melphalan and Cell-Transformation--Viral

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.

Topics: Active Transport, Cell Nucleus; Adaptor Proteins, Signal Transducing; Antineoplastic Agents, Alkylating; Apoptosis; Apoptosis Regulatory Proteins; B-Lymphocytes; bcl-2-Associated X Protein; Caspases; Cell Transformation, Viral; Cells, Cultured; DNA-Binding Proteins; Enzyme Activation; Gene Expression Regulation; Herpesvirus 4, Human; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; Melphalan; Mitochondria; Neoplasm Proteins; Nuclear Proteins; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Proline; Protein Interaction Mapping; Protein Isoforms; Protein Processing, Post-Translational; Protein Structure, Tertiary; Proto-Oncogene Proteins; Pyridines; Reactive Oxygen Species; RNA Interference; RNA, Small Interfering; Transcriptional Activation; Tumor Protein p73; Tumor Suppressor Proteins; Xeroderma Pigmentosum Group A Protein

Lymphoblastoid cell lines (LCLs) were established from 83 patients with ovarian cancer by transformation of peripheral B lymphocytes with Epstein-Barr virus. Comparing the melphalan resistance of different groups of LCLs using the mean Do obtained from clonogenic survival assays, LCLs from melphalan-treated patients were significantly more resistant than LCLs from patients not treated with this drug. However, prior treatment of the patient with ionizing radiation was not associated with increased in vitro resistance of the LCL to this agent. In melphalan-treated patients where LCLs were established serially, the melphalan Do increased after further melphalan treatment in vivo and decreased when no further treatment was given. No correlation was found between age of donor and LCL resistance to any of the above agents. A group of 15 LCLs previously established from non-tumour donors was less resistant to melphalan than the LCLs from patients with ovarian cancer. In a group of 29 patients with advanced disease in whom the clinical response was known, LCL resistance to melphalan appeared to be associated with poor clinical response to cross-linking agents. These results suggest that B cell populations undergo long term, but not necessarily permanent, increases in resistance to melphalan.

Topics: B-Lymphocytes; Cell Line; Cell Survival; Cell Transformation, Viral; Cells, Cultured; Chlorambucil; Cross-Linking Reagents; Dose-Response Relationship, Drug; Drug Resistance; Female; Herpesvirus 4, Human; Humans; Melphalan; Ovarian Neoplasms; Prognosis; Time Factors