Background Maintenance of genome integrity is crucial for the propagation of the genetic information. Cdt1 is a major component of the pre-replicative complex, which controls once per cell cycle DNA replication. Upon DNA damage, Cdt1 is rapidly targeted for degradation. This targeting has been suggested to safeguard genomic integrity and prevent re-replication while DNA repair is in progress. Cdt1 is deregulated in tumor specimens, while its aberrant expression is linked with aneuploidy and promotes tumorigenesis in animal models. The induction of lesions in DNA is a common mechanism by which many cytotoxic anticancer agents operate, leading to cell cycle arrest and apoptosis. Methodology/Principal Finding In the present study we examine the ability of several anticancer drugs to target Cdt1 for degradation. We show that treatment of HeLa and HepG2 cells with MMS, Cisplatin and Doxorubicin lead to rapid proteolysis of Cdt1, whereas treatment with 5-Fluorouracil and Tamoxifen leave Cdt1 expression unaffected. Etoposide affects Cdt1 stability in HepG2 cells and not in HeLa cells. RNAi experiments suggest that Cdt1 proteolysis in response to MMS depends on the presence of the sliding clamp PCNA. Conclusion/Significance Our data suggest that treatment of tumor cells with commonly used chemotherapeutic agents induces differential responses with respect to Cdt1 proteolysis. Information on specific cellular targets in response to distinct anticancer chemotherapeutic drugs in different cancer cell types may contribute to the optimization of the efficacy of chemotherapy.
References
[1]
Maiorano D, Moreau J, Mechali M (2000) XCDT1 is required for the assembly of pre-replicative complexes in Xenopus laevis. Nature 404: 622–625.
[2]
Nishitani H, Lygerou Z, Nishimoto T, Nurse P (2000) The Cdt1 protein is required to license DNA for replication in fission yeast. Nature 404: 625–628.
[3]
Wong PG, Glozak MA, Cao TV, Vaziri C, Seto E, et al. (2010) Chromatin unfolding by Cdt1 regulates MCM loading via opposing functions of HBO1 and HDAC11-geminin. Cell Cycle 9: 4351–4363.
[4]
Nishitani H, Taraviras S, Lygerou Z, Nishimoto T (2001) The human licensing factor for DNA replication Cdt1 accumulates in G1 and is destabilized after initiation of S-phase. J Biol Chem 276: 44905–44911.
[5]
Sakaue-Sawano A, Kurokawa H, Morimura T, Hanyu A, Hama H, et al. (2008) Visualizing spatiotemporal dynamics of multicellular cell-cycle progression. Cell 132: 487–498.
[6]
Wohlschlegel JA, Dwyer BT, Dhar SK, Cvetic C, Walter JC, et al. (2000) Inhibition of eukaryotic DNA replication by geminin binding to Cdt1. Science 290: 2309–2312.
[7]
Xouri G, Lygerou Z, Nishitani H, Pachnis V, Nurse P, et al. (2004) Cdt1 and geminin are down-regulated upon cell cycle exit and are over-expressed in cancer-derived cell lines. Eur J Biochem 271: 3368–3378.
[8]
Xouri G, Squire A, Dimaki M, Geverts B, Verveer PJ, et al. (2007) Cdt1 associates dynamically with chromatin throughout G1 and recruits Geminin onto chromatin. Embo J 26: 1303–1314.
[9]
Tada S, Li A, Maiorano D, Mechali M, Blow JJ (2001) Repression of origin assembly in metaphase depends on inhibition of RLF-B/Cdt1 by geminin. Nat Cell Biol 3: 107–113.
[10]
Li X, Zhao Q, Liao R, Sun P, Wu X (2003) The SCF(Skp2) ubiquitin ligase complex interacts with the human replication licensing factor Cdt1 and regulates Cdt1 degradation. J Biol Chem 278: 30854–30858.
[11]
Liu E, Li X, Yan F, Zhao Q, Wu X (2004) Cyclin-dependent kinases phosphorylate human Cdt1 and induce its degradation. J Biol Chem 279: 17283–17288.
[12]
Sugimoto N, Tatsumi Y, Tsurumi T, Matsukage A, Kiyono T, et al. (2004) Cdt1 phosphorylation by cyclin A-dependent kinases negatively regulates its function without affecting geminin binding. J Biol Chem 279: 19691–19697.
[13]
Arias EE, Walter JC (2006) PCNA functions as a molecular platform to trigger Cdt1 destruction and prevent re-replication. Nat Cell Biol 8: 84–90.
