Molecular mechanism of upregulation of survivin transcription by the AT-rich DNA-binding ligand, Hoechst33342: evidence for survivin involvement in drug resistance.
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.
Wu
Jianguo
J
Department of Pharmacology, Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Apontes
Pasha
P
Song
Lei
L
Liang
Ping
P
Yang
Lily
L
Li
Fengzhi
F
D020856:AT Rich Sequence; D001562:Benzimidazoles / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D045744:Cell Line, Tumor; D018983:DNA Footprinting; D004268:DNA-Binding Proteins / Q000378:metabolism; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D006801:Humans; D051034:Inhibitor of Apoptosis Proteins; D008024:Ligands; D008156:Luciferases / Q000235:genetics; D008869:Microtubule-Associated Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D009363:Neoplasm Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D000077022:Survivin; D015533:Transcriptional Activation; D015854:Up-Regulation
C107489:BIRC5 protein, human; D001562:Benzimidazoles; D004268:DNA-Binding Proteins; D051034:Inhibitor of Apoptosis Proteins; D008024:Ligands; D008869:Microtubule-Associated Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D000077022:Survivin; D008156:Luciferases; C017807:bisbenzimide ethoxide trihydrochloride