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Long-term p21 and p53 dynamics regulate the frequency of mitosis events and cell cycle arrest following radiation damage

Abstract

Radiation exposure of healthy cells can halt cell cycle temporarily or permanently. In this work, we analyze the time evolution of p21 and p53 from two single cell datasets of retinal pigment epithelial cells exposed to several levels of radiation, and in particular, the effect of radiation on cell cycle arrest. Employing various quantification methods from signal processing, we show how p21 levels, and to a lesser extent p53 levels, dictate whether the cells are arrested in their cell cycle and how frequently these mitosis events are likely to occur. We observed that single cells exposed to the same dose of DNA damage exhibit heterogeneity in cellular outcomes and that the frequency of cell division is a more accurate monitor of cell damage rather than just radiation level. Finally, we show how heterogeneity in DNA damage signaling is manifested early in the response to radiation exposure level and has potential to predict long-term fate.

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Fig. 1: Visualization as a function of time of the x-ray irradiation dataset at 0 Gy, 0.5 Gy, 1 Gy, 2 Gy, 4 Gy, and 8 Gy.
Fig. 2: Parameterizations revealed by dynamic time warping method between detrended p53 and p21 time series.
Fig. 3: 2D representations of the p21 and p53 temporal data for the x-ray radiation dataset.
Fig. 4: The moving averages for the expression levels of geminin, p53, and p21 are plotted and centered around mitosis (at t = 0) for the x-ray irradiation dataset.
Fig. 5: The moving averages for the expression levels of geminin, p53, and p21 are plotted and centered around mitosis (at t = 0) for the x-ray irradiation dataset for cells that showed signs of division, but reversed course.
Fig. 6: Swarm plots of mean values of p21 and p53 for x-ray data.
Fig. 7: The histograms for time to first division, number of total divisions observed over the 5-days period, the mean levels of p53, and mean levels of p21 over that period are presented.

Code availability

The availability of the code to reproduce the results as well as the algorithms made in this work are made available at https://github.com/phongatran/p21p53/.

Data availability

The data that support the findings of this study are available upon request through the corresponding author.

References

  1. Dhillon AS, Hagan S, Rath O, Kolch W. MAP kinase signalling pathways in cancer. Oncogene 2007;26:3279–90.

    Article  PubMed  CAS  Google Scholar 

  2. Vogelstein B, Lane D, Levine AJ. Surfing the p53 network. Nature 2000;408:307–10.

    Article  PubMed  CAS  Google Scholar 

  3. Lakin ND, Jackson SP. Regulation of p53 in response to DNA damage. Oncogene 1999;18:7644–55.

    Article  PubMed  CAS  Google Scholar 

  4. Efeyan A, Comb WC, Sabatini DM. Nutrient-sensing mechanisms and pathways. Nature 2015;517:302–10.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  5. Lee P, Chandel NS, Simon MC. Cellular adaptation to hypoxia through hypoxia inducible factors and beyond. Nat Rev Mol Cell Biol. 2020;21:268–83.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  6. Purvis JE, Lahav G. Encoding and decoding cellular information through signaling dynamics. Cell 2013;152:945–56.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Kolch W, Halasz M, Granovskaya M, Kholodenko BN. The dynamic control of signal transduction networks in cancer cells. Nat Rev Cancer. 2015;15:515–27.

    Article  PubMed  CAS  Google Scholar 

  8. Albeck JG, Mills GB, Brugge JS. Frequency-Modulated Pulses of ERK activity transmit quantitative proliferation signals. Mol Cell. 2013;49:249–61.

    Article  PubMed  CAS  Google Scholar 

  9. Reyes J, Chen JY, Stewart-Ornstein J, Karhohs KW, Mock CS, Lahav G. Fluctuations in p53 signaling allow escape from cell-cycle arrest. Mol Cell. 2018;71:581–91.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. Hafner A, Reyes J, Stewart-Ornstein J, Tsabar M, Jambhekar A, Lahav G. Quantifying the central dogma in the p53 pathway in live single cells. Cell Syst. 2020;10:495–505.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. Bothma JP, Norstad MR, Alamos S, Garcia HG. LlamaTags: A versatile tool to image transcription factor dynamics in live embryos. Cell 2018;173:1810–1822. e16

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  12. Yang JM, Chi WY, Liang J, Takayanagi S, Iglesias PA, Huang CH. Deciphering cell signaling networks with massively multiplexed biosensor barcoding. Cell 2021;184:6193–206.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  13. Tsabar M, Mock CS, Venkatachalam V, Reyes J, Karhohs KW, Oliver TG, et al. A Switch in p53 dynamics marks cells that escape from DSB-induced cell cycle arrest. Cell Rep. 2020;33:107995.

