Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

SP600125 suppresses Cdk1 and induces endoreplication directly from G2 phase, independent of JNK inhibition

Oncogene volume 29, pages 1702–1716 (2010)Cite this article

Subjects

Abstract

Cell cycle controls ensure that DNA replication (S phase) follows mitosis resulting in two precise copies of the genome. A failure of the control mechanisms can result in multiple rounds of DNA replication without cell division. In endoreplication, cells with replicated genomes bypass mitosis, then replicate their DNA again, resulting in polyploidy. Endoreplication from G2 phase lacks all hallmarks of mitosis. Using synchronized cells, we show that the c-Jun N-terminal kinase (JNK) inhibitor, SP600125, prevents the entry of cells into mitosis and leads to endoreplication of DNA from G2 phase. We show that cells proceed from G2 phase to replicate their DNA in the absence of mitosis. This effect of SP600125 is independent of its suppression of JNK activity. Instead, the inhibitory effect of SP600125 on mitotic entry predominantly occurs upstream of Aurora A kinase and Polo-like kinase 1, resulting in a failure to remove the inhibitory phosphorylation of Cdk1. Importantly, our results directly show that the inhibition of Cdk1 activity and the persistence of Cdk2 activity in G2 cells induces endoreplication without mitosis. Furthermore, endoreplication from G2 phase is independent of p53 control.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

Similar content being viewed by others

References

  • Andreassen PR, Margolis RL . (1994). Microtubule dependency of p34cdc2 inactivation and mitotic exit in mammalian cells. J Cell Biol 127: 789–802.

    Article  CAS  PubMed  Google Scholar 

  • Arias EE, Walter JC . (2007). Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells. Genes Dev 21: 497–518.

    Article  CAS  PubMed  Google Scholar 

  • Bain J, McLauchlan H, Elliott M, Cohen P . (2003). The specificities of protein kinase inhibitors: an update. Biochem J 371: 199–204.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bain J, Plater L, Elliott M, Shpiro N, Hastie CJ, McLauchlan H et al. (2007). The selectivity of protein kinase inhibitors: a further update. Biochem J 408: 297–315.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Barr FA, Sillje HH, Nigg EA . (2004). Polo-like kinases and the orchestration of cell division. Nat Rev Mol Cell Biol 5: 429–440.

    Article  CAS  PubMed  Google Scholar 

  • Bennett BL, Sasaki DT, Murray BW, O'Leary EC, Sakata ST, Xu W et al. (2001). SP600125, an anthrapyrazolone inhibitor of Jun N-terminal kinase. Proc Natl Acad Sci USA 98: 13681–13686.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bulavin DV, Saito S, Hollander MC, Sakaguchi K, Anderson CW, Appella E et al. (1999). Phosphorylation of human p53 by p38 kinase coordinates N-terminal phosphorylation and apoptosis in response to UV radiation. EMBO J 18: 6845–6854.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Busby EC, Leistritz DF, Abraham RT, Karnitz LM, Sarkaria JN . (2000). The radiosensitizing agent 7-hydroxystaurosporine (UCN-01) inhibits the DNA damage checkpoint kinase hChk1. Cancer Res 60: 2108–2112.

    CAS  PubMed  Google Scholar 

  • Cai D, Latham Jr VM, Zhang X, Shapiro GI . (2006). Combined depletion of cell cycle and transcriptional cyclin-dependent kinase activities induces apoptosis in cancer cells. Cancer Res 66: 9270–9280.

    Article  CAS  PubMed  Google Scholar 

  • Crosio C, Fimia GM, Loury R, Kimura M, Okano Y, Zhou H et al. (2002). Mitotic phosphorylation of histone H3: spatio-temporal regulation by mammalian Aurora kinases. Mol Cell Biol 22: 874–885.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Damiens E, Baratte B, Marie D, Eisenbrand G, Meijer L . (2001). Anti-mitotic properties of indirubin-3′-monoxime, a CDK/GSK-3 inhibitor: induction of endoreplication following prophase arrest. Oncogene 20: 3786–3797.

    Article  CAS  PubMed  Google Scholar 

  • Davis FM, Tsao TY, Fowler SK, Rao PN . (1983). Monoclonal antibodies to mitotic cells. Proc Natl Acad Sci USA 80: 2926–2930.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Di Fiore B, Pines J . (2007). Emi1 is needed to couple DNA replication with mitosis but does not regulate activation of the mitotic APC/C. J Cell Biol 177: 425–437.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Diffley JF, Labib K . (2002). The chromosome replication cycle. J Cell Sci 115: 869–872.

