Abstract
Many severely hypoxic cells fail to initiate DNA replication, but the mechanism underlying this observation is unknown. Specifically, although the ataxia-telangiectasia-rad3 related (ATR) kinase has been shown to be activated in hypoxic cells, several studies have not been able to document down-stream consequences of ATR activation in these cells. By clearly defining the DNA replication initiation checkpoint in hypoxic cells, we now demonstrate that ATR is responsible for activating this checkpoint. We show that the hypoxic activation of ATR leads to the phosphorylation-dependent degradation of the cdc25a phosphatase. Downregulation of cdc25a protein by ATR in hypoxic cells decreases CDK2 phosphorylation and activity, which results in the degradation of cdc6 by APC/CCdh1. These events do not occur in hypoxic cells when ATR is depleted, and the initiation of DNA replication is maintained. We therefore present a novel mechanism of cdc6 regulation in which ATR can have a central role in inhibiting the initiation of DNA replication by the regulation of cdc6 by APC/CCdh1. This model provides insight into the biology and therapy of hypoxic tumors.
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References
Benmaamar R, Pagano M . (2005). Involvement of the SCF complex in the control of Cdh1 degradation in S-phase. Cell Cycle 4: 1230–1232.
Borlado LR, Mendez J . (2008). CDC6: from DNA replication to cell cycle checkpoints and oncogenesis. Carcinogenesis 29: 237–243.
Brown EJ, Baltimore D . (2000). ATR disruption leads to chromosomal fragmentation and early embryonic lethality. Genes Dev 14: 397–402.
Busino L, Donzelli M, Chiesa M, Guardavaccaro D, Ganoth D, Dorrello NV et al. (2003). Degradation of Cdc25A by beta-TrCP during S phase and in response to DNA damage. Nature 426: 87–91.
Cook JG, Park CH, Burke TW, Leone G, DeGregori J, Engel A et al. (2002). Analysis of Cdc6 function in the assembly of mammalian prereplication complexes. Proc Natl Acad Sci USA 99: 1347–1352.
Cortez D, Glick G, Elledge SJ . (2004). Minichromosome maintenance proteins are direct targets of the ATM and ATR checkpoint kinases. Proc Natl Acad Sci USA 101: 10078–10083.
Cortez D, Wang Y, Qin J, Elledge SJ . (1999). Requirement of ATM-dependent phosphorylation of brca1 in the DNA damage response to double-strand breaks. Science 286: 1162–1166.
Costanzo V, Shechter D, Lupardus PJ, Cimprich KA, Gottesman M, Gautier J . (2003). An ATR- and Cdc7-dependent DNA damage checkpoint that inhibits initiation of DNA replication. Mol Cell 11: 203–213.
de Oliveira PE, Zhang L, Wang Z, Lazo JS . (2009). Hypoxia-mediated regulation of Cdc25A phosphatase by p21 and miR-21. Cell Cycle 8: 3157–3164.
Gardner LB, Corn PG . (2008). Hypoxic regulation of mRNA expression. Cell Cycle 7: 1916–1924.
Gardner LB, Li F, Yang X, Dang CV . (2003). Anoxic fibroblasts activate a replication checkpoint that is bypassed by E1a. Mol Cell Biol 23: 9032–9045.
Gardner LB, Li Q, Park MS, Flanagan WM, Semenza GL, Dang CV . (2001). Hypoxia inhibits G1/S transition through regulation of p27 expression. J Biol Chem 276: 7919–7926.
Graeber TG, Peterson JF, Tsai M, Monica K, Fornace Jr AJ, Giaccia AJ . (1994). Hypoxia induces accumulation of p53 protein, but activation of a G1-phase checkpoint by low-oxygen conditions is independent of p53 status. Mol Cell Biol 14: 6264–6277.
Green SL, Freiberg RA, Giaccia AJ . (2001). p21(Cip1) and p27(Kip1) regulate cell cycle reentry after hypoxic stress but are not necessary for hypoxia-induced arrest. Mol Cell Biol 21: 1196–1206.
Green SL, Giaccia AJ . (1998). Tumor hypoxia and the cell cycle: implications for malignant progression and response to therapy. Cancer J Sci Am 4: 218–223.
Guardavaccaro D, Kudo Y, Boulaire J, Barchi M, Busino L, Donzelli M et al. (2003). Control of meiotic and mitotic progression by the F box protein beta-Trcp1 in vivo. Dev Cell 4: 799–812.
Hall JR, Kow E, Nevis KR, Lu CK, Luce KS, Zhong Q et al. (2007). Cdc6 stability is regulated by the Huwe1 ubiquitin ligase after DNA damage. Mol Biol Cell 18: 3340–3350.
Hammer S, To KK, Yoo YG, Koshiji M, Huang LE . (2007). Hypoxic suppression of the cell cycle gene CDC25A in tumor cells. Cell Cycle 6: 1919–1926.
Hammond EM, Denko NC, Dorie MJ, Abraham RT, Giaccia AJ . (2002). Hypoxia links ATR and p53 through replication arrest. Mol Cell Biol 22: 1834–1843.
Hammond EM, Dorie MJ, Giaccia AJ . (2003a). ATR/ATM targets are phosphorylated by ATR in response to hypoxia and ATM in response to reoxygenation. J Biol Chem 278: 12207–12213.
Hammond EM, Dorie MJ, Giaccia AJ . (2004). Inhibition of ATR leads to increased sensitivity to hypoxia/reoxygenation. Cancer Res 64: 6556–6562.
Hammond EM, Giaccia AJ . (2004). The role of ATM and ATR in the cellular response to hypoxia and re-oxygenation. DNA Repair (Amst) 3: 1117–1122.
Hammond EM, Green SL, Giaccia AJ . (2003b). Comparison of hypoxia-induced replication arrest with hydroxyurea and aphidicolin-induced arrest. Mutat Res 532: 205–213.
Hateboer G, Wobst A, Petersen BO, Le Cam L, Vigo E, Sardet C et al. (1998). Cell cycle-regulated expression of mammalian CDC6 is dependent on E2F. Mol Cell Biol 18: 6679–6697.
Heffernan TP, Simpson DA, Frank AR, Heinloth AN, Paules RS, Cordeiro-Stone M et al. (2002). An ATR- and Chk1-dependent S checkpoint inhibits replicon initiation following UVC-induced DNA damage. Mol Cell Biol 22: 8552–8561.
Heffernan TP, Unsal-Kacmaz K, Heinloth AN, Simpson DA, Paules RS, Sancar A et al. (2007). Cdc7-Dbf4 and the human S checkpoint response to UVC. J Biol Chem 282: 9458–9468.
Jiang W, McDonald D, Hope TJ, Hunter T . (1999). Mammalian Cdc7-Dbf4 protein kinase complex is essential for initiation of DNA replication. EMBO J 18: 5703–5713.
Kaufmann WK, Cleaver JE, Painter RB . (1980). Ultraviolet radiation inhibits replicon initiation in S phase human cells. Biochim Biophys Acta 608: 191–195.
Koshiji M, Kageyama Y, Pete EA, Horikawa I, Barrett JC, Huang LE . (2004). HIF-1alpha induces cell cycle arrest by functionally counteracting Myc. EMBO J 23: 1949–1956.
Lau E, Zhu C, Abraham RT, Jiang W . (2006). The functional role of Cdc6 in S-G2/M in mammalian cells. EMBO Rep 7: 425–430.
Machida YJ, Dutta A . (2007). The APC/C inhibitor, Emi1, is essential for prevention of rereplication. Genes Dev 21: 184–194.
Mailand N, Diffley JF . (2005). CDKs promote DNA replication origin licensing in human cells by protecting Cdc6 from APC/C-dependent proteolysis. Cell 122: 915–926.
Mailand N, Falck J, Lukas C, Syljuasen RG, Welcker M, Bartek J et al. (2000). Rapid destruction of human Cdc25A in response to DNA damage. Science 288: 1425–1429.
Martin L, Kimball SR, Gardner LB . (2010). Regulation of the unfolded protein response by eif2bdelta isoforms. J Biol Chem 285: 31944–31953.
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.
Montagnoli A, Valsasina B, Brotherton D, Troiani S, Rainoldi S, Tenca P et al. (2006). Identification of Mcm2 phosphorylation sites by S-phase-regulating kinases. J Biol Chem 281: 10281–10290.
Olcina M, Lecane PS, Hammond EM . (2010). Targeting hypoxic cells through the DNA damage response. Clin Cancer Res 16: 5624–5629.
Olive PL, Banath JP, Durand RE . (2002). The range of oxygenation in SiHa tumor xenografts. Radiat Res 158: 159–166.
Reimann JD, Freed E, Hsu JY, Kramer ER, Peters JM, Jackson PK . (2001). Emi1 is a mitotic regulator that interacts with Cdc20 and inhibits the anaphase promoting complex. Cell 105: 645–655.
Schmaltz C, Hardenbergh PH, Wells A, Fisher DE . (1998). Regulation of proliferation-survival decisions during tumor cell hypoxia. Mol Cell Biol 18: 2845–2854.
Sivaprasad U, Machida YJ, Dutta A . (2007). APC/C--the master controller of origin licensing? Cell Div 2: 8.
Sotillo E, Garriga J, Padgaonkar A, Kurimchak A, Cook JG, Grana X . (2009). Coordinated activation of the origin licensing factor CDC6 and CDK2 in resting human fibroblasts expressing SV40 small T antigen and cyclin E. J Biol Chem 284: 14126–14135.
Stoeber K, Mills AD, Kubota Y, Krude T, Romanowski P, Marheineke K et al. (1998). Cdc6 protein causes premature entry into S phase in a mammalian cell-free system. EMBO J 17: 7219–7229.
Vassin VM, Wold MS, Borowiec JA . (2004). Replication protein A (RPA) phosphorylation prevents RPA association with replication centers. Mol Cell Biol 24: 1930–1943.
Vaupel P, Schlenger K, Knoop C, Hockel M . (1991). Oxygenation of human tumors: evaluation of tissue oxygen distribution in breast cancers by computerized O2 tension measurements. Cancer Res 51: 3316–3322.
Vigo E, Muller H, Prosperini E, Hateboer G, Cartwright P, Moroni MC et al. (1999). CDC25A phosphatase is a target of E2F and is required for efficient E2F-induced S phase. Mol Cell Biol 19: 6379–6395.
Zou L, Elledge SJ . (2003). Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 300: 1542–1548.
Acknowledgements
We appreciate the kind gifts of reagents from John Dillfey and Stephen Elledge. Luca Busino, Michele Pagano,Vincenzo D’Angiolella provided reagents and helpful advice. This work was supported in part by DK08164 and the Feldstein Foundation. LBG is the Saul J Farber Assistant Professor of Medicine.
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Martin, L., Rainey, M., Santocanale, C. et al. Hypoxic activation of ATR and the suppression of the initiation of DNA replication through cdc6 degradation. Oncogene 31, 4076–4084 (2012). https://doi.org/10.1038/onc.2011.585
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DOI: https://doi.org/10.1038/onc.2011.585
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