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Cellular responses to etoposide: cell death despite cell cycle arrest and repair of DNA damage

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Abstract

The topoisomerase IIα inhibitor etoposide is a ‘broad spectrum’ anticancer agent and a potent inducer of DNA double strand breaks. DNA damage response of mammalian cells usually involves cell cycle arrest and DNA repair or, if unsuccessful, cell death. We investigated these processes in the human colon cancer cell line HT-29 treated with three different etoposide regimens mimicking clinically relevant plasma concentrations of cancer patients. Each involved a period of drug-free incubation following etoposide exposure to imitate the decline of plasma levels between the cycles of chemotherapy. We found a massive induction of double strand breaks that were rapidly and nearly completely fixed long before the majority of cells underwent apoptosis or necrosis. An even greater percentage of cells lost clonogenicity. The occurrence of double strand breaks was accompanied by a decrease in the levels of Ku70, Ku86 and DNA-PKcs as well as an increase in the level of Rad51 protein. Twenty-four hours after the first contact with etoposide we found a pronounced G2/M arrest, regardless of the duration of drug exposure, the level of double strand breaks and the extent of their repair. During the subsequent drug-free incubation period, the loss of clonogenicity correlated well with the preceding G2/M arrest as well as with the amount of cell death found several days after exposure. However, it correlated neither with early apoptosis or necrosis nor with any of the other investigated parameters. These results suggest that the G2/M arrest is an important determinant in the cytostatic action of etoposide and that the removal of DNA double strand breaks is not sufficient to ensure cell survival.

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References

  1. Walker JV, Nitiss JL (2002) DNA topoisomerase II as a target for cancer chemotherapy. Cancer Invest 20:570–589

    Article  PubMed  CAS  Google Scholar 

  2. Gieseler F, Bauer E, Nuessler V, Clark M, Valsamas S (1999) Molecular effects of topoisomerase II inhibitors in AML cell lines: correlation of apoptosis with topoisomerase II activity but not with DNA damage. Leukemia 13:1859–1863

    Article  PubMed  CAS  Google Scholar 

  3. Wang Y, Zhou R, Liliemark J, Gruber A, Lindemalm S, Albertioni F, Liliemark E (2001) In vitro topo II–DNA complex accumulation and cytotoxicity of etoposide in leukaemic cells from patients with acute myelogenous and chronic lymphocytic leukaemia. Leuk Res 25:133–140. doi:10.1016/S0145-2126(00)00103-X

    Article  PubMed  CAS  Google Scholar 

  4. Baguley BC, Ferguson LR (1998) Mutagenic properties of topoisomerase-targeted drugs. Biochim Biophys Acta 1400:213–222. doi:10.1016/S0167-4781(98)00137-7

    PubMed  CAS  Google Scholar 

  5. Felix CA (2001) Leukemias related to treatment with DNA topoisomerase II inhibitors. Med Pediatr Oncol 36:525–535

    Article  PubMed  CAS  Google Scholar 

  6. Felix CA, Kolaris CP, Osheroff N (2006) Topoisomerase II and the etiology of chromosomal translocations. DNA Repair (Amst) 5:1093–1108. doi:10.1016/j.dnarep.2006.05.031

    Article  CAS  Google Scholar 

  7. Mistry AR, Felix CA, Whitmarsh RJ, Mason A, Reiter A, Cassinat B, Parry A, Walz C, Wiemels JL, Segal MR, Ades L, Blair IA, Osheroff N, Peniket AJ, Lafage-Pochitaloff M, Cross NC, Chomienne C, Solomon E, Fenaux P, Grimwade D (2005) DNA topoisomerase II in therapy-related acute promyelocytic leukemia. N Engl J Med 352:1529–1538

    Article  PubMed  CAS  Google Scholar 

  8. Ohshima A, Miura I, Chubachi A, Hashimoto K, Nimura T, Utsumi S, Takahashi N, Hayashi Y, Seto M, Ueda R, Miura AB (1996) 11q23 aberration is an additional chromosomal change in de novo acute leukemia after treatment with etoposide and mitoxantrone. Am J Hematol 53:264–266