[14]
Higa LA, Banks D, Wu M, Kobayashi R, Sun H, et al. (2006) L2DTL/CDT2 interacts with the CUL4/DDB1 complex and PCNA and regulates CDT1 proteolysis in response to DNA damage. Cell Cycle 5: 1675–1680.
[15]
Nishitani H, Sugimoto N, Roukos V, Nakanishi Y, Saijo M, et al. (2006) Two E3 ubiquitin ligases, SCF-Skp2 and DDB1-Cul4, target human Cdt1 for proteolysis. Embo J 25: 1126–1136.
[16]
Senga T, Sivaprasad U, Zhu W, Park JH, Arias EE, et al. (2006) PCNA is a cofactor for Cdt1 degradation by CUL4/DDB1-mediated N-terminal ubiquitination. J Biol Chem 281: 6246–6252.
[17]
Sansam CL, Shepard JL, Lai K, Ianari A, Danielian PS, et al. (2006) DTL/CDT2 is essential for both CDT1 regulation and the early G2/M checkpoint. Genes Dev 20: 3117–3129.
[18]
Arias EE, Walter JC (2005) Replication-dependent destruction of Cdt1 limits DNA replication to a single round per cell cycle in Xenopus egg extracts. Genes Dev 19: 114–126.
[19]
Davidson IF, Li A, Blow JJ (2006) Deregulated replication licensing causes DNA fragmentation consistent with head-to-tail fork collision. Mol Cell 24: 433–443.
[20]
Thomer M, May NR, Aggarwal BD, Kwok G, Calvi BR (2004) Drosophila double-parked is sufficient to induce re-replication during development and is regulated by cyclin E/CDK2. Development 131: 4807–4818.
[21]
Zhong W, Feng H, Santiago FE, Kipreos ET (2003) CUL-4 ubiquitin ligase maintains genome stability by restraining DNA-replication licensing. Nature 423: 885–889.
[22]
Vaziri C, Saxena S, Jeon Y, Lee C, Murata K, et al. (2003) A p53-dependent checkpoint pathway prevents rereplication. Mol Cell 11: 997–1008.
[23]
Liontos M, Koutsami M, Sideridou M, Evangelou K, Kletsas D, et al. (2007) Deregulated overexpression of hCdt1 and hCdc6 promotes malignant behavior. Cancer Res 67: 10899–10909.
[24]
Tatsumi Y, Sugimoto N, Yugawa T, Narisawa-Saito M, Kiyono T, et al. (2006) Deregulation of Cdt1 induces chromosomal damage without rereplication and leads to chromosomal instability. J Cell Sci 119: 3128–3140.
[25]
Petropoulou C, Kotantaki P, Karamitros D, Taraviras S (2008) Cdt1 and Geminin in cancer: markers or triggers of malignant transformation? Front Biosci 13: 4485–4494.
[26]
Higa LA, Mihaylov IS, Banks DP, Zheng J, Zhang H (2003) Radiation-mediated proteolysis of CDT1 by CUL4-ROC1 and CSN complexes constitutes a new checkpoint. Nat Cell Biol 5: 1008–1015.
[27]
Hu J, McCall CM, Ohta T, Xiong Y (2004) Targeted ubiquitination of CDT1 by the DDB1-CUL4A-ROC1 ligase in response to DNA damage. Nat Cell Biol 6: 1003–1009.
[28]
Ralph E, Boye E, Kearsey SE (2006) DNA damage induces Cdt1 proteolysis in fission yeast through a pathway dependent on Cdt2 and Ddb1. EMBO Rep 7: 1134–1139.
[29]
Havens CG, Walter JC (2009) Docking of a specialized PIP Box onto chromatin-bound PCNA creates a degron for the ubiquitin ligase CRL4Cdt2. Mol Cell 35: 93–104.
[30]
Hu J, Xiong Y (2006) An evolutionarily conserved function of proliferating cell nuclear antigen for Cdt1 degradation by the Cul4-Ddb1 ubiquitin ligase in response to DNA damage. J Biol Chem 281: 3753–3756.
[31]
Guarino E, Shepherd ME, Salguero I, Hua H, Deegan RS, et al. (2011) Cdt1 proteolysis is promoted by dual PIP degrons and is modulated by PCNA ubiquitylation. Nucleic Acids Res 39: 5978–5990.
