It is known that expression of survivin in cancer is associated with cancer progression and drug resistance (6
). Inhibition of survivin expression or survivin function appears to be important for cancer treatment (19
). Current studies indicate that various transcriptional factors and/or signaling molecules appear to transcriptionally and post-transcriptionally control the expression of survivin (20
). Importantly, growing evidence reveals that the transcriptional and/or post-transcriptional regulation of survivin expression appears to be different in cancer cells versus normal cells (20
). This important insight could provide exciting opportunities for cancer therapy without or with low toxicity to normal cells and tissues. Additionally, given the multiple subcellular localizations and multiple functions of survivin (4
), both understanding survivin transcriptional control at the molecular level and finding an easy way to modulate survivin transcription, has significant translational implications for the development of novel approaches for cancer treatment. We recently reported that a GC-rich sequence-selective DNA-binding antitumor agent, hedamycin, transcriptionally downregulates survivin expression through abrogation of Sp-1 or Sp1-like proteins, which bind to a 21-bp GC-rich motif in the survivin core promoter region and, that downregulation of survivin transcription by hedamycin, is associated with the enhancement of hedamycin's effectiveness to induce cancer cell death (8
). In the current study, we have characterized the effect of Hoechst33342, an AT-rich sequence-selective DNA-binding ligand, on the regulation of survivin gene transcription. We found that in contrast to the inhibition of survivin transcription by hedamycin, Hoechst33342 increases survivin protein, mRNA and promoter activity (). Importantly, this opposite or inverse modulation of survivin promoter activity by hedamycin compared to Hoechst33342 is achieved using an equi-cytotoxic concentration of the ligands, respectively. An equi-toxic concentration allowed us to make the unambiguous conclusion that the opposite effect on survivin expression is attributable to the opposite actions of the hedamycin and Hoechst33342. Specifically, Hoechst33342 upregulates survivin promoter activity at concentrations of 5, 10 and 20
μM (E), while hedamycin in the equi-cytotoxic range of concentrations () of 10, 25 and 50
nM strikingly downregulates survivin promoter activity (8
). Using survivin promoter-luciferase reporter assay, in vivo
footprinting, and EMSA experiments, we identified a 28-bp AT-rich DNA element (H369W) in which Hoechst33342 interacted with and abrogated DNA–protein interactions at this locus (–). Our in vivo
footprinting experiments revealed that Hoechst33342 treatment sensitizes the H369W AT-rich DNA element to DMS-mediated piperidine digestion, suggesting the abrogation of the DNA–protein interaction within the region (). This in vivo
data were further confirmed by the in vitro
DNA–protein interaction experiments (–). Using U937 cell nuclear extracts, we demonstrated that the cold canonical Gfi-1 binding DNA motif could compete with the H369W DNA–protein complex while the scramble DNA failed to do so (B). Consistently, the canonical Gfi-1 transcriptional repressor-binding site is highly conserved in the 28-bp AT-rich DNA element (H369W, A). This may explain the high efficiency of cold canonical Gfi-1 binding DNA oligonucleotides to compete with the H369W–protein complexes in the EMSA experiment (B). It is likely that Gfi-1 or Gfi1-like proteins bind to the H369W AT-rich DNA element before Hoechst33342 treatment. This was further confirmed by gel supershift assay experiments (C). However, we notice that while cold canonical Gfi-1 or H369W oligonucleotides effectively competed with the DNA–protein complexes (B), anti-Gfi-1 antibody was only able to supershift the upper band but not the lower band (C). A couple of possibilities may account for this inconsistency. First, the anti-Gfi-1 antibody we used in this study may not be able to recognize Gfi1-like protein. Second, the proteins in the lower band may be irrelevant to Gfi-1 or Gfi1-like proteins. In any case, the lower DNA–protein complex band shown in C is unlikely a non-specific DNA–protein complex since cold non-specific/scramble DNA could not compete with this DNA–protein complex but both cold canonical Gfi-1 and H369W oligonucleotides could do so (B). Nevertheless, the involvement of the identified 28-bp AT-rich DNA element (H369W) in survivin gene regulation was further supported by the fact that Hoechst33342 could upregulate survivin promoter activity from the pLuc-957 survivin promoter-luciferase construct containing the H369W motif but not from the pLuc-839 construct lacking H369W motif (D).
