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Blood, 15 May 2008, Vol. 111, No. 10, pp. 5258-5259. This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Kaluz, S. Articles by Stanbridge, E. J. Search for Related Content PubMed PubMed Citation Articles by Kaluz, S. Articles by Stanbridge, E. J. Related Collections Related Articles in Blood Online Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  CORRESPONDENCE Comment on the role of FIH in the inhibitory effect of bortezomib on hypoxia-inducible factor-1 To the editor: Recently, Shin et al reported that the proteasomal inhibitor bortezomib inhibits the hypoxic response by stimulating the factor-inhibiting HIF-1 (FIH)–mediated repression of hypoxia-inducible factor-1 (HIF-1).1 The essence of this study is that bortezomib enhances the FIH function and the repression is dependent on asparagine 803 (N803). Both conclusions are in contradiction to previously published data,2,3 Shin et al suggested that this contradiction could be the consequence of a cell-type specific effect and/or the higher concentrations of bortezomib used in these previous studies. We tested the effect of various concentrations of bortezomib on the activity of the wild type and N803A mutant Gal4– HIF-1 C-terminal activation domain (CAD) in various cell lines. No inhibitory effect was observed at subnanomolar concentrations in any of 4 cell lines tested, including the HEK 293 cell line used in the Shin et al studies (data not shown). More importantly, we found that in all 4 cell lines activities of the wild type and the N803A mutant HIF-1 CAD constructs were strictly coregulated by bortezomib (Figure 1). Both constructs were invariably inhibited by higher concentrations ( 10 nM), whereas 1 nM had a moderate cell type specific effect: inhibition (HEK 293 and M006) and activation (JEG and MCF-7; Figure 1). View larger version (28K): [in this window] [in a new window]   Figure 1. Dose-dependent effect of bortezomib on activity of the wild-type and N803A mutant HIF-1 CAD in various cell lines. The Gal4– HIF-1 CAD constructs were cotransfected with a Gal4 reporter plasmid pFR-Luc (Stratagene, La Jolla, CA) using Effectene transfection reagent (QIAGEN, Valencia, CA). After exposure to the transfection mixture for 16 hours, the cells were trypsinized, replated, allowed to adhere for 5 hours, pretreated with bortezomib for 30 minutes, and exposed to normoxia or 0.5% O2 hypoxia (in the presence of bortezomib) for 24 hours. Activities are expressed as the ratio of luciferase activity/protein concentration in arbitrary units (AU) and each of the bars represents the mean value (± SD) from 3 independent experiments.   Mechanistically, Shin et al concluded that the inhibitory effect of bortezomib was due to stimulation of the physical interaction between FIH and HIF-1 CAD. However, bortezomib treatment had no effect on cellular localization of HIF-1 (mainly nuclear) and FIH (cytoplasmic).1 In our opinion, transiently enhanced interaction between 2 proteins that eventually end up in different cellular compartments cannot explain the potent inhibitory effect. Should FIH be implicated, before being released, HIF-1 would have to be inactivated (presumably permanently) by hydroxylation of N803. It is not clear, however, how this hydroxylation could efficiently proceed under conditions of hypoxia, the major physiologic inhibitor of FIH, without a significant up-regulation of FIH. Shin et al failed to discuss the conclusion of Birle and Hedley that FIH does not play a role in the inhibitory effect of bortezomib.3 In addition, their statement that "demonstration that CAD activity was regulated by FIH expression or knock-down even at an O2 tension of 1%, indicating that FIH regulates CAD even in hypoxia" was misassigned to our studies.2 Their statement that "bortezomib inhibited HIF-1 more so than proteasome" cannot be justified exclusively by the absence of stabilized HIF-1 or CITED2 (cAMP-responsive element–binding protein [CBP]/p300-interacting transactivators with glutamic acid [E] and aspartic acid [D]–rich tail 2) in bortezomib-treated samples (bortezomib inhibits chymotrypsin-like activity of the proteasome with Ki = 0.6 nM).4 Nevertheless, Shin et al speculated that the HIF-inhibitory effect of subnanomolar concentrations of bortezomib "may not be attributable to proteasome inhibition." Bortezomib is considered a highly specific inhibitor of proteasome and before being approved for clinical use, extensive screening found no other intracellular targets.4 Although we are not clear about the underlying cause(s), the discrepancies brought up in this Letter argue against the universal appeal of the mechanism outlined by Shin et al. More work is required before the mechanism of inhibition of HIF function by bortezomib is fully understood. Authorship Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: Stefan Kaluz, Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, CA 92697-4025; e-mail: skaluz{at}uci.edu. Stefan Kaluz, Milota Kaluzová, and Eric J. Stanbridge References Shin DH, Chun YS, Lee DS, Huang LE, Park JW. Bortezomib inhibits tumor adaptation to hypoxia by stimulating the FIH-mediated repression of hypoxia-inducible factor-1. Blood. 2008;111:3131–3136.[Abstract/Free Full Text] Kaluz S, Kaluzová M, Stanbridge EJ. Proteasomal inhibition attenuates transcriptional activity of hypoxia-inducible factor 1 (HIF-1) via specific effect on the HIF-1 CAD. Mol Cell Biol. 2006;26:5895–5907.[Abstract/Free Full Text] Birle DC, Hedley DW. Suppression of the hypoxia-inducible factor-1 response in cervical carcinoma xenografts by proteasome inhibitors. Cancer Res. 2007;67:1735–1743.[Abstract/Free Full Text] Kisselev AF, Goldberg AL. Proteasome inhibitors: from research tools to drug candidates. Chem Biol. 2001;8:739–758.[CrossRef][Medline] [Order article via Infotrieve] CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? Related Articles in Blood Online: Response: Oxygen-dependent effect of bortezomib on FIH-mediated repression of HIF-1 Dong Hoon Shin, Yang-Sook Chun, and Jong-Wan Park Blood 2008 111: 5259-5261. [Full Text] [PDF] Bortezomib inhibits tumor adaptation to hypoxia by stimulating the FIH-mediated repression of hypoxia-inducible factor-1 Dong Hoon Shin, Yang-Sook Chun, Dong Soon Lee, L. Eric Huang, and Jong-Wan Park Blood 2008 111: 3131-3136. [Abstract] [Full Text] [PDF] This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Kaluz, S. Articles by Stanbridge, E. J. Search for Related Content PubMed PubMed Citation Articles by Kaluz, S. Articles by Stanbridge, E. J. Related Collections Related Articles in Blood Online Social Bookmarking                   What's this?

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