    Article  PubMed  CAS  Google Scholar 

  9. Sandoval C, Pui CH, Bowman LC, Heaton D, Hurwitz CA, Raimondi SC, Behm FG, Head DR (1993) Secondary acute myeloid leukemia in children previously treated with alkylating agents, intercalating topoisomerase II inhibitors, and irradiation. J Clin Oncol 11:1039–1045

    PubMed  CAS  Google Scholar 

  10. Schiavetti A, Varrasso G, Maurizi P, Castello MA (2001) Two secondary leukemias among 15 children given oral etoposide. Med Pediatr Oncol 37:148–149

    Article  PubMed  CAS  Google Scholar 

  11. Zhang Y, Rowley JD (2006) Chromatin structural elements and chromosomal translocations in leukemia. DNA Repair (Amst) 5:1282–1297. doi:10.1016/j.dnarep.2006.05.020

    Article  CAS  Google Scholar 

  12. Liu LF, Rowe TC, Yang L, Tewey KM, Chen GL (1983) Cleavage of DNA by mammalian DNA topoisomerase II. J Biol Chem 258:15365–15370

    PubMed  CAS  Google Scholar 

  13. Tanaka T, Halicka HD, Traganos F, Seiter K, Darzynkiewicz Z (2007) Induction of ATM activation, histone H2AX phosphorylation and apoptosis by etoposide: relation to cell cycle phase. Cell Cycle 6:371–376

    PubMed  CAS  Google Scholar 

  14. Dartsch DC, Gieseler F (2007) Repair of idarubicin-induced DNA damage: a cause of resistance? DNA Repair (Amst) 6:1618–1628. doi:10.1016/j.dnarep.2007.05.007

    Article  CAS  Google Scholar 

  15. Fan JR, Peng AL, Chen HC, Lo SC, Huang TH, Li TK (2008) Cellular processing pathways contribute to the activation of etoposide-induced DNA damage responses. DNA Repair (Amst) 7:452–463. doi:10.1016/j.dnarep.2007.12.002

    Article  CAS  Google Scholar 

  16. Agner J, Falck J, Lukas J, Bartek J (2005) Differential impact of diverse anticancer chemotherapeutics on the Cdc25A-degradation checkpoint pathway. Exp Cell Res 302:162–169. doi:10.1016/j.yexcr.2004.08.035

    Article  PubMed  CAS  Google Scholar 

  17. Hartlerode AJ, Scully R (2009) Mechanisms of double-strand break repair in somatic mammalian cells. Biochem J 423:157–168. doi:10.1042/BJ20090942

    Article  PubMed  CAS  Google Scholar 

  18. Pardo B, Gomez-Gonzalez B, Aguilera A (2009) DNA repair in mammalian cells: DNA double-strand break repair: how to fix a broken relationship. Cell Mol Life Sci 66:1039–1056. doi:10.1007/s00018-009-8740-3

    Article  PubMed  CAS  Google Scholar 

  19. Shrivastav M, De Haro LP, Nickoloff JA (2008) Regulation of DNA double-strand break repair pathway choice. Cell Res 18:134–147. doi:10.1038/cr.2007.111

    Article  PubMed  CAS  Google Scholar 

  20. Sonoda E, Hochegger H, Saberi A, Taniguchi Y, Takeda S (2006) Differential usage of non-homologous end-joining and homologous recombination in double strand break repair. DNA Repair (Amst) 5:1021–1029. doi:10.1016/j.dnarep.2006.05.022

    Article  CAS  Google Scholar 

  21. Flygare J, Benson F, Hellgren D (1996) Expression of the human RAD51 gene during the cell cycle in primary human peripheral blood lymphocytes. Biochim Biophys Acta 1312:231–236

    Article  PubMed  Google Scholar 

  22. Mao Z, Bozzella M, Seluanov A, Gorbunova V (2008) DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells. Cell Cycle 7:2902–2906