    CAS  PubMed  Google Scholar 

  • Du L, Lyle CS, Obey TB, Gaarde WA, Muir JA, Bennett BL et al. (2004). Inhibition of cell proliferation and cell cycle progression by specific inhibition of basal JNK activity: evidence that mitotic Bcl-2 phosphorylation is JNK-independent. J Biol Chem 279: 11957–11966.

    Article  CAS  PubMed  Google Scholar 

  • Duncia JV, Santella III JB, Higley CA, Pitts WJ, Wityak J, Frietze WE et al. (1998). MEK inhibitors: the chemistry and biological activity of U0126, its analogs, and cyclization products. Bioorg Med Chem Lett 8: 2839–2844.

    Article  CAS  PubMed  Google Scholar 

  • Edgar BA, Orr-Weaver TL . (2001). Endoreplication cell cycles: more for less. Cell 105: 297–306.

    Article  CAS  PubMed  Google Scholar 

  • Fotedar A, Cannella D, Fitzgerald P, Rousselle T, Gupta S, Doree M et al. (1996). Role for cyclin A dependent kinase in DNA replication in human S phase cell extracts. J Biol Chem 271: 31627–31637.

    Article  CAS  PubMed  Google Scholar 

  • Georgatos SD, Pyrpasopoulou A, Theodoropoulos PA . (1997). Nuclear envelope breakdown in mammalian cells involves stepwise lamina disassembly and microtubule-drive deformation of the nuclear membrane. J Cell Sci 110: 2129–2140.

    CAS  PubMed  Google Scholar 

  • Glover DM, Hagan IM, Tavares AA . (1998). Polo-like kinases: a team that plays throughout mitosis. Genes Dev 12: 3777–3787.

    Article  CAS  PubMed  Google Scholar 

  • Hamilton BT, Snyder JA . (1982). Rapid completion of mitosis and cytokinesis in PtK cells following release from nocodazole arrest. Eur J Cell Biol 28: 190–194.

    CAS  PubMed  Google Scholar 

  • Han Z, Boyle DL, Chang L, Bennett B, Karin M, Yang L et al. (2001). c-Jun N-terminal kinase is required for metalloproteinase expression and joint destruction in inflammatory arthritis. J Clin Invest 108: 73–81.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hendzel MJ, Wei Y, Mancini MA, Van Hooser A, Ranalli T, Brinkley BR et al. (1997). Mitosis-specific phosphorylation of histone H3 initiates primarily within pericentromeric heterochromatin during G2 and spreads in an ordered fashion coincident with mitotic chromosome condensation. Chromosoma 106: 348–360.

    Article  CAS  PubMed  Google Scholar 

  • Hirota T, Kunitoku N, Sasayama T, Marumoto T, Zhang D, Nitta M et al. (2003). Aurora-A and an interacting activator, the LIM protein Ajuba, are required for mitotic commitment in human cells. Cell 114: 585–598.

    Article  CAS  PubMed  Google Scholar 

  • Hochegger H, Dejsuphong D, Sonoda E, Saberi A, Rajendra E, Kirk J et al. (2007). An essential role for Cdk1 in S phase control is revealed via chemical genetics in vertebrate cells. J Cell Biol 178: 257–268.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Itzhaki JE, Gilbert CS, Porter AC . (1997). Construction by gene targeting in human cells of a ‘conditional’ CDC2 mutant that rereplicates its DNA. Nat Genet 15: 258–265.

    Article  CAS  PubMed  Google Scholar 

  • Jascur T, Brickner H, Salles-Passador I, Barbier V, El Khissiin A, Smith B et al. (2005). Regulation of p21(WAF1/CIP1) stability by WISp39, a Hsp90 binding TPR protein. Mol Cell 17: 237–249.

    Article  CAS  PubMed  Google Scholar 

  • Jin S, Tong T, Fan W, Fan F, Antinore MJ, Zhu X et al. (2002). GADD45-induced cell cycle G2-M arrest associates with altered subcellular distribution of cyclin B1 and is independent of p38 kinase activity. Oncogene 21: 8696–8704.

    Article  CAS  PubMed  Google Scholar 

  • Kiang L, Heichinger C, Watt S, Bahler J, Nurse P . (2009). Cyclin-dependent kinase inhibits reinitiation of a normal S-phase program during G2 in fission yeast. Mol Cell Biol 29: 4025–4032.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Knockaert M, Greengard P, Meijer L . (2002). Pharmacological inhibitors of cyclin-dependent kinases. Trends Pharmacol Sci 23: 417–425.