    Article  CAS  Google Scholar 

  14. Engeland K. Cell cycle regulation: p53-p21-RB signaling. Cell Death \ Differ. 2022;29:946–60.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  15. Lane DP. p53, guardian of the genome. Nature 1992;358:15–6.

    Article  PubMed  CAS  Google Scholar 

  16. Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes the rapid degradation of p53. Nature 1997;387:296–9.

    Article  PubMed  CAS  Google Scholar 

  17. Kim DH, Rho K, Kim S. A theoretical model for p53 dynamics: identifying optimal therapeutic strategy for its activation and stabilization. Cell Cycle. 2009;8:3707–16.

    Article  PubMed  CAS  Google Scholar 

  18. Bar-Or RL, Maya R, Segel LA, Alon U, Levine AJ, Oren M. Generation of oscillations by the p53-Mdm2 feedback loop: A theoretical and experimental study. Proc Natl Acad Sci USA. 2000;97:11250–5.

    Article  CAS  Google Scholar 

  19. Wagner J, Ma L, Rice JJ, Hu W, Levine AJ, Stolovitzky GA. p53-Mdm2 loop controlled by a balance of its feedback strength and effective dampening using ATM and delayed feedback. IEE Proc Syst Biol. 2005;152:109–17.

    Article  CAS  Google Scholar 

  20. Zhang T, Brazhnik P, Tyson JJ. Exploring mechanisms of the DNA-damage response: p53 pulses and their possible relevance to apoptosis. Cell Cycle. 2007;6:85–94.

    Article  PubMed  Google Scholar 

  21. Geva-Zatorsky N, Rosenfeld N, Itzkovitz S, Milo R, Sigal A, Dekel E, et al. Oscillations and variability in the p53 system. Mol Syst Biol. 2006;2:33–2006.

    Article  CAS  Google Scholar 

  22. Batchelor E, Mock CS, Bhan I, Loewer A, Lahav G. Recurrent Initiation: A Mechanism for Triggering p53 Pulses in Response to DNA Damage. Mol Cell. 2008;30:277–89.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. Stewart-Ornstein J, Lahav G p53 dynamics in response to DNA damage vary across cell lines and are shaped by efficiency of DNA repair and activity of the kinase ATM. Sci Signal. 2017;10:1–10.

  24. Christophorou MA, Ringshausen I, Finch AJ, Swigart LB, Evan GI. The pathological response to DNA damage does not contribute to p53-mediated tumour suppression. Nature 2006;443:214–7.

    Article  PubMed  CAS  Google Scholar 

  25. Efeyan A, Serrano M. p53: guardian of the genome and policeman of the oncogenes. Cell Cycle. 2007;6:1006–10.

    Article  PubMed  CAS  Google Scholar 

  26. Xue W, Zender L, Miething C, Dickins RA, Hernando E, Krizhanovsky V, et al. Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas. Nature 2007;445:656–60.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  27. Collado M, Serrano M. Senescence in tumours: Evidence from mice and humans. Nat Rev Cancer. 2010;10:51–7.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. Nardella C, Clohessy JG, Alimonti A, Pandolfi PP. Pro-senescence therapy for cancer treatment. Nat Rev Cancer. 2011;11:503–11.

    Article  PubMed  CAS  Google Scholar 

  29. Beauséjour CM, Krtolica A, Galimi F, Narita M, Lowe SW, Yaswen P, et al. Reversal of human cellular senescence: Roles of the p53 and p16 pathways. EMBO J. 2003;22:4212–22.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Dirac AMG, Bernards R. Reversal of Senescence in Mouse Fibroblasts through Lentiviral Suppression of p53* 210. J Biol Chem. 2003;278:11731–4.

    Article  PubMed  CAS  Google Scholar 

  31. Wade Harper J, Adami GR, Wei N, Keyomarsi K, Elledge SJ. The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 1993;75:805–16.

    Article  Google Scholar 

  32. Xiong Y, Hannon GJ, Zhang H, Casso D, Kobayashi R, Beach D. p21 is a universal inhibitor of cyclin kinases. Nature 1993;366:701–4.

    Article  PubMed  CAS  Google Scholar 

  33. Brugarolas J, Chandrasekaran C, Gordon JI, Beach D, Jacks T, Hannon GJ. Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature 1995;377:552–7.

