HPV E7 inhibition and Rb overexpression affects the activity of the Kpnβ1 and Kpnα2 promoters.

<p>A, B. HPV16 E7 siRNA was used to inhibit E7 expression in CaSki cells and Western blot analysis performed to analyse HPV E7 (A) and Rb (B) levels after HPV E7 knock-down. C, D. Kpnβ1 (C) and Kpnα2 (D) promoter activities were measured after E7 siRNA transfection, and were found to be significantly inhibited after HPV E7 inhibition. E, F. Rb was overexpressed in CaSki cells and Kpnβ1 (E) and Kpnα2 (F) promoter activities were found to be significantly inhibited. Results shown are the mean ± SE of experiments in quadruplicate performed at least two times (* p<0.05).</p

Kpnβ1 and Kpnα2 protein levels in normal, transformed and cancer cells correlate with respective promoter activities.

<p>A. Western Blot analysis of Kpnβ1 and Kpnα2 protein levels in normal WI38, transformed SVWI38, and cervical cancer CaSki cells reveals increased expression in the cancer and transformed cells. B, C. −2013 to +100 Kpnβ1 promoter activity (B) and −1900 to +69 Kpnα2 promoter activity (C) was measured in cell lysates prepared from transfected WI38, SVWI38 and CaSki cells, and was observed to be significantly higher in the transformed and cancer cells compared to the normal cells (*p<0.05). TK-Renilla-Luc was used as a control for transfection efficiency and luciferase activity is expressed relative to Renilla luciferase. Results shown are the mean ± SD of experiments performed in triplicate and repeated at least two times.</p

E2F/DP1 binds and activates the Kpnβ1 and Kpnα2 promoters <i>in vivo</i>.

<p>A, B. Samples of sonicated chromatin were immunoprecipitated with an α-E2F2 antibody, an α-DP1 antibody, or no antibody, as indicated. DNA isolated from immunoprecipitated material was amplified by PCR using primers spanning the E2F sites present in the Kpnβ1 (A) or Kpnα2 (B) promoters. Amplified fragments were analysed by electrophoresis on a 2% agarose gel. Experiments were performed in both SVWI38 and CaSki cell lines. C. Western Blot showing Kpnβ1 and Kpnα2 protein levels after Dp1 inhibition using siRNA. β-tubulin was used as a control for protein loading. D, E. Kpnβ1 and Kpnα2 promoter activities after Dp1 inhibition. Results shown are the mean ± SE of experiments in quadruplicate performed at least two times (* p<0.05).</p

Functional E2F sites in the Kpnβ1 (−637 to −271) and Kpnα2 (−180 to −24) promoter regions contribute to high promoter activity in transformed and cancer cells.

<p>A. Mutation of the five putative E2F sites in the Kpnβ1 (−637 to +100) promoter was carried out by site-directed mutagenesis. Mutation of the three distal E2F sites lead to a significant decrease in Kpnβ1 (−637 to +100) promoter activity, as indicated by luciferase assays. Mutation of the three functional E2F sites simultaneously did not lead to any further decrease in promoter activity. B. Mutation of the three E2F sites in SVWI38 and CaSki cells had a more profound impact on Kpnβ1 promoter activity compared to that in WI38 cells (numbers indicate fold repression). C. Mutation of the single E2F site in the Kpnα2 promoter completely abolished promoter activity. D. Mutation of the E2F site in SVWI38 and CaSki cells had a more profound impact on Kpnα2 promoter activity compared to that in WI38 cells. Experiments are shown as the mean ± SE of experiments in quadruplicate performed at least two times (* p<0.05).</p

Activity of Kpnβ1 and Kpnα2 promoter deletion constructs in WI38, SVWI38 and CaSki cells.

<p>A. Luciferase reporter constructs containing deleted fragments of the human Kpnβ1 promoter were transiently transfected into WI38 and SVWI38 cells, respectively. Luciferase activity was significantly higher in the SVWI38 cells compared to normal WI38 cells for all constructs tested. The −637 to +100 Kpnβ1 promoter fragment contained significantly more promoter activity than the −271 to +100 fragment in transformed cells (*p<0.05). B. Activity of Kpnβ1 promoter deletion constructs in CaSki cells. C. Luciferase reporter constructs containing deleted fragments of the human Kpnα2 promoter were transiently transfected into WI38 and SVWI38 cells, respectively. Deletion of the −180 to −24 promoter region resulted in significantly diminished promoter activity. D. Activity of Kpnα2 promoter deletion constructs in CaSki cells.</p