    PubMed  CAS  Google Scholar 

  23. Daley JM, Wilson TE (2005) Rejoining of DNA double-strand breaks as a function of overhang length. Mol Cell Biol 25:896–906. doi:10.1128/MCB.25.3.896-906.2005

    Article  PubMed  CAS  Google Scholar 

  24. Hansen L, Lundin C, Spang-Thomsen M, Petersen L, Helleday T (2003) The role of Rad51 in etoposide (VP-16) resistance in small cell lung cancer. Int J Cancer 105:472–479. doi:10.1002/ijc.11106

    Article  PubMed  CAS  Google Scholar 

  25. Iliakis G, Wang H, Perrault AR, Boecker W, Rosidi B, Windhofer F, Wu W, Guan J, Terzoudi G, Pantelias G (2004) Mechanisms of DNA double strand break repair and chromosome aberration formation. Cytogenet Genome Res 104:14–20. doi:10.1159/000077461

    Article  PubMed  CAS  Google Scholar 

  26. Wang H, Zeng Z-C, Bui T, Sonoda E, Takata M, Takeda S, Iliakis G (2001) Efficient rejoining of radiation-induced DNA double-strand breaks in vertebrate cells deficient in genes of the RAD52 epistasis group. Oncogene 20:2212–2224

    Article  PubMed  CAS  Google Scholar 

  27. Fiorenza MT, Bevilacqua A, Bevilacqua S, Mangia F (2001) Growing dictyate oocytes, but not early preimplantation embryos, of the mouse display high levels of DNA homologous recombination by single-strand annealing and lack DNA nonhomologous end joining. Dev Biol 233:214–224. doi:10.1006/dbio.2001.0199

    Article  PubMed  CAS  Google Scholar 

  28. Goedecke W, Eijpe M, Offenberg HH, van Aalderen M, Heyting C (1999) Mre11 and Ku70 interact in somatic cells, but are differentially expressed in early meiosis. Nat Genet 23:194–198. doi:10.1038/13821

    Article  PubMed  CAS  Google Scholar 

  29. Hamer G, Roepers-Gajadien HL, van Duyn-Goedhart A, Gademan IS, Kal HB, van Buul PP, Ashley T, de Rooij DG (2003) Function of DNA-protein kinase catalytic subunit during the early meiotic prophase without Ku70 and Ku86. Biol Reprod 68:717–721. doi:10.1095/biolreprod.102.008920

    Article  PubMed  CAS  Google Scholar 

  30. Hopfner K, Putnam C, Tainer J (2002) DNA double-strand break repair from head to tail. Curr Opin Struct Biol 12:115–122. doi:10.1016/S0959-440X(02)00297-X

    Article  PubMed  CAS  Google Scholar 

  31. Khanna K, Jackson S (2001) DNA double-strand breaks: signaling, repair and the cancer connection. Nat Genet 27:247–254. doi:10.1038/85798

    Article  PubMed  CAS  Google Scholar 

  32. Van Dyck E, Stasiak A, Stasiak A, West S (1999) Binding of double strand breaks in DNA by human Rad52 protein. Nature 398:728–731. doi:10.1038/19560

    Article  PubMed  Google Scholar 

  33. Heyer W-D, Li X, Rolfsmeier M, Zhang X-P (2006) Rad54: the Swiss army knife of homologous recombination? Nucl Acid Res 34:4115–4125. doi:10.1093/nar/gkl481

    Article  CAS  Google Scholar 

  34. Morin PJ, Vogelstein B, Kinzler KW (1996) Apoptosis and APC in colorectal tumorigenesis. Proc Natl Acad Sci USA 93:7950–7954

    Article  PubMed  CAS  Google Scholar 

  35. Aydiner A, Koyuncu H, Tas F, Topuz E, Disci R (2000) Crossover clinical study comparing the pharmacokinetics of etoposide (75 mg) administered as 25-mg capsules three times a day versus once a day. Int J Clin Pharm Res 20:21–30

