Figure 11.

Functional analysis of the Sp1 sites in the SCPO4 and SCPO5 DNA element in YM155-mediated inhibition of survivin promoter activity: A. Luciferase activity assays suggest the most proximate area in the survivin promoter plays a critical role in mediation of YM155’s inhibitory effect on survivin gene activity. Subconfluent EKVX and PC-3 cells were transfected overnight with the survivin core promoter-luciferase constructs with the indicated length as shown. Cells were then treated with YM155 or DMSO (control) for 24 hours, followed by the luciferase activity assay. B. Forced expression of Sp1 neutralized YM155-mediated inhibition of survivin promoter activity. Subconfluent PC-3 cells were cotransfected with both pLuc-269 and the Sp1 expression vectors or control vectors overnight. Cells were then treated with YM155 or DMSO (control) for 24 hours as shown, followed by luciferase activity assay. C. Mutation of the identified Sp1 sites diminished survivin promoter activity. PC-3 cells were transfected with wild type (pLuc197) or Sp1 site-mutated pluc197 alone (4m3, 5m6), or in combination (4m3/5m6) as shown. The transfected cells were grown in complete cell cultural medium for 16 hours, followed by serum starvation treatment for 24 hours. Cells were then lysed for the luciferase activity assay. Data in A, B, and C were normalized with the internal Renilla luciferase activity (internal control). The resultant data are presented in histograms as arbitrary units. Each bar is the mean ± SD derived from three independent assays run in triplicate.