[32]
Jin J, Arias EE, Chen J, Harper JW, Walter JC (2006) A family of diverse Cul4-Ddb1-interacting proteins includes Cdt2, which is required for S phase destruction of the replication factor Cdt1. Mol Cell 23: 709–721.
[33]
Shibata E, Abbas T, Huang X, Wohlschlegel JA, Dutta A (2011) Selective ubiquitylation of p21 and Cdt1 by UBCH8 and UBE2G ubiquitin-conjugating enzymes via the CRL4Cdt2 ubiquitin ligase complex. Mol Cell Biol 31: 3136–3145.
[34]
Zhou BB, Bartek J (2004) Targeting the checkpoint kinases: chemosensitization versus chemoprotection. Nat Rev Cancer 4: 216–225.
[35]
Gasnereau I, Ganier O, Bourgain F, de Gramont A, Gendron MC, et al. (2007) Flow cytometry to sort mammalian cells in cytokinesis. Cytometry A 71: 1–7.
[36]
Spella M, Britz O, Kotantaki P, Lygerou Z, Nishitani H, et al. (2007) Licensing regulators Geminin and Cdt1 identify progenitor cells of the mouse CNS in a specific phase of the cell cycle. Neuroscience 147: 373–387.
[37]
Kondo T, Kobayashi M, Tanaka J, Yokoyama A, Suzuki S, et al. (2004) Rapid degradation of Cdt1 upon UV-induced DNA damage is mediated by SCFSkp2 complex. J Biol Chem 279: 27315–27319.
[38]
Pines J, Hunter T (1990) Human cyclin A is adenovirus E1A-associated protein p60 and behaves differently from cyclin B. Nature 346: 760–763.
[39]
Wang J, Pabla N, Wang CY, Wang W, Schoenlein PV, et al. (2006) Caspase-mediated cleavage of ATM during cisplatin-induced tubular cell apoptosis: inactivation of its kinase activity toward p53. Am J Physiol Renal Physiol 291: F1300–1307.
[40]
Wang D, Lippard SJ (2005) Cellular processing of platinum anticancer drugs. Nat Rev Drug Discov 4: 307–320.
[41]
Nitiss JL (2009) Targeting DNA topoisomerase II in cancer chemotherapy. Nat Rev Cancer 9: 338–350.
[42]
Bender RP, Jablonksy MJ, Shadid M, Romaine I, Dunlap N, et al. (2008) Substituents on etoposide that interact with human topoisomerase IIalpha in the binary enzyme-drug complex: contributions to etoposide binding and activity. Biochemistry 47: 4501–4509.
[43]
Wilstermann AM, Bender RP, Godfrey M, Choi S, Anklin C, et al. (2007) Topoisomerase II - drug interaction domains: identification of substituents on etoposide that interact with the enzyme. Biochemistry 46: 8217–8225.
[44]
Bravou V, Nishitani H, Song SY, Taraviras S, Varakis J (2005) Expression of the licensing factors, Cdt1 and Geminin, in human colon cancer. Int J Oncol 27: 1511–1518.
[45]
Karakaidos P, Taraviras S, Vassiliou LV, Zacharatos P, Kastrinakis NG, et al. (2004) Overexpression of the replication licensing regulators hCdt1 and hCdc6 characterizes a subset of non-small-cell lung carcinomas: synergistic effect with mutant p53 on tumor growth and chromosomal instability–evidence of E2F-1 transcriptional control over hCdt1. Am J Pathol 165: 1351–1365.
[46]
Arentson E, Faloon P, Seo J, Moon E, Studts JM, et al. (2002) Oncogenic potential of the DNA replication licensing protein CDT1. Oncogene 21: 1150–1158.
[47]
Seo J, Chung YS, Sharma GG, Moon E, Burack WR, et al. (2005) Cdt1 transgenic mice develop lymphoblastic lymphoma in the absence of p53. Oncogene 24: 8176–8186.
[48]
Ponti D, Costa A, Zaffaroni N, Pratesi G, Petrangolini G, et al. (2005) Isolation and in vitro propagation of tumorigenic breast cancer cells with stem/progenitor cell properties. Cancer Res 65: 5506–5511.
[49]
Sharma SV, Lee DY, Li B, Quinlan MP, Takahashi F, et al. (2010) A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations. Cell 141: 69–80.
Musch T, Oz Y, Lyko F, Breiling A (2010) Nucleoside drugs induce cellular differentiation by caspase-dependent degradation of stem cell factors. PLoS One 5: e10726.
