Molecular mechanism of upregulation of survivin transcription by the AT-rich DNA-binding ligand, Hoechst33342: evidence for survivin involvement in drug resistance
Open Access
- 11 March 2007
- journal article
- research article
- Published by Oxford University Press (OUP) in Nucleic Acids Research
- Vol. 35 (7), 2390-2402
- https://doi.org/10.1093/nar/gkm149
Abstract
We have previously shown that hedamycin, a GC-rich DNA-binding antitumor agent, downregulates survivin transcription (Wu et al. (2005) Molecular mechanism of inhibition of survivin transcription by the GC-rich sequence selective DNA-binding antitumor agent, hedamycin: evidence of survivin downregulation associated with drug sensitivity. J. Biol. Chem., 280, 9745–9751). Here, we report that treatment of cancer cells with Hoechst33342, an AT-rich DNA-binding ligand, upregulated survivin protein, mRNA and promoter activity. Functional analysis of survivin promoter-luciferase constructs followed by in vivo footprinting experiments identified a 28-bp AT-rich DNA element (−908 to −881, designated as H369W) that mediates a major effect of Hoechst33342 on the upregulation of survivin promoter activity. Electrophoresis mobility shift assay (EMSA) experiments showed that Hoechst33342 binds to H369W and abrogates H369W–protein interactions. Intriguingly, there is a highly conserved DNA-binding motif for growth factor independence 1 (Gfi-1), a transcriptional repressor protein, in the H369W DNA element. Accordingly, EMSA experiments demonstrated that either the cold canonical Gfi-1-binding DNA oligonucleotide or the cold H369W specifically competes with H369W–protein complexes. Consistently, anti-Gfi-1 antibody is able to supershift the H369W–protein complex on the EMSA gel. Lastly, our data reveal that upregulation of survivin by Hoechst33342 is involved in cancer drug resistance. We propose that hindrance of H369W–Gfi-1 interactions in the survivin promoter, initiated by Hoechst33342, contributes to upregulation of survivin transcription, and as a consequence, hampers Hoechst33342's cytotoxicity.Keywords
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