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Leptomycin B (leptomycin + b)
Selected AbstractsCo-treatment with deoxycholic acid and azoxymethane accelerates secretion of HMGB1 in IEC6 intestinal epithelial cellsCELL PROLIFERATION, Issue 5 2009K. Fujii Objectives:, High-mobility group box 1 (HMGB1) is a nuclear protein that acts as a ligand of the receptor for advanced glycation end products (RAGE) and its expression enhances progression of cancer. However, the mechanism underlying HMGB1 secretion is still unclear. In this study, we examined the effect of deoxycholic acid (DCA), a promoter of colon carcinogenesis, on HMGB1 secretion. Materials and Methods:, We used an in vitro transformation model comprised of IEC6 intestinal epithelial cells treated with azoxymethane (AOM) and/or DCA. HMGB1 expression and secretion were examined by Western and Northern blot analyses, and ELISA. Intracellular translocation of HMGB1 was examined by protein fractionation. Results:, AOM + DCA-treated IEC6 cells showed upregulation of HMGB1 mRNA expression and increased level of HMGB1 protein in culture medium, but decreased level of HMGB1 protein in the nucleus. AOM + DCA treatment increased level of histone H4 acetylation, which induced translocation of HMGB1 from the nucleus to the cytoplasm and increased HMGB1 secretion. Leptomycin B inhibited extranuclear translocation and secretion of the HMGB1 protein. Conclusion:, These findings suggest that DCA affects intracellular localization and secretion of HMGB1. [source] Determinants of the nucleocytoplasmic shuttling of muscle glycogen synthaseFEBS JOURNAL, Issue 12 2005Emili Cid Muscle glycogen synthase (MGS) presents a nuclear speckled pattern in primary cultured human muscle and in 3T3-L1 cells deprived of glucose and with depleted glycogen reserves. Nuclear accumulation of the enzyme correlates inversely with cellular glycogen content. Although the glucose-induced export of MGS from the nucleus to the cytoplasm is blocked by leptomycin B, and therefore mediated by CRM1, no nuclear export signal was identified in the sequence of the protein. Deletion analysis shows that the region comprising amino acids 555,633 of human MGS, which encompasses an Arg-rich cluster involved in the allosteric activation of the enzyme by Glc6P, is crucial for its nuclear concentration and aggregation. Mutation of these Arg residues, which desensitizes the enzyme towards Glc6P, interferes with its nuclear accumulation. In contrast, the known phosphorylation sites of MGS that regulate its activity are not involved in the control of its subcellular distribution. Nuclear human MGS colocalizes with the promyelocytic leukaemia oncoprotein and p80-coilin, a marker of Cajal bodies. The subnuclear distribution of MGS is altered by incubation with transcription inhibitors. These observations suggest that, in addition to its metabolic function, MGS may participate in nuclear processes. [source] Intracellular redistribution and modification of proteins of the Mre11/Rad50/Nbs1 DNA repair complex following irradiation and heat-shockJOURNAL OF CELLULAR PHYSIOLOGY, Issue 2 2004Joshua D. Seno Mre11, Rad50, and Nbs1form a tight complex which is homogeneously distributed throughout the nuclei of mammalian cells. However, after irradiation, the Mre11/Rad50/Nbs1 (M/R/N) complex rapidly migrates to sites of double strand breaks (DSBs), forming foci which remain until DSB repair is complete. Mre11 and Rad50 play direct roles in DSB repair, while Nbs1 appears to be involved in damage signaling. Hyperthermia sensitizes mammalian cells to ionizing radiation. Radiosensitization by heat shock is believed to be mediated by an inhibition of DSB repair. While the mechanism of inhibition of repair by heat shock remains to be elucidated, recent reports suggest that the M/R/N complex may be a target for inhibition of DSB repair and radiosensitization by heat. We now demonstrate that when human U-1 melanoma cells are heated at 42.5 or 45.5°C, Mre11, Rad50, and Nbs1 are rapidly translocated from the nucleus to the cytoplasm. Interestingly, when cells were exposed to ionizing radiation (12 Gy of X-rays) prior to heat treatment, the extent and kinetics of translocation were increased when nuclear and cytoplasmic fractions of protein were analyzed immediately after treatment. The kinetics of the translocation and subsequent relocalization back into the nucleus when cells were incubated at 37°C from 30 min to 7 h following treatment were different for each protein, which suggests that the proteins redistribute independently. However, a significant fraction of the translocated proteins exist as a triple complex in the cytoplasm. Treatment with leptomycin B (LMB) inhibits the translocation of Mre11, Rad50, and Nbs1 to the cytoplasm, leading us to speculate that the relocalization of the proteins to the cytoplasm occurs via CRM1-mediated nuclear export. In addition, while Nbs1 is rapidly phosphorylated in the nuclei of irradiated cells and is critical for a normal DNA damage response, we have found that Nbs1 is rapidly phosphorylated in the cytoplasm, but not in the nucleus, of heated irradiated cells. The phosphorylation of cytoplasmic Nbs1, which cannot be inhibited by wortmannin, appears to be a unique post-translational modification in heated, irradiated cells, and coupled with our novel observations that Mre11, Rad50, and Nbs1 translocate to the cytoplasm, lend further support for a role of the M/R/N complex in thermal radiosensitization and inhibition of DSB repair. J. Cell. Physiol. 