    CAS  Google Scholar 

  36. Eksborg S, Strandler H-S, Edsmyr F, Näslund I, Tahvanainen P (1985) Pharmacokinetic study of IV infusions of adriamycin. Eur J Clin Pharmacol 28:205–212

    Article  PubMed  CAS  Google Scholar 

  37. Greene R, Collins J, Jenkins J, Speyer J, Myers C (1983) Plasma pharmacokinetics of adriamycin and adriamycinol: implications for the design of in vitro experiments and treatment protocols. Cancer Res 43:3417–3421

    PubMed  CAS  Google Scholar 

  38. Holthuis J, Postmus P, Van Oort W, Hulshoff B, Verleun H, Sleijfer D, Mulder N (1986) Pharmacokinetics of high dose etoposide (VP 16–213). Eur J Cancer Clin Oncol 22:1149–1155

    Article  PubMed  CAS  Google Scholar 

  39. Mross K, Mayer U, Hamm K, Burk K, Hossfeld D (1990) Pharmacokinetics and metabolism of iodo-doxorubicin and doxorubicin in humans. Eur J Clin Pharmacol 39:507–513

    Article  PubMed  CAS  Google Scholar 

  40. van der Gaast A, Vlastuin M, Kok T, Splinter T (1992) What is the optimal dose and duration of treatment with etoposide ? II. Comparative pharmacokinetic study of three schedules: 1 × 100 mg, 2 × 50 mg, and 4 × 25 mg of oral etoposide daily for 21 days. Semin Oncol 19:8–12

    PubMed  Google Scholar 

  41. Zhu G, Gilchrist R, Borley N, Chng H, Morgan M, Marshall J, Camplejohn R, Muir G, Hart I (2004) Reduction of TSG101 protein has a negative impact on tumor cell growth. Int J Cancer 109:541–547. doi:10.1002/ijc.20014

    Article  PubMed  CAS  Google Scholar 

  42. Dartsch D, Schaefer A, Boldt S, Kolch W, Marquardt H (2002) Comparison of anthracycline-induced death of human leukemia cells: programmed cell death versus necrosis. Apoptosis 7:537–548. doi:10.1023/A:1020647211557

    Article  PubMed  CAS  Google Scholar 

  43. Singh N, McCoy M, Tice R, Schneider E (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191. doi:10.1016/0014-4827(88)90265-0

    Article  PubMed  CAS  Google Scholar 

  44. Collins AR, Oscoz AA, Brunborg G, Gaivao I, Giovannelli L, Kruszewski M, Smith CC, Stetina R (2008) The comet assay: topical issues. Mutagenesis 23:143–151. doi:10.1093/mutage/gem051

    Article  PubMed  CAS  Google Scholar 

  45. Baldwin EL, Osheroff N (2005) Etoposide, topoisomerase II and cancer. Curr Med Chem Anticancer Agents 5:363–372

    Article  PubMed  CAS  Google Scholar 

  46. Chen G, Yang L, Rowe T, Halligan B, Tewey K, Liu L (1984) Nonintercalative antitumor drugs interfere with the breakage reunion reaction of mammalian DNA topoisomerase II. J Biol Chem 259(21):13560–13566

    PubMed  CAS  Google Scholar 

  47. Hande K (1998) Etoposide: four decades of development of a topoisomerase II inhibitor. Eur J Cancer 34:1514–1521. doi:10.1016/S0959-8049(98)00228-7

    Article  PubMed  CAS  Google Scholar 

  48. Brown JM, Wilson G (2003) Apoptosis genes and resistance to cancer therapy: what does the experimental and clinical data tell us? Cancer Biol Ther 2:477–490

    PubMed  CAS  Google Scholar 

  49. Brown JM, Wouters BG (1999) Apoptosis, p53, and tumor cell sensitivity to anticancer agents. Cancer Res 59:1391–1399

    PubMed  CAS  Google Scholar 

  50. Lo Nigro C, Arnolfo E, Taricco E, Fruttero A, Russi EG, Lucio F, Ribero S, Comino A, Merlano M, Ungari S (2007) The cisplatin-irradiation combination suggests that apoptosis is not a major determinant of clonogenic death. Anticancer Drugs 18:659–667. doi:10.1097/CAD.0b013e328087388f

