M2, a novel anthracenedione, elicits a potent DNA damage response that can be subverted through checkpoint kinase inhibition to generate mitotic catastrophe |
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Authors: | Evison Benny J Pastuovic Mile Bilardi Rebecca A Forrest Robert A Pumuye Paul P Sleebs Brad E Watson Keith G Phillips Don R Cutts Suzanne M |
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Institution: | aDepartment of Biochemistry, La Trobe University, Victoria 3086, Australia;bThe Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia |
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Abstract: | Pixantrone is a promising anti-cancer aza-anthracenedione that has prompted the development of new anthracenediones incorporating symmetrical side-chains of increasing length varying from two to five methylene units in each pair of drug side-chains. A striking relationship has emerged in which anthracenedione-induced growth inhibition and apoptosis was inversely associated with side-chain length, a relationship that was attributable to a differential ability to stabilise the topoisomerase II (TOP2) cleavage complex. Processing of the complex to a DNA double strand break (DSB) flanked by γH2AX in nuclear foci is likely to occur, as the generation of the primary lesion was antecedent to γH2AX induction. M2, bearing the shortest pair of side-chains, induced TOP2-mediated DSBs efficiently and activated cell cycle checkpoints via Chk1 and Chk2 phosphorylation, implicating the involvement of ATM and ATR, and induced a protracted S phase and subsequent G2/M arrest. The inactive analogue M5, containing the longest pair of side-chains, only weakly stimulated any of these responses, suggesting that efficient stabilisation of the TOP2 cleavage complex was crucial for eliciting a strong DNA damage response (DDR). An M2 induced DDR in p53-defective MDA-MB-231 cells was abrogated by UCN-01, a ubiquitous inhibitor of kinases including Chk1, in a response associated with substantial mitotic catastrophe and strong synergy. The rational selection of checkpoint kinase inhibitors may significantly enhance the therapeutic benefit of anthracenediones that efficiently stabilise the TOP2 cleavage complex. |
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Keywords: | Abbreviations: AN-9 pivaloyloxymethyl butyrate ATM ataxia telangiectasia mutated ATR ataxia telangiectasia mutated-related CC closed Chk1 checkpoint kinase 1 Chk2 checkpoint kinase 2 CI combination index DDR DNA damage response DNA-PK DNA protein kinase DSB double strand break γH2AX phosphorylated H2AX IC50 50% inhibitory concentration Kapp apparent binding constant kDNA kinetoplast DNA MTT 3-(4 5-dimethyl-2-thiazolyl)-2 5-diphenyl-2H-tetrazolium bromide MX mitoxantrone NAC N-acetyl-cysteine NC decatenated nicked OTM olive tail moment PIX pixantrone PMSF phenylmethylsulfonyl fluoride ROS reactive oxygen species Sur suramin TOP2 topoisomerase II |
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