199: 157,170, 2004© 2004 Wiley-Liss, Inc. [source] Nuclear accumulation of the ankyrin repeat protein ANK1 enhances the auxin-mediated transcription accomplished by the bZIP transcription factors BZI-1 and BZI-2THE PLANT JOURNAL, Issue 6 2009Stefan Böttner Summary The tobacco (Nicotiana tabacum) basic leucine zipper (bZIP) transcription factor BZI-1 has been implicated in auxin-mediated gene regulation. Yeast two-hybrid analysis has led to the identification of two BZI-1 protein interaction partners: the heterodimerizing bZIP factor BZI-2 and an ankyrin repeat domain protein, ANK1. Analysis in transgenic plants confirms that low levels of functional BZI-1, BZI-2 and ANK1 result in reduced auxin responses. This finding indicates that the three proteins act in the same functional context. The in vivo interaction of ANK1 and BZI-1 has been confirmed by protoplast two-hybrid analysis, as well as by bimolecular fluorescence complementation (BiFC) studies. Whereas YFP-BZI-1 has been found to be localized in the nucleus, YFP-ANK1 resides in the cytosol. Nevertheless, the inhibition of nuclear export with the inhibitor leptomycin B (LMB) and the co-expression with BZI-1, as well as treatment with auxin, results in the accumulation of YFP-ANK1 in the nucleus. Whereas BZI-1 is a weak activator, BZI-1/BZI-2 heterodimers efficiently support transcription. Importantly, conditions that lead to the accumulation of ANK1 in the nucleus, such as the expression of an ANK1 protein fused to a nuclear localization sequence (NLS) or auxin treatment, lead to a significant enhancement of BZI-1/BZI-2-mediated transcription. We therefore propose a mechanism in which the nuclear accumulation of ANK1 enhances BZI-1/BZI-2-mediated transcription in an auxin-dependent manner, presumably facilitated by protein,protein interaction. In summary, this study defines novel components in auxin-dependent signalling and transcriptional control. [source] Expression of the nuclear export protein chromosomal region maintenance/exportin 1/Xpo1 is a prognostic factor in human ovarian cancerCANCER, Issue 8 2008Aurelia Noske MD Abstract BACKGROUND The human nuclear export protein chromosomal region maintenance/exportin 1/Xpo1 (CRM1) mediates the nuclear export of proteins and messenger RNAs and, thus, is an important regulator of subcellular distribution of key molecules. Whereas cell-biologic studies have suggested a fundamental role for CRM1 in the regulation of mitosis, the expression of this protein in human tumor tissue has not been investigated to date. METHODS In this study, the expression of CRM1 was analyzed in a cohort of 88 ovarian tumors and 12 ovarian cell lines for the first time to the authors' knowledge. RESULTS Immunohistochemistry revealed increased nuclear (52.7%) and cytoplasmic (56.8%) expression of CRM1 in 74 carcinomas compared with the expression revealed in borderline tumors and benign lesions. Similarly, CRM1 expression was increased in ovarian cancer cell lines compared with human ovarian surface epithelial cells. Cytoplasmic CRM1 expression was related significantly to advanced tumor stage (P = .043), poorly differentiated carcinomas (P = .011), and higher mitotic rate (P = .008). Nuclear CRM1 was associated significantly with cyclooxygenase-2 (COX-2) expression (P = .002) and poor overall survival (P = .01). Because it was demonstrated previously that blocking of CRM1 by leptomycin B (LMB) contributes to the inhibition of nuclear export, the authors used a set of mechanistic assays to study the effects of CRM1 inhibition in cancer cells. Treatment of OVCAR-3 cells with LMB revealed a significant reduction of cell proliferation and increased apoptosis as well as suppressed interleukin-1,-induced COX-2 expression. CONCLUSIONS The current results indicated that CRM1 is expressed in a subpopulation of ovarian carcinomas with aggressive behavior and is related to poor patient outcome. A correlation also was demonstrated between CRM1 and COX-2 expression in ovarian cancer tissue. Furthermore, the treatment of ovarian cancer cells with LMB revealed a reduction in COX-2 expression. Therefore, the authors suggest that CRM1 may be an interesting biomarker for the assessment of patient prognosis and a molecular target for anticancer treatment. Cancer 2008. © 2008 American Cancer Society. [source] | ||||||||||