    Article  PubMed  CAS  Google Scholar 

  34. Bornstein G, Bloom J, Sitry-Shevah D, Nakayama K, Pagano M, Hershko A. Role of the SCFSkp2 ubiquitin ligase in the degradation of p21Cip1 in S phase. J Biol Chem. 2003;278:25752–7.

    Article  PubMed  CAS  Google Scholar 

  35. Overton KW, Spencer SL, Noderer WL, Meyer T, Wang CL. Basal p21 controls population heterogeneity in cycling and quiescent cell cycle states. Proc Natl Acad Sci. 2014;111:E4386–E4393.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. Barr AR, Cooper S, Heldt FS, Butera F, Stoy H, Mansfeld J, et al. DNA damage during S-phase mediates the proliferation-quiescence decision in the subsequent G1 via p21 expression. Nat Commun. 2017;8:1–17.

    Article  CAS  Google Scholar 

  37. Spencer SL, Cappell SD, Tsai FC, Overton KW, Wang CL, Meyer T. The proliferation-quiescence decision is controlled by a bifurcation in CDK2 activity at mitotic exit. Cell 2013;155:369.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  38. Moosmüller C, Tralie C, Kooshkbaghi M, Belkhatir Z, Pouryahya M, Reyes J, et al. Periodicity scoring of time series encodes dynamical behavior of the tumor suppressor p53. IFAC-PapersOnLine. 2021;54:488–95. https://www.sciencedirect.com/science/article/pii/S2405896321005814

    Article  Google Scholar 

  39. Sakoe H, Chiba S. Dynamic programming algorithm optimization for spoken word recognition. IEEE Trans Acoust. 1978;26:43–9.

    Article  Google Scholar 

  40. Berndt DJ, Clifford J Using dynamic time warping to find patterns in time series. In: KDD Workshop. 1994. p. 359–70.

  41. Chiarella C, He XZ, Hommes C. A dynamic analysis of moving average rules. J Econ Dyn Control. 2006;30:1729–53.

    Article  Google Scholar 

  42. Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, Osawa H, et al. Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 2008;132:487–98.

    Article  PubMed  CAS  Google Scholar 

  43. Batchelor E, Loewer A, Lahav G. The ups and downs of p53: understanding protein dynamics in single cells. Nat Rev Cancer. 2009;9:371–7.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  44. Van Der Maaten L, Hinton G. Visualizing data using t-SNE. J Mach Learn Res. 2008;9:2579–625.

    Google Scholar 

  45. Coleman KE, Grant GD, Haggerty RA, Brantley K, Shibata E, Workman BD, et al. Sequential replication-coupled destruction at G1/S ensures genome stability. Genes Dev. 2015;29:1734–46.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  46. Krenning L, Feringa FM, Shaltiel IA, vandenBerg J, Medema RH. Transient activation of p53 in G2 phase is sufficient to induce senescence. Mol Cell. 2014;55:59–72.

    Article  PubMed  CAS  Google Scholar 

  47. Ryl T, Kuchen EE, Bell E, Shao C, Flórez AF, Mönke G, et al. Cell-cycle position of single MYC-driven cancer cells dictates their susceptibility to a chemotherapeutic drug. Cell Syst. 2017;5:237–50.

    Article  PubMed  CAS  Google Scholar 

  48. Granada AE, Jiménez A, Stewart-Ornstein J, Blüthgen N, Reber S, Jambhekar A, et al. The effects of proliferation status and cell cycle phase on the responses of single cells to chemotherapy. Mol Biol Cell. 2020;31:845–57.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  49. El-Deiry WS, Harper JW, O’Connor PM, Velculescu VE, Canman CE, Jackman J, et al. WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res. 1994;54:1169–74.

    PubMed  CAS  Google Scholar 

  50. Brown JP, Wei W, Sedivy JM. Bypass of senescence after disruption of p21 CIP1/WAF1 gene in normal diploid human fibroblasts. Science 1997;277:831–4.

    Article  PubMed  CAS  Google Scholar 

  51. Fang L, Igarashi M, Leung J, Sugrue MM, Lee SW, Aaronson SA. p21Waf1/Cip1/Sdi1 induces permanent growth arrest with markers of replicative senescence in human tumor cells lacking functional p53. Oncogene 1999;18:2789–97.

    Article  PubMed  CAS  Google Scholar 

  52. Arnoff TE, El-Deiry WS. CDKN1A/p21WAF1, RB1, ARID1A, FLG, and HRNR mutation patterns provide insights into urinary tract environmental exposure carcinogenesis and potential treatment strategies. Am J Cancer Res. 2021;11:5452.

