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Molecular Cancer

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Open Access 01-12-2008 | Research

Soluble histone H2AX is induced by DNA replication stress and sensitizes cells to undergo apoptosis

Authors: Ying Liu, Joshua A Parry, Anna Chin, Stefan Duensing, Anette Duensing

Published in: Molecular Cancer | Issue 1/2008

Abstract

Background

Chromatin-associated histone H2AX is a key regulator of the cellular responses to DNA damage. However, non-nucleosomal functions of histone H2AX are poorly characterized. We have recently shown that soluble H2AX can trigger apoptosis but the mechanisms leading to non-chromatin-associated H2AX are unclear. Here, we tested whether stalling of DNA replication, a common event in cancer cells and the underlying mechanism of various chemotherapeutic agents, can trigger increased soluble H2AX.

Results

Transient overexpression of H2AX was found to lead to a detectable fraction of soluble H2AX and was associated with increased apoptosis. This effect was enhanced by the induction of DNA replication stress using the DNA polymerase α inhibitor aphidicolin. Cells manipulated to stably express H2AX did not contain soluble H2AX, however, short-term treatment with aphidicolin (1 h) resulted in detectable amounts of H2AX in the soluble nuclear fraction and enhanced apoptosis. Similarly, soluble endogenous H2AX was detected under these conditions. We found that excessive soluble H2AX causes chromatin aggregation and inhibition of ongoing gene transcription as evidenced by the redistribution and/or loss of active RNA polymerase II as well as the transcriptional co-activators CBP and p300.

Conclusion

Taken together, these results show that DNA replication stress rapidly leads to increased soluble H2AX and that non-chromatin-associated H2AX can sensitize cells to undergo apoptosis. Our findings encourage further studies to explore H2AX and the cellular pathways that control its expression as anti-cancer drug targets.

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Bassing CH, Chua KF, Sekiguchi J, Suh H, Whitlow SR, Fleming JC, Monroe BC, Ciccone DN, Yan C, Vlasakova K, Livingston DM, Ferguson DO, Scully R, Alt FW: Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX. Proc Natl Acad Sci U S A. 2002, 99: 8173-8178. 10.1073/pnas.122228699PubMedCentralCrossRefPubMed

Rogakou EP, Pilch DR, Orr AH, Ivanova VS, Bonner WM: DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J Biol Chem. 1998, 273: 5858-5868. 10.1074/jbc.273.10.5858CrossRefPubMed

Sedelnikova OA, Pilch DR, Redon C, Bonner WM: Histone H2AX in DNA damage and repair. Cancer Biol Ther. 2003, 2: 233-235.CrossRefPubMed

Fernandez-Capetillo O, Lee A, Nussenzweig M, Nussenzweig A: H2AX: the histone guardian of the genome. DNA Repair (Amst). 2004, 3: 959-967. 10.1016/j.dnarep.2004.03.024CrossRef

Celeste A, Difilippantonio S, Difilippantonio MJ, Fernandez-Capetillo O, Pilch DR, Sedelnikova OA, Eckhaus M, Ried T, Bonner WM, Nussenzweig A: H2AX haploinsufficiency modifies genomic stability and tumor susceptibility. Cell. 2003, 114: 371-383. 10.1016/S0092-8674(03)00567-1CrossRefPubMed

Celeste A, Petersen S, Romanienko PJ, Fernandez-Capetillo O, Chen HT, Sedelnikova OA, Reina-San-Martin B, Coppola V, Meffre E, Difilippantonio MJ, Redon C, Pilch DR, Olaru A, Eckhaus M, Camerini-Otero RD, Tessarollo L, Livak F, Manova K, Bonner WM, Nussenzweig MC, Nussenzweig A: Genomic instability in mice lacking histone H2AX. Science. 2002, 296: 922-927. 10.1126/science.1069398CrossRefPubMed

Redon C, Pilch D, Rogakou E, Sedelnikova O, Newrock K, Bonner W: Histone H2A variants H2AX and H2AZ. Curr Opin Genet Dev. 2002, 12: 162-169. 10.1016/S0959-437X(02)00282-4CrossRefPubMed

Burma S, Chen BP, Murphy M, Kurimasa A, Chen DJ: ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J Biol Chem. 2001, 276: 42462-42467. 10.1074/jbc.C100466200CrossRefPubMed

Ward IM, Chen J: Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J Biol Chem. 2001, 276: 47759-47762. 10.1074/jbc.M009785200CrossRefPubMed

10.

Park EJ, Chan DW, Park JH, Oettinger MA, Kwon J: DNA-PK is activated by nucleosomes and phosphorylates H2AX within the nucleosomes in an acetylation-dependent manner. Nucleic Acids Res. 2003, 31: 6819-6827. 10.1093/nar/gkg921PubMedCentralCrossRefPubMed

11.

