title: Melphalan-induced apoptosis of EBV-transformed B cells through upregulation of TAp73 and XAF1 and nuclear import of XPA.
abstract: Melphalan (Mel) is widely used to treat patients with hematologic cancer, including multiple myeloma, but its mechanism of action in EBV-transformed B cells is poorly described. In this study, we demonstrate a novel mechanism by which transcriptionally active p73 (TAp73) induces translocation of X-linked inhibitor of apoptosis protein-associated factor 1 (XAF1) and xeroderma pigmentosum group A (XPA) during apoptosis caused by Mel treatment. We observed that Mel induced significant generation of reactive oxygen species (ROS) and subsequent apoptosis, as well as an early phosphorylation of p38 MAPK that preceded expression of the mitochondria membrane potential disruption-related molecules and the cleavage of caspases. In particular, Mel led to upregulation of TAp73, XAF1, and Puma and induced XPA nuclear import and translocation of Bax into mitochondria. Mel-induced apoptosis was inhibited by pretreatment with the ROS scavenger 4-amino-2,4-pyrrolidine-dicarboxylic acid (APDC) and the p38 MAPK inhibitor SB203580. We supposed that ROS generation might be the first event in Mel-induced apoptosis, because APDC blocked the increase in ROS, p38 MAPK, and TAp73, but SB203580 did not block ROS generation. Moreover, Mel elicited activation of ATR, and APDC inhibited phosphorylation of ATR but not SB203580. APDC and SB203580 completely blocked XPA and Bax translocation. We conclude that Mel promotes TAp73-mediated XAF1 and Puma expression via ROS generation and ATR/p38 MAPK pathway activation, thereby triggering apoptosis. Our results provide evidence of a novel alternate regulatory mechanism of TAp73 and reveal that Mel may be a therapeutic drug for curing EBV-related malignancies.
authors.affiliation: Department of Anatomy and Research Center for Tumor Immunology, Inje University College of Medicine, Busan 614-735, Republic of Korea;
authors.forename: Ga Bin
authors.initials: GB
authors.lastname: Park
authors.forename: Yeong-Seok
authors.initials: YS
authors.lastname: Kim
authors.forename: Daejin
authors.initials: D
authors.lastname: Kim
authors.forename: Seonghan
authors.initials: S
authors.lastname: Kim
authors.forename: Hyun-Kyung
authors.initials: HK
authors.lastname: Lee
authors.forename: Dae-Ho
authors.initials: DH
authors.lastname: Cho
authors.forename: Wang Jae
authors.initials: WJ
authors.lastname: Lee
authors.forename: Dae Young
authors.initials: DY
authors.lastname: Hur
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D048868:Adaptor Proteins, Signal Transducing; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis / Q000235:genetics; D001402:B-Lymphocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism / Q000821:virology; D020169:Caspases / Q000378:metabolism; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D004789:Enzyme Activation / Q000187:drug effects; D005786:Gene Expression Regulation / Q000187:drug effects; D004854:Herpesvirus 4, Human / Q000502:physiology*; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000235:genetics; D008558:Melphalan / Q000494:pharmacology; D008928:Mitochondria / Q000378:metabolism; D009363:Neoplasm Proteins / Q000096:biosynthesis* / Q000235:genetics; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D018384:Oxidative Stress; D010766:Phosphorylation / Q000187:drug effects; D011392:Proline / Q000031:analogs & derivatives / Q000494:pharmacology; D025941:Protein Interaction Mapping; D020033:Protein Isoforms / Q000502:physiology; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics; D011725:Pyridines / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D017382:Reactive Oxygen Species / Q000378:metabolism; D015533:Transcriptional Activation; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000502:physiology; D051760:Xeroderma Pigmentosum Group A Protein / Q000378:metabolism*; D051028:bcl-2-Associated X Protein / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: C097299:4-aminopyrrolidine-2,4-dicarboxylic acid; D048868:Adaptor Proteins, Signal Transducing; D018906:Antineoplastic Agents, Alkylating; D051017:Apoptosis Regulatory Proteins; C494826:BAX protein, human; C438378:BBC3 protein, human; D004268:DNA-Binding Proteins; D007093:Imidazoles; D047908:Intracellular Signaling Peptides and Proteins; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; D011725:Pyridines; D034741:RNA, Small Interfering; D017382:Reactive Oxygen Species; C000606460:TP73 protein, human; D000072160:Tumor Protein p73; D025521:Tumor Suppressor Proteins; C417551:XAF1 protein, human; C497314:XPA protein, human; D051760:Xeroderma Pigmentosum Group A Protein; D051028:bcl-2-Associated X Protein; D011392:Proline; D048051:p38 Mitogen-Activated Protein Kinases; D020169:Caspases; C093642:SB 203580; D008558:Melphalan