2362

Survivin, a novel member of the inhibitor of apoptosis (IAP) protein family, is aberrantly expressed in cancer cells, and its expression is associated with tumorigenesis, cancer progression, radiation/drug resistance and shorter patient survival. Survivin is also expressed in certain human adult tissues and cells, and has been shown to play a role in physiology. However, targeting survivin for cancer treatment did not show obvious toxicity to normal tissues and cells. This implies that the mechanism for the regulation and function of survivin may actually be different in cancer cells versus normal cells. Therefore, studies of the transcriptional regulation of survivin will be important for development of novel approaches for cancer treatment. We have previously shown that Hedamycin, a GC-rich DNA-binding antitumor agent, downregulates survivin transcription (Wu et al., JBC, 280, 9745-51, 2005). 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 motif for Gfi-1 repressor proteins in H369W DNA element. Accordingly, EMSA experiments demonstrated that either the cold consensus Gfi-1-binding DNA oligonucleotide or the cold H369W specifically competes H369W-protein complexes. Consistently, anti-Gfi-1 antibody is able to supershift the H369W-protein complex on the EMSA gel. Lastly, our data showed that upregulation of survivin expression by Hoechst33342 is involved in drug resistance. We propose that the alteration of H369W DNA-protein interactions in the survivin promoter region contributes to the effect of Hoechst33342 on the upregulation of survivin transcription.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA