1/Cip1 Tet-ON cells versus non-induced (red dots, -ve: control experiment with no MCM2 antibody; blue dots, + ve: experiment with MCM2 antibody) (P < 0.05, p < 0.01, p < 0.005, t-test, error bars indicate mean ± SDs, n = 3 experiments). (e) Re-replication and DNA damage was significantly lesser in Saos2 cells infected with p21PCNA mutant. i. Comet assays showed DNA breaks in cells infected with the indicated constructs (see also Fig. 4b, d, f) (p < 0.0001, ANOVA, error bars indicate mean ± SDs, n = 3 experiments). Red lines in magnifications of insets label comet (moment) tails for length comparison. ii. FACS analysis of the corresponding treatments. iii. DNA damage is p21WAF1/Cip1 dependent in Saos2 cells treated with TGF-β (n = 3 experiments). iv. p21WAF1/Cip1 dependent DNA damage, in Saos2 cells treated with TGF-β, is exerted via Cdc6/Cdt1 mediated replication stress. (Empty vector: pMSCV, p21PCNA: mutant p21WAF1/Cip1 harboring Q144, M147, F150 substitutions to A in its PIP degron motif). Source data can be found in Supplementary Table 25.

Supplementary Figure 2

(a) Silencing of p21WAF1/Cip1 in induced (ii.) Saos2- and (iii.) Li Fraumeni- p21WAF1/Cip1 Tet-ON cells alleviates replication stress, DNA damage, senescence induction and enhanced resistance to chemotherapeutic drugs. Timeline of the experimental procedure is also depicted (i.). cells (p = NS, t-test or ANOVA, error bars indicate mean ± SDs, n = 3 experiments). Bars: 20 μm (IF), 30 μm (comet). (b) PCNA staining patterns reveal that sustained p21WAF1/Cip1 expression, in cells with non-functional p53, “traps” cells mainly in early S-phase. IF analysis for assessing PCNA staining patterns in non-induced and 96 h induced cells. Histograms depict average of observed patterns in the induction conditions employed (mean ± SDs, n = 3 experiments). Scale bars: 10 μm. (c) Absence of nascent ssDNA in Saos2 p21WAF1/Cip1 Tet-ON expressing cells. p21WAF1/Cip1 expression was induced for 96 h with 1 μg ml−1 doxycycline. The newly synthesized DNA was labeled for 20 min with 10 μM BrdU. 2 mM HU and 5 μM ATRi were added after the BrdU pulse as indicated for 2 h. After the indicated treatments, cells were fixed and stained with antibodies against BrdU without DNA denaturation to selectively detect nascent-strand ssDNA. Bars: 40 μm. (d) P21WAF1/Cip1 mediated DNA damage is processed by MUS81 resolvase. i. IF staining of DDR markers (53BP1 and γH2AX) in Saos2 p21WAF1 Tet-ON induced cells for 96 h, with or without anti-MUS81 siRNA targeting. Histogram depicts quantification of 53BP1 and γH2AX foci/cell (p < 0.01, t-test, error bars indicate mean ± SDs, n = 3 experiments). Bars: 20 μm. ii. DNA damage assessed by comet assay after prolonged expression in Li–Fraumeni p21WAF1 Tet-ON cells (p < 0.01, t-test, error bars indicate mean ± SDs, n = 3 experiments). Bars: 50 μm. iii. Silencing of the homologous repair recombinase Rad51 resulted in decreased γH2AX levels in Li–Fraumeni p21WAF1 Tet-ON cells (p < 0.0001, ANOVA, error bars indicate mean ± SDs, n = 3 experiments). (e) Sustained expression of p21WAF1/Cip1 in cells with non-functional p53 leads to restoration of Cdk2 activity in “escaped” cells. i. Following an initial decline (days 2-12) Cdk2 activity is increased in “escaped” cells (after day 20) (p < 0.01, t-test, error bars indicate mean ± SDs, n = 3 experiments). ii. Expression levels of p21WAF1/Cip1 in the “escaped” (i) Saos2 and (ii) Li–Fraumeni p21WAF1 Tet-ON cells were similar or even higher sometimes (see Fig. 7g) to those observed in the initial phase of p21WAF1/Cip1 induction. (f) Potential mechanisms involved in p73 down regulation in the “escaped” Saos2- and Li–Fraumeni-p21 cells. (see Fig. 7g) i. Absence of p73 promoter methylation and genetic loss at TP73 locus (1p36.33) (see Supplementary Fig. S5, S6; Supplementary Table 4). Representative result from real-time PCR followed by high resolution melting (HRM) analysis is depicted (n = 3 experiments). Ctl DNA: SssI methylated and unmethylated control DNA. ii. Bioinformatic analysis employing Ingenuity software revealed potential factors that regulate p73 expression and activity. EGR-1 (Early Growth Response-1) is a potent transcriptional up-regulator of p7351. In turn, p73 can also transcriptionally induce EGR-1exprresion, forming a positive feed-back loop. HECW2 (HECT, C2 and WW Domain Containing E3 Ubiquitin Protein Ligase 2) expression stabilizes p73 protein levels via mono-ubiquitination1, while PRKACB (Protein Kinase A Catalytic Subunit β) decreases p73 transactivation and intramolecular interaction capacity2. iii. TP73 gene locus organization and structure of p73 protein with HECW2 and PRKACB interacting domains. Yellow rectangles: transcribed non translated TP73 exons; Blue rectangles: transcribed TP73 exons; Green rectangle: P1 promoter of TP73 gene; Blue ovals: EGR-1 biding sites. TDA: transactivation domain; DBD: DNA binding domain; OD: oligomerization domain; SAM: sterile alpha-motif domain. ivv. Analysis of EGR-1, HECW2 and PRKACB expression status in “escaped” Saos2 p21WAF1/Cip1 cells at mRNA (iv.) and protein (v.) level validated results obtained from high-throughput transcriptome analysis (Supplementary Fig. S3). CREB phosphorylation was examined as a proof-of-concept for PRKACB activity. vi. Analysis of EGR-1 at mRNA and protein level in “escaped” Li–Fraumeni p21WAF1/Cip1 cells (fold difference mean ± SDs, n = 3 experiments). vii. Potential mechanism for p73 downregulation in “escaped” cells. Decreased levels of EGR-1 possibly represent the main reason for low p73 expression49. Additionally, high levels of PRKACB decreases p73 transactivation and intramolecular interaction abilities2, counteracting the ability of high HECW2 expression to stabilize p73 via mono-ubiquitination1. High PRKACB levels may contribute further to p73 down-regulation by interfering with the positive feed-back loop between ERG-1 and p732. (g) Serial-section immunohistochemical (IHC) analysis showed co-expression of p21WAF1/Cip1, Ki67 and Cdc6/Cdt1 in atypical cancer cells in head and neck carcinomas, urothelial carcinomas and precancerous lesions. Actin serves as a loading control. Source data can be found in Supplementary Table 25.