    Article  PubMed  CAS  Google Scholar 

  51. Russell J, Ling CC (2003) Studies with cytotoxic agents suggest that apoptosis is not a major determinant of clonogenic death in neuroblastoma cells. Eur J Cancer 39:2234–2238. doi:10.1016/S0959-8049(03)00488-X

    Article  PubMed  CAS  Google Scholar 

  52. Bertrand R, Sarang M, Jenkin J, Kerrigan D, Pommier Y (1991) Differential induction of secondary DNA fragmentation by topoisomerase II inhibitors in human tumor cell lines with amplified c-myc expression. Cancer Res 51:6280–6285

    PubMed  CAS  Google Scholar 

  53. Gieseler F, Nussler V, Brieden T, Kunze J, Valsamas S (1998) Intracellular pharmacokinetics of anthracyclines in human leukemia cells: correlation of DNA-binding with apoptotic cell death. Int J Clin Pharmacol Ther 36:25–28

    PubMed  CAS  Google Scholar 

  54. Li TK, Liu LF (2001) Tumor cell death induced by topoisomerase-targeting drugs. Annu Rev Pharmacol Toxicol 41:53–77. doi:10.1146/annurev.pharmtox.41.1.53

    Article  PubMed  Google Scholar 

  55. Long BH, Musial ST, Brattain MG (1985) Single- and double-strand DNA breakage and repair in human lung adenocarcinoma cells exposed to etoposide and teniposide. Cancer Res 45:3106–3112

    PubMed  CAS  Google Scholar 

  56. Chatterjee S, Trivedi D, Petzold S, Berger N (1990) Mechanism of epipodophyllotoxin-induced cell death in poly(adenosine diphosphate-ribose) synthesis-deficient V79 Chinese hamster cell lines. Cancer Res 50:2713–2718

    PubMed  CAS  Google Scholar 

  57. Sung PA, Libura J, Richardson C (2006) Etoposide and illegitimate DNA double-strand break repair in the generation of MLL translocations: new insights and new questions. DNA Repair (Amst) 5:1109–1118. doi:10.1016/j.dnarep.2006.05.018

    Article  CAS  Google Scholar 

  58. Chuang SM, Wang LH, Hong JH, Lin YW (2008) Induction of Rad51 protein levels by p38 MAPK decreases cytotoxicity and mutagenicity in benzo[a]pyrene-exposed human lung cancer cells. Toxicol Appl Pharmacol 230:290–297. doi:10.1016/j.taap.2008.03.001

    Article  PubMed  CAS  Google Scholar 

  59. Averbeck D, Averbeck S (1998) DNA photodamage, repair, gene induction and genotoxicity following exposures to 254 nm UV and 8-methoxypsoralen plus UVA in a eukaryotic cell system. Photochem Photobiol 68:289–295. doi:10.1111/j.1751-1097.1998.tb09683.x

    Article  PubMed  CAS  Google Scholar 

  60. Cohen Y, Dardalhon M, Averbeck D (2002) Homologous recombination is essential for RAD51 up-regulation in Saccharomyces cerevisiae following DNA crosslinking damage. Nucleic Acids Res 30:1224–1232

    Article  PubMed  CAS  Google Scholar 

  61. Klein HL (2008) The consequences of Rad51 overexpression for normal and tumor cells. DNA Repair (Amst) 7:686–693. doi:10.1016/j.dnarep.2007.12.008

    Article  CAS  Google Scholar 

  62. Karran P (2000) DNA double strand break repair in mammalian cells. Curr Opin Genet Dev 10:144–150. doi:10.1016/S0959-437X(00)00069-1