    PubMed  PubMed Central  CAS  Google Scholar 

  53. Shiohara M, El-Deiry WS, Wada M, Nakamaki T, Takeuchi S, Yang R, et al. Absence of WAF1 mutations in a variety of human malignancies. Blood 1994;84:3781–4.

    Article  PubMed  CAS  Google Scholar 

  54. McKenzie KE, Siva A, Maier S, Runnebaum IB, Seshadri R, Sukumar S. Altered WAF1 genes do not play a role in abnormal cell cycle regulation in breast cancers lacking p53 mutations. Clin Cancer Res J Am Assoc Cancer Res. 1997;3:1669–73.

    CAS  Google Scholar 

  55. Deng C, Zhang P, Harper JW, Elledge SJ, Leder P. Mice lacking p21CIP1/WAF1 undergo normal development, but are defective in G1 checkpoint control. Cell 1995;82:675–84.

    Article  PubMed  CAS  Google Scholar 

  56. Kastenhuber ER, Lowe SW. Putting p53 in context. Cell 2017;170:1062–78.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  57. Li R, Waga S, Hannon GJ, Beach D, Stillman B. Differential effects by the p21 CDK inhibitor on PCNA-dependent DNA replication and repair. Nature 1994;371:534–7.

    Article  PubMed  CAS  Google Scholar 

  58. Cazzalini O, Scovassi AI, Savio M, Stivala LA, Prosperi E. Multiple roles of the cell cycle inhibitor p21CDKN1A in the DNA damage response. Mutat Res Mutat Res. 2010;704:12–20.

    Article  PubMed  CAS  Google Scholar 

  59. Ruan S, Okcu MF, Ren JP, Chiao P, Andreeff M, Levin V, et al. Overexpressed WAF1/Cip1 renders glioblastoma cells resistant to chemotherapy agents 1, 3-bis (2-chloroethyl)-1-nitrosourea and cisplatin. Cancer Res. 1998;58:1538–43.

    PubMed  CAS  Google Scholar 

  60. Hsu CH, Altschuler SJ, Wu LF. Patterns of early p21 dynamics determine proliferation-senescence cell fate after chemotherapy. Cell 2019;178:361–73.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  61. Gillies TE, Pargett M, Minguet M, Davies AE, Albeck JG. Linear integration of ERK activity predominates over persistence detection in Fra-1 regulation. Cell Syst. 2017;5:549–63.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  62. Abraham U, Schlichting JK, Kramer A, Herzel H. Quantitative analysis of circadian single cell oscillations in response to temperature. PLoS One. 2018;13:e0190004.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  63. Gabriel CH, Del Olmo M, Zehtabian A, Jäger M, Reischl S, van Dijk H, et al. Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells. Nat Commun. 2021;12:1–15.

    Article  CAS  Google Scholar 

  64. Sanaki-Matsumiya M, Matsuda M, Gritti N, Nakaki F, Sharpe J, Trivedi V, et al. Periodic formation of epithelial somites from human pluripotent stem cells. Nat Commun. 2022;13:1–14.

    Article  CAS  Google Scholar 

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Acknowledgements

The authors thank Professor Galit Lahav from Havard Medical School for making the data available to use in this study as well as the support of Memorial Sloan Kettering Cancer Center that made this work possible.

Funding

This study was supported by AFOSR grant (FA9550-17-1-0435), a grant from National Institutes of Health (R01-AG048769), MSK Cancer Center Support Grant/Core Grant (P30 CA008748), and a grant from Breast Cancer Research Foundation (BCRF-17-193). C.M. is supported by NSF DMS grant 2111322. J.R. received support from CONACyT/Fundacion Mexico en Harvard (404476), and Harvard Graduate Merit Fellowship.

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All authors participated in the study concept and design. APT, CT, JR, CM, ZB, JD, and AT performed development of the methodologies and analysis of the results. JR and AL performed biological interpretation of the obtained results. APT, CT, JR, CM, ZB, and AT performed initial writing and review of the paper. APT, JR, and AT performed the revision of the paper. All authors read and approved the final paper.

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Correspondence to Allen R. Tannenbaum.

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Tran, A.P., Tralie, C.J., Reyes, J. et al. Long-term p21 and p53 dynamics regulate the frequency of mitosis events and cell cycle arrest following radiation damage. Cell Death Differ (2022). https://doi.org/10.1038/s41418-022-01069-x

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