The nuclear transporter exportin-1 (XPO1) is highly expressed in mantle cell

The nuclear transporter exportin-1 (XPO1) is highly expressed in mantle cell lymphoma (MCL) cells, and is believed to be associated with the pathogenesis of this disease. to influence patient survival in MCL, and the SINE XPO1 antagonist KPT-185 effectively activates p53-mediated transcription and apoptosis, which would provide a novel strategy for the therapy of MCL. mutations occur in 15C20% of the cases of MCL,(13) and wild-type p53 is usually inactivated by upstream gene amplification of (10%), homozygous deletion of (15C20%), the overexpression of human homolog of murine double minute 2 (MDM2) (5%), or gene deletion (25C30%).(14C16) All of these abnormalities essentially lead to the loss of p53 tumor suppressor activity. The nuclear export of p53 is usually cooperatively mediated by MDM2 and XPO1.(17) MDM2 activates the nuclear export transmission (NES) in p53 through its E3 ubiquitin ligase activity, leading a conformational switch in p53 that exposes p53’s NES domain name. Following ubiquitination, XPO1 Dovitinib Dilactic acid recognizes p53’s NES and exports the protein from the nucleus to the cytoplasm, where it is usually unable to execute transcriptional activity to regulate cell fate. As we mentioned previously, XPO1 is usually highly expressed in MCL cells,(8) which may limit p53-mediated transcriptional activity, and hence the ability of p53 to trigger apoptosis.(18) It has been reported that wild-type p53 is usually abnormally sequestered in the cytoplasm in certain human tumor cells.(19,20) Novel small-molecule, drug-like, potent, and covalent XPO1-selective inhibitors of nuclear export (SINE) compounds were recently designed. These compounds selectively hole to Dovitinib Dilactic acid the Cys528 of XPO1, thereby inhibiting XPO1 binding to the NES domains of its valuables protein.(21) The SINE KPT-185 has been shown to induce apoptosis in MCL cells.(8) The inhibition of XPO1 is believed to maintain the nuclear localization, and hence function, of p53.(1C3) Furthermore, XPO1 is involved in the nuclear export of numerous proteins including p21, p27, p73, nucleophosmin-1, PP2A, FOXO, -catenin/APC, topoisomerase II, and IB.(1) This would suggest that the biological significance of p53 activation in XPO1 inhibition-induced apoptosis in MCL cells is highly unspecified and thus in need of further elucidation. Accordingly, we examined the pathophysiological significance of XPO1’s influence on p53 cellular localization and functional activity and its potential as a therapeutic target for enhancing MCL cell apoptosis. Materials and Methods Reagents The selective XPO1 inhibitor KPT-185 was Dovitinib Dilactic acid synthesized and provided by Karyopharm (Karyopharm, Natick, MA, USA). The selective small-molecule antagonist of MDM2, Nutlin-3a was purchased from Cayman Chemical Organization (Ann Arbor, MI, USA). Cells and cell culture A total of 16 lymphoid cell lines, including six MCL cell lines, were cultured in RPMI-1640 medium made up of 20% heat-inactivated FBS (Table ?(Table1).1). Z-138 and JVM-2 have wild-type p53, whereas MINO, JeKo-1, MAVER-1, and NCEB-1 have defective ( the., missense mutated or deleted) p53.(22) The Z-138 and JVM-2 cells were transduced with LAMC2 retroviruses encoding either p53-specific shRNA (nucleotides 611C629, Genbank NM000546) or scrambled shRNA and stable shRNA-expressing cells were generated.(23) The cell were harvested in log-phase growth, seeded at a density of 2 105 cells/mL and uncovered to the indicated compounds. Table 1 Effective doses of KPT-185 and Nutlin-3a for inducing 50% killing in lymphoid cells, as assessed by annexin V positivity, comparative to the cell lines’ p53 mutational status E-TCL1 transgenic mice (kindly provided by Dr. Carlo M. Croce, Ohio State University or college, Columbus, Oh yea, USA) with different p53 experience (TCL1-Tg:p53WT/WT or TCL1-Tg:p53R172H/R172H) were generated. E-TCL1 transgenic mice spontaneously develop CD19+/CD5+/CD23+/? B-lineage lymphoid malignancies, and have been used as a mouse model for chronic lymphocytic leukemia that typically shows CD19+/CD5+/CD23+ immunophenotype.(24) The immunological profile of MCL cells is usually defined by CD19+/CD5+/CD23?; p53R172H is usually comparative to human p53R175H, one of the hotspot mutations in human cancers. B-lymphoma cells were collected from affected spleens, cultured in RPMI-1640 medium made up of 20% FBS at 2 106 cells/mL, and were uncovered to the indicated compounds. Heparinized peripheral blood and pleural effusion samples were.