Supplementary Figure 3 Differentially expressed genes whose expression status affects cancer according to literature in Saos2- and Li–Fraumeni-p21 cells.

Expression status of genes associated with cancer progression (see also Supplemental Table 8). (a) Timeline of experimental planning of transcriptome analyses. (b) Principal Component Analysis (PCA) of the differentially expressed genes depicting the majorly different gene expression signatures over the (19540 in Saos2- and 25376 in Li–Fraumeni cells) transcripts analysed. (c) Validation of representative factors in “escaped” (ON) cells versus non-induced (OFF) Saos2 and Li–Fraumeni cells. Representative real-time RT-PCR analyses, validating the high-throughput expression analysis (p < 0.01, t-test, error bars indicate mean ± SDs, n = 3 experiments). (d) Relative expression levels given as log-2-ratios of differentially expressed genes (p < 0.05) of the “escaped” vs OFF-cells, whose expression status (up or down-regulated) is reported to promote carcinogenesis. Arrow (←) denotes genes conferring cancer stemness (see also Supplemental Tables 8Aa, 8Ba). Lysates from non-induced and escaped Saos2 p21WAF1/Cip1 Tet-ON cells were immunoblotted to verify representatively the expression of the LGR5 cancer related stemness gene. (e) Differentially expressed genes whose expression status either promotes or suppresses cancer according to literature. Relative expression levels given as log-2-ratios of differentially expressed genes (p < 0.05) of the “escaped” vs OFF-cells. The lengths of the “encircled” lines depict the intensity of expression. Uncropped images of blots are shown in Supplementary Fig. S9. Source data can be found in Supplementary Table 25.

Supplementary Figure 4 “Escaped” Saos2 p21WAF1/Cip1 cells exhibit increased genomic instability relative to non-induced cells.

(a) Timeline of experimental planning of genomic analyses. (b) Overview of all array-CGH (aCGH) analyses results. In total 41 aberrations were found involving all chromosomes (except 9, 12, 14 and 15). The aberrations included 19 gains and 22 losses (Supplemental Table 5). The majority of aberrations were concentrated in chromosomes 3, 10 and X (Supplemental Table 5). [reference (Ref) genome is from un-induced (0 d) Saos2 p21WAF1/Cip1 cells]. (c) Novel clonal rearrangements distinguish the “escaped” Saos2 p21WAF1/Cip1 (ON) from OFF cells [arrows indicate lost (in OFF cells) or rearranged (in “escaped”-ON cells) chromosomes]. The p21WAF1/Cip1-OFF cells (control), were mainly hypotriploid (51-56 chromosomes) and shared most of the characteristic structural chromosome aberrations of the parental Saos2 cell line3. Compared to these cells, the “escaped” ones remained hypo-triploid but displayed at least 10 novel clonal structural or numerical aberrations affecting chromosomes 2, 3, 5, 8, 11, 13, 14, 20, 21 and X. Large portions of chromosomes X and 13 were lost in 90% of the “escaped” cells, confirming the aCGH findings. Furthermore, differential imbalances of chromosomes 5 and X between Saos2 p21WAF1/Cip1 ON cells and the controls were observed. In “escaped” (ON) cells, an additional inverted duplication of 5p was also present in 90% of the examined nuclei. (d) The Saos2 p21WAF1/Cip1 ON cells exert significantly higher rates (two fold) of random structural CIN/chromosome as compared to controls. (CIN:chromosomal instability). (e) Genomic distribution of breakpoints of random structural chromosome anomalies. Telomeric regions were found to be most frequently affected by fusions, translocations and tandem duplications of large chromosome segments. As unidentified ones were categorized the non-telomeric, non centromeric genomic rearrangements in which the cytogenetic bands of their breakpoints remained obscure. (f) “Escaped” Saos2 p21WAF1/Cip1 cells exhibit increased karyotypic aberrations relative to non-induced cells. Comparative pseudo-colored M-FISH/SKY karyograms of 10 non-induced (OFF) Saos2 p21WAF1/Cip1 cells (588 chromosomes) and 10 “escaped” (ON) ones (639 chromosomes), for the evaluation of whole genome structural CIN at the 350 chromosome band level. Arrows (and dashed rectangles) indicate representative non-clonal random structural rearrangements (unique anomalies encountered in a single cell). The “escaped” p21WAF1/Cip1 expressing cells (ON) displayed significantly higher rates of genome wide, random structural chromosomal rearrangements. ON cells (upper panel): Cells #1 and #7, from the Saos2 p21WAF1/Cip1 OFF panel, represent a minor subclone (20%) of this population because they share a distinctive clonal rearrangement affecting a derivative chromosome X and a deletion of 12p. Cells #3 and #5, belong to a second subclone of the control cells that is characterized by a deletion of a rearranged chromosome 19. The remaining non-induced (OFF) p21WAF1/Cip1 cells#2, #4, #6, #8 and #10, display a homogeneous karyotypic constitution and represent the major clone. Cell #9 is a polyploid product of whole genome endoreduplication of the major clone of Saos2 p21WAF1/Cip1 OFF cells. “Escaped”-OFF cells (lower panel): Cells #1 and #6 differ from the majority of the “escaped” (ON) population as they share a clonal inverted duplication of the long arm of chromosome 21. In addition, cells #2, #4 and #9, have lost a marker translocation der(9)t(5;9) that was replaced by a deletion 9p and acquired clonally an extra translocated der(22)t(20;22). A unique subclonal finding in Cells #3 and #10, of the “escaped” (ON) cells is the persistence of der(9)t(5;9). Cells #5 and #7 represent two different endoreduplicated ON subclones, characterized by unique structural abnormalities of chromosomes 7, 15 and 6 respectively. The karyotypic constitution of cell #8, resembles that of the control population and justifies the presence of an additional subclone that does not exceed the 10% of the “escaped” (ON) cells. (CIN:chromosomal instability).

Supplementary Figure 5 Correlation between aCGH replicates and corroboration with the cytogenetically detectable novel clonal alterations in Saos2 p21 cells ( see also Fig. 8f).

