title: E2F1-dependent pathways are involved in amonafide analogue 7-d-induced DNA damage, G2/M arrest, and apoptosis in p53-deficient K562 cells.
abstract: The E2F1 gene well known is its pivotal role in regulating the entry from G1 to S phase, while the salvage antitumoral pathway which implicates it, especially in the absence of p53, is not fully characterized. We therefore attempted to identify the up- and down-stream events involved in the activation of the E2F1-dependent pro-apoptotic pathway. For this purpose, a amonafide analogue, 7-d (2-(3-(2-(Dimethylamino)ethylamino)propyl)-6-(dodecylamino)-1H-benzo[de]isoquinoline-1,3(2H)-dione) was screened, which exhibited high antitumor activity against p53-deficient human Chronic Myelogenous Leukemia (CML) K562 cells. Analysis of flow cytometry and western blots of K562 cells treated with 7-d revealed an appreciable G2/M cycle arrest and apoptosis in a dose and time-dependent manner via p53-independent pathway. A striking increase in "Comet tail" formation and γ-H2AX expression showed that DNA double strand breaks (DSB) were caused by 7-d treatment. ATM/ATR signaling was reported to connect E2F1 induction with apoptosis in response to DNA damage. Indeed, 7-d-induced G2/M arrest and apoptosis were antagonized by ATM/ATR signaling inhibitor, Caffeine, which suggested that ATM/ATR signaling was activated by 7-d treatment. Furthermore, the increased expression of E2F1, p73, and Apaf-1 and p73 dissociation from HDM2 was induced by 7-d treatment, however, knockout of E2F1 expression reversed p73, Apaf-1, and p21(Cip1/WAF1) expression, reactivated cell cycle progression, and inhibited 7-d-induced apoptosis. Altogether our results for the first time indicate that 7-d mediates its growth inhibitory effects on CML p53-deficient cells via the activation of an E2F1-dependent mitochondrial and cell cycle checkpoint signaling pathway which subsequently targets p73, Apaf-1, and p21(Cip1/WAF1).
authors.affiliation: State Key Laboratory of Bioreactor Engineering & Shanghai Key Laboratory of Chemical Biology, School of Pharmacy, East China University of Science and Technology, #268, 130 Meilong Road, Shanghai 200237, PR China.
authors.forename: Yiquan
authors.initials: Y
authors.lastname: Li
authors.forename: Jin
authors.initials: J
authors.lastname: Shao
authors.forename: Ke
authors.initials: K
authors.lastname: Shen
authors.forename: Yufang
authors.initials: Y
authors.lastname: Xu
authors.forename: Jianwen
authors.initials: J
authors.lastname: Liu
authors.forename: Xuhong
authors.initials: X
authors.lastname: Qian
mesh_terms: D000225:Adenine; D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1 / Q000235:genetics / Q000378:metabolism; D064007:Ataxia Telangiectasia Mutated Proteins; D015153:Blotting, Western; D002110:Caffeine / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D020552:Comet Assay; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D004305:Dose-Response Relationship, Drug; D050687:E2F1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D059565:G2 Phase Cell Cycle Checkpoints; D055786:Gene Knockout Techniques / Q000379:methods; D045325:HCT116 Cells; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D020014:K562 Cells; D059566:M Phase Cell Cycle Checkpoints; D061986:MCF-7 Cells; D053644:Naphthalimides / Q000494:pharmacology*; D063065:Organophosphonates; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051381:Rats; D015398:Signal Transduction; D013997:Time Factors; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: C577144:2-(3-(2-(dimethylamino)ethylamino)propyl)-6-(dodecylamino)-1H-benzo(de)isoquinoline-1,3(2H)-dione; C506592:APAF1 protein, human; D000970:Antineoplastic Agents; D053477:Apoptotic Protease-Activating Factor 1; C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D053644:Naphthalimides; D063065:Organophosphonates; D016159:Tumor Suppressor Protein p53; C037020:amonafide; D002110:Caffeine; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D000225:Adenine