NF-κB Hyper-Activation by HTLV-1 Tax Induces Cellular Senescence, but Can Be Alleviated by the Viral Anti-Sense Protein HBZ
Figure 4
Inhibition of NF-κB prevents the stabilization of p27 protein and the up-regulation of p21 mRNA, but not the trans-activation of p21 promoter by Tax.
(A) P21 and p27 protein levels in Ad-tTa-transduced HeLa-G (CTRL), ΔN-I-κBα L6, and ΔN-I-κBα H5 cells. Times (0, 2, 4 and 6 hours) after the addition of protein synthesis inhibitor, cycloheximide (100 µg/ml), are indicated. Immunoblots for p21 and p27 were done with all samples loaded in one gel. The comparable loading of protein samples in each set of 4 time points was confirmed by an actin immunoblot, which was sometimes done separately before the p21 and p27 blots were performed. (B) P21 and p27 protein levels in Ad-Tax-transduced parental HeLa-G (CTRL), ΔN-I-κBα L6, and ΔN-I-κBα H5 cells. Immunoblots were carried out as in (A) except the amount of lysate used in each lane of the control (CTRL) was one fifth that of the ΔN-I-κBα cell lines because of the dramatic p21 and p27 up-regulation by Tax in the former. (C) Quantitative analysis of p21 mRNA in Ad-tTa- (open bars) versus Ad-Tax- (solid bars) transduced HeLa-G (CTRL), ΔN-I-κBα L6, and ΔN-I-κBα H5 cells. (D) Transcriptional activation of p21-Luc reporter (−2.4 kB to +16 of p21 promoter fused to Luc) by Ad-tTa versus Ad-Tax in HeLa-G (CTRL), ΔN-I-κBα L6, and ΔN-I-κBα H5 cells. DNA transfection, luciferase assays and data analyses are as in Fig. 3A.