Inhibition of Melanoma Angiogenesis by Telomere Homolog Oligonucleotides

Figure 2

T-oligo treatment increases E2F1 expression/activity and decreases HIF-1$\alpha$ DNA binding activity in MM-AN cells. Cells were treated with 40 $\mu$M T-oligo or diluent alone and harvested at various times. (a) The pellets were examined by qRT-PCR for E2F1 mRNA level, shown as a percent of time 0 levels (mean $\pm$ SEM) for 2 separate experiments in triplicate. (b) The pellets were also examined by western blot analysis for E2F1 protein levels. Actin expression was used as an internal loading control. (c) Densitometric analysis of E2F1 protein expression after loading adjustment by actin, represented as a percent of time 0 levels. Results are pooled data (mean $\pm$ SEM) from three independent experiments. (d) The DNA binding activity of E2F1 was analyzed by EMSA. No difference in E2F1 DNA binding activity was detected between the treatment groups at 16 hours (lane 1 versus 2) but E2F1 DNA binding activity doubled in T-oligo treated cells at 32 hours (lane 3 versus 4). Specificity of bands was confirmed by preincubating the nuclear protein of T-oligo-treated cells harvested at 32 hours with $\times$25 cold probe (lane 4 versus 5) and by supershift of E2F1 protein/DNA and E2F1 competing antibody complex (lane 4 versus 6). (e) Quantification of the band intensity of DNA binding activity is represented as relative density untis (RDU) for both treatment groups at 16 and 32 hours after treatment. E2F1 EMSA was repeated 2 times with similar results. (f) Nuclear protein was isolated from cells and processed for electromobility shift assay (EMSA) for evaluation of HIF-1$\alpha$ DNA binding activity. Specificity of the bands was confirmed by preincubating the nuclear protein extract of cells treated with diluent for 32 hours with $\times$25 cold probe (not labelled with ³²P) and mutant HIF-1$\alpha$ consensus sequence for 20 minutes before incubating the nuclear extracts with ³²P-labeled consensus oligonucleotides. HIF-1$\alpha$ EMSA was repeated 2 times with similar results. (g) Densitometric analysis of the protein/DNA complex bands for HIF-1$\alpha$ is graphed as relative densitometric units (RDU).