Paull TT, Rogakou EP, Yamazaki V, Kirchgessner CU, Gellert M, Bonner WM: A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage. Curr Biol. 2000, 10: 886-895. 10.1016/S0960-9822(00)00610-2CrossRefPubMed

12.

Celeste A, Fernandez-Capetillo O, Kruhlak MJ, Pilch DR, Staudt DW, Lee A, Bonner RF, Bonner WM, Nussenzweig A: Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks. Nat Cell Biol. 2003, 5: 675-679. 10.1038/ncb1004CrossRefPubMed

13.

Stucki M, Clapperton JA, Mohammad D, Yaffe MB, Smerdon SJ, Jackson SP: MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell. 2005, 123: 1213-1226. 10.1016/j.cell.2005.09.038CrossRefPubMed

14.

Fernandez-Capetillo O, Mahadevaiah SK, Celeste A, Romanienko PJ, Camerini-Otero RD, Bonner WM, Manova K, Burgoyne P, Nussenzweig A: H2AX is required for chromatin remodeling and inactivation of sex chromosomes in male mouse meiosis. Dev Cell. 2003, 4: 497-508. 10.1016/S1534-5807(03)00093-5CrossRefPubMed

15.

Xie A, Puget N, Shim I, Odate S, Jarzyna I, Bassing CH, Alt FW, Scully R: Control of sister chromatid recombination by histone H2AX. Mol Cell. 2004, 16: 1017-1025. 10.1016/j.molcel.2004.12.007CrossRefPubMed

16.

Liu Y, Tseng M, Perdreau SA, Rossi F, Antonescu C, Besmer P, Fletcher JA, Duensing S, Duensing A: Histone H2AX is a mediator of gastrointestinal stromal tumor cell apoptosis following treatment with imatinib mesylate. Cancer Res. 2007, 67: 2685-2692. 10.1158/0008-5472.CAN-06-3497CrossRefPubMed

17.

Gunjan A, Verreault A: A Rad53 kinase-dependent surveillance mechanism that regulates histone protein levels in S. cerevisiae. Cell. 2003, 115: 537-549. 10.1016/S0092-8674(03)00896-1CrossRefPubMed

18.

Bonner WM, Wu RS, Panusz HT, Muneses C: Kinetics of accumulation and depletion of soluble newly synthesized histone in the reciprocal regulation of histone and DNA synthesis. Biochemistry. 1988, 27: 6542-6550. 10.1021/bi00417a052CrossRefPubMed

19.

Bonner WM, Mannironi C, Orr A, Pilch DR, Hatch CL: Histone H2A.X gene transcription is regulated differently than transcription of other replication-linked histone genes. Mol Cell Biol. 1993, 13: 984-992.PubMedCentralPubMed

20.

Siino JS, Nazarov IB, Svetlova MP, Solovjeva LV, Adamson RH, Zalenskaya IA, Yau PM, Bradbury EM, Tomilin NV: Photobleaching of GFP-labeled H2AX in chromatin: H2AX has low diffusional mobility in the nucleus. Biochem Biophys Res Commun. 2002, 297: 1318-1323. 10.1016/S0006-291X(02)02383-5CrossRefPubMed

21.

Steger DJ, Workman JL: Transcriptional analysis of purified histone acetyltransferase complexes. Methods. 1999, 19: 410-416. 10.1006/meth.1999.0877CrossRefPubMed

22.

Hahn S: Structure and mechanism of the RNA polymerase II transcription machinery. Nat Struct Mol Biol. 2004, 11: 394-403. 10.1038/nsmb763PubMedCentralCrossRefPubMed

23.

Jackson DA, Hassan AB, Errington RJ, Cook PR: Visualization of focal sites of transcription within human nuclei. Embo J. 1993, 12: 1059-1065.PubMedCentralPubMed

24.

Wansink DG, Schul W, van der Kraan I, van Steensel B, van Driel R, de Jong L: Fluorescent labeling of nascent RNA reveals transcription by RNA polymerase II in domains scattered throughout the nucleus. J Cell Biol. 1993, 122: 283-293. 10.1083/jcb.122.2.283CrossRefPubMed

25.

Janknecht R, Hunter T: Transcription. A growing coactivator network. Nature. 1996, 383: 22-23. 10.1038/383022a0CrossRefPubMed

26.

Lee KB, Wang D, Lippard SJ, Sharp PA: Transcription-coupled and DNA damage-dependent ubiquitination of RNA polymerase II in vitro. Proc Natl Acad Sci U S A. 2002, 99: 4239-4244. 10.1073/pnas.072068399PubMedCentralCrossRefPubMed

27.

von Mikecz A, Zhang S, Montminy M, Tan EM, Hemmerich P: CREB-binding protein (CBP)/p300 and RNA polymerase II colocalize in transcriptionally active domains in the nucleus. J Cell Biol. 2000, 150: 265-273. 10.1083/jcb.150.1.265PubMedCentralCrossRefPubMed

28.

