Abstract
This study provides evidence for the importance of p21CDKN1A for the repair of replication-mediated DNA double-strand breaks (DSBs) induced by topoisomerase I. We report that defects of p21CDKN1A and p53 enhance camptothecin-induced histone H2AX phosphorylation (γH2AX), a marker for DNA DSBs. In human colon carcinoma HCT116 cells with wild-type (wt) p53, γH2AX reverses after camptothecin removal. By contrast, γH2AX increases after camptothecin removal in HCT116 cells deficient for p53 (p53−/−) or p21CDKN1A (p21−/−) as the cells reach the late-S and G2 phases. Since p21−/− cells exhibit similar S-phase arrest as wt cells in response to camptothecin and aphidicolin does not abrogate the enhanced γH2AX formation in p21−/− cells, we conclude that enhanced γH2AX formation in p21−/− cells is not due to re-replication. The cell cycle checkpoint abrogator and Chk1/Chk2 inhibitor 7-hydroxystaurosporine (UCN-01) also increases camptothecin-induced γH2AX formation and inhibits camptothecin-induced p21CDKN1A upregulation in HCT116 wt cells. TUNEL (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling) assays demonstrate that γH2AX formation in late S and G2 cells following CPT treatment corresponds to DNA breaks. However, these breaks are not related to apoptotic DNA fragmentation. We propose that p21CDKN1A prevents the collapse of replication forks damaged by stabilized topoisomerase I cleavage complexes.
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Abbreviations
- BSA:
-
bovine serum albumin
- FBS:
-
fetal bovine serum
- DMSO:
-
dimethylsulfoxide
- DSB:
-
DNA-double strand break
- IR:
-
ionizing radiation
- LSC:
-
laser scanning cytometry
- p53−/−:
-
deficient for p53
- p21−/−:
-
deficient for p21CDKN1A
- PBS:
-
phosphate-buffered saline
- PVDF:
-
polyvynilidene difluoride
- TBS:
-
Tris-buffered saline
- TBS-T:
-
Tris-buffered saline with Tween 20
- TCA:
-
trichloroacetic acid
- TUNEL:
-
terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling
- UCN-01:
-
7-hydroxystaurosporine
- wt:
-
wild type
References
Abraham RT . (2004). DNA Repair (Amst) 3: 883–887.
Antony S, Theruvathu JA, Brooks PJ, Lesher DT, Redinbo M, Pommier Y . (2004). Nucleic Acids Res 32: 5685–5692.
Banin S, Moyal L, Shieh S, Taya Y, Anderson CW, Chessa L et al. (1998). Science 281: 1674–1677.
Brown EJ, Baltimore D . (2003). Genes Dev 17: 615–628.
Bunz F, Dutriaux A, Lengauer C, Waldman T, Zhou S, Brown JP et al. (1998). Science 282: 1497–1501.
Busby EC, Leistritz DF, Abraham RT, Karnitz LM, Sarkaria JN . (2000). Cancer Res 60: 2108–2112.
Canman CE, Lim DS, Cimprich KA, Taya Y, Tamai K, Sakaguchi K et al. (1998). Science 281: 1677–1679.
Caspari T . (2000). Curr Biol 10: R315–R317.
Chehab NH, Malikzay A, Stavridi ES, Halazonetis TD . (1999). Proc Natl Acad Sci USA 96: 13777–13782.
Dancey J, Sausville EA . (2003). Nat Rev Drug Discov 2: 296–313.
Fan S, Smith ML, Rivet II DJ, Duba D, Zhan Q, Kohn KW et al. (1995). Cancer Res 55: 1649–1654.
Foiani M, Ferrari M, Liberi G, Lopes M, Lucca C, Marini F et al. (1998). Biol Chem 379: 1019–1023.
Fujimori A, Gupta M, Hoki Y, Pommier Y . (1996). Mol Pharmacol 50: 1472–1478.
Furuta T, Takemura H, Liao ZY, Aune GJ, Redon C, Sedelnikova OA et al. (2003). J Biol Chem 278: 20303–20312.
Godthelp BC, Wiegant WW, van Duijn-Goedhart A, Scharer OD, van Buul PP, Kanaar R et al. (2002). Nucleic Acids Res 30: 2172–2182.