    Article  CAS  PubMed  Google Scholar 

  • Kumagai A, Dunphy WG . (1996). Purification and molecular cloning of Plx1, a Cdc25-regulatory kinase from Xenopus egg extracts. Science 273: 1377–1380.

    Article  CAS  PubMed  Google Scholar 

  • L'Italien L, Tanudji M, Russell L, Schebye XM . (2006). Unmasking the redundancy between Cdk1 and Cdk2 at G2 phase in human cancer cell lines. Cell Cycle 5: 984–993.

    Article  CAS  PubMed  Google Scholar 

  • Laronne A, Rotkopf S, Hellman A, Gruenbaum Y, Porter AC, Brandeis M . (2003). Synchronization of interphase events depends neither on mitosis nor on cdk1. Mol Biol Cell 14: 3730–3740.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lee J, Kim JA, Barbier V, Fotedar A, Fotedar R . (2009). DNA damage triggers p21WAF1-dependent Emi1 down-regulation that maintains G2 arrest. Mol Biol Cell 20: 1891–1902.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lee JC, Laydon JT, McDonnell PC, Gallagher TF, Kumar S, Green D et al. (1994). A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 372: 739–746.

    Article  CAS  PubMed  Google Scholar 

  • MacCorkle RA, Tan TH . (2004). Inhibition of JNK2 disrupts anaphase and produces aneuploidy in mammalian cells. J Biol Chem 279: 40112–40121.

    Article  CAS  PubMed  Google Scholar 

  • Machida YJ, Dutta A . (2007). The APC/C inhibitor, Emi1, is essential for prevention of rereplication. Genes Dev 21: 184–194.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Margolis RL, Lohez OD, Andreassen PR . (2003). G1 tetraploidy checkpoint and the suppression of tumorigenesis. J Cell Biochem 88: 673–683.

    Article  CAS  PubMed  Google Scholar 

  • Marumoto T, Zhang D, Saya H . (2005). Aurora-A—a guardian of poles. Nat Rev Cancer 5: 42–50.

    Article  CAS  PubMed  Google Scholar 

  • Mendez J, Stillman B . (2000). Chromatin association of human origin recognition complex, cdc6, and minichromosome maintenance proteins during the cell cycle: assembly of prereplication complexes in late mitosis. Mol Cell Biol 20: 8602–8612.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Mingo-Sion AM, Marietta PM, Koller E, Wolf DM, Van Den Berg CL . (2004). Inhibition of JNK reduces G2/M transit independent of p53, leading to endoreduplication, decreased proliferation, and apoptosis in breast cancer cells. Oncogene 23: 596–604.

    Article  CAS  PubMed  Google Scholar 

  • Minshull J, Blow JJ, Hunt T . (1989). Translation of cyclin mRNA is necessary for extracts of activated Xenopus eggs to enter mitosis. Cell 56: 947–956.

    Article  CAS  PubMed  Google Scholar 

  • Miyamoto-Yamasaki Y, Yamasaki M, Tachibana H, Yamada K . (2007). Induction of endoreduplication by a JNK inhibitor SP600125 in human lung carcinoma A 549 cells. Cell Biol Int 31: 1501–1506.

    Article  CAS  PubMed  Google Scholar 

  • Niculescu III AB, Chen X, Smeets M, Hengst L, Prives C, Reed SI . (1998). Effects of p21(Cip1/Waf1) at both the G1/S and the G2/M cell cycle transitions: pRb is a critical determinant in blocking DNA replication and in preventing endoreduplication. Mol Cell Biol 18: 629–643.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nigg EA . (1998). Polo-like kinases: positive regulators of cell division from start to finish. Curr Opin Cell Biol 10: 776–783.

    Article  CAS  PubMed  Google Scholar 

  • Nurse P . (1990). Universal control mechanism regulating onset of M-phase. Nature 344: 503–508.