Bassing CH, Suh H, Ferguson DO, Chua KF, Manis J, Eckersdorff M, Gleason M, Bronson R, Lee C, Alt FW: Histone H2AX: a dosage-dependent suppressor of oncogenic translocations and tumors. Cell. 2003, 114: 359-370. 10.1016/S0092-8674(03)00566-XCrossRefPubMed

29.

Ikura T, Tashiro S, Kakino A, Shima H, Jacob N, Amunugama R, Yoder K, Izumi S, Kuraoka I, Tanaka K, Kimura H, Ikura M, Nishikubo S, Ito T, Muto A, Miyagawa K, Takeda S, Fishel R, Igarashi K, Kamiya K: DNA damage-dependent acetylation and ubiquitination of H2AX enhances chromatin dynamics. Mol Cell Biol. 2007, 27: 7028-7040. 10.1128/MCB.00579-07PubMedCentralCrossRefPubMed

30.

Lu C, Zhu F, Cho YY, Tang F, Zykova T, Ma WY, Bode AM, Dong Z: Cell apoptosis: requirement of H2AX in DNA ladder formation, but not for the activation of caspase-3. Mol Cell. 2006, 23: 121-132. 10.1016/j.molcel.2006.05.023PubMedCentralCrossRefPubMed

31.

Wu D, Ingram A, Lahti JH, Mazza B, Grenet J, Kapoor A, Liu L, Kidd VJ, Tang D: Apoptotic release of histones from nucleosomes. J Biol Chem. 2002, 277: 12001-12008. 10.1074/jbc.M109219200CrossRefPubMed

32.

Bartkova J, Horejsi Z, Koed K, Kramer A, Tort F, Zieger K, Guldberg P, Sehested M, Nesland JM, Lukas C, Orntoft T, Lukas J, Bartek J: DNA damage response as a candidate anti-cancer barrier in early human tumorigenesis. Nature. 2005, 434: 864-870. 10.1038/nature03482CrossRefPubMed

33.

Ekholm-Reed S, Mendez J, Tedesco D, Zetterberg A, Stillman B, Reed SI: Deregulation of cyclin E in human cells interferes with prereplication complex assembly. J Cell Biol. 2004, 165: 789-800. 10.1083/jcb.200404092PubMedCentralCrossRefPubMed

34.

Mailand N, Bekker-Jensen S, Faustrup H, Melander F, Bartek J, Lukas C, Lukas J: RNF8 ubiquitylates histones at DNA double-strand breaks and promotes assembly of repair proteins. Cell. 2007, 131: 887-900. 10.1016/j.cell.2007.09.040CrossRefPubMed

35.

Wang B, Elledge SJ: Ubc13/Rnf8 ubiquitin ligases control foci formation of the Rap80/Abraxas/Brca1/Brcc36 complex in response to DNA damage. Proc Natl Acad Sci U S A. 2007, 104: 20759-20763. 10.1073/pnas.0710061104PubMedCentralCrossRefPubMed

36.

Kolas NK, Chapman JR, Nakada S, Ylanko J, Chahwan R, Sweeney FD, Panier S, Mendez M, Wildenhain J, Thomson TM, Pelletier L, Jackson SP, Durocher D: Orchestration of the DNA-damage response by the RNF8 ubiquitin ligase. Science. 2007, 318: 1637-1640. 10.1126/science.1150034PubMedCentralCrossRefPubMed

37.

Huen MS, Grant R, Manke I, Minn K, Yu X, Yaffe MB, Chen J: RNF8 transduces the DNA-damage signal via histone ubiquitylation and checkpoint protein assembly. Cell. 2007, 131: 901-914. 10.1016/j.cell.2007.09.041PubMedCentralCrossRefPubMed

38.

Zhao GY, Sonoda E, Barber LJ, Oka H, Murakawa Y, Yamada K, Ikura T, Wang X, Kobayashi M, Yamamoto K, Boulton SJ, Takeda S: A critical role for the ubiquitin-conjugating enzyme Ubc13 in initiating homologous recombination. Mol Cell. 2007, 25: 663-675. 10.1016/j.molcel.2007.01.029CrossRefPubMed

39.

Mendez J, Stillman B: 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. 2000, 20: 8602-8612. 10.1128/MCB.20.22.8602-8612.2000PubMedCentralCrossRefPubMed

Title: Soluble histone H2AX is induced by DNA replication stress and sensitizes cells to undergo apoptosis
Authors: Ying Liu
Joshua A Parry
Anna Chin
Stefan Duensing
Anette Duensing
Publication date: 01-12-2008
Publisher: BioMed Central
Published in: Molecular Cancer / Issue 1/2008
Electronic ISSN: 1476-4598
DOI: https://doi.org/10.1186/1476-4598-7-61

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