Goldwasser F, Shimizu T, Jackman J, Hoki Y, O'Connor PM, Kohn KW et al. (1996). Cancer Res 56: 4430–4437.
Graves PR, Yu L, Schwarz JK, Gales J, Sausville EA, O'Connor PM et al. (2000). J Biol Chem 275: 5600–5605.
Gudkov AV, Komarova EA . (2003). Nat Rev Cancer 3: 117–129.
Guo CY, D'Anna JA, Li R, Larner JM . (1999). Radiat Res 151: 125–132.
Gupta M, Fan S, Zhan Q, Kohn KW, O'Connor PM, Pommier Y . (1997). Clin Cancer Res 3: 1653–1660.
Han Z, Wei W, Dunaway S, Darnowski JW, Calabresi P, Sedivy J et al. (2002). J Biol Chem 277: 17154–17160.
Hayward RL, Macpherson JS, Cummings J, Monia BP, Smyth JF, Jodrell DI . (2003). Clin Cancer Res 9: 2856–2865.
Hinz JM, Helleday T, Meuth M . (2003). Carcinogenesis 24: 249–253.
Hirao A, Kong YY, Matsuoka S, Wakeham A, Ruland J, Yoshida H et al. (2000). Science 287: 1824–1827.
Hoeijmakers JH . (2001). Nature 411: 366–374.
Huang X, Okafuji M, Traganos F, Luther E, Holden E, Darzynkiewicz Z . (2004). Cytometry A 58: 99–110.
Huberman JA . (1981). Cell 23: 647–648.
Kachnic LA, Wu B, Wunsch H, Mekeel KL, DeFrank JS, Tang W et al. (1999). J Biol Chem 274: 13111–13117.
Kohn EA, Ruth ND, Brown MK, Livingstone M, Eastman A . (2002). J Biol Chem 277: 26553–26564.
Kohn KW, Pommier Y . (2005). Biochem Biophys Res Commun 331: 816–827.
Larminat F, Germanier M, Papouli E, Defais M . (2004). Biol Cell 96: 545–552.
Levesque AA, Kohn EA, Bresnick E, Eastman A . (2005). Oncogene 24: 3786–3796.
Liu LF, Desai SD, Li TK, Mao Y, Sun M, Sim SP . (2000). Ann N Y Acad Sci 922: 1–10.
Liu W, Zhang R . (1998). Int J Oncol 12: 793–804.
Melchionna R, Chen XB, Blasina A, McGowan CH . (2000). Nat Cell Biol 2: 762–765.
Melo J, Toczyski D . (2002). Curr Opin Cell Biol 14: 237–245.
Monks A, Harris ED, Vaigro-Wolff A, Hose CD, Connelly JW, Sausville EA . (2000). Invest New Drugs 18: 95–107.
Motoyama N, Naka K . (2004). Curr Opin Genet Dev 14: 11–16.
Nelson WG, Kastan MB . (1994). Mol Cell Biol 14: 1815–1823.
O'Connell MJ, Walworth NC, Carr AM . (2000). Trends Cell Biol 10: 296–303.
Pommier Y, Barcelo J, Rao VA, Sordet O, Jobson AG, Thibaut L et al. (2006). Prog Nucleic Acid Res Mol Biol (in press).
Pommier Y, Cherfils J . (2005). Trends Pharmacol Sci 28: 136–145.
Pommier Y, Kohlhagen G, Laco GS, Kroth H, Sayer JM, Jerina DM . (2002). J Biol Chem 277: 13666–13672.
Pommier Y, Kohlhagen G, Pourquier P, Sayer JM, Kroth H, Jerina DM . (2000). Proc Natl Acad Sci USA 97: 2040–2045.
Pommier Y, Redon C, Rao VA, Seiler JA, Sordet O, Takemura H et al. (2003). Mutat Res 532: 173–203.
Pommier Y, Sordet O, Rao A, Zhang H, Kohn KW . (2005). Curr Pharm Des 11: 2855–2872.
Pourquier P, Pommier Y . (2001). Adv Cancer Res 80: 189–216.