    Article  PubMed  CAS  Google Scholar 

  63. Han Z, Malik N, Carter T, Reeves WH, Wyche JH, Hendrickson EA (1996) DNA-dependent protein kinase is a target for a CPP32-like apoptotic protease. J Biol Chem 271:25035–25040. doi:10.1074/jbc.271.40.25035

    Article  PubMed  CAS  Google Scholar 

  64. Kim S, Kim D, Han J, Jeong C, Chung B, Kang C, Li G (1999) Ku autoantigen affects the susceptibility to anticancer drugs. Cancer Res 59:4012–4017

    PubMed  CAS  Google Scholar 

  65. Smith G, Jackson S (1999) The DNA-dependent protein kinase. Genes Dev 13:916–934

    Article  PubMed  CAS  Google Scholar 

  66. Song Q, Lees-Miller SP, Kumar S, Zhang Z, Chan DW, Smith GC, Jackson SP, Alnemri ES, Litwack G, Khanna KK, Lavin MF (1996) DNA-dependent protein kinase catalytic subunit: a target for an ICE-like protease in apoptosis. EMBO J 15:3238–3246

    PubMed  CAS  Google Scholar 

  67. Zhu P, Zhang D, Chowdhury D, Martinvalet D, Keefe D, Shi L, Lieberman J (2006) Granzyme A, which causes single-stranded DNA damage, targets the double-strand break repair protein Ku70. EMBO Rep 7:431–437. doi:10.1038/sj.embor.7400622

    Article  PubMed  CAS  Google Scholar 

  68. Gama V, Yoshida T, Gomez JA, Basile DP, Mayo LD, Haas AL, Matsuyama S (2006) Involvement of the ubiquitin pathway in decreasing Ku70 levels in response to drug-induced apoptosis. Exp Cell Res 312:488–499. doi:10.1016/j.yexcr.2005.11.016

    Article  PubMed  CAS  Google Scholar 

  69. Lee SE, Moore JK, Holmes A, Umezu K, Kolodner RD, Haber JE (1998) Saccharomyces Ku70, mre11/rad50 and RPA proteins regulate adaptation to G2/M arrest after DNA damage. Cell 94:399–409. doi:10.1016/S0092-8674(00)81482-8

    Article  PubMed  CAS  Google Scholar 

  70. Zhang Y, Hefferin ML, Chen L, Shim EY, Tseng HM, Kwon Y, Sung P, Lee SE, Tomkinson AE (2007) Role of Dnl4-Lif1 in nonhomologous end-joining repair complex assembly and suppression of homologous recombination. Nat Struct Mol Biol 14:639–646. doi:10.1038/nsmb1261

    Article  PubMed  CAS  Google Scholar 

  71. Frank-Vaillant M, Marcand S (2002) Transient stability of DNA ends allows nonhomologous end joining to precede homologous recombination. Mol Cell 10:1189–1199. doi:10.1016/S1097-2765(02)00705-0

    Article  PubMed  CAS  Google Scholar 

  72. Ira G, Pellicioli A, Balijja A, Wang X, Fiorani S, Carotenuto W, Liberi G, Bressan D, Wan L, Hollingsworth NM, Haber JE, Foiani M (2004) DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1. Nature 431:1011–1017. doi:10.1038/nature02964

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

We would like to thank Prof. Dr. Frank Gieseler from the University Hospital Luebeck for many helpful discussions. Our work was supported by the Ernst und Elfriede Griebel’s Foerderungs- und Unterstuetzungsstiftung, Hamburg. This paper is written in memory of Michael R. Clark.

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The authors declare that they have no conflict of interest.

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  1. Institute of Pharmacy, Hamburg University, Bundesstr. 45, 20146, Hamburg, Germany

    Ilona Schonn, Jana Hennesen & Dorothee C. Dartsch

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  1. Ilona Schonn
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Schonn, I., Hennesen, J. & Dartsch, D.C. Cellular responses to etoposide: cell death despite cell cycle arrest and repair of DNA damage. Apoptosis 15, 162–172 (2010). https://doi.org/10.1007/s10495-009-0440-9

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