[reference (Ref) genome is from un-induced (0 d) Saos2 p21WAF1/Cip1 cells].

Supplementary Figure 6

Correlation between aCGH and deep sequencing in Saos2 (a) and Li–Fraumeni (b) cells (Next Generation Sequencing: NGS) analyses. Data from all replicates for each application were averaged before comparison.

Supplementary Figure 7

Novel chromosomal rearrangements and microhomology regions related to breakpoints in (a) Saos2 and (b) Li–Fraumeni cells. Circos diagrams depicting novel chromosomal rearrangements in “escaped” Saos2 (a) and Li–Fraumeni (b) p21WAF1/Cip1 Tet-ON expressing cells, respectively, revealed by deep sequencing (human chromosomes are located at the perimeter). Data from two biological replicates are depicted. Circos in the middle show shared chromosomal rearrangements by the two Saos2 (a) and Li–Fraumeni (b) p21WAF1/Cip1 Tet-ON biological replicates. Breakpoints employing micro-homologies ≥4bp in Saos2 (a) and ≥3bp in Li–Fraumeni (b) p21WAF1/Cip1 Tet-ON cells, respectively, are also presented below each circus diagram. Cytogenetic analyses (see also Supplementary Fig S4) confirming NGS data on breakpoints in the “escaped” Saos2 p21WAF1/Cip1 Tet-ON expressing cells are also shown. Asterisk (a) denotes breakpoint that does not encompass a micro-homology. Continuous red line denotes position of breakpoints.

Supplementary Figure 8

Relative gene expression levels (log-2 ratios) at 12, 48, 96-hs after p21WAF1/Cip1 -induction as well as “escaped” versus OFF cells in (a) Saos2 and relative gene expression levels (log-2 ratios) at 10 days after p21WAF1/Cip1 -induction as well as “escaped” versus OFF in (b) Li–Fraumeni cells. (c) Proposed model. (a) A: Relative expression of all measured genes (19540) at each depicted time-point as compared to non-induced cells (OFF). The correlogram at the bottom which presents the Pearson correlation coefficient among the 4 datasets illustrates that the overall gene-expression of the “escaped” population is non-correlated (0 correlation coefficient) to the three prior time points, which amongst them present a high degree of correlation. B: Relative expression of genes presenting differential expression (p < 0.05) in the “escaped” cells in relation to OFF (553 genes). The correlogram at the bottom illustrates the absence of correlation between the “escaped” population with the three early time points (12, 48, 96 hs). C: Relative expression of commonly differentially expressed genes (42) (p < 0.05) at each time-point versus OFF. Special interest present the 16 out of 42 marked genes whose expression levels are reversed at the “escaped” population in comparison to the previous time-points. (b) The same heatmaps are presented for Li–Fraumeni cells. A: Relative expression of all measured genes (25367) at each depicted time-point as compared to non-induced cells (OFF). B: Relative expression of genes presenting differential expression (p < 0.05) in the “escaped” cells in relation to OFF (3507 genes). C: Relative expression of commonly differentially expressed genes (538) (p < 0.05) at each time-point versus OFF. Special interest present the 154 out of 538 marked genes whose expression levels are reversed at the “escaped” population in comparison to 10-days. (c) Proposed model depicting prolonged p53-independent p21 oncogenic action (for additional mechanistic explanations see discussion). Under “physiological” conditions, MDM2 degrades p534,10. Dashed lines depict ineffective pathway.

Supplementary Figure 9 Unprocessed blots/gels employed in the current manuscript.

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Dividing Saos2 p21WAF1/Cip1 Tet-OFF cells. (AVI 27738 kb)

Senescent Saos2 p21WAF1/Cip1 Tet-ON cells. (AVI 32065 kb)

Senescent and dying Saos2 p21WAF1/Cip1 Tet-ON cells. (AVI 22761 kb)

Re-replicating Saos2 p21WAF1/Cip1 Tet-ON cells. (AVI 15198 kb)

Escaped and diving Saos2 p21WAF1/Cip1 Tet-ON cells. (AVI 26181 kb)

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Galanos, P., Vougas, K., Walter, D. et al. Chronic p53-independent p21 expression causes genomic instability by deregulating replication licensing. Nat Cell Biol 18, 777–789 (2016). https://doi.org/10.1038/ncb3378

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