    Article  CAS  PubMed  Google Scholar 

  • O'Farrell PH . (2001). Triggering the all-or-nothing switch into mitosis. Trends Cell Biol 11: 512–519.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Qian YW, Erikson E, Taieb FE, Maller JL . (2001). The polo-like kinase Plx1 is required for activation of the phosphatase Cdc25C and cyclin B-Cdc2 in Xenopus oocytes. Mol Biol Cell 12: 1791–1799.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Roberts EC, Shapiro PS, Nahreini TS, Pages G, Pouyssegur J, Ahn NG . (2002). Distinct cell cycle timing requirements for extracellular signal-regulated kinase and phosphoinositide 3-kinase signaling pathways in somatic cell mitosis. Mol Cell Biol 22: 7226–7241.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Roshak AK, Capper EA, Imburgia C, Fornwald J, Scott G, Marshall LA . (2000). The human polo-like kinase, PLK, regulates cdc2/cyclin B through phosphorylation and activation of the cdc25C phosphatase. Cell Signal 12: 405–411.

    Article  CAS  PubMed  Google Scholar 

  • Schmidt M, Budirahardja Y, Klompmaker R, Medema RH . (2005). Ablation of the spindle assembly checkpoint by a compound targeting Mps1. EMBO Rep 6: 866–872.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Sclafani RA, Holzen TM . (2007). Cell cycle regulation of DNA replication. Annu Rev Genet 41: 237–280.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Seki A, Coppinger JA, Du H, Jang CY, Yates III JR, Fang G . (2008). Plk1- and beta-TrCP-dependent degradation of Bora controls mitotic progression. J Cell Biol 181: 65–78.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Storchova Z, Pellman D . (2004). From polyploidy to aneuploidy, genome instability and cancer. Nat Rev Mol Cell Biol 5: 45–54.

    Article  CAS  PubMed  Google Scholar 

  • Uetake Y, Sluder G . (2007). Cell-cycle progression without an intact microtuble cytoskeleton. Curr Biol 17: 2081–2086.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Vassilev LT, Tovar C, Chen S, Knezevic D, Zhao X, Sun H et al. (2006). Selective small-molecule inhibitor reveals critical mitotic functions of human CDK1. Proc Natl Acad Sci USA 103: 10660–10665.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang M, Atayar C, Rosati S, Bosga-Bouwer A, Kluin P, Visser L . (2009). JNK is constitutively active in mantle cell lymphoma: cell cycle deregulation and polyploidy by JNK inhibitor SP600125. J Pathol 218: 95–103.

    Article  CAS  PubMed  Google Scholar 

  • Walter AO, Seghezzi W, Korver W, Sheung J, Lees E . (2000). The mitotic serine/threonine kinase Aurora2/AIK is regulated by phosphorylation and degradation. Oncogene 19: 4906–4916.

    Article  CAS  PubMed  Google Scholar 

  • Yu Q, La Rose J, Zhang H, Takemura H, Kohn KW, Pommier Y . (2002). UCN-01 inhibits p53 up-regulation and abrogates gamma-radiation-induced G(2)-M checkpoint independently of p53 by targeting both of the checkpoint kinases, Chk2 and Chk1. Cancer Res 62: 5743–5748.

    CAS  PubMed  Google Scholar 

  • Zhao ZS, Lim JP, Ng YW, Lim L, Manser E . (2005). The GIT-associated kinase PAK targets to the centrosome and regulates Aurora-A. Mol Cell 20: 237–249.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We dedicate this paper in fond memory of Arun Fotedar, a colleague and friend, whose laughter infected us all. We thank Kristian Helin (European Institute of Oncology, Italy) for Cdc6 antibody, Hideo Nishitani (Kyushu University) for Cdt1 antibody and Robert J Schultz (NCI, Bethesda, MD) for UCN-01. This work was supported by grants from the National Institutes of Health to RF (CA108947 and CA101810) and to RM (GM068107 and GM088716).

Author information

Authors and Affiliations

  1. Sidney Kimmel Cancer Center, San Diego, CA, USA

    J A Kim, J Lee, R L Margolis & R Fotedar

  2. Burnham Institute for Medical Research,, La Jolla, CA, USA

    R L Margolis & R Fotedar

Authors
  1. J A Kim
    View author publications

    Search author on:PubMed Google Scholar

  2. J Lee
    View author publications

    Search author on:PubMed Google Scholar

  3. R L Margolis
    View author publications

    Search author on:PubMed Google Scholar

  4. R Fotedar
    View author publications

    Search author on:PubMed Google Scholar

Corresponding author

Correspondence to R Fotedar.

Additional information

Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kim, J., Lee, J., Margolis, R. et al. SP600125 suppresses Cdk1 and induces endoreplication directly from G2 phase, independent of JNK inhibition. Oncogene 29, 1702–1716 (2010). https://doi.org/10.1038/onc.2009.464

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/onc.2009.464

Keywords

This article is cited by

Search

Advanced search

Quick links