Raderschall E, Bazarov A, Cao J, Lurz R, Smith A, Mann W et al. (2002). J Cell Sci 115: 153–164.
Rao A, Fan AM, Meng L, Doe CL, North PS, Hickson I et al. (2005). Mol Cell Biol 25: 8925–8937.
Redon C, Pilch D, Rogakou E, Sedelnikova O, Newrock K, Bonner W . (2002). Curr Opin Genet Dev 12: 162–169.
Rogakou EP, Nieves-Neira W, Boon C, Pommier Y, Bonner WM . (2000). J Biol Chem 275: 9390–9395.
Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM . (1998). J Biol Chem 273: 5858–5868.
Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S . (2004). Annu Rev Biochem 73: 39–85.
Sato S, Fujita N, Tsuruo T . (2002). Oncogene 21: 1727–1738.
Senderowicz AM . (2002). Oncologist 7: 12–19.
Senderowicz AM . (2003). Oncogene 22: 6609–6620.
Shao RG, Cao CX, Pommier Y . (2004). Acta Pharmacol Sin 25: 756–762.
Shao RG, Cao CX, Shimizu T, O'Connor PM, Kohn KW, Pommier Y . (1997a). Cancer Res 57: 4029–4035.
Shao RG, Cao CX, Zhang H, Kohn KW, Wold MS, Pommier Y . (1999). EMBO J 18: 1397–1406.
Shao RG, Shimizu T, Pommier Y . (1997b). Exp Cell Res 234: 388–397.
Shi Z, Azuma A, Sampath D, Li YX, Huang P, Plunkett W . (2001). Cancer Res 61: 1065–1072.
Shieh SY, Ahn J, Tamai K, Taya Y, Prives C . (2000). Genes Dev 14: 289–300.
Shiloh Y . (2003). Nat Rev Cancer 3: 155–168.
Stivala LA, Riva F, Cazzalini O, Savio M, Prosperi E . (2001). Oncogene 20: 563–570.
Strumberg D, Pilon AA, Smith M, Hickey R, Malkas L, Pommier Y . (2000). Mol Cell Biol 20: 3977–3987.
Syljuasen RG, Sorensen CS, Hansen LT, Fugger K, Lundin C, Johansson F et al. (2005). Mol Cell Biol 25: 3553–3562.
Taylor WR, Stark GR . (2001). Oncogene 20: 1803–1815.
Tornusciolo DR, Schmidt RE, Roth KA . (1995). Biotechniques 19: 800–805.
Tse AN, Schwartz GK . (2004). Cancer Res 64: 6635–6644.
Vaziri C, Saxena S, Jeon Y, Lee C, Murata K, Machida Y et al. (2003). Mol Cell 11: 997–1008.
Waga S, Stillman B . (1998). Mol Cell Biol 18: 4177–4187.
Waldman T, Lengauer C, Kinzler KW, Vogelstein B . (1996). Nature 381: 713–716.
Wan S, Capasso H, Walworth NC . (1999). Yeast 15: 821–828.
Ward IM, Chen J . (2001). J Biol Chem 276: 47759–47762.
Weinert T . (1998). Cell 94: 555–558.
Wu J, Yin MB, Hapke G, Toth K, Rustum YM . (2002). Mol Pharmacol 61: 742–748.
Wyllie FS, Haughton MF, Bond JA, Rowson JM, Jones CJ, Wynford-Thomas D . (1996). Oncogene 12: 1077–1082.
Yu Q, La Rose J, Zhang H, Takemura H, Kohn KW, Pommier Y . (2002). Cancer Res 62: 5743–5748.
Zhivotovsky B, Kroemer G . (2004). Nat Rev Mol Cell Biol 5: 752–762.
Acknowledgements
This research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
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Furuta, T., Hayward, R., Meng, LH. et al. p21CDKN1A allows the repair of replication-mediated DNA double-strand breaks induced by topoisomerase I and is inactivated by the checkpoint kinase inhibitor 7-hydroxystaurosporine. Oncogene 25, 2839–2849 (2006). https://doi.org/10.1038/sj.onc.1209313
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DOI: https://doi.org/10.1038/sj.onc.1209313
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