The possibility that Gfi-1 may suppress survivin gene transcription is suggested in the literature as well. For example, it has been demonstrated that Gfi-1 restricts hematopoietic stem cell proliferation (22–24
) and consistently, survivin is known to be involved in the promotion of cell proliferation (6
). It is possible that Gfi-1 inhibits cell proliferation through the suppression of survivin gene transcription. Thus, one explanation for the Hoechst33342-mediated increase survivin promoter activity is that the interaction of Hoechst33342 with the H369W AT-rich DNA element results in the dissociation of Gfi-1 or Gfi-1-like proteins from H369W, enhancing the permissiveness for survivin transcription. Here, we should point out that the characterized 28-bp AT-rich DNA elements may not be the only DNA element involved in Hoechst33342's effects on the upregulation of survivin promoter activity, although this DNA element appears to play a major role. For example, based on the functional data shown in B, the DNA fragment between pLuc-1332 and pLuc-1242 appears to be involved in the ligand's effect on survivin promoter activity as well. Consistent with this notion, there is an AT-rich DNA sequence within this region (−1303
It was previously reported that Hoechst33342 could affect the initiation of RNA polymerase II activity by altering the formation of the TATA-box binding protein (TBP) within the TATA box motif from the adenovirus-major-late-promoter in EMSA experiments (25
). The presence of Hoechst33342 (26.7
μM) decreased the amount of the control complex and increased the presence of lower molecular weight species, suggesting the degradation of nuclear TBP and/or the release of other transcription factors from the complex (25
). However, the above finding is unlikely to explain Hoechst33342's effects on survivin transcription. This is because the survivin promoter is a GC-rich promoter without a TATA-box motif. While there is no TATA-box motif within the survivin core promoter, several AT-rich elements exist upstream of the GC-rich core promoter region of survivin. One of these AT-rich elements is the 28-bp cis
-acting motif identified in this report (designated as H369W). Interestingly, in addition to the previous finding that Hoechst33342 interferes with the TBP/TATA-box motif complex formation to suppress gene transcription (25
), our experiments suggest an alternative ligand–DNA–protein interaction model in which Hoechst33342 actually displaces Gfi-1 or Gfi-1-like transcription suppressor proteins from the 28-bp AT-rich DNA element. However, we should point out that while our study identified a role of the 28-bp AT-rich DNA element in survivin gene transcription, this study has not excluded potential roles of other AT-rich sequences in survivin transcriptional controls. Furthermore, in the EMSA experiment both distamycin and DAPI at their equal concentrations to that of Hoechst33342 (10–20
nM) were unable to compete with the DNA–protein complexes (B). This does not exclude the possibility that at higher concentrations, distamycin or DAPI would still be unable to do so. Our experiments indicate that distamycin, at very high concentrations (10–400
μM), enhances survivin promoter activity (not shown), with the highest increase in survivin promoter activity at 400
μM. But this is in striking contrast to the dynamic pattern for Hoechst33342 in this study (E). Additionally, DAPI at the tested concentrations of 2, 10, 20, 100 and 1000
nM showed no effect on survivin promoter activity. These observations argue for the differential specificity for these ligand actions.
Hoechst33342 has been reported to be a cell death-inducing agent (26
). However, our experiments show that, compared to hedamycin, Hoechast33342 appears to be much less potent. Our data shown in indicate that hedamycin is at least over a hundred times more effective than Hoechst33342
at inducing cancer cell death. This is consistent with our observation that hedamycin downregulates but Hoechst33342 upregulates the expression of survivin. To determine whether upregulation of survivin by Hoechst33342 indeed contributes to Hoechst33342 resistance, we took advantage of our previously characterized survivin antagonists (14
) to counter the induction of survivin by Hoechst33342 during treatment (). Consistent with our previous finding that taxol/paclitaxel upregulates survivin, which increases cell viability and drug resistance (9
), forced expression of survivin antagonists sensitized cells to death induced by Hoechst33342 (). Given that survivin requires its Baculovirus IAP Repeat (BIR) domain to inhibit apoptosis and promote cell division, the sensitization of cells to drug-induced death could be either due to the induction of apoptosis or through forcing cells into a state (such as growth arrest), in which cells are easily attacked by antitumor agents. Nevertheless, using a low concentration of Hoechst33342 that does not induce cell death but substantially upregulates the expression of survivin, we demonstrated that Hoechst33342
at this concentration alleviates hedamycin-induced cell death, apparently a consequence of the induction of survivin expression by Hoechst33342. Together, these observations suggest that upregulation of the drug-resistant factor survivin by Hoechst33342 diminishes the effectiveness of the Hoechst33342 ligand to induce cell death.
Finally, it would be interesting to know if the identified 28-bp DNA element has general roles in transcriptional control of other genes. A comprehensive sequence search of the whole human genome indicated that the 28-bp AT-rich DNA element is highly conserved (≥20
bp of identify) in the putative promoter region of the CDCA2 (cell division cycle associated 2) gene as well as in the first intron of 17 additional human genes. Examples of these genes include the syndecan-binding protein (Syntenin/SDCBP), which was found to promote cell migration in metastatic breast and gastric cancer cells (27
), and the target of the myb1-like2 (TOM1L2) gene. Together, these findings argue that the identified AT-rich DNA element may play an important role in the regulation of gene transcription in general.
In conclusion, in this report, we have exposed a novel molecular mechanism by which Hoechst33342 upregulates survivin transcription. Our finding may provide new opportunities for the development of novel approaches and/or new ligands to modulate the expression of the